Anda di halaman 1dari 778

S E C O N D E D I T I O N

Metabolic
and
Therapeutic
Aspects
of
Amino Acids
in
ClinIcal Nutrition
1382_C00.fm Page 2 Tuesday, October 7, 2003 5:54 PM
S E C O N D E D I T I O N

Metabolic
and
Therapeutic
Aspects
of
Amino Acids
in
ClinIcal Nutrition

Edited by
Luc a. Cynober

CRC PR E S S
Boca Raton London New York Washington, D.C.
1382_C00.fm Page 4 Tuesday, October 7, 2003 5:54 PM

Library of Congress Cataloging-in-Publication Data

Metabolic & therapeutic aspects of amino acids in clinical nutrition / edited by Luc A.
Cynober. --2nd ed.
p. ; cm.
Rev. ed of: Amino acid metabolism and therapy in health and nutritional disease. 1995.
Includes bibliographical references and index.
ISBN 0-8493-1382-1 (alk. paper)
1. Amino acids--Metabolism. 2. Amino acids--Pathophysiology. 3. Amino acids in
human nutrition. I. Title: Metabolic and therapeutic aspects of amino acids in clinical
nutrition. I. Cynober, Luc A. II. Amino acid metabolism and therapy in health and
nutritional disease.
[DNLM: 1. Amino acids--metabolism. 2. Amino Acids--therapeutic use. 3. Amino
Acids--administration & dosage. QU 60 M5867 2003]
QP561.A4615 2003
612.3'98—dc22 2003047263

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with
permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish
reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials
or for the consequences of their use.

Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical,
including photocopying, microfilming, and recording, or by any information storage or retrieval system, without prior
permission in writing from the publisher.

All rights reserved. Authorization to photocopy items for internal or personal use, or the personal or internal use of specific
clients, may be granted by CRC Press LLC, provided that $1.50 per page photocopied is paid directly to Copyright clearance
Center, 222 Rosewood Drive, Danvers, MA 01923 USA. The fee code for users of the Transactional Reporting Service is
ISBN 0-8493-1382-1/04/$0.00+$1.50. The fee is subject to change without notice. For organizations that have been granted
a photocopy license by the CCC, a separate system of payment has been arranged.

The consent of CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works,
or for resale. Specific permission must be obtained in writing from CRC Press LLC for such copying.

Direct all inquiries to CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431.

Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for
identification and explanation, without intent to infringe.

Visit the CRC Press Web site at www.crcpress.com

© 2004 by CRC Press LLC

No claim to original U.S. Government works


International Standard Book Number 0-8493-1382-1
Library of Congress Card Number 2003047263
Printed in the United States of America 1 2 3 4 5 6 7 8 9 0
Printed on acid-free paper
1382_C00.fm Page 5 Tuesday, October 7, 2003 5:54 PM

Dedication

Prof. Bernard Beaufrère Prof. Peter Reeds

The second edition of this book is dedicated to the memories of


Prof. Bernard Beaufrère and Prof. Peter Reeds.
Both were first-rate scientists, and their contributions to the field
of amino acid and protein metabolism were considerable.
They were life-loving people and are sorely missed
by their many friends across the world.

Luc A. Cynober
Editor
1382_C00.fm Page 6 Tuesday, October 7, 2003 5:54 PM
1382_C00.fm Page 7 Tuesday, October 7, 2003 5:54 PM

Preface
When CRC Press urged me a year ago to supervise a second edition of this book, I accepted
for several reasons. First, this book remained the only one devoted exclusively to the
metabolic and therapeutic aspects of amino acids in clinical nutrition. Second, in the 9
years since the first edition, a number of very important advances had been made in the
field. Third, some new topics had emerged with recent work, for example, on amino acids
and aging, and on arginine as a nutraceutical in cardiovascular disease. There were also
interesting new findings concerning taurine and other sulfur amino acids. Additionally, I
had reached the conclusion that the aftereffects of sports were a catabolic disorder and so
deserved a chapter. Finally, technical considerations concerning amino acid measurements,
which were lacking in the first edition, could now be addressed. So a second edition was
in order.
Most of the authors had already contributed to the first edition. They have done a
fine job once more, not only refining their earlier contributions, but also thoroughly
updating them to make this book a most valuable store of information and insight. Some
of the original authors were unable to contribute again for various reasons, but new
authors bring fresh ideas and new viewpoints. I warmly thank all the contributors.
Despite all possible efforts, some topics (AIDS, transplantation, etc.) remain unad-
dressed in this edition. I may be able to correct this in a third edition. Who knows?
Lastly, I am most grateful for the secretarial assistance of Solange Ngon in the prep-
aration of the book. Her perseverance in handling hundreds of e-mails, opening obstinate
attachments, and dealing with the different versions of the manuscripts was decisive for
a successful outcome.

Luc A. Cynober
Paris
1382_C00.fm Page 8 Tuesday, October 7, 2003 5:54 PM
1382_C00.fm Page 9 Tuesday, October 7, 2003 5:54 PM

About the Editor


Luc A. Cynober, Ph.D., is head of the Department of Clinical Biochemistry, Hotel-Dieu
Hospital, Paris, and is professor of nutrition and head of the Biological Nutrition Labo-
ratory at the School of Pharmacy, University of Paris, France.
Dr. Cynober obtained his Pharm.D. degree in 1979 from the School of Pharmacy, Paris
XI University. In 1985, he received his Ph.D. degree in biological and pharmaceutical
sciences from the same university.
Dr. Cynober is a member of the European Society of Parenteral and Enteral Nutrition,
the American Society of Parenteral and Enteral Nutrition, the French-Speaking Society of
Clinical Biology, and the French Society of Biochemistry and Molecular Biology, among
others.
He served from 1992 to 2000 as officer in the executive committee of the European
Society of Parenteral and Enteral Nutrition, and since 2001 as chairman of the French-
Speaking Society of Enteral and Parenteral Nutrition. He served (1987–1992) as editor-in-
chief of Nutrition Clinique & Métabolisme. He is presently (since 1998) editor-in-chief of
Current Opinion in Clinical Nutrition and Metabolic Care.
Among other awards, he has received the Pharmacy Academy Award for his Ph.D.
thesis and the International Research Award of the French Society of Clinical Biology.
Dr. Cynober has presented over 70 invited lectures at international and national
meetings and over 150 guest lectures at universities and institutes. He has published more
than 200 research papers. His current major research interests relate to amino acid metab-
olism and therapy in critical illness and aging.
1382_C00.fm Page 10 Tuesday, October 7, 2003 5:54 PM
1382_C00.fm Page 11 Tuesday, October 7, 2003 5:54 PM

Contributors List
Naji N. Abumrad Denis Breuillé
Vanderbilt University Medical Center Nestlé Research Center
Nashville, Tennesee Vers-Chez-Les-Blanc, Switzerland

Jorge E. Albina Philip C. Calder


Rhode Island Hospital and Brown Medical University of Southampton
School Southampton, United Kingdom
Providence, Rhode Island
Antonio C.L. Campos
Birgit Alteheld Federal University of Parana
University of Bonn Brazil
Bonn, Germany
Noël Cano
Adrian Barbul School of Pharmacy and Clinique
Sinai Hospital and Johns Hopkins Medical Résidence due Parc
Institutions Marseille, France
Baltimore, Maryland
Marika Collin
Vickie E. Baracos University of Helsinki
University of Alberta Helsinki, Finland
Edmonton, Alberta, Canada
Luc A. Cynobar
Bernard Beaufrére (Deceased) Hôtel-Dieu Hospital
Centre de Recherche en Nutrition Paris, France
Humaine d’Auverfue
Clermont-Ferrand, France Nicole M. Daignault
Emory University School of Medicine
Luc Bertrand Atlanta, Georgia
Institute of Cellular Pathology and
Université Catholique de Louvain S.W.M. Olde Damink
Brussels, Belgium Academic Hospital
Maastricht, The Netherlands
Petra G. Boelens
VU University Medical Center Dominique Darmaun
Amsterdam, The Netherlands Hôtel-Dieu Hospital
Nantes, France
Yves Boirie
Centre de Recherche en Nutrition
Humaine d’Auverfue
Clermont-Ferrand, France
1382_C00.fm Page 12 Wednesday, October 8, 2003 1:55 PM

Patrick David Daniel P. Griffith


Hôtel-Dieu Hospital Emory University School of Medicine
Paris, France Atlanta, Georgia

Jean-Pascal De Bandt George K. Grimble


Université Paris 5 University of Surrey Roehampton
Paris, France London, United Kingdom

Cornelis H.C. Dejong Svend Høime Hansen


Academic Hospital Rigshospitalet
Maastricht, The Netherlands Copenhagen University Hospital
Copenhagen, Denmark
Nicolaas E.P. Deutz Dieter Häussinger
Academic Hospital Heinrich Heine University
Maastricht, The Netherlands Dusseldorf, Germany

Ketan Dhatariya Milan Holecek


Mayo Clinic and Foundation Charles University School of Medicine
Rochester, Minnesota Hradec Králové, Czech Republic

Peter F. Dubbelhuis Louis Hue


Academic Medical Center Institute of Cellular Pathology and
Amsterdam, The Netherlands Université Catholique de Louvain
Brussels, Belgium
Filippo Rossi Fanelli
University ‘La Sapienza’ Karel W. Hulsewe
Rome, Italy Academic Hospital
Maastricht, The Netherlands
Charles J. Foulks
Gaetano Iapichino
Texas A&M University Health Science
Universitá degli studi di Milano
Center
Milan, Italy
Temple, Texas
Katsuhisa Inoue
Peter Fürst Medical College of Georgia
University of Bonn Augusta, Georgia
Bonn, Germany
R. Jalan
Malliga E. Ganapathy University College Medical School and
Medical College of Georgia UCLH Hospitals
Augusta, Georgia London, United Kingdom

Vadivel Ganapathy Asker E. Jeukendrup


Medical College of Georgia University of Birmingham
Augusta, Georgia Birmingham, United Kingdom

Michael Gleeson Kenneth A. Kudsk


Loughborough University The University of Wisconsin–Madison
Leicestershire, United Kingdom Madison, Wisconsin
1382_C00.fm Page 13 Tuesday, October 7, 2003 5:54 PM

Xavier M. Leverve Nathalie Neveux


Université Joseph Fourier Hôtel-Dieu Hospital
Grenoble, France Paris, France

Yvette Luiking Itzhak Nissim


Academic Hospital University of Pennsylvania School of
Maastricht, The Netherlands Medicine
Philadelphia, Pennsylvania
Michelle Mackenzie
University of Alberta Christiane Obled
Edmonton, Alberta, Canada Unité de Nutrition et Métabolisme
Protéique, INRA
Eric J. Mahoney Saint Genes Champanelle, France
Brown Medical School
Rhode Island Hospital Rudolf Oehler
Providence, Rhode Island University of Vienna
Vienna, Austria
Willy J. Malaisse
Brussels Free University Isabelle Papet
Brussels, Belgium Unité de Nutrition et Métabolisme
Protéique, INRA
Michael M. Meguid Saint Genes Champanelle, France
Upstate Medical University
Syracuse, New York Phillippe Patureau Mirand
Unité de Nutrition et Métabolisme
Alfred J. Meijer Protéique
Academic Medical Center Clermont-Ferrand, France
Amsterdam, The Netherlands
Puttur D. Prasad
Sidney M. Morris, Jr. Medical College of Georgia
University of Pittsburgh School of Augusta, Georgia
Medicine
Pittsburgh, Pennsylvania Danilo Radrizzani
Universitá degli studi
Laurent Mosoni Milan, Italy
Unité de Nutrition et Métabolisme
Protéique, INRA David K. Rassin
Clermont-Ferrand, France The University of Texas Medical Branch
at Galveston
Maurizio Muscaritoli Galveston, Texas
University ‘La Sapienza’
Rome, Italy D. Rémond
Unité de Nutrition et Métabolisme
K. Sreekumaran Nair Protéique
Mayo Clinic and Foundation Clermont-Ferrand, France
Rochester, Minnesota
Erich Roth
Thomas V. Nattakom University of Vienna
Memorial Medical Center Vienna, Austria
Las Cruces, New Mexico
1382_C00.fm Page 14 Tuesday, October 7, 2003 5:54 PM

Gordon S. Sacks Yasuo Wakabayashi


The University of Wisconsin–Madison Kyoto Prefectural University of Medicine
Madison, Wisconsin Kyoto, Japan

Peter B. Soeters Stéphane Walrand


Academic Hospital Centre de Recherche en Nutrition
Maastricht, The Netherlands Humaine d’Auverfue
Clermont-Ferrand, France
Peter Stehle
University of Bonn Tomas Welbourne
Bonn, Germany Louisiana State University/HSC
Shreveport, Louisiana
John F. Tharakan
Massachusetts Institute of Technology Jan Wernerman
Cambridge, Massachusetts Huddinge University Hospital
Karolinska Institutet
Paul A.M. van Leeuwen Stockholm, Sweden
VU University Medical Center
Amsterdam, The Netherlands Guoyao Wu
Texas A&M University
Heikki Vapaatalo College Station, Texas
University of Helsinki
Helsinki, Finland Parveen Yaqoob
The University of Reading
Stephan vom Dahl Reading, United Kingdom
Heinrich Heine University
Düsseldorf, Germany Vernon R. Young
Massachusetts Institute of Technology
Anton J.M. Wagenmakers Cambridge, Massachusetts
Maastricht University and University
Hospital Thomas R. Ziegler
Maastricht, The Netherlands Emory University School of Medicine
Atlanta, Georgia
1382_C00.fm Page 15 Tuesday, October 7, 2003 5:54 PM

Contents
Introduction ....................................................................................................................................1
John M. Kinney

Physiology and Physiopathology

Part I: Introduction to amino acid metabolism

Chapter 1 Measurement of amino acid concentrations in biological fluids


and tissues using ion exchange chromatography ...........................................17
Nathalie Neveux, Patrick David, and Luc Cynober

Chapter 2 Measurement of amino acid concentrations in biological fluids


and tissues using reversed-phase HPLC-based methods ..............................29
Birgit Alteheld, Peter Stehle, and Peter Fürst

Chapter 3 Approaches to studying amino acid metabolism: from quantitative


assays to flux assessment using stable isotopes ..............................................45
Dominique Darmaun and Luc Cynober

Chapter 4 Cellular uptake of amino acids: systems and regulation...............................63


Vadivel Ganapathy, Katsuhisa Inoue, Puttur D. Prasad, and Malliga E. Ganapathy

Part II: Physiology

Section A: Metabolism

Chapter 5 Amino acid metabolism and gluconeogenesis.................................................83


Xavier M. Leverve

Chapter 6 Contribution of amino acids to ketogenesis.....................................................97


Milan Holecek
ˇ

Chapter 7 Ureagenesis and ammoniagenesis: an update ............................................... 111


Alfred J. Meijer
1382_C00.fm Page 16 Tuesday, October 7, 2003 5:54 PM

Chapter 8 Metabolism of branched-chain amino acids in man.....................................123


Anton J.M. Wagenmakers

Chapter 9 The glutamate crossway ....................................................................................135


Yasuo Wakabayashi

Chapter 10 Arginine metabolism in mammals...................................................................153


Guoyao Wu and Sidney M. Morris, Jr.

Chapter 11 Glutamine metabolism .......................................................................................169


Rudolf Oehler and Erich Roth

Section B: Control of and by amino acids

Chapter 12 Insulin and the regulation of amino acid catabolism and protein
turnover ................................................................................................................185
Jean-Pascal De Bandt

Chapter 13 Control of amino acid metabolism by counterregulatory hormones.........201


Jan Wernerman

Chapter 14 Nitric oxide...........................................................................................................211


Eric J. Mahoney and Jorge E. Albina

Chapter 15 Control of amino acid metabolism by lipids, ketone bodies,


and glucose substrates .......................................................................................241
Yves Boirie, Stéphane Walrand, and Bernard Beaufrère

Chapter 16 Amino acid signaling and the control of protein metabolism ....................253
Alfred J. Meijer and Peter F. Dubbelhuis

Chapter 17 The role of amino acids in the control of proteolysis ...................................275


Stephan vom Dahl and Dieter Häussinger

Chapter 18 Anabolic effects and signaling pathways triggered by amino acids


in the liver ............................................................................................................291
Louis Hue and Luc Bertrand

Chapter 19 Amino acids and immune function .................................................................305


Philip C. Calder and Parveen Yaqoob

Chapter 20 Amino acid-mediated insulin secretion ..........................................................321


Willy J. Malaisse
1382_C00.fm Page 17 Tuesday, October 7, 2003 5:54 PM

Part III: Amino acid metabolism in disease

Chapter 21 Cancer-associated cachexia: altered metabolism of protein


and amino acids ..................................................................................................339
Michelle Mackenzie and Vickie E. Baracos

Chapter 22 Diabetes mellitus .................................................................................................355


Ketan Dhatariya and K. Sreekumaran Nair

Chapter 23 Acidosis and amino acid metabolism..............................................................375


Tomas Welbourne and Itzhak Nissim

Chapter 24 Muscle protein and amino acid metabolism with respect to


age-related sarcopenia ........................................................................................389
Stéphane Walrand and Yves Boirie

Chapter 25 Gastrointestinal disease......................................................................................405


Peter B. Soeters, Karel W. Hulsewe, Nicolaas E.P. Deutz, Yvette Luiking,
and Cornelis H.C. Dejong

Chapter 26 Amino acids and ammonia in liver disease ...................................................419


Cornelius H.C. Dejong, S.W.M. Olde Damink, R. Jalan, Nicolaas E.P. Deutz,
and Peter B. Soeters

Requirements and Supply

Part IV: Amino acid requirements

Chapter 27 Nutritional essentiality of amino acids and amino acid


requirements in healthy adults .........................................................................439
Vernon R. Young and John F. Tharakan

Chapter 28 Neonatal requirements for amino acids..........................................................471


David K. Rassin

Chapter 29 Amino acid requirements in the elderly .........................................................483


Phillippe Patureau Mirand, L. Mosoni, and D. Rémond

Chapter 30 Amino acid requirements in sport ...................................................................497


Michael Gleeson and Asker E. Jeukendrup
1382_C00.fm Page 18 Tuesday, October 7, 2003 5:54 PM

Part V: Amino acid supply in diseases

Section A: Quantitative and qualitative aspects

Chapter 31 Quantitative and qualitative amino acid intake by the parenteral


route.......................................................................................................................519
Gaetano Iapichino, Danilo Radrizzani, and Luc A. Cynober

Chapter 32 Quantitative and qualitative aspects of nitrogen supply in enteral


nutrition in relation to free amino acids and peptides.................................529
George K. Grimble

Chapter 33 Branched-chain amino and keto acids in renal failure.................................557


Noël Cano

Chapter 34 Glutamine-supplemented diets in enteral nutrition .....................................577


Petra G. Boelens and Paul A.M. van Leeuwen

Chapter 35 The use of arginine in clinical practice............................................................595


Naji N. Abumrad and Adrian Barbul

Chapter 36 Glutamine and glutamine-containing dipeptides..........................................613


Peter Fürst and Peter Stehle

Chapter 37 Ornithine a-ketoglutarate ..................................................................................633


Luc A. Cynober

Section B: Formulas devoted to specific situations

Chapter 38 Amino acid support in patients with catabolic illness .................................649


Nicole M. Daignault, Daniel P. Griffith, Thomas V. Nattakom, and Thomas R. Ziegler

Chapter 39 Sulfur-containing amino acids and glutathione in diseases ........................667


Christiane Obled, Isabelle Papet, and Denis Breuillé

Chapter 40 Amino acid requirement in cancer...................................................................689


Maurizio Muscaritoli, Filippo Rossi Fanelli, Michael M. Meguid,
and Antonio C.L. Campos

Chapter 41 Amino acid solutions for acute renal failure..................................................705


Charles J. Foulks

Chapter 42 Amino acids to support gut function and morphology...............................717


Gordon S. Sacks and Kenneth A. Kudsk
1382_C00.fm Page 19 Tuesday, October 7, 2003 5:54 PM

Section C: Nutraceutics

Chapter 43 L-arginine-enriched diets in cardiovascular diseases ...................................729


Marika Collin and Heikki Vapaatalo

Chapter 44 Taurine homeostasis and its importance for physiological functions........739


Svend Høime Hansen

Index .............................................................................................................................................749
1382_C00.fm Page 20 Tuesday, October 7, 2003 5:54 PM
1382_C00 Intro.fm Page 1 Tuesday, October 7, 2003 5:56 PM

Introduction

John M. Kinney

A review of the history of proteins and amino acids at the beginning of the twentieth
century makes it apparent that the physiology of the protein was moving toward the
physiology of amino acids. The recognition of individual amino acids had developed for
over 100 years when Rose reported threonine in 1936.1 Yet the chemical identification of
individual amino acids involved an immense amount of time and skill, which usually
required the isolation of a material that could be weighed. This situation underwent major
changes in the 1940s and 1950s when new methodology changed the measurement of
amino acids to rapid and automated procedures.
Advances of the 1940s were associated with the stimulus of the wartime search for
antibiotics such as gramicidin. During this period came the discovery that certain bacteria
could be used quantitatively to measure the presence of specific amino acids. Fruton2 has
summarized the advances of this period. Martin and Synge introduced paper chromatog-
raphy with the use of ninhydrin. This was followed by the column chromatography of
Moore and Stein, using starch and then resins for ion exchange chromatography. This
technique was quickly enhanced by the addition of a fraction collector, producing an
automated system for separation of a mixture of amino acids. The next advance was high-
performance reversed-phase liquid chromatography (HPLC). Subsequent refinements and
advances in the measurement of amino acids are discussed in the early chapters of this
volume.
Balance studies to determine the amount of protein required to achieve nitrogen
balance in normal adults occupied much attention in the first third of the 1900s. However,
as attention turned to the necessary intake of amino acids rather than protein, the work
of W.C. Rose over 20 years was definitive in establishing the intake of individual amino
acids required for the growth and health of the laboratory rat.
This led to data in 1957 from balance studies in man that listed the essential amino
acid requirements for a healthy human diet.3 These studies included the identification of
threonine but did not include histidine and arginine, which were felt to be nonessential.
A consistent finding was that even when the estimates for amino acid requirement were
based on the highest levels of each essential amino acid indicated by previous studies,
the total was still quite low relative to estimates of the total protein needs.
Adult protein requirements had not been a high-priority area of research between
1920 and 1960 since protein needs seemed to be readily met in practice. However, the U.S.
Space Agency in 1960 became concerned about how astronauts remaining in space for
long periods should be fed. Remarkably detailed long-term studies were conducted at
Berkeley by Calloway and coworkers and at MIT by Scrimshaw and coworkers.1 Despite
some variability in results, there was general agreement that the requirements for nitrogen
balance considerably exceeded the theoretical estimate for optimum amino acid intake,

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 1
1382_C00 Intro.fm Page 2 Tuesday, October 7, 2003 5:56 PM

2 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

suggesting that adults used even high-class proteins with no more than approximately
60% efficiency. The FAO/WHO/UNU report in 1985 concluded that 52.5 g of first-class
protein would meet the needs of 97% of the adult population. Various groups were unable
to obtain nitrogen balance on such small intakes.4
Vernon Young of MIT urged repeatedly that it was unsafe to accept the 1985 interna-
tional standards for adult amino acid requirements, based solely on the results of nitrogen
balance studies. Young and his colleagues began a major research program using an
approach that focused on an indirect measure of the balance of individual amino acids.
The details of these methods, which involved C13-labeled amino acids, have been carefully
described in Chapter 27.
A general conclusion from such studies was that the adult requirement for individual
essential amino acids (leucine, lysine, and threonine) appeared to be considerably greater
than the standards derived from earlier balance studies. Because of the errors in balance
studies and the complexity of the isotope studies, final conclusions on amino acid require-
ments have remained controversial, yet there has been growing acceptance that the former
estimates were too low.
Many hormones and cytokines are involved in the regulation of amino acid metabo-
lism. The leading anabolic hormone, insulin, continues to be of particular interest in
relation to muscle tissue. Insulin deficiency has long been known to be associated with
muscle wasting while visceral protein is affected less or not at all. However, the insulin
action on muscle tissue continues to revolve around whether protein synthesis is stimu-
lated or protein breakdown is inhibited.
Muscle protein synthesis has been shown in vitro to involve gene transcription, mes-
senger RNA translation, and activation of preexisting enzymes. This is followed by an
increase in tissue RNA content, of both ribosomal RNA and messenger RNA. In contrast
to in vitro findings, the in vivo studies in rats are less clear but generally support the effect
of insulin as stimulating muscle protein synthesis. The situation in man is even less clear.
Wolfe4 has summarized the errors in interpretation of using a tracer amino acid such as
phenylalanine in a limb balance study to determine synthesis and breakdown directly
from the rates of appearance and disappearance in the blood. He noted that combining a
direct measure of the fractional synthesis rate and the arteriovenous balance/intramus-
cular pool during hyperinsulinemia in the human leg revealed that insulin was associated
with more efficient reutilization of intracellular amino acids. The systemic infusion of
insulin causes a reduction in blood amino acid concentrations. In this circumstance, insulin
does not stimulate muscle protein synthesis. When this insulin-induced hypoaminoaci-
demia is avoided by an appropriate infusion of amino acids, insulin is seen to stimulate
muscle protein synthesis.
The ubiquitin–proteosome system is primarily responsible for protein breakdown in
resting muscle. An acute increase in insulin concentration, such as occurs after a meal,
has little effect on this system. This is in contrast to long-term regulation of the ubi-
quitin–proteosome pathway, where a stimulated production of mRNA encoding ubiliq-
uitin and proteosome subunits is associated with an increased transcription of the ubiq-
uitin gene.
In contrast to the ubiquitin–proteosome pathway, the lysosomal breakdown of protein
is responsive to acute changes in insulin concentration. Yet lysosomes do not normally
play an important role in myofibrillar breakdown. This is consistent with findings that
local hyperinsulinemia does not increase protein breakdown in resting muscle. Wolfe4
summarizes the actions of insulin on muscle protein as follows: at rest the basal insulin
concentration seems to play a role in curtailing the ubiquitin–proteosome pathway of
muscle protein breakdown, but acute increases in insulin (as after a meal) may have little
effect on muscle protein breakdown. This would be different if the individual is in a
1382_C00 Intro.fm Page 3 Tuesday, October 7, 2003 5:56 PM

Introduction 3

stressed state or is studied immediately after exercise where the lysosomal pathway of
breakdown contributes significantly to the breakdown process and is therefore responsible
for the suppressive action of insulin.
The relative rates of growth and protein synthesis are higher during the neonatal
period than at any other stage of postnatal life, and more rapid gains occur in skeletal
muscle than in other parts of the protein mass. This feeding-induced stimulus of protein
synthesis is most dramatic in skeletal muscle and decreases profoundly with development.
Insulin is recognized as a key factor in the regulation of skeletal muscle protein synthesis.
Insulin sensitivity and responsiveness of amino acid disposal decrease with development.
The response of protein synthesis to insulin infusion declines with development in parallel
with the developmental decline in the stimulation of muscle protein synthesis by feeding.
However, whether insulin and amino acids interact at submaximal doses to stimulate
skeletal muscle protein synthesis remains uncertain.
O’Connor et al.5 utilized a pancreatic-amino acid clamp in fasted neonatal pigs to
block insulin secretion, while glucose and glucagon were maintained at fasting levels.
Insulin was infused at different amounts, and at each insulin dose, amino acids were
clamped at either the fasting or fed level. The results showed that insulin and amino acids
act independently to stimulate protein synthesis in skeletal muscle of the neonate. Davis
et al.6 used this model to show that exogenous insulin growth factor-1 (IGF-1) stimulates
protein synthesis in skeletal muscle and other insulin-sensitive tissues of the neonate,
presumably because IGF-1 acts on the same signaling pathway as insulin leading to
translation initiation.
Calbet and MacLean7 measured the insulin and glucagon responses to the rate of
appearance of amino acids after ingestion of different isonitrogenous solutions in humans.
The insulin response was closely related to the increase in plasma levels of leucine,
isoleucine, valine, phenylalanine, and arginine, regardless of the rate of gastric emptying.
Consumption of a protein-containing meal enhances the fractional rate of synthesis of
total mixed proteins in skeletal muscle. The feeding-induced stimulation of protein syn-
thesis requires the hormone insulin and an adequate supply of amino acids. The relative
contribution of these regulatory factors to the increase in protein synthesis is controversial,
particularly since some amino acids independently influence protein synthesis by enhanc-
ing insulin release. Anthony et al.8 have obtained evidence in the rat that transient increases
in serum insulin are permissive for the leucine-induced stimulation of protein synthesis
in skeletal muscle. This rise in insulin contributes to the hyperphosphorylation of trans-
lational factors and suggests that the signaling pathway involving mammalian target of
rapamycin (mTOR) may be a convergence point for both leucine- and insulin-mediated
effects on certain steps in translation initiation. However, unknown steps in mRNA trans-
lation appear to be rate controlling in the stimulus of protein synthesis by leucine.
Protein synthesis and degradation are closely regulated in vivo, and each is affected
by physiological and pathophysiological conditions such as fasting, feeding, exercise,
disease, and aging. Davis and Reeds9,10 have reviewed the advantages and problems of
different isotopic methods for quantifying protein turnover in vivo, considering the meth-
ods of tracer dilution in contrast to methods involving tracer incorporation. These authors
found that measurements made on the basis of labeling plasma and breath are well suited
for the measurement of body amino acid oxidation and balance, yet underestimate protein
turnover. They also note that leucine may be the most useful labeled amino acid, for
measuring both whole-body and muscle protein synthesis, because of the close isotopic
equilibrium between muscle-free and tRNA-bound leucine pools. The authors analyze the
use of tracer infusion and the flooding dose technique for measuring the incorporation of
tracer amino acids into tissue protein. The best estimate of the aminoacyl-tRNA precursor
pool for the constant infusion method will depend upon the organ or tissue under study.
1382_C00 Intro.fm Page 4 Tuesday, October 7, 2003 5:56 PM

4 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Wolfe and Miller11 edited a group of papers devoted to the influence of the dietary
pattern and composition of ingested protein on protein metabolism. The rise in muscle
protein sysnthesis occurs relatively quickly with the rise in extracellular amino acid con-
centration. The latency and duration of amino acid stimulation of muscle protein were
reported to be important since the system becomes refractory after 2 h of constant elevation
of amino acid concentration. A study by Tipton et al.12 demonstrated that an acute response
of net muscle protein balance could reflect the 24-h balance after exercise and amino acid
ingestion.
In contrast to the rapid absorption and delivery after ingestion of free amino acids,
intact proteins are digested at variable rates, a factor that appears to independently
regulate postprandial protein gain. These differences are further modified by transient
elevations in substrates and hormones, as well as factors independent of the food source,
such as exercise, age, or metabolic stress. The splanchnic area, which sees very rapid
turnover of protein metabolism, seems to respond to almost all nutritional perturbations,
whereas the peripheral muscle protein mass, which is slowly renewed, appears to be only
slightly affected by acute dietary factors.
Liu and Barrett13 discuss what information can be obtained from whole-body protein
turnover studies, utilizing tracer infusion methods. A comparison of the flux data for
protein, triglycerides, and glycogen reveals that protein is unique in that there is no storage
form that is not already serving another significant purpose. Despite having a higher
energy requirement to synthesize a protein than to store energy as either carbohydrates
or fat, the turnover rate of the body’s protein pool is substantially higher than that of
either of the other two principal fuels. However, the fraction of amino acids liberated from
body protein via one or another proteolytic pathway that is subsequently oxidized fully
is substantially less than that fraction for either fat or carbohydrates. Thus, a selective
advantage appears to accrue to the organism when the building blocks of proteins are
used sparingly as oxidative fuel, whereas the protein pool per se turns over rapidly at
considerable energy cost to the body.
Protein synthesis and degradation are each regulated by multiple hormonal as well
as nutritional factors, and the protein balance of individual tissues, as well as the whole
body, changes constantly. Three peptide hormones, insulin, insulin growth factor-1, and
growth hormone, affect body protein metabolism acutely and have been studied exten-
sively. Recent findings for each hormone have been reviewed for the whole body and
individual organs by Liu and Barrett.13 These investigators note that for insulin there
appears to be a dissociation between the doses that affect protein synthesis vs. those that
affect protein degradation. It is suggested that proteolysis is more sensitive than synthesis
to small changes in plasma insulin within its physiological range. Inasmuch as insulin
and IGF-1 exert anabolic actions on both protein synthesis and proteolysis, evidence is
reviewed suggesting that the cellular signals that mediate insulin and IGF-1 action in
muscle, although similar, diverge significantly despite the fact that each hormone can exert
a major influence on both synthesis and degradative pathways. Growth hormone increases
lean body mass while decreasing fat mass, although the mechanisms remain unclear.
Biolo et al.14 have reviewed kinetic studies in clinical states to search for relationships
between rates of protein synthesis and breakdown. It appears that when protein synthesis
is primarily suppressed, protein degradation is found unchanged or even slightly
decreased. Where protein breakdown is primarily accelerated, the rate of synthesis is
unchanged or even increased. Apparent discrepancies among various studies of chronic
disease may arise from the many factors influencing protein metabolism. When the effects
of inflammatory mediators and stress hormones start to overwhelm the factors that tend
to decrease protein synthesis, the rate of turnover will increase along with a net increase
in the rate of protein breakdown.
1382_C00 Intro.fm Page 5 Tuesday, October 7, 2003 5:56 PM

Introduction 5

The process of proteolysis has been recognized for many years. However, it was
formerly considered to be a nonselective process mainly involved in basal protein turnover,
the elimination of abnormal proteins, and perhaps the regulation of certain key enzymes.
More recently, the characterization and regulation of proteolysis have revealed the exist-
ence of three separate systems, the most prominent being the ubiquitin–proteosome sys-
tem.15 The details and metabolic steps of this system have been presented by Hasselgren.16
The ubiquitin–proteosome pathway is the major nonlysosomal process responsible
for the breakdown of most short- and long-lived proteins in mammalian cells. Abnormal
proteins, which are misfolded, oxidized, or mutant, are very good substrates of this system.
There are two main steps in the pathway: the covalent attachment of a polyubiquitin chain
to the substrate and the specific recognition of this signal and degradation of the tagged
protein by the 16S proteosome. This ubiquitinylation not only is a degradation signal but
also directs proteins to a variety of fates, including roles in DNA repair, protein translo-
cation, or modulating the structure or activity of the target proteins.17 In order to be
efficiently degraded, the substrate must be bound to a polyubiquitin degradation signal
that comprises at least four ubiquitin moieties.
Conaway and co-workers18 discuss new roles for ubiquitin in the regulation of RNA
polymerase II (Pol II) transcription that are being discovered at an accelerating pace. It
is evident that the integral role of ubiquitin in Pol II transcription also involves the
complex and multifaceted nature of ubiquitin’s participation in this process. These
authors emphasize that future investigations can be expected to reveal a fundamental
and perhaps general role for ubiquitin in some of the most basic aspects of transcription
activation and repression.
The proteosome is a self-compartmentalizing protease, as substrates must enter the
catalytic chamber within, in order to be degraded into peptides. Multiple active sites are
confined within the small chamber. The proteosome hydrolyzes most peptide bonds and
generates peptides that are typically 3 to 22 amino acids long and do not conserve
biological properties, except for antigen presentation. Recent evidence indicates that sev-
eral proteosome-dependent pathways can be involved in the breakdown of a single sub-
strate.19 This might explain how a cell could rapidly modulate the half-life of various
proteins in response to the cell environment.20 In order for the myofibrillar proteins actin
and myosin to be ubiquinated and degraded by the proteosome, the myofilaments must
first be released from the sarcomere by the calcium-dependent protease calpain.19
Muscle cachexia has been induced by severe injury, sepsis, and cancer where there is
increased gene expression and activity of the calcium/calpain and ubiqitin–proteosome
proteolytic pathways. Despite certain exceptions, most proteins require ubiquination
before catabolism. In cancer cachexia, this process is controlled by a tumor-produced
sulfated glycoprotein.21 A proteolysis-inducing factor (PIF) detected in the urine of weight-
losing cancer patients has been detected in gastrointestinal tumors.22 In vivo studies have
shown that PIF induces catabolism of skeletal muscle, while visceral protein reserves are
preserved. PIF also appears to be involved in the inflammatory response observed in
cachexia, related to increased production of interleukin (IL)-6, IL-8, and C-reactive protein
and a decreased production of transferrin.23
Eicosapentaenoic acid (EPA) has been shown to decrease protein catabolism with
activation of the ubiquitin–proteosome pathway. Animal evidence of reducing protein
catabolism by EPA in starvation suggests that a similar mechanism may be involved in
conditions other than cancer.24
The cascade of various neurologic stimuli that can contribute to loss of appetite and
the catabolism of starvation has been reviewed by Nandi and co-workers.25 Recent studies
have focused on the interactions between Ca++ and the proinflammatory cytokines (in
particular tumor necrosis factor-a) and the activation of transcription factors such as
1382_C00 Intro.fm Page 6 Tuesday, October 7, 2003 5:56 PM

6 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

nuclear factor-kB for the stimulation of major proteolytic pathways in cachexia.


Langhans26 has reviewed interrelationships between hypermetabolism and the catabolism
of lipid and protein in cachexia, emphasizing steps that hold promise for future pharma-
ceutical therapy.
Proteins are synthesized by translation of their mRNAs, a process carried out by the
ribosome. The process is divided into stages: translation, elongation of the nascent peptide,
and termination of peptide synthesis. The rate of elongation and termination is determined
by the amount of aminoacylated tRNA available to decode the mRNA and by the activities
of various translation factors. However, evidence is emerging that the nascent peptide
itself, within the ribosome that is making it, can profoundly affect mRNA translation.27
A supply of a complete complement of essential amino acids is a prerequisite for
maintenance of optimal rates of protein synthesis in both liver and skeletal muscle.28
Degradation of even a single essential amino acid causes a decrease in the synthesis of
essentially all cellular proteins through an inhibition of the initiation phase of mRNA
translation. However, the synthesis of all proteins is not repressed equally. Specific subsets
of proteins, in particular those encoded by mRNAs containing an F-terminal oligopyrim-
idine (TOP) motif, are affected to a much greater extent than most proteins. The specific
decrease in TOP mRNA translation is a result of an inhibition of the ribosomal protein S6
kinase, S6K1, and a concomitant decline in S6 phosporylation. Interestingly, many TOP
mRNAs encode proteins involved in mRNA translation, such as elongation factors eEFIA
and eEF2, as well as the ribosomal proteins. Thus, deprivation of essential amino acids
not only directly and rapidly represses global mRNA translation but also potentially
results in a reduction in the capacity to synthesize protein.
Over the past several decades, biologists have unraveled some of the ways cells
communicate with each other. The long-distance messages that the cells exchange involve
hundreds of different proteins, such as hormones and growth factors. The receivers are a
multitude of specialized receptors on the surface of target cells. Beyond these fundamen-
tals, however, the picture of cellular communication gets sketchy. There are very few cases
in which scientists understand all the intricate biochemical steps that flow from message
received to action inside the cell.28
Protein kinases and phosphorylation play a key role in many intracellular signaling
pathways. Because kinases catalyze the addition of a phosphate group to another protein
or enzyme, investigators often look for proteins that are rapidly phosphorylated, in the
hope that they will be part of a cell’s response to an extracellular message.
The presence and importance of amino acid transport and receptor proteins have been
biochemically identified for some years. However, it is only in the past decade that
definitive reports have appeared regarding the structure and function of these proteins.
cDNAs have recently been reported that encode proteins capable of the activities of the
Na+-dependent systems (A and N) and the Na+-independent systems (T and asc). This
new information will make possible the exploration of how individual transport proteins
in apical and basolateral peptide transport activities are coordinated with other transport
factors to achieve cellular, tissue, and whole-body amino acid flux capacity.29
Major progress has been made in the potential role of the excitatory receptors AMPA
(preferentially gated low-conductance ion channels permeable to Na and K and voltage
independent) and NMDA (large-conductance ion channels permeable to the Ca ion).
Characterization of these channels is important for both molecular composition and turn-
over in the synaptic membrane. Hypoxic-ischemic brain injury in animals is followed by
the lethal overstimulation of glutamate receptors. This research holds great promise for
understanding and treating hypoxic-ischemia encephalopathy in particular, as well as
addictive behavior and other neurologic disorders.
1382_C00 Intro.fm Page 7 Tuesday, October 7, 2003 5:56 PM

Introduction 7

Amino acids serve as substrates for many functions besides the obvious role of protein
synthesis. These include gluconeogenesis and ureagenesis. However, they can also serve
as regulators of cell metabolism. Chapter 16 in this volume discusses ways in which amino
acid signaling can influence protein metabolism. The focus begins with the discovery of
Haussinger et al.30 that cell swelling, associated with the Na+-dependent influx of some
amino acids into cells or with the intracellular accumulation of amino acid catabolites,
such as glutamate and aspartate, exerts both anabolic and anticatabolic effects.
Later work has revealed the existence of an amino acid-stimulated signal transduction
pathway that shares components and acts in concert with a signaling pathway that is
stimulated by insulin. It is this pathway that simultaneously controls autophagic protein
degradation and protein synthesis, but in opposite directions. A central player in the
intracellular pathways discussed by these authors is mTOR. The mechanism by which
amino acids activate mTOR remains unknown, although several possibilities are presented.
The fact that amino acid signaling can behave with insulin-like actions in regard to
protein synthesis and also promote autophagic protein degradation provides a unique
challenge. The authors point out that the amino acid signaling pathway differs from
glutamate signaling via glutamate receptors in the central nervous system. Here these
receptors gate cation channels, and their activation causes depolarization and an increase
in cytosolic Ca ++ , together with interacting with phosphatidylinositol 3-kinase
(PI 3-kinase).
In amino acid signaling, the authors speculate that mTOR may also sense amino acid-
induced increases in cell volume. The importance of defining this pathway may shed light
on cancer growth and perhaps lead to treatment with agents such as rapamycin and its
analogues, which might not only inhibit protein synthesis but at the same time accelerate
autophagic protein degradation.
The anabolic effects of amino acids are further explored in Chapter 18. Hue and
Bertrand observe that in addition to their mass effect on protein synthesis, certain amino
acids inhibit autophagy in the liver. They contrast the effects of insulin, glutamine, and
leucine on protein synthesis in isolated liver cells. The central role of mTOR in the control
of protein synthesis by nutrients and energy is such that it has been proposed to act as
an ATP sensor of the cell. After presenting the detailed anabolic response of glutamine
associated with Na+ movement, they report that leucine transport does not depend on
Na+ and therefore does not cause cell swelling or glutamate-activated protein phosphatase
(GAPP) activation. The work of these authors suggests that two separate phosphatases
are involved, one upstream of mTOR and one downstream. They also note that AMPK,
a protein kinase activated by hypoxia, is able to inactivate other enzymes in the anabolic
pathway, perhaps explaining the inhibition of protein synthesis observed during oxygen
deprivation.
In recent years the intestine has received increasing attention as an organ of unique
metabolic activity involving particular amino acids. Chapter 42 discusses the intestine
from the separate perspectives of the mucosa, the local immune system, and the control
of the vasculature. This presentation focuses on the dietary provision of glutamine,
glutamate, ornithine, arginine, and glutathione and the importance of each for intestinal
integrity.
Evidence is presented that enteral glutamine is important to maintain mucosal thick-
ness, villus height, and cell count, as well as normal permeability. Of particular interest
is the idea that while increased permeability can be measured with lactulose, mannitol,
or xylose, this does not imply increased permeability to bacteria or endotoxin molecules,
which are significantly larger in size. Oral glutathione (GSH) as well as GSH monoesters
provide effective transport and conversion into intracellular GSH, while intact GSH is
poorly transported into enterocytes.
1382_C00 Intro.fm Page 8 Tuesday, October 7, 2003 5:56 PM

8 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Secretory immunoglobulin A (sIgA) is produced through the interaction of sensitized


B and T cells in the lamina propria below the mucosa and moves to the mucosal surfaces
of the gastrointestinal and respiratory tracts to prevent bacterial and viral adherence to
epithelial cell layers and mucosal penetration. Glutamine has direct effects in supporting
the function of B and T cells within the mucosa to provide defenses against intraluminal
infectious agents. Provision of intravenous nutrition that lacks glutamine has shown that
the intestinal changes have implications on extraintestinal sites like the respiratory tract
as well. Thus, the concept of a common mucosal immune system (MALT) has been
proposed, which is active in defense against both bacterial and viral infections. Evidence
is presented that intracellular stores of GSH promote the production of interleukin (IL)-2
and interferon (IFN)-gamma, thus affecting IgA synthesis and mucosal protection.
Sacks and Kudsk present evidence that changes occur in an intracellular adhesion
molecule (ICAM)-1, which is responsible for adhesion between the endothelium and
polymorphonuclear neutrophils (PMNs). This can produce deleterious effects as the gut
serves as a priming bed for circulating neutrophils. Both IL-10 and IL-4 normally inhibit
ICAM-1 expression, and both cytokines decrease in response to parenteral nutrition. A
study in mice involved administration of isotopic ICAM-1 antibody and myeloperoxidase
(MPO), an enzyme found primarily in PMNs. While on intravenous nutrition, the expres-
sion of intestinal ICAM-1 significantly increased along with increases in MPO levels and
was quickly reversed with chow refeeding.
Isotopic albumin was used to measure vascular permeability in various tissues and
lung, while PMN accumulation was assessed with MPO. A significantly higher vascular
permeability was seen in lung and liver while on intravenous nutrition. Pulmonary tissues
showed marked increases in expression of CD18, a marker for PMN cell priming, while
no increases were seen in the enterally fed mice. Glutamine (GLN)-supplemented intra-
venous nutrition produced major increases in survival for 72 h after 15 min of mesenteric
artery occlusion compared to those not receiving GLN. The results of GLN were thought
to possibly reflect prevention of mucosal changes associated with increased permeability
or a decreased production of oxygen free radicals by PMNs. Other studies have suggested
that ischemia-reperfusion activates protein kinase cascades that regulate the expression
of proinflammatory genes. Excessive gene activation can lead to decreased gut-associated
lymphoid tissue (GALT) and ultimately multiple organ failure. The amino acid GLN is of
special interest since it has been shown to have beneficial effects on all three aspects —
mucosa, immunity, and vascularity — of the intestine.
Some benefits of GLN may result from an influence on arginine production. Arginine
has been reported to facilitate mucosal recovery after ischemia-reperfusion by preventing
the reduction in mucosal blood flow, presumably by providing a source of nitric oxide
(NO).
Inflammation is the response of the immune system to the damage caused by chem-
icals, physical insults, or pathogenic organisms. Although painful, inflammation is usually
a healing response. This response can spiral out of control, leading to shock and death,31,32
or it can proceed to a chronic state associated with debilitating disease. Only recently has
the great significance of inflammation been recognized in the etiology of atherosclerosis.33
The points of control in inflammation involve signaling pathways between circulating
white cells and their inflammatory products, such as hormones, cytokines, and others.34
Some biochemical agents can serve as important signals while under other circumstances
they may play deleterious roles. Notable in this regard is the molecule NO, which arises
from the amino acid arginine.
Chapter 14 on nitric oxide separates the biology and mechanism of action from a
detailed discussion of pathophysiology in mammalian cells. The sheer number and diver-
sity of roles attributed to NO are astounding: the control of blood pressure, emptying of
1382_C00 Intro.fm Page 9 Tuesday, October 7, 2003 5:56 PM

Introduction 9

the stomach, penile erection, release of neurotransmitters and hormones, learning and
memory, pain sensation, and protection of cells from intracellular parasites are some of
the roles. Many of the reports seem contradictory. NO is reported to both promote and
suppress apoptosis; it is both an oxidant and an antioxidant; it is cytostatic yet promotes
tumor growth. What can explain this?
Lane and Gross35 have reviewed many of the factors that contribute to this remarkable
diversity of function. NO is unlike any other cellular messenger. It is a lipophilic, reactive,
free radical, gaseous molecule, representing the combination of two of the most abundant
atoms in our atmosphere. It is a free radical with an unpaired electron in its outer orbit,
yet it does not react readily. This slow reactivity, combined with its lipophilic nature,
allows NO to diffuse rapidly through most cells and tissues with little consumption or
reaction. Instead of binding selectively to a specific receptor, it engages in chemical reac-
tions with multiple protein targets. These may be enzymes, receptors, structural proteins,
or transcription factors. Whether the net effect of NO in a given biological system is
beneficial or deleterious to an organism is determined by the relative availability of
alternative protein targets and the duration and amount of NO produced.
The large diversity of functions may be further explained by NO being an array of
three interchangeable species (NO*, NO+, and NO–), each with a distinct spectrum of
chemical and biological activities. Synthesis of NO is mediated by a family of three
mammalian gene products termed nitric oxide synthases (NOSs). All three catalyze the
oxidation of one of the guanidinonitrogens of L-arginine, yielding NO and L-citrulline.
Each of the three NOS isoforms differs in its tissue distribution, subcellular localization,
and mode of regulation. The two constitutive forms (neuronal and endothelial) are regu-
lated predominantly by changing levels of intracellular Ca++. The remaining isoform is
the inducible NOS (iNOS), resulting from exposure to immunostimulants, which is con-
tinuous and high output, and Ca++ independent. It is regulated at the transcriptional
level, and once the protein is expressed, a large continuous flux of NO ensues that is
limited only by substrate availability.
Asymmetrical dimethylarginine (ADMA) is an endogenously produced inhibitor of
nitric oxide synthase, whereas symmetrical dimethylarginine (SDMA) competes with
arginine for transport. The metabolism of these two compounds is largely unknown.
However, ADMA is subject to enzymatic degradation by an enzyme that is highly
expressed in the liver. Nijveldt et al.36 have conducted studies in the rat demonstrating
that the liver plays an important role by removing ADMA from the systemic circulation.
Stuhlinger et al.37 present evidence indicating that ADMA is elevated in many disorders
and appears to be associated with endothelial dysfunction. There is great interest in
whether therapy that improves insulin resistance will also improve endothelial function
while lowering ADMA.38 Nijveldt et al.39 have reported that high plasma ADMA concen-
tration is an independent risk factor of mortality in intensive care patients.
Professor Cynober deserves special commendation for assembling modern contribu-
tions to such an ever-broadening field. The subject of the book embraces science in the
depths of the cell, the integration of tissues and organs, and, ultimately, the future of
optimum nutrition in human health and disease.

References
1. Tanford, C. and Reynolds, J., Nature’s Robots: A History of Proteins, Oxford University Press,
Oxford, 2001, 30, 304 pp.
2. Fruton, J.S., Proteins, Enzymes, Genes: The Interplay of Chemistry and Biology, Yale University
Press, New Haven, CT, 1999, 5, pp. 212–215.
1382_C00 Intro.fm Page 10 Tuesday, October 7, 2003 5:56 PM

10 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

3. Carpenter, K.J., Vitamins and amino acids 1910–1950, in Protein and Energy: A Study of
Changing Ideas in Nutrition, Cambridge University Press, Cambridge, U.K., 1994, pp. 119–141.
4. Wolfe, R.R., Effects of insulin on muscle tissue, Curr. Opin. Clin. Nutr. Metab. Care, 3, 67–71,
2000.
5. O’Connor, P.M.J., Bush, J.A., Suryawan, A., Nguyen H.V., and Davis, T.A., Insulin and amino
acids independently stimulate skeletal muscle protein synthesis in neonatal pigs, Am. J.
Physiol., 284, E110–E119, 2003.
6. Davis, T.A. et al., Acute IGF-I infusion stimulates protein synthesis in skeletal muscle and
other tissues of neonatal pigs, Am. J. Physiol., 283, E638–E647, 2002.
7. Calbet, J.A.L. and MacLean, D.A., Plasma glucagon and insulin responses depend on the
rate of appearance of amino acids after ingestion of different protein solutions in humans,
J. Nutr., 132, 2174–2182, 2002.
8. Anthony, J.C., Lang, C.H., Crozier, S.J., Anthony, T.G., MacLean, D.A., Kimbal, S.R. and
Jefferson, L.S., Contribution of insulin to the translational control of protein synthesis in
skeletal muscle by leucine, Am. J. Physiol., 282, E1092–E1101, 2002.
9. Reeds, P.J. and Davis, T.A., Of Flux and Flooding: The Advantages and Problems of Different
Isotopic Methods for Quantifying Protein Turnover In Vivo: 1. Methods Based on the Dilution of a
Tracer, Lippincott Williams & Wilkins, Baltimore, 1999, pp. 23–28.
10. Davis, T.A. and Reeds, P.J., Of Flux and Flooding: The Advantages and Problems of Different
Isotopic Methods for Quantifying Protein Turnover In Vivo: 11. Methods Based on the Incorporation
of a Tracer, Lippincott Williams & Wilkins, Baltimore, 2001, pp. 51–56.
11. Wolfe, R.R. and Miller, S.L., Supplement: protein metabolism in response to ingestion pattern
and composition of proteins, J. Nutr., 132, 3207S–3218S, 2002.
12. Tipton, K.D., Borsheim, E., Wolf, S.E., Sanford, A.P., and Wolfe, R.R., Acute response of net
muscle protein balance reflects 24-h balance after exercise and amino acid ingestion, Am. J.
Physiol., 284, E76–E89, 2003.
13. Liu, Z. and Barrett, E.J., Human protein metabolism: its measurement and regulation, Am.
J. Physiol., 283, El 105–El 112, 2002.
14. Biolo, G., Antonione, R., Barazzoni, R., Zanetti, M., and Guarnieri, G., Mechanisms of altered
protein turnover in chronic diseases: a review of human kinetic studies, Curr. Opin. Clin.
Nutr. Metab. Care, 6, 55–63, 2003.
15. Attaix, D., Combaret, L., Pouch, M.N., and Taillandier, D., Regulation of proteolysis, Curr.
Opin. Clin. Nutr. Metab. Care, 4, 45–49, 2001.
16. Hasselgren, P.O., Molecular regulation of muscle wasting, Sci. Med., 230–239, 2002.
17. Ciechanover, A., Orian, A., and Schwartz, A.L., Ubiquitin-mediated proteolysis: biological
regulation via destruction, Bioessays, 22, 442–451, 2000.
18. Conaway, R.C., Brower, C.S., and Conaway, J.W., Emerging roles of ubiquitin in transcription
regulation, Sci. Compass, 296, 1254–1258, 2002.
19. Tanahashi, N., Murakami, Y., Minami, Y., et al., Hybrid proteasomes: induction by interferon-
gamma and contribution to ATP-dependent proteolysis, J. Biol. Chem., 275, 14336–14345, 2000.
20. Jang, J.S. and Choi, Y.H., Proteolytic degradation of the retinoblastoma family protein, p 107:
a putative: cooperative role of calpain and proteasome, Int. J. Mol. Med., 4, 487–492, 1999.
21. Lorite, M.J., Smith, J.J., Arnold, J.A., et al., Activation of ATP-ubiquitin-dependent proteolysis
in skeletal muscle in vivo and murine myoblasts in vitro by a proteolysis-inducing factor
(PIF), Br. J. Cancer, 85, 297–302, 2001.
22. Cabal-Manzano, R., Bhargava, P., Torres-Durate, A., et al., Proteolysis inducing factor is
expressed in tumours of patients with gastrointestinal cancers and correlates with weight
loss, Br. J. Cancer, 94, 1599–1601, 2001.
23. Watchom, T.M., Waddell, I.D., Dowidar, N., and Ross, J.A., Proteolysis inducing factor reg-
ulates hepatic gene expression via the transcription factors NF-B and STAT3, FASEB J., 15,
562–564, 2001.
24. Tisdale, M.J., Biochemical mechanisms of cellular catabolism, Curr. Opin. Clin. Nutr. Metab.
Care, 5, 401–405, 2002.
25. Nandi, J., Meguid, M., Inui, A., Xu, Y., Makarenko, I.G., Tada, T., and Chen, C., Central
mechanisms involved with catabolism, Curr. Opin. Clin. Nutr. Metab. Care, 5, 407–418, 2002.
1382_C00 Intro.fm Page 11 Tuesday, October 7, 2003 5:56 PM

Introduction 11

26. Langhans, W., Peripheral mechanisms involved with catabolism, Curr. Opin. Clin. Nutr.
Metab. Care, 5, 419–426, 2002.
27. Sachs, M.S. and Geballe, A.P., Sense and sensitivity: controlling the ribosome, Science, 297,
1820–1821, 2002.
28. Kimball, S.R., Regulation of global and specific mRNA translation by amino acids, J. Nutr.,
883–886, 2002.
29. Matthews, J.C. and Anderson, K.J., Recent advances in amino acid transporters and excitatory
amino acid receptors, Curr. Opin. Clin. Nutr. Metab. Care, 5, 77–84, 2002.
30. Haussinger, D., Hallbrucker, C., vom Dahl, S., et al., Cell volume is a major determinant of
proteolysis control in liver, FEBS Lett., 283, 70–72, 1991.
31. Cohen, J., The immunopathogenesis of sepsis, Nature, 420, 885, 2002.
32. Hotchkiss, R.S. and Karl, I.E., The pathophysiology and treatment of sepsis, N. Engl. J. Med.,
348, 138–148, 2003.
33. Libby, P., Inflammation in atherosclerosis, Nature, 420, 868, 2002.
34. Nathan, C., Points of control in inflammation, Nature, 420, 846, 2002.
35. Lane, P. and Gross, S.S., Nitric oxide: promiscuous and duplicitous, Sci. Med., 96–107, 2002.
36. Nijveldt, R.J., Terrlink, T., Siroen, M.P.C., Vanlambalgen, A.A., Rauwerda, J.A., and
VanLeeuwen, P.A.M., The liver is an important organ in the metabolism of asymmetrical
dimethylarginine (ADMA), Clin. Nutr., 22, 17–22, 2003.
37. Stuhlinger, M.C., Abbasi, F., Chu, J.W., et al., Relationship between insulin resistance and an
endogenous nitric oxide synthase inhibitor, JAMA, 287, 1420–1426, 2002.
38. Nash, D.T., Insulin resistance, ADMA levels, and cardiovascular disease, JAMA, 287,
1451–1452, 2002.
39. Nijveldt, R.J., Teerlink, T., VanDeffloven, B., Siroen, M.P.C., Kuik, D.J., Rauwerda, J.A., and
VanLeeuwen, P.A.M., Asymmetrical dimethylarginine (ADMA) in critically ill patients: high
plasma ADMA concentration is an independent risk factor of ICU mortality, Clin. Nutr., 22,
23–30, 2003.
1382_C00 Intro.fm Page 12 Tuesday, October 7, 2003 5:56 PM
1382_C01.fm Page 13 Tuesday, October 7, 2003 5:57 PM

Physiology and Physiopathology


1382_C01.fm Page 14 Tuesday, October 7, 2003 5:57 PM
1382_C01.fm Page 15 Tuesday, October 7, 2003 5:57 PM

Part I

Introduction to amino acid metabolism


1382_C01.fm Page 16 Tuesday, October 7, 2003 5:57 PM
1382_C01.fm Page 17 Tuesday, October 7, 2003 5:57 PM

chapter one

Measurement of amino acid


concentrations in biological fluids
and tissues using ion exchange
chromatography
Nathalie Neveux
Hôtel-Dieu Hospital
Patrick David
Hôtel-Dieu Hospital
Luc Cynober
Hôtel-Dieu Hospital

Contents
Introduction....................................................................................................................................18
1.1 Sampling and storage of the biological material for analysis ......................................19
1.1.1 Blood..........................................................................................................................19
1.1.2 Urine ..........................................................................................................................19
1.1.3 Other biological fluids and tissues .......................................................................20
1.2 Deproteinization of biological samples ............................................................................20
1.3 Principle of ion exchange chromatography.....................................................................20
1.3.1 Factors influencing the separation........................................................................21
1.3.1.1 Resin............................................................................................................21
1.3.1.2 Buffers .........................................................................................................22
1.3.1.3 pH ................................................................................................................22
1.3.1.4 Temperature ...............................................................................................22
1.3.2 Detection ...................................................................................................................22
1.3.3 Data processing, internal standards, and calibration solutions.......................24
1.4 Interferences ..........................................................................................................................24
1.5 Conclusion .............................................................................................................................25
References .......................................................................................................................................26

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 17
1382_C01.fm Page 18 Tuesday, October 7, 2003 5:57 PM

18 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Introduction
Quantification of free amino acids present in biological fluids and tissues is an important
tool in biomedical and nutritional research and in the diagnosis of various disease states,
especially metabolic deficiencies. The main diagnostic application of free amino acid
profiling is for blood, urine, and amniotic fluid1–3 (Table 1.1). Other biological fluids such
as breast milk, saliva, synovial fluid, cerebrospinal fluid, and tears are analyzed much less
frequently.2,7 Amino acids can also be measured in numerous cells and tissues (e.g., in
liver and muscles).2
The classical amino acid analysis techniques involve separation of amino acids by ion
exchange chromatography (IEC), followed by postcolumn continuous reaction with nin-
hydrin. Originally, the analysis was performed at a constant temperature on a two-column
system, each column requiring a separate sample injection; the acidic and neutral amino
acids were separated using two elution buffers on one column, and the basic amino acids
using a single buffer on the other column.
Since the first reports8,9 describing the separation of plasma amino acids on a sul-
fonated polystyrene resin column, followed a few years later by the first automated amino
acid analyzer developed by Spackman et al.,10 many improvements have been made,
essentially to obtain faster and more sensitive analysis. Thus, time required for a complete
amino acid analysis of physiological fluids using an automatic amino acid analyzer has
decreased considerably, from 4 days in 195811 to less than 2 h today (including the column
regeneration time). In parallel, the sensitivities of these analyses have gradually increased,
and thresholds of 50 to 100 pmol and 1 pmol of amino acid have been reached using

Table 1.1 Amino Acid Concentrations in Adult Human Plasma, Urine, and Cerebrospinal
Fluid (CSF) Determined by Ion Exchange Chromatography
Amino Acids Plasma [4]a Urine [5]b CSF [6]a
Taurine 55 ± 13 16–180 (72) 6.8 ± 1.7
Aspartate 3 ±1 2–7 (4) 0.6 ± 0.3
Threonine 140 ± 33 7–29 (13) 27.7 ± 4.7
Serine 114 ± 19 21–50 (30) 24.5 ± 4.4
Asparagine 41 ± 10 5.4 ± 1.4
Glutamate 24 ± 15 <12 11.3 ± 6.4
Glutamine 586 ± 84 20–76 (36) 444 ± 84
Proline 168 ± 60 <9
Glycine 230 ± 52 43–173 (107) 4.7 ± 1.5
Alanine 333 ± 74 16–68 (30) 23.2 ± 5.1
Citrulline 38 ±8 <4 1.5 ± 0.5
Valine 233 ± 43 3–13 (5) 15 ± 2.8
Cysteine 52 + 11 6–34 (13) 0.1 + 0.1
Methionine 25 ±4 2–16 (6) 1.9 ± 0.7
Isoleucine 62 ± 14 <4 3.9 ± 1.0
Leucine 123 ± 25 2–11 (5) 10.1 ± 2.1
Tyrosine 59 ± 12 2–23 (10) 6.4 ± 1.5
Phenylalanine 57 ±9 2–19(7) 6.5 ± 1.2
Ornithine 55 ± 16 <5 3.7 ± 1.0
Histidine 82 ± 10 26–153 (79) 11.9 ± 1.7
Lysine 188 ± 32 7–58 (17) 21.7 ± 3.7
3-MH 3 ±2 19–47 (32)
Arginine 80 ± 20 <5 18.3 ± 3.2
a Mean ± SD, mmol/l.
b Range (and mean), mmol/mol of creatinine.
3-MH: 3-Methylhistidine.
1382_C01.fm Page 19 Tuesday, October 7, 2003 5:57 PM

Chapter one: Measurement of amino acid concentrations in biological fluids and tissues 19

colorimetric detection and fluorescence, respectively. Further improvements have followed


the development of automatic sample loading, flow-cell design and electronic stabilization
of the detectors, reduction of the signal-to-noise ratio, function timing, and data process-
ing. Most often, a computer is used not only to operate the apparatus but also to carry
out diagnostic tests such as measuring pressure limits or monitoring buffer volumes.

1.1 Sampling and storage of the biological material for analysis


1.1.1 Blood
There are differences between some amino acid concentrations in plasma and serum,
probably linked to platelet disruption during clotting,1 and plasma samples are generally
preferred. The choice of the anticoagulant is important; heparin appears to be most often
used, although when in excess, it can cause hemolysis and consequently release ninhydrin-
positive substances from red blood cells.12 Problems may also arise with EDTA, which can
contain contaminants that cause the appearance of unusual peaks.12–14
After blood centrifugation, the plasma must be immediately sampled to avoid con-
tamination by intracellular amino acids.12,15 The plasma then needs to be deproteinized as
quickly as possible (within 1 h of blood sampling).16,17 The deproteinized sample can be
stored at –70˚C until analysis without any loss of amino acid, for several months2 to at
least 1 year.18
However, if the deproteinization cannot be carried out rapidly after blood samples
have been obtained, an alternative consists of immediately freezing the separated plasma
at –30˚C for 30 h. The frozen plasma can then be stored at –70˚C for 4 weeks before protein
removal and analysis.19 If the specimens cannot be stored at –70˚C but only at –20˚C, it is
preferable to deproteinize them, because at –20˚C amino acids are more stable in depro-
teinated plasma than in native plasma.20,21 Other reports22,23 have also recommended that
samples be stored at neutral as well as deproteinized and deep-frozen.
Capillary blood sampling from newborns is made at the heel or finger. After removal
of the first drop, at least three spots are taken on paper cards. The cards are then dried at
room temperature, and disks of controlled size are punched out and placed in diluent
buffer. After centrifugation, the supernatant, which contains free amino acids, is treated
and analyzed as for plasma.24 Blood collected on filter paper can be stored for up to 2 weeks
at room temperature (providing it is completely dry) and for at least 21 weeks at +4 to
–70˚C.25

1.1.2 Urine
As urinary amino acid content undergoes circadian variation, 24-h urine must be used for
quantitative analysis of amino acids.2 It is essential to avoid fecal and bacterial contami-
nation, which may increase or decrease the concentrations of many amino acids in urine.25
It is usual to add a preservative such as chloroform or thymol to the container. The sample
should be stored at temperatures not exceeding 4˚C, but it is preferable to freeze it at –20˚C
or below, especially if analysis cannot be performed within 24 h. Repeated freezing and
thawing increases amino acid levels and should be avoided.2 When the sample has to be
transported, particularly at ambient temperature, changes in the amino acid composition
may occur if no bactericidal agent is added. In normal situations, the low level of urinary
proteins does not interfere with amino acid profiling. In pathological states, if the presence
of an abnormal amount of proteins is measured or suspected, samples need to be depro-
teinized. In practice, it is better to deproteinize in any case, simply because this ensures
that samples have a constant pH.
1382_C01.fm Page 20 Tuesday, October 7, 2003 5:57 PM

20 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

1.1.3 Other biological fluids and tissues


The same general recommendations as those for plasma are made for the storage of other
biological fluids: if the sample contains proteins, it should be rapidly deproteinized and
stored at –70˚C if it cannot be analyzed immediately.
Amino acid determination in cerebrospinal fluid is carried out first to diagnose non-
ketotic hyperglycinemia and second to measure the neurotransmitters g-aminobutyric acid
(GABA), aspartic, and glutamic acids in research studies. In the latter case, it may be better
not to deproteinize, because the neurotransmitters are present predominantly as conju-
gates and may be degraded during protein removal.26
Tissues and biopsies should be placed in liquid nitrogen immediately after sampling.

1.2 Deproteinization of biological samples


Proteins must be removed from biological fluids before they are applied to an ion exchange
resin column, because they stick to the resin, causing peak spreading and increased column
back pressure.
Numerous techniques can be used to deproteinize biological fluids before amino acid
analysis. Among these, physical techniques such as high-speed centrifugation, dialysis,
and ultrafiltration are currently not often used because, although they can achieve 100%
recovery,27 they do not completely remove proteins.28 Ultrafiltration procedures, although
simple, are not widely applied in practice because they decrease the retention times during
amino acid separation by ion exchange chromatography.2 This results in distorted sepa-
rations of critical pairs of amino acids, particularly serine–threonine and tyrosine–phenyl-
alanine.
The most widely used method for the deproteinization of a biological sample (plasma,
urine, cerebrospinal, and amniotic fluids) before ion exchange analysis is chemical pre-
cipitation with sulfosalicylic acid (SSA). It is preferable to use as little of the precipitating
reagent as possible. This represents 20 to 50 mg of the substance per milliliter of sample.
When larger amounts are used, the ion exchange separation of amino acids is distorted.29
SSA is added directly either as a powder or as a solution. Samples containing SSA should
be stirred immediately and, after 10 min, centrifuged to remove the precipitated proteins.
After SSA treatment, the acid does not have to be removed from the supernatant before
analysis, and the pH, between 1.0 and 2.0, is almost ideal for subsequent ion exchange
chromatography. Because precipitation may cause amino acid losses (from 2 to 20%), it is
important to add an internal standard.20,27 With picric acid, recoveries are better, but the
acid has to be removed before analysis by ion exchange chromatography.9,12
Deproteinization with perchloric acid or trichloracetic acid30 is usually used for tissues
because they are homogenized in these acids, but this is also possible with SSA (10:1,
w/v).31

1.3 Principle of ion exchange chromatography


The free amino acids are separated by high-performance ion exchange chromatography
with postcolumn derivatization. The resolution is based on differential interaction with a
negatively charged stationary phase (cation exchanger). The amino acids are eluted with
a gradient of acidic buffers applied stepwise. As they elute from the column, amino acids
are mixed with the derivatizing solution and sent through a reaction coil, and the reaction
products are detected continuously. Amino acids are identified and quantified by relating
the peaks to an internal standard and to a standard mixture analyzed at the start of every
new analysis series (defined as the use of a new ninhydrin solution; see below).
1382_C01.fm Page 21 Tuesday, October 7, 2003 5:57 PM

Chapter one: Measurement of amino acid concentrations in biological fluids and tissues 21

Buffers Valves

Buffer Sample
pump loader
3

Sample

4
Chart recorder/
data handling
5
Column

Regeneration
buffer

Ninhydrin Ninhydrin Mixing Reaction Detector


reservoir pump manifold coil

Figure 1.1 Schematic representation of a cation exchange amino acid analyzer.

The main components of a cation exchange amino acid analyzer are set out in
Figure 1.1.

1.3.1 Factors influencing the separation


To obtain a good chromatographic separation, it is necessary to have the right combination
of resin, buffers, pH, and temperature.

1.3.1.1 Resin
The properties of the resin largely influence the performance of the analysis and especially
the separation of the amino acids. The resin consists of small beads of polystyrene, sul-
fonated to provide a negative electrical charge, and reacted with divinylbenzene to achieve
around 8 to 10% cross-linkage.32,33 The optimal cross-linkage is about 8%.8,34 At higher
values, the resolution is poor, while at lower values, the resin distorts under pressure.
The charged groups (SO3–) are associated with mobile counter-ions, either Na+ or, more
frequently, Li+. Amino acids are injected in the column at an acidic pH (about 2.2) at which
ionization of the carboxyl group is suppressed and most amino acids have a net positive
charge. They enter the pores of the resin and displace some of the bound Li+. Separation
is primarily due to differences in the pKa of amino acids and follows the general order:
acidic, neutral, basic.8,35 At pH 2.2, the most basic amino acids (histidine, lysine, and
arginine) are bound most strongly, and the most acidic (aspartic and glutamic acids) least
strongly. Increasing the pH of the mobile phase toward the isoelectric point of a given
amino acid causes loss of charge and desorption of that amino acid. Increasing the strength
of the eluting buffers decreases the interaction with the resin and also causes desorption.
Particle size of the resin is also a critical factor because it determines the diffusion
time needed to reach equilibrium. Since 1958 the advantages of decreasing particle size
1382_C01.fm Page 22 Tuesday, October 7, 2003 5:57 PM

22 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

for increased speed and resolution in amino acid analysis have been demonstrated.36 In
practice, the use of increasingly fine resin beds has allowed the operating temperature,
column length, and diameter to be reduced, which in turn affords greater sensitivity and
speed of analysis. The particles are spherical with diameters ranging from 5 to 10 mm.

1.3.1.2 Buffers
To obtain a satisfactory analysis of physiological fluids, four or five buffers are required.
Currently, the most frequently used buffers are lithium citrate buffers, which have super-
seded sodium citrate buffers and have allowed the resolution of asparagine, glutamic acid,
and glutamine.37 Thiodiglycol is added as an antioxidant to prevent loss of methionine.28
Addition of organic solvents such as propanol improves the resolution of threonine and
serine.25
Chemical contamination is a recurring problem, arising from impurities in
commercial38 and extemporaneous buffers, water, and glassware, the major source of
contamination being the water used to prepare the buffer solutions.28 The use of a water
purification system is essential for the preparation of buffers, which must be membrane-
filtered before use.38 Microbial synthesis is another possible source of amino acid contam-
ination: adding a preservative such as phenol or caprylic acid inhibits bacterial growth.
The introduction of precolumn filters and buffer refrigeration in modern analyzers also
limits contamination of the column.2,38
Another problem linked to the buffer solutions is the presence of high ammonia
concentrations that may affect the baseline. This problem can be solved by introducing
an extra column between buffer outlets and the column inlet.39
The flow rate of the buffers is also critical: if it is too fast, there is asymmetry and
tailing of the peaks, loss of resolution, and increased back pressure. Precise control of both
timing of buffer change and flow rates is included in the modern analyzer.

1.3.1.3 pH
pH is critical for resolution. When it is too high, the amino acid peaks elute prematurely,
with poor resolution, and if it is too low, they elute late and are wider. The most sensitive
amino acids are those first eluted (see Figure 1.2). To avoid errors of identification with
automatic integration, the pH of the sample must be close to that of the calibrating standard
mixture.

1.3.1.4 Temperature
The separation of amino acids is dependent on temperature, partly because the pH of
citrate buffers increases with temperature, and partly because of altered affinity of amino
acids for the resin. Generally, the temperature of the column is held below 40˚C until
glutamine has eluted (see Figure 1.2) to minimize its loss and is subsequently increased
stepwise.1

1.3.2 Detection
The classical amino acid analysis technique involves separation of amino acids by cation
exchange chromatography, followed by postcolumn derivatization by a continuous reac-
tion with ninhydrin.40 The column effluent is mixed with an acetate buffer containing
reduced ninhydrin (hydrindantin) and then heated to 130 to 135˚C in a Teflon® reaction
coil. Primary amino acids form a purple chromophore and the imino amino acids (proline
and hydroxyproline) a yellow complex with absorbance peaks at 570 and 440 nm, respec-
tively. Absorbance is measured at both wavelengths, which can help in peak identification
1382_C01.fm Page 23 Tuesday, October 7, 2003 5:57 PM

Chapter one: Measurement of amino acid concentrations in biological fluids and tissues 23

Figure 1.2 Chromatographic chart of amino acids using IEC-based method. (A) Calibration solution.
(B) Plasma profile. (C) Muscle profile. (D) 3-methylhistidine (short program). All these chromato-
grams were recorded on a Jeol AminoTac JLC-500V machine, except for D, which was recorded on
a Hitachi L-8500A machine.
1382_C01.fm Page 24 Tuesday, October 7, 2003 5:57 PM

24 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

because the ratio A570/A440 is specific to each amino acid. Inspection of this ratio helps to
detect interference, e.g., by antibiotics (see below). As the reaction is sensitive to oxygen,
buffers and reagent must be maintained under nitrogen. To avoid the problem of ninhydrin
reagent instability, which limits its use to 1 to 2 weeks after preparation, extemporaneous
automatic mixing of the detection reagent is available on some modern apparatus.4
Postcolumn derivatization using fluorescent reactions offers an alternative to the use
of ninhydrin. Both orthophthalaldehyde (OPA) and fluorescamine have been shown to
react rapidly with primary amines to form highly fluorescent products.41–43 However, OPA
is most frequently used because it has the best fluorescent yield and is soluble and stable
in the aqueous reaction buffer (unlike fluorescamine).44 For primary amino acids, OPA is
at least 10 times more sensitive than ninhydrin, but there is a low response to cysteine.2
Hence, this type of derivatization is of particular interest for the analysis of small (i.e.,
50 mg) human biopsies (e.g., from muscles). However, except for its increased sensitivity
(1 pmol), postcolumn derivatization with OPA offers no improvement over the classical
ion exchange ninhydrin procedure. Furthermore, secondary amines do not form fluores-
cent derivatives with OPA unless oxidative reagents are present in the reaction mixture,
which necessitate two postcolumn pumps.

1.3.3 Data processing, internal standards, and calibration solutions


Computerized data processing is an essential feature of modern analyzers. Data are col-
lected and integrated, and amino acids are identified by comparison of their retention
times with a calibration solution. Results may be displayed, stored, and retrieved.
A major problem in amino acid analysis is the reliability of the commercially prepared
standard solutions used for calibration. In these solutions, concentration of several amino
acids may be significantly different from the stated concentrations.18,45 Preparation of a
standard by dissolving salts of individual amino acids can solve this problem, and for
maximum accuracy, use of calibration solutions from several sources is recommended. It
is noteworthy that glutamine is not incorporated into commercial standards because of
its instability in solution. Thus, an aqueous solution of this amino acid must be prepared
fresh for each analytical batch, and an aliquot added to the calibration mixture before
analysis.1
Precision and accuracy are improved by adding an internal standard that serves as a
control for the analytical procedure and allows correction for losses.46 By definition, the
substance chosen as the internal standard must not be present at a detectable level in the
physiological fluid being analyzed. Also, the retention time of the internal standard(s)
must not interfere with the retention time of the physiological amino acids. Since charac-
teristics of resin, buffers, etc., vary from one analyzer to another, the most suitable internal
standards may be different. For example, with our Hitachi L-8500A we use acetyl–lysine,
whereas with our Jeol AminoTac JLC-500V we use glucosaminic acid and aminoethyl–cys-
teine as internal standards.
Also, for specific diagnosis, we may be interested in a single amino acid, e.g., plasma
homocysteine as a marker of cardiovascular risk47 or urinary 3-methylhistidine (3-MH) as
a marker of muscle myofibrillar protein breakdown.48 Thus, short programs are used (so
as not to wait 1 h for 3-MH). In that case, retention times of all amino acids are modified,
and the appropriate internal standard may be different.

1.4 Interferences
Ninhydrin-positive drugs or drug metabolites are responsible for interfering peaks in
both plasma and urine samples, and these are most troublesome in urine. Among the
1382_C01.fm Page 25 Tuesday, October 7, 2003 5:57 PM

Chapter one: Measurement of amino acid concentrations in biological fluids and tissues 25

Table 1.2 Performance (Between-Run Reproducibility) of IEC-Based Apparatus over the


Last 20 Years
1980 1995 2000
Chromakon 500 Hitachi L-8500A Jeol AminoTac
Amino Acids [51] [4] JLC-500Va
Taurine 11.1 1.5 2.3
Aspartate 12.5 12.0 9.5
Threonine 9.3 3.1 2.3
Serine 9.5 2.3 2.2
Asparagine 5.1 1.2
Glutamate 10.2 7.9 3.2
Glutamine 11.1 4.7 1.6
Proline 17.6 1.5 3.6
Glycine 9.3 1.8 1.9
Alanine 7.9 2.8 1.9
Citrulline 10.0 7.5 4.3
Valine 9.4 1.8 1.9
Cysteine 2.1 2.2
Methionine 10.3 1.4 3.8
Isoleucine 10.0 1.5 1.9
Leucine 8.2 1.4 1.8
Tyrosine 11.6 1.4 2.3
Phenylalanine 8.9 1.9 1.8
Ornithine 11.6 1.3 2.3
Histidine 9.6 3.0 4.0
Lysine 12.7 1.8 2.7
Arginine 7.6 7.3 4.4

Note: Data are expressed in CV %.


a Neveux et al., unpublished data.

numerous drugs involved are antibiotics (e.g., benzylpenicillin, cefotaxime, ampicillin)


and therapeutic amines or amino acid derivatives such as N-acetylcysteine, dopamine,
or D-penicillamine.2,28,49

1.5 Conclusion
It is now almost 50 years since the first automated procedure for the analysis of amino
acids by ion exchange chromatography was described.8 Although other methods have
attempted to rival this system, it still remains the most common method used for quan-
titative amino acid profiling,45,50 the analytical performance having improved dramatically
in recent years (Table 1.2). However, some problems persist independently of the instru-
mentation. These arise in sample preparation, treatment, and storage, which are frequently
neglected preanalytic stages where artifacts or specious results may originate. Cation
exchange chromatography is the method of choice when precise profiling of amino acid
in biological fluids and tissues and quantitative data are required. The separation of
underivatized amino acids is carried out by means of a stepped series of lithium citrate
buffers with ninhydrin or OPA detection. Automation and computerization have been
largely developed and are applied to automated sample delivery, automated and com-
puterized gradient formation, and quantification of the data obtained. The alternative is
reversed-phase high-performance liquid chromatography (RP-HPLC) methods with pre-
column derivatization (see Chapter 2). Advantages and drawbacks of ion exchange chro-
matography compared with reversed-phase HPLC methods are given in Table 1.3. It is
1382_C01.fm Page 26 Tuesday, October 7, 2003 5:57 PM

26 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 1.3 Advantages and Drawbacks of IEC-Based Apparatus Compared to RP-HPLC with
Precolumn Derivatization

Advantages
• Allows the measurement of all amino acids
• Well standardized
• Best analytical performance (lowest within-run and between-run reproducibility CVs)
• Excellent after-sales service and methodological assistance from the supplier
• Low cost per assay (a column allows 2000 analyses and buffers are cheap)

Drawbacks
• High cost of the machine (at least twice the price of an HPLC system)
• Longer analysis time (although decreasing, it is still twice the time required with RP-HPLC)
• Fully dedicated machine (cannot be used for other applications)

clear that a fully automated apparatus based on the ion exchange method is the best option
for a laboratory with a high amino acid analysis throughput, such as in a hospital.

References
1. Armstrong, M.D. and Stave, U., A study of plasma free amino acid levels. II. Normal values
for children and adults, Metabolism, 22, 561, 1973.
2. Deyl, Z., Hyanek, J., and Horakova, M., Profiling of amino acids in body fluids and tissues
by means of liquid chromatography, J. Chromatogr., 379, 177, 1986.
3. Wuu, J.A., Wen, L.Y., Chuang, T.Y., and Chang, G.G., Amino acid concentrations in serum
and aqueous humor from subjects with extreme myopia or senile cataract, Clin. Chem., 34,
1610, 1988.
4. Le Boucher, J., Charret, C., Coudray-Lucas, C., Giboudeau, J., and Cynober, L., Amino acid
determination in biological fluids by automated ion-exchange chromatography: performance
of Hitachi L-8500A, Clin. Chem., 43, 1421, 1997.
5. Parvy, P.R., Bardet, J.I., Rabier, D.M., and Kamoun, P.P., Age-related reference values for free
amino acids in first morning urine specimens, Clin. Chem., 34, 2092, 1988.
6. Gjessing, L.R., Gjesdahl, P., and Sjaastad, O., The free amino acids in human cerebrospinal
fluid, J. Neurochem., 19, 1807, 1972.
7. Puck, A., Liappis, N., and Hildenbrand, G., Ion exchange column chromatographic investi-
gation of free amino acids in tears of healthy adults, Ophthalmic Res., 16, 284, 1984.
8. Moore, S. and Stein, W.H., Chromatography of amino acids on sulfonated polystyrene resins,
J. Biol. Chem., 192, 663, 1951.
9. Stein, W.H. and Moore, S., The free amino acids in human blood plasma, J. Biol. Chem.,
211,915, 1954.
10. Spackman, D.H., Stein, W.H., and Moore, S., Automatic recording apparatus for use in the
chromatography of amino acids, Anal. Chem., 30, 1190, 1958.
11. Moore, S. and Stein, W.H., Chromatographic determination of amino acids by the use of
automatic recording equipment, Meth. Enzymol., 6, 819, 1958.
12. Perry, T.L. and Hansen, S., Technical pitfalls leading to errors in the quantitation of plasma
amino acids, Clin. Chim. Acta, 25, 55, 1969.
13. Parvy, Ph., Bardet, J., and Kamoun, P., EDTA in vacutainer tubes can interfere with plasma
amino acid analysis, Clin. Chem., 29, 735, 1983.
14. Upton, J.D. and Hindmarsh, P., More pitfalls in human plasma amino acid analysis, Clin.
Chem., 36, 157, 1990.
15. Hill, A., Casey, R., and Zaleski, W.A., Difficulties and pitfalls in the interpretation of screening
tests for the detection of inborn errors of metabolism, Clin. Chim. Acta, 72, 1, 1976.
16. Sahai, S. and Uhlhaas, S., Stability of amino acids in human plasma, Clin. Chim. Acta, 148,
255, 1985.
1382_C01.fm Page 27 Tuesday, October 7, 2003 5:57 PM

Chapter one: Measurement of amino acid concentrations in biological fluids and tissues 27

17. Schaefer, A., Piquard, F., and Haberey, P., Plasma amino-acids analysis: effects of delayed
samples preparation and of storage, Clin. Chim. Acta, 164, 163, 1987.
18. de Jonge, L.H. and Breuer, M., Evaluation of systematic errors due to deproteinization,
calibration and storage of plasma for amino acid assay by ion-exchange chromatography,
J Chromatogr. B, 677, 61, 1996.
19. Hubbard, R.W. and Mejia, A., Human plasma preservation for amino acid analysis by
immediate low temperature freezing, Clin. Biochem., 28, 318, 1995.
20. Thornber, M.J., Buchanan, N., and Manchester, K.L., Preparation of plasma samples for amino
acid analysis by equilibrium dialysis, Biochem. Med., 19, 71, 1978.
21. Ukida, M., Schäfer, K., and Bode, J.Ch., Effect of storage at –20˚C on the concentration of
amino acids in plasma, J. Clin. Chem. Clin. Biochem., 19, 1193, 1981.
22. Kornhuber, M.E., Balabanova, S., Heiligensetzer, G.V., Kornhuber, C., Zettlmeissl, H., and
Kornhuber, A.W., Stability of human blood serum amino acids after storage at different pH
and temperature conditions, Clin. Chim. Acta, 197, 189, 1991.
23. Van Eijk, H.M.H., Dejong, C.H.C., Deutz, N.E.P., and Soeters, P.B., Influence of storage
conditions on normal plasma amino-acid concentrations, Clin. Nutr., 13, 374, 1994.
24. Abdulrazzaq, Y.M. and Ibrahim, A., Determination of amino acids by ion-exchange chroma-
tography on filter paper spotted blood samples stored at different temperatures and for
different periods: comparison with capillary and venous blood, Clin. Biochem., 34, 399, 2001.
25. Walker, V. and Mills, G.A., Quantitative methods for amino acid analysis in biological fluids,
Ann. Clin. Biochem., 32, 28, 1995.
26. Ferraro, T.N., Manyam, B.V., and Hare, T.A., Further characterization of in-vitro conditions
appropriate for GABA determination in human CSF: impact of acid deproteinization and
freeze/thaw, J. Neurochim., 41, 1057, 1983.
27. Gerritsen, T., Rehberg, M.L., and Waisman, H.A., On the determination of free amino acids
in serum, Anal. Biochem., 11, 460, 1965.
28. Williams, A.P., General problems associated with the analysis of amino acids by automated
ion-exchange chromatography, J. Chromatogr., 379, 177, 1986.
29. Mondino, A., Automatic ion-exchange chromatography of amino acids: experimental studies
for optimising resin column dimensions, J. Chromatogr., 50, 260, 1970.
30. Manchester, K.L., Influence of extraction procedure on size of free amino acid pool of heart
muscle, Anal. Biochem., 102, 300, 1980.
31. Fürst, P. and Kuhn, K.S., The application of muscle biopsy in the study of amino acid and
protein metabolism, in Methods for Investigation of Amino Acid and Protein Metabolism, El-
Khoury, A.E., Ed., CRC Press, Boca Raton, FL, 1999, p. 147.
32. Yokoyama, Y., Watanabe, M., Horikoshi, S., and Sato, H., Sulfoacylated macro-porous poly-
styrene-divinylbenzene low-capacity cation exchanger selective for amino acids, Anal. Sci.,
18, 59, 2002.
33. Klingenberg, A. and Seubert, A., Sulfoacylated poly(styrene-divinylbenzene) copolymers as
resins for cation chromatography: comparison with sulfonated, dynamically coated and silica
gel cation exchangers, J. Chromatogr. A, 946, 91, 2002.
34. Long, C.L. and Geiger, J.W., Automatic analysis of amino acids: effect of resin cross-linking
and operational variables on resolution, Anal. Biochem., 29, 265, 1969.
35. Ersser, R.S. and Davey, J.F., Liquid chromatographic analysis of amino acids in physiological
fluids: recent advances, Med. Lab. Sci., 48, 59, 1991.
36. Benson, J.R., Improved ion-exchange resins, Meth. Enzymol., 47, 19, 1977.
37. Vega, A. and Nunn, P.B., A lithium buffer system for single-column amino acid analysis,
Anal. Biochem., 32, 446, 1969.
38. James, L.B., Amino acid analysis: buffers and artifacts, J. Chromatogr., 436, 80, 1988.
39. Bergström, J., Fürst, P., Noree, L.-O., and Vinnars, E., Intracellular free amino acid concen-
tration in human muscle tissue, J. Appl. Physiol., 36, 693, 1974.
40. Samejima, K., Dairman, W., and Udenfriend, S., Condensation of ninhydrin with aldehydes
and primary amines to yield highly fluorescent ternary products. 1. Studies on the mecha-
nism of the reaction and some characteristics of the condensation product, Anal. Biochem.,
42, 222, 1971.
1382_C01.fm Page 28 Tuesday, October 7, 2003 5:57 PM

28 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

41. Roth, M., Fluorescence reaction for amino acids, Anal. Chem., 43, 880, 1971.
42. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leimgruber, W., and Weigele, M., Fluores-
camine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the
picomole range, Science, 178, 871, 1972.
43. Stein, S., Bohlen, P., Stone, J., Dairman, W., and Udenfriend, S., Amino acid analysis with
fluorescamine at the picomole level, Arch. Biochem. Biophys., 155, 202, 1973.
44. Benson, J.R. and Hare, P.E., O-phthalaldehyde: fluorogenic detection of primary amines in
the picomole range: comparison with fluorescamine and ninhydrin, Proc. Natl. Acad. Sci.
U.S.A., 72, 619, 1975.
45. Parvy, Ph., Bardet, J., Rabier, D., Gasquet, M., and Kamoun, P., Intra- and inter-laboratory
quality control for assay of amino acids in biological fluids: 14 years of the French experience,
Clin. Chem., 39, 1831, 1993.
46. Gardner, M.L.G., A comparison of internal and external standardization in amino acid
analysis, Anal. Biochem., 150, 174, 1985.
47. Malinow, M.R., Plasma homocyst(e)inemia and arterial occlusive disease: a mini-review, Clin.
Chem., 41, 173, 1995.
48. Ballard, F.J. and Tomas, F.M., 3-Methylhistidine as a measure of skeletal muscle protein
breakdown in human subjects: the case for its continued use, Clin. Sci., 65, 209, 1983.
49. Parvy, P., Modification des concentrations des acides aminés plasmatiques et urinaires in-
duites par des thérapeutiques, Ann. Biol. Clin., 40, 23, 1982.
50. Rattenbury, J.M. and Townsend, J.C., Establishment of an external quality-assessment scheme
for amino acid analyses: results from assays of samples distributed during two years, Clin.
Chem., 36, 217, 1990.
51. Cynober, L., Coudray-Lucas, C., Ziegler, F., and Giboudeau, J., High performance ion-ex-
change chromatography of amino-acids in biological fluids using Chromakon 500: perform-
ance of the apparatus, J. Autom. Chem., 7, 201, 1985.
1382_C02.fm Page 29 Tuesday, October 7, 2003 6:01 PM

chapter two

Measurement of amino acid


concentrations in biological fluids
and tissues using reversed-phase
HPLC-based methods
Birgit Alteheld
University of Bonn
Peter Stehle
University of Bonn
Peter Fürst
University of Bonn

Contents
Introduction....................................................................................................................................29
2.1 Analytical techniques for plasma and other tissue material ........................................30
2.2 Measurement of urinary amino acids...............................................................................34
2.3 Determination of protein-bound glutamine ....................................................................36
2.4 Quality control ......................................................................................................................36
2.5 Analytical pitfalls to consider ............................................................................................36
2.6 Reference ranges...................................................................................................................38
2.7 Conclusion .............................................................................................................................40
References .......................................................................................................................................41

Introduction
More than 50 years ago, Moore and Stein reported quantitative analyses of amino acids
by using sulfonated polystyrene resin columns;1,2 the method was automated some years
later by Spackman et al.3 It was stated, “a protein free filtrate corresponding to 4 mL of
plasma constitutes an appropriate sample size for an analysis,” and further “quantitative
determination of amino acids is made simpler and more rapid, the more complex mixtures
of blood plasma, urine and mammalian tissues can be analysed in 2 days.”3 Since these
statements, much has happened. Better resins, automated sample injectors, steering with

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 29
1382_C02.fm Page 30 Tuesday, October 7, 2003 6:01 PM

30 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

microprocessors, computing integrators, and computer evaluations facilitate ultrarapid


analyses in less than 13 min in 10 mL of ultrafiltrate.4
At present, many laboratories still use the original cation exchange method, yet mod-
ified and modernized.5,6 In Chapter 1 the classical procedure for amino acid analysis is
described; it involves separation of amino acids by ion exchange chromatography followed
by derivatization with ninhydrin. The disadvantage of this method includes the
low-mobile-phase flow rate, thereby limiting the rate of sample analyses. A further draw-
back is the relatively poor sensitivity for free amino acids. Sensitive detection methods
are highly desirable, considering the remarkable achievements related to research on
amino acid metabolism of individual tissues and cells. The substantial progress made in
the field of analytical biochemistry is exemplified by the explosion of new information
about the potential uses of reversed-phase high-performance liquid chromatography (RP-
HPLC). Numerous reports emphasize the use of RP-HPLC in amino acid analysis. Indeed,
the application of RP-HPLC reduces the time required for analysis and increases the
sensitivity for quantification of amino acids.7
There are other methods, employing gas chromatography, combined gas chromato-
graphy–mass spectrometry, and mass spectrometry, used mainly for the tandem determi-
nation of individual amino acids or small groups of amino acids and related metabolites.
Capillary zone electrophoresis is an extremely sensitive method that is until later applied
only to protein hydrolysates, though its implication in biological fluids is certainly prom-
ising.8 The interested reader is referred to available reviews in order to deal with these
exceptional and auspicious methods.9 The present compilation is restrictively devoted to
describing recent advances with RP-HPLC of free amino acids, especially in biological
material.

2.1 Analytical techniques for plasma and other tissue material


According to our experience, five automated or semiautomated precolumn derivatization
methods are generally used for determination of free amino acids in biological fluids,
including muscle tissue, by using 9-fluorenylmethyl chloroformate (FMOC-Cl), phenyl
isothiocyanate (PITC), o-phthaldialdehyde (OPA), 1-dimethylaminonaphthalene-5-sul-
phonyl chloride (dansyl-Cl), and ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate
(SBD-F) for thiols. The superior sensitivity and practicability favors the use of three of
them, namely, OPA, dansyl-Cl, and SBD-F.
Because of instability of the OPA adducts, automated on-line derivatization is required
when using this method in general practice. Reliable automated assessments of plasma,
muscle, and liver free amino acids are facilitated with the method in 250 mL of plasma or in
biopsy specimens of about 1 mg of tissue in about 40 min.4,10 A representative chromatogram
is given in Figure 2.1. The major disadvantage of the OPA method lies in the fact that only
primary amines form adducts. This means measurements of proline and hydroxyproline are
not feasible with this method. Cyst(e)ine also cannot be measured with this derivatization
because of quenching of the fluorescence (Table 2.1).
OPA derivatization also allows detection of the rare amino acid N-6-trimethyllysine
(TML) that is formed by posttranslational methylation of lysine. A detection limit of 3.5
pmol could be achieved that requires a volume of 10 mL of plasma for the sample
preparation procedure for TML extraction.11
An ultrarapid and sensitive OPA/3-mercaptopropionic acid method has been devel-
oped by using 3-mm particle-size reversed-phase columns, enabling separation of 26 major
tissue free physiological amino acids in the lower picomole range in 12.7 min (still the valid
world record).4 Ultrasensitive applications are the micro- and narrow-bore methods,
1382_C02.fm Page 31 Tuesday, October 7, 2003 6:01 PM

Chapter two: Measurement of amino acid concentrations in biological fluids and tissues 31

Figure 2.1 Typical chromatograms of OPA derivatized (A) amino acid standard and (B) human plasma.
Chromatographic conditions: column, Spherisorb ODS II (3 mm) (150 ¥ 4.5 mm, I.D.); fluorescence
detection, lex = 330 nm, lem = 450 nm; injection volume, 20 mL; standard, 10 pmol/amino acid.

employing RP-HPLC columns with internal diameters of 1.0 and 1.8 mm, respectively. This
method requires special and individual care and thus is not suitable for routine applications.
The sensitivity of the analysis is about 25 fmol and 1 pmol of amino acid per injection,
respectively, and the interassay reproducibility and reliability range between 4 and 8%
(C.V.). The separation of 23 major free amino acids can be accomplished in 22 min. The
limit of detection is about 5 and 150 fmol at a signal-to-noise ratio (S/N) of 2.5, respectively.
For the narrow-bore application, a tissue specimen of 1 mg wet weight is an appropriate
sample size. The great advantage of narrow-bore chromatography compared to conven-
tional column technology is the considerably reduced consumption of expensive and pol-
luting organic solvents, the actual use of such reagents with the narrow-bore method being
only 15 to 20% of that with conventional RP-HPLC (Figure 2.2) (see Fürst et al.7).
Precolumn derivatization with FMOC-Cl permits the fluorimetric detection of primary
and secondary amino acids as stable FMOC adducts, while determination of free
1382_C02.fm Page 32 Tuesday, October 7, 2003 6:01 PM

32 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 2.1 HPLC Analyses of Free Amino Acids and Thiols: Comparison of Five Derivatization
Methods
SBD-F
Parameter OPAa FMOC-Clb PITCa Dansyl-Cla (Thiols)c
Limit of sensitivity, 0.8 0.2 5.0 1.5 0.1–0.3
pmol (S/N = 2.5)
Error of the method 1.0–4.7 0.7–4.9 3.6–7.0 1.7–4.5 2.0–3.2
(C.V., %)
(based on duplicate
determinations)
Reproducibility 0.4–2.2 0.9–3.8 2.6–5.5 1.5–4.1 2.0–6.6
(interassay, C.V., %)
Stable adducts N Y Y Y Y
Detection of N/N Y/N Y/Y Y/Y N/Y
secondary
amines/cystine
Laborious – +++ ++ + +
Problematic amino Asp/Trp Trp, His Orn, Trp, His, His, Asn
acids Cystine

Note: Y = yes; N = no; – = not; + = slightly; ++ = moderate; +++ = very.


a Reprinted from Fürst, P. et al., J. Chromatogr., 499, 557–569, 1990.
b Reprinted from Bank, R.A. et al., Anal. Biochem., 240, 167–176, 1996.
c Reprinted from Kuhn, K.S. et al., Clin. Chem., 46, 1003–1005, 2000.
Source: Reprinted from Fürst, P. et al., J. Chromatogr., 499, 557–569, 1990; Bank, R.A. et al., Anal. Biochem., 240,
167–176, 1996; Kuhn, K.S. et al., Clin. Chem., 46, 1003–1005, 2000.

tryptophan and cyst(e)ine is not possible with FMOC-Cl, because the fluorescence of the
adducts is quenched. The FMOC-Cl method suffers from the disadvantage that an excess
of strong fluorescent reagent has to be extracted manually with pentane as FMOC-OH, in
order to stop the derivatization reaction and to avoid spontaneous hydrolysis of the FMOC
adducts. This laborious manual extraction procedure prevents the wide acceptance of this
method. This shortcoming, however, might be overcome by using specially designed
autosamplers or a combination of the FMOC-Cl and OPA methods.7
A recent modification in the FMOC-Cl method improved recovery of the so far critical
amino acid histidine by using a different pH for the derivatization (pH 11.4 instead of the
originally described pH 7.7). A threefold improvement in sensitivity could be achieved
by the selection of a higher-emission wavelength (630 nm instead of 313 nm) (Table 2.1).12
Application of the PITC method, although less sensitive, is useful in clinical chemistry,
where sample availability is rarely a problem. Determination of free cyst(e)ine is not
practicable because of poor linearity and reproducibility (Table 2.1). In addition, we
observed rapid deterioration of the column when analyzing tissue material (e.g., muscle).
In our experience, a maximum of 150 physiological analyses per column could be per-
formed in spite of the use of rigorous sample preparation and suitable guard columns
containing the same resin.7 This is a serious shortcoming of the PITC method.
Dansyl-Cl is a well-known fluorogenic reagent for the determination of primary and
secondary amines. Adducts are formed at room temperature in the dark. In contrast to
the PITC method, the dansyl-Cl technique shows excellent linearity for cyst(e)ine and also
for cyst(e)ine-containing short-chain dipeptides (Figure 2.3). Hence, the dansyl-Cl method
appears to offer a suitable quantitative approach for measuring free and peptide-bound
cyst(e)ine in biological material by RP-HPLC techniques.
1382_C02.fm Page 33 Tuesday, October 7, 2003 6:01 PM

Chapter two: Measurement of amino acid concentrations in biological fluids and tissues 33

Figure 2.2 Narrow-bore HPLC of (A) a standard mixture and (B) TCA-precipitated rat plasma
sample after derivatization with OPA-3-MPA. Chromatographic conditions: column, Spherisorb
ODS II (3 mm) (200 ¥ 1.8 mm, I.D.); fluorescence detection, lex = 330 nm, lem = 450 nm; injection
volume, 1 mL; standard, 1 pmol/amino acid.

Furthermore, derivatization with dansyl-Cl enables the determination of the thiol


glutathione (Mercaptopropionic acid) in reduced (GSH) and oxidized (GSSG) forms, which
are important intracellular antioxidants and measured for calculation of redox potentials.13
Red blood cells contain an approximately 500 times higher GSH concentration than
plasma, so that minor hemolysis (0.1 to 1%) can result in erroneously high plasma values.
GSH concentrations might change in the sample due to oxidation or degradation by
l-glutamyltranspeptidase. Samples should therefore be preserved by addition of
serin–borate (inhibits enzymatic degradation), bathophenanthroline disulfonate (BPDS)
(inhibits oxidation), and iodacetic acid for alkylation of GSH.13 Some thiols like homo-
cysteine, mixed disulfides, and, to a lesser extent, cysteine are unstable when stored
without prior deproteinization.14
Glutathione can be measured alternatively with SBD-F derivatization (vide infra). This
method is especially suitable for assessment for tissue glutathione.
Derivatization with SBD-F was first used for determination of homocysteine.15 Line-
arity was shown up to 500 mmol/L with a detection limit of 0.3 pmol per injection. Total
imprecision was determined with plasma samples and revealed a coefficient of variation
of 3.0%; the within-run component of imprecision was 2.3%. Recently, SBD-F has also been
used to analyze glutathione both in plasma and in tissue samples.16 SBD-F possesses major
advantages for measuring thiols: high reactivity with the thiol compounds, a low detection
limit, high stability of the derivatives, and lack of native fluorescence or fluorescent by-
products.17 A routinely manageable method for the separation of GSH, Cys, g-Glu-Cys,
and Cys-Gly in small tissue specimens is reported, with detection limits of 0.3 pmol for
Cys, 0.2 pmol for g-Glu-Cys, and 0.1 pmol for Cys-Gly and GSH (Table 2.1).16
1382_C02.fm Page 34 Tuesday, October 7, 2003 6:01 PM

34 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 2.3 Elution profile of a human plasma sample after derivatization with dansyl-Cl. Chromato-
graphic conditions: column, Spherisorb ODS II (3 mm) (125 ¥ 4.5 mm, I.D.); fluorescence detection,
lex = 330 nm, lem = 550 nm; injection volume, 20 mL.

The results obtained with HPLC methods compare favorably with those derived from
conventional ion exchange amino acid analyses (Table 2.2). According to our experience,
the separation remains satisfactory for at least 600 OPA and FMOC-Cl, 300 dansyl-Cl, and
150 SBD-F analyses. Careful control of factors and limitations inherent to the various
methodologies is a prerequisite for proper identification and appropriate quantitation. The
sensitivity, errors of the methods, advantages and disadvantages, and problems with
certain “difficult” amino acids are summarized in Table 2.1.

2.2 Measurement of urinary amino acids


There are few reports of controlled analyses of amino acids in urine by HPLC. Precolumn
OPA derivatization has been applied to urine.18 Due to the presence of numerous primary
and secondary amines and aromatic compounds, the baseline is usually severely disturbed
and quantitative evaluation of the data is difficult because of the large range of concen-
trations within the sample. PITC methods have also been used, yet aromatic compounds
with absorption at 254 nm cause interferences, especially in the front part of the chromato-
gram.19,20 A separate extraction on ion exchange resin20 or electrochemical detection may
eliminate disturbing interferences,21 but this is at the expense of increased baseline noise
and instrument instability. The major drawback concerning assessment of urinary free
amino acids is that there is substantial background noise resulting from interfering sub-
stances and the columns can only be used for a rather small number of analyses.
How to collect the sample is an essential question. The excretion rate of amino acids
varies independently of creatinine over 24 h. Therefore, when using creatinine as a reference
in random urine samples, a higher variation is to be expected than in 24-h collections.22 It
1382_C02.fm Page 35 Tuesday, October 7, 2003 6:01 PM

Chapter two: Measurement of amino acid concentrations in biological fluids and tissues 35

Table 2.2 Free Amino Acid Concentrations in Human Plasma (mmol/L) Determined by HPLC
(Precolumn Derivatization with OPA, FMOC-Cl, and PITC) and Ion Exchange Chromatography
(Ninhydrin Postcolum Derivatization)
HPLC
Ion exchange OPA
Amino acid (ninhydrin) (automated) FMOC-Cl PITC
Glu 23.0 ± 8.4 16.4 ± 4.7 19.2 ± 4.0 11.6 ± 3.9
Asn 38.4 ± 7.1 38.4 ± 10.9 41.2 ± 7.0 43.8 ± 9.5
Ser 119.9 ± 10.6 99.4 ± 26.6 103.3 ± 18.3 102.0 ± 13.5
Glu 574.5 ± 31.8 556.7 ± 31.5 547.3 ± 51.1 493.1 ± 56.9
Gly 262.2 ± 46.6 214.5 ± 34.7 207.1 ± 29.5 191.8 ± 23.4
Thr 114.7 ± 30.3 96.5 ± 30.1 102.1 ± 20.3 116.6 ± 20.8
His 85.7 ± 23.2 85.5 ± 16.4 69.7 ± 20.5 79.8 ± 20.3
Ala 353.5 ± 105.1 319.0 ± 99.4 303.7 ± 70.7 339.9 ± 51.5
Tau 60.7 ± 8.8 38.9 ± 9.8 38.7 ± 4.4 59.4 ± 16.8
Arg 84.8 ± 7.2 74.3 ± 29.9 74.9 ± 19.6 77.5 ± 11.6
Tyr 66.8 ± 17.2 48.1 ± 17.0 51.4 ± 11.1 54.7 ± 5.0
Val 198.7 ± 36.9 209.1 ± 40.6 185.3 ± 43.2 222.2 ± 28.3
Met 28.7 ± 12.4 35.0 ± 7.5 25.3 ± 7.0 25.9 ± 2.8
Ile 54.2 ± 12.1 51.1 ± 6.6 50.4 ± 13.8 68.7 ± 10.3
Phe 53.7 ± 12.9 52.2 ± 12.3 47.5 ± 8.2 60.3 ± 4.8
Trp 45.8 ± 17.4 34.5 ± 7.6 — —
Leu 114.9 ± 24.7 112.7 ± 24.2 104.2 ± 29.2 141.6 ± 16.8
Orn 80.0 ± 23.2 81.4 ± 15.1 78.5 ± 10.8 —
Lys 195.9 ± 44.7 182.7 ± 39.4 178.1 ± 40.7 202.7 ± 43.6
Pro 205.3 ± 40.8 — 193.3 ± 50.6 240.8 ± 48.1

Note: Results are means ± S.D. (n = 10).


Source: Reprinted from Fürst, P. et al., J. Chromatogr., 499, 557–569, 1990. With permission.

is important to avoid fecal and bacterial contaminations since they increase concentrations
of many amino acids.23,24 It is recommended that a suitable preservative be added to the
specimen container, like toluene, chloroform, or thymol. Samples should be stored at –20˚C
or lower until analysis. Repeated freezing and thawing increase amino acid concentra-
tions.25
Special attention should also be paid to the excretion of 3-methylhistidine (3-MeHis),
which has been suggested as a suitable marker amino acid for measurements of myofibril-
lar protein breakdown.26 An automated method for the determination of urinary 3-MeHis
has been recently described.27 There is considerable controversy on the interpretation of
3-MeHis excretion rates and whether nonmuscle or smooth muscle sources of 3-MeHis
may contribute significantly to urinary 3-MeHis.28,29 This suggests not only the need for
considerable caution in the interpretation of 3-MeHis data but also the need for further
investigation. Meanwhile, its value for monitoring the response to treatment in severe
trauma and acute illnesses has been encouraging.29,30
Urinary hydroxyproline (Hyp) might be a good marker of bone turnover, since it is
released during collagen breakdown. Thus, increased Hyp excretion indicates enhanced
bone turnover, like in Paget’s disease and malignancies.31,32 RP-HPLC methods have been
reported by using derivatizing agents PITC, dansyl-Cl, OPA, and FMOC-Cl.33,34 Recently,
a new procedure has been developed for quantification of Hyp by prior elimination of all
primary amino acids with nitrous acid to the corresponding hydroxyl acids. Amino acids
are transformed into N-nitroso derivatives that are treated in a following step with HBr
for denitrosation. Finally, the remaining secondary amino acids, including Hyp, are deriva-
tized preferentially with dansyl-Cl.
1382_C02.fm Page 36 Tuesday, October 7, 2003 6:01 PM

36 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Pyridinoline and deoxypyridinoline are 3-hydroxypyridinium derivatives, present


only in mature collagen. Urinary excretion of these pyridinoline cross-links determined
are increased during normal growth and in disorders with increased bone resorption.34–36

2.3 Determination of protein-bound glutamine


Glutamine is considered to be a conditionally essential amino acid during episodes of
catabolic stress and malnutrition. Knowledge of glutamine contents of natural proteins is
thus of utmost importance. Quantitative assessment of protein-bound glutamine is, how-
ever, hampered by glutamic acid formation during acid hydrolysis, invalidating subse-
quent distinction between glutamine and glutamic acid residues.
Reliable assessment of the true glutamine content might be obtained using laborious
and cost-intensive biotechnological methods (cDNA technology) or might be acquired
from sequence analyses (purified protein fragments are requested).
Recently, an easy and rapid procedure for the determination of glutamine in isolated
proteins has been developed. It involves a prehydrolysis reaction of glutamine residues
with bis-(1,1-trifluoroacetoxy)-iodobenzene (BTI) to yield acid-stable L-2,4-diaminobutyric
acid (DABA), protein hydrolysis using the microwave technique.37 Subsequent amino acid
analyses are performed with dansyl-Cl derivatization of amino acids as described above.
The RP-HPLC analysis of a human muscle specimen is shown in Figure 2.4. DABA and
the proteic amino acids could be simultaneously measured with high sensitivity (2.0
pmol/injection; S/N = 3.1) and good reproducibility (C.V. = 2.3%, measured as interassay
variability of the derivatization plus chromatographic procedure from analyses of 20
standard solutions). The linearity between DABA formation and glutamine concentration
was excellent (r = 0.9996).

2.4 Quality control


The important issue of the quality of analytical results is the scope of discussion in Chapter
1. It is, however, pertinent to note that the majority of efforts concerning quality assurance
are devoted to ion exchange with ninhydrin. The French external scheme operative since
1978, including 49 laboratories, reports on intra- and interlaboratory quality control in
biological fluids in 94% from ion exchange and in 6% from gas chromatography methods.38
A quality assurance scheme has been operating in the U.K.; the combined results from 26
(23 used ion exchange and 4 HPLC) laboratories were published. ERNDIM is the European
Research Network for Evaluation and Improvement of Screening, Diagnosis and Treat-
ment of Inherited Disorders of Metabolism; it reflects a system of quality control of
laboratory measurements. ERNDIM was founded in 1994 in Maastricht, The Netherlands.
The section Quantitative Amino Acids includes 160 participants, mainly working with ion
exchange chromatography. Indeed, it is desirable to establish a European quality assurance
scheme for amino acid analyses in biological fluids with HPLC as soon as possible.
Considering the concerted action of research efforts as harmonized by the European Union,
this would be a highly realistic approach. Validation and recovery data for useful com-
parisons are warranted.

2.5 Analytical pitfalls to consider


Successful normalization or partial correction of an abnormal amino acid pattern is often
taken as a guarantee for adequate amino acid utilization. However, the limitations inherent
in methodology and in data interpretation are sadly often neglected. There are many
factors that influence amino acid concentrations. Particularly important points to consider
1382_C02.fm Page 37 Tuesday, October 7, 2003 6:01 PM

Chapter two: Measurement of amino acid concentrations in biological fluids and tissues 37

Cys-Gly
240

MPG
M illivolts 180

GSH
Cys

Hcy
120

Glu-Cys
60

1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50

Cys Time (min)

B
960

720
M illivolts

Cys-Gly

480
Hcy

MPG
Glu-Cys

GSH

240

1.50 3.00 4.50 6.00 7.50 9.00 10.50 12.00 13.50

Time (min)

Figure 2.4 Typical chromatogram of (A) a standard mixture of thiols and (B) human plasma sample
after derivatization with SBD-F. Chromatographic conditions: column, Hypersil ODS II (3 mm) (150
¥ 4.6 mm, I.D.); fluorescence detection, lex = 385 nm, lem = 515 nm; injection volume, 20 mL.

are when to take the sample, how to perform the sampling, and what kind of sample will
be analyzed.
Diurnal variations in amino acid levels should be contemplated.39 It is important to
emphasize that alterations in amino acid rhythmicity are easily induced by the onset of
an acute illness or during chronic disease states.39 Thus, adequate control observations are
especially pertinent during any study of amino acid absorption after feeding a test meal
or an amino acid load or infusion.
The state of nutrition must also be considered. Many patients are first seen when acutely
ill, often at times of diminished food intake. Under such conditions it is necessary to
segregate the amino acid imbalance caused by secondary illness from that associated with
the primary disease and the effects of nutritional supplementation.
Apparent steady state: In most cases samples are collected after an overnight fast. Slight
variations in sampling time after an overnight fast may not seriously affect plasma amino
acid concentrations.40 Postprandial sampling or sampling during ongoing parenteral nutri-
tion during a given phase of treatment may provide a more adequate picture of the amino
acid steady state. Postprandial changes in total homocysteine (tHcy) concentrations
include a modest decrease in the first few hours, followed by an increase after 8 h; therefore,
fasting blood samples are recommended to minimize any effects of meals.41,42
Physical activity40 — rarely controlled — as well as alterations that have been reported
throughout the menstrual cycle43 might also be considered.
1382_C02.fm Page 38 Tuesday, October 7, 2003 6:01 PM

38 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 2.5 The amino acid concentrations in plasma and muscle as a function of sex and age in chronic
uremic patients compared with data derived either from mixed healthy controls or from age- and sex-
matched controls. (Results adapted from Alvestrand, A. et al., Clin. Nephrol., 18, 297–305, 1982.)

Technical errors: Repeated venipuncture lowers the values of taurine and glutamic acid
and arginine since arginase is released during the clotting process.44 The majority of amino
acid levels in serum are not only considerably higher than in plasma but also more
variable.40 The choice of anticoagulant can also be important,45 as discussed in Chapter 1.
Immediate and adequate deproteinization is important to avoid certain errors and artifacts,
such as enhanced deamidation of glutamine46 and significant losses of disulfide amino
acids.44,47 Glutamic acid and aspartatic acid rise slowly and glutamine and asparagine fall
equivalently in samples stored at –20˚C.40,46–48 Additional loss of cyst(e)ine is reported.40,46–48
Contamination with sweat,49 from platelets and leukocytes44,45 or hemolysis,40,44 will result
in errors and artifacts that can jeopardize the reliability of analyses.
Age and sex are two further important factors affecting amino acid concentrations.50,51
Measurements of free amino acids are usually made at a single point in time, and it is
upon such a determination that one needs to decide whether a subject manifests normal
or abnormal amino acid metabolism. Thus, an adequately controlled database is of the
utmost importance in evaluating the results. As exemplified in Figure 2.5, patient data
may reveal a completely normal level of an amino acid when compared with healthy
controls, but the value is, as a matter of fact, significantly and markedly reduced when
the comparison is made with adequate age- and sex-matched controls. In contrast, an
apparently increased value is in reality completely normal when age- and sex-matched
controls are used for comparison.

2.6 Reference ranges


Reference data for plasma, muscle, and erythrocytes are presented in Table 2.3 simulta-
neously obtained from healthy subjects. As emphasized, age and sex are important factors,
Table 2.3 Simultaneous Measurements of Free Amino Acid Patterns of Plasma, Muscle, and Erythrocytes in 27 Healthy Human Subjects
Plasma Muscle RBC Gradient Gradient Gradient
mmol/L PlW mmol/L ICW mmol/L ICW RBC/Plasma Muscle/plasma Muscle/RBC
Chapter two:

Essential
Histidine 87± 3 592 ± 54 120 ± 18 1.3 ± 0.05 6.4 ± 0.51 5.0 ± 0.44
Isoleucine 63 ± 3 68 ± 4 71 ± 3 1.0 ± 0.03 1.0 ± 0.05 1.1 ± 0.05
Leucine 120 ± 5 133 ± 6 137 ± 6 1.1 ± 0.04 1.1 ± 0.04 1.0 ± 0.05
Lysine 195 ± 9 994 ± 77 177 ± 5 0.9 ± 0.04 5.0 ± 0.42 5.6 ± 0.45
Methionine 25 ± 1 41 ± 6** 20 ± 3 0.8 ± 0.04 1.6 ± 0.28** 2.0 ± 0.26**
Phenylalanine 53 ± 2 62 ± 3 62 ± 2 1.1 ± 0.05 1.1 ± 0.06 1.0 ± 0.05
Threonine 128 ± 5 571 ± 31 157 ± 6 1.1 ± 0.06 4.3 ± 0.2 4.0 ± 0.24
1382_C02.fm Page 39 Tuesday, October 7, 2003 6:01 PM

Tyrosine 60 ± 4 87 ± 4 82 ± 5 1.3 ± 0.06 1.4 ± 0.07 1.1 ± 0.04


Valine 220 ± 8 253 ± 11 248 ± 9 1.1 ± 0.03 1.1 ± 0.04 1.1 ± 0.03

Non-essential
Alanine 316 ± 17 2249 ± 96 419 ± 16 1.3 ± 0.05 6.8 ± 0.3 5.5 ± 0.2
Arginine 86 ± 3 633 ± 46 258 ± 23 2.8 ± 0.28 6.8 ± 0.5 4.3 ± 1.2
Asparagine 47 ± 2 266 ± 11 155 ± 4 3.1 ± 0.10 5.4 ± 0.3 1.8 ± 0.07
Citrulline 34 ± 1 170 ± 13 47 ± 2 1.3 ± 0.05 4.7 ± 0.4 3.8 ± 0.31
Glutamic acid 32 ± 4 4015 ± 249 446 ± 17 14.0 ± 2.65 123 ± 14 8.6 ± 1.42
Glutamine 655 ± 17 20050 ± 514 758 ± 15 1.1 ± 0.04 29 ± 0.9 26.9 ± 0.8
Glycine 248 ± 13 1304 ± 75 544 ± 21 2.2 ± 0.11 5.0 ± 0.3 2.5 ± 0.14
Ornithine 66 ± 4 493 ± 50 271 ± 18 4.4 ± 0.42 5.3 ± 0.8 2.2 ± 0.16
Serine 114 ± 4 584 ± 24 211 ± 6 1.8 ± 0.05 5.0 ± 0.2 2.9 ± 0.12
Taurine 49 ± 3 19194 ± 676 196 ± 28 4.0 ± 1.14 385 ± 17 184 ± 22
Carnosine 6130 ± 370
 EAA 857 ± 27 2831 ± 91 1034 ± 23 1.1 ± 0.04 3.1 ± 0.15 2.8 ± 0.15
 NEAA* 1446 ± 38 29101 ± 696 2345 ± 41 1.5 ± 0.05 18.1 ± 0.56 11.8 ± 0.38
 BCAA 402 ± 15 457 ± 19 456 ± 16 1.1 ± 0.04 1.1 ± 0.04 1.0 ± 0.04
 AA 2303 ± 58 31902 ± 729 3380 ± 53 1.4 ± 0.04 12.6 ± 0.43 9.1 ± 0.25

Note: Values are means ± SEM. ** in 5 subjects only; PLW, plasma water; ICW, intracellular water; RBC, erythrocytes; Â, sum of; EAA, essential amino
Measurement of amino acid concentrations in biological fluids and tissues

acids; NEAA, non-essential amino acids; BCAA, branched-chain amino acids. * Taurine, citrulline and ornithine are not included in the sum.
Source: Reprinted from Divino Filho, J.C. et al., Clin Nutr., 16, 299–305, 1997.
39
1382_C02.fm Page 40 Tuesday, October 7, 2003 6:01 PM

40 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 2.4 Free Amino Acids in Plasma and in Muscle in Young and Elderly Males
Plasma amino acids Muscle amino acids
(mmol/L) mmol/L of intracellular water)
Elderly men Young men Elderly men Young men
(n = 10) (n = 14) (n = 10) (n = 12)
Taurine 99.2 ± 13.7 84.1 ± 8.6 19.46 ± 2.20 15.34 ± 1.03
Aspartate 7.4 ± 0.6 9.6 ± 2.1 1.83 ± 0.13 1.18 ± 0.13**
Serine 136.2 ± 9.7 120.0 ± 7.4 1.16 ± 0.13 1.45 ± 0.28
Asparagine 53.0 ± 2.7 63.4 ± 6.1 0.36 ± 0.06 0.57 ± 0.11
Glutamate 46.2 ± 6.8 34.1 ± 3.5 4.84 ± 0.47 3.93 ± 0.21
Glutamine 808.4 ± 56.0 696.5 ± 34.3 23.62 ± 2.55 18.47 ± 1.45
Proline 235.2 ± 45.0 192.4 ± 15.2 1.22 ± 0.19 1.04 ± 0.11
Glycine 256.1 ± 25.4 258.2 ± 15.1 2.21 ± 0.29 1.18 ± 0.19
Alanine 396.0 ± 28.5 346.7 ± 30.2 3.57 ± 0.44 2.51 ± 0.28*
Citrulline 48.6 ± 5.7 39.7 ± 7.6 0.25 ± 0.06 0.05 ± 0.01**
Ornithine 116.3 ± 7.4 96.9 ± 8.1 0.56 ± 0.07 0.40 ± 0.06
Histidine 106.9 ± 10.5 77.0 ± 5.2** 0.61 ± 0.08 0.44 ± 0.03*
Arginine 113.7 ± 19.8 81.6 ± 7.3 0.89 ± 0.07 0.51 ± 0.04***
Threonine 162.9 ± 18.6 129.9 ± 9.8 0.89 ± 0.07 0.81 ± 0.04
Valine 279.5 ± 25.0 205.6 ± 22.2* 0.44 ± 0.03 0.39 ± 0.04
Methionine 28.2 ± 2.2 31.9 ± 5.1 0.05 ± 0.01 0.05 ± 0.01
Isoleucine 70.1 ± 5.7 62.2 ± 5.3 0.11 ± 0.01 0.09 ± 0.01
Leucine 143.7 ± 9.2 121.3 ± 11.6 0.22 ± 0.01 0.17 ± 0.02*
Tyrosine 99.1 ± 7.7 60.0 ± 5.0** 0.05 ± 0.01 0.06 ± 0.01
Phenylalanine 62.4 ± 4.5 62.6 ± 5.4 0.07 ± 0.02 0.06 ± 0.01
Lysine 220.2 ± 18.5 165.9 ± 14.1* 1.49 ± 0.13 0.88 ± 0.09***
ÂNEAA 2.43 ± 0.16 2.15 ± 0.09 45.33 ± 3.02 31.03 ± 1.87***
ÂEAA 0.97 ± 0.07 0.78 ± 0.06* 3.18 ± 0.22 2.52 ± 0.13*
ÂNEAA/ÂEAA 2.59 ± 0.10 2.82 ± 0.16 18.61 ± 1.11 12.54 ± 0.79***
Tyr/Phe 1.60 ± 0.06 1.03 ± 0.12*** 0.83 ± 0.11 1.31 ± 0.34
Gly/Val 0.96 ± 0.12 1.55 ± 0.26 5.06 ± 0.58 5.29 ± 0.55

Note: Young, 20–36 years; elderly, 52–77 years. Mean values ± SE are shown. * 0.01 < P < 0.05; ** 0.001 < P
< 0.01; *** P < 0.001. EAA and NEAA, essential and non-essential amino acids, respectively.
Source: Reprinted from Möller, P. et al., Clin. Sci. (Lond.), 56, 427–432, 1979.

significantly affecting amino acid concentrations. Table 2.4 and Table 2.5 present repre-
sentative data for plasma and muscle in young and elderly females and males, respectively.
Great caution should be exercised at interpretation of normal ranges. Physiological
deviations are to be considered since amino acid concentrations are affected by many
variables. As pointed out previously, an adequately controlled database, i.e., age- and sex-
matched control material, is of essential importance for adequate data interpretation.

2.7 Conclusion
Quantitative analysis of amino acids in biological fluids is a critical issue: success depends
upon fastidious attention to details not only concerning analytical technique, but also the
tiniest details related to sampling and preparation procedures. The presented HPLC meth-
ods have many practical advantages over the classical ion exchange method; reduced
analysis time and cost, improved sensitivity, and more robust instrumentation are a few.
Along with good separation of amino acids, data interpretation of abnormalities is a major
task that requires considerable experience and knowledge not only in analytical chemistry
but also in the fields of pathophysiology and medicine. Keeping with this background,
1382_C02.fm Page 41 Tuesday, October 7, 2003 6:01 PM

Chapter two: Measurement of amino acid concentrations in biological fluids and tissues 41

mmol/L) and in Muscle (mmol/L Intracellular Water) in


Table 2.5 Free Amino Acids in Plasma (m
Young and Elderly Females
Plasma Muscle
Elderly females Young females Elderly females Young females
n = 91 n = 11 n = 91 n = 11
65.6 ± 1.8 years) 27.9 ± 1.65 years 65.6 ± 1.8 years 27.9 ± 1.65 years
Taurine 108.2 ± 11.1 108.2 ± 15.4 21.20 ± 2.02 24.00 ± 2.40
Aspartate 19.1 ± 1.8 17.9 ± 1.9 1.98 ± 0.20 2.45 ± 0.21
Serine 177.2 ± 8.6 188.2 ± 13.5 1.15 ± 0.11 1.42 ± 0.19
Asparagine 64.9 ± 4.7 63.8 ± 5.3 0.52 ± 0.05 0.46 ± 0.06
Glutamate 31.2 ± 1.9* 42.6 ± 4.1 4.97 ± 0.34 5.15 ± 0.36
Glutamine 707.4 ± 32.4 637.2 ± 31.6 21.47 ± 1.38 20.88 ± 1.04
Proline 225.1 ± 30.6 233.1 ± 25.0 0.80 ± 0.07* 1.47 ± 0.24
Glycine 363.4 ± 39.4 331.7 ± 32.4 1.86 ± 0.17 2.33 ± 0.27
Alanine 449.5 ± 38.2 388.1 ± 36.1 2.85 ± 0.10 3.58 ± 0.38
Citrulline 65.0 ± 7.2* 35.8 ± 3.5 0.12 ± 0.03 0.13 ± 0.03
Ornithine 114.1 ± 7.7 114.8 ± 12.9 0.45 ± 0.05 0.44 ± 0.04
Histidine 100.6 ± 2.8* 113.8 ± 3.5 0.35 ± 0.02* 0.58 ± 0.06
Arginine 88.0 ± 7.8 72.4 ± 6.7 1.05 ± 0.37 0.95 ± 0.07
Threonine 188.2 ± 6.0 208.8 ± 12.9 0.84 ± 0.04* 1.06 ± 0.09
Valine 263.4 ± 15.5 264.2 ± 9.6 0.24 ± 0.01 0.30 ± 0.04
Methionine 30.0 ± 1.7 28.9 ± 2.1 0.02 ± 0.01 0.05 ± 0.01
Isoleucine 63.0 ± 4.5 69.3 ± 3.9 0.08 ± 0.01 0.11 ± 0.02
Leucine 145.3 ± 9.0 159.3 ± 8.2 0.13 ± 0.01* 0.22 ± 0.03
Tyrosine 91.8 ± 5.6 84.6 ± 5.6 0.14 ± 0.01 0.15 ± 0.01
Phenylalanine 77.1 ± 4.5 68.0 ± 4.0 0.7 ± 0.01 0.10 ± 0.01
Lysine 202.4 ± 9.5 223.3 ± 13.6 1.08 ± 0.10* 1.63 ± 0.19
ÂTAA, mmol/L 3.57 ± 0.14 3.42 ± 0.17 40.20 ± 1.89 43.54 ± 2.59
ÂNEAA, mmol/L 2.43 ± 0.11 2.31 ± 0.14 37.61 ± 1.80 39.93 ± 2.39
ÂEAA, mmol/L 1.14 ± 0.05 1.14 ± 0.04 2.59 ± 0.12+ 3.61 ± 0.32
ÂNEAA/ÂEAA 2.13 ± 0.05 1.96 ± 0.05 14.28 ± 0.94* 11.11 ± 1.43
ÂBCAA, mmol/L 0.47 ± 0.03 0.49 ± 0.02 0.45 ± 0.03 0.62 ± 0.09

Note: Young, 20–35 years; elderly, 57–75 years. Mean values ± SE are shown. * p < .05; + p < .01. ÂTAA =
sum of total amino acids; ÂNEAA = sum of nonessential amino acids; ÂEAA = sum of essential amino
acids; ÂBCAA = sum of branched-chain amino acids.
1 No biopsy specimens obtained from patient 1.
Source: Reprinted from Moller, P. et al., Gerontology, 29, 1–8, 1983.

there would be a strong case to establish centralized specialist laboratories containing both
analytical and interpretative expertise at the same place.

References
1. Moore, S. and Stein, W.H., Chromatography of amino acids on sulfonated polystyrene resins,
J. Biol. Chem., 192, 663–681, 1951.
2. Stein, W.H. and Moore, S., The free amino acids of human blood plasma, J. Biol. Chem., 211,
915–926, 1954.
3. Spackman, D.H., Stein, W.H., and Moore, S., Automatic recording apparatus for use in the
chromatography of amino acids, Anal. Chem., 30, 1190, 1958.
4. Graser, T.A., Godel, H.G., Albers, S., Foldi, P., and Fürst, P., An ultra rapid and sensitive
high-performance liquid chromatographic method for determination of tissue and plasma
free amino acids, Anal. Biochem., 151, 142–152, 1985.
1382_C02.fm Page 42 Tuesday, October 7, 2003 6:01 PM

42 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

5. Kedenburg, C.P., A lithium buffer system for accelerated single-column amino acid analysis
in physiological fluids, Anal. Biochem., 40, 35–42, 1971.
6. Rattenbury, J.M. and Townsend, J.C., Establishment of an external quality-assessment scheme
for amino acid analyses: results from assays of samples distributed during two years, Clin.
Chem., 36, 217–224, 1990.
7. Fürst, P., Pollack, L., Graser, T.A., Godel, H., and Stehle, P., Appraisal of four pre-column
derivatization methods for the high-performance liquid chromatographic determination of
free amino acids in biological materials, J. Chromatogr., 499, 557–569, 1990.
8. Bergman, T., Agerberth, B., and Jornvall, H., Direct analysis of peptides and amino acids
from capillary electrophoresis, FEBS. Lett., 283, 100–103, 1991.
9. Walker, V. and Mills, G.A., Quantitative methods for amino acid analysis in biological fluids,
Ann. Clin. Biochem., 32 (Pt. 1), 28–57, 1995.
10. Ziegler, F., Le Boucher, J., Coudray-Lucas, C., and Cynober, L., Plasma amino-acid determi-
nations by reversed-phase HPLC: Improvement of the orthophthaladehyde method and
comparison with ion exchange chromatography, J. Autom. Chem., 14, 145–149, 1992.
11. Kohse, K.P., Graser, T.A., Godel, H.G., Rössle, C., Franz, H.E., and Fürst, P., High-performance
liquid chromatographic determination of plasma free trimethyllysine in humans, J. Chro-
matogr., 344, 319–324, 1985.
12. Bank, R.A., Jansen, E.J., Beekman, B., and te Koppele, J.M., Amino acid analysis by reverse-
phase high-performance liquid chromatography: improved derivatization and detection
conditions with 9-fluorenylmethyl chloroformate, Anal. Biochem., 240, 167–176, 1996.
13. Jones, D.P., Carlson, J.L., Samiec, P.S., Sternberg, P., Jr., Mody, V.C., Jr., Reed, R.L., and Brown,
L.A., Glutathione measurement in human plasma: evaluation of sample collection, storage
and derivatization conditions for analysis of dansyl derivatives by HPLC, Clin. Chim. Acta,
275, 175–184, 1998.
14. Smith, K.L., Bradley, L., Levy, H.L., and Korson, M.S., Inadequate laboratory technique for
amino acid analysis resulting in missed diagnoses of homocystinuria, Clin. Chem., 44,
897–898, 1998.
15. Vester, B. and Rasmussen, K., High performance liquid chromatography method for rapid
and accurate determination of homocysteine in plasma and serum, Eur. J. Clin. Chem. Clin.
Biochem., 29, 549–554, 1991.
16. Kuhn, K.S., Krasselt, A.I., and Fürst, P., Glutathione and glutathione metabolites in small
tissue samples and mucosal biopsies, Clin. Chem., 46, 1003–1005, 2000.
17. Imai, K., Toyo’oka, T., and Watanabe, Y., A novel fluorogenic reagent for thiols: ammonium
7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate, Anal. Biochem., 128, 471–473, 1983.
18. Turnell, D.C. and Cooper, J.D., Rapid assay for amino acids in serum or urine by pre-column
derivatization and reversed-phase liquid chromatography, Clin. Chem., 28, 527–531, 1982.
19. Cohen, S.A. and Strydom, D.J., Amino acid analysis utilizing phenylisothiocyanate deriva-
tives, Anal. Biochem., 174, 1–16, 1988.
20. Davey, J.F. and Ersser, R.S., Amino acid analysis of physiological fluids by high-performance
liquid chromatography with phenylisothiocyanate derivatization and comparison with ion-
exchange chromatography, J. Chromatogr., 528, 9–23, 1990.
21. Sherwood, R.A., Titheradge, A.C., and Richards, D.A., Measurement of plasma and urine
amino acids by high-performance liquid chromatography with electrochemical detection
using phenylisothiocyanate derivatization, J. Chromatogr., 528, 293–303, 1990.
22. Tsai, M.Y., Marshall, J.G., and Josephson, M.W., Free amino acid analysis of untimed and 24-
h urine samples compared, Clin. Chem., 26, 1804–1808, 1980.
23. Levy, H.L., Madigan, P.M., and Lum, A., Fecal contamination in urine amino acid screening:
artifactual cause of hyperaminoaciduria, Am. J. Clin. Pathol., 51, 765–768, 1969.
24. Edwards, M.A., Grant, S., and Green, A., A practical approach to the investigation of amino
acid disorders, Ann. Clin. Biochem., 25 (Pt. 2), 129–141, 1988.
25. Deyl, Z., Hyanek, J., and Horakova, M., Profiling of amino acids in body fluids and tissues
by means of liquid chromatography, J. Chromatogr., 379, 177–250, 1986.
26. Young, V.R. and Munro, H.N., N-gamma-methylhistidine (3-methylhistidine) and muscle
protein turnover: an overview, Fed. Proc., 37, 2291–2300, 1978.
1382_C02.fm Page 43 Tuesday, October 7, 2003 6:01 PM

Chapter two: Measurement of amino acid concentrations in biological fluids and tissues 43

27. Neuhauser, M. and Furst, P., An automatic method for determination of urinary 3-methyl-
histidine: normal values, Anal. Biochem., 92, 294–304, 1979.
28. Millward, D.J., Bates, P.C., Brown, J.G., Rosochacki, S.R., and Rennie, M.J., Protein degrada-
tion and the regulation of protein balance in muscle, Ciba Foundation Symposium, 75,
307–329, 1979.
29. Sjolin, J., Stjernstrom, H., Arturson, G., Andersson, E., Friman, G., and Larsson, J., Exchange
of 3-methylhistidine in the splanchnic region in human infection, Am. J. Clin. Nutr., 50,
1407–1414, 1989.
30. Neuhauser, M., Bergstrom, J., Chao, L., Holmstrom, J., Nordlund, L., Vinnars, E., and Furst,
P., Urinary excretion of 3-methylhistidine as an index of muscle protein catabolism in post-
operative trauma: the effect of parenteral nutrition, Metabolism, 29, 1206–1213, 1980.
31. Smith, R. and Elia, M., The significance of urinary hydroxyproline and 3-methylhistidine
changes in growth, starvation and injury, in Amino Acid Analysis, Rattenbury, J.M., Ed., Ellis
Horwood Ltd., Chichester, U.K., 1981, pp. 225–236.
32. Kataoka, H., Nabeshima, N., Nagao, K., and Makita, M., Selective and sensitive determina-
tion of urinary total proline and hydroxyproline by gas chromatography with flame photo-
metric detection, Clin. Chim. Acta, 214, 13–20, 1993.
33. Umagat, H., Kucera, P., and Wen, L.-F., Total amino acid analysis using pre-column fluores-
cence derivatization., J. Chromatogr., 239, 463–474, 1982.
34. Bettica, P., Moro, L., Robins, S.P., Taylor, A.K., Talbot, J., Singer, F.R., and Baylink, D.J., Bone-
resorption markers galactosyl hydroxylysine, pyridinium crosslinks, and hydroxyproline
compared, Clin. Chem., 38, 2313–2318, 1992.
35. Demers, L.M., New biochemical marker for bone disease: is it a breakthrough? Clin. Chem.,
38, 2169–2170, 1992.
36. Black, D., Duncan, A., and Robins, S.P., Quantitative analysis of the pyridinium crosslinks
of collagen in urine using ion-paired reversed-phase high-performance liquid chromatogra-
phy, Anal. Biochem., 169, 197–203, 1988.
37. Kuhn, K.S., Stehle, P., and Fürst, P., Quantitative analyses of glutamine in peptides and
proteins, J. Agric. Food Chem., 44, 1808–1811, 1996.
38. Parvy, P., Bardet, J., Rabier, D., Gasquet, M., and Kamoun, P., Intra- and interlaboratory
quality control for assay of amino acids in biological fluids: 14 years of the French experience,
Clin. Chem., 39, 1831–1836, 1993.
39. Wurtman, R.J., Diurnal rhythm in mammalian protein metabolism, in Mammalian Protein
Metabolism, Munro Academic Press, New York, 1970, pp. 445–479.
40. Armstrong, M.D. and Stave, U., A study of plasma free amino acid levels. I. Study of factors
affecting validity of amino acid analyses, Metabolism, 22, 549–560, 1973.
41. Ubbink, J.B., Vermaak, W.J., van der Merwe, A., and Becker, P.J., The effect of blood sample
aging and food consumption on plasma total homocysteine levels, Clin. Chim. Acta, 207,
119–128, 1992.
42. Guttormsen, A.B., Schneede, J., Fiskerstrand, T., Ueland, P.M., and Refsum, H.M., Plasma
concentrations of homocysteine and other aminothiol compounds are related to food intake
in healthy human subjects, J. Nutr., 124, 1934–1941, 1994.
43. Craft, I.L. and Wise, I.S., Changes in amino acid metabolism during the menstrual cycle,
J. Obstet. Gynaecol. Br. Commonw., 76, 928, 1969.
44. Rauser, G., Jelinek, B., Samuels, A.S., and Kinugasa, K., Free amino acids in the blood of
man and animals. I. Method of study and the effects of venipuncture and food intake on
blood free amino acids, in Amino Acid Pools, Holden Elsevier, Amsterdam, 1962, pp. 350–372.
45. Perry, T.L. and Hansen, S., Technical pitfalls leading to errors in the quantitation of plasma
amino acids, Clin. Chim. Acta, 25, 53–58, 1969.
46. Jacob, R. and Barrett, E., Chromatographic analysis of glutamine in plasma, J. Chromatogr.,
229, 188–192, 1982.
47. Dickinson, J.C., Rosenblum, H., and Hamilton, P.B., Ion exchange chromatography of the
free amino acids in the plasma of the new born infant, Pediatrics, 36, 2, 1965.
48. Wolfe, M.S.de, Baskurt, S., and Cochrane, W.A., Automatic amino acid analysis of blood
serum and plasma, Clin. Biochem., 1, 75, 1967.
1382_C02.fm Page 44 Tuesday, October 7, 2003 6:01 PM

44 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

49. Hamilton, P.B., Amino acids on hands, Nature, 205, 284, 1965.
50. Armstrong, M.D. and Stave, U., A study of plasma free amino acid levels. II. Normal values
for children and adults, Metabolism, 22, 561–569, 1973.
51. Möller, P., Bergström, J., Eriksson, S., Fürst, P., and Hellström, K., Effect of aging on free
amino acids and electrolytes in leg skeletal muscle, Clin. Sci. (Lond.), 56, 427–432, 1979.
52. Divino Filho, J.C., Bergström, J., Stehle, P., and Fürst, P., Simultaneous measurements of free
amino acid patterns of plasma, muscle and erythrocytes in healthy human subjects, Clin
Nutr., 16, 299–305, 1997.
53. Möller, P., Alvestrand, A., Bergström, J., Fürst, P., and Hellstrom, K., Electrolytes and free
amino acids in leg skeletal muscle of young and elderly women, Gerontology, 29, 1–8, 1983.
54. Alvestrand, A., Fürst, P., and Bergström, J., Plasma and muscle free amino acids in uremia:
influence of nutrition with amino acids, Clin. Nephrol., 18, 297–305, 1982.
1382_C03.fm Page 45 Tuesday, October 7, 2003 6:08 PM

chapter three

Approaches to studying amino acid


metabolism: from quantitative
assays to flux assessment using
stable isotopes
Dominique Darmaun
Hôtel-Dieu Hospital, Nantes
Luc Cynober
Hôtel-Dieu Hospital, Paris

Contents
Introduction....................................................................................................................................46
3.1 Plasma amino acid concentrations and the significance of their variations ..............46
3.1.1 Interpreting normoaminoacidemia.......................................................................47
3.1.2 Interpreting hyperaminoacidemia ........................................................................47
3.1.3 Interpreting hypoaminoacidemia .........................................................................48
3.2 Measurement of arteriovenous differences......................................................................48
3.2.1 Blood flow measurements......................................................................................49
3.2.2 Regional balances of amino acids.........................................................................50
3.3 Use of stable isotopes to assess amino acid metabolism in vivo ..................................51
3.3.1 Assessment of whole-body protein kinetics .......................................................51
3.3.1.1 The 15N-glycine method ...........................................................................51
3.3.1.2 The L-13C-leucine method ........................................................................51
3.3.1.3 The phenylalanine–tyrosine method .....................................................55
3.3.2 Use of stable isotopes to assess protein metabolism in specific tissues.........56
3.3.2.1 Assessing the fractional uptake of amino acids in the
splanchnic bed...........................................................................................56
3.3.2.2 Combined use of stable isotopes and arteriovenous differences .....56
3.3.2.3 Assessing the fractional synthesis rate of specific proteins...............56
3.3.2.4 Future developments................................................................................57
References .......................................................................................................................................57

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 45
1382_C03.fm Page 46 Tuesday, October 7, 2003 6:08 PM

46 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Introduction
Critical illness is accompanied by vast alterations in protein turnover resulting in wasting
of lean body mass. Body protein indeed contributes 10 to 13% of weight loss in these
situations.1 Although the amount of body protein that can be lost without serious delete-
rious effects is not known,2 a loss of 8 to 17% of body protein results in a loss of fitness
in healthy persons,1 and a 50% loss is fatal.3 Most of this nitrogen loss arises from skeletal
muscle,4,5 with alanine and glutamine accounting for 35 to 46% of peripheral nitrogen
output. Alanine is used by the liver for gluconeogenesis. Glutamine is used by several
visceral organs. It is a source of ammonia for the kidney to aid in acid–base balance
homeostasis. In intestinal mucosa, glutamine serves as a primary oxidizable fuel source.
Glutamine also serves as an energy source for immunologically active cells and other
rapidly proliferating cells such as fibroblasts.5,6 More-recent evidence points to a major
role of glutamine as a source of carbon for gluconeogenesis as well.7,8 Concomitant with
this efflux of amino acids from muscle is an increase in the rate of hepatic synthesis of
various plasma proteins.
Classically, nitrogen (N) balance has been used as a tool to study protein metabolism
and assess nitrogen requirements under steady-state conditions. N balance is calculated
as the difference between the nitrogen in and the nitrogen out. This method, however, is
plagued by several intrinsic flaws: (1) The most important of these relates to the fact that
illness is usually associated with non-steady-state conditions, and hence many of the
assumptions used for estimating nitrogen balance are not applicable. (2) N intake is likely
to be overestimated because of inadequate assessment of the food consumed. (3) The
component of nitrogen out is likely to be underestimated because of methodological
problems in collecting and measuring nitrogen losses from the body.9 This includes esti-
mates of losses through the urine, feces, and integuments (skin, hair, nails, sweat, etc.).
For example, under normal physiologic conditions, the integumental losses are usually
very small and relatively insignificant. After severe burn injury, however, these losses
increase dramatically and were estimated by Waxman et al.10 to be equivalent to 0.19 ¥
body surface area ¥ percent burn size. Thus, in an adult man suffering a 50% burn, this
loss would amount to 9.0 g of nitrogen per day in the first week and 4.5 g per day
subsequently.11 (4) Furthermore, nitrogen balance does not yield any insight into the
dynamics of protein turnover (e.g., protein synthesis and breakdown, as well as amino
acid oxidation). A positive nitrogen balance is indicative of a relative excess in the rate of
protein synthesis over that of breakdown. A negative nitrogen balance suggests that
synthesis is lower than breakdown, while a nil nitrogen balance indicates that the subject
is in equilibrium with synthesis relatively equivalent to breakdown.5 Finally, nitrogen
balance does not shed any light on the relative contribution of the different organs to
nitrogen output.

3.1 Plasma amino acid concentrations and the significance of their


variations
Plasma amino acids can be measured by various methods, the most popular being ion
exchange chromatography and reversed-phase high-performance liquid chromatography
(see Chapters 1 and 2, respectively).
Although there is considerable literature dealing with the plasma concentrations of
amino acids under various physiological and pathological conditions, it is frequently
stated that interpreting plasma amino acid concentrations is difficult, or even impossible.
The basis for this statement is that the plasma pool of free amino acids is very small
compared to the intracellular pool of free amino acids, which is in turn of little importance
1382_C03.fm Page 47 Tuesday, October 7, 2003 6:08 PM

Chapter three: Approaches to studying amino acid metabolism 47

with regard to the protein-bound amino acid pool, the three being in equilibrium. In
addition, some amino acids, especially the acidic ones, are present in large amounts in
erythrocytes. The contribution of this pool to tissue exchanges remains a subject of debate.12
Finally, amino acids are subject to multiple interorgan exchanges, which further confuses
the interpretation of static plasma values.
However, catheterization and stable isotope-based techniques have clearly indicated
(see infra) that the concentration of a given amino acid in plasma is the end result of its
rate of appearance (Ra) in plasma minus its rate of disappearance (Rd), where Ra is mainly
a function of muscle production æ provided it is measured at a time when no amino acids
arise from the intestinal absorption of a meal or from amino acid infusion æ and Rd
depends on uptake by splanchnic tissues, mainly the liver.

3.1.1 Interpreting normoaminoacidemia


There are many situations where plasma amino acid concentrations are normal with Ra
= Rd, such as the interprandial state where Ra and Rd are normal, the mild catabolic states
where Ra and Rd are increased equally,13 and the intermediate stage of starvation where
they are both equally decreased.14 In these situations, it can be considered that the pro-
duction of amino acids matches their consumption, and thus, the determination of plasma
amino acids gives little information.

3.1.2 Interpreting hyperaminoacidemia


Hyperaminoacidemia can result from a decrease in Rd with Rd < Ra or from an increase
in Ra with Ra > Rd.
The decrease in Rd is typically observed during multiorgan failure; in the terminal
phase of septic shock, hyperalaninemia is observed, resulting primarily from a collapse
in the uptake of alanine by the liver.15
An excessive rise in Ra can be observed during the administration of an inappropriate
parenteral nutrition regimen. Hyperaminoacidemia can be global as the result of a too
rapid perfusion rate, or more or less selective when the solution is qualitatively inadequate
for the patient. It is reasonable to assume that the most appropriate solution for a given
patient is that which induces the least imbalance in plasma amino acid concentrations,
reflecting a correct utilization of the amino acids perfused.16 The study of the pharmaco-
kinetics of perfused amino acids is probably one of the most promising fields for the
future, as described in Chapter 31 and in recent reviews.17,18
Hyperphenylalaninemia merits further discussion because, in the context of clinical
nutrition, it can result from either an increase in Ra or a decrease in Rd. As a matter of
fact, phenylalanine is poorly metabolized in peripheral tissues and is mainly metabolized
in the liver so that any increase in its plasma concentration reflects either protein hyper-
catabolism or hepatocellular insufficiency. In burn patients, plasma phenylalanine corre-
lates positively with 3-methylhistine excretion and negatively with N balance.19 Therefore,
the plasma phenylalanine level can be considered a suitable marker of N turnover in
situations of stress. Its determination offers the advantage of not requiring urine collection,
which is always difficult even in intensive care units (ICUs). When liver function is altered,
plasma phenylalanine increases sharply. This has been exploited in the context of liver
transplantation; the occurrence of hyperphenylalaninemia in the first hour following rep-
erfusion is associated with complications (primary graft dysfunction, cellular necrosis) in
the 48 h following the transplantation.20
The use of variations in the concentrations of a given amino acid as a marker of organ
function could be expanded. For example, since kidneys actively metabolize citrulline into
1382_C03.fm Page 48 Tuesday, October 7, 2003 6:08 PM

48 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

arginine, hypercitrullinemia is a constant feature of chronic renal failure21; however, to the


best of our knowledge, this type of approach has not been explored. In the same way,
tumor consumption of amino acids can specifically modify the plasma amino acid pattern.
This could be of use in the diagnosis and follow-up of cancer patients (see Chapter 40 for
more details).

3.1.3 Interpreting hypoaminoacidemia


Hypoaminoacidemia can result from an increase in Rd or a decrease in Ra.
An increase in Rd is typical of stress situations, resulting from the increase in hepatic
uptake, primarily of gluconeogenic amino acids.22 In this situation, Ra is also increased,
but this is insufficient for maintaining amino acid homeostasis and Ra < Rd. For example,
in burn patients, plasma alanine decreases by 50% and glutamine by even more.23,24 In
addition, the drop in plasma alanine is more profound in patients with sepsis or those
who subsequently die than in those who survive.25 The same has been found for
glutamine.23 This underlines the relationship between morbidity and metabolic exhaus-
tion, i.e., the body’s incapacity to mobilize the required stores to cope with stress.
A decrease in Ra can result either from a decrease in protein intake or from a decrease
in mobilizable amino acid stores. This is typical of states of chronic undernutrition and
mainly concerns essential amino acids.
Also, a decreased Ra may be observed in the case of organ failure in a tissue that plays
a major role in the production of a given amino acid. For instance, Crenn et al.26 found
that plasma citrulline is low in patients with short bowel syndrome, compared with control
subjects, with a correlation between residual intestinal mass and plasma citrulline level.
Similar observations have been reported during the rejection of intestinal transplants27 or
after intestinal irradiation.28
Finally, a decrease in Ra can result from therapeutic manipulations. For example,
Cynober et al.29 have shown that ornithine a-ketoglutarate (OKG) administration to burn
patients results in a significantly greater decline in the plasma amino acid concentrations,
compared to burn patients receiving the control regimen. In a further trial,30 it was shown
that OKG decreases muscle amino acid output (see Chapter 37 for more details). Similarly,
Mjaaland et al.31 demonstrated that human growth hormone (hGH) therapy decreases
both the concentration of amino acids in venous plasma and their efflux from the forearm.
The correct interpretation of this data is that the decrease in plasma concentrations is due
to a decrease in muscle output in response to OKG or hGH, without any significant change
in their splanchnic uptake. Obviously, in this case a single determination of plasma amino
acids cannot be interpreted and even confuses the issue.

3.2 Measurement of arteriovenous differences


This section describes some of the methods utilized in the laboratory of Abumrad et al.
for the past decade in the conscious dog model.32–34 This approach offers several advan-
tages. The size of the animal is such that it allows for easy surgical preparation and for
withdrawal of adequate amounts of blood without untoward effects. These measurements
can be easily combined with determinations of hemodynamic status. Finally, ethical con-
siderations for the use of invasive techniques in humans limit the ability of the investiga-
tors to control for all the necessary variables to eliminate bias in the experimental design.
Details regarding animal preparations, preoperative care, operative procedures, and
postoperative care have already been published.33 In brief, the animals undergo placement
of silastic catheters in any or all of the various arteries (femoral, carotid, or vertebral) or
veins (femoral, portal, hepatic, internal jugular, coronary, etc.) under general endotracheal
1382_C03.fm Page 49 Tuesday, October 7, 2003 6:08 PM

Chapter three: Approaches to studying amino acid metabolism 49

anesthesia at least 3 weeks prior to the day of the study. Catheters are filled with heparinized
saline, knotted, and placed in subcutaneous pouches. When needed, the animals are also
fitted with tracheostomy or with placement of intracerebroventricular catheters in either the
third or fourth ventricles. Simultaneously, Doppler flow cuffs of various diameters (e.g.,
6 to 7 mm for the portal vein, 3 mm for the hepatic artery, 5 to 6 mm for the iliac artery,
etc.) are usually placed around the vessels of interest, and the leads are then tunneled and
secured to the abdominal incision as those of the silastic catheters. On the day of the study,
the catheter ends are usually exteriorized by making a small skin incision using local
anesthesia (1% lidocaine). The catheters are flushed with saline and used for blood sampling.

3.2.1 Blood flow measurements


Blood flow measurements are carried out in one of several methods. The first, Doppler
technique, is based on the recorded shift in a pulsed sound signal according to the method
of Hartley et al.35 In this method, blood volume flow is estimated by the equation

Volume flow (Q) = Vavg ¥ A (3.1)

where Vavg is the average velocity of the blood and A is the average diameter of the
vessel lumen. This method has two major distinct advantages. The first relates to the
ability to make separate estimates of arterial and venous blood flows. The second relates
to the consistency in the measurements across specific organs that can be obtained.
A second method is based on the use of dye dilution techniques. Both indocyanine
green (ICG) and para-aminohippurate (PAH) dyes can be utilized. ICG may be used for
estimating splanchnic blood flows according to the method of Leevy et al.36 Continuous
infusion of ICG is given at 0.1 mg/m2/min, blood samples are taken any time after 45 min
(after establishment of steady-state levels), and plasma ICG is estimated by spectropho-
tometric methods at 810 nm. Similarly, we have utilized PAH for estimation of kidney
plasma flow. This method involves the use of a primed (0.3 mg/kg), continuous
(0.3 mg/kg/min) infusion of PAH. Estimate of organ plasma flow is estimated according
to the equation

Infusion rate
organ plasma flow = (3.2)
artery dye - vein dye

Another technique frequently used for estimation of pulmonary plasma (or blood
flows) is the use of thermodilution according to the method of Fegler.37 Modifications of
this method entail the manual injection of 5.0 ml of cold saline (0.9%) into the right atrium
and recording the change in blood temperature in the pulmonary artery. This method
bears a high coefficient of correlation with dye dilution techniques and electromagnetic
flow meter. The use of radiolabeled microspheres has been proposed for estimates of
regional blood flow in the hindlimbs, kidneys, brain, etc., as was described by Heyman
et al.38 Several radiolabeled microspheres have been used: 141cerium, 85strontium, and
46scandium are usually injected into the left atrium. Blood is drawn at a constant rate via

the femoral artery before, during, and after the microsphere injections. At the end of the
experiment, tissues are sampled for radioisotope determination using a multichannel
pulse-height analyzer. All samples are counted on the same day. Regional blood flow is
then estimated according to

(regional # microspheres in cpm / g) ¥ (reference blood flow in ml / min)


(3.3)
reference blood number of micropheres in cpm
1382_C03.fm Page 50 Tuesday, October 7, 2003 6:08 PM

50 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Several assumptions need to be satisfied for appropriate estimates of blood flow using
this method. Several studies, however, have shown good coefficients of correlation with
other established methods such as electromagnetic flow meter, dye dilution, etc.

3.2.2 Regional balances of amino acids


In general, the net balance of any amino acid across an organ can be estimated by the
following:

net balance = (arteryamino acid – veinamino acid) (3.4)

As would be anticipated, a positive net balance indicates net retention of the amino
acid, while a negative balance indicates net release. In none of these circumstances, how-
ever, do the methods allow the differentiation between a significant change in either
protein synthesis over that of protein breakdown. Such a shortcoming can be overcome
by the constant infusion of a labeled (either radioactive or stable isotope) amino acid.
Although several amino acids have been utilized, those that are carbon labeled are pre-
ferred, as they allow the determination of the rate of production of either 14CO2 or 13CO2
across the organ in question. Estimates of the net uptake or release of the labeled amino
acid across the organ will allow for the estimation of the unidirectional entry (utilization,
estimate of protein synthesis) or exit (loss, protein breakdown) according to the following
formulas:

(artery labeled a.a. - vein labeled a.a ) ¥ blood flow


Unidirectional entry of amino acid = (3.5)
SA labeled a.a

( vein *CO2 - artery *CO2 ) ¥ blood flow


Net oxidation of amino acid = (3.6)
SA labeled a.a

Rate of protein breakdown across an organ = net balance – unidirectional utilization (3.7)

In summary, it is quite clear that the combined use of labeled radio- or stable isotopes
in conjunction with arteriovenous techniques allows the investigator to estimate the rel-
ative regional contribution of protein synthesis and breakdown and amino acid oxidation
to the overall metabolism of the specific amino acid in question during periods of stress,
illness, etc.
Such methods have been used for estimating the relative contribution of various
organs to overall metabolism of various organs to amino acid metabolism. Several studies
by Nair et al. in man,39 Helland et al. in pigs,40 and Lobley et al. in cattle41 estimate the
contribution of muscle protein synthesis to the overall rate of whole-body protein synthesis
to be approximately 25 to 30%. Previous studies from the Abumrad33,34 laboratory showed
that the splanchnic organs are responsible for about 30% of the total flux of leucine. Most
of these studies have utilized state-of-the-art methodologies for estimating arteriovenous
differences of amino acids in combination with various techniques for measurements of
blood flow as outlined above.
Finally, measurement of arteriovenous differences has resolved the problem of the
origin of 3-methylhistidine; 3-methylhistidine comes mainly from muscle. In trauma
patients, the increase in the urinary output of this amino acid is solely due to increasing
muscle myofibrillar protein catabolism.42,43
1382_C03.fm Page 51 Tuesday, October 7, 2003 6:08 PM

Chapter three: Approaches to studying amino acid metabolism 51

3.3 Use of stable isotopes to assess amino acid metabolism in vivo


3.3.1 Assessment of whole-body protein kinetics
Because metabolism is a dynamic process, tracer methods are theoretically ideally suited
to follow the fate of amino acids in vivo.

3.3.1.1 The 15N-glycine method


15
The N-glycine method was the first stable isotope method used for quantitation of protein
turnover in vivo.44,45 Briefly, when 15N-glycine is administered at a constant rate (i), the 15N
content (enrichment) in urinary urea reaches steady state after 48 to 72 h. It is assumed
that glycine’s nitrogen (N) is evenly distributed in the body’s nitrogen metabolic pool and
that the fraction of the 15N dose administered that is eventually excreted equals the fraction
of the nitrogen pool turnover excreted as urea; whole-body N turnover (Q) is quantitated
by measuring steady-state N enrichment in urea (Eurea):

Q = i/Eurea

Assuming that the body N pool is at steady state, the amount of N entering the pool,
i.e., the sum of dietary N intake (I) and N released from protein breakdown (B), equals
the amount leaving the pool and directed into either protein synthesis (S) or oxida-
tion/excretion to urea (U). Thus,

B = Q – I and S = Q – U

The method relies on robust analytical techniques — precise determination of low 15N
enrichments (down to <0.01%) on large amounts (several micromoles) of urinary urea N
by gas isotope ratio mass spectrometry (IRMS) after conversion to N2 gas45 — and is
noninvasive. Yet it shares some of the practical difficulties of the N balance technique and
relies on a host of unproven assumptions. For instance, there is no such thing as a nitrogen
metabolic pool since there are both anatomical and biochemical barriers to glycine: (1)
because glycine is poorly transported across certain cell membranes, it does not freely
enter all tissues of the body,46,47 and (2) its a-amino N does not end up evenly distributed
among the 20 free amino acids.47 Some of these obstacles may be overcome by using a
mixture of 15N-labeled amino acids.48 In addition, the ideal end product in which 15N
determination should be performed æ urinary urea, urinary ammonium, or both æ
remains a matter of debate.49 Finally, in order to shorten the measurement period, a
technique involving the single administration of a single tracer bolus has been proposed.49

3.3.1.2 The L-13C-leucine method


The l-13C-leucine method50 emerged when new developments in gas chromatogra-
phy–mass spectrometry (GCMS) made it possible to determine relatively high isotopic
enrichments (>0.2%, usually 1 to 5%) not in micromoles of end products in liters of urine,
but rather in nanomolar amounts of precursors, that is, amino acids themselves, in micro-
liters of plasma.45 When pure L[1-13C]-leucine is infused (Figure 3.1) at a constant rate (i),
a plateau of 13C-leucine enrichment is reached in 2 to 3 h in plasma (Ep), and the appear-
ance rate (Ra) of leucine can be calculated from the dilution of the labeled leucine in
plasma at steady state:

Ra = i [Ei/Ep – 1]
1382_C03.fm Page 52 Tuesday, October 7, 2003 6:08 PM

52 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 3.1 The 13C-leucine model.

where Ei is the isotopic enrichment in the tracer infused. As leucine is an essential amino
acid, its only sources are protein breakdown (B) and dietary intake (I):

Ra = B + I

If the total rate of respiratory CO2 excretion (V CO2) and the 13C enrichment of breath
CO2 are quantitated using indirect calorimetry and IRMS, respectively, the rate of leucine
oxidation (Ox) is measured. Under conditions of steady state, leucine Ra equals its disap-
pearance rate (Rd). Since leucine may only be oxidized or utilized for protein synthesis,
the nonoxidized fraction of leucine Rd is thus attributed to leucine incorporation into
protein synthesis (S):

S = Rd – Ox

Leucine fluxes can be extrapolated to whole-body protein fluxes, based on the average
leucine content of body protein (8 g of leucine per 100 g of protein).50
The model relies on several assumptions, many of which have been tested and
validated:

1. There is no reason to believe that any enzymatic system or membrane carrier


distinguishes between 13C- and 12C-leucine. For instance, although the red blood
cell membrane is poorly equipped with amino acid carriers, 13C-leucine freely
equilibrates between erythrocytes and plasma.46
2. 13C-Leucine, once incorporated into protein, may be released into plasma over the
course of the experiment. This tracer recycling51 results in artificially high plasma
leucine enrichments and, consequently, underestimation of leucine Ra, causing as
much as a 30% error for a 24-h-long infusion, but is thought to be minimal when
infusions are less than 4 to 8 h.51
3. It is now proven that plasma 13C-leucine enrichment overestimates intracellular
leucine enrichment. Because a-ketoisocaproate (KIC) is solely derived from intra-
cellular transamination of leucine (Figure 3.2) and since transamination is extreme-
ly fast within cells, several authors have proposed to measure the 13C enrichment
of plasma free KIC instead of leucine.52,53 Plasma 13C-KIC enrichment is indeed a
good estimate of 13C enrichment in the muscle tissue tRNA-bound leucine, from
which leucine is drawn for protein synthesis.54 Moreover, the steady-state 13C
enrichment of the leucine incorporated in plasma apolipoprotein B — a protein
with a very fast turnover rate — was similar to that of plasma KIC during an
1382_C03.fm Page 53 Tuesday, October 7, 2003 6:08 PM

Chapter three: Approaches to studying amino acid metabolism 53

Figure 3.2 Relationships between leucine and a-ketoisocaproate (KIC) during an infusion of L-[l-13C]
leucine. (From Matthews, D.E. et al., Metabolism, 31, 1105, 1982. With permission.)

infusion of 13C-leucine.55 Plasma 13C-KIC enrichment usually reaches 80% of


plasma 13C-leucine enrichment; the difference is attributed to the fraction of leucine
that is released by protein breakdown inside cells but does not appear in the general
circulation.
4. Recovery of metabolically produced 13C is not quantitative, due to loss of labeled
bicarbonate in hidden, slowly overturning pools such as bone or utilization of CO2
in the gluconeogenic pathway. Recovery is affected by rates of energy expenditure
and total CO2 production — e.g., rising from 74 to 82% in the transition from the
fasted to the fed state — but not by the route of tracer delivery.56 Ideally, recovery
of 13CO2 in breath should be determined in any given experimental setting. Several
groups have shown that an infusion of 13CO3HNa can be performed in the few
hours immediately preceding the infusion of labeled leucine. An additional ad-
vantage of this approach is the fact that indirect calorimetry is no longer necessary
to quantify overall CO2 production, since the rate of CO2 production can be deter-
mined using isotope dilution equations.57–59
5. As leucine is an essential amino acid, B = Ra is the fasting state. Rates of protein
breakdown (B) are calculated by subtracting intake (I) from total rate of appearance
(B = Ra – I), when I is accurately known in a parenterally fed patient. When a
patient is fed orally, however, part of dietary leucine undergoes first-pass extraction
in the splanchnic bed and never reaches systemic blood. This problem can be
circumvented by adding a second (e.g., 2H3-labeled) leucine tracer to the enteral
regimen.60 Yet this approach assumes that the leucine present as bound residues
1382_C03.fm Page 54 Tuesday, October 7, 2003 6:08 PM

54 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

in peptides/protein in the regimen has the same fate as the free leucine tracer in
the intestinal lumen. This may not be the case, as recent studies using intrinsically
13C-labeled proteins have found that proteins with different rates of hydrolysis

in the intestinal lumen clearly have different effects on whole-body protein


metabolism.61,62
6. Although leucine and phenylalanine have different target organs in the body —
leucine is predominantly oxidized in muscle and phenylalanine in liver — their
Ra values yield similar estimates of whole-body protein breakdown, even in situ-
ations such as cortisol-induced hypercatabolism,63 which supports the use of leu-
cine Ra as a valid index of whole-body proteolysis.
7. The assessment of protein synthesis is indirect, derived from the difference between
two measured parameters (total Ra and oxidation) and is therefore the weak point
of the method. However, inhibition of protein synthesis using the drug emetine
resulted in nearly complete suppression of nonoxidative disposal in dogs.64

Overall, the 13C-leucine method has survived two decades of intense scrutiny and is
now considered the method of reference to obtain fair estimates of whole-body protein
metabolism.
A major strength of this method is that it uses a stochastic model and relies on simple,
robust, mathematical analysis. Although compartmental models have been developed for
leucine metabolism,65 they have not been widely used, presumably because of the com-
plexity of their mathematical analysis.
However, the 13C-leucine method provides no insight into other important aspects of
protein metabolism:

1. Although the model has only been validated under conditions of steady state,
many physiological situations (such as oral food intake) cannot be described in
terms of steady state, and the constraints of the model have forced most investi-
gators to use a simplified experimental design such as continuous feeding.50
2. The leucine method does not explore the metabolism of nonessential amino acids.
This can, however, be achieved by a simultaneous infusion of 13C-leucine and a
labeled nonessential amino acid tracer, e.g., L [2- 15 N ]-glutamine.63 Because
glutamine is a nonessential amino acid, two endogenous sources contribute to its
Ra in the postabsorptive state: (1) protein breakdown and (2) de novo synthesis.
The contribution of proteolysis to glutamine Ra can be estimated based on leucine
Ra — an index of proteolysis — and the average glutamine and leucine content
of body protein (0.5 mol of glutamine for each mole of leucine). This approach
relies on the assumption that the release of amino acids through protein breakdown
is directly proportional to their relative abundance in body protein. Protein break-
down indeed only accounts for 13% of glutamine Ra.66 The fraction of glutamine
Ra not accounted for by proteolysis is attributed to de novo synthesis,63 with >30%
of its N derived from branched-chain amino acids in postabsorptive humans.67
Although this approach has yielded further insight into glutamine interorgan
exchange, it should be borne in mind that the measured glutamine Ra only mea-
sures a fraction of whole-body glutamine production, due to the compartmentation
of glutamine between the extra- and intracellular milieus.68,69 Similarly, although
the endogenous rates of production of most essential amino acids correlate with
their relative abundance in body protein,70 the rates of appearance of several
nonessential amino acids were found to be paradoxically low, barely exceeding
what can be accounted for by protein breakdown. This is particularly true for
1382_C03.fm Page 55 Tuesday, October 7, 2003 6:08 PM

Chapter three: Approaches to studying amino acid metabolism 55

glutamate,68 arginine,71 or proline.72 The relatively low de novo synthesis rates


measured for these presumably nonessential amino acids imply either (1) that the
model used for measuring their turnover rate is overly simplistic, or (2) alterna-
tively, that these amino acids indeed are not truly nonessential and can become
conditionally essential under some circumstances.73
3. While the human genome codes for the synthesis of several thousand different
proteins, the 13C-leucine method only assesses the overall picture, i.e., the end
result of the synthesis and breakdown of a host of proteins with widely different
half-lives, ranging from a few minutes for hepatic enzymes to several weeks in
the case of myofibrillar proteins. This can be overcome by measuring the synthesis
rate of specific tissue proteins (see below).

3.3.1.3 The phenylalanine–tyrosine method


As stated above, the leucine method is applicable to other essential amino acids as well,
among which is phenylalanine. One shortcoming of the 13C-leucine method is that it
requires collection of both blood and breath and three analytical instruments: GCMS,
IRMS, and indirect calorimetry for plasma 13C-leucine, breath 13CO2, and CO2 production,
respectively. In contrast, the first, irreversible step in phenylalanine oxidation produces
tyrosine rather than CO2. Upon constant infusion of 2H5-phenylalanine, oxidation of the
tracer will produce 2H4-tyrosine, provided the deuteriums are located on the phenyl ring.74
Both labeled phenylalanine and labeled tyrosine can be quantitated by GCMS. Phenyl-
alanine oxidation can be calculated based on (1) the appearance of 2H4 in tyrosine and
(2) total tyrosine Ra, which requires the simultaneous infusion of another tracer of tyrosine
(e.g., 13C-tyrosine), just like measurement of total CO2 production is needed when leucine
oxidation is quantitated. As conversion to tyrosine and incorporation into protein are the
only significant routes of phenylalanine disposal, the nonoxidized fraction of phenyl-
alanine Ra is attributed to phenylalanine incorporation into protein.75
Moreover, labeled phenylalanine has been used in combination with measurement of
arteriovenous gradients to assess muscle protein metabolism.75,76 Indeed, contrary to leu-
cine, phenylalanine cannot be oxidized in muscle. Therefore, its extraction in skeletal
muscle solely reflects its utilization for muscle protein synthesis. Thus, if catheters are
inserted into the artery and a deep vein of the forearm, and if blood flow (F) through the
forearm is measured, then the extraction coefficient (ER) of phenylalanine can be deter-
mined as the fraction of the labeled phenylalanine disappearing between the arterial and
venous sites:

ER = (*Phea – *Phev)/*Phea)

where *Phea and *Phev are the concentrations of 2H5-labeled phenylalanine in arterial and
deep venous blood, respectively. The total rate of phenylalanine release from the forearm
equals Phea ¥ F, where Phev is the concentration of unlabeled phenylalanine in venous
plasma. This rate of release is the sum of the phenylalanine that crosses the forearm
unaltered, i.e., phenylalanine that is not extracted,

F ¥ Phea ¥ (1 – ER)

plus the phenylalanine released by muscle proteolysis (Bm). Thus,

Phev ¥ F = (1 – ER)(Bm + F ¥ Phea)


1382_C03.fm Page 56 Tuesday, October 7, 2003 6:08 PM

56 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

It follows that

Bm = [Phev ¥ F/(l – ER)] – [F ¥ Phea]

In turn, the arteriovenous balance for unlabeled phenylalanine measures the net
difference between local proteolysis and local protein synthesis (Sm), which can therefore
be calculated as:75

Sm = F (Phea – Phev) – Bm

3.3.2 Use of stable isotopes to assess protein metabolism in specific tissues


3.3.2.1 Assessing the fractional uptake of amino acids in the splanchnic bed
As the splanchnic bed plays a prominent role in the disposal of most amino acids, several
groups have used a dual-label approach to quantify the fraction of a given amino acid
that undergoes uptake in the splanchnic territory. For instance, an intravenous infusion
of deuterium-labeled leucine (2H3-leucine) has been combined with the concomitant infu-
sion of 13C-leucine through a nasogastric tube to assess the rate of first-pass splanchnic
extraction in the splanchnic bed. This approach relies on the assumptions that (1) 100%
of the enterally administered labeled amino acid undergoes intestinal absorption, and
(2) 2H3- and 13C-labeled leucine are handled similarly in the body. Both of these assump-
tions have been validated. This method was used to demonstrate that approximately 25%
of enterally administered leucine is taken up in the first pass in the splanchnic bed of
healthy adult humans in the postabsorptive state77,78 (with only 2 to 3% of the leucine
oxidized in the first pass). In contrast, a much higher fraction (ª42 to 50%) of dietary
leucine is taken up in the splanchnic territory in premature infants79 or elderly subjects.80
Fractional splanchnic uptake varies considerably from one amino acid to the next, as it
amounts to 50 to 70% for glutamine81,82 and >90% for glutamate.83

3.3.2.2 Combined use of stable isotopes and arteriovenous differences


Although the combined use of stable isotope infusion and arteriovenous gradient methods
has been applied less extensively in humans than in animals, this approach has, for
instance, been used to demonstrate that muscle contributes over 50% of the overall endog-
enous production of alanine and glutamine in healthy humans.84 Similarly, the contribution
of the human kidney in the production of glucose from glutamine carbon was quantitated
by this approach.85

3.3.2.3 Assessing the fractional synthesis rate of specific proteins


In the last decade, the measurement of labeled amino acid incorporation into specific
proteins has been greatly facilitated by the advent of a new technique combining the ease
of separation of GCMS with the high sensitivity of IRMS, namely, gas chromatogra-
phy–mass spectrometry–combustion–isotope ratio mass spectrometry (GC-C-IRMS),
whereby the leucine peak eluted from the gas chromatograph is combusted on line to CO2
in a furnace and subsequently analyzed for 13CO2/12CO2 by an on-line IRMS.86 In this
approach, the isotope enrichment is measured both in the precursor pool (e.g., intracellular
free leucine) and product, i.e., the protein of interest. The fractional synthesis rate (FSR)
of the protein of interest is calculated as

FSR = 24 ¥ 100 ¥ (Eboundt2 – Eboundt1)/(Eprecursor¥ Dt)


1382_C03.fm Page 57 Tuesday, October 7, 2003 6:08 PM

Chapter three: Approaches to studying amino acid metabolism 57

where Eboundt2 and Eboundt1 are the isotope enrichments (mole percent excess) measured
in protein-bound leucine at times t2 and t1 (hours), respectively, over the course of the
isotope infusion study; Eprecursor is the steady-state isotope enrichment in the precursor
amino acid pool used for protein synthesis; Dt is the time interval (hours) between the
two sampling points t2 and t1; and 24 and 100 convert FSR to percent per day. The major
difficulty in this approach lies in the choice of the appropriate precursor pool. Although
tRNA-bound leucine would be the ideal precursor pool, determination of isotope enrich-
ment in the tRNA pool has been hampered by technical difficulties54 and is therefore rarely
performed. The use of plasma KIC may be an alternative for liver and muscle87 but is not
optimal in other tissues, such as small intestine, where free leucine enrichment should be
measured.88,89
This approach has been applied to circulating plasma proteins such as albumin,
fibrinogen, or apolipoproteins,55 or to the synthesis rate of a nutritionally significant pep-
tide abundant in erythrocyte such as glutathione.90 In vivo studies have, for instance, shown
that insulin deficiency decreases albumin FSR, while it stimulates fibrinogen FSR.91 This
illustrates the fact that a given regulatory factor can affect various proteins in opposite
directions and the need for measurement of the synthesis of specific proteins.
The performance of a muscle biopsy has allowed the determination of the FSR of
mixed muscle protein, as well as of specific contractile proteins such as myosin heavy
chain in both young and elderly subjects.92,93 Similarly, the performance of a duodenal or
colonic biopsy, using an endoscopic procedure at the end of a 4- to 6-h infusion of labeled
leucine, allows for the determination of intestinal protein synthesis. This approach showed
gut protein synthesis to be extremely rapid (ª40 to 80% per day), compared with muscle
protein synthesis (ª1% per day).88,89,94

3.3.2.4 Future developments


In the long run, developments in the techniques of nuclear magnetic resonance (NMR)
may allow amino acid metabolism to be followed live and in situ in a specific tissue and
in a nonsampling fashion. Such a noninvasive approach of protein kinetics has only been
accomplished in isolated tissues in vitro95 so far.

References
1. Kinney, J.M. and Elwyn, D.H., Protein metabolism and injury, Annu. Rev. Nutr., 3, 433, 1983.
2. Wilmore, D.W., Catabolic illness: strategies for enhancing recovery, N. Engl. J. Med., 325, 695,
1991.
3. Mirtallo, J.M., Assessing the nutritional needs of the critically ill patient, DICP Ann. Pharma-
ceut., 24, 520, 1990.
4. Hasselgren, P.O., Pedersen, P., Sax, H.C., Warner, B.W., and Fisher, J.E., Current concepts of
protein turnover and amino acid transport in liver and skeletal muscle during sepsis, Arch.
Surg., 123, 992, 1988.
5. Goldstein, S.A. and Elywn, D.H., The effects of injury and sepsis on fuel utilization, Annu.
Rev. Nutr., 9, 445–473, 1989.
6. Abumrad, N.N., Morse, E.L., Lochs, H., Williams, P.E., and Adibi, S.A., Possible sources of
glutamine for parenteral nutrition: impact on glutamine metabolism, Am. J. Physiol., 257,
E228, 1989.
7. Nurjhan, N., Bucci, A., Perriello, G., Stumvoll, M., Dailey, G., Bier, D.M., Toft, I., Jenssen,
T.G., and Gerich, J.E., Glutamine: a major gluconeogenic precursor and vehicle for interorgan
carbon transfer in man, J. Clin. Invest., 95, 272, 1995.
8. Hankard, R.G., Haymond, M.W., and Darmaun, D., Role of glutamine as a glucose precursor
in fasting humans, Diabetes, 46, 1535, 1997.
1382_C03.fm Page 58 Tuesday, October 7, 2003 6:08 PM

58 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

9. Manatt, M.W. and Garcia, P.A., Nitrogen balance: concepts and techniques, in Modern Methods
in Protein Nutrition and Metabolism, Nissen, S., Ed., Academic Press, San Diego, 1992, pp. 9–66.
10. Waxman, K., Robello, T., Pinderski, L., O’Neal, K., and Khan, N., Protein loss across burn
wounds, J. Trauma, 27, 136, 1987.
11. Goldstein, S.A. and Elywn, D.H., The effects of injury and sepsis on fuel utilization, Annu.
Rev. Nutr., 9, 445, 1989.
12. Wernerman, J. and Rooyackers, O., Methods, who cares? Clin. Nutr., 19, 145, 2000.
13. Hammarqvist, F., Wernerman, J., Ali, R., Von Der Decken, A., and Vinnars, E., Effects of
glutamine supplementation to total parenteral nutrition after elective abdominal surgery
spares free glutamine in muscle and counteracts the fall in muscle protein synthesis, Ann.
Surg., 209, 455, 1989.
14. Goodman, M.N., Larsen, P.R., Kaplan, M.M., Aoki, T.T., Young, V.R., and Ruderman, N.B.
Starvation in the rat. II. Effect of age and obesity on protein sparing and fuel metabolism,
Am. J. Physiol., 239, E277, 1980.
15. Cerra, F.B., Hypermetabolism, organ failure, and metabolic support, Surgery, 101, 14, 1987.
16. Cynober, L., Coudray-Lucas, C., Lim, S.K., Dumas, F., and Giboudeau, J., Intérêt et limites
du dosage des acides aminés plasmatiques en nutrition, Nutr. Clin. Métabol., 6, 83, 1992.
17. Cynober, L.A., Plasma amino acid levels with a note on membrane transport: characteristics,
regulation and metabolic significance, Nutrition, 18, 761, 2002.
18. Cynober, L., Lessons from pharmacokinetics in the design of new nutrition formulas for
critically ill patients, in Nutrition in Critical Care, Cynober, L. and Moore, F.A., Eds., Karger,
Basel, Switzerland, 2003, in press.
19. Coudray-Lucas, C., Cynober, L., Lioret, N., Saizy, R., Baux, S., and Giboudeau, J., Origins of
hyperphenylalaninemia in burn patients, Clin. Nutr., 4, 179, 1985.
20. Fath, H.H., Ascher, N.L., Konstantinides, F.N., Bloomer, J., Sharp, H., Najarian, J.S., and Cerra,
F.B., Metabolism during hepatic transplantation: indicators of allograft function, Surgery, 96,
664, 1984.
21. Barbul, A., Arginine: biochemistry, physiology and therapeutic implications, J. Parenter. En-
teral Nutr., 10, 227, 1986.
22. Cynober, L., Amino acid metabolism in thermal burns, J. Parenter. Enteral Nutr., 13, 193, 1989.
23. Parry-Billings, M., Evans, J., Calder, P.C., and Newsholme, E.A., Does glutamine contribute
to immunosuppression after major burns? Lancet, 336, 523, 1990.
24. Cynober, L., Nguyen, D.F., Blondé, F., Saizy, R., and Giboudeau, J., Plasma and urinary amino
acid pattern, in severe burn patients: evolution throughout the healing period, Am. J. Clin.
Nutr., 36, 416, 1982.
25. Cynober, L., Nguyen, D.F., Saizy, R., Blondé, F., and Giboudeau, J., Plasma amino acid levels
in the first few days after burn injury and their predictive value, Intensive Care Med., 9, 325,
1983.
26. Crenn, P., Coudray-Lucas, C., Thuillier, F., Cynober, L., and Messing, B., Post absorptive
plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal
failure in humans, Gastroenterology, 119, 1496, 2000.
27. Gondolesi, G., Fishbein T., Chehade, M., Tschernia, A., Magid, M., Kaufman, S., Raymond,
K., Sansaricq, C., and LeLeiko, N., Serum citrulline is a potential marker for rejection of
intestinal allografts, Transplant Proc., 34, 918, 2002.
28. Lutgens, L., Cleutjens, J., Gueulette, J., Berger, M., Wouters, B., Von Myenfeldt, M., Lambin,
P., and Deutz, N., Small bowel epithelial radiation damage can be quantified and monitored
by plasma citrulline levels, Clin. Nutr., 202 (Suppl. 1), 64, 2002.
29. Cynober, L., Saizy, R., Nguyen, D.F., Lioret, N., and Giboudeau, J., Effect of enterally admin-
istered ornithine alpha-ketoglutarate on plasma and urinary amino acids levels after burn
injury, J. Trauma, 24, 590, 1984.
30. Cynober, L., Blonde, F., Lioret, N., Coudray-Lucas, C., Saizy, R., and Giboudeau, J., Arterio-
venous differences in amino acids, glucose, lactate and fatty acids in the burn patients: effect
of ornithine a-ketoglutarate, Clin. Nutr., 5, 221, 1986.
1382_C03.fm Page 59 Tuesday, October 7, 2003 6:08 PM

Chapter three: Approaches to studying amino acid metabolism 59

31. Mjaaland, M., Unneberg, K., Larsson, J., Nilsson, L., and Revhaug, A., Growth hormone after
abdominal surgery attenuated forearm glutamine, alanine, 3-methylhistidine and total amino
acid efflux in patients receiving total parenteral nutrition, Ann. Surg., 217, 413, 1993.
32. Abumrad, N.N., Darmaun, D., and Cynober, L.A., Approaches to studying amino acid
metabolism: from quantitative assays to flux assessment using stable isotopes, in Amino Acid
Metabolism and Therapy in Health and Nutritional Disease, CRC Press, Boca Raton, FL, 1995,
pp. 15–30.
33. Abumrad, N.N., William, P.E., Frexis-Steed, M., Geer, R., Flakoll, P., Cersosimo, E., et al.,
Inter-organ metabolism of amino acids in vivo, Diabetes Metab. Rev., 5, 213, 1989.
34. Williams, P.E., Flakoll, P.J., Frexes-Steed, M., and Abumrad, N.N., Surgical models to measure
organ amino acid metabolism, in Modern Methods in Protein Nutrition and Metabolism, Nissen,
S., Ed., Academic Press, San Diego, 1992, pp. 167–194.
35. Hartley, C.J., Lewis, R.M., and Cole, J.S., Synchronized pulsed Doppler blood flow and
ultrasonic dimension measurements in conscious dog, Ultrasound Med. Biol., 4, 99, 1978.
36. Leevy, C.M., Mendenhall, C.L., Lesko, W., and Howard, M.M., Estimation of hepatic blood
flow with ICG, J. Clin. lnvest., 71, 1169, 1962.
37. Fegler, Measurement of cardiac output in anesthetized animals by thermodilution methods,
Q. J. Exp. Physiol., 39, 153, 1954.
38. Heyman, M.A., Payne, B.D., Hoffman, J.I., and Rudolph, A.M., Blood flow measurements
with radionuclide-labelled particles, Prog. Cardiavasc. Dis., 20, 55, 1977.
39. Nair, K.S., Halliday, D., and Graggs, R.S., Leucine incorporation into mixed skeletal muscle
proteins in humans, Am. J. Physiol., 254, E208, 1988.
40. Helland, S.J., Grisdate-Hellan, B., and Nissen, S., Infusion and sampling site effects on two-
pool model estimates of leucine metabolism, Am. J. Physiol., 254, E414, 1988.
41. Lobley, G.I., Milne, V., Lovie, M., Reeds, P.J., and Rennie, K., Whole body and tissue protein
synthesis in cattle, Br. J. Physiol., 43, 491, 1980.
42. Prockop, D.J. and Kivirikko, K.I., Relationship of hydroxyproline excretion in urine to col-
lagen metabolism, biochemistry and clinical applications, Ann. Intern. Med., 1243, 1967.
43. Sjolin, J., Sternstrom, H., Arturson, G., Andersson, E.G., Friman, G., and Larsson, J., Exchange
of 3-methylhistidine in the splanchnic region in human infection, Am. J. Clin. Nutr., 50, 1407,
1989.
44. Picou, D. and Taylor-Roberts, T., The measurement of total synthesis and catabolism and
nitrogen turnover in infants in different nutritional states and receiving different amounts
of dietary protein, Clin. Sci., 36, 283, 1969.
45. Wolfe, R.R., Radioactive and Stable Isotope Tracers in Biomedicine, Wiley-Liss, New York, 1992.
46. Darmaun, D., Froguel, P., Rongier, M., and Robert, J.J., Amino acid exchange between plasma
and erythrocytes in vivo in humans, J. Appl. Physiol., 67, 2383, 1989.
47. Fern, E.S., Garlick, P.J., and Waterlow, J.C., Apparent compartmentation of body nitrogen in
one human subject: its consequences in measuring the rate of whole body protein synthesis
with 15N, Clin. Sci., 68, 271, 1985.
48. Plauth, C., Heine, W., Wurtzke, K.D., Krienke, L., Töwe, J., Massute, G., and Windischmann,
C., 15N tracer kinetic studies on the validity of various 15N tracer substances for determining
whole body protein parameters in very small preterm infants, J. Pediatr. Gastroenterol. Nutr.,
6, 400, 1987.
49. Waterlow, J.C., Garlick, P.J., and Millward, D.J. Protein Turnover in Mammalian Tissues and in
the Whole Body, North Holland, Amsterdam, 1978.
50. Matthews, D.E., Motil, K.J., Rohrbaugh, D.K., Burke, J.F., Young, V.R., and Bier, D.M., Mea-
surement of leucine metabolism in man from a primed, continuous infusion of L-[1-13C]
leucine, Am. J. Physiol., 238, E473, 1980.
51. Schwenk, W.F., Tsalikian, E., Beaufrère, B., and Haymond, M.W., Recycling of an amino acid
label with prolonged isotope infusion: implication for kinetic studies, Am. J. Physiol., 248,
E482, 1985.
52. Matthews, D.E., Schwarz, H.P., Yang, R.D., Motil, K.J., Young, V.R., and Bier, D.M., Relation-
ship of plasma leucine and a-ketoisocaproate during a L-[1-13C] leucine infusion in man: a
method for measuring intracellular leucine tracer enrichment, Metabolism, 31, 1105, 1982.
1382_C03.fm Page 60 Tuesday, October 7, 2003 6:08 PM

60 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

53. Schwenk, W.F., Beaufrère, B., and Haymond, M.W., Use of reciprocal pool specific activities
to model leucine metabolism in humans, Am. J. Physiol., 249, E646, 1985.
54. Watt, P.W., Lindsay, Y., Chien, P.A.F., Gibson, J.N.A., Scrimgeour, C.M., Taylor, D.J., and
Rennie, M.J., Isolation of aminoacyl tRNA and its site of labelling with stable isotope tracer
used in studies of human tissue protein synthesis, Proc. Natl. Acad. Sci. U.S.A., 88, 5892, 1991.
55. Parhofer, K.G., Hugh, P., Barrett, R., Bier, D.M., and Schonfeld, G., Determination of kinetic
parameters of apolipoprotein B metabolism using amino acids labeled with stable isotopes,
J. Lipid Res., 32, 1311, 1991.
56. Hoerr, R.A., Yu, Y.M., Wagner, D.A., Burke, J.F., and Young, V.R., Recovery of 13C in breath
from Na13CO3 infused by gut and vein: effect of feeding, Am. J. Physiol., 257, E426, 1989.
57. Kien, L., Isotopic dilution of CO2 as an estimate of CO2 production during substrate oxida-
tion studies, Am. J. Physiol., 257, E296, 1989.
58. Van Goudoever, J.B., Wattimena, J.D.L., Carnielli, V.P., Sulkers, E.J., Degenhart, H.J., and
Sauer, P.J.J., Effect of dexamethasone on protein metabolism in infants with bronchopul-
monary dysplasia, J. Pediatr., 124, 112, 1994.
59. Spear, M.L., Darmaun, D., Sager, B.K., Parsons, W.R., and Haymond, M.W., Use of [13C]bi-
carbonate infusion for measurement of CO2 production, Am. J. Physiol., 268, E1123, 1995.
60. Cortiella, J., Matthews, D.E., Hoerr, R.A., Bier, D.M., and Young, V.R., Leucine kinetics at
graded intakes in young men: quantitative fate of dietary leucine, Am. J. Clin. Nutr., 48, 998,
1988.
61. Boirie, Y., Gachon, P., Corny, S., Fauquant, J., Maubois, J.L., and Beaufrère, B., Acute post-
prandial changes in leucine metabolism as assessed with an intrinsically labeled milk protein,
Am. J. Physiol., 271, E1083, 1996.
62. Boirie, Y., Dangin, M., Gacon, P., Vasson, M.P., Maubois, J.L., and Beaufrère, B., Slow and
fast dietary proteins differently modulate postprandial protein accretion, Proc. Natl. Acad.
Sci. U.S.A., 94, 14930, 1997.
63. Darmaun, D., Matthews, D.E., and Bier, D.M., Physiological hypercortisolemia increases
proteolysis, glutamine, and alanine production, Am. J. Physiol., 255, E366, 1988.
64. Schwenk, W.F., Rubanyi, E., and Haymond, M.W., The effect of a protein synthetic inhibitor
on in vivo estimates of protein synthesis in dogs, Am. J. Physiol., 252, E595, 1987.
65. Cobelli, C., Saccomani, M.P., Tessari, P., Biolo, G., Luzi, L., and Matthews, D.E., Compart-
mental model of leucine kinetics in humans, Am. J. Physiol., 261, E539, 1991.
66. Kuhn, K.S., Schuhmann, K., Stehle, P., Darmaun, D., and Fürst, P., Determination of glutamine
in muscle protein facilitates accurate assessment of proteolysis and de novo synthesis-
derived endogenous glutamine production, Am. J. Clin. Nutr., 70, 484, 1999.
67. Darmaun, D. and Déchelotte, P., Role of leucine as a precursor of glutamine a-amino nitrogen
in vivo in humans, Am. J. Physiol., 260, E326, 1991.
68. Darmaun, D., Matthews, D.E., and Bier, D.M., Glutamine and glutamate kinetics in humans,
Am. J. Physiol., 251, E117, 1986.
69. Van Acker, B.A., Hulsewe, K.W., Wagenmakers, A.J., Deutz, N.E., Van Kreel, B.K., Halliday,
D., Matthews, D.E., Soeters, P.B., and Von Meyenfeldt, M.F., Absence of glutamine isotopic
steady state: implications for the assessment of whole-body glutamine production rate, Clin.
Sci. (Lond.), 95, 339, 1998.
70. Bier, D.M., Intrinsically difficult problems: the kinetics of body proteins and amino acids in
man, Diabetes Metab. Rev., 5, 111, 1989.
71. Castillo, L., Chapman, T.E., Sanchez, M., Yu, Y.M., Burke, J.F., Ajami, A.M., Vogt, J., and
Young, V.R., Plasma arginine and citrulline kinetics in adults given adequate and arginine-
free diets, Proc. Natl. Acad. Sci. U.S.A., 90, 7749, 1993.
72. Jaksic, T., Wagner, D.A., Burke, J.F., Young, V.R., Jaksic, T., Wagner, D.A., Burke, J.F., and
Young, V.R., Proline metabolism in adult male burned patients and healthy control subjects,
Am. J. Clin. Nutr., 54, 408, 1991.
73. Young, V.R. and El Khoury, A.E., The notion of nutritional essentiality of amino acids,
revisited, with a note on the indispensable amino acid requirements in adults, in Amino Acid
Metabolism and Therapy in Health and Nutritional Disease, CRC Press, Boca Raton, FL, 1995,
pp. 191–232.
1382_C03.fm Page 61 Tuesday, October 7, 2003 6:08 PM

Chapter three: Approaches to studying amino acid metabolism 61

74. Clarke, J.T.R. and Bier, D.M., The conversion of phenylalanine to tyrosine in man. Direct
measurement by continuous intravenous tracer infusions of L-[ring 2H5] phenylalanine and
L-[l-13C] tyrosine in the postabsorptive state, Metabolism, 31,999, 1982.
75. Thompson, G.N., Pacy, P.J., Merritt, H., Ford, G.C., Read, M.A., Cheng, K.N., and Halliday,
D., Rapid measurement of whole body and forearm protein turnover using a [2H5] phenyl-
alanine model, Am. J. Physiol., 256, E63l, 1989.
76. Barrett, E.J., Revkin, J.H., Young, L.H., Zaret, B.L., Jacob, R., and Gelfand, G.A., An isotopic
method for measurement of muscle protein synthesis and degradation in vivo, Biochem. J.,
245, 223, 1987.
77. Hoerr, R.A., Matthews, D.E., Bier, D.M., and Young, V.R., Effects of protein restriction and
acute refeeding on leucine and lysine kinetics in young men, Am. J. Physiol., 264, E567, 1993.
78. Horber, F.F. and Haymond, M.W., Human growth hormone prevents the protein catabolic
side effects of prednisone in humans, J. Clin. Invest., 86, 265, 1990.
79. Darmaun, D., Roig, J.C., Auestad, N., Sager, B.K., and Neu, J., Glutamine metabolism in very
low birth weight infants, Pediatr. Res., 41, 391, 1997.
80. Boirie, Y., Gachon, P., and Beaufrère, B., Splanchnic and whole body leucine kinetics in young
and elderly men, Am. J. Clin. Nutr., 65, 4879, 1997.
81. Hankard, R.G., Darmaun, D., Sager, B.K., D’Amore, D., Parsons, W.R., and Haymond, M.W.,
Response of glutamine metabolism to exogenous glutamine in humans, Am. J. Physiol., 269,
E663, 1995.
82. Matthews, D.E., Mariano, M.A., and Campbell, R.G., Splanchnic bed utilization of glutamine
and glutamic acid in humans, Am. J. Physiol., 264, E848, 1993.
83. Battezzati, A., Brillon, D.J., and Matthews, D.E., Oxidation of glutamic acid by the splanchnic
bed in humans, Am. J. Physiol., 269, E269, 1995.
84. Perriello, G., Jorde, R., Nurjhan, N., Stumvoll, M., Dailey, G., Jenssen, T., Bier, D.M., and
Gerich, J.E., Estimation of glucose-alanine-lactate-glutamine cycles in postabsorptive hu-
mans: role of skeletal muscle, Am. J. Physiol., 269, E443, 1995.
85. Stumvoll, M., Meyer, C., Perriello, G., Kreider, M., Welle, S., and Gerich, J.E., Human kidney
and liver gluconeogenesis: evidence for organ substrate selectivity, Am. J. Physiol., 274, E817,
1998.
86. Yarasheski, K.E., Smith, K., Rennie, M.J., and Bier, D.M., Measurement of muscle protein
fractional synthetic rate by capillary gas chromatography/combustion isotope ratio mass
spectrometry, Biol. Mass Spectrom., 21, 486, 1992.
87. Barazzoni, R., Meek, S.E., Ekberg, K., Wahren, J., and Nair, K.S., Arterial KIC as marker of
liver and muscle intracellular leucine pools in healthy and type 1 diabetic humans, Am. J.
Physiol., 272, E238, 1999.
88. Marchini, J.S., Nguyen, P., Deschamps, Y., Maugère, P., Krempf, M., and Darmaun, D., Effect
of intravenous glutamine on duodenal mucosa protein synthesis in healthy growing dogs,
Am. J. Physiol., 276, E747, 1999.
89. Bouteloup-Demange, C., Boirie, Y., Déchelotte, P., Gachon, P., and Beaufrère, B., Gut mucosal
protein synthesis in fed and fasted humans, Am. J. Physiol., 274, E541, 1998.
90. Reid, M. and Jahoor, F., Methods for measuring glutathione concentration and rate of syn-
thesis, Curr. Opin. Clin. Nutr. Metab. Care, 3, 385, 2000.
91. De Feo, P., Gan-Gaisano, M., and Haymond, M.W., Differential effects of insulin deficiency
on albumin and fibrinogen synthesis in humans, J. Clin. Invest., 88, 883, 1991.
92. Welle, S., Thornton, C., Jozefowics, R., and Statt, M., Myofibrillar protein synthesis in young
and old men, Am. J. Physiol., 264, E693, 1993.
93. Balagopal, P., Schimke, J.C., Ades, P., Adey, D., and Nair, K.S., Age effect on transcript levels
and synthesis rate of muscle MHC and response to resistance exercise, Am. J. Physiol., 280,
E203, 2001.
94. Nakshabendi, I., McKee, R., Downie, S., Russell, R., and Rennie, M., Rates of small intestinal
mucosal protein synthesis in human jejunum and ileum, Am. J. Physiol., 277, E1028, 1999.
95. Meynial-Denis, D., Renou, J.P., and Arnal, M., Heteronuclear 15N/1H spin echo NMR: an
in vitro quantitative analysis of leucine transamination in rat skeletal muscle, Biochem. Biophys.
Res. Commun., 160, 1033, 1989.
1382_C03.fm Page 62 Tuesday, October 7, 2003 6:08 PM
1382_C04.fm Page 63 Tuesday, October 7, 2003 6:11 PM

chapter four

Cellular uptake of amino acids:


systems and regulation
Vadivel Ganapathy
Medical College of Georgia
Katsuhisa Inoue
Medical College of Georgia
Puttur D. Prasad
Medical College of Georgia
Malliga E. Ganapathy
Medical College of Georgia

Contents
Introduction....................................................................................................................................63
4.1 Classification of amino acid transport systems...............................................................64
4.2 SLC 1 family..........................................................................................................................68
4.3 SLC 6 family..........................................................................................................................69
4.4 SLC 7 family..........................................................................................................................70
4.5 SLC 16 family........................................................................................................................71
4.6 SLC 38 family........................................................................................................................71
4.7 Heterodimeric amino acid transporters ...........................................................................73
4.8 Conclusions ...........................................................................................................................74
Acknowledgments ........................................................................................................................75
References .......................................................................................................................................75

Introduction
Amino acids perform a variety of functions in mammalian cells. They not only serve as
the building blocks for protein synthesis but also play additional important roles in
neurotransmission, production and storage of metabolic energy, nitrogen metabolism, and
synthesis of hormones, purine and pyrimidine nucleotides, and glutathione. The intra-
cellular pool of amino acids is derived not only from endogenous production (biosynthesis
as well as protein degradation) but also from transfer across the plasma membrane. Due

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 63
1382_C04.fm Page 64 Tuesday, October 7, 2003 6:11 PM

64 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

to their ionic nature, amino acids do not permeate biological membranes by diffusion to
any great extent. Transfer across biological membranes therefore requires participation of
specific transport proteins. This transfer process does not necessarily have to serve only
the entry of amino acids into cells. This process also mediates the exit of certain amino
acids in a tissue-specific manner. In a number of cases, the transfer involves exchange
across the membrane, a process that couples the entry of certain amino acids into cells
and the exit of different amino acids from the cells. A multitude of transport systems is
known to facilitate the transfer of amino acids across the plasma membrane in mammalian
cells. These transport systems exhibit variable but in many instances overlapping substrate
specificity and are not expressed uniformly in all cell types. The differential expression of
amino acid transport systems coupled with their variable substrate specificity has physi-
ological significance because different tissue types vary markedly in their requirements
for specific amino acids to suit their specific physiological functions. In addition to the
differences in substrate specificity, the amino acid transport systems also differ in their
energetics and hence in their transport mechanism. Some transport systems are passive,
functioning either as uniporters or exchangers without involving any driving force,
whereas others are active, driven by transmembrane ion gradients. In many cases, multiple
ion gradients are involved in the energization process, thus enhancing the concentrative
capacity of the transport systems.

4.1 Classification of amino acid transport systems


Prior to the cloning era, the nomenclature of amino acid transport systems was dominated
by the classification system developed by Halvor Christensen.1–3 Even though most of the
work from Christensen’s laboratory involved amino acid transport in nonepithelial cells,
his original classification system was later expanded by him and other investigators in
the field to include amino acid transport in polarized epithelial cells as well.4–6 Polarized
cells such as the intestinal and kidney epithelial cells and placental syncytiotrophoblast
utilize amino acid transport systems not only to modulate the intracellular pool of amino
acids, but also to effect vectorial transfer of amino acids from one side of the cell to the
other. This means that while different types of nonepithelial cells express different amino
acid transport systems in their plasma membrane, polarized cells express different amino
acid transport systems within the two domains of the plasma membrane, namely, the
apical membrane and the basolateral membrane. This adds a new dimension to the
complexity of amino acid transport systems in polarized epithelial cells. The classification
system developed by Christensen relied primarily on substrate specificity. The system was
later improved to include information on whether a transmembrane Na+ gradient plays
a role in the function of the transport systems.7 According to this newer nomenclature,
the Na+ gradient-dependent transport systems are denoted by uppercase letters, whereas
the Na+-independent transport systems are denoted by lowercase letters (Table 4.1 to
Table 4.3). In many instances, the names of the transport systems themselves provide clues
to the identity of the substrates handled by the transport systems, and the notations in
superscript indicate the ionic nature of the amino acid substrates. For example, system A
refers to a Na+ gradient-dependent transport system that prefers alanine and other small
neutral amino acids as substrates, and system B0,+ refers to a Na+-dependent transport
system that exhibits a broad substrate specificity, including neutral (indicated by 0 in the
superscript) as well as cationic (indicated by + in the superscript) amino acids as substrates.
On the other hand, system l (originally known as system L) refers to a Na+-independent
transport system that shows preference for leucine and other hydrophobic bulky amino
acids, system t (originally known as system T) refers to a Na+-independent transport
system that accepts tryptophan and other aromatic amino acids as substrates, and system
Table 4.1 Amino Acid Transporters Coded by Single Genes
Solute Carrier Christensen Alternative Ion Transport
Classification Classification cDNA Identity Names Dependence Mechanism
Chapter four:

SLC1 ASC ASCT1 SAAT1 Na+ 1 Na+–AA symport/1Na+–AA antiport


ASCT2 ATB0
X–AG EAAT1 GLAST Na+, K+, H+ 3 Na+–1 H+–AA
symport/1 K+ antiport
EAAT2 GLT-1
EAAT3 EAAC1
EAAT4
1382_C04.fm Page 65 Tuesday, October 7, 2003 6:11 PM

EAAT5

SLC6 B0,+ ATB0,+ Na+, Cl¯ 2 Na+–1 Cl––AA symport


b TAUT 2 or 3 Na+–1 Cl––AA symport
GLY GLYT1 2 Na+–1 Cl––AA symport
GLYT2 3 Na+–1 Cl––AA symport

SLC7 y+ CAT1 None AA uniport


CAT2
CAT3

SLC16 t (T) TAT1 None AA uniport


Cellular uptake of amino acids: systems and regulation

SLC38 A ATA1 GlnT, SAT1, NAT2, SA2 Na+ 1 Na+–AA symport


ATA2 SAT2, SA1
ATA3 NAT3
N SN1 NAT1 Na+, H+ Na+–AA symport/H+ antiport
SN2
PAT1 LYAAT-1 H+ 1 H+–AA symport
PAT2

Note: AA = amino acid.


65
1382_C04.fm Page 66 Tuesday, October 7, 2003 6:11 PM

66 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 4.2 Substrate Specificity and Gene Locus of Single-Gene Amino Acid Transporters
cDNA Identity Human Gene Locus Primary Physiological Substrates
ASCT1 2p13–15 Ala, Ser, Cys
ASCT2 19q13.3 Ala, Ser, Cys, Thr, Gln, Asn

EAAT1 5p13 Anionic amino acids (Glu, Asp)


EAAT2 11p13 Anionic amino acids (Glu, Asp)
EAAT3 9p24 Anionic amino acids (Glu, Asp)
EAAT4 19p13.1 Anionic amino acids (Glu, Asp)
EAAT5 1p32 Anionic amino acids (Glu, Asp)

ATB0,+ Xq23–24 Neutral and cationic amino acids

TAUT 3p25 Taurine, b-Ala

GLYT1 1p33 Gly, Sarcosine


GLYT2 11p15.1–15.2 Gly

CAT1 13q12 Cationic amino acids (Arg, Lys, Orn)


CAT2 8p22 Cationic amino acids (Arg, Lys, Orn)
CAT3 Xq12 Cationic amino acids (Arg, Lys, Orn)

TAT1 6q21–22 Aromatic amino acids (Trp, Phe, Tyr)

ATA1 12q12 Linear neutral amino acids (Pro)


ATA2 12q12 Linear neutral amino acids (Pro)
ATA3 12q12 Linear neutral amino acids (Pro)

SN1 3p21.3 Gln, Asn, His


SN2 Xp11.23 Gln, Asn, His, Ser, Gly

PAT1 5q33.1 Gly, Ala, Pro


PAT2 Not known Gly, Ala, Pro, Ser

x-c refers to a Na+-independent transport system that prefers the anionic form of the
disulfide-containing amino acid cystine as the substrate. Some systems transport certain
amino acids in a Na+-independent manner but other amino acids in a Na+-dependent
manner. The newly improved classification method also allows for accurate identification
of such a system. For example, system y+L refers to a transport system that transports
cationic amino acids in a Na+-independent manner and neutral amino acids in a Na+-
dependent manner.
Successful cloning of several of the plasma membrane amino acid transporters in
recent years has led to the expansion of Christensen’s original classification to incorporate
the new information on the structure, substrate specificity, and transport mechanism of
these transporters. Interestingly, the new information on the molecular nature of these
amino acid transporters has not weakened in any way the original classification. What is
new is the realization that many of the transport systems, which were once thought to be
single transport systems according to the original classification, based on substrate spec-
ificity and ion coupling, actually consist of several subtypes. These subtypes, coded by
separate genes, are expressed differentially in different tissues but mediate similar trans-
port function in terms of substrate specificity and ion coupling. Another new revelation
as a result of cloning studies is that in many cases, transport systems that are distinct in
Chapter four:

Table 4.3 Heterodimeric Amino Acid Transporters Coded by Two Different Genes
Light Chain (SLC7) Heavy Chain (SLC3)
Christensen cDNA Human cDNA Human Amino Acid Ion Transport
Classification Identity Gene Locus Identity Gene Locus Substrates Dependence Mechanism
l (L) LAT1 16q24.3 4F2hc (CD98) 11q12 Neutral AA None AA exchange
LAT2 14q11.1 4F2hc (CD98) 11q12 Neutral AA None AA exchange
1382_C04.fm Page 67 Tuesday, October 7, 2003 6:11 PM

y+L y+LAT1 14q11.1 4F2hc (CD98) 11q12 Neutral and Na+ (only for neutral Na+–AAo/AA+
cationic AA AA) exchange
y+LAT2 16q21 4F2hc (CD98) 11q12 Neutral and Na+ (only for neutral Na+–AAo/AA+
cationic AA AA) exchange

bo,+ bo,+AT 19q12–13.1 rBAT (NBAT, D2H) 2p21 Neutral and None AA exchange
cationic AA

x–c xCT 4q28 4F2hc (CD98) 11q12 Cystine, Glu None Cystine/Glu
exchange

asc Asc1 19q12–13.1 4F2hc (CD98) 11q12 Ala, Ser, Cys None AA exchange
Asc2 — Not known Ala, Ser, Cys None Not known
Cellular uptake of amino acids: systems and regulation

ag AGT1 8q21.1 Not known Asp, Glu None Not known

Note: AA = amino acid; AAo = zwitterionic amino acid; AA+ = cationic amino acid.
67
1382_C04.fm Page 68 Tuesday, October 7, 2003 6:11 PM

68 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

substrate specificity and ion coupling fall within a single group based on homology in
amino acid sequence of the transport proteins. Thus, within a given single group consisting
of members of similar amino acid sequences, the members differ in substrate specificity,
ion coupling, and transport mechanism. This new nomenclature based on the structure
of the transporter proteins rather than on the transport function is called solute carrier
classification. Recent cloning studies have also led to the discovery that while many of
the transport systems do consist of single-gene products, a growing number of transport
systems function as heterodimers whose subunits are coded by separate genes. Interest-
ingly, based on amino acid sequence, the heavy chains of these heterodimeric transporters
fall into one solute carrier group, whereas the light chains fall into another solute carrier
group. According to this solute carrier classification, the plasma membrane amino acid
transporters thus far identified in mammalian cells at the molecular level are grouped into
six distinct families: SLC (solute carrier) 1, SLC 3, SLC 6, SLC 7, SLC 16, and SLC 38 (Table
4.1 to Table 4.3). Of these, SLC 1, SLC 6, SLC 16, and SLC 38 comprise solely amino acid
transporters coded by single genes. In contrast, SLC 7 comprises amino acid transporters
coded by single genes as well as the light chains of amino acid transporters that function
as heterodimers. SLC 3 consists solely of the heavy chains of the heterodimeric amino acid
transporters. Tables 4.1 to 4.3 summarize the available information on solute carrier clas-
sification, Christensen’s original classification, identity of the cloned amino acid trans-
porter cDNAs, alternative names, substrate specificity, ion coupling, transport mechanism,
and chromosomal localization of the genes for the plasma membrane amino acid trans-
porters identified thus far in mammalian cells at the molecular level. Readers are referred
to recent reviews for a detailed analysis of the structure, function, and regulation of various
amino acid transporters in mammalian cells.5,8–15 In this chapter, we provide a short
overview of these transporters.

4.2 SLC 1 family


The SLC 1 family consists of transport systems referred to as ASC and X-AG according to
Christensen’s classification. ASC is a Na+-coupled transport system expressed widely in
mammalian tissues that accepts alanine, serine, and cysteine as the preferred substrates.
Two subtypes of this transport system have been identified thus far, namely, ASCT1 and
ASCT2.16–19 Both subtypes function as Na+-dependent amino acid exchangers rather than
as uniporters. ASCT1 is specific for alanine, serine, and cysteine, whereas ASCT2 accepts,
in addition to these three amino acids, glutamine and asparagine as substrates.19 Since
ASCT2 accepts glutamine as a high-affinity substrate and is also capable of mediating the
influx as well as the efflux of this amino acid depending on the metabolic profile of the
cell types, it has been proposed that this transporter may play a critical role not only in
glutamine release in the brain, placenta, and liver as a part of the glutamine–glutamate
cycle that occurs in these tissues, but also in glutamine uptake in tumor cells to support
cell proliferation.12 An interesting aspect of ASCT2 that is independent of its transport
activity is its function as a receptor for certain types of retroviruses.20,21 ASCT2 is also
referred to as ATB0 because the functional characteristics of ASCT2 resemble those of system
B0 described in the brush border membrane of intestinal and kidney epithelial cells.19
Successful cloning of ASCT2 from intestine and cell lines of intestinal origin22 and dem-
onstration of localization of this transporter in the brush border membrane of intestinal
and kidney epithelial cells23 support this notion. However, the notion of ASCT2 being
identical to system B0 is not universally accepted. Many investigators in the field believe
that system B0 still remains to be identified at the molecular level.11 One of the primary
reasons for the lack of widespread acceptance of the notion is that ASCT2 behaves as a
Na+-dependent amino acid exchanger, whereas system B0 is believed to be a Na+-dependent
1382_C04.fm Page 69 Tuesday, October 7, 2003 6:11 PM

Chapter four: Cellular uptake of amino acids: systems and regulation 69

amino acid uniporter. Thus, the issue of whether ASCT2 is identical to system B0 remains
unresolved. The function and expression of ASCT2 in some tissues and cell lines is up-
regulated by epidermal growth factor,24 growth hormone,25 and protein kinase C.26
System X-AG mediates the cellular influx of anionic amino acids (glutamate and aspar-
tate) in a Na+-coupled manner. Since glutamate is an excitatory amino acid in the brain,
the transporters responsible for X-AG activity are referred to as EAATs (excitatory amino
acid transporters). There are five subtypes of X-AG (EAAT1 to EAAT5). All of these subtypes
are highly concentrative with the ability to accumulate glutamate inside the cells several
hundred-fold higher than outside. This is facilitated by transmembrane gradients of Na+,
K+, and H+ and membrane potential as driving forces. The transport mechanism involves
the influx of 3 Na+, 1 H+, and 1 glutamate, coupled to the efflux of 1 K+.27 Each EAAT
subtype is expressed differentially in a tissue-specific manner. EAAT1, EAAT2, and EAAT4
are mainly brain specific, and EAAT5 is almost exclusively expressed in the retina. EAAT3
is expressed in the brain as well as in peripheral tissues, including the absorptive cells of
the intestine and kidney. In the brain, EAAT1 and EAAT2 are present primarily in glial
cells, whereas EAAT3, EAAT4, and EAAT5 are present in neurons. The function of EAAT3
is subject to regulation by osmotic stress and amino acid deprivation.28 Exposure of
EAAT3-expressing cells to hyperosmotic conditions leads to a large increase in glutamate
influx associated with enhanced expression of EAAT3 mRNA. In contrast, amino acid
deprivation enhances EAAT3 activity by a process not linked to changes in EAAT3 mRNA
levels. The remarkable ability of EAAT3 to concentrate glutamate inside the cells makes
this transporter ideally suited for osmoregulation. The cells exposed to hyperosmotic
conditions up-regulate the expression of EAAT3 to increase the intracellular concentrations
of glutamate and consequently reverse the cell volume changes induced by hyperosmotic
conditions. The concentrative ability is not unique to EAAT3. Other subtypes of EAAT
also possess this characteristic. But it remains to be seen whether other EAAT subtypes
also respond to hyperosmotic insult in a similar manner. The transport function of EAATs
is also subject to regulation by different isoforms of protein kinase C, but without involving
changes in the steady-state levels of respective mRNA.29,30 The effects of protein kinase C
are due to changes either in the cell surface expression of the transporter protein or in the
catalytic efficiency of the transporter. Interestingly, the activation of protein kinase C does
not have the same effect on all EAAT subtypes. EAATs are also subject to regulation by
phosphatidylinositol 3-kinase (PI 3-kinase).30 Recent studies have unraveled a new mode
of regulation of certain subtypes of EAAT.31,32 This occurs through protein–protein inter-
action involving specific intracellular proteins, known as GTRAPs (glutamate transporter-
associated proteins). The transport function of EAAT3 is enhanced by GTRAP3 to
GTRAP18, and the process involves an increase in the substrate affinity of the transporter.31
GTRAP41 and GTRAP48 increase the transport activity of EAAT4 either through an
increase in the catalytic rate of the transporter or through an increase in the cell surface
availability of the transporter.32

4.3 SLC 6 family


The SLC 6 gene family is also known as the neurotransmitter transporter gene family
because it consists of transporters for neurotransmitters such as serotonin, dopamine,
norepinephrine, and g-aminobutyrate. With regard to amino acids as substrates, systems
B0,+, b, and GLY belong to this family. B0,+ is a transporter that mediates the cellular
uptake of a broad spectrum of zwitterionic amino acids and cationic amino acids in a
Na+- and Cl--coupled process. Na+ as well as Cl- are obligatory for the transport function.
The cloned transporter, known as ATB0,+, exhibits exactly the same functional charac-
teristics with regard to substrate specificity and ionic requirements as those described
1382_C04.fm Page 70 Tuesday, October 7, 2003 6:11 PM

70 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

originally for systems B0,+.33 ATB0,+ is expressed primarily in the lung, breast, and colon.
The Na+:Cl-:amino acid stoichiometry for the transport process is 2:1:1. An interesting
feature of ATB0,+ is its ability to transport D-amino acids.34,35
System b refers to a Na+- and Cl--coupled amino acid transport activity that is specific
for taurine and other b-amino acids. The cloned transporter, TAUT, exhibits functional
features that are identical to those described for system b.36 TAUT is expressed widely in
mammalian tissues, especially in those tissues with very high levels of intracellular taurine
(e.g., retina, heart). TAUT is also responsible for the intestinal absorption of dietary taurine
and for the renal reabsorption of taurine from the glomerular filtrate. The most notable
characteristic of TAUT is the regulation of its expression in response to hyperosmotic
stress.37 One of the well-recognized biological functions of taurine is its role as an osmolyte.
TAUT operates with a Na+:Cl-:taurine stoichiometry of 2 to 3:1:1, which empowers the
transporter thermodynamically to mediate uphill transport of taurine. The remarkable
ability of TAUT to concentrate taurine inside the cells against a concentration gradient
makes this transporter ideal for osmoregulation. As is the case with EAAT3, the expression
of TAUT is up-regulated when cells are exposed to hyperosmotic conditions. The resultant
increase in intracellular levels of taurine aids in the reversal of cell volume changes induced
by the hyperosmotic conditions. The expression of TAUT is also up-regulated by nitric
oxide38 and tumor necrosis factor-a,39 but down-regulated by the tumor repressor protein
p53.40
System GLY refers to a transport activity that mediates the Na+- and Cl--dependent
uptake of glycine. Cloning studies have revealed that this system consists of two subtypes,
GLYT1 and GLYT2.41 Both subtypes are obligatorily dependent on Na+ and Cl- for their
transport function, and both accept glycine as a substrate. However, the two subtypes can
be differentiated based on their interaction with sarcosine (N-methylglycine). GLYT1
accepts sarcosine as a substrate, whereas GLYT2 does not. GLYT1 is expressed in the brain
as well as in peripheral tissues, including liver, lung, kidney, and placenta. In contrast,
the expression of GLYT2 is restricted to brain. In the brain, GLYT1 is present in glial cells
and GLYT2 is present in neuronal cells. In addition to these differences in substrate
selectivity and tissue distribution pattern, GLYT1 and GLYT2 also differ in transport
mechanism in terms of coupling to Na+.42 GLYT1 operates with a Na+:Cl-:glycine stoichi-
ometry of 2:1:1, and therefore the transport process results in the transfer of one positive
charge into the cells per transport cycle. In contrast, GLYT2 operates with a Na+:Cl-:glycine
stoichiometry of 3:1:1, and this results in the transfer of two positive charges into the cells
per transport cycle. Therefore, GLYT2 is much more concentrative than GLYT1. This may
have physiological relevance in terms of the handling of glycine in the brain by the glial
cells vs. the neuronal cells. The mode of glycine transport via GLYT1 can be reversed from
influx to efflux even under physiological conditions if intracellular concentrations of
glycine and Na+ build up to a certain level. Such a reversal in the direction of glycine
transport may not occur in the case of GLYT2. This means that under certain conditions,
glycine may be released from glial cells via GLYT1 and transferred into neurons via GLYT2.

4.4 SLC 7 family


This family consists of system y+, which transports the cationic amino acids arginine and
lysine. Ornithine is also a substrate for this system. There are three subtypes within this
family for which transport function has been demonstrated. These subtypes are referred
to as CAT (cationic amino acid transporter) 1, CAT2, and CAT3.5,8,9 All three subtypes
mediate the cellular uptake of cationic amino acids by a process that is facilitated by the
inside-negative membrane potential. The expression of CAT1 is widespread in mammalian
1382_C04.fm Page 71 Tuesday, October 7, 2003 6:11 PM

Chapter four: Cellular uptake of amino acids: systems and regulation 71

tissues with the notable exception of liver. CAT2 consists of two alternative splice variants,
CAT2A and CAT2B. CAT2A is specifically expressed in the liver, and CAT2B is expressed
primarily in immune cells such as macrophages. CAT3 shows species-dependent varia-
tions in tissue expression.43 In the rat, CAT3 expression is brain specific, whereas in humans
this subtype is expressed not only in the brain but also in various peripheral tissues. In
terms of transport function, CAT1, CAT2B, and CAT3 exhibit high affinity for their sub-
strates. In contrast, CAT2A shows a 10-fold lower affinity comparatively toward its sub-
strates. There are also significant differences between CAT2A and CAT2B in terms of
voltage dependence.44 There has been a long-standing interest in CATs because of their
involvement in the supply of arginine to nitric oxide synthases for generation of nitric
oxide.45 CAT1 plays an essential role in nitric oxide production in endothelial cells where
this transporter appears to function in concert with endothelial nitric oxide synthase
(NOS3). Similarly, CAT2 is functionally coupled to inducible nitric oxide synthase (NOS2)
in activated macrophages.46 CAT1 is linked to cell proliferation, and its expression is
induced in regenerating liver and in proliferating immune cells.5,8,9,45 Thus, normal liver
expresses only CAT2A, but regenerating liver expresses high levels of CAT1. The relation-
ship between cell proliferation and induction of CAT1 may be related to the role of arginine
as the precursor for polyamine biosynthesis. There is also evidence that CAT1 activity is
modulated by protein kinase C47 and cytoskeletal proteins.48 On the other hand, the
expression of CAT2 is induced by bacterial lipopolysaccharide and proinflammatory
cytokines such as interleukin-1, tumor necrosis factor-a, and interferon g.45 These agents
are known to enhance nitric oxide production in macrophages, and accordingly, they
induce the expression of CAT2 and NOS2 in a coordinated manner. Recent findings
indicate that CAT2 expression is enhanced in peripheral blood mononuclear cells from
patients with sepsis, a pathological condition associated with increased production of
nitric oxide.49 Antiinflammatory cytokines such as interleukin-10 suppress the expression
of CAT2 induced by proinflammatory cytokines.50

4.5 SLC 16 family


This family consists of monocarboxylate transporters that handle lactate and pyruvate.
Recent studies have shown that the amino acid transport system t belongs to this gene
family.51,52 System t refers to a Na+-independent amino acid transport system that mediates
the cellular uptake of tryptophan and other aromatic amino acids such as phenylalanine
and tyrosine. It was originally described in human erythrocytes.4 The recently cloned
amino acid transporter TAT1 exhibits functional characteristics resembling those of system
t.51,52 TAT1 mediates the cellular uptake of aromatic amino acids. It is expressed in the
intestine, kidney, liver, and placenta. Interestingly, even though TAT1 is structurally related
to monocarboxylate transporters that transport their substrates in a H+-coupled manner,
the amino acid transport via TAT1 is not associated with H+ cotransport.

4.6 SLC 38 family


The SLC 38 family consists of amino acid transport systems A and N. System A refers to
a Na+-coupled transport activity that prefers alanine and other small neutral amino acids
as substrates, whereas system N refers to a Na+-coupled transport activity that prefers as
substrates those amino acids containing nitrogen in their side chain (glutamine, aspar-
agine, and histidine). There are three subtypes within system A, referred to as ATA (amino
acid transporter A) 1, ATA2, and ATA3.53–58 A unique characteristic of system A is its ability
1382_C04.fm Page 72 Tuesday, October 7, 2003 6:11 PM

72 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

to transport the amino acid model substrate a-(methylamino)isobutyric acid (MeAIB). All
three subtypes of system A mediate the transport of MeAIB in a Na+-coupled manner
with a Na+:MeAIB stoichiometry of 1:1. H+ is not a cotransported ion for ATAs, but the
function of these transporters is markedly influenced by pH. The physiological substrates
of ATAs include alanine and other small linear neutral amino acids. Glutamine is also an
excellent substrate for ATAs. Bulky neutral amino acids such as leucine are excluded by
system A subtypes. ATA1 and ATA2 cannot be differentiated simply based on transport
characteristics because they exhibit similar substrate selectivity, substrate affinity, Na+
dependence, and sensitivity to pH. ATA3 differs from ATA1 and ATA2 in substrate affinity
and substrate selectivity. ATA3 transports not only neutral amino acids but also cationic
amino acids. Furthermore, ATA3 exhibits lower affinity for its substrates than ATA1 and
ATA2. All three subtypes of ATA differ markedly in their tissue distribution pattern. The
expression of ATA1 is restricted to the brain and placenta, whereas ATA2 is expressed in
almost every tissue. In contrast, ATA3 is expressed most exclusively in the liver.
System N consists of two subtypes, SN1 and SN2.59–63 Both subtypes transport
glutamine, asparagine, and histidine in a Na+-dependent manner. Interestingly, a trans-
membrane H+ gradient also plays a role in the transport mechanism. The transport of
amino acid substrate and Na+ occurs in one direction, whereas the transport of H+ occurs
in the opposite direction. Even though the Na+:amino acid:H+ stoichiometry is 2:1:1 and
the transport process is associated with membrane depolarization, it appears that the
actual mechanism of the transport process is more complex than originally thought.64 The
actual transport process itself is electroneutral, involving the movement of one Na+ and
the amino acid substrate in one direction, coupled to the movement of one H+ in the
opposite direction. Irrespective of the ion coupling, the transport process mediated by
SN1 and SN2 leads to intracellular alkalization when these transporters catalyze the influx
of amino acid substrates. However, SN1 and SN2 can also function in the reverse mode,
in which Na+ and amino acid substrate move out of the cell and H+ moves into the cell.
In this mode, the efflux of amino acid substrates via these transporters is associated with
intracellular acidification.
The physiological and clinical significance of systems A and N is partly related to the
ability of these two systems to mediate the transport of glutamine. This amino acid plays
a crucial role in the metabolic functions of the liver, brain, skeletal muscle, and placenta.
The different subtypes of systems A and N function in the cellular uptake or release of
glutamine as a part of the glutamine–glutamate cycle and glutamine cycle that occur
between or within these organs. SN1 and SN2 are expressed in all of these organs. ATA1
is expressed in the brain and placenta, ATA2 is expressed ubiquitously, and ATA3 is
expressed primarily in the liver. The potential for the reversal of the direction of glutamine
transport via SN1and SN2 makes them suitable to participate in the intercellular glutamine
cycle that occurs between the periportal and perivenous hepatocytes in the liver and in
the glutamine release from the astrocytes associated with the glutamine–glutamate cycle
in the brain. In the liver, periportal hepatocytes take up glutamine, whereas perivenous
hepatocytes release glutamine. Since glutaminase activity is high in periportal hepatocytes
and glutamine synthetase activity is high in perivenous hepatocytes, the direction of
glutamine concentration gradient differs between these two cell populations, favoring
glutamine uptake in the former and glutamine release in the latter. In addition, the
magnitude of the H+ gradient also differs between these two cell populations. Periportal
hepatocytes synthesize urea, which requires not only ammonia but also HCO3-. These
cells thus have an effective means of HCO3- disposal. In contrast, perivenous hepatocytes
remove ammonia via glutamine synthesis rather than urea synthesis, and this process
does not involve HCO3-. Thus, perivenous hepatocytes do not have an effective means of
HCO3- disposal. Therefore, it is likely that the intracellular pH in periportal hepatocytes
1382_C04.fm Page 73 Tuesday, October 7, 2003 6:11 PM

Chapter four: Cellular uptake of amino acids: systems and regulation 73

is acidic compared to the intracellular pH in perivenous hepatocytes. An acidic intracel-


lular pH in periportal hepatocytes is expected to facilitate the transport function of SN1
and SN2 in the direction of glutamine uptake. Similarly, an alkaline intracellular pH in
perivenous hepatocytes is expected to facilitate the transport function of SN1 and SN2 in
the direction of glutamine release. In the brain, glutamine release from astrocytes is
mediated by SN1 and SN2. In these cells, there exists a high inside-to-outside concentration
gradient for glutamine because of the abundant glutamine synthetase activity in these
cells. In addition, synaptic transmission results in an increase in intracellular pH in astro-
cytes. The resultant outward-directed gradients for glutamine and pH favor glutamine
release coupled to H+ influx. In glutamatergic neurons, extracellular glutamine is the
immediate source of glutamate. The presynaptic glutamatergic neurons take up glutamine
that is released from the astrocytes and convert it into glutamate. This uptake step is
mediated by ATA1. Since ATA1-mediated transport occurs only in the influx mode, this
transporter is uniquely suited for this purpose. Thus, the amino acid transport systems A
and N participate in the brain in the glutamine–glutamate cycle. Similar functions of
various subtypes of systems A and N are likely in the glutamine–glutamate cycle that
occurs between placenta and fetal liver and in the uptake and release of glutamine that
occurs in the skeletal muscle.
The activity of system A is regulated by various hormones.15 However, most of the
work on the regulation of this transport system was carried out prior to the molecular
identification of the transporters responsible for this transport activity. Recent studies
show that ATA2 is the subtype that is subject to regulation by hormones such as insulin65
and by amino acid deprivation66,67 and hyperosmolality.68 There is also evidence indicating
that the three known subtypes of system A are affected differentially by cAMP.69
The SLC38 gene family also consists of at least two amino acid transporters whose
transport function is coupled to cotransport of H+. These transporters, known as PAT1
and PAT2, are expressed differentially in mammalian tissues. They mediate electrogenic
transport of small neutral amino acids into the cells.70–72 The H+:amino acid stoichiometry
for the transport process is 1:1. PAT1 is expressed predominantly in the intestinal tract.
Moderate expression is evident in the brain, colon, kidney, liver, lung, placenta, and testis.
PAT2 is expressed mainly in the heart and lung. Both transporters recognize small neutral
amino acids (glycine, alanine, and proline) as substrates, but with variable affinity. PAT1
is a low-affinity transport system compared to PAT2. A H+-coupled transport activity for
small neutral amino acids has been described at the brush border membrane of the
intestinal cell line Caco-2.73 Recent studies have shown that PAT1 is responsible for this
transport activity.72

4.7 Heterodimeric amino acid transporters


A growing number of amino acid transporters consist of two subunits, a light chain and
a heavy chain. These transporters are called heterodimeric amino acid transporters.13,14
This group includes systems l, y+L, b0,+, x-c, asc, and ag (Table 4.3). Two different heavy
chains, known as rBAT and 4F2hc, have been identified thus far. These heavy chains are
glycosylated, and they interact with the corresponding light chains and direct their traf-
ficking to the plasma membrane. The heavy chains themselves do not exhibit transport
activity but may influence the transport function of the heterodimer.74 rBAT interacts with
the light chain b0,+AT to constitute the Na+-independent amino acid transport system b0,+.
This transport system is expressed mostly in the intestine and kidney and is responsible
for the transport of neutral amino acids and cationic amino acids across the brush border
membrane of the absorptive cells in these tissues. Cystine is also a substrate for this system.
4F2hc interacts with a variety of light chains to constitute different amino acid transport
1382_C04.fm Page 74 Tuesday, October 7, 2003 6:11 PM

74 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

systems. LAT1 and LAT2 are the light chains that interact with 4F2hc and form the two
known subtypes of amino acid transport system l. System l mediates the Na+-independent
transport of neutral amino acids. y+LAT1 and y+LAT2 are the light chains that interact
with 4F2hc to form the two known subtypes of amino acid transport system y+L. This
system mediates the transport of cationic amino acids in a Na+-independent manner and
the transport of neutral amino acids in a Na+-dependent manner. System x-c consists of
4F2hc and the light chain xCT. This transport system is Na+ independent and mediates
the cellular uptake of cystine coupled to the efflux of glutamate. System asc consists of
two subtypes. One of the subtypes consists of 4F2hc and the light chain Asc1. This system
mediates the Na+-independent transport of small neutral amino acids. The other subtype
consists of the light chain Asc2 and a hitherto unidentified heavy chain.75 Similarly, the
light chain of system ag has been identified (AGT1), but the nature of the heavy chain is
not yet known.76 The substrate specificity and transport properties of this newly identified
system ag do not conform to the functional features of any of the previously characterized
amino acid transport systems. At the structural level, the light chains of all of the known
heterodimeric amino acid transporters exhibit significant similarity. Since these light chains
also show sequence homology to the cationic amino acid transporters, they are considered
members of the SLC7 gene family. The two known heavy chains, rBAT and 4F2hc, are
structurally related to each other, but do not show any sequence homology to the light
chains. Therefore, the heavy chains are grouped under a separate solute carrier gene family
(SLC3). It is clear that there are additional heavy chains yet to be identified at the molecular
level. An interesting feature of these heterodimeric amino acid transporters is that they
all function as obligatory exchangers. They mediate the entry of amino acids into cells by
a process that is coupled to the exit of amino acids from the cells. This can be either homo-
exchange or hetero-exchange. Nonetheless, some of these transporters can facilitate the
vectorial transport of certain amino acids in polarized cells when functionally coupled to
other amino acid transporters.5,11,13
In terms of regulation, system x-c has received considerable attention in recent years.
The activity of system x-c is up-regulated by oxidative stress induced by nitric oxide,77
oxidized low-density lipoproteins,78 oxygen,79 and other oxidants.80 This regulation is
related to the role of system x-c in the influx of cystine into the cells where it is reduced
to cysteine and used for the synthesis of the antioxidant glutathione.

4.8 Conclusions
Most of the amino acid transport systems that are known to exist in mammalian cells based
on functional studies have now been cloned and characterized at the molecular level. The
information on the identity and structure of the genes coding for these transport systems
will undoubtedly help the investigators in the area of amino acid transport to generate
useful tools such as cDNAs and antibodies to usher this area of research in newer directions.
Detailed investigations of the various isoforms of different amino acid transport systems
in mammalian cells in terms of the regulatory aspects of their expression and function at
the gene and protein levels should now be feasible. There are, however, a few of the
classically identified amino acid transport systems that have not yet been cloned. This
includes the transport systems IMINO (a Na+-coupled transport system for imino acids
and glycine), X-A (a Na+-coupled transport system selectively for aspartate), and x-G (a Na+-
independent transport system selectively for glutamate). There is a very high likelihood
that these transport systems will be cloned and characterized at the molecular level in the
coming years.
1382_C04.fm Page 75 Tuesday, October 7, 2003 6:11 PM

Chapter four: Cellular uptake of amino acids: systems and regulation 75

Acknowledgments
This work is supported by the National Institutes of Health grant GM65344.

References
1. Christensen, H.N., Interorgan amino acid nutrition, Physiol. Rev., 62, 1193, 1982.
2. Christensen, H.N., On the strategy of kinetic discrimination of amino acid transport systems,
J. Membr. Biol., 84, 97, 1985.
3. Christensen, H.N., Distinguishing amino acid transport systems of a given cell or tissue,
Meth. Enzymol., 173, 576, 1989.
4. Christensen, H.N., Role of amino acid transport and counter-transport in nutrition and
metabolism, Physiol. Rev., 70, 43, 1990.
5. Palacin, M. et al., Molecular biology of mammalian plasma membrane amino acid transport-
ers, Physiol. Rev., 78, 969, 1998.
6. Stevens, B.R., Amino acid transport in intestine, in Mammalian Amino Acid Transport: Mech-
anisms and Control, Kilberg, M.S. and Haussinger, D., Eds., Plenum, New York, 1992, p. 149.
7. Bannai, S. et al., Amino acid transport systems, Nature, 311, 308, 1984.
8. Deves, R. and Boyd, C.A., Transporters for cationic amino acids: discovery, structure and
function, Physiol. Rev., 78, 487, 1998.
9. Closs, E.I., Expression, regulation and function of carrier proteins for cationic amino acids,
Curr. Opin. Nephrol. Hypertens., 11, 99, 2002.
10. Ganapathy, V., Ganapathy, M.E., and Leibach, F.H., Intestinal transport of peptides and amino
acids, in Current Topics in Membranes, Gastrointestinal Transport, Molecular Physiology, Barrett,
K.E. and Donowitz, M., Eds., Academic Press, San Diego, 2001, p. 379.
11. Broer, S., Adaptation of plasma membrane amino acid transport mechanisms to physiological
demands, Pflugers Arch. Eur. J. Physiol., 444, 457, 2002.
12. Bode, B.P., Recent molecular advances in mammalian glutamine transport, J. Nutr., 131,
2475S, 2001.
13. Chillaron, J. et al., Heteromeric amino acid transporters: biochemistry, genetics, and physi-
ology, Am. J. Physiol., 281, F995, 2001.
14. Kanai, Y. and Endou, H., Heterodimeric amino acid transporters: molecular biology and
pathological and pharmacological relevance, Curr. Drug Metab., 2, 339, 2001.
15. McGivan, J.D. and Pastor-Anglada, M., Regulatory and molecular aspects of mammalian
amino acid transport, Biochem. J., 299, 321, 1994.
16. Arriza, J.L. et al., Cloning and expression of a human neutral amino acid transporter with
structural similarity to the glutamate transporter gene family, J. Biol. Chem., 268, 15329, 1993.
17. Shafqat, S. et al., Cloning and expression of a novel Na+-dependent neutral amino acid
transporter structurally related to mammalian Na+/glutamate cotransporters, J. Biol. Chem.,
268, 15351, 1993.
18. Utsunomiya-Tate, N., Endou, H., and Kanai, Y., Cloning and functional characterization of
a system ASC-like Na+-dependent neutral amino acid transporter, J. Biol. Chem., 271, 14883,
1996.
19. Kekuda, R. et al., Cloning of the sodium-dependent, broad-scope, neutral amino acid trans-
porter B0 from a human placental choriocarcinoma cell line, J. Biol. Chem., 271, 18657, 1996.
20. Rasco, J.E.J. et al., The RD114/simian type D retrovirus receptor is a neutral amino acid
transporter, Proc. Natl. Acad. Sci. U.S.A., 96, 2129, 1999.
21. Tailor, C.R. et al., A sodium-dependent neutral amino acid transporter mediates infections
of feline and baboon endogenous retroviruses and simian type D retroviruses, J. Virol., 73,
4470, 1999.
22. Kekuda, R. et al., Molecular and functional characterization of intestinal Na+-dependent
neutral amino acid transporter B0, Am. J. Physiol., 272, G1473, 1997.
23. Avissar, N.E. et al., Na+-dependent neutral amino acid transporter ATB0 is a rabbit epithelial
cell brush-border protein, Am. J. Physiol., 281, C963, 2001.
1382_C04.fm Page 76 Tuesday, October 7, 2003 6:11 PM

76 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

24. Torres-Zamorano, V. et al., Tyrosine phosphorylation- and epidermal growth factor-depen-


dent regulation of the sodium-coupled amino acid transporter B0 in the human placental
choriocarcinoma cell line JAR, Biochim. Biophys. Acta, 1356, 258, 1997.
25. Iannoli, P. et al., Human growth hormone induces system B transport in short bowel syn-
drome, J. Surg. Res., 69, 150, 1997.
26. Pan, M. and Stevens, B.R., Differentiation and protein kinase C-dependent regulation of
alanine transport via system B, J. Biol. Chem., 270, 3582, 1995.
27. Zerangue, N. and Kavanaugh, M.P., Flux coupling in a neuronal glutamate transporter,
Nature, 383, 634, 1996.
28. McGivan, J.D. and Nicholson, B., Regulation of high-affinity glutamate transport by amino
acid deprivation and hyperosmotic stress, Am. J. Physiol., 277, F498, 1999.
29. Gegelashvili, G. et al., Regulation of glutamate transporters in health and disease, Prog. Brain
Res., 132, 267, 2001.
30. Robinson, B., Regulated trafficking of neurotransmitter transporters: common notes but
different melodies, J. Neurochem., 80, 1, 2002.
31. Lin, C.I. et al., Modulation of the neuronal glutamate transporter EAAC1 by the interacting
protein GTRAP3-18, Nature, 410, 84, 2001.
32. Jackson, M. et al., Modulation of the neuronal glutamate transporter EAAT4 by two inter-
acting proteins, Nature, 410, 89, 2001.
33. Sloan, J.L. and Mager, S., Cloning and functional expression of a human Na+ and Cl--
dependent neutral and cationic amino acid transporter B0,+, J. Biol. Chem., 274, 23740, 1999.
34. Hatanaka, T. et al., Na+- and Cl--coupled active transport of nitric oxide synthase inhibitors
via the amino acid transporter ATB0,+, J. Clin. Invest., 107, 1035, 2001.
35. Hatanaka, T. et al., Transport of D-serine via the amino acid transporter ATB0,+ expressed in
the colon, Biochem. Biophys. Res. Commun., 291, 291, 2002.
36. Ramamoorthy, S. et al., Functional characterization and chromosomal localization of a cloned
taurine transporter from human placenta, Biochem. J., 300, 893, 1994.
37. Schaffer, S., Takahashi, K., and Azuma, J., Role of osmoregulation in the actions of taurine,
Amino Acids, 19, 527, 2000.
38. Bridges, C.C. et al., Regulation of taurine transporter expression by NO in cultured human
retinal pigment epithelial cells, Am. J. Physiol., 281, C1825, 2001.
39. Mochizuki, T., Satsu, H., and Shimizu, M., Tumor necrosis factor alpha stimulates taurine
uptake and transporter gene expression in human intestinal Caco-2 cells, FEBS Lett., 517, 92,
2002.
40. Han, X., Patters, A.B., and Chesney, R.W., Transcriptional repression of taurine transporter
gene (TauT) by p53 in renal cells, J. Biol. Chem., 277, 39266, 2002.
41. Zafra, F., Aragon, C., and Gimenez, C., Molecular biology of glycinergic neurotransmission,
Mol. Neurobiol., 14, 117, 1997.
42. Roux, M.J. and Supplisson, S., Neuronal and glial glycine transporters have different sto-
ichiometries, Neuron, 25, 373, 2000.
43. Vekony, N. et al., Human cationic amino acid transporter hCAT3 is preferentially expressed
in peripheral tissues, Biochemistry, 40, 12387, 2001.
44. Nawrath, H. et al., Voltage dependence of L-arginine transport by hCAT2A and hCAT2B
expressed in oocytes from Xenopus laevis, Am. J. Physiol., 279, C1336, 2000.
45. Closs, E.I. and Graf, P., Cationic amino acid transporters (CATs): targets for the manipulation
of NO-synthase activity? Pharmaceut. Biotechnol., 12, 229, 1999.
46. Nicholson, B. et al., Sustained nitric oxide production in macrophages requires the arginine
transporter CAT2, J. Biol. Chem., 276, 15881, 2001.
47. Graf, P., Forstermann, U., and Closs, E.I., The transport activity of the human cationic amino
acid transporter hCAT1 is downregulated by activation of protein kinase C, Br. J. Pharmacol.,
132, 1193, 2001.
48. Zharikov, S.I. and Block, E.R., Association of L-arginine transporters with fodrin: implications
for hypoxic inhibition of arginine uptake, Am. J. Physiol., 278, L111, 2000.
49. Reade, M.C. et al., Increased cationic amino acid flux through a newly expressed transporter
in cells overproducing nitric oxide from patients with septic shock, Clin. Sci., 102, 645, 2002.
1382_C04.fm Page 77 Tuesday, October 7, 2003 6:11 PM

Chapter four: Cellular uptake of amino acids: systems and regulation 77

50. Huang, C.J. et al., Interleukin-10 inhibition of nitric oxide biosynthesis involves suppression
of CAT2 transcription, Nitric Oxide, 6, 79, 2002.
51. Kim, D.K. et al., Expression cloning of a Na+-independent aromatic amino acid transporter
with structural similarity to H+/monocarboxylate transporters, J. Biol. Chem., 276, 17221, 2001.
52. Kim, D.K. et al., The human T-type amino acid transporter-1: characterization, gene organi-
zation, and chromosomal location, Genomics, 79, 95, 2002.
53. Varoqui, H. et al., Cloning and functional identification of a neuronal glutamine transporter,
J. Biol. Chem., 275, 4049, 2000.
54. Wang, H. et al., Cloning and functional expression of ATA1, a subtype of amino acid trans-
porter A, from human placenta, Biochem. Biophys. Res. Commun., 273, 1175, 2000.
55. Sugawara, M. et al., Cloning of an amino acid transporter with functional characteristics and
tissue expression pattern identical to that of system A, J. Biol. Chem., 275, 16473, 2000.
56. Hatanaka, T. et al., Primary structure, functional characteristics and tissue expression pattern
of human ATA2, a subtype of amino acid transport system A, Biochim. Biophys. Acta, 1467,
1, 2000.
57. Sugawara, M. et al., Structure and function of ATA3, a new subtype of amino acid transport
system A, primarily expressed in the liver and skeletal muscle, Biochim. Biophys. Acta, 1509,
7, 2000.
58. Hatanaka, T. et al., Evidence for the transport of neutral as well as cationic amino acids by
ATA3, a novel and liver-specific subtype of amino acid transport system A, Biochim. Biophys.
Acta, 1510, 10, 2001.
59. Chaudhry, F.A. et al., Molecular analysis of system N suggests novel physiological roles in
nitrogen metabolism and synaptic transmission, Cell, 99, 769, 1999.
60. Gu, S. et al., Identification and characterization of an amino acid transporter expressed
differentially in liver, Proc. Natl. Acad. Sci. U.S.A., 97, 3230, 2000.
61. Fei, Y.J. et al., Primary structure, genomic organization, and functional and electrogenic
characteristics of human system SN1, a Na+ and H+-coupled glutamine transporter, J. Biol.
Chem., 275, 23707, 2000.
62. Nakanishi, T. et al., Cloning and functional characterization of a new subtype of the amino
acid transport system N, Am. J. Physiol., 281, C1757, 2001.
63. Nakanishi, T. et al., Structure, function, and tissue expression pattern of human SN2, a
subtype of the amino acid transport system N, Biochem. Biophys. Res. Commun., 281, 1343,
2001.
64. Broer, A. et al., Regulation of the glutamine transporter SN1 by extracellular pH and intra-
cellular sodium ions, J. Physiol., 539, 3, 2002.
65. Hyde, R., Peyrollier, K., and Hundal, H.S., Insulin promotes the cell surface recruitment of
the SAT2/ATA2 system A amino acid transporter from an endosomal compartment in
skeletal muscle cells, J. Biol. Chem., 277, 13628, 2002.
66. Ling, R. et al., Involvement of transporter recruitment as well as gene expression in the
substrate-induced adaptive regulation of amino acid transport system A, Biochim. Biophys.
Acta, 1512, 15, 2001.
67. Hyde, R. et al., Subcellular localization and adaptive up-regulation of the system A (SAT2)
amino acid transporter in skeletal muscle cells and adipocytes, Biochem. J., 355, 563, 2001.
68. Alfieri, R.R. et al., Osmotic regulation of ATA2 mRNA expression and amino acid transport
system A activity, Biochem. Biophys. Res. Commun., 283, 174, 2001.
69. Hatanaka, T. et al., Differential influence of cAMP on the expression of the three subtypes
(ATA1, ATA2, and ATA3) of the amino acid transport system A, FEBS Lett., 505, 317, 2001.
70. Sagne, C. et al., Identification and characterization of a lysosomal transporter for small
neutral amino acids, Proc. Natl. Acad. Sci. U.S.A., 98, 7206, 2001.
71. Boll, M. et al., Functional characterization of two novel mammalian electrogenic proton-
dependent amino acid cotransporters, J. Biol. Chem., 277, 22966, 2002.
72. Chen, Z. et al., Structure, function, and immunolocalization of a proton-coupled amino acid
transporter (hPAT1) in the human intestinal cell line Caco-2, J. Physiol., 546, 349, 2003.
73. Thwaites, D.T. and Stevens, B.C., H+/zwitterionic amino acid symport at the brush border
membrane of human intestinal epithelial (Caco-2) cells, Exp. Physiol., 84, 275, 1999.
1382_C04.fm Page 78 Tuesday, October 7, 2003 6:11 PM

78 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

74. Rajan, D.P. et al., Differential influence of the 4F2 heavy chain and the protein related to b0,+
amino acid transport on substrate affinity of the heteromeric b0,+ amino acid transporter,
J. Biol. Chem., 275, 14331, 2000.
75. Chairoungdua, A. et al., Identification and characterization of a novel member of the het-
erodimeric amino acid transporter family presumed to be associated with an unknown heavy
chain, J. Biol. Chem., 276, 49390, 2001.
76. Matsuo, H. et al., Identification of a novel Na+-independent acidic amino acid transporter
with structural similarity to the member of a heterodimeric amino acid transporter family
with unknown heavy chains, J. Biol. Chem., 277, 21017, 2002.
77. Bridges, C.C. et al., Structure, function, and regulation of human cystine/glutamate trans-
porter in retinal pigment epithelial cells, Invest. Ophthalmol. Vis. Sci., 42, 47, 2001.
78. Sato, H. et al., Increase in cystine transport activity and glutathione levels in mouse peritoneal
macrophages exposed to oxidized low-density lipoprotein, Biochem. Biophys. Res. Commun.,
215, 154, 1995.
79. Bannai, S. et al., Induction of cystine transport activity in human fibroblasts by oxygen, J. Biol.
Chem., 264, 18480, 1989.
80. Tomi, M. et al., Induction of xCT gene expression and L-cystine transport activity by diethyl
maleate at the inner blood-retinal barrier, Invest. Ophthalmol. Vis. Sci., 43, 774, 2002.
1382_C05.fm Page 79 Tuesday, October 7, 2003 6:14 PM

Part II

Physiology
1382_C05.fm Page 80 Tuesday, October 7, 2003 6:14 PM
1382_C05.fm Page 81 Tuesday, October 7, 2003 6:14 PM

section A

Metabolism
1382_C05.fm Page 82 Tuesday, October 7, 2003 6:14 PM
1382_C05.fm Page 83 Tuesday, October 7, 2003 6:14 PM

chapter five

Amino acid metabolism


and gluconeogenesis
Xavier M. Leverve
Université Joseph Fourier

Contents
Introduction....................................................................................................................................83
5.1 Gluconeogenesis at the cellular level: liver has a predominant but
nonexclusive role..................................................................................................................84
5.1.1 From amino acids to oxaloacetate ........................................................................86
5.1.2 From oxaloacetate to glucose ................................................................................86
5.1.3 Hormonal control of gluconeogenesis .................................................................87
5.2 Gluconeogenesis: interorgan cooperation ........................................................................88
5.3 Gluconeogenesis and pathological states.........................................................................90
5.4 Gluconeogenesis or glucose recycling? ............................................................................90
5.4.1 De novo gluconeogenesis ........................................................................................91
5.4.2 Glucose recycling: the futile cycles.......................................................................91
5.5 Conclusion .............................................................................................................................91
References .......................................................................................................................................92

Introduction
In terms of mass, glucose synthesis is probably the most important biosynthetic process
in living systems. It is a general feature of plants, microorganisms, and animals, and the
ability of heterotrophic cells to synthesize glucose or glycogen from lactate, pyruvate, and
also from nearly all amino acids present in proteins is one of the most fundamental
biosynthetic pathways of cellular metabolism. Classically, in highly complex organisms
such as mammals, and therefore in man, this function of glucose synthesis is mainly
devoted to the liver. While the kidney has also long been recognized as a gluconeogenic
organ, it has only recently been described that the gut is a gluconeogenic organ as well.
In fact, many other tissues, including skeletal muscle, are enzymatically equipped with
phosphoenolpyruvate carboxykinase, allowing glucose 6-phosphate synthesis. Hence,
these organs may also be considered as involved in the gluconeogenic pathway in a broad
sense, even if not able to release substantial amounts of glucose. In man, the main

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 83
1382_C05.fm Page 84 Tuesday, October 7, 2003 6:14 PM

84 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 5.1 Origin of Hepatic Glucose Production in Postabsorptive


Healthy Man
Hepatic glucose output 100%
Glycogenolysis 75%
Gluconeogenesis 25%
Lactate 16%
Amino acids (mainly alanine) 6%
Glycerol 2%
Pyruvate 1%

Note: Liver glucose production from the different gluconeogenic substrates is


expressed as the percentage of total glucose production (10 to
12 mmol/kg/min).
Source: Data are from Newsholme, E.A. and Leech, A.R., Biochemistry for Medical
Sciences, John Wiley & Sons, Chichester, U.K., 1990; Windmueller, H.G. and
Spaeth, A.E., J. Biol. Chem., 255, 107–112, 1980; Fevrannini, E. et al., Diabetes, 34,
580–588, 1985. With permission.

substrates for gluconeogenesis are lactate, pyruvate, amino acids, and, to a lesser extent,
glycerol (Table 5.1; see Newsholme and Leech1 for a review). Since gluconeogenesis from
lactate is predominantly related to a recycling of carbons (Cori’s glucose–lactate cycle),
the net synthesis of glucose is essentially provided from amino acid sources and to a minor
extent from glycerol. Hence, in the absence of exogenous intakes of carbohydrates, the
oxidized glucose is replaced by newly synthesized molecules coming from the muscle
mass, and amino acids play a major role in glucose homeostasis.
Glucose is a major fuel in man, although its storage is limited (the glycogen storage is
consumed within 12 to 24 h) when compared to the lipid stores permitting weeks or months
of starvation.1 Hence, when exogenous carbohydrates are not sufficient, two mechanisms
permit the maintenance of glucose homeostasis: a decrease in glucose oxidation and an
enhancement of the synthesis of new glucose molecules from either liver glycogen or
gluconeogenic precursors2–4 (Table 5.2). Interestingly, the human body is able to self-satisfy
entirely its need of glucose through endogenous synthesis, conversely to lipids since essen-
tial fatty acid cannot be synthesized. If the relationships between amino acids and glucose
metabolism are predominantly considered through the role of amino acids as net gluco-
neogenic substrates, glucose and other carbohydrates are also precursors for nonessential
amino acid synthesis by transamination. Gluconeogenesis connects carbohydrate, lipid,
and amino acid metabolism in such a manner that it is not possible to study amino acid
gluconeogenesis without considering also lactate, glycerol, and lipid metabolism.

5.1 Gluconeogenesis at the cellular level: liver has a predominant


but nonexclusive role
It is classically believed that complete gluconeogenesis, i.e., glucose release, occurs only
in the liver and kidney,5,6 since only these tissues express the glucose-6-phosphatase gene.
However, it was recently shown that this gene is also expressed in the small intestine in
rats and humans.7–10 In fasting and diabetes the small intestine may contribute up to 20
to 25% of whole-body endogenous glucose production. In this organ, glutamine appears
to be the main gluconeogenic substrate, and therefore, glucose synthesis requires phos-
phoenolpyruvate carboxykinase (PEPCK), which is strongly induced in this tissue in some
conditions, such as long-term fasting and insulinopenia. Interestingly, glycerokinase is
also expressed in the small intestine, allowing the use of glycerol as a gluconeogenic
substrate together with glutamine. Alanine is substantially released by the gut, and since
1382_C05.fm Page 85 Tuesday, October 7, 2003 6:14 PM

Chapter five: Amino acid metabolism and gluconeogenesis 85

Table 5.2 Glucose, Lactate, Alanine, and Glutamine Metabolism in Healthy Man at Basal
Postabsorptive Steady State
Total hepatic glucose output = total glucose uptake @ 10–12 mmol/kg/min
Glucose oxidation @ 5–7 mmol/kg/min
Brain glucose oxidation @ 4–5 mmol/kg/min
Glucose recycling via glycolysis @ 5.5 mmol/kg/min
Splanchnic @ 2.8 mmol/kg/min
Nonsplanchnic @ 2.4 mmol/kg/min
Muscle @ 2 mmol/kg/min
Blood cells, renal medulla @ 0.4 mmol/kg/min
Lactate turnover @ 4–5.5 mmol/kg/min
Alanine turnover @ 4–5 mmol/kg/min
Glutamine turnover @ 5–6 mmol/kg/min

Note: Averaged values of glucose metabolism in postabsorptive healthy man expressed as micromole
per kilogram of body mass per minute (mmol/kg/min). Although glucose oxidation concerns
about half of total glucose turnover, brain glucose oxidation accounts almost entirely for the
total glucose oxidation. The remaining half of glucose output is metabolized via recycling
pathways (lactate and alanine) divided into splanchnic and nonsplanchnic routes. The fluxes of
glucose oxidation, glucose recycling, lactate, alanine, and glutamine are of the same order of
magnitude: about 5 mmol/kg of body mass per hour.
Source: Data are from Cersosimo, E. et al., Metabolism, 49, 676–683, 2000; Cersosimo, E. et al., Diabetes,
49, 1186–1193, 2000; Mithieux, G., Curr. Opin. Clin. Nutr. Metab. Care, 4, 267–271, 2001; Pilkis, S.J. and
Claus, T.H., Annu. Rev. Nutr., 11, 465–515, 1991; Pilkis, S.J. and Granner, D.K., Annu. Rev. Physiol., 54,
885–909, 1992; Ferre, P. et al., Reprod. Nutr. Dev., 26, 619–631, 1986; Girard, J. et al., Reprod. Nutr. Dev.,
25, 303–319, 1985; Wahren, J. et al., J. Clin. Invest., 51, 1870–1878, 1972; Felig, P., Annu. Rev. Biochem.,
44, 933–955, 1975; Dechelotte, P. et al., Am. J. Physiol., 260, G677–G682, 1991.

this amino acid is a major precursor for liver gluconeogenesis, the gut appears to play an
important role in the gluconeogenesis from amino acids either directly (glutamine) or
indirectly (alanine).
Contrary to the classical view, PEPCK seems to be present in many tissues, including
the gut, as mentioned above, but also muscle and heart. Hence, all these organs are able
to achieve a net synthesis of phosphoenolpyruvate from oxaloacetate; therefore, they can
synthesize six-carbon intermediates, such as glucose 6-phosphate, from lactate. Interest-
ingly, this pathway is probably of major importance in muscle since it permits the restoring
of glycogen storage from lactate (and not from amino acids) in conditions where glycogen
is low and lactate is high, e.g., after intense muscular work.11
Theoretically, the lack of glucose-6-phosphatase prevents any glucose release from
muscle. In fact, in particular conditions of very active glycogenolysis, such a net release
of glucose can occur.12 Indeed, despite the absence of this enzyme, a very small amount
of nonphosphorylated glucose is released during hydrolysis of glycogen when this path-
way is highly activated. Renal gluconeogenesis is believed to be significant only after long-
term starvation. In this condition, this glucose formation is related to the need for an
increased proton excretion in urine, which is achieved when nitrogen is excreted predom-
inantly as ammonia from glutamine hydrolysis in the kidney, rather than urea synthesized
by the liver. Hence, in this particular condition, renal gluconeogenesis is mainly provided
by glutamine.1,13 Interestingly, a recent work has investigated the gluconeogenesis from
13C-lactate in patients during the anhepatic phase occurring in liver transplant surgery.14

This exceptional physiological condition of the complete absence of any liver function has
permitted the assessment of nonhepatic gluconeogenesis in a completely unequivocal
manner. In this particular condition, it was shown that as much as 50% of glucose synthesis
was achieved in the kidneys from labeled lactate.
The gluconeogenic pathway is classically described from pyruvate to glucose,
although several gluconeogenic pathways should actually be considered according to the
1382_C05.fm Page 86 Tuesday, October 7, 2003 6:14 PM

86 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

precursor. Hence, gluconeogenesis from either pyruvate or lactate is different, since in one
case reducing equivalents must be exported from the mitochondrion to the matrix, whereas
in the latter NADH is provided directly in the cytosol. Moreover, pathways from alanine,
glycerol, or fructose are different.1 When amino acids are the gluconeogenic precursors,
oxaloacetate is a common intermediate, regardless of the amino acid substrate. Thus, the
amino acid gluconeogenic pathway can be divided into two parts: upstream and down-
stream of oxaloacetate.

5.1.1 From amino acids to oxaloacetate


As shown in Figure 5.1, amino acid enters the pathway at the site of pyruvate or of Krebs
cycle intermediates (2-oxoglutarate, succinyl-CoA, or fumarate) or directly via oxaloace-
tate. Entry of metabolites into the Krebs cycle via acetyl-CoA cannot lead to net oxaloac-
etate formation and therefore to glucose synthesis. Indeed, the Krebs cycle involves two
decarboxylative steps, while acetyl-CoA is a two-carbon metabolite. This is the case for
all substrates metabolized solely via acetyl-CoA, i.e., fatty acids, leucine, and lysine. Some
amino acids lead to both acetyl-CoA and pyruvate or citric acid cycle intermediates
(isoleucine, threonine, tryptophan, phenylalanine, or tyrosine); therefore, they are both
glucogenic and ketogenic. Among the different amino acids, the two most important for
gluconeogenesis are alanine and glutamine. In the liver, it is reported that alanine metab-
olism is largely controlled by its transport across the plasma membrane.15 However, in
experimental conditions of a very high rate of gluconeogenesis from alanine, it was
reported that the transamination step could be controlling.16 This point is of interest
because the cytosolic 2-oxoglutarate concentration is related to mitochondrial membrane
potential and then to cellular energy status. After transamination into pyruvate, alanine
is carboxylated into oxaloacetate by pyruvate carboxylase, a step also important for glu-
coneogenesis from lactate. Hence, the two major substrates for glucose synthesis, i.e.,
lactate and alanine, compete at the site of pyruvate carboxylase. Since the regulations of
alanine and lactate pathways have been studied separately so far, not much is known
about the reciprocal effect of lactate on alanine metabolism.

5.1.2 From oxaloacetate to glucose


This part of gluconeogenesis involves several steps shared with glycolysis (the near-
equilibrium steps), while three steps are far from equilibrium and are therefore irreversible.
These steps, catalyzed by pyruvate kinase, phosphofructokinase, and gluco (hexo) kinase,
need different reactions to reverse the flux; they are, respectively, pyruvate carboxylase
and PEPCK, fructose-1,6-biphosphatase, and glucose-6-phosphatase.1 For these three
steps, the presence of both forward and reverse enzymes creates a substrate cycling when
they are active simultaneously. This plays a major regulatory role, although it dissipates
some energy. The phosphoenolpyruvate (PEP)/pyruvate cycling is of crucial regulatory
importance in alanine, glutamine, and lactate gluconeogenesis, and the three main
enzymes, pyruvate carboxylase, pyruvate kinase, and PEPCK, share a large part of the
gluconeogenic flux control.17 Among the numerous factors involved in this regulation,
three should be emphasized: hormones (glucagon and insulin), acetyl-CoA concentration
(via free CoA to acetyl-CoA ratio), and cellular energy state (via phosphate potentials). It
should be emphasized that fatty acid oxidation increases acetyl-CoA, inhibits pyruvate
dehydrogenase, and activates pyruvate carboxylase in such a manner that the net result
is a simultaneous decrease in carbohydrate oxidation and an activation of gluconeogenesis.
Thus, fatty acid oxidation activates gluconeogenesis from lactate and alanine, as well as
from other amino acids entering the pathway at the site of pyruvate.
1382_C05.fm Page 87 Tuesday, October 7, 2003 6:14 PM

Chapter five: Amino acid metabolism and gluconeogenesis 87

glucose

glucose-6-phosphate
alanine leucine
cysteine lysine
glycine phenylalanine
serine fructose-6-phosphate tyrosine
threonine
tryptophan fructose-1,6-disphosphate
acetoacetate

pyruvate phosphoenolpyruvate

isoleucine
leucine
acetylCoA threonine
aspartate oxaloacetate tryptophan
asparagine

malate citrate

phenylalanine
tyrosine fumarate isocitrate

CO 2 arginine
glutamate
succinate α-ketoglutarate glutamine
histidine
proline
succinyl-CoA

CO 2

isoleucine
methionine
valine

Figure 5.1 Shown are amino acids as gluconeogenic precursors and their relationships with Krebs
cycle intermediates. Amino acid metabolism is related to carbohydrate metabolism via the Krebs
cycle. Amino acids can be divided into three groups according to the site of their entry into the
pathway: acetyl-CoA, Krebs cycle intermediates, or pyruvate. (1) The entry via acetoacetate or acetyl-
CoA cannot lead to synthesis of net glucose precursors since the presence of two decarboxylative
steps at the cycle does not permit net synthesis of oxaloacetate from acetyl-CoA (a two-carbon
compound). Such a group of amino acids are precursors for ketone body synthesis The two amino
acids leucine and lysine, which enter the pathway at this site only, are not precursors for glucose
synthesis. (2) The majority of the gluconeogenic amino acids (12/18) enter the pathway at the site
of one of the Krebs cycle intermediates. (3) Six amino acids are metabolized via pyruvate, and among
them, alanine is the major gluconeogenic amino acid, sharing the gluconeogenic pathway with
lactate metabolism. The synthesis of glucose from oxaloacetate is mainly regulated via three substrate
cycles: the phosphoenolpyruvate/pyruvate, the fructose-1,6-bisphosphate/fructose-6-phosphate,
and the glucose 6-phosphate/glucose cycles.

5.1.3 Hormonal control of gluconeogenesis


The hormonal control of gluconeogenesis could be divided into transcriptional and post-
transcriptional regulation. The posttranscriptional regulation is achieved by protein phos-
phorylation mechanisms, which are related to either cAMP or calcium changes. Four
enzymes of the pathway are concerned: 6-phosphofructo-1-kinase, fructose-1,6-biphos-
phatase, fructose-2,6-biphosphatase, and pyruvate kinase. When phosphorylated, pyruvate
1382_C05.fm Page 88 Tuesday, October 7, 2003 6:14 PM

88 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

kinase is inhibited, although fructose-2,6-biphosphatase is activated. Hence, the net result


is a decrease in fructose-2,6-biphosphate concentration and in the PEP/pyruvate recycling,
leading to an enhancement of gluconeogenesis. It can be noted that 6-phosphofructokinase
and fructose-1,6-biphosphatase18 also can be phosphorylated, although this does not affect
the enzyme activity,19 the meaning of such phosphorylation being unknown as yet.
The effects are mediated by four different hormones: glucagon, insulin, catechola-
mines, and cortisol. Glucagon is long known to stimulate gluconeogenesis from different
substrates by a rise in cAMP.20,21 It is a dominant hormone during fasting. Similarly,
b-adrenergic hormones increase cAMP, whereas a-adrenergic hormones act via intracel-
lular free calcium changes. The larger effect of the cAMP rise, as compared to that of the
calcium increase, may be due to the phosphorylation of both pyruvate kinase and fructose-
2,6-biphosphatase by cAMP, whereas calcium affects only fructose-2,6-biphosphatase. Cor-
tisol has no direct effect on phosphorylation, but it is known to amplify the effect of the
other hormones. Insulin is the dominant hormone during the fed state, but its effect is
located on muscle and adipose tissues, and the role of insulin on hepatic gluconeogenesis
is still a matter of controversy. Indeed, insulin addition is not responsible for an increased
glycogen deposition in the postprandial phase, and it seems that this hormone acts mainly
against the hyperglycemic effect of the other hormones by decreasing their effect on the
cAMP rise.22 The effect is probably due to an increased cAMP breakdown.23 It should be
added that posttranscriptional effects of hormones can also be mediated via changes in
energy metabolism, i.e., ATP-to-ADP ratio or transfer of reducing equivalents across the
mitochondrial membrane.24
The transcriptional regulation is involved in prolonged fasted states or in diseases
such as diabetes or acute illness, although it has been shown in early sepsis that the
hormonal effect of a-adrenergic stimulation by the a-agonist phenylephrine on gluconeo-
genesis was partly abolished, whereas ureagenesis stimulation was unchanged.25 This
transcriptional regulation concerns a glucagon-related cAMP rise leading to an increase
in several gluconeogenic enzymes (glucose-6-phosphatase, PEPCK, and possibly fructose-
1,6-biphosphatase) and a decrease in glycolytic enzymes (glucokinase, 6-phosphofructo-
1-kinase, and pyruvate kinase).23,26,27 Insulin or food intake has an opposite effect.27 This
cAMP effect on gene expression is of great importance in glucose homeostasis of the
newborn, since after birth PEPCK mRNA must be built for the first time. The rise in
glucagon immediatly after the birth plays a crucial role in energy metabolism at birth.28–30
It is of interest to note that the negative regulation on gene expression appears to be
dominant. Hence, the inhibitory effect of glucagon (cAMP) on glycolytic enzyme tran-
scription is stronger than the positive effect of insulin on these enzymes, whereas the
negative effect of insulin on gluconeogenic enzyme synthesis is stronger than that (posi-
tive) of glucagon.27

5.2 Gluconeogenesis: interorgan cooperation


The physiological need for hepatic gluconeogenesis is related to the condition where there
is a lack of exogenous glucose, i.e., the fasting state. In this condition, the muscle amino
acid balance is negative, and alanine and glutamine represent two thirds of the total
released amino acids.31–33 Such high alanine production is correlated with the use of amino
acid by the liver for glucose synthesis (Table 5.2). However, the question arises about the
origin of alanine and glutamine, since the muscle protein content of these two amino acids
is about 10%, whereas the net muscle production is about 60%. Hence, it appears that
these amino acids are synthesized in the muscle cells and simply not released after
muscular protein degradation.32 Alanine is probably formed from glucose carbon skeleton
via pyruvate and from ammonia as the result of leucine or other branched-chain amino
1382_C05.fm Page 89 Tuesday, October 7, 2003 6:14 PM

Chapter five: Amino acid metabolism and gluconeogenesis 89

acid catabolism.34–37 As mentioned above, fatty acid oxidation reduces pyruvate oxidation
and enhances its use for gluconeogenesis; the same result can be achieved in muscle by
leucine oxidation, since its metabolism leads also to acetyl-CoA formation. Although the
muscle is not a gluconeogenic organ stricto sensu, such a mechanism of inhibition of
pyruvate oxidation when leucine is oxidized may contribute to the increase in muscle
alanine formation, which in turn affects the liver gluconeogenesis rate. Because alanine,
coming from glucose in muscle cells, is directly taken up by the liver mainly to resynthesize
glucose, Felig38 proposed 20 years ago the concept of an alanine–glucose cycle by reference
to Cori’s cycle (see Section 5.4). Indeed, it has been shown in man that glucose infusion
involves a decrease in both glucose and urea production, whereas the rate of appearance
of labeled alanine in the plasma, as well as the glucose synthesis from alanine, was
enhanced.39
Glutamine is also synthesized in muscle cells, but the carbon skeleton is provided
from the catabolism of other amino acids such as branched-chain amino acids or aspartic
acid; ammonia also results, at least for one part, from the branched-chain amino acid
catabolism.33,35–37,40 Glutamine is probably also a major gluconeogenic amino acid in vivo
at the level of the gut itself and indirectly as a precursor for alanine synthesis. It is actively
taken up by the gut and converted into pyruvate, which is either oxidized or transaminated
to form alanine released in the portal blood and potentially taken up by the liver.41,42
Therefore, if we consider the complete carbon pathway, glutamine is clearly also a quan-
titative substrate for gluconeogenesis, mainly after a first conversion into alanine. The
quantitative role of the other gluconeogenic amino acids in the hepatic glucose synthesis
rate is probably not very high, although glycine can be actively converted to glucose. It
is possible that pyruvate used in muscle for alanine synthesis is not only derived from
glucose but also from other gluconeogenic amino acids. This fact is of importance, since
when derived from glucose, this pathway represents a simple carbon recycling, whereas
in the second case, i.e., from other amino acids, there is a net neoglucose formation from
protein (see Section 5.4). It is difficult to estimate the exact contribution of alanine to
gluconeogenesis in vivo. Indeed, some studies using stable isostopes have measured ala-
nine, glutamine, and glucose turnover in man (see Table 5.2), and it has been shown that
alanine turnover represents about one third of glucose turnover, whereas glutamine flux
is about 40%.33,35–37,40 It is difficult to assess precisely the percentage of alanine synthesized
from glutamine in gut and taken up by the liver, since it is not possible to obtain in humans
portal blood samples. Assuming that two thirds of glutamine are completely oxidized in
the gut,3,41–43 it can be estimated, in terms of carbon equivalents, that alanine plus glutamine
contribution to hepatic glucose synthesis in overnight fasted healthy subjects may reach
about 25% of the total glucose synthesis. In fasted dogs, it has been shown that after an
oral glucose load, the liver glycogen was restored by exogenous glucose for 50% and by
endogenous substrates (lactate and alanine) for the other 50%.44 Moreover, alanine or
lactate was mainly coming from the gut.
During exercise, there is an accelerated rate of muscular glucose uptake, which must
be linked to major changes in gluconeogenesis/glycolysis regulation in order to prevent
dramatic changes in blood glucose concentration. Glycogenolysis and gluconeogenesis
are both involved in the increase in hepatic glucose production, glycogenolysis being
predominant in short-term exercise and gluconeogenesis being the major pathway in
prolonged exercise or during recovery. When the gluconeogenic pathway is enhanced by
exercise, the use of alanine as a hepatic substrate for glucose is increased. This effect is
mainly due to the increase in hepatic fractional extraction but also to a more efficient
channeling of alanine metabolism into glucose.45 During the recovery phase, the glucose
formation from alanine is further enhanced, since in addition to the increased alanine
extraction by the liver, there is a rise in alanine supply to the liver.45
1382_C05.fm Page 90 Tuesday, October 7, 2003 6:14 PM

90 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

5.3 Gluconeogenesis and pathological states


It has long been known that gluconeogenesis is enhanced in severe illness, injury, and
diabetes, whereas it is decreased in long-term starvation.46 In stressed patients, the occur-
rence of an increased gluconeogenesis together with hyperglycemia is known as insulin
resistance. The increased gluconeogenesis is not suppressible by infusion of high amounts
of glucose.47 The splanchnic glucose output is increased by a factor of 2,47–49 whereas the
glucose formation from the kidney is not enhanced as in prolonged starvation. Wolfe and
coworkers50 have shown that the large increase in glucose production observed in burn
patients (24 µmol/kg/min), compared to controls (12 µmol/kg/min), was related to an
increase in both recycling and nonrecycling glucose production. The glucose oxidized,
when expressed as a percentage of total glucose uptake, was significantly decreased. The
increased use of amino acids for gluconeogenesis has been recognized for a long time,
since stress and the hypercatabolic state are associated with an increase in urinary nitrogen
loss. In fact, it was shown that in burn patients the entire increment in glucose production
could be accounted for by the increase in gluconeogenic precursors uptake by the
liver.48,49,51 Simultaneously, with such increased splanchnic glucose output and turnover,
some data showed, in these patients, a low respiratory quotient, demonstrating a small
contribution of glucose oxidation to total body oxygen consumption. This suggests that
the nonoxidative pathways (glucose–alanine or glucose–lactate) are primarily involved in
the increased glucose turnover, even when muscle amino acid contribution to gluconeo-
genesis is enhanced.48

5.4 Gluconeogenesis or glucose recycling?


The term gluconeogenesis means new glucose synthesis from a nonglucose compound. Thus,
we call gluconeogenesis the glucose formation from either fructose, glycerol, lactate, pro-
pionate, or amino acids, although the source and the pathways are quite different. In fact,
physiologically there are two kinds of gluconeogenesis, depending on the origin of the
carbons and not on their metabolic fate.52,53 Indeed, glucose can be formed from precursors
belonging to biochemical families other than carbohydrates. For example, ruminants absorb
very small amounts of glucose, and conversely, to nonruminant animals (like humans),
gluconeogenesis is maximally stimulated in the fed state and depressed during the fasting
state. The nutrients are first metabolized by bacteria, and fatty acids containing an odd
number of carbons are powerful gluconeogenic substrates via propionate.54 This is an
example of a true gluconeogenesis de novo since glucose is made from odd fatty acids. This
situation is comparable to the gluconeogenesis from amino acids, where proteins from
muscle are used to make new glucose molecules. On the other hand, when there is carbon
recycling, for instance, in the Cori’s cycle between glucose and lactate, there is only a partial
glucose hydrolysis (from six to three carbon intermediates), and the resynthesis occurs from
the hydrolysis products in another tissue.52 In this example, there is no net glucose synthesis,
but only a recycling of carbons between glucose and lactate, which consumes energy but
does not lead to net glucose accumulation, since the hydrolysis of one glucose into two
lactates is needed to rebuild a new glucose from the two lactates. In view of this fact,
gluconeogenesis from alanine is either a pathway of net gluconeogenesis if carbons are
coming from some amino acids resulting from muscle proteolysis or only a pathway of
carbon recycling if these carbons are coming from muscle glucose hydrolysis.

5.4.1 De novo gluconeogenesis


Another approach may relate true de novo gluconeogenesis and the total amount of oxi-
dized glucose. Indeed, in postabsorptive steady state, i.e., when the pools of the main
1382_C05.fm Page 91 Tuesday, October 7, 2003 6:14 PM

Chapter five: Amino acid metabolism and gluconeogenesis 91

carbohydrates are constant — glucose, lactate, pyruvate, glycogen content — the glucose,
which is fully oxidized as CO2 and H2O, must be replaced by a true neo-glucose. Hence,
the measurement of oxidized carbohydrates gives an estimation of the true glucose syn-
thesis. In this fasting situation, gluconeogenesis from muscle protein amino acids can
account almost entirely (glycerol excepted) for such a true glucose formation; i.e., it is
equal to the net glucose oxidation. Hence, in the absence of exogenous intakes and in a
steady-state situation, the net glucose oxidation is an estimation of the net muscle protein
amino acid amount used for glucose synthesis.

5.4.2 Glucose recycling: the futile cycles


When considering the recycling of glucose, the situation is opposite. There is neither net
glucose oxidation nor newly synthesized glucose molecules, but only waste of energy in
the form of heat with increased oxygen consumption and CO2 production (Figure 5.2).
Although in the previous situation the muscle mass was decreasing parallel to glucose
formation, in this situation the lipid mass is used parallel to glucose recycling (see Figure
5.2). Indeed, the major metabolic fuel for liver is fatty acid, and consequently, acetyl-CoA
rise inhibits carbohydrate oxidation. Hence, the ATP consumption for liver gluconeogen-
esis (from lactate or alanine) is provided by fatty acid oxidation (b-oxidation ATP),
whereas peripheral glucose hydrolysis gives glycolytic ATP. Hence, these metabolic con-
siderations lead to different approaches of the two main glucose recycling cycles: glu-
cose–lactate cycle and glucose–alanine cycle. On the one hand, in terms of yield, there is
a clear loss of energy efficacy during recycling since two ATP are produced from one
glucose hydrolyzed into pyruvate, whereas six ATP are needed to make one glucose from
either two pyruvate, two lactate, or two alanine. Thus, two thirds of oxygen consumption
for ATP synthesis is directly lost as heat. On the other hand, when considering the
qualitative aspects instead of the quantitative, it appears that glucose hydrolysis into
pyruvate and finally into lactate (in insulin resistance, anoxic, or stressed tissue) or to
alanine or glutamine (in muscle to bring ammonia to the liver as a nontoxic form) provides
glycolytic anaerobic (or “nonrobic”) ATP to cells, whereas these compounds were made
with fatty acid aerobic ATP in the liver. Hence, one can say that these two main glucose
cycles allow the transfer of ATP made aerobically in the liver from lipid stores to glycolytic
anaerobic ATP made from glucose in peripheral cells. This is achieved with a decrease
in efficacy, but when considering the mass of lipid stores compared to the size of carbo-
hydrate and amino acid stores, it might be an advantage in these fasted states or acute
illnesses.

5.5 Conclusion
Gluconeogenesis is the pathway allowing glucose formation; however, regarding the
multiple precursors, this pathway is actually multiple. It is very important to consider
that glucose metabolism is divided in two parts: oxidative and nonoxidative routes. This
means that in the former case new molecules of glucose must be built from amino acids
provided from protein degradation, while in the latter it is only a carbon recycling with
energy dissipation. Lactate and pyruvate correspond solely to recycling; alanine and
glutamine correspond to both recycling and new synthesis, although recycling is probably
predominant. Other amino acids and all essential amino acids are involved in the neo-
synthetic pathway.
Contrary to the classical view, the liver is not the unique organ in glucose synthesis,
the kidney plays a major role, and the gut is probably an important source of neo-glucose
as well.
1382_C05.fm Page 92 Tuesday, October 7, 2003 6:14 PM

92 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

H 2 O + CO2

oxidation
recycling recycling
glucose
LIVER MUSCLE
glucose
glucose
glucose 2 ADP
2 ADP 6 ADP

PERIPHERIC β oxidation VALINE


or other AA
CELL 2 ATP
6 ADP 2 ATP
PYRUVATE PYRUVATE
PYRUVATE

LACTATE GLUTAMINE
ALANINE

LACTATE ALANINE

ALANINE

AMMONIA
GLUTAMINE GLUTAMINE

GUT

Figure 5.2 Gluconeogenesis: net glucose synthesis and substrate recycling. Liver glucose
production can be divided in either de novo synthesized glucose or substrate recycling.
Lactate and alanine are the main glucose precursors, although other gluconeogenic amino
acids and glycerol are also involved. Glucose recycling via lactate or alanine creates a
futile cycle between three- and six-carbon compounds. Although it dissipates directly, two
thirds of the energy (six ATP are needed to build one glucose from two lactate or two
alanine, whereas two ATP only are produced when splitting glucose into lactate or ala-
nine), the liver energy source is mainly coming from fatty acids. Hence, glucose recycling
provides glycolytic ATP to peripheric cells, whereas this ATP comes from lipid stores.
Glutamine plays a special role as glucose precursor; it is poorly involved per se, but since
it is converted into alanine in enterocytes, it is taken up by the liver. Moreover, in muscle,
glutamine is formed from an amino acid carbon skeleton (mainly branched chain), explain-
ing that such glucose synthesis is mainly a true de novo glucose synthesis rather than
recycling.

References
1. Newsholme, E.A. and Leech, A.R., Biochemistry for Medical Sciences, John Wiley & Sons,
Chichester, U.K., 1990.
2. Ferrannini, E., Bjorkman, O., Reichard, G.A., Jr., Pilo, A., Olsson, M., Wahren, J., and
DeFronzo, R.A., The disposal of an oral glucose load in healthy subjects: a quantitative study,
Diabetes, 34, 580–588, 1985.
3. Windmueller, H.G. and Spaeth, A.E., Respiratory fuels and nitrogen metabolism in vivo in
small intestine of fed rats: quantitative importance of glutamine, glutamate, and aspartate,
J. Biol. Chem., 255, 107–112, 1980.
4. Ahlborg, G. and Wahren, J., Brain substrate utilization during prolonged exercise, Scand. J.
Clin. Lab. Invest., 29, 397–402, 1972.
5. Cersosimo, E., Garlick, P., and Ferretti, J., Regulation of splanchnic and renal substrate supply
by insulin in humans, Metabolism, 49, 676–683, 2000.
1382_C05.fm Page 93 Tuesday, October 7, 2003 6:14 PM

Chapter five: Amino acid metabolism and gluconeogenesis 93

6. Cersosimo, E., Garlick, P., and Ferretti, J., Renal substrate metabolism and gluconeogenesis
during hypoglycemia in humans, Diabetes, 49, 1186–1193, 2000.
7. Mithieux, G., New data and concepts on glutamine and glucose metabolism in the gut, Curr.
Opin. Clin. Nutr. Metab. Care, 4, 267–271, 2001.
8. Mithieux, G., New knowledge regarding glucose-6 phosphatase gene and protein and their
roles in the regulation of glucose metabolism, Eur. J. Endocrinol., 136, 137–145, 1997.
9. Minassian, C., Tarpin, S., and Mithieux, G., Role of glucose-6 phosphatase, glucokinase, and
glucose-6 phosphate in liver insulin resistance and its correction by metformin, Biochem.
Pharmacol., 55, 1213–1219, 1998.
10. Rajas, F., Bruni, N., Montano, S., Zitoun, C., and Mithieux, G., The glucose-6 phosphatase
gene is expressed in human and rat small intestine: regulation of expression in fasted and
diabetic rats, Gastroenterology, 117, 132–139, 1999.
11. Greenhalf, P.L., Hultman, E., and Harris, R.C., Carbohydrate metabolism, in Principle of
Exercise Biochemistry, Pootsmans, J.R., Ed., Karger, Basel, Switzerland, 1993, pp. 89–136.
12. Wicklmayr, M. and Dietze, G., On the mechanism of glucose release from the muscle of
juvenile diabetics in acute insulin deficiency, Eur. J. Clin. Invest., 8, 81–86, 1978.
13. Newsholme, P., Procopio, J., Lima, M.M., Pithon-Curi, T.C., and Curi, R., Glutamine and
glutamate: their central role in cell metabolism and function, Cell Biochem. Funct., 21, 1–9,
2003.
14. Joseph, S.E., Heaton, N., Potter, D., Pernet, A., Umpleby, M.A., and Amiel, S.A., Renal glucose
production compensates for the liver during the anhepatic phase of liver transplantation,
Diabetes, 49, 450–456, 2000.
15. Groen, A.K., Sips, H.J., Vervoorn, R.C., and Tager, J.M., Intracellular compartmentation and
control of alanine metabolism in rat liver parenchymal cells, Eur. J. Biochem., 122, 87–93, 1982.
16. Burelle, Y., Fillipi, C., Peronnet, F., and Leverve, X., Mechanisms of increased gluconeogenesis
from alanine in rat isolated hepatocytes after endurance training, Am. J. Physiol., 278, E35–E42,
2000.
17. Groen, A.K., van Roermund, C.W., Vervoorn, R.C., and Tager, J.M., Control of gluconeogen-
esis in rat liver cells: flux control coefficients of the enzymes in the gluconeogenic pathway
in the absence and presence of glucagon, Biochem. J., 237, 379–389, 1986.
18. Riou, J.P., Claus, T.H., Flockhart, D.A., Corbin, J.D., and Pilkis, S.J., In vivo and in vitro
phosphorylation of rat liver fructose-1,6-bisphosphatase, Proc. Natl. Acad. Sci. U.S.A., 74,
4615–4619, 1977.
19. Mendicino, J., Leibach, F., and Reddy, S., Role of enzyme interactions in the regulation of
gluconeogenesis: phosphorylation of fructose 1,6-bisphosphatase and phosphofructokinase
by kidney protein kinase, Biochemistry, 17, 4662–4669, 1978.
20. Exton, J.H., Mallette, L.E., Jefferson, L.S., Wong, E.H., Friedmann, N., Miller, T.B., Jr., and
Park, C.R., The hormonal control of hepatic gluconeogenesis, Recent Prog. Horm. Res., 26,
411–461, 1970.
21. Hers, H.G. and Hue, L., Gluconeogenesis and related aspects of glycolysis, Annu. Rev.
Biochem., 52, 617–653, 1983.
22. Katz, J. and McGarry, J.D., The glucose paradox: is glucose a substrate for liver metabolism?
J. Clin. Invest., 74, 1901–1909, 1984.
23. Pilkis, S.J., el-Maghrabi, M.R., and Claus, T.H., Hormonal regulation of hepatic gluconeo-
genesis and glycolysis, Annu. Rev. Biochem., 57, 755–783, 1988.
24. Halestrap, A.P., Quinlan, P.T., Whipps, D.E., and Armston, A.E., Regulation of the mitochon-
drial matrix volume in vivo and in vitro: the role of calcium, Biochem. J., 236, 779–787, 1986.
25. Ohtake, Y. and Clemens, M.G., Interrelationship between hepatic ureagenesis and gluconeo-
genesis in early sepsis, Am. J. Physiol., 260, E453–E458, 1991.
26. Pilkis, S.J. and Claus, T.H., Hepatic gluconeogenesis/glycolysis: regulation and struc-
ture/function relationships of substrate cycle enzymes, Annu. Rev. Nutr., 11, 465–515, 1991.
27. Pilkis, S.J. and Granner, D.K., Molecular physiology of the regulation of hepatic gluconeo-
genesis and glycolysis, Annu. Rev. Physiol., 54, 885–909, 1992.
1382_C05.fm Page 94 Tuesday, October 7, 2003 6:14 PM

94 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

28. Ferre, P., Decaux, J.F., Issad, T., and Girard, J., Changes in energy metabolism during the
suckling and weaning period in the newborn, Reprod. Nutr. Dev., 26, 619–631, 1986.
29. Girard, J., Duee, P.H., Ferre, P., Pegorier, J.P., Escriva, F., and Decaux, J.F., Fatty acid oxidation
and ketogenesis during development, Reprod. Nutr. Dev., 25, 303–319, 1985.
30. Girard, J., La gluconeogenèse: une voie métabolique essentielle au maintien de l'homéostasie
glucidique du nouveau-né, Méd. Sci., 9, 297–306, 1993.
31. Wahren, J., Felig, P., Cerasi, E., and Luft, R., Splanchnic and peripheral glucose and amino
acid metabolism in diabetes mellitus, J. Clin. Invest., 51, 1870–1878, 1972.
32. Felig, P., Amino acid metabolism in man, Annu. Rev. Biochem., 44, 933–955, 1975.
33. Dechelotte, P., Darmaun, D., Rongier, M., Hecketsweiler, B., Rigal, O., and Desjeux, J.F.,
Absorption and metabolic effects of enterally administered glutamine in humans, Am. J.
Physiol., 260, G677–G682, 1991.
34. Chang, T.W. and Goldberg, A.L., The metabolic fates of amino acids and the formation of
glutamine in skeletal muscle, J. Biol. Chem., 253, 3685–3693, 1978.
35. Darmaun, D., Métabolisme de la glutamine in vivo chez l'homme: implications pour la
nutrition artificielle, Nutr. Clin. Métabol., 4, 203–214, 1990.
36. Darmaun, D. and Dechelotte, P., Role of leucine as a precursor of glutamine alpha-amino
nitrogen in vivo in humans, Am. J. Physiol., 260, E326–E329, 1991.
37. Darmaun, D., Role of nutrients in the regulation of in vivo protein metabolism in humans,
Acta Paediatr. Suppl., 88, 92–94, 1999.
38. Felig, P., The glucose-alanine cycle, Metabolism, 22, 179–207, 1973.
39. Royle, G.T., Molnar, J.A., Wolfe, M.H., Wolfe, R.R., and Burke, J.F., Urea, glucose and alanine
kinetics in man: effects of glucose infusion, Clin. Sci., 62, 553–556, 1982.
40. Darmaun, D., Role of glutamine depletion in severe illness, Diabetes Nutr. Metab., 13, 25–30,
2000.
41. Hanson, P.J. and Parsons, S., Metabolism and transport of glutamine and glucose in vascu-
larly perfused small intestine rat, Biochem. J., 166, 509–519, 1977.
42. Haymond, M.W. and Miles, J.M., Branched chain amino acids as a major source of alanine
nitrogen in man, Diabetes, 31, 86–89, 1982.
43. Windmueller, H.G. and Spaeth, A.E., Identification of ketone bodies and glutamine as the
major respiratory fuels in vivo for postabsorptive rat small intestine, J. Biol. Chem., 253, 69–76,
1978.
44. Mitrakou, A., Jones, R., Okuda, Y., Pena, J., Nurjhan, N., Field, J.B., and Gerich J.E., Pathway
and carbon sources for hepatic glycogen repletion in dogs, Am. J. Physiol., 260, E194–E202,
1991.
45. Wasserman, D.H., Williams, P.E., Lacy, D.B., Green, D.R., and Cherrington, A.D., Importance
of intrahepatic mechanisms to gluconeogenesis from alanine during exercise and recovery,
Am. J. Physiol., 254, E518–E525, 1988.
46. Streja, D.A., Steiner, G., Marliss, E.B., and Vranic, M., Turnover and recycling of glucose in
man during prolonged fasting, Metabolism, 26, 1089–1098, 1977.
47. Long, C.L., Kinney, J.M., and Geiger, J.W., Nonsuppressability of gluconeogenesis by glucose
in septic patients, Metabolism, 25, 193–201, 1976.
48. Gelfand, R.A., Glickman, M.G., Jacob, R., Sherwin, R.S., and DeFronzo, R.A., Removal of
infused amino acids by splanchnic and leg tissues in humans, Am. J. Physiol., 250, E407–E413,
1986.
49. Wilmore, D.W., Aulick, H.L., and Goodwin, C.W., Glucose metabolism following severe
surgery, Acta Chir. Scand., 498 (Suppl.), 43–47, 1979.
50. Wolfe, R.R., Durkot, M.J., Allsop, J.R., and Burke, J.F., Glucose metabolism in severely burned
patients, Metabolism, 28, 1031–1039, 1979.
51. Wilmore, D.W., Goodwin, C.W., Aulick, L.H., Powanda, M.C., Mason, A.D., Jr., and Pruitt,
B.A., Jr., Effect of injury and infection on visceral metabolism and circulation, Ann. Surg.,
192, 491–504, 1980.
1382_C05.fm Page 95 Tuesday, October 7, 2003 6:14 PM

Chapter five: Amino acid metabolism and gluconeogenesis 95

52. Leverve, X.M., Lactic acidosis: a new insight? Minerva Anestesiol., 65, 205–209, 1999.
53. Leverve, X.M., Energy metabolism in critically ill patients: lactate is a major oxidizable
substrate, Curr. Opin. Clin. Nutr. Metab. Care, 2, 165–169, 1999.
54. Demigne, C., Yacoub, C., Morand, C., and Remesy, C., Interactions between propionate and
amino acid metabolism in isolated sheep hepatocytes, Br. J. Nutr., 65, 301–317, 1991.
1382_C05.fm Page 96 Tuesday, October 7, 2003 6:14 PM
1382_C06.fm Page 97 Tuesday, October 7, 2003 6:16 PM

chapter six

Contribution of amino acids


to ketogenesis
Milan Holecˇek
Charles University School of Medicine

Contents
Abbreviations.................................................................................................................................98
Introduction....................................................................................................................................98
6.1 General pathways of metabolism of ketone bodies .......................................................98
6.1.1 Synthesis of KB ........................................................................................................98
6.1.2 Utilization of ketone bodies.................................................................................100
6.2 Ketogenesis from amino acids .........................................................................................100
6.2.1 Contribution of amino acids to the total production of ketone bodies ..........101
6.2.2 Ketogenesis from leucine .....................................................................................101
6.2.2.1 Skeletal muscle ........................................................................................101
6.2.2.2 Adipose tissue .........................................................................................102
6.2.2.3 Liver ..........................................................................................................102
6.2.2.4 Brain ..........................................................................................................103
6.3 Physiology and pathophysiology of ketogenesis from amino acids.........................104
6.3.1 Ketogenesis from amino acids in hyperketonemic states...............................104
6.3.1.1 Starvation .................................................................................................104
6.3.1.2 Diabetes mellitus.....................................................................................105
6.3.2 Ketogenesis from amino acids in stress illness ................................................106
6.4 Conclusions .........................................................................................................................106
Acknowledgments ......................................................................................................................107
References .....................................................................................................................................107

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 97
1382_C06.fm Page 98 Tuesday, October 7, 2003 6:16 PM

98 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Abbreviations
BCAA branched-chain amino acids (valine, leucine, and isoleucine)
BCKA branched-chain keto acids
CoA coenzyme A
FFA free fatty acids
HMG-CoA b-hydroxy-b-methylglutaryl-CoA
KB ketone bodies
KIC a-ketoisocaproic acid
KIV a-ketoisovaleric acid
KMV a-keto-b-methylvaleric acid
TAG triacylglycerols

Introduction
Physiologically important ketone bodies (KB) are represented by acetoacetate and
b-hydroxybutyrate. Minute quantities of acetone generated in the body by spontaneous
decarboxylation of acetoacetic acid are not metabolized and are a byproduct released by
lungs and kidneys. KB are an important fuel for the brain, heart, skeletal muscle, intestine,
and kidney both in physiological and pathological conditions. Production and utilization
of KB is markedly activated in starvation, a high-fat diet, strenuous exercise, and uncon-
trolled diabetes, and during development. Apart from acting as an important energy
substrate, KB can provide acetyl-CoA for synthesis of lipids in several tissues and play a
regulatory role in the metabolism of other substrates. Hyperketonemia results in the
sparing of glucose oxidation by the brain due to cerebral utilization of KB and inhibits
lipolysis in adipose tissue. Several studies suggested an inhibitory effect of KB on protein
breakdown.1
Ketogenesis occurs mainly in the liver from long-chain fatty acids derived from adi-
pose tissue. Other potential endogenous precursors of KB are short-chain fatty acids (e.g.,
acetate and butyrate) formed by gut flora and ketogenic amino acids (phenylalanine,
tyrosine, tryptophan, isoleucine, leucine, and lysine). These compounds are believed to
make insignificant contributions. However, five of six ketogenic amino acids are indis-
pensable, and their activated catabolism in reactions of ketogenesis may significantly affect
protein metabolism and the whole status of the human or animal. Unfortunately, there is
little attention given to understanding the regulation and clinical importance of ketogen-
esis from amino acids, and the available data are scattered throughout a limited number
of original research papers.

6.1 General pathways of metabolism of ketone bodies


The blood concentration of KB is given by the difference in their release to the blood and
uptake by extrahepatic tissues. In overnight fasted humans and rats it is around 0.1 to
0.4 mmol/l. At this point, the pathways and factors controlling KB synthesis from fatty
acids and pathways of KB utilization will be briefly described (see also Figure 6.1). The
pathways in which ketogenic amino acids contribute to ketogenesis are discussed later.

6.1.1 Synthesis of KB
Ketogenesis from fatty acids in overnight fasted humans is about 0.2 to 0.4 mmol/min,2
occurs almost entirely in the liver, and is controlled at several sites.
1382_C06.fm Page 99 Tuesday, October 7, 2003 6:16 PM

Chapter six: Contribution of amino acids to ketogenesis 99

ADIPOSE TISSUE LIVER BRAIN, MUSCLE, KIDNEY,


HEART
TAG FFA FFA ATP
1 CoA
2

Acyl-CoA ATP

Acetyl-CoA Citric acid cycle


Acetyl-CoA
3
CoA

Acetoacetyl-CoA 2 Acetyl-CoA
Acetyl-CoA
4 3
CoA CoA
HMG-CoA Acetoacetyl-CoA
5 Succinate
Acetyl-CoA 7
Succinyl-CoA

Acetoacetate Acetoacetate
NADH+H + NADH+H +
6 6
NAD + NAD +
β-Hydroxybutyrate β-Hydroxybutyrate

Figure 6.1 Pathways of ketogenesis from fatty acids and ketone bodies utilization. TAG, triacyl-
glycerols; 1, hormone sensitive lipase (E.C.3.1.1.3); 2, acyl-CoA synthase (E.C.6.2.1.3); 3, acetoacetyl-
CoA thiolase (E.C.2.3.1.9.); 4, HMG-CoA synthase (E.C.4.1.3.5.); 5, HMG-CoA lyase (E.C.4.1.3.4.); 6,
b-hydroxybutyrate dehydrogenase (E.C.1.1.1.30); 7, 3-oxoacid-CoA-transferase (E.C.2.8.3.5).

Lipolysis and release of free fatty acids (FFA) from white adipose tissue to the blood
is the first step. The enzyme regulating lipolysis is hormone-sensitive lipase, which can
be activated by glucagon, epinephrine, norepinephrine, ACTH, thyroid-stimulating hor-
mone, growth hormone, and glucocorticoids. Insulin antagonizes the effect of lipolytic
hormones.
Fatty acids taken up by the liver in a concentration-dependent manner, after conver-
sion to the acyl-CoA, either are reesterified or enter the mitochondria via the carnitine
shuttle to be oxidized (second control point). In the majority of catabolic situations,
b-oxidation in mitochondrial matrix predominates. The rate of b-oxidation is regulated
through levels of fatty acyl-CoA, carnitine, and malonyl-CoA. Malonyl-CoA acts as a
potent inhibitor of carnitine acyltransferase I, the enzyme catalyzing the initial step in
fatty acid oxidation. As malonyl-CoA is an intermediate in synthesis of fatty acids, b-oxi-
dation is inhibited when fatty acid synthesis is active.
After b-oxidation, the resultant acetyl-CoA may be either converted to KB or con-
densed with oxaloacetate and enter the citric acid cycle (third control point). Ketogenesis
from acetyl-CoA occurs in mitochondria when the rate of supply of acetyl-CoA outstrips
the capacity for its oxidation by the citric acid cycle (e.g., increased lipolysis) and all
enzymes of pathway for ketogenesis are available (only in the liver). At the first step, two
molecules of acetyl-CoA condense (catalyzed by thiolase) to form one molecule of ace-
toacetyl-CoA (acetoacetyl-CoA also arises directly during the course of b-oxidation). Then
the ketogenesis pathway involves the condensation of acetoacetyl-CoA with acetyl-CoA
to b-hydroxy-b-methylglutaryl-CoA (HMG-CoA) catalyzed by HMG-CoA synthase. It
should be noted that the activity of HMG-CoA synthase is located in two different com-
partments, cytosol and mitochondria,and the genes for mitochondrial and cytosolic HMG-
CoA synthase are differently regulated.3,4 Control of ketogenesis is exerted by transcrip-
tional regulation of mitochondrial HMG-CoA synthase. Fasting, cAMP, and fatty acids
increase its transcriptional rate, while refeeding and insulin repress it.5 The HMG-CoA
produced in cytosol is the starting point of the isoprenoid pathway with cholesterol as
the main end product. HMG-CoA produced in mitochondria is split by HMG-CoA lyase
1382_C06.fm Page 100 Tuesday, October 7, 2003 6:16 PM

100 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

to acetoacetic acid and acetyl-CoA. Part of the acetoacetic acid is converted into b-hydrox-
ybutyric acid. The interconversion of acetoacetate and b-hydroxybutyrate is controlled by
the mitochondrial ratio of [NAD+] to [NADH]. The [NAD+]/[NADH] ratio may increase
as result of rapid b-oxidation.

6.1.2 Utilization of ketone bodies


Ketone body utilization occurs rapidly in most extrahepatic tissues, particularly in the
brain, heart, skeletal muscle, and kidneys. The main pathway involves activation of ace-
toacetate to acetoacetyl-CoA by 3-oxoacid-CoA-transferase, an enzyme that appears to be
present in sufficient quantities in all tissues except liver. Acetoacetyl-CoA is split to acetyl-
Co by thiolase. Acetyl-CoA is oxidized in the citric acid cycle (major fate) or used for
synthesis of lipids. Biosynthesis of lipids from KB has been demonstrated in the developing
brain, adipose tissue, and lactating mammary gland.
In the liver, in contrast to other tissues, all enzymes essential for synthesis of acetyl
CoA from KB, the reaction necessary for oxidation of KB, are not expressed. The alternative
possibility of utilization of KB in liver is synthesis of cholesterol in the cytosol. This
pathway is not very active, and most KB synthesized in the liver are released to the
bloodstream and utilized in other tissues.
It appears that the main factor influencing the rate of KB utilization in extrahepatic
tissues is their concentration in the blood. If the blood KB level rises, oxidation of KB
increases. The pathway of KB oxidation is saturated at a KB concentration of approxi-
mately 12 mmol/l.

6.2 Ketogenesis from amino acids


Ketogenic amino acids are those yielding either acetoacetate or one of its precursors, acetyl-
CoA or acetoacetyl-CoA (phenylalanine, tyrosine, tryptophan, isoleucine, leucine, and
lysine). Amino acids whose catabolism yields pyruvate or one of the intermediates of the
citric acid cycle are glucogenic. Some amino acids have both ketogenic and glucogenic
functions (phenylalanine, tyrosine, tryptophan, and isoleucine). The only amino acids that
are exclusively ketogenic are leucine and lysine, because they cannot function as carbon
sources for the net synthesis of glucose. The final step in catabolism of leucine is break-
down of HMG-CoA to acetoacetate and acetyl-CoA. This explains its strong ketogenic
effect. Several studies have demonstrated that ketogenesis from amino acids, particularly
from leucine and isoleucine, is not a specific feature of hepatic tissue and also occurs in
the brain, skeletal muscle, adipose tissue, kidneys, heart, and gut.
The first step in ketogenesis from amino acids is their transamination followed by
elimination of the a-amino group in the form of urea. Most of the aminotransferase activity
is located in the liver. However, branched-chain amino acid (BCAA) aminotransferase, the
enzyme regulating the initial step in catabolism of BCAA (valine, leucine, and isoleucine),
has the highest activity in skeletal muscle. Acute regulation of transamination occurs by
the concentration of substrate and therefore is dependent mainly on the net rate of protein
breakdown or protein intake by food. Chronic regulation is by changing the activities of
the enzymes concerned. This may be achieved by glucagon, glucocorticoids, or other
humoral factors. It is unique in the catabolism of lysine that neither of its amino groups
undergoes transamination as the first step in their catabolism.
After the a-amino group has been removed from the amino acid, the remaining carbon
skeleton is available for conversion to intermediates of energy metabolism. Control of the
breakdown of the C-skeleton of amino acids depends on the nature of a-ketoacid
produced.
1382_C06.fm Page 101 Tuesday, October 7, 2003 6:16 PM

Chapter six: Contribution of amino acids to ketogenesis 101

6.2.1 Contribution of amino acids to the total production of ketone bodies


Studies trying to estimate the contribution of ketogenic amino acids to the total KB pro-
duction are rare. Thomas et al.6 demonstrated that in rats starved for 3 h, 4.4% of KB carbon
is derived from the metabolism of leucine. The specific radioactivity of blood KB was four-
to fivefold higher after injection of labeled leucine than after injection of lysine or phenyl-
alanine. A similar result was obtained by Kulaylat et al.,7 who measured the hepatic
conversion of leucine to KB in overnight fasted dogs using continuous infusion of
L-U-[14C]-leucine and by determination of the appearance of [14C]-KB across the liver.
Authors calculated that hepatic conversion of leucine to KB accounted for 3.5% of net
hepatic production of KB.
It may be deduced from these studies that during the postprandial state (i.e., within
12 h of eating), the contribution of all ketogenic amino acids to ketogenesis is around 10%.
Considering the importance of ketogenic amino acids, particularly of leucine, in protein
economy, these amounts should not be considered negligible.

6.2.2 Ketogenesis from leucine


This chapter will mainly concentrate on leucine, as leucine is undoubtedly the most
significant ketogenic amino acid6 and because limited information about the contribution
of others exists. In an effort to elucidate the pathways of ketogenesis from leucine, we
should consider specific features in the metabolism of BCAA. The first step in BCAA
catabolism is reversible transamination leading to the production of corresponding
branched-chain keto acids (BCKA). Leucine is converted to a-ketoisocaproic acid (KIC),
isoleucine to a-keto-b-methylvaleric acid (KMV), and valine to a-ketoisovaleric acid (KIV).
The BCKA formed in this reaction then undergo irreversible decarboxylation catalyzed
by BCKA dehydrogenase to form thioesters of coenzyme A, and then through a series of
reactions is converted to acetoacetate and acetyl-CoA (leucine), propionyl-CoA and acetyl-
CoA (isoleucine), and succinyl-CoA (valine). In view of the importance of succinyl-CoA
in combustion of KB (see Figure 6.1), we can speculate about mutual relationships in
catabolism of valine (yielding succinyl-CoA) and KB generated from leucine and isoleu-
cine. This speculation is supported by the observation of Meguid et al.,8 who have shown
that an excess of leucine increases oxidation of valine both in vivo in humans and in isolated
epitrochlearis muscles from rats. Similarly, Block and Harper9 demonstrated that the
depression in plasma isoleucine and valine concentrations in rats fed by a high leucine
meal was associated with 50% increase in whole-body valine oxidation.
The activity of the first enzyme in BCAA metabolism, BCAA aminotransferase, is high
in muscle, heart, and kidney and very low in liver. The rate of transamination depends
primarily on the activity of the enzyme, and concentrations of substrates and products of
transamination. BCKA dehydrogenase is a multienzyme complex located in mitochondria
regulated by reversible phosphorylation (inactivation) and dephosphorylation (activa-
tion). Its activity is highest in the liver, while it is low in muscle, adipose tissue, and the
brain.10 Several studies provide strong evidence that these differences in BCKA dehydro-
genase activity are the basis of interorgan cooperation in BCAA metabolism. Therefore,
several tissues are involved and cooperate in ketogenesis from leucine.

6.2.2.1 Skeletal muscle


Due to a high activity of BCAA aminotransferase, the skeletal muscle has a remarkable
capability to transaminate BCAA to their ketoanalogues and is considered the initial site
of BCAA catabolism. BCAA are considered as essential donors of nitrogen in synthesis of
glutamine and alanine. Increased availability of BCAA enhances glutamine synthetase
1382_C06.fm Page 102 Tuesday, October 7, 2003 6:16 PM

102 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

activities and release of alanine and glutamine from muscle.11,12 It can be suggested that
increased protein breakdown and activated catabolism of BCAA in muscle in stress illness
such as sepsis, burn injury, and trauma enable the body to provide for its increased need
for glutamine and alanine.
As the BCKA dehydrogenase activity in skeletal muscle is low, a significant portion
of BCKA generated in BCAA aminotransferase reaction is released to the blood and then
metabolized in other tissues.10,13 Therefore, under physiological conditions, a limited
amount of BCAA is catabolized in muscle to KB. These are mostly oxidized to CO2 and
water, and only a smaller part is released to the blood.14,15 Nevertheless, these data clearly
demonstrate that skeletal muscle may be a peripheral site of ketogenesis from amino acids.
KIC released to the blood may be an important precursor of KB in other tissues, particu-
larly in the liver.

6.2.2.2 Adipose tissue


Adipose tissue is very active in utilization of BCAA, particularly in a well-nourished state.
The capability of adipose tissue to concentrate BCAA is considerably high, even higher
than that of skeletal muscle. Leucine concentration in intracellular water of adipose tissue
is fourfold, while in skeletal muscle it is only 25 to 50% greater than that in extracellular
fluid.16,17 Available evidence suggests that in adipose tissue the aminotransferase activity
exceeds maximal rates of leucine oxidation and that the BCKA dehydrogenase activity is
the rate-limiting step controlling leucine degradation.18 Therefore, KIC formed from leu-
cine may accumulate to such an extent that 15% leaks to the blood.19
Degradation of leucine in adipose tissue is stimulated by insulin20 and coupled to
production of alanine and glutamine, like in the muscle. Generated acetyl-CoA and aceto-
acetate can be oxidized for energy or used for the synthesis of long-chain fatty acids and
cholesterol.21 In fact, in fed animals, the products of leucine catabolism are utilized for
fatty acid synthesis, while in fasted animals they might play a role in the development of
ketoacidosis. The significant contribution of leucine to ketogenesis in adipose tissue also
clearly demonstrates an increased amount of acetoacetate released by epididymal adipose
tissue of mice after addition of leucine to the incubation medium.22

6.2.2.3 Liver
The main end product of catabolism of leucine in liver is acetoacetate, together with
smaller amounts of b-hydroxybutyrate and acetyl-CoA. From rates of 14CO2 production
from [1-14C]-KIC and [U-14C]-KIC by isolated hepatocytes, it has been shown that only
10% of flux of KIC through BCKA dehydrogenase was oxidized to CO2; the reminder was
released as acetoacetate.23
Low activity of hepatic BCAA aminotransferase and high activity of BCKA dehydro-
genase implicate the important role of BCKA, delivered mainly from skeletal muscle and
adipose tissue, in hepatic ketogenesis. The hypothesis that the main substrate for hepatic
ketogenesis from amino acids is BCKA, particularly KIC, delivered by the bloodstream is
supported by a number of studies. Livesey and Lund24 demonstrated that the liver can
extract a quantity of BCKA equivalent to that released by muscle. Spydevold and
Hokland 25 demonstrated using perfused liver of rat that KB were the main product
released when liver was provided with KIC (ketoleucine) or KMV (ketoisoleucine). Kulay-
lat et al.7 measured hepatic conversion of leucine to KB by continuous infusion of U-[14C]-
leucine and by determination of the appearance of [14C]-KB in dogs fasted for 3 days. The
results showed that the rate of production of [14C]-KB exceeded the rate of unidirectional
uptake of labeled leucine by the liver. This finding clearly indicates that some leucine
carbon converted to KB must have been derived from one of the metabolites of leucine,
1382_C06.fm Page 103 Tuesday, October 7, 2003 6:16 PM

Chapter six: Contribution of amino acids to ketogenesis 103

most likely KIC, produced in extrahepatic tissues. The crucial position of the liver in
ketogenesis from KIC is also demonstrated by the observations of Krebs and Lund,26 who
showed that KIC supported a rate of ketogenesis in isolated hepatocytes from 48-h starved
rats almost 100-fold higher than leucine in diaphragm muscle.
As BCAA aminotransferase reaction is reversible and its direction is determined by a
number of factors, a portion of BCKA delivered to liver from extrahepatic sources can be
reaminated to BCAA. Abumrad et al.27 demonstrated that after infusion of KIC into the
gut of postabsorptive dogs, 59% of the absorbed KIC was taken up by the liver and one-
third of this was transaminated to leucine. It can be suggested that partition of BCKA
between oxidation and reamination is an important regulatory mechanism supporting
either ketogenesis or resynthesis of leucine from KIC in the liver.

6.2.2.4 Brain
Undoubtedly, the brain is the tissue for which KB synthesized mostly in the liver and
delivered to the brain by the bloodstream are the most important energy fuel when there
is a lack of glucose, primarily in starvation. During prolonged fasting, more than half of
the brain’s energy supply is derived from KB. Recent findings show that leucine is an
important substrate of ketogenesis in astrocytes. KB produced by astrocytes might be used
as substrates for neuronal oxidative metabolism in situations of enhanced synaptic activity
and hypoxia.28 There are no data demonstrating the net release of KB from the brain.
It can be summarized that several tissues are involved in ketogenesis from leucine
and isoleucine. The interrelationships among muscle, liver, and adipose tissue are sche-
matically proposed in Figure 6.2. The principal source of KB synthesized from leucine and
released to the bloodstream is the liver. The main precursor seems to be the KIC delivered
to the liver mainly from skeletal muscle and adipose tissue. KB synthesized in the liver
are released to the blood due to the lack of enzymes, which enable conversion of KB to
acetyl-CoA. On the contrary, most of the KB produced in muscle or adipose tissue are
utilized in their place of origin, and only a smaller portion is released to the blood.

ADIPOSE TISSUE MUSCLE

Leu Leu PROTEINS Leu

KIC
KIC LIPIDS KIC

KB H2O + CO2 H2O + CO2 KB

Leu Leu
KIC KIC
KB KB
LIVER

Leu PROTEINS

KIC

KB H2O + CO2

KB

Figure 6.2 The cooperativity of skeletal muscle, adipose tissue, and liver in ketogenesis from leucine.
KIC, a-ketoisocaproic acid (ketoleucine); KB, ketone bodies.
1382_C06.fm Page 104 Tuesday, October 7, 2003 6:16 PM

104 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

6.3 Physiology and pathophysiology of ketogenesis from amino acids


As pathways of ketogenesis from fatty acids and amino acids are different and differently
regulated, it may be assumed that ketogenesis from amino acids has different physiological
importance than that from fatty acids. While ketogenesis from fatty acids is activated
mainly in starvation and inhibited in satiety, ketogenesis from leucine is stimulated by
food intake. Several studies indicate that adipose tissue of fed animals converts leucine
to acetoacetate at a higher rate than in fasted ones.18,22 Block et al.29 have demonstrated a
strong correlation between ambient leucine concentrations and the activation state of
skeletal muscle BCKA dehydrogenase in rats fed four levels of dietary protein.

6.3.1 Ketogenesis from amino acids in hyperketonemic states


Several studies clearly demonstrate that hyperketonemia may significantly affect the keto-
genesis not only from fatty acids but also from amino acids. Paul and Adibi30 demonstrated
an almost linear increase in the rate of leucine decarboxylation by gastrocnemius muscle
homogenate when acetoacetate in the incubation medium was varied from 1 to 20 mM,
while a decrease was observed in homogenates of liver. The inhibitory effect of KB on
degradation of leucine intermediates in rat liver mitochondria was also observed by
Landaas.31 In another study,15 KB inhibited alanine release and 14CO2 production from
[1-14C]-BCAA by rat hemidiaphragms. Inhibition of leucine oxidation was also observed
during infusion of lipids.32 Hyperketonemia also affects ketogenesis from amino acids by
changes in protein turnover and delivery of leucine into body fluids.33 This occurs
undoubtedly in starvation and diabetes mellitus, the conditions in which marked hyper-
ketonemia is a common finding.

6.3.1.1 Starvation
Response to starvation follows three phases. The initial one (about 3 days in man and
1 day in rats) is concerned with the maintenance of glucose production (gluconeogenic
phase). The second phase (3 days to 2 months in man and 1 to 5 days in rat) is one of
protein conservation in which metabolism switches away from glucose to fatty acid and
KB oxidation. The decisive amount of KB produced is undoubtedly from b-oxidation of
fatty acids and acetyl-CoA overproduction in the liver promoted by the lack of glucose,
rise in glucagon, and decrease in insulin levels. Rous et al.22 have also found release of
greater quantities of KB by adipose tissue of 24-h fasted mice than of fed mice. The third
phase (premortal) is characterized by accelerated protein breakdown because energy stores
are exhausted.
The results of studies evaluating the effect of starvation on ketogenesis from amino
acids are not uniform. Thomas et al.6 observed that in rats starving for 48 h, 2.3% of KB
carbon is derived from metabolism of leucine, while in animals starving only for 3 h, the
corresponding value is 4.4%. Considering that starvation for 2 days caused a fourfold
increase in total blood KB, the results indicate a net increase in conversion of leucine to
KB. The main source of KB derived from leucine is probably the liver. Kulaylat et al.7
demonstrated that contribution of leucine to hepatic ketogenesis in dogs increases from
3.5% in overnight fasted animals to 10% in those fasted for 3 days. However, the changes
in leucine or KIC oxidation in isolated perfused liver of rats starving for 3 days are not
significant.34
The quantity of KB formed from leucine during fasting in adipose tissue is inhibited.
The amount of actetoacetate and 14CO2 released by adipose tissue of fasted mice incubated
in buffer containing [1-14C]-leucine was lower than that of fed mice.22 In epididymal fat
1382_C06.fm Page 105 Tuesday, October 7, 2003 6:16 PM

Chapter six: Contribution of amino acids to ketogenesis 105

pads of rats fasted for 44 to 48 h, both the transamination of leucine and its subsequent
decarboxylation decreased.18
The effect of starvation on leucine conversion to KB in skeletal muscle was examined
by Palmer et al.,35 who demonstrated after addition of leucine to hemidiaphragms of 40-h
starved rats a higher release of KB than that from hemidiaphragms of fed rats. The
increased rates of KB release in the starved state were not a consequence of increased
leucine catabolism, as the rates of 14CO2 production from [U-14C]-leucine and [1-14C]-
leucine were lower in hemidiaphragms from starved than from fed rats. These results
indicate that leucine-stimulated production of KB in skeletal muscle of starved rats is
predominantly due to incomplete oxidation of leucine. This hypothesis is in agreement
with a marked decrease in BCKA dehydrogenase activity, indicating decreased leucine
oxidation, in gastrocnemius muscle and the heart of rats starving for 2 and 4 days, while
an increase was observed on the sixth day of starvation.36 Wagenmakers et al.37 demon-
strated that starvation decreased the activity state of BCKA dehydrogenase in the quad-
riceps muscle, although actual activity did not change. This finding is in agreement with
our observation of the gradual increase in the amount of BCKA dehydrogenase in skeletal
muscle and heart during starvation, which indicates an increasing capacity of these tissues
to oxidize leucine.36 In another study, starvation for 4 days increased the rate of leucine
decarboxylation by homogenates of skeletal muscle but was without effect on the rate of
leucine decarboxylation by the kidney and liver homogenates.30
It is not easy to summarize these contradictory reports and provide a reasonable
conclusion. It is clear that increased production of KB due to fatty acid oxidation in the
protein conservation phase of starvation is not accompanied by a corresponding increase
in ketogenesis from amino acids. It appears that enhanced ketogenesis from leucine during
starvation is more related to decreased utilization of KB in liver and muscle than to
increased catabolism of leucine. Leucine oxidation is activated in the premortal phase of
starvation.

6.3.1.2 Diabetes mellitus


Several factors contribute to diabetic hyperketonemia. The lack of insulin and excess in
anti-insulin hormones cause the inability of muscle and adipose tissue to take up glucose
from the blood, stimulating lipolysis and massive efflux of free fatty acids from adipose
tissue to the liver. These hormonal changes also enhance the ketogenic capacity of the
liver, and a higher proportion of acetyl-CoA produced in b-oxidation is converted into
KB. The hyperketonemia is amplified by limitation in the ability of extrahepatic tissue to
remove KB from the blood.
Ketogenesis from amino acids, at least from leucine, contributes significantly to hyper-
ketonemia in diabetes. In dogs fasted for 3 days with selective insulin deficiency induced
by a peripheral intravenous infusion of somatostatin and intraportal glucagon, the con-
tribution of leucine carbon to hepatic production of KB increased from 10 to 15%.7 This
finding is in agreement with observations of elevated activity of BCKA dehydrogenase in
skeletal muscle,30,38,39 kidney,30 and liver40,41 of diabetic rats. Further studies showed that
diabetes induced by streptozotocin treatment increased BCKA dehydrogenase activity in
skeletal muscle by inhibiting specific kinase.42 The elevated rates of KIC oxidation in
hindquarters of diabetic rats were depressed by the addition of acetoacetate to the perfu-
sion medium.43
The observations obtained in animals are in good agreement with observations in
insulin-dependent diabetics. In type I (insulin-dependent) diabetics, increased turnover
and oxidation rates of leucine that decreased with insulin therapy were reported.44–46
However, in type II diabetes, the values of leucine oxidation were not different from those
1382_C06.fm Page 106 Tuesday, October 7, 2003 6:16 PM

106 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

of nondiabetic controls.47 One possible explanation is the difference in plasma insulin


concentration and insulin resistance.

6.3.2 Ketogenesis from amino acids in stress illness


Despite the importance of KB as an alternative fuel, several studies demonstrated signif-
icant depression of KB levels in severe illness, particularly in sepsis.48,49 In addition, KB
levels failed to rise in trauma patients during carbohydrate restriction.49 The inhibition of
ketogenesis from fatty acids in catabolic states is probably related to elevated insulin levels
due to increased production of catabolic hormones (cortisol, glucagon, and catechola-
mines) and to the metabolic response mediated by cytokines, particularly tumor necrosis
factor-a (TNF-a).50,51
Several studies evaluating changes in whole-body leucine and protein metabolism in
metabolic stress states, e.g., trauma and sepsis, or after administration of glucocorticoids
or pro-inflammatory cytokines demonstrated an increased rate in whole-body oxidation
of leucine.52,53 However, the stimulatory effect of cytokines on whole-body leucine oxida-
tion was not observed by others.54
It appears that in stress illness most of the leucine is completely oxidized in skeletal
muscle. Enhanced rates of leucine oxidation and BCKA dehydrogenase activity in skeletal
muscle showed endotoxins, cytokines, glucocorticoids, and acidosis.29,55–57 Unfortunately,
there are no studies evaluating the proportions of complete (to CO2 and water) and
incomplete (to KB) oxidation of leucine in muscle in stress conditions.
Most studies devoted to metabolism of ketogenic amino acids in the liver indicate
decreased ketogenesis, particularly from leucine. Pailla et al.58 demonstrated using isolated
rat hepatocytes that both TNF-a and interleukin (IL)-6 inhibit KB synthesis from KIC. In
further experiments, they demonstrated that inhibitory action of the cytokines on ketoge-
nesis is not related to changes in nitric oxide production or protein synthesis.51 Using
isolated perfused rat liver, we demonstrated that administration of endotoxin or TNF-a
decreases the flux of KIC through the BCKA dehydrogenase.56,59 Similar results were also
observed when endotoxin or TNF-a was added in the perfusion medium.53 These changes,
which also indicate an increased capacity of hepatic tissue to reaminate BCKA to BCAA,
were confirmed using an isolated perfused rat liver technique by adding KIC (leucine
precursor) to the perfusion medium. In effluent of hepatic tissue of endotoxemic rats, a
significantly higher leucine concentration was detected than in controls.59As severe illness
is commonly associated with anorexia, the activated resynthesis of BCAA in hepatic tissue
should be considered an important adaptive response of the body that can resupply
essential BCAA and prevent the rapid development of negative nitrogen balance.60 Sig-
nificant decrease in flux of KIC through hepatic BCKA dehydrogenase and decreased
b-hydroxybutyrate production from KIC were also observed in rats with cirrhosis.60–62

6.4 Conclusions
There are apparent species differences in BCAA metabolism, and the results obtained
mostly from laboratory animals should be carefully examined if valid also for humans.
The most important difference between humans and rats in BCAA catabolism is the lower
activity of hepatic BCKA dehydrogenase in humans.63 However, considering that the
capability of the liver to utilize KB is low in both humans and animals and that the majority
of studies evaluating changes in whole-body leucine metabolism in humans or dogs are
in good agreement with results obtained using small laboratory animals, the described
pathways of ketogenesis from leucine should also be valid for humans.
1382_C06.fm Page 107 Tuesday, October 7, 2003 6:16 PM

Chapter six: Contribution of amino acids to ketogenesis 107

The results of several laboratories indicate that there is a significant potential for
formation of KB from ketogenic amino acids, at least from leucine. The ketogenesis from
lysine, tyrosine, phenylalanine, and tryptophane occurs mainly in the liver, whereas keto-
genesis from isoleucine and leucine occurs partly in extrahepatic tissues and partly in the
liver. As the contribution of individual tissues to ketonemia is determined not only by the
rate of KB production, but also by the presence or absence of key enzymes of KB utilization,
the contribution of KB synthesized in extrahepatic tissues is low. The liver should be
considered the most important source of blood KB derived from amino acids because of
the high activity of BCKA dehydrogenase (the key enzyme in BCAA catabolism), delivery
of significant amounts of KIC from extrahepatic tissues, and incomplete set of enzymes
for oxidation of KB.
Increased ketogenesis from amino acids in starvation is caused by incomplete oxida-
tion of leucine in muscle and liver rather than by its increased catabolism. The exact
reasons for this increase in the ratio of leucine conversion to KB and leucine oxidation is
not well defined. In insulin-dependent diabetes, the higher rate of ketogenesis from leucine
is associated with its increased oxidation. In stress conditions, activated leucine utilization
in skeletal muscle is not coupled with increased KB release to the blood. Decreased
ketogenesis from leucine in liver is related to enhanced resynthesis of BCAA from BCKA.

Acknowledgments
The author gratefully acknowledges the support of the Grant Agency of the Czech Repub-
lic (grants 306/94/1873, 306/98/0046, and 305/01/0578), the Internal Grant Agency of
Charles University (grants 152/95 and 276/98C), and the Internal Grant Agency of Min-
istry of Health of the Czech Republic (grants 3772-3 and 6793-3).

References
1. Sherwin, R., Hendler, R., and Felig, P., Effect of ketone infusions on amino acid and nitrogen
metabolism in man, J. Clin. Invest., 55, 1382–1390, 1975.
2. Balasse, E.E. and Féry, F., Ketone body production and disposal: effect of fasting, diabetes,
and exercise, Diabetes Metab. Rev., 5, 247–270, 1989.
3. Gil, G., Goldstein, J.L., and Brown, M.S., Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme
A synthase from the hamster. II. Isolation of the gene and characterization of the 5' flanking
region, J. Biol. Chem., 26, 3717–3724, 1986.
4. Gil-Gómez, G., Ayté, J., and Hegardt, F.G., The rat mitochondrial 3-hydroxy-3-methylglutar-
yl-CoA synthase gene contains elements that mediate its multihormonal regulation and
tissue specificity, Eur. J. Biochem., 213, 773–779, 1993.
5. Hegardt, F.G., Transcriptional regulation of mitochondrial HMG-CoA synthase in the control
of ketogenesis, Biochimie, 80, 803–806, 1998.
6. Thomas, L.K., Ittmann, M., and Cooper, C., The role of leucine in ketogenesis in starved rats,
Biochem. J., 204, 399–403, 1982.
7. Kulaylat, M.N., Frexes-Steed, M., Geer, R., Williams, P.E., and Abumrad, N.N., The role of
leucine in hepatic ketogenesis, Surgery, 103, 351–360, 1988.
8. Meguid, M.M., Schwarz, H., Matthews, D.E., Karl, I.E., Young, V.R., and Bier, D.M., In vivo
and in vitro branched-chain amino acid interactions, in Amino Acids: Metabolism and Medical
Applications, Blackburn, G.L., Grant, J.P., and Young, V.R., Eds., John Wright, Boston, 1983,
pp. 147–154.
9. Block, K.P. and Harper, A.E., Valine metabolism in vivo: effects of high dietary levels of
leucine and isoleucine, Metabolism, 33, 559–566, 1984.
10. Harper, A.E., Miller, R.H., and Block, K.P., Branched-chain amino acid metabolism, Annu.
Rev. Nutr., 4, 409–454, 1984.
1382_C06.fm Page 108 Tuesday, October 7, 2003 6:16 PM

108 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

11. King, P.A., Goldstein, L., and Newsholme, E.A., Glutamine synthetase activity of muscle in
acidosis, Biochem. J., 216, 523–525, 1983.
12. Elia, M. and Livesey, G., Effects of ingested steak and infused leucine on forelimb metabolism
in man and the fate of the carbon skeletons and amino groups of branched-chain amino
acids, Clin. Sci., 64, 517–526, 1983.
13. Harkema, J.M., Gorman, M.W., Bieber, L.L., and Chaudry, I.H., Metabolic interaction between
skeletal muscle and liver during bacteremia, Arch. Surg., 123, 1415–1419, 1988.
14. Odessey, R. and Goldberg, A.L., Oxidation of leucine by rat skeletal muscle, Am. J. Physiol.,
223, 1376–1383, 1972.
15. Palmer, T.T., Caldecourt, M.A., Warner, J.P., and Sugden, M.C., Modulation of branched-
chain amino acid oxidation in rat hemidiaphragms in vitro by glucose and ketone bodies,
Biochem. Int., 11, 407–413, 1985.
16. Hutson, S.M., Zapalowski, C., Cree, T.C., and Harper, A.E., Regulation of leucine and
a-ketoisocaproic acid metabolism in skeletal muscle: effect of starvation and insulin, J. Biol.
Chem., 255, 2418–2426, 1980.
17. Minemura, T., Lacy, W.W., and Crofford, O.B., Regulation of the transport and metabolism
of amino acids in isolated fat cells, J. Biol. Chem., 245, 3872–3881, 1970.
18. Tischler, M.E. and Goldberg, A.L., Leucine catabolism and production of glutamine and
alanine by adipose tissue, in Metabolism and Clinical Implications of Branched Chain Amino and
Ketoacids, Walser, M. and Williamson, J.R., Eds., Elsevier/North Holland, New York, 1981,
pp. 283–288.
19. Tischler, M.E. and Goldberg, A.L., Leucine degradation and release of glutamine and alanine
by adipose tissue, J. Biol. Chem., 255, 8074–8081, 1980.
20. Rosenthal, J., Angel, A., and Farkas, J., Metabolic fate of leucine: a significant sterol precursor
in adipose tissue and muscle, Am. J. Physiol., 226, 411–418, 1974.
21. Meikle, A.W. and Klain, G.J., Effect of fasting and fasting-refeeding on conversion of leucine
into CO2 and lipids in rats, Am. J. Physiol., 222, 1246–1250, 1972.
22. Rous, S., Bas, S., and Sengupta, S., Contribution of leucine in the fatty acid synthesis and
ketogenesis in mice adipose tissue, Int. J. Biochem., 11, 337–340, 1980.
23. Corkey, B., Martin-Requero A., Walajtys-Rode, E., Williams, R.J., and Williamson, J.R., Reg-
ulation of the branched chain a-ketoacid pathway in liver, J. Biol. Chem., 257, 9668–9676, 1982.
24. Livesey, G. and Lund, P., Enzymic determination of branched-chain amino acids and 2-oxo-
acids in rat tissues, Biochem. J., 188, 705–713, 1980.
25. Spydevold, O. and Hokland, B., Release of leucine and isoleucine metabolites by perfused
skeletal muscle and liver of rat, Int. J. Biochem., 15, 985–990, 1983.
26. Krebs, H.A. and Lund, P., Aspects of the regulation of the metabolism of branched-chain
amino acids, Adv. Enzyme Regul., 15, 375–394, 1976.
27. Abumrad, N.N., Robinson, R.P., Gooch, B.R., and Lacy, W.W., The effect of leucine infusion
on the substrate flux across the human forearm, J. Surg. Res., 32, 453–463, 1982.
28. Guzman, M. and Blazquez, C., Is there an astrocyte-neuron ketone body shuttle? Trends
Endocrinol. Metab., 12, 169–173, 2001.
29. Block, K.P., Aftring, R.P., Mehard, W.B., and Buse M.G., Modulation of rat skeletal muscle
branched-chain a-keto acid dehydrogenase in vivo: effects of dietary protein and meal
consumption, J. Clin. Invest., 79, 1349–1358, 1987.
30. Paul, H.S. and Adibi, S.A., Leucine oxidation in diabetes and starvation, effects of ketone
bodies on branched-chain amino acid oxidation in vitro, Metabolism, 27, 185–200, 1978.
31. Landaas, S., Inhibition of branched-chain amino acid degradation by ketone bodies, Scand.
J. Clin. Lab. Invest., 37, 411–418, 1977.
32. Beaufrère, B., Tessari, P., Cattalini, M., Miles, J., and Haymond, M.W., Apparent decreased
oxidation and turnover of leucine during infusion of medium-chain triglycerides, Am. J.
Physiol., 249, E175–E182, 1985.
33. Crowe, P.J., Royle, G.T., Wagner, D., and Burke, J.F., Does hyperketonemia affect protein of
glucose kinetics in postabsorptive or traumatized man? J. Surg. Res., 47, 313–318, 1989.
34. Holecek,
ˇ M., Šprongl, L., and Tilser,
ˇ I., Metabolism of branched-chain amino acids in starved
rats: the role of hepatic tissue, Physiol. Res., 50, 25–33, 2001.
1382_C06.fm Page 109 Tuesday, October 7, 2003 6:16 PM

Chapter six: Contribution of amino acids to ketogenesis 109

35. Palmer, T.N., Gossain, S., and Sugden, M.C., Partial oxidation of leucine in skeletal muscle,
Biochem. Mol. Biol. Int., 29, 255–262, 1993.
36. Holecek,
ˇ M., Effect of starvation on branched-chain a-keto acid dehydrogenase activity in
rat heart and skeletal muscle, Physiol. Res., 5, 19–24, 2001.
37. Wagenmakers, A.J., Schepens J.T., and Veerkamp J.H., Effect of starvation and exercise on
actual activity of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues,
Biochem. J., 223, 815–821, 1984.
38. Buse, M.G., Herlong, H.F., and Weigand, D.A., The effect of diabetes, insulin and the redox
potential on leucine metabolism by isolated rat hemidiaphragm, Endocrinology, 98, 1166–1175,
1976.
39. Aftring, R.P., Miller, W.J., and Buse, M.G., Effects of diabetes and starvation on skeletal muscle
branched-chain a-keto acid dehydrogenase activity, Am. J. Physiol., 254, E292–E300, 1988.
40. May, M.E., Mancusi, J.J., Aftring, R.P., and Buse, M.G., Effects of diabetes on oxidative
decarboxylation of branched-chain keto acids, Am. J. Physiol., 239, E215–E222, 1980.
41. Xu, M., Nakai, N., Ishigure, K., Nonami, T., Nagasaki, M., Obayashi, M., Li, Z., Sato, Y.,
Fujitsuka, N., Murakami, T., and Shimomura, Y., The a-ketoisocaproate catabolism in human
and rat livers, Biochem. Biophys. Res. Commun., 276, 1080–1084, 2000.
42. Holecek,
ˇ M., Paul, H.S., and Adibi, S.A., Activation of muscle branched-chain keto acid
dehydrogenase (BCKAD) in diabetic and tumor necrosis factor treated rats, Clin. Nutr., 11
(Special Suppl.), 84–85, 1992.
43. Zapalowski, C., Hutson, S.M., and Harper, A.E., Effects of starvation and diabetes on leucine
and valine metabolism in the perfused rat hindquarter, in Metabolism and Clinical Implications
of Branched Chain Amino and Ketoacids, Walser, M. and Williamson, J.R., Eds., Elsevier/North
Holland, New York, 1981, pp. 239–244.
44. Nair, K.S., Ford G.C., and Halliday, D., Effect of intravenous insulin treatment on in vivo
whole body leucine kinetics and oxygen consumption in insulin-deprived type I diabetic
patients, Metabolism, 36, 491–495, 1987.
45. Umpleby, A.M., Boroujerdi, M.A., Brown, P.M., Carson, E.R., and Sonksen, P.H., The effect
of metabolic control on leucine metabolism in type I (insulin-dependent) diabetic patients,
Diabetologia, 29, 131–141, 1986.
46. Inchiostro, S., Biolo, G., Bruttomesso, D., Fongher, C., Sabadin, L., Carlini, M., Duner, E.,
Tiengo, A., and Tessari, P., Effects of insulin and amino acid infusion on leucine and phenyl-
alanine kinetics in type 1 diabetes, Am. J. Physiol., 262, E203–E210, 1992.
47. Staten, M.A., Matthews, D.E., and Bier, D.M., Leucine metabolism in type II diabetes mellitus,
Diabetes, 35, 1249–1253, 1986.
48. O’Donnell, T.F., Clowe, G.H.A., Blackburn, G.L., Ryan, P.D., Benotti, P.N., and Miller, J.D.B.,
Proteolysis associated with a deficit of peripheral energy fuel substrates in septic man,
Surgery, 80, 192–200, 1976.
49. Birkhahn, R.H., Long, C.L., Fitkin, D.L., Busnardo, A.C., Geiger, J.W., and Blakemore, W.S.,
A comparison of the effects of skeletal trauma and surgery on the ketosis of starvation in
man, J. Trauma, 21, 513–519, 1981.
50. De Bandt, J.P., Lim, S.K., Plassart, F., Lucas, C.C., Rey, C., Poupon, R., Gibodeau, J., and
Cynober, L., Independent and combined actions of interleukin-1b, tumor necrosis factor a,
and glucagons on amino acid metabolism in the isolated perfused rat liver, Metabolism, 43,
822–829, 1994.
51. Pailla, K., El-Mir, M.Y., Cynober, L., and Blonde-Cynober, F., Cytokine-mediated inhibition
of ketogenesis is unrelated to nitric oxide or protein synthesis, Clin. Nutr., 20, 313–317, 2001.
52. Garcia-Martinez, C., Lloyera, M., Lopez-Soriano, F.J., delSanto, B., and Argiles, J.M.,
Lipopolysaccharide (LPS) increases the in vivo oxidation of branched-chain amino acids in
the rat: a cytokine-mediated effect, Mol. Cell. Biochem., 148, 9–15, 1995.
53. Holecek,
ˇ M., Šprongl, L., Skopec, F., Andrys,
´ C., and Pecka, M., Leucine metabolism in TNF-
a- and endotoxin-treated rats: contribution of hepatic tissue, Am. J. Physiol., 273, E1052–E1058,
1997.
1382_C06.fm Page 110 Tuesday, October 7, 2003 6:16 PM

110 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

54. Flores, E.A., Bistrian, B.R., Pomposelli, J.J., Dinarello, C.A., Blackburn, G.L., and Istfan, NW.,
Infusion of tumor necrosis factor/cachectin promotes muscle catabolism in the rat: a syner-
gistic effect with interleukin 1, J. Clin. Invest., 83, 1614–1622, 1989.
55. Nawabi, M.D., Block, K.P., Chakrabarti, M.C., and Buse, M.G., Administration of endotoxin,
tumor necrosis factor, or interleukin 1 to rats activates skeletal muscle branched-chain a-keto
acid dehydrogenase, J. Clin. Invest., 85, 256–263, 1990.
56. Holecek,
ˇ M., Leucine metabolism in fasted and tumor necrosis factor-treated rats, Clin. Nutr.,
15, 91–93, 1996.
57. England, B.K., Greiber, S., Mitch, W.E., Bowers, B.A., Herring, W.J., McKean, M., Ebb, R.G.,
Price, S.R., and Danner, D.J., Rat muscle branched-chain ketoacid dehydrogenase activity
and mRNAs increase with extracellular acidemia, Am. J. Physiol., 268, C1395–C1400, 1995.
58. Pailla, K., Lim, S.K., De Bandt, J.P., Aussel, C., Giboudeau, J., Troupel, S., Cynober, L., and
Blonde-Cynober, F., TNF-a and IL-6 synergistically inhibit ketogenesis from fatty acids and
a-ketoisocaproate in isolated rat hepatocytes, J. Parenter. Enteral Nutr., 22, 286–290, 1998.
59. Holecek,
ˇ M., Šprongl, L., Tichy,
´ M., and Pecka, M., Leucine metabolism in rat liver after a
bolus injection of endotoxin, Metabolism, 47, 681–685, 1998.
60. Holecek,
ˇ M., Relation between glutamine, branched-chain amino acids, and protein metab-
olism, Nutrition, 18, 130–133, 2002.
61. Blonde-Cynober, F., Plassart, F., De Bandt, J.P., Rey, C., Lim, S.K., Moukarbel, N., Ballet, F.,
Poupon, R., Giboudeau, J., and Cynober, L., Metabolism of a-ketoisocaproic acid in isolated
perfused liver of cirrhotic rats, Am. J. Physiol., 268, E298–E304, 1995.
62. Holecek,
ˇ M., Tilser,
ˇ I., Skopec, F., and Šprongl, L., Leucine metabolism in cirrhotic rats,
J. Hepatol., 24, 209–216, 1996.
63. Khatra, B.S., Chawla, R.K., Sevell, C.W., and Rudman, D., Distribution of branched-chain
a-keto acid dehydrogenase in primate tissues, J. Clin. Invest., 59, 558–564, 1977.
1382_C07.fm Page 111 Tuesday, October 7, 2003 6:18 PM

chapter seven

Ureagenesis and ammoniagenesis:


an update
Alfred J. Meijer
Academic Medical Center, Amsterdam

Contents
Introduction.................................................................................................................................. 111
7.1 Control mechanisms ..........................................................................................................112
7.1.1 Regulation of carbamoyl-phosphate synthase..................................................112
7.1.2 Transport of ornithine, citrulline, and aspartate across the
mitochondrial membrane.....................................................................................114
7.1.3 Urea synthesis and pH homeostasis ..................................................................115
7.1.4 The periportal/pericentral glutamine cycle......................................................115
7.1.5 Metabolite channeling and urea synthesis........................................................116
7.1.6 Interaction of fatty acids with amino acid metabolism ..................................117
7.1.7 Interaction of glucose with urea synthesis........................................................117
7.1.8 Cell hydration and urea synthesis......................................................................118
7.2 Conclusions .........................................................................................................................118
References .....................................................................................................................................119

Introduction
The synthesis of urea occurs by a complex pathway that is localized both in the mitochon-
dria and in the cytosol of the liver cell (Figure 7.1). Synthesis of citrulline from NH4+,
HCO3–, ATP, and ornithine, catalyzed by carbamoyl-phosphate synthase and ornithine
carbamoyltransferase, is a mitochondrial process, whereas synthesis of urea from ATP,
citrulline, and aspartate via argininosuccinate synthase, argininosuccinate lyase, and argi-
nase I is a cytosolic process. Because of this dual localization, the transport of ornithine
into the mitochondria and the efflux of citrulline, aspartate, and ATP from the mitochon-
dria are essential steps in the pathway. Moreover, N-acetylglutamate, the essential activator
of carbamoyl-phosphate synthase, is synthesized in the mitochondria, whereas the com-
pound is degraded in the cytosol, which makes mitochondrial N-acetylglutamate efflux
another essential step in ureagenesis.

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 111
1382_C07.fm Page 112 Tuesday, October 7, 2003 6:18 PM

112 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

cytosol mitochondrion
arg
acetate
+
glu AGA ? AGA glu
acetylCoA

UREA orn orn


+ 2ATP
HCO3-
CP NH3

arg

fum 1
as

AMP ATP Pi
cit cit

asp asp
NH3
2

glu glu αOG


NAD(P)+ NAD(P)H + H+
3
amino acids amino acids

Figure 7.1 Urea synthesis. AGA, N-acetylglutamate; glu, glutamate; asp, aspartate; CP, carbamoyl
phosphate; orn, ornithine; cit, citrulline; as, argininosuccinate; aOG, a-oxoglutarate; 1, ornithine/cit-
rulline translocator; 2, glutamate/aspartate translocator; 3, glutamate translocator. The putative
translocator for N-acetylglutamate is indicated by the question mark.

The regulation of urea synthesis has been reviewed extensively in the past.1–3 In the
previous volume of this series we discussed some features of the short-term regulation of
urea synthesis4; in the present chapter, only recent developments in this field are high-
lighted. For the transcriptional regulation of the enzymes participating in urea synthesis,
refer to recent reviews,2,3 and for a discussion of disorders in urea synthesis, refer to
Brusilow and Horwich.2

7.1 Control mechanisms


7.1.1 Regulation of carbamoyl-phosphate synthase
We previously pointed out repeatedly1,4 that discussions on the factors controlling flux
through the ornithine cycle should take into account that neither amino acid degradation
nor carbamoyl-phosphate synthase is sensitive to inhibition by their products, ammonia
and carbamoyl-phosphate, respectively. Thus, at constant substrate supply, any change in
the activity of one of the enzymes of the ornithine cycle, any change in N-acetylglutamate
synthase, or any change in the mitochondrial transport steps will influence neither orni-
thine cycle flux nor amino acid degradation. Such changes will only affect the steady-state
concentration of ammonia and of carbamoyl phosphate in the mitochondrial matrix. This
statement is best illustrated by the accumulation of ammonia in severe deficiencies of the
enzymes of the ornithine cycle and by the production of orotic acid when the deficiency
is distal to that of carbamoyl-phosphate synthase.1,2
Because of the lack of product inhibition by ammonia of amino acid degradation, we
previously argued that in vivo flux through the ornithine cycle is entirely controlled by
1382_C07.fm Page 113 Wednesday, October 8, 2003 1:13 PM

Chapter seven: Ureagenesis and ammoniagenesis: an update 113

the rate of amino acid degradation.1 The capacity of the enzymes of the ornithine cycle is
more than sufficient to handle the amount of ammonia formed by amino acid degradation
in vivo under normal conditions. Perhaps the best evidence supporting this statement is
the fact that urea synthesis rates were near normal in asymptomatic carriers with ornithine
carbamoyltransferase (OCT) deficiency with an enzyme activity below normal.5 Another
illustration can be found in a recent study from the same group with spf mutant mice, in
which the activity of OCT in the liver was only 5% of that of control mice: a relatively
small increase in OCT activity by adenovirus-mediated gene transfer was sufficient for
complete correction of the defect in urea synthesis in the mutant mice.6
In the regulation of carbamoyl-phosphate synthase activity, N-acetylglutamate is of
crucial importance. As we indicated in the past, the intramitochondrial synthesis of
N-acetylglutamate from glutamate and acetylCoA is part of a mechanism to buffer the
intramitochondrial (and thus the intrahepatic) concentration of ammonia.1,4,7 When the
amino acid supply to the liver increases, flux through carbamoyl-phosphate synthase
increases because of two reasons. First, there is an increase in portal ammonia (derived
from amino acid breakdown in enterocytes, glutamine in particular8,9) and also in the rate
of intrahepatic production of ammonia (formed either by deamidation of some amino
acids or by glutamate dehydrogenase). Second, there is an increase in synthesis of N-acetyl-
glutamate. Thus, not only is there an increase in substrate (i.e., ammonia) supply but
simultaneously there is an increase in the number of active, already existing carbamoyl-
phosphate synthase molecules. This results in a sigmoidal relationship between the con-
centration of ammonia and the rate of urea synthesis and allows large changes in ornithine
cycle flux at relatively constant ammonia concentrations.7 Such a mechanism makes perfect
sense, and it is surprising that in a recent review this mechanism was not considered and
a role of N-acetylglutamate in the short-term regulation of urea synthesis was even ques-
tioned.3 Perhaps some of the confusion is caused by the fact that intrahepatic N-acetyl-
glutamate levels do not necessarily reflect the intramitochondrial concentration of this
compound. It is also important to stress that the concentration of carbamoyl-phosphate
synthase in the mitochondrial matrix is extremely high and similar to that of N-acetyl-
glutamate in the mitochondrial matrix, so that deviations from normal Michaelis–Menten
kinetics may be expected.1
Recently, the gene of the mouse and human liver N-acetylglutamate synthase was
cloned.10,11 As expected, the gene is highly expressed in the liver and small intestine; in
the latter tissue, the enzyme plays an important role in the synthesis of citrulline from
glutamine, which, after entry into the bloodstream, is subsequently converted into arginine
by the kidneys.1–3,8 The protein contains a putative mitochondrial target sequence at its
aminoterminus,10 and its activity is stimulated by arginine,10,11 as predicted by Kawamoto
and coworkers.12 Even though the capacity of arginase in the liver is extremely high,
changes in its activity influence the cytosolic concentration of arginine and, assuming that
these changes are also reflected in the mitochondria,1 directly affect the synthesis of
N-acetylglutamate and thus the steady-state concentration of ammonia.
As indicated in the Introduction, degradation of N-acetylglutamate occurs in the
cytosol. Whether regulation of N-acetylglutamate levels can also occur at the level of its
degradation is not known, but a recent paper13 suggests that this may be the case: it was
shown that the increased rate of urea production in hypothyroid rats was accompanied
by a rise in N-acetylglutamate in the liver, which was caused not only by increased
synthesis but also by decreased degradation of the compound. Decreased degradation
may be caused by decreased activity of the cytosolic deacylase or by decreased transport
of N-acetylglutamate from the mitochondria. Only in the latter case, this will result in
increased intramitochondrial N-acetylglutamate, because N-acetylglutamate transport is
unidirectional, out of the mitochondria only.14
1382_C07.fm Page 114 Tuesday, October 7, 2003 6:18 PM

114 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

7.1.2 Transport of ornithine, citrulline, and aspartate across the mitochondrial


membrane
Important progress has been made with the recent cloning of the genes encoding the
mitochondrial translocators for ornithine/citrulline, glutamate/aspartate, and glutamate.
The gene encoding the ornithine/citrulline translocator was cloned in 1999, and a
defect in this gene is the underlying cause for the so-called HHH (hyperornithinemia,
hyperammonemia, and homocitrullinemia) syndrome,15 as was postulated several years
ago.16–18 The transporter catalyzes an electroneutral exchange between cytosolic ornithine+
and mitochondrial citrulline plus one H+.19 This proton is produced in the ornithine car-
bamoyltransferase reaction,1 and its removal from the mitochondrial matrix by the orni-
thine/citrulline exchange presumably accounts for the well-known activation by ornithine
of flux through carbamoyl-phosphate synthase in intact but not broken mitochondria,
because acidification of the mitochondrial matrix would inhibit the production of HCO3–
by mitochondrial carbonic anhydrase.1 Such mitochondrial acidification is likely to occur
in HHH patients and presumably accounts for the morphological abnormalities of liver
mitochondria seen in this disorder.3
A second mitochondrial transport system that was cloned recently is the
glutamate/aspartate translocator.20,21 This transporter catalyzes the electrophoretic
exchange between glutamic acid (the protonated form) and aspartate; it is unidirectional,
out of the mitochondria, energy dependent, and driven by the mitochondrial proton
gradient.22
The hepatocyte contains both cytosolic and mitochondrial aspartate aminotransferase.
When the mitochondrial enzyme is used for aspartate synthesis, the efflux of aspartate
from the mitochondria becomes essential for urea synthesis.23 In principle, both transam-
ination pathways are available. However, when their relative contributions were studied
with 15NH4Cl (by measurement of the degree of labeling with 15N of the urea molecule),
they strongly depended on the experimental conditions.9,24–27
The protein responsible for aspartate transport in the liver is called citrin, and a
defect in this transporter is the underlying cause of adult-onset type II citrullinemia.20,21
The structure of citrin is similar to that of aralar1 encoded by another gene.28,29 While
citrin is found predominantly (albeit not exclusively) in the liver, aralar1 is found
mainly in the heart, skeletal muscle, brain, and pancreatic b-cells.20,28–31 This suggests
that citrin is mainly involved in liver-specific processes such as urea synthesis and
gluconeogenesis from lactate.21,31 On the other hand, aralar1 is involved in the operation
of the malate/aspartate shuttle responsible for the mitochondrial oxidation of glycolytic
NADH, which is of eminent importance for ATP production in muscle, brain, and
pancreatic b-cells.21,30 Interestingly, both citrin and aralar1 require Ca++ at the external
face of the mitochondrial inner membrane.21 This agrees entirely with the suggestion
made years ago that in hepatocytes transport of cytosolic-reducing equivalents to the
mitochondria via the malate–aspartate shuttle was stimulated by Ca++ at the level of
the glutamate/aspartate translocator.32
The third mitochondrial transport system, important for urea synthesis, which was
recently cloned, is the translocator for glutamate.33 This protein transports glutamate,
derived from cytosolic amino acid transaminations, to the mitochondria (Figure 7.1), in
association with a proton. There are two isoforms of this protein that differ in kinetic
properties: isoform 1 has a lower affinity for glutamate but a higher Vmax than isoform
2. Both forms are ubiquitously expressed in all tissues. Expression of the mRNA of
isoform 1 predominates in most tissues except in the brain, where both forms are equally
expressed.33
1382_C07.fm Page 115 Tuesday, October 7, 2003 6:18 PM

Chapter seven: Ureagenesis and ammoniagenesis: an update 115

7.1.3 Urea synthesis and pH homeostasis


Atkinson and colleagues34,35 were the first to focus attention on the fact that synthesis of
urea consumes equal amounts of NH4+ and HCO3– (up to an amount of about 1 mol each
in man per day, assuming a daily protein intake of 100 g), and that its function is to remove
not only NH4+ but also bicarbonate. NH4+ and bicarbonate ions arise from the positively
charged amino groups and the negatively charged carboxylate groups during amino acid
oxidation. Because no more than 5% of the daily bicarbonate production can be removed
by urinary excretion, it was proposed that ureagenesis must inevitably play an important
role in the regulation of pH homeostasis.34,35 Although this issue has been extremely
controversial and the idea was challenged by many (cf. Meijer et al.,1 Brusilow and Hor-
wich,2 and Häussinger36 for a discussion of this issue), the chemistry of urea synthesis
(i.e., consumption of equal amounts of ammonia and bicarbonate ions) cannot be denied.
There is general agreement that urea synthesis decreases in metabolic acidosis in order to
save bicarbonate ions for neutralization of the excess of protons, and several mechanisms
contribute to this decrease (see Meijer et al.1 and Meijer4 for reviews). Under these condi-
tions, NH4+ ions that are not converted into urea are excreted into the urine, with glutamine
as the nontoxic carrier of ammonia in the circulation. The glutamine may be produced by
the pericentral hepatocytes37 but can also be produced elsewhere in the body.1 The con-
troversy, however, is about the prediction that metabolic alkalosis is expected when urea
synthesis is compromised, e.g., in patients with cirrhosis or with inborn errors of urea
synthesis. However, this is not always found2,38 (contrast with Häussinger et al.39). Accord-
ing to Shangraw and Jahoor,38 the rate of urea production in man in vivo is only a few
percent of the total bicarbonate flux, and a change in urea synthesis will have a negligible
effect on the acid–base balance. Unfortunately, however, the bicarbonate flux they referred
to was the rate of CO2 production in vivo, not the rate of HCO3– production.40 As indicated
above, HCO3– ions mainly arise from the negatively charged carboxylate groups of amino
acids during amino acid oxidation. Oxidation of (neutral) fat and carbohydrate produces
CO2, not HCO3–. Studies on the issue have so far never considered the fact that a decrease
in urea synthesis can only cause alkalosis by an increase in the concentration of HCO3– if
the rate of amino acid oxidation, and thus the rate of HCO3– production, is unchanged. If
amino acid oxidation in patients with cirrhosis or with an ornithine cycle enzyme defi-
ciency would decrease, metabolic alkalosis would be compensated for because less HCO3–
would be produced. This situation is analogous but opposite that in metabolic acidosis
where urea synthesis decreases, and proteolysis and amino acid oxidation in the muscle
increases,41 and the increased HCO3– production counteracts acidosis.1
A role for urea metabolism in pH control can also be found in nonmammalian cells.
For example, a high rate of urea synthesis in the liver (i.e., HCO3– consumption) allows
certain fish to survive alkaline environments of pH as high as 10.42–44 Oppositely, intra-
cellular degradation of urea by urease after H+-dependent membrane transport of urea
allows Helicobacter pylori to survive the acidic environment of the stomach.45

7.1.4 The periportal/pericentral glutamine cycle


The presence of periportal glutaminase and pericentral glutamine synthase results in
intercellular glutamine cycling.37,46 This is not a waste of energy: urea synthesis in peri-
portal hepatocytes has a low affinity for ammonia, and any ammonia escaping urea
synthesis will be scavenged by glutamine synthase in pericentral hepatocytes because this
enzyme has a very high affinity for ammonia in addition to a high capacity. Glutaminase
activity is strongly inhibited when the pH falls because the activation by ammonia, its
essential activator, is exquisitely sensitive to small pH changes within the physiological
1382_C07.fm Page 116 Tuesday, October 7, 2003 6:18 PM

116 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

range.47 In acidosis, hepatic glutaminase flux decreases with relatively little change in
glutamine synthase flux.48 A possible source of glutamate for pericentral glutamine syn-
thesis is ornithine (in addition to plasma glutamate), because ornithine aminotransferase
colocalizes with the glutamine synthase-containing cells and is not present in the periportal
cells (see Meijer4 and Watford49 for literature), and because ornithine aminotransferase is
induced by high-protein feeding.50 Interestingly, pericentral cells contain some arginase II
(which is the kidney isoenzyme), but do not contain the other enzymes of the ornithine
cycle.51 It was suggested that this may help to supply the pericentral hepatocytes with
ornithine from circulating arginine. However, whether the ornithine supply is sufficient
to drive pericentral glutamine synthesis remains to be seen.49
The net balance of glutamine across the liver strongly depends on the metabolic
conditions. There is net consumption of glutamine after protein feeding and in uncon-
trolled diabetes, sepsis, and short-term starvation; there is a net release of glutamine from
the liver after long-term starvation and, as discussed above, in metabolic acidosis (see
Curthoys and Watford8 and Watford49 for reviews). However, it must be stressed again
that flux through periportal glutaminase and pericentral glutamine synthase may be
considerable, even though the net balance of glutamine across the liver is zero.8,37,46,49

7.1.5 Metabolite channeling and urea synthesis


Previously, kinetic evidence obtained with intact and permeabilized mitochondria and
with permeabilized hepatocytes strongly supported the possibility of channeling of metab-
olites between the enzymes of the ornithine cycle, even across the mitochondrial inner
membrane, and the concept was coined that the ornithine cycle is a metabolon spanning
the mitochondrial membrane (reviewed by Watford52 and Meijer4). In agreement with this
concept, Cohen and colleagues53,54 provided immunocytochemical evidence that the cyto-
plasmic enzymes argininosuccinate synthase and argininosuccinate lyase in hepatocytes
are located in the immediate vicinity of the cytoplasmic surface of the mitochondrial outer
membrane. However, until now, evidence directly supporting the occurrence of channeling
of ornithine cycle intermediates in hepatocytes, in perfused liver, or in vivo has not yet
been provided.
On the basis of experiments with isolated liver mitochondria, incubated with high
concentrations of glutamine (when flux through glutaminase was very high), we previ-
ously concluded that ammonia was directly channeled from glutaminase to carbamoyl-
phosphate synthase and did not escape the mitochondria.55 It was proposed that the
activation of glutaminase by ammonia in this way increased the affinity of carbamoyl-
phosphate synthase for ammonia.55 However, experiments by others carried out with
isolated rat hepatocytes and with rat liver perfused with [15N]glutamine, labeled in the
amide group [5-15N], did not support this conclusion.56,57 According to these data, 15NH3
derived from [5-15N]glutamine completely mixed with the ammonia in the incubation
medium, and the isotopic enrichment of [15N]citrulline was identical to that of [15N]NH3
in the medium. However, this conclusion must be considered with caution for two reasons.
First, the flux through glutaminase in the experiments of Meijer55 was at least 10-fold
higher than that in the experiments of Nissim and coworkers.56,57 Second, metabolite
channeling is never 100% tight,58 and after a certain period of time, depending on the
degree of channeling, complete equilibration of label with the extramitochondrial pool of
ammonia can be expected. The data in Nissim et al.56,57 would have been more convincing
if the degree of labeling of citrulline and of ammonia had been analyzed in the first few
minutes after [5-15N]glutamine addition rather than after 40 to 70 min.
1382_C07.fm Page 117 Wednesday, October 8, 2003 1:15 PM

Chapter seven: Ureagenesis and ammoniagenesis: an update 117

7.1.6 Interaction of fatty acids with amino acid metabolism


Fatty acid oxidation provides both ATP for urea production and acetyl-CoA for the syn-
thesis of N-acetylglutamate.1 Indeed, defects in fatty acid oxidation are often accompanied
by hyperammonemia, in addition to hypoglycemia.1 It has now been shown that at least
in isolated liver mitochondria, high concentrations of long-chain fatty acids are also able
to inhibit carbamoyl-phosphate synthase in an irreversible manner by acylation of the
enzyme protein on cysteine residues.59 It was proposed that this mechanism reduces amino
acid utilization during long-term starvation and that it contributes to the nitrogen-sparing
effect in long-term starvation.59 Because the acylation of carbamoyl-phosphate synthase
was counteracted by N-acetylglutamate and MgATP,59 this may provide a mechanism to
prevent inactivation of the enzyme during short-term activation when amino acids are
needed for gluconeogenesis.
Another development in the interaction between fatty acid metabolism and amino
acid metabolism and urea synthesis has been the observation that activation of PPARa
(peroxisome proliferator-activated receptor a, a fatty acid-activated nuclear receptor
required for the expression of many genes involved in fatty acid oxidation) inhibits mRNA
expression of enzymes of amino acid catabolism and of urea synthesis. By contrast,
disruption of PPARa results in an increase in these messengers.60 It has been proposed
that PPARa activation (like acylation of carbamoyl-phosphate synthase, discussed above)
contributes to the nitrogen-sparing effect of long-term starvation when ketone bodies
replace glucose as a fuel for the brain.60
Interestingly, mice with juvenile visceral statosis (JVS) (caused by systemic carnitine
deficiency with accumulation of fat in the liver, hyperammonemia, and hypoglycemia)
also have a decreased expression of all the genes of the ornithine cycle.61 Although it is
tempting to conclude that PPARa activation may be part of the mechanism, this possibility
was considered to be less likely.61 The hyperammonemia is not caused by a fall in N-acetyl-
glutamate (because the carnitine deficiency is not complete) but rather by the diminished
activity of the enzymes of the ornithine cycle.61

7.1.7 Interaction of glucose with urea synthesis


The interaction between glucose and urea synthesis is another area of interest because
glucose has a nitrogen-sparing effect on metabolism; it reduces urea synthesis indepen-
dently of changes in plasma amino acid concentrations.62,63 Mechanisms underlying this
effect are direct inhibition by glucose of gluconeogenesis from amino acids63 and inhibition
of glucagon production.64 Conversely, in hypoglycemia, glucagon is the primary mediator
in the coordination of the increased fluxes through the ornithine cycle and through the
proteolytic pathway in muscle.65 Because glucagon does not directly affect muscle pro-
teolysis, it is likely that in hypoglycemia the increased catabolism of amino acids in the
liver results in decreased plasma amino acid concentrations and, in this way, accelerates
net protein breakdown in the muscle (cf. Chapter 16).65 Mechanisms by means of which
glucagon can activate ureagenesis in addition to its well-known stimulation of gluconeo-
genesis have been reviewed previously.1 These include increased amino acid transport
across the plasma membrane, stimulation of hepatic autophagic proteolysis (cf. Chapter
16), increased degradation of some amino acids (e.g., glutamine), stimulation of carbam-
oyl-phosphate synthase activity by increased N-acetylglutamate levels in the mitochon-
dria, and increased synthesis of enzymes involved in urea synthesis.1
1382_C07.fm Page 118 Tuesday, October 7, 2003 6:18 PM

118 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

7.1.8 Cell hydration and urea synthesis


Changes in cell volume have profound effects on hepatic metabolism66 (cf. Chapters 16 to
18). In general, increases and decreases in cell volume exert anabolic and catabolic effects,
respectively. Experiments with the isolated perfused liver showed that the effects of cell
volume changes on urea synthesis are complex: cell swelling increased urea synthesis
from amino acids but decreased urea synthesis with ammonia as the substrate.66 Neither
the mechanism nor the physiological meaning of these opposing effects is entirely clear.
In a study with healthy men, Ivarsen et al.67 examined the effect of furosemide-induced
dehydration on the rate of synthesis of urea from infused alanine and found that moderate
dehydration down-regulated urea synthesis and its activation by glucagon. These data
are consistent with the effects of cell swelling on urea synthesis from amino acids in the
perfused liver (see above), but again, the mechanism and meaning of these effects are not
clear: dehydration of liver cells decreases protein synthesis and increases protein degra-
dation during an amino acid load,66 so that stimulation rather than down-regulation of
urea synthesis would be expected under these conditions.

7.2 Conclusions
Research on ureagenesis in the past decades has made it clear that flux through the
ornithine cycle (i.e., the rate of urea production) is primarily determined by substrate
supply, i.e., the rate of supply of ammonia from the intestine to the liver and the intrahe-
patic rate of amino acid degradation. Mechanisms involved in the control of the activity
of the ornithine cycle itself are essentially mechanisms of control of substrate concentra-
tion, i.e., mechanisms to keep the intrahepatic ammonia concentration constant. This is
logical because, as first pointed out by Krebs and coworkers,68 ammonia is not only toxic
for the brain but is also an essential metabolite in intermediary metabolism, so that its
concentration must be kept within certain limits.
Short-term regulation is primarily exerted at the level of carbamoyl-phosphate syn-
thase, in which N-acetylglutamate plays a crucial role. In addition, there is the long-term
regulation of urea synthesis via changes in the amount of the enzymes involved in the
synthesis of urea (not reviewed in this chapter). Mechanisms controlling the transcription
of these genes clearly form an important research area for the years to come.2,3 In connec-
tion to this, the mechanisms responsible for the peculiar pericentral localization of
glutamine synthase69 are another area of great interest.
The transport proteins responsible for the transfer of ornithine, citrulline, and aspartate
across the mitochondrial membrane also play important roles in urea synthesis. An intrigu-
ing question yet to be answered is the importance of metabolite channeling in the func-
tioning of the ornithine cycle and whether it does, indeed, enhance the efficiency of this
process. Is aspartate, transported out of the mitochondria, directly channeled into argin-
inosuccinate synthase? If so, how can this aspartate then be discriminated from the aspar-
tate engaged in the transfer of reducing equivalents across the mitochondrial membrane
or in the mitochondrial transfer of carbon during gluconeogenesis from lactate? It would
also be important to know how transport of N-acetylglutamate out of the mitochondria
is achieved. Is it by simple diffusion, or is it mediated by an as yet unknown translocator
or by one of the known translocators?
The relevance of regulation of urea synthesis by changes in cell volume is not entirely
clear. It is more likely that changes in flux through the ornithine cycle by changes in cell
volume are caused by a change in substrate supply, i.e., by an effect on protein synthesis
and degradation (cf. Chapter 16), either in the liver itself or in extrahepatic tissues (e.g.,
muscle).
Clearly, these are important issues that deserve to be explored in the near future.
1382_C07.fm Page 119 Wednesday, October 8, 2003 1:16 PM

Chapter seven: Ureagenesis and ammoniagenesis: an update 119

References
1. Meijer, A.J., Lamers, W.H., and Chamuleau, R.A.F.M., Nitrogen metabolism and ornithine
cycle function, Physiol. Rev., 70, 701–748, 1990.
2. Brusilow, S.W. and Horwich, A.L., Urea cycle enzymes, in The Metabolic and Molecular Basis
of Inherited Disease, Scriver, C.R., Baudet, A.L., Sly, W.S., Valle, D., Childs, B., Kinzler, K.W.,
and Vogelstein, B., Eds., McGraw-Hill, New York, 2001, pp. 1909–1963.
3. Morris, S.M., Jr., Regulation of enzymes of the urea cycle and arginine metabolism, Annu.
Rev. Nutr., 22, 87–105, 2002.
4. Meijer, A.J., Ureagenesis and ammoniagenesis, in Amino Acid Metabolism and Therapy in Health
and Nutritional Disease, Cynober, L., Ed., CRC Press, Boca Raton, FL, 1995, pp. 57–66.
5. Yudkoff, M., Daikhin, Y., Nissim, I., Jawad, A., Wilson, J., and Batshaw, M., In vivo nitrogen
metabolism in ornithine transcarbamylase deficiency, J. Clin. Invest., 98, 2167–2173, 1996.
6. Batshaw, M.L., Robinson, M.B., Ye, X., Pabin, C., Daikhin, Y., Burton, B.K., Wilson, J.M., and
Yudkoff, M., Correction of ureagenesis after gene transfer in an animal model and after liver
transplantation in humans with ornithine transcarbamylase deficiency, Pediatr. Res., 46,
588–593, 1999.
7. Meijer, A.J., Lof, C., Ramos I.C., and Verhoeven A.J., Control of ureogenesis, Eur. J. Biochem.,
148, 189–196, 1985.
8. Curthoys, N.P. and Watford, M., Regulation of glutaminase activity and glutamine metabo-
lism, Annu. Rev. Nutr., 15, 133–159, 1995.
9. Yang, D., Hazey, J.W., David, F., Singh, J., Rivchum, R., Streem, J.M., Halperin, M.L., and
Brunengraber, H., Integrative physiology of splanchnic glutamine and ammonium metabo-
lism, Am. J. Physiol. Endocrinol. Metab., 278, E469–E476, 2000.
10. Caldovic, L., Morizono, H., Yu, X., Thompson, M., Shi, D., Gallegos, R., Allewell, N.M.,
Malamy, M.H., and Tuchman, M., Identification, cloning and expression of the mouse
N-acetylglutamate synthase gene, Biochem. J., 364, 825–831, 2002.
11. Caldovic, L., Morizono, H., Gracia Panglao, M., Gallegos, R., Yu, X., Shi, D., Malamy, M.H.,
Allewell, N.M., and Tuchman, M., Cloning and expression of the human N-acetylglutamate
synthase gene, Biochem. Biophys. Res. Commun., 299, 581–586, 2002.
12. Kawamoto, S., Sonoda, T., Ohtake, A., and Tatibana, M., Stimulatory effect of arginine on
acetylglutamate synthesis in isolated mitochondria of mouse and rat liver, Biochem. J., 232,
329–334, 1985.
13. Hayase, K., Naganuma, Y., Koie, M., and Yoshida, A., Role of N-acetylglutamate turnover
in urea synthesis by rats treated with the thyroid hormone, Biosci. Biotechnol. Biochem., 62,
535–539, 1998.
14. Meijer, A.J., Van Woerkom, G.M., Wanders, R.J., and Lof, C., Transport of N-acetylglutamate
in rat-liver mitochondria, Eur. J. Biochem., 124, 325–330, 1982.
15. Camacho, J.A., Obie, C., Biery, B., Goodman, B.K., Hu, C.A., Almashanu, S., Steel, G., Casey,
R., Lambert, M., Mitchell, G.A., and Valle, D., Hyperornithinaemia-hyperammonaemia-ho-
mocitrullinuria syndrome is caused by mutations in a gene encoding a mitochondrial orni-
thine transporter, Nat. Genet., 22, 151–158, 1999.
16. Fell, V., Pollitt, R.J., Sampson, G.A., and Wright, T., Ornithinemia, hyperammonemia, and
homocitrullinuria: a disease associated with mental retardation and possibly caused by
defective mitochondrial transport, Am. J. Dis. Child., 127, 752–756, 1974.
17. Hommes, F.A., Ho, C.K., Roesel, R.A., Coryell, M.E., and Gordon, B.A., Decreased transport
of ornithine across the inner mitochondrial membrane as a cause of hyperornithinaemia,
J. Inherit. Metab. Dis., 5, 41–47, 1982.
18. Inoue, I., Saheki, T., Kayanuma, K., Uono, M., Nakajima, M., Takeshita, K., Koike, R., Yuasa,
T., Miyatake, T., and Sakoda, K., Biochemical analysis of decreased ornithine transport
activity in the liver mitochondria from patients with hyperornithinemia, hyperammonemia
and homocitrullinuria, Biochim. Biophys. Acta, 964, 90–95, 1988.
19. Indiveri, C., Tonazzi, A., Stipani, I., and Palmieri, F., The purified and reconstituted orni-
thine/citrulline carrier from rat liver mitochondria: electrical nature and coupling of the
exchange reaction with H+ translocation, Biochem. J., 327, 349–355, 1997.
1382_C07.fm Page 120 Tuesday, October 7, 2003 6:18 PM

120 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

20. Kobayashi, K., Sinasac, D.S., Iijima, M., Boright, A.P., Begum, L., Lee, J.R., Yasuda, T., Ikeda,
S., Hirano, R., Terazono, H., Crackower, M.A., Kondo, I., Tsui, L.C., Scherer, S.W., and Saheki,
T., The gene mutated in adult-onset type II citrullinaemia encodes a putative mitochondrial
carrier protein, Nat. Genet., 22, 159–163, 1999.
21. Palmieri, L., Pardo, B., Lasorsa, F.M., del Arco, A., Kobayashi, K., Iijima, M., Runswick, M.J.,
Walker, J.E., Saheki, T., Satrustegui, J., and Palmieri, F., Citrin and aralar1 are Ca(2+)-stimu-
lated aspartate/glutamate transporters in mitochondria, EMBO J., 20, 5060–5069, 2001.
22. LaNoue, K.F., Meijer, A.J., and Brouwer, A., Evidence for electrogenic aspartate transport in
rat liver mitochondria, Arch. Biochem. Biophys., 161, 544–550, 1974.
23. Meijer, A.J., Gimpel, J.A., Deleeuw, G., Tischler, M.E., Tager, J.M., and Williamson, J.R.,
Interrelationships between gluconeogenesis and ureogenesis in isolated hepatocytes, J. Biol.
Chem., 253, 2308–2320, 1978.
24. Patterson, B.W., Carraro, F., Klein, S., and Wolfe, R.R., Quantification of incorporation of
[15N]ammonia into plasma amino acids and urea, Am. J. Physiol., 269, E508–E515, 1995.
25. Brosnan, J.T., Brosnan, M.E., Charron, R., and Nissim, I., A mass isotopomer study of urea
and glutamine synthesis from 15N-labeled ammonia in the perfused rat liver, J. Biol. Chem.,
271, 16199–16207, 1996.
26. Weijs, P.J., Calder, A.G., Milne, E., and Lobley, G.E., Conversion of [15N]ammonia into urea
and amino acids in humans and the effect of nutritional status, Brit. J. Nutr., 76, 491–499, 1996.
27. Brosnan, J.T., Brosnan, M.E., Yudkoff, M., Nissim, I., Daikhin, Y., Lazarow, A., Horyn, O.,
and Nissim, I., Alanine metabolism in the perfused rat liver: studies with (15)N, J. Biol. Chem.,
276, 31876–31882, 2001.
28. del Arco, A. and Satrustegui, J., Molecular cloning of Aralar, a new member of the mitochon-
drial carrier superfamily that binds calcium and is present in human muscle and brain, J. Biol.
Chem., 273, 23327–23334, 1998.
29. Del Arco, A., Agudo, M., and Satrustegui, J., Characterization of a second member of the
subfamily of calcium-binding mitochondrial carriers expressed in human non-excitable tis-
sues, Biochem. J., 345, 725–732, 2000.
30. del Arco, A., Morcillo, J., Martinez-Morales, J.R., Galian, C., Martos, V., Bovolenta, P., and
Satrustegui, J., Expression of the aspartate/glutamate mitochondrial carriers aralar1 and
citrin during development and in adult rat tissues, Eur. J. Biochem., 269, 3313–3320, 2002.
31. Begum, L., Jalil, M.A., Kobayashi, K., Iijima, M., Li, M.X., Yasuda, T., Horiuchi, M., del Arco,
A., Satrustegui, J., and Saheki, T., Expression of three mitochondrial solute carriers, citrin,
aralar1 and ornithine transporter, in relation to urea cycle in mice, Biochim. Biophys. Acta,
1574, 283–292, 2002.
32. Leverve, X.M., Verhoeven, A.J., Groen, A.K., Meijer, A.J., and Tager, J.M., The malate/aspar-
tate shuttle and pyruvate kinase as targets involved in the stimulation of gluconeogenesis
by phenylephrine, Eur. J. Biochem., 155, 551–556, 1986.
33. Fiermonte, G., Palmieri, L., Todisco, S., Agrimi, G., Palmieri, F., and Walker, J.E., Identification
of the mitochondrial glutamate transporter: bacterial expression, reconstitution, functional
characterization, and tissue distribution of two human isoforms, J. Biol. Chem., 277,
19289–19294, 2002.
34. Atkinson, D.E. and Camien, M.N., The role of urea synthesis in the removal of metabolic
bicarbonate and the regulation of blood pH, Curr. Top. Cell. Regul., 21, 261–302, 1982.
35. Atkinson, D.E. and Bourke, E., Metabolic aspects of the regulation of systemic pH, Am. J.
Physiol., 252, F947–F956, 1987.
36. Häussinger, D., Liver regulation of acid-base balance, Miner. Electrolyte Metab., 23, 249–252,
1997.
37. Häussinger, D., Sies, H., and Gerok, W., Functional hepatocyte heterogeneity in ammonia
metabolism. The intercellular glutamine cycle, J. Hepatol., 1, 3–14, 1985.
38. Shangraw, R.E. and Jahoor, F., Effect of liver disease and transplantation on urea synthesis
in humans: relationship to acid-base status, Am. J. Physiol., 276, G1145–G1152, 1999.
39. Häussinger, D., Steeb, R., and Gerok, W., Ammonium and bicarbonate homeostasis in chronic
liver disease, Klin. Wochenschr., 68, 175–182, 1990.
1382_C07.fm Page 121 Tuesday, October 7, 2003 6:18 PM

Chapter seven: Ureagenesis and ammoniagenesis: an update 121

40. Hoerr, R.A., Yu, Y.M., Wagner, D.A., Burke, J.F., and Young, V.R., Recovery of 13C in breath
from NaH13CO3 infused by gut and vein: effect of feeding, Am. J. Physiol., 257, E426–E438,
1989.
41. May, R.C., Hara, Y., Kelly, R.A., Block, K.P., Buse, M.G., and Mitch, W.E., Branched-chain
amino acid metabolism in rat muscle: abnormal regulation in acidosis, Am. J. Physiol., 252,
E712–E718, 1987.
42. Randall, D.J., Wood, C.M., Perry, S.F., Bergman, H., Maloiy, G.M., Mommsen, T.P., and Wright,
P.A., Urea excretion as a strategy for survival in a fish living in a very alkaline environment,
Nature, 337, 165–166, 1989.
43. Lindley, T.E., Scheiderer, C.L., Walsh, P.J., Wood, C.M., Bergman, H.L., Bergman, A.L.,
Laurent, P., Wilson, P., and Anderson, P.M., Muscle as the primary site of urea cycle enzyme
activity in an alkaline lake-adapted tilapia, Oreochromis alcalicus grahami, J. Biol. Chem.,
274, 29858–29861, 1999.
44. Saha, N., Kharbuli, Z.Y., Bhattacharjee, A., Goswami, C., and Häussinger, D., Effect of alka-
linity (pH 10) on ureogenesis in the air-breathing walking catfish, Clarias batrachus, Comp.
Biochem. Physiol. A Mol. Integr. Physiol., 132, 353–364, 2002.
45. Weeks, D.L., Eskandari, S., Scott, D.R., and Sachs, G., A H+-gated urea channel: the link
between Helicobacter pylori urease and gastric colonization, Science, 287, 482–485, 2000.
46. Häussinger, D., Nitrogen metabolism in liver: structural and functional organization and
physiological relevance, Biochem. J., 267, 281–290, 1990.
47. Verhoeven, A.J., van Iwaarden, J.F., Joseph, S.K., and Meijer, A.J., Control of rat-liver glutam-
inase by ammonia and p, Eur. J. Biochem., 133, 241–244, 1983.
48. Watford, M., Chellaraj, V., Ismat, A., Brown, P., and Raman, P., Hepatic glutamine metabolism,
Nutrition, 18, 301–303, 2002.
49. Watford, M., Glutamine and glutamate metabolism across the liver sinusoid, J. Nutr., 130,
983S–987S, 2000.
50. Boon, L., Geerts, W.J., Jonker, A., Lamers, W.H., and van Noorden, C.J., High protein diet
induces pericentral glutamate dehydrogenase and ornithine aminotransferase to provide
sufficient glutamate for pericentral detoxification of ammonia in rat liver lobules, Histochem.
Cell Biol., 111, 445–452, 1999.
51. O’Sullivan, D., Brosnan, J.T., and Brosnan, M.E., Hepatic zonation of the catabolism of
arginine and ornithine in the perfused rat liver, Biochem. J., 330, 627–632, 1998.
52. Watford, M., Channeling in the urea cycle: a metabolon spanning two compartments, Trends
Biochem. Sci., 14, 313–314, 1989.
53. Cohen, N.S. and Kuda, A., Argininosuccinate synthetase and argininosuccinate lyase are
localized around mitochondria: an immunocytochemical study, J. Cell Biochem., 60, 334–340,
1996.
54. Cohen, N.S., Cheung, C.-W., and Raijman L., The urea cycle, in Channelling in Intermediary
Metabolism, Agius, L. and Sherratt, H.S.A., Eds., Portland Press, London, 1997, pp. 183–199.
55. Meijer, A.J., Channeling of ammonia from glutaminase to carbamoyl-phosphate synthetase
in liver mitochondria, FEBS Lett., 191, 249–251, 1985.
56. Nissim, I., Yudkoff, M., and Brosnan, J.T., Regulation of [15N]urea synthesis from
[5-15N]glutamine: role of pH, hormones, and pyruvate, J. Biol. Chem., 271, 31234–31242, 1996.
57. Nissim, I., Brosnan, M.E., Yudkoff, M., and Brosnan, J.T., Studies of hepatic glutamine
metabolism in the perfused rat liver with (15)N-labeled glutamine, J. Biol. Chem., 274,
28958–28965, 1999.
58. Cornish-Bowden, A., Kinetic consequences of channelling, in Channelling in Intermediary
Metabolism, Agius, L. and Sherratt, H.S.A., Eds., Portland Press, London, 1997, pp. 53–70.
59. Corvi, M.M., Soltys, C.L., and Berthiaume, L.G., Regulation of mitochondrial carbamoyl-
phosphate synthetase 1 activity by active site fatty acylation, J. Biol. Chem., 276, 45704–45712,
2001.
60. Kersten, S., Mandard, S., Escher, P., Gonzalez, F.J., Tafuri, S., Desvergne, B., and Wahli, W.,
The peroxisome proliferator-activated receptor alpha regulates amino acid metabolism,
FASEB J., 15, 1971–1978, 2001.
1382_C07.fm Page 122 Tuesday, October 7, 2003 6:18 PM

122 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

61. Saheki, T., Li, M.X., and Kobayashi, K., Antagonizing effect of AP-1 on glucocorticoid induc-
tion of urea cycle enzymes: a study of hyperammonemia in carnitine-deficient, juvenile
visceral steatosis mice, Mol. Genet. Metab., 71, 545–551, 2000.
62. Vilstrup, H., Effects of glucose on alanine-derived urea synthesis, Clin. Physiol., 4, 495–507,
1984.
63. Jahoor, F. and Wolfe, R.R., Regulation of urea production by glucose infusion in vivo, Am.
J. Physiol., 253, E543–E550, 1987.
64. Hamberg, O. and Vilstrup, H., Effects of glucose on hepatic conversion of aminonitrogen to
urea in patients with cirrhosis: relationship to glucagon, Hepatology, 19, 45–54, 1994.
65. Grofte, T., Wolthers, T., Jorgensen, J.O., Poulsen, P.L., Vilstrup, H., and Moller, N., Hepatic
amino- to urea-N clearance and forearm amino-N exchange during hypoglycemic and eug-
lycemic hyperinsulinemia in normal man, J. Hepatol., 30, 819–825, 1999.
66. Häussinger, D., The role of cellular hydration in the regulation of cell function, Biochem. J.,
313, 697–710, 1996.
67. Ivarsen, P., Greisen, J., and Vilstrup, H., Acute effects of moderate dehydration on the hepatic
conversion of amino nitrogen into urea nitrogen in healthy men, Clin. Sci. (Lond.), 101,
339–344, 2001.
68. Krebs, H.A., Hems, R., and Lund, P., Some regulatory mechanisms in the synthesis of urea
in the mammalian liver, Adv. Enzyme Regul., 11, 361–377, 1973.
69. Christoffels, V.M., Sassi, H., Ruijter, J.M., Moorman, A.F., Grange, T., and Lamers, W.H., A
mechanistic model for the development and maintenance of portocentral gradients in gene
expression in the liver, Hepatology, 29, 1180–1192, 1999.
1382_C08.fm Page 123 Tuesday, October 7, 2003 6:20 PM

chapter eight

Metabolism of branched-chain
amino acids in man
Anton J.M. Wagenmakers
Maastricht University and University Hospital

Contents
Introduction..................................................................................................................................123
8.1 BCAA metabolism in the postabsorptive state .............................................................124
8.2 BCAA metabolism after ingestion of protein-containing meals ................................125
8.3 BCAA metabolism during one-leg knee-extension exercise: a model with
exceptionally high net protein degradation rates.........................................................126
8.4 Are BCAA a source for both the carbon and nitrogen of alanine and
glutamine? Studies with isotopic tracers .......................................................................128
8.5 The glucose–alanine cycle: a changing concept ............................................................128
8.6 The role of alanine aminotransferase and deamination of BCAA in TCA cycle
anaplerosis during exercise ..............................................................................................129
8.7 Lessons from patients with McArdle’s disease.............................................................131
8.8 Conclusion ...........................................................................................................................132
References ....................................................................................................................................132

Introduction
The branched-chain amino acids (BCAA) — leucine, valine, and isoleucine — are three of
the nine essential amino acids in mammals. Together, they make up 40% of the daily
requirement for essential amino acids in man. They serve among others as building blocks
for tissue proteins, and as carbon and nitrogen precursors for the synthesis of the most
abundant nonessential amino acids in muscle and plasma (glutamine, alanine, and
glutamate); they play an important role in the synthesis of tricarboxylic acid (TCA) cycle
intermediates in contracting skeletal muscle during workload transitions; they are oxi-
dized to acetyl-CoA and CO2 ; and they (especially leucine) are important signal molecules
that exert control on insulin production (pancreas) and on the translation initiation phase
of protein synthesis (liver and skeletal muscle). As such, a thorough understanding of the
metabolism of the BCAA is essential for a proper understanding of the cellular control
mechanisms of energy production, fuel selection, and protein metabolism (maintenance

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 123
1382_C08.fm Page 124 Tuesday, October 7, 2003 6:20 PM

124 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

of muscle mass), both in healthy subjects and in patients with cachexia due to nutritional
and metabolic diseases.
The aim of this chapter is to present an update of the literature that has appeared
since the previous publication of this chapter in 1995.1 Few new publications have
appeared on the following topics: (1) oxidative pathways of BCAA and tissue distribution
of enzyme activities and (2) effect of diet, starvation, exercise, and hormones on the activity
of the enzymes catalyzing steps 1 to 3 in the BCAA degradative pathway. One exception
is the effect of cytokines on enzyme activities. For details, refer to Chapter 6, as stress and
trauma metabolism are out of the scope of this chapter. The minimal progress in current
knowledge on these topics is disappointing in light of the earlier conclusion1 that we need
to extend this knowledge for a proper understanding of, first, the physiological basis of
the interorgan collaboration in BCAA metabolism and of, second, the complex role that
these compounds play in the regulation of energy and protein metabolism at the cellular
and whole-body level. Therefore, we advise graduate students and others with a devel-
oping interest in BCAA metabolism to first read Chapter 5 of the 1995 book.1 Further
useful readings are the four books on BCAA metabolism that have been published so
far,2–5 and for an overview of the important early in vivo animal studies (e.g., on BCAA
antagonism), refer to the review of Harper and colleagues.6 In this chapter we will focus
on a few selected new aspects and physiological concepts of BCAA metabolism that have
been further developed or emerged since 1995.1 The focus of this review is on new studies
in man.

8.1 BCAA metabolism in the postabsorptive state


Most of the essential amino acids are degraded by the liver, but the BCAA are primarily
oxidized in skeletal muscle and probably also in adipose tissue.1 This conclusion was
originally based on data from rat muscles incubated in vitro.7,8
These muscles are in net protein breakdown (protein synthesis < protein degradation),
especially when insulin is omitted from the incubation or perfusion medium. This implies
that all the amino acids are released from the existing muscle proteins and that those
amino acids that are not metabolized in muscle will be released in proportion to their
relative occurrence in muscle protein, while a discrepancy will be found when amino acids
are transaminated, oxidized, or synthesized. Such comparisons revealed that rat skeletal
muscle can oxidize only six amino acids: the three BCAA, glutamate, aspartate, and
asparagine (Figure 8.1).7–9 Glutamine and alanine were released in excess of the relative
occurrence in muscle protein. This suggests that de novo synthesis of these amino acids
occurs.7–9 In 1972 Ruderman and Lund7 were the first to observe that addition of BCAA
to the perfusion medium of rat hindquarters increased the release of alanine and
glutamine. The relationship between the metabolism of BCAA on the one hand and the
release of alanine and glutamine on the other has since been the subject of many studies.
Most of this relationship today has been firmly established.9 In the BCAA aminotransferase
reaction the amino group is donated to a-ketoglutarate to form glutamate and a branched-
chain a-keto acid (Figure 8.1). In the reaction catalyzed by glutamine synthase, glutamate
reacts with ammonia to form glutamine. Alternatively, glutamate may donate the amino
group to pyruvate to form alanine and regenerate a-ketoglutarate. These reactions provide
a mechanism for the elimination of amino groups from muscle in the form of the nontoxic
nitrogen carriers alanine and glutamine (Figure 8.1).
Muscle amino acid metabolism has also been investigated in man in vivo by measuring
the exchange of amino acids across a forearm or a leg (arteriovenous difference multiplied
by blood flow gives the net exchange of amino acids).10–15 As muscle is the largest and
most active tissue in the limbs, the assumption that limb exchange primarily reflects
1382_C08.fm Page 125 Wednesday, October 8, 2003 1:25 PM

Chapter eight: Metabolism of branched-chain amino acids in man 125

Figure 8.1 Schematic presentation of the interactions of amino acids with the TCA cycle. The trans-
amination products of leucine, isoleucine, and valine are a-ketoiscocaproic acid (a-KIC), a-oxo-
methylvaleric acid (a-KMV), and a-ketoisovaleric acid (a-KIV). Glucose 6-P, glucose-6-phosphate.
(This figure is reproduced, with permission, from Van Hall et al., 1999, Clin. Sci., 97, 557, © the
Biochemical Society and the Medical Research Society (UK).)

muscle metabolism seems reasonable. After overnight fasting there is net breakdown of
muscle proteins as protein synthesis is slightly lower than protein degradation.16 This
again (as explained for the incubated rat muscles) implies that those amino acids that are
not metabolized in muscle will be released in proportion to their relative occurrence in
muscle protein, while a discrepancy will be found when amino acids are transaminated,
oxidized, or synthesized. Human limbs at rest after overnight fasting released much more
glutamine (48% of total amino acid release) and alanine (25%)15 than would be anticipated
from the relative occurrence in muscle protein (glutamine, 8%; alanine, 9%).15 This implies
that glutamine with two N-atoms per molecule is dominant for the amino acid N-release
from human muscle. The BCAA (19% relative occurrence in muscle protein), glutamate
(7%), and aspartate and asparagine (together, 9%), on the other hand, are not released or
in lower amounts than their relative occurrence. Glutamate, in fact, is always taken up
from the circulation by human skeletal muscle.10–15 This implies that in human skeletal
muscle after overnight fasting the BCAA, glutamate, aspartate, and asparagine originating
from net breakdown of muscle proteins and glutamate taken up from the circulation are
metabolized and used for de novo synthesis of glutamine and alanine (Figure 8.1). All other
amino acids are released in proportion to their relative occurrence in muscle protein,
implying that little or no metabolism occurs.15

8.2 BCAA metabolism after ingestion of protein-containing meals


Several studies have investigated the fate of BCAA ingested orally either as a mixed meal
containing carbohydrate, fat, and protein or as a pure protein meal.12,19–22 Following inges-
tion of mixed protein-containing meals, small amounts of most amino acids are taken up
by muscle and most other tissues, as there is net protein deposition in the fed state (protein
synthesis > protein degradation), which compensates for the net losses in the overnight
fasting period.16,17 The high plasma insulin concentration that is seen in the postprandial
state, in combination with the increase in the intracellular concentration or availability
(arterial blood concentration) of the essential amino acids, has been shown to both
1382_C08.fm Page 126 Tuesday, October 7, 2003 6:20 PM

126 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

stimulate protein synthesis (in liver and muscle at the initiation phase of mRNA trans-
lation18) and reduce protein degradation at the whole-body level and in skeletal muscle.16,17
An excessively large uptake of BCAA and glutamate is seen in muscle in the 4-h period
after ingestion of a protein meal12 and a mixed meal,19 and also after ingestion of a large
steak.20 Together, BCAA and glutamate then cover more than 90% of the muscle amino
acid uptake. The BCAA originate from dietary protein. After digestion of dietary protein,
most of the resulting BCAA escape from uptake and metabolism in gut and liver12 as a
consequence of the low BCAA aminotransferase activity in these tissues.1 In agreement
with this conclusion Hagenfeldt et al.21 found that 55% of a constant intravenous leucine
infusion (2.5 h at a rate of 300 mmol/min) in postabsorptive human subjects was taken
up by the peripheral tissues, 25% by the splanchnic region, and 10% by the brain. The
source of the glutamate is less clear. The diet only seems to deliver a minor proportion as
both a [15N]- and [13C]-glutamate tracer ingested orally were almost quantitatively removed
in the first pass through the splanchnic area (gut and liver).22,23 However, the splanchnic
area in man is producing significant amounts of glutamate after protein ingestion,12 after
overnight fasting,10 and after prolonged starvation.10 Equivalent amounts of glutamate are
taken up by muscle in all these conditions.10–15,19,20 After ingestion of a large steak, the
muscle release of glutamine more than doubles, while the alanine release is strongly
reduced to only 10% of the overnight fasted value.20 In the 4-h period after ingestion of a
mixed meal,19 the dominance of glutamine in carrying N out of skeletal muscle also was
much larger than after overnight fasting. Glutamine then accounted for 71% of the amino
acid release and 82% of the N-release from muscle.19 In summary, these data show that
after consumption of protein-containing meals, BCAA and glutamate are produced by the
splanchnic bed and taken up by muscle, and their carbon skeletons are primarily used
for de novo synthesis of glutamine.

8.3 BCAA metabolism during one-leg knee-extension exercise: a


model with exceptionally high net protein degradation rates
Van Hall et al.15 measured the exchange of amino acids across the leg muscles during 90
min of one-leg knee-extensor kicking exercise in six healthy trained human subjects. One-
leg knee-extensor exercise is primarily confined to the quadriceps muscle group (only
3 kg of active muscle, while there is 9 kg of muscle in a human leg). The maximal oxygen
consumption of the active muscle is about 800 ml/min/kg of muscle, which is two- to
threefold higher than during two-legged cycling on a normal cycle ergometer (which is
performed with a much larger muscle mass). This implies that the metabolic rate of the
active muscle during one-leg knee-extensor exercise is much greater than with whole-
body exercise. Such high metabolic rates have also been reached during electrical stim-
ulation of rat muscles, and in that case, the contractions lead to a reduction of the energy
charge and a decrease of the rate of muscle protein synthesis.24 A recent study25 has
disclosed the mechanism behind this observation. Increases in muscle AMP concentration
via activation of the AMP-activated protein kinase (AMPK) signaling cascade have been
shown to suppress protein synthesis in rat skeletal muscle through down-regulation of
the mammalian target of rapamycin (mTOR) signaling pathway. This is one of the impor-
tant pathways controlling the translation initiation phase of protein synthesis (see Chapter
16 for more details). The understanding of the AMPK signaling cascade recently has
received a lot of attention in exercise physiology. Activation of AMPK, among others,
leads to the phosphorylation of enzymes that exert control on glucose and fatty acid
transport and oxidation.26 AMPK is activated during exercise both by an increase of the
AMP concentration and by a reduction of the phosphocreatine/creatine ratio.26 These
1382_C08.fm Page 127 Tuesday, October 7, 2003 6:20 PM

Chapter eight: Metabolism of branched-chain amino acids in man 127

Figure 8.2 The leg amino acid exchange observed during one-leg knee-extensor leg exercise — a
model with exceptionally high net protein degradation rates. For a detailed explanation of the
experimental conditions and methods used, see Van Hall et al.14,15

mechanisms seem to explain that one-legged knee-extensor exercise with its high meta-
bolic rate in comparison to the rest situation (and in comparison to normal dynamic
exercise such as running or cycling) leads to a large net protein degradation rate (protein
degradation >>> protein synthesis) (Figure 8.2).15 The conclusion that net protein degra-
dation was increased was based on the 10-fold increase in the release of amino acids that
are not metabolized (nmAA), transaminated, and oxidized, in human muscle.15 These
amino acids are only used for protein synthesis and produced by protein degradation,
and therefore, a net release indicates that protein degradation exceeds protein synthesis.15
As one-leg knee-extensor exercise appears to lead to the largest physiological imbal-
ance between muscle protein synthesis and protein degradation that has ever been
observed in the scientific literature in any other physiological condition, the amino acid
exchange data obtained in this model are uniquely suited to investigate the metabolism
of the large amounts of BCAA and glutamate that are released from muscle protein. It is
clear that BCAA and glutamate are vividly metabolized by human skeletal muscle, as
they, despite the high production rate by net protein degradation during one-leg exercise,
were taken up in substantial amounts (≥10-fold the resting uptake) from the circulation
(Figure 8.2). Data on aspartate were not obtained in these human experiments, but in
incubated rat muscles aspartate is rapidly transaminated to oxaloacetate and not released
to the medium.27 During one-leg knee-extensor exercise the alanine is released in an
amount about equal to its relative occurrence,15 and the amount released is not higher than
that in the resting state (Figure 8.2). Glutamine, on the other hand, is released in amounts
that are much larger than its relative occurrence,15 and the amount released is ninefold
higher than that in the resting state (Figure 8.2). Together, these data indicate that the
carbon skeletons of the BCAA, glutamate, aspartate, asparagine, and other amino acids
that are partly oxidized in muscle15 are not used for the synthesis of alanine but are solely
used for conversion into tricarboxylic acid cycle intermediates and glutamine (see Section
8.4 and Figure 8.1).
1382_C08.fm Page 128 Tuesday, October 7, 2003 6:20 PM

128 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

8.4 Are BCAA a source for both the carbon and nitrogen of alanine
and glutamine? Studies with isotopic tracers
The next question is whether the carbon and nitrogen atoms of the BCAA can be used for
complete synthesis of both glutamine and alanine (Figure 8.1). Studies with [15N]-leucine
have shown that the amino group of the BCAA is indeed incorporated in humans in vivo
in the a-amino nitrogen of alanine28 and glutamine.29As glutamate is central in all ami-
notransferase reactions in muscle (Figure 8.1), this implies that the amino group of all six
mentioned amino acids (BCAA, glutamate, aspartate, and asparagine) is interchangeable
and can be incorporated in the a-amino nitrogen of alanine and glutamine. The source of
ammonia in glutamine synthesis (incorporated in the amide nitrogen) was not known in
19951 and still is not known today. Two deamination reactions (purine nucleotide cycle
and glutamate dehydrogenase) theoretically may deaminate the six metabolized amino
acids as discussed before,1,9 but the experimental evidence is still lacking.
In vitro muscle incubations and perfusions with [U-14C]-amino acids and in vivo exper-
iments in rats with [1,2-13C]-leucine have led to the general consensus that the carbon
skeletons of the six metabolized amino acids (Figure 8.1) are used for de novo synthesis of
TCA cycle intermediates and glutamine.1 Carbon from the BCAA enters the TCA cycle
both as acetyl-CoA (leucine and isoleucine) and as succinyl-CoA (isoleucine and valine)
and is then via a-ketoglutarate converted to glutamate amd glutamine (Figure 8.1). No,
or very little, radioactivity was found in lactate, pyruvate, and alanine during incubation
of rat diaphragms30 and perfusion of rat hindquarters1 with [U-14C]-valine. This implies
that there is no active pathway in muscle for conversion of TCA cycle intermediates into
pyruvate. It also implies that the carbon skeleton of the six amino acids that are converted
to TCA cycle intermediates (Figure 8.1) cannot be used for complete oxidation (which is
only possible when carbon enters the TCA cycle as acetyl-CoA, as is the case for leucine
and for part of the isoleucine molecule) or for pyruvate and alanine synthesis. Therefore,
the only fate of these carbon skeletons is synthesis of TCA cycle intermediates and
glutamine (Figure 8.1). The question then is what are the sources of the carbon atoms of
alanine? The remaining sources are muscle glycogen and blood glucose converted by
glycolysis into pyruvate (Figure 8.1). In agreement with this conclusion, Chang and
Goldberg30 reported that over 97% of the carbons of the alanine, pyruvate, and lactate
released by incubated diaphragms were derived from exogenous glucose. In a recent
whole-body tracer study in man,31 42% of the alanine released by muscle was reported to
originate from blood glucose.

8.5 The glucose–alanine cycle: a changing concept


The conclusion of the previous section slightly changes the concept of the glucose–alanine
cycle as proposed by Felig and colleagues32 and as explained today in most textbooks.
According to the original formulation of the glucose–alanine cycle the pyruvate used for
alanine production in muscle was derived from either glycolysis of blood glucose or
pyruvate derived from metabolism of other muscle protein-derived amino acids. The
alanine is then released to the blood and converted to glucose via gluconeogenesis in the
liver. Carbon derived from muscle protein and amino acids in this way was suggested to
be a major source used to help maintain blood glucose concentrations after overnight
fasting and during prolonged starvation. The implication, however, of the above conclu-
sions is that all pyruvate is either derived from glycolysis of blood glucose or from
breakdown of muscle glycogen followed by glycolysis. In a recent tracer study in man,31
42% of the alanine released by muscle was reported to originate from blood glucose. This
suggests that more than half of the alanine released by muscle is formed from pyruvate
1382_C08.fm Page 129 Wednesday, October 8, 2003 1:18 PM

Chapter eight: Metabolism of branched-chain amino acids in man 129

generated by muscle glycogen breakdown. This route in fact may provide a mechanism
to slowly mobilize the large muscle glycogen stores (350 to 800 g)9 during starvation, such
that these stores can be used to help maintain the blood glucose concentration and function
as fuel in tissues that critically depend on glucose availability such as brain, red blood
cells, and kidney cortex. The amino acids liberated during starvation by increased net
rates of protein degradation16,17 are not converted to alanine but to glutamine, which also
is a precursor for gluconeogenesis in the liver in the postabsorptive state (for references,
see Wagenmakers9). Glutamine also is a precursor for gluconeogenesis in the kidney, but
renal gluconeogenesis only starts to be significant (>10% of total glucose output) in man
after starvation for more than 60 h (for references, see Wagenmakers9). Protein-derived
amino acids metabolized in muscle thus still can help to maintain blood glucose concen-
tration during starvation, but by a different route than suggested in the original formu-
lation of the glucose–alanine cycle. Recent tracer studies in man indeed confirm that
glutamine is more important than alanine as a gluconeogenic precursor after overnight
starvation,33 and that glutamine is more important than alanine as a vehicle for transport
of protein-derived carbon and nitrogen from muscle through plasma to the sites of glu-
coneogenesis or further metabolism.31

8.6 The role of alanine aminotransferase and deamination of BCAA


in TCA cycle anaplerosis during exercise
During one- and two-legged cycling exercise at intensities between 40 and 70% of Wmax,
only two amino acids change substantially in concentration in the muscle free amino acid
pool, i.e., glutamate and alanine (for references, see Wagenmakers9,16 and van Hall et al.14).
Glutamate decreases by 50 to 70% within 10 min of exercise, while alanine at that point
in time is increased by 50 to 60%. The low concentration of glutamate is maintained when
exercise is continued for periods up to 90 min or until exhaustion, while alanine slowly
returns to resting levels. Substantial amounts of alanine, furthermore, are released into
circulation during the first 30 min of exercise.14 Alanine release is reduced again when
exercise is continued and the muscle glycogen stores are gradually emptied.14,15
The functionality of the rapid fall in muscle glutamate concentration during the first
minutes of moderate-intensity exercise is conversion of its carbon skeleton via the alanine
aminotransferase reaction (glutamate + pyruvate ´ a-ketoglutarate + alanine) into a-keto-
glutarate and other TCA cycle intermediates (Figure 8.1). The sum concentration of the
most abundant TCA cycle intermediates in skeletal muscle has been shown to increase
5- to 10-fold within 5 min of the start of exercise both in rat muscle and in human muscle
(for references, see Wagenmakers9 and Gibala et al.34). The increase, furthermore, is pro-
portional to the exercise intensity in human muscle.35 We have previously suggested9 that
the increase in concentration of TCA cycle intermediates during the first minutes of
exercise is needed to increase the flux in the TCA cycle and make ATP production by
aerobic substrate oxidation match the increased ATP demand when going from rest to
exercise (for details of the theoretical basis of this conclusion, see Wagenmakers9).
In skeletal muscle, the rate of aerobic energy production, and therefore the flux in the
TCA cycle, can increase more than 80-fold going from rest to exercise. The understanding
of the mechanisms that lead to this massive increase within minutes of the start of exercise
is one of the most interesting and most complex academic challenges of the biochemistry
of exercise of the last decades.
Theoretically, other reactions could make contributions to the synthesis of TCA cycle
intermediates during the first minutes of exercise. Glutamate dehydrogenase (glutamate
+ NAD+ ´ a-ketoglutarate + NH4+ + NADH) can be excluded as a major contributing
1382_C08.fm Page 130 Tuesday, October 7, 2003 6:20 PM

130 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

reaction, as ammonia production rates were very low in the first 10 min of exercise, when
the large decrease in muscle glutamate concentration was observed.14 For the same reason,
purine nucleotide cycling, suggested by Aragón and Lowenstein36 to be the major anaple-
rotic mechanism during exercise, can be excluded. The alanine aminotransferase reaction
is a near-equilibrium reaction. At the start of exercise the rate of glycolysis and thus of
pyruvate formation is high, as indicated by a temporary increase of the muscle pyruvate
concentration (for references, see Wagenmakers9). The increase in muscle pyruvate con-
centration automatically forces the alanine aminotransferase reaction toward a new equi-
librium with production of a-ketoglutarate and alanine from pyruvate (continuously
supplied by glycolysis) and glutamate (falling in concentration).
Felig and Wahren11 have shown that the rate of release of alanine from muscle
depended on the exercise intensity and suggested a direct relation between the rate of
formation of pyruvate from glucose and alanine release. This led to the suggestion that
the glucose–alanine cycle also operated during exercise: glucose taken up by muscle from
the blood via glycolysis is converted to pyruvate and then via transamination to alanine
to subsequently serve as substrate for gluconeogenesis in the liver and help to maintain
blood glucose concentration during exercise. Here we propose that the alanine aminotrans-
ferase reaction primarily functions for de novo synthesis of a-ketoglutarate and TCA cycle
intermediates at the start of exercise. The augmented glycolysis during exercise thus
appears to serve a dual function. More pyruvate is generated (1) to function as a substrate
for pyruvate dehydrogenase and subsequent oxidation and (2) to force the alanine ami-
notransferase reaction toward production of a-ketoglutarate and TCA cycle intermediates,
and thus to increase TCA cycle activity and the capacity to oxidize acetyl-CoA derived
from pyruvate and fatty acid oxidation.
Another metabolic need for a high net flux through the alanine aminotransferase
reaction during exercise is the increase in the oxidation rate of BCAA that occurs, among
others, due to activation of the branched-chain a-keto acid dehydrogenase complex.9,37
This also leads to an increase in the rate of the BCAA aminotransferase reaction (leucine
+ a-ketoglutarate ´ a-ketoisocaproate (KIC) + glutamate). In case of leucine transamina-
tion and oxidation, acetyl-CoA is the end product, and therefore, leucine transamination
would lead to a net loss of TCA cycle intermediates when the glutamate would not be
converted back to a-ketoglutarate via the alanine aminotransferase reaction. Previously,
we suggested that leucine transamination via its draining effect on the TCA cycle may
play a role in the gradual decrease in TCA cycle intermediates and the development of
fatigue in muscles with a low glycogen content and, therefore, low capacity for alanine
production.9,16
An alternative mechanism that may generate TCA cycle intermediates during more
prolonged exercise leading to glycogen depletion is increased deamination rates of amino
acids in muscle. A gradually increasing ammonia production has been observed during
prolonged one-leg knee-extension exercise by van Hall et al. (Figure 8.2).14 As there was
no net breakdown of adenine nucleotides to IMP in that study (ATP Æ ADP Æ AMP Æ
IMP + NH4+), the conclusion was drawn that the ammonia production must have origi-
nated from net deamination of amino acids either via purine nucleotide cycling or via
glutamate dehydrogenase (source is unknown today). Deamination of amino acids in
contrast to transamination does not use a-ketoglutarate as an amino group acceptor
(Figure 8.1). As TCA cycle intermediates are formed in the degradation routes of valine,
isoleucine, aspartate, and glutamate (Figure 8.1), deamination will lead to net synthesis
of TCA cycle intermediates. Deamination of leucine leads to acetyl-CoA formation and,
therefore, does not influence the TCA cycle intermediate concentration. As explained
above (Section 8.3), one-leg knee-extensor exercise is an exercise model with exceptionally
high net rates of protein degradation. The amino acid exchange observed under these
1382_C08.fm Page 131 Tuesday, October 7, 2003 6:20 PM

Chapter eight: Metabolism of branched-chain amino acids in man 131

conditions (Figure 8.2) indicated that BCAA and glutamate released by the net breakdown
of muscle protein and taken up from the circulation were either deaminated (thus leading
to synthesis of TCA cycle intermediates) or used for the synthesis of vast amounts of
glutamine (Figure 8.2). Removal of amino groups from muscle in the form of glutamine
provides another mechanism for net synthesis of TCA cycle intermediates,15 as is illus-
trated by the following net reactions (see Figure 8.1 for the complete metabolic pathways):

2 glutamate Æ glutamine + a-ketoglutarate

Valine + isoleucine Æ succinyl-CoA + glutamine

Aspartate + isoleucine Æ oxaloacetate + glutamine

Valine + leucine Æ 2 acetyl-CoA + glutamine

Therefore, BCAA and glutamate released by the net breakdown of muscle protein and
taken up from the circulation seem to be used for net synthesis of TCA cycle intermediates
and glutamine during prolonged one-legged exercise. The extent to which these reactions
were accelerated was even larger when the exercise was performed by knee-extensor
muscles with a low glycogen content.15 It is far from clear whether dynamic whole-body
exercise as practiced by athletes during competition (cycling or running) leads to net
protein breakdown in muscle and helps to provide carbon skeletons for synthesis of TCA
cycle intermediates via the indicated reactions. Different stable isotope tracers used to
measure protein synthesis and degradation in laboratory conditions gave different
answers; different answers also were obtained for changes observed at the whole-body
level and muscle level (for references, see Wagenmakers9,16). Whole-body measurements
with L-[1-13C]-leucine suggest that there is increased whole-body net protein breakdown
during 1 to 6 h of cycling exercise at intensities of 30 to 50% VO2max, but with other tracers
(urea and [2H5-ring]-phenylalanine), no such increases have been observed.16 Carraro et
al.39 did not find an effect of cycling exercise at 40% VO2max on muscle protein synthesis
measured directly from the incorporation of [13C]-leucine. Furthermore, carbohydrate
ingestion as practiced by endurance athletes during competition reduces net protein
breakdown and amino acid oxidation and upgrades the relative importance of the alanine
aminotransferase reaction for TCA cycle anaplerosis.

8.7 Lessons from patients with McArdle’s disease


Patients with McArdle’s disease cannot substantially increase the glycolytic rate during
exercise due to the glycogen breakdown defect in muscle, and they therefore do not
increase muscle pyruvate. The arterial alanine and muscle alanine concentrations do not
increase in these patients during exercise.35,38 The muscle glutamate concentration is only
about 30% of normal and decreases only little during incremental exercise.35 This implies
that the anaplerotic capacity of the alanine aminotransferase reaction in these patients is
substantially reduced in comparison to that of healthy subjects. Sahlin and colleagues35
indeed observed that patients with McArdle’s disease could only marginally increase the
concentration of TCA cycle intermediates during incremental exercise. The maximal work
rate and oxygen consumption of these patients during cycling exercise is between 40 and
50% of the maximum predicted for their age and build. In ultraendurance exercise without
carbohydrate ingestion, healthy subjects have to reduce the work rate to about the same
level when the glycogen stores have been emptied, suggesting that muscle glycogen
1382_C08.fm Page 132 Tuesday, October 7, 2003 6:20 PM

132 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

indeed is needed to maintain high work rates potentially by means of its ability to establish
and maintain high concentrations of TCA cycle intermediates.
An excessive release of ammonia and glutamine and an excessive net breakdown of
muscle protein (several-fold more than in one-leg knee-extensor exercise in healthy sub-
jects) also were observed during two-legged cycling in patients with McArdle’s disease,38
indicating that deamination of BCAA and glutamate and synthesis of glutamine and TCA
cycle intermediates from glutamate and BCAA also provide alternative mechanisms of
TCA cycle anaplerosis in this muscle disease with zero glycogen availability and low
pyruvate concentrations. The fact that high exercise intensities cannot be maintained by
these patients and in glycogen-depleted muscles seems to indicate that these alternative
anaplerotic reactions are not as effective as the alanine aminotransferase reaction and only
allow muscular work at 40 to 50% of Wmax.

8.8 Conclusion
Since 1995 little new information has been obtained on the activity of the enzymes involved
in BCAA degradation, their tissue distribution, and regulation by physiological stresses
and hormones. Maybe a disproportionate effort goes into the modern large-scale genomic
and proteomic techniques as the regulation of enzyme activities will always continue to
form the basis for our understanding of the interorgan collaboration in the handling of
metabolites and of short-term cellular control mechanisms. Today our understanding of
BCAA metabolism in man has primarily been extended by a number of studies investi-
gating the exchange of amino acids across tissue beds using arteriovenous cannulations
and muscle biopsies and with studies investigating the fate of the amino group and carbon
skeleton with stable isotope tracers. The most important new insights are that the main
six amino acids metabolized in human muscle are the BCAA, glutamate, aspartate, and
asparagine. Only leucine and part of isoleucine generate acetyl-CoA and can be oxidized.
The other carbon skeletons are used solely for de novo synthesis of TCA cycle intermediates
and glutamine. The carbon atoms of the alanine released by muscle originate from glyc-
olysis of blood glucose and from glycogen breakdown. During the first 30 min of endur-
ance exercise, the alanine aminotransferase reaction functions to establish and maintain
high concentrations of TCA cycle intermediates and counteract the drain of the leucine
transaminase reaction. Deamination of amino acids and glutamine synthesis present alter-
native anaplerotic mechanisms in glycogen-depleted muscles but only allow exercise at
40 to 50% of Wmax. One-leg knee-extensor exercise leads to an excessive rate of net muscle
protein degradation. The liberated amino acids are used for synthesis of TCA cycle inter-
mediates and glutamine. Interactions between the metabolism of the BCAA, glutamate,
and aspartate and the TCA cycle clearly play a central role in the energy metabolism of
the exercising muscle.

References
1. Wagenmakers, A.J.M., Metabolism of branched-chain amino acids, in Amino Acid Metabolism
and Therapy in Health and Nutritional Disease, Cynober L.A., Ed., CRC Press, Boca Raton, 1995,
chap. 5.
2. Walser, M. and Williamson, J.R., Metabolism and Clinical Implications of Branched Chain Amino
and Ketoacids, Elsevier/North-Holland, Amsterdam, 1981.
3. Adibi, S.A., Fekl, W., Langenbeck, U., and Schauder, P., Branched Chain Amino and Keto Acids
in Health and Disease, S. Karger, Basel, Switzerland, 1984.
4. Odessey, R., Problems and Potential of Branched-Chain Amino Acids in Physiology and Medicine,
Elsevier Science Publishers, Amsterdam, 1986.
1382_C08.fm Page 133 Tuesday, October 7, 2003 6:20 PM

Chapter eight: Metabolism of branched-chain amino acids in man 133

5. Harris, R.A. and Sokatch, J.R., Branched chain amino acids, Methods Enzymol., 166, 1988.
6. Harper, A.E., Miller, R.H., and Block, K.P., Branched-chain amino acid metabolism, Annu.
Rev. Nutr., 4, 409, 1984.
7. Ruderman, N.B. and Lund, P., Amino acid metabolism in skeletal muscle: regulation of
glutamine and alanine release in the perfused rat hindquarter, Israel J. Med. Sci., 8, 295, 1972.
8. Goldberg, A.L. and Chang, T.W., Regulation and significance of amino acid metabolism in
skeletal muscle, Fed. Proc., 37, 2301, 1978.
9. Wagenmakers, A.J.M., Muscle amino acid metabolism at rest and during exercise: role in
human physiology and metabolism, Ex. Sport Sci. Rev., 26, 287, 1998.
10. Marliss, E.B., Aoki, T.T., Pozefsky, T., Most, A.S., and Cahill, G.F., Muscle and splanchnic
glutamine and glutamate metabolism in postabsorptive and starved man, J. Clin. Invest., 50,
814, 1971.
11. Felig, P. and Wahren, J., Amino acid metabolism in exercising man, J. Clin. Invest., 50, 2703,
1971.
12. Wahren, J., Felig, P., and Hagenfeldt, L., Effect of protein ingestion on splanchnic and leg
metabolism in normal man and patients with diabetes mellitus, J. Clin. Invest., 57, 987, 1976.
13. Eriksson, L.S., Broberg, S., Björkman, O., and Wahren, J., Ammonia metabolism during
exercise in man, Clin. Physiol., 5, 325, 1985.
14. van Hall, G., Saltin, B., van der Vusse, G.J., Söderlund, K., and Wagenmakers, A.J.M., Deam-
ination of amino acids as a source for ammonia production during prolonged exercise in
man, J. Physiol., 489, 251, 1995.
15. van Hall, G., Saltin, B., and Wagenmakers, A.J.M., Muscle protein degradation and amino
acid metabolism during prolonged knee-extensor exercise in humans, Clin. Sci., 97, 557, 1999.
16. Wagenmakers, A.J.M., Protein and amino acid metabolism in human muscle, Adv. Exp. Biol.
Med., 441, 307, 1998.
17. Wagenmakers, A.J.M., Tracers to investigate protein and amino acid metabolism in human
subjects, Proc. Nutr. Soc., 58, 987, 1999.
18. Kimball, S.R., Regulation of global and specific mRNA translation by amino acids, J. Nutr.,
132, 883, 2002.
19. Elia, M., Schlatmann, A., Goren, A., and Austin, S., Amino acid metabolism in muscle and
in the whole body of man before and after ingestion of a single mixed meal, Am. J. Clin.
Nutr., 49, 1203, 1989.
20. Elia, M. and Livesey, G., Effects of ingested steak and infused leucine on forearm metabolism
in man and the fate of amino acids in healthy subjects, Clin. Sci., 64, 517, 1983.
21. Hagenfeldt, L., Eriksson, S., and Wahren, J., Influence of leucine on arterial concentrations
and regional exchange of amino acids in healthy subjects, Clin. Sci., 59, 173, 1980.
22. Matthews, D.E., Marano, M.A., and Campbell, R.G., Splanchnic bed utilization of glutamine
and glutamic acid in humans, Am. J. Physiol., 264, E848, 1993.
23. Battezzati, A., Brillon, D.J., and Matthews, D.E., Oxidation of glutamic acid by the splanchnic
bed in humans, Am. J. Physiol., 269, E269, 1995.
24. Bylund-Fellenius, A.-C., Ojamaa, K.M., Flaim, K.E., Li, J.B., Wassner, S.J., and Jefferson, L.S.,
Protein synthesis versus energy state in contracting muscles of perfused rat hindlimb, Am.
J. Physiol., 246, E297, 1984.
25. Bolster D.R., Crozier S.J., Kimball, S.R., and Jefferson, L.S., AMP-activated protein kinase
suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian
target of rapamycin (mTOR) signaling, J. Biol. Chem., 277, 23977, 2002.
26. Hardie, D.G., Carling, D., and Carlson, M., The AMP-activated/SNF1 protein kinase sub-
family: metabolic sensors of the eukaryotic cell? Annu. Rev. Biochem., 67, 821, 1998.
27. Chang, T.W. and Goldberg, A.L., The metabolic fates of amino acids and the formation of
glutamine in skeletal muscle, J. Biol. Chem., 253, 3685, 1978.
28. Haymond, M.W. and Miles, J.M., Branched-chain amino acids as a major source of alanine
nitrogen in man, Diabetes, 31, 86, 1982.
29. Darmaun, D. and Déchelotte, P., Role of leucine as a precursor of glutamine a-amino nitrogen
in vivo in humans, Am. J. Physiol., 260, E326, 1991.
1382_C08.fm Page 134 Tuesday, October 7, 2003 6:20 PM

134 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

30. Chang, T.W. and Goldberg, A.L., The origin of alanine produced in skeletal muscle, J. Biol.
Chem., 253, 3677, 1978.
31. Perriello, G., Jorde, R., Nurjhan, N., Stumvoll, N., Dailey, G., Jenssen, T.G., Bier, D.M., and
Gerich, J.E., Estimation of glucose-alanine-lactate-glutamine cycles in postabsorptive hu-
mans: role of skeletal muscle, Am. J. Physiol., 269, E443, 1995.
32. Felig, P., Pozefsky, T., Marliss, E., and Cahill, G.F., Alanine: a key role in gluconeogenesis,
Science, 167, 1003, 1970.
33. Nurjhan, N., Bucci, A., Perriello, G., Stumvoll, N., Dailey, G., Bier, D.M., Toft, I., Jenssen,
T.G., and Gerich, J.E., Glutamine: a major gluconeogenic precursor and vehicle for interorgan
carbon transport in man, J. Clin. Invest., 95, 272, 1995.
34. Gibala, M.J., Tarnapolski, M.A., and Graham, T.E., Tricarboxylic acid cycle intermediates in
human muscle at rest and during prolonged cycling, Am. J. Physiol., 272, E239, 1997.
35. Sahlin, K., Jorfeldt, L., Henriksson, K.G., Lewis, S.R., and Haller, R.G., Tricarboxylic acid
cycle intermediates during incremental exercise in healthy subjects and in patients with
McArdle’s disease, Clin. Sci., 88, 687, 1995.
36. Aragón, J.J. and Lowenstein, J.M., The purine nucleotide cycle: comparison of the levels of
citric acid cycle intermediates with the operation of the purine nucleotide cycle in rat skeletal
muscle during exercise and recovery from exercise, Eur. J. Biochem., 110, 371, 1980.
37. van Hall, G., MacLean, D.A., Saltin, B., and Wagenmakers, A.J.M., Mechanisms of activation
of muscle branched-chain a-keto acid dehydrogenase during exercise in man, J. Physiol., 494,
899, 1996.
38. Wagenmakers, A.J.M., Coakley, J.H, and Edwards, R.H.T., Metabolism of branched-chain
amino acids and ammonia during exercise: clues from McArdle’s disease, Int. J. Sports Med.,
11, 101, 1990.
39. Carraro, F., Stuart, C.A., Hartl, W.H., Rosenblatt, J., and Wolfe, R.R., Effect of exercise and
recovery on muscle protein synthesis in human subjects, Am. J. Physiol., 259, E470, 1990.
1382_C09.fm Page 135 Tuesday, October 7, 2003 6:23 PM

chapter nine

The glutamate crossway


Yasuo Wakabayashi
Kyoto Prefectural University of Medicine

Contents
Introduction..................................................................................................................................135
9.1 Enzymes and tissues involved.........................................................................................135
9.1.1 Distribution of enzymes in the body .................................................................135
9.1.2 Ontogenic development of enzymes..................................................................138
9.1.3 Nutritional and hormonal effects on enzymes.................................................140
9.1.4 Tissue-selective expression of enzymes .............................................................141
9.2 Pioneering experiments led to fundamental findings .................................................142
9.2.1 Nutritional studies with isotopes .......................................................................142
9.2.2 Perfusion studies of tissues..................................................................................143
9.3 Overall flow of metabolites and significance in human nutrition.............................144
9.3.1 Derivation from animal studies ..........................................................................144
9.3.2 Implication from human clinical studies...........................................................145
References .....................................................................................................................................147

Introduction
In this chapter, metabolism of glutamate (or glutamine) as a precursor for arginine and
proline is discussed. Interplay between organs, regulation, and nutritional, hormonal, and
molecular aspects will also be considered. For healthy humans and adult mammals, these
four amino acids are nutritionally dispensable.1 However, supplementation of arginine
and glutamine is essential for cultured cells.2 Thus, the dietary dispensability of arginine
and glutamine does not mean that individual cells constituting the body can form both
amino acids by themselves.

9.1 Enzymes and tissues involved


9.1.1 Distribution of enzymes in the body
Figure 9.1 and Table 9.1 summarize distribution and activity of enzymes involved in this
pathway on tissue basis. The enzymes can be categorized into three subgroups based upon

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 135
1382_C09.fm Page 136 Tuesday, October 7, 2003 6:23 PM

136 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 9.1 Organs involved in synthesis and degradation of arginine and proline. Typical interorgan
relationships are shown. Citrulline synthesis from glutamate definitely requires the small intestine.
Conversions of citrulline to arginine, arginine to proline, or proline to glutamate are not exclusively
dominated by the kidney and liver. Enzymes denoted by Arabic numbers are (with abbreviations
in parenthesis) as follows: 1, phosphate-dependent glutaminase (GLNase); 2, pyrroline-5-carboxylate
synthase (PCS); 3, ornithine aminotransferase (OAT); 4, ornithine transcarbamylase (OTC); 5, argin-
inosuccinate synthetase (ASS); 6, argininosuccinate lyase (ASL); 7, chemically spontaneous and
reversible reaction, although the equilibrium strongly favors P5C formation; 8, pyrroline-5-carbox-
ylate reductase (PCR); 9, arginase (ARGase); 10, carbamyl-phosphate synthetase (CPS); 11, N-acetyl-
glutamate synthase (AGS); 12, proline oxidase (PROox); 13, pyrroline-5-carboxylate dehydrogenase
(PCDH). Abbreviations for specific amino acid intermediates: GSA, glutamic-g-semialdehyde; P5C,
pyrroline-5-carboxylate; Cit, citrulline; ASA, argininosuccinate; Orn, ornithine; AGA, N-acetyl-
glutamate.

the extent of ubiquity. The most ubiquitous enzymes are ornithine aminotransferase (OAT)
and pyrroline-5-carboxylate reductase (PCR). The second enzymes are moderately ubiq-
uitous, but there is some convergency. They are phosphate-dependent glutaminase
(GLNase), glutamine synthetase (GS), argininosuccinate synthetase (ASS), argininosucci-
nate lyase (ASL), pyrroline-5-carboxylate dehydrogenase (PCDH), and arginase (ARGase).
The third enzymes display extreme polarity. Several tissues are unable to detect activity.
They are pyrroline-5-carboxylate synthase (PCS), carbamoyl-phosphate synthetase (CPS),
ornithine transcarbamoylase (OTC), N-acetylglutamate synthase (AGS), and proline oxi-
dase (PROox). One will notice that the small intestine is the only tissue where all the
enzymes required to construct citrulline or ornithine from glutamate or glutamine are
assembled, particularly PCS, CPS, OTC, and AGS. On the contrary, enzymes from citrulline
to arginine, ASS and ASL, are widespread, though relative abundance is found in the liver
and kidney.3 This is in accord with Eagle’s observation that all the human cell lines studied
lived satisfactorily with citrulline in place of arginine.2 Enzymes for proline and arginine
Table 9.1 Distribution of Enzymes Involved in Arginine, Proline, Glutamine, and Glutamate Interconversion
Tissue GLNase GS PCS OAT CPS OTC ASS
Small intestine 19 252 117 10 100 68
Liver 5 244 0 188 284 4110 1386
Chapter nine:

Kidney 24 221 0 166 8 5 677


Brain 16 337 1 13 4 0 51
Thymus 6 22 8 0 2
Lung 18 0 15 0 2 22
Spleen 6 166 0 15 0 0 11
Muscle 1 10 0 13 0 0
Pancreas 12 40 3 2
Salivary gland 1 10 12
1382_C09.fm Page 137 Tuesday, October 7, 2003 6:23 PM

Mammary gland 3
References (95, 96) (97) (56) (31) (98) (98) (99)
The glutamate crossway

Tissue ASL AGS PCR PROox PCDH ARGase


Small intestine 6 31 28 0.0 0.4 129
Liver 308 327 33 5.3 33.5 2545
Kidney 41 0 24 2.0 14.5 57
Brain 10 0 15 0.4 0.4 3
Thymus 3 20 0.3 2
Lung 10 14 7 0.1 0.4 5
Spleen 14 7 19 0.0 0.4 1
Muscle 5 0 5 0.5 0.4 0
Pancreas 0 10 0.0 0.9 103
Salivary gland 8 57 0.0 0.4 170
Mammary gland 5 0.8 163
References (100) (101) (102) (102) (102) (103)

Note: Original data were rounded so that figures may expedite comparisons. This distribution profile is intended to reflect whether these
enzymes are distributed ubiquitously or limited to specific tissues. Activities are expressed as pmol/mg/min with PCS and AGS;
nmol/mg/min with GS; mmol/g/min with ARGase, GLNase, PCR, PROox, and PCDH; mmol/g/h with CPS, OTC, ASL, and OAT;
and nmol/mg/h with ASS. Abbreviations of enzymes are the same as in Figure 9.1 except that GS denotes glutamine synthetase.
Muscle represents skeletal muscle except that the heart muscle was reported with OAT, PROox, and ASL. The mammary gland
was taken from pregnant or lactating animals.
137
1382_C09.fm Page 138 Tuesday, October 7, 2003 6:23 PM

138 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

degradation, PROox, PCDH, and ARGase, are sparse in many tissues. ARGase is a mixture
of isozymes, type I and type II. Type I is a cytosolic enzyme found almost exclusively in
the liver as a part of urea cycle enzymes, whereas type II is in the mitochondria and
distributed, more or less, in almost all extrahepatic tissues.4 Intracellular location of
enzymes is in either cytosol or mitochondria. PCS, PROox, and GLNase are bound to
mitochondrial inner membranes.5 GLNase has two isozymes, liver type and kidney type.
The liver type is found exclusively in the liver and supplies ammonia to the urea cycle,
and the kidney type is ubiquitous and essential for pH homeostasis in the kidneys. PCDH,
OAT, OTC, CPS, and AGS are in mitochondrial matrix. Human PCDH has been identified
as aldehyde dehydrogenase isozyme IV. Besides pyrroline-5-carboxylate (P5C), adipic and
glutaric semialdehyde are other substrates.6 OTC and CPS activities in the liver are much
higher than those in the small intestine on a tissue mass basis. However, concentrations
per mitochondria basis, as estimated by the electron microscope immunocytochemistry
technique, differ only by twofold. In addition, CPS and OTC mRNAs are enriched twice
as high in the liver as in the small intestine when an identical amount of mRNA or poly(A)-
rich RNA from the tissues was analyzed by dot blot or Northern hybridization or in vitro
translation.7 Therefore, enrichment of cells by the mitochondria and the population in
given tissues of the cells expressing enzyme activities explain the discrepancy. GS, ASS,
ASL, and PCR are in cytosol. Erythrocyte PCR exhibits vigorous conversion of P5C to
proline with concomitant oxidation of NADPH2 to NADP+. Formed NADP+ stimulates
the pentose phosphate pathway to produce a large amount of phosphoribosylpyrophos-
phate (PRPP). PRPP will be efficiently used for salvaging purine bases.8
An inefficient kinetic property of OAT for ornithine synthesis had been argued.
Strecker9 showed minor conversion from P5C to ornithine with partially purified rat liver
enzyme and obtained an equilibrium constant of 70, which suggested that OAT works
much easier toward formation of P5C but not ornithine. McGivan et al.10 showed that
liver mitochondria failed to synthesize ornithine from P5C, unless rotenone was incorpo-
rated in the system to prevent P5C from being consumed by PCDH and NADH dehydro-
genase. In the meantime, Herzfeld and Raper11 reported that a mitochondria fraction of
young rat small intestine was effective in converting P5C to ornithine, although the rate
was one sixth that of the reverse reaction. Nonetheless, they were unable to detect ornithine
production from P5C by similar fractions of submaxillary gland and liver under similar
conditions.11 Since purified OAT proteins from the liver, kidney, and small intestine are
identical by kinetic, electrophoretic, and immunological criteria,12 the small intestine is
unique in its high OAT and low PCDH content, which permits ornithine synthesis once
P5C is supplied by PCS. When [14C]glutamate was injected into mice fed an arginine-free
diet, [14C] was incorporated into tissue arginine and ornithine. However, administration
of gabaculine, a potent suicide inhibitor of OAT, reduced the incorporation to one-third
of control. This provides direct evidence for involvement of OAT in arginine and ornithine
synthesis in vivo from glutamate.13 In the meantime, Ross et al.14 demonstrated conversion
of [14C]glutamate to [14C]ornithine using cell-free extract of rat intestinal mucosa.

9.1.2 Ontogenic development of enzymes


Developmental patterns of relevant enzymes are twofold. Typically, synthetic enzymes
are premature, whereas degradative enzymes are gradual.
PCS activity in fetus small intestine immediately before birth is one fifth that of adult
rats.15 The activity on postnatal day 1 is 180 pmol/mg/min, a value similar to that of
adults and is triplicated to 540 pmol/mg/min at day 14 and reduced to an adult level at
day 24. Thus, PCS of small intestine is high during suckling period and reduces with the
beginning of weaning. Wu et al.16 showed that the glucocorticoid surge at this postnatal
1382_C09.fm Page 139 Tuesday, October 7, 2003 6:23 PM

Chapter nine: The glutamate crossway 139

period is the trigger for the enhancement of PCS activity in the small intestine, and the
enhanced activity of PCS determines the high output of citrulline from this tissue.16
Development of GLNase in the small intestine is similar to the profile of PCS.17 The highest
peak is shown in the midst of the weaning period. OAT in small intestine develops with
a prominent activation by about 400% during the suckling period.11 The crucial role of
this peak for the intestinal supply of ornithine and citrulline in the normal development
was shown by a gene-targeted knockout technology. OAT-null mice developed hypoargin-
inemia, hypocitrullinemia, hypoornithinemia, and hyperammonemia during the early
postnatal period. Without arginine supplementation, the mice died immediately after
birth, but with daily supplementation of arginine, the mice survived. Interestingly, the
adult mice developed hyperornithinemia and retinal degeneration similar to human
Gyrate atrophy (i.e., OAT deficiency).18 Therefore, the role of OAT in the developing period
favors ornithine synthesis and that in adult degradation. Liver OAT stays low until day
18 and then increases to an adult level after weaning. Also, liver ARGase develops with
a similar profile.11 CPS in the small intestine expresses the highest activity at birth, and
then activity monotonously decreases to an adult level.19 OTC in the small intestine is
more than 150% of adult activity at the 2nd postnatal day and approaches adult activity
at the 18th day.11 The mRNA level of CPS in the small intestine is highest at the 21st
gestational day; after birth, it decreases to an adult level gradually.20 On the contrary, the
mRNA of liver CPS continues to increase gradually to an adult level after birth. Thus, the
mRNA profiles explain the developmental changes in CPS activities of both tissues. mRNA
of OTC in the small intestine is highest at the 21st gestational day and accords to its activity
profile, except that the activity immediately before birth is too low for the highest mRNA
level at this stage. Also, the prenatal activity of liver OTC seems to be lower for the
abundant mRNA for the enzyme.11,20
PCR activity in the small intestine has two peaks, highest at birth and day 24.11 GLNase
protein and mRNA have their peak at postnatal day 19, and then mRNA drops, while
protein remains almost at the adult level.21 Activity of AGS in the fetus intestine is 14
pmol/mg/min and expresses a sudden peak of 64 pmol/mg/min at the first postnatal
day. It drops to 20 pmol/mg/min at day 3 and then gradually increases to an adult level.15
The AGS profile of the liver is different. The activity in fetuses is only one twentieth that
of adults. It increases gradually after birth to an adult level for 10 weeks. Immunocy-
tochemical staining of ASS and ASL proteins in the kidneys revealed that both proteins
were detectable at the 15th gestational day and the density increased up to gestational
day 23. mRNAs for ASS and ASL were 2 and 7% of adult levels, respectively, at the 15th
day. The mRNAs increased toward delivery when 50% or more of an adult level was
expressed.22
The significant role of the small intestine in citrulline-to-arginine conversion in the
perinatal period was visualized by a study where spatiotemporal expression patterns of
mRNAs of OAT, CPS, OTC, ASS, and ASL, and proteins of CPS and ASS were investi-
gated.23 The mRNAs of ASS and ASL were found only in the upper villi; those of OAT,
CPS, and OTC were condensed in the crypts. ASS protein was located in the upper villi
up to the weaning period but was almost lost in the adult villi. CPS protein was found
over all layers. Other enzymological studies quantified the synthesis of arginine in the
postnatal intestines and established the significance of this phenomenon.24–26 Overexpres-
sion of ARGase I in the developing intestine, therefore, provokes a serious effect: the
promoter and enhancer element of the rat intestinal fatty acid-binding protein (FABPi)
gene fused to ARGase I structural elements of rat was constructed to generate ARGase I
overexpressing transgenic mice.27 The enterocytes of the mice expressed ectopic ARGase
I extensively, and the suckling mice became arginine deficient. Typical phenotypes were
hypoargininemia, about one third of controls, and retardation of hair, muscle growth, and
1382_C09.fm Page 140 Tuesday, October 7, 2003 6:23 PM

140 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

development of the lymphoid tissues. Specifically, an impairment of transition from the


pro-B cells to pre-B cells in the bone marrow was elucidated. Daily injection of arginine
prevented these completely. This observation demonstrated that the synthesis of arginine
by the intestine of suckling mice is essential for normal development, and also that
extraintestinal conversion of citrulline to arginine is premature at early postnatal period.

9.1.3 Nutritional and hormonal effects on enzymes


The molecular mass of purified OTC from the liver and small intestine (38,000 Da), their
pI (isoelectric point) (7.3), the identical reactivity against antibody against the liver enzyme
as detected by Western hybridization, the identical size of mRNA as visualized on North-
ern hybridization using cDNA for the liver OTC, and the specific activities of the pure
preparations (928 to 966 mmol/min/mg) indicate that both proteins are the same gene
product.28 However, intestinal OTC activity decreased from 8.8 to 5.7 mmol/min/mg DNA
when 15% casein diet was changed to 60%, whereas that of the liver increased from 0.8
to 1.3 mmol/min/mg DNA. Moreover, mRNA abundance as probed by [32P]-labeled OTC
cDNA in the liver increased from 930 to 2300 cpm/100 mg RNA, whereas that in the small
intestine decreased from 370 to 210 by the protein surplus.29 A 70% casein diet compared
with 5% increased OAT activity by 10-fold in liver, whereas there was no change in the
small intestine and kidney.12 mRNA levels for CPS and OTC in the liver responded to
glucagon positively by 1.5 to 2.2 times, but those in the small intestine did not.20 A 50%
casein diet compared with 8% did not affect CPS activity in the small intestine at all, while
it augmented the liver CPS by threefold.19 Nonetheless, reduction in caloric intake without
change in protein intake enhanced intestinal CPS activity twofold, with mRNA level
unchanged, which suggested posttranscriptional activation of CPS in the intestine. At the
same time, the activity in the liver increased by five times with concomitant threefold
increases of mRNA and protein.30 The apparently opposite regulations on OTC, CPS, and
OAT in the liver and small intestine are consistent with the role of the organs for the
synthesis of metabolites and their breakdown (urea synthesis), respectively. OAT activity
in the female kidney is twice as high as that in the male. Ovariectomy erases the difference.
Estrogen enhances kidney OAT in both sexes.31 Lyons and Pitot32 investigated responses
of OAT activity and the relative rate of its synthesis in vivo as estimated by pulse-labeling
rats with [3H]leucine combined with immunoprecipitation of OAT proteins. Triiodothy-
ronine (T3) injection into rats increased activity and synthesis by twofold in the kidney
but not in the liver. Glucagon enhanced activity in the liver by 2-fold and its synthesis by
10-fold, but had no effect on the kidney enzyme. Thyroidectomy reduced activity and
synthesis by 50% only in the kidney. This treatment abolished the induction of kidney
OAT by estrogen. Adrenalectomy had no effect on the basal activity and synthesis in the
liver and kidney.
The kidney arginine synthetase (ASS + ASL) increased threefold in rats fed an 80%
casein diet compared with an 8% diet.33 mRNA abundance of ASS and ASL in the kidney
became 2.5 times as rich as control after starvation for 6 days and doubled after transition
from 27 to 60% casein. ASS was boosted by dibutyryl cAMP by 2.3-fold, but failed to
respond to dexamethasone. ASL did not respond to either compound.34 Interestingly,
activities of ASS and ASL of HeLa, KB, and L cell strains were multiplied up to 15-fold
when these cells were grown in arginine-deficient media.35 ARGase in the liver increases
its activity and mRNA abundance coordinately after starvation, a high-protein diet, and
dexamethasone treatment.36 Also, glucagon augments ARGase activity.
PCR in the developing small intestine is elevated up to fivefold by starvation and cortisol,
while liver PCR is not.37 PCS is regulated by the negative feedback control sensitive to
intramitochondrial ornithine.38 Lysine, arginine, proline, and citrulline are ineffective. Also,
1382_C09.fm Page 141 Tuesday, October 7, 2003 6:23 PM

Chapter nine: The glutamate crossway 141

glucose is the crucial substrate to provide NADPH, via the pentose phosphate pathway, to
PCS as the mandatory reductant in the reaction.39 Intestinal GLNase does not respond to
acid–base changes, whereas kidney GLNase increased four times by NH4Cl-induced meta-
bolic acidosis.17 PROox activity in adrenalectomized rat livers is 50% of controls, and hydro-
cortisone restored normal activity.40 A 60% protein diet compared with 25% enhanced liver
activity by twofold, but had no effect to the kidney, brain, and heart activity.41
Skeletal muscle is the major tissue to synthesize and release glutamine into systemic
circulation.42 Administration of dexamethasone to rats at a dose that imitates corticosteroid
levels under severe stress (0.5 mg/kg/day) caused loss of muscular weight and increased
GS activity in the plantaris muscle by 40% with concomitant rise of its mRNA by five times.
This rise was accompanied by a 2.5 times enrichment of GS mRNA in total translatable
mRNA. A dose up to 5.0 mg resulted in, by far, prominent augmentation of activity and
mRNA content. However, such treatment did not affect heart GS.43 Inhibition and activation
of GS by exercise and neural denervation, respectively, are other regulatory factors.44

9.1.4 Tissue-selective expression of enzymes


The mechanism of tissue-selective expression of genes mostly depends on how regulatory
elements, including promoters of those genes, can find appropriate transcription factors
(TFs) in the particular cells. There are two types of TFs, ubiquitous and tissue-specific
ones. Many TFs are required for tissue-selective expression of OTC and CPS.
A recombinant gene, a 1.3-kb segment of the 5' flanking region of rat OTC gene fused
to rat OTC cDNA, was made to generate the transgenic mice. The transgene was expressed
only in liver and small intestine. However, the hepatic transgene expression was much
lower than the endogenous gene expression.45 The lower expression was normalized when
a 1.6-kb segment about 11 kb upstream from the transcription start site was appended to
the recombinant. This far upstream region contains two hepatocyte nuclear factor (HNF)-4
binding sites and two CCAAT/enhancer binding protein (C/EBP) sites. By a cotransfection
analysis in a nonhepatic cell line, the combination of HNF-4 and C/EBP, both of which
are liver-specific TFs, normalized the expression in the liver of the recombinant OTC
gene.46 Also, two more elements were found in the promoter region to which HNF-4 and
chicken ovalbumin upstream promoter-transcription factor (COUP-TF) can bind to acti-
vate and suppress, respectively, the promoter activity of OTC gene.47 HNF-4 is selectively
expressed in the liver and intestine, whereas chicken ovalbumin upstream promoter-
transcription factor (COUP-TF) is ubiquitous.48 Therefore, the repression of OTC promoter
by the competitive binding of COUP-TF against HNF-4 may be involved in empty expres-
sion of OTC in nonhepatic tissues.
The crucial role of HNF-4a was demonstrated by introducing liver-specific destruction
of HNF-4a in mice.49 The liver-specific HNF-4a-null mice survived, although they devel-
oped mild hyperammonemia and low plasma urea. Western blot showed that HNF-4a
and OTC were almost lost in the liver, whereas CPS, ASS, ASL, and ARGase were normally
expressed. The residual OTC activity was 5% of the control. However, CPS and OTC
proteins in the intestine remained intact. Therefore, intestinal OTC and CPS might com-
pensate, at least partly, the role of the liver urea cycle. A dual role in ammonia detoxifi-
cation and citrulline synthesis by the small intestine will be an intriguing matter to be
elucidated.
Interplay between promoter and far upstream elements was also revealed with the
CPS gene.50 Transgenic mice were analyzed for the expression of the reporter gene: the
first transgene was a 1.8-kb fragment immediately upstream from the transcription start
site of a rat CPS gene fused to a reporter gene (CAT), the second one was a 12-kb fragment
instead of 1.8-kb one, and the third one was a minimized promoter and 469-bp enhancer
1382_C09.fm Page 142 Tuesday, October 7, 2003 6:23 PM

142 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

sequence at 6.3 kb upstream. No CAT activity was found in the livers of the transgenic
mouse of the first group. In the second group, CAT activity was high in the liver and
moderate in the intestine; there was no activity in the spleen and kidney. mRNA of CAT
was distributed in the liver at a periportal location, as was mRNA of endogenous CPS.
The mRNA was also found in the epithelium of the small intestine, and there was more
density in the crypt region, which was the same for the endogenous CPS expression. The
developmental changes of the CAT mRNA in the liver and intestine of the second trans-
gene were the same as those of the endogenous CPS. Dexamethasone treatment of the
transgenic mouse resulted in a severalfold increase of CAT mRNA in the liver but not
intestine, reproducing exactly the expression of endogenous CPS. The third transgene was
expressed only in the periportal area. Therefore, the far upstream enhancer, 469 bp, at –6.3
kb must determine the periportal location of CPS in the liver. Within this enhancer
sequence several regulatory elements like CRE, GRE, HNF-3, and HNF-4, or elements
with homology to such, are identified. The 469-bp enhancer is crucial for the hormone-
dependent expression of CPS gene in hepatocyte and requires a GAG box (which contains
a direct repeat of the nonamer, GAGGAGGGG) in the promoter region.51
ASS and ASL genes have sequences in the promoter proximal region for Sp1 or NF-Y,
this explains the widespread expressions of the enzymes.48 An octameric element in the
distal promoter region of ASS is similar to that found in the albumin gene, and therefore,
it may promote expression of ASS in the liver.
Observations on regulations of gene expression at posttranscriptional levels are accu-
mulating. A study of villus-crypt axis distribution of mRNA and protein of CPS in the
developing intestine of the rat and human found mosaicism in cellular expression of CPS
protein under uniform expression of CPS mRNA.52 The cells with mRNA but without
protein suggest cell-specific posttranscriptional processing of mRNA. Diversity in the
5¢-untranslated region (UTR) of mRNA transcripts brought an important effect in the
tissue-specific expression of ASS protein in endothelial cells.53 A strict colocalization of
ASS and ASL in a segregated subcellular compartment of endothelial cells, plasmalemmal
caveolae, was demonstrated and construed for the presence of tight channeling of citrulline
and arginine for nitric oxide synthase.54
Recently, an intriguing question as to the consistency of mRNA abundance in tissues
with the activity of the product was presented for PCS. Full-length human cDNA encoding
PCS was cloned and found to encode a bifunctional protein for g-glutamyl kinase and
g-glutamyl phosphate reductase activities.55 The mature peptides for PCS activity exist in
two isoforms, long and short ones. The short isoform is shorter by only two amino acids
interposed in the kinase domain of the long one, but this minimum difference confers the
ability of regulation by ornithine on the short isoform. Northern blot showed that most
human tissues had a single predominant mRNA transcript of 3.6 kb. A consensus sequence
for the feedback inhibition by proline in plants was conserved, although no inhibition by
proline of mammalian enzyme has been reported. The possibility that the short isozyme
works in the intestine for citrulline synthesis while the long isozyme works in the periph-
ery for proline synthesis is attractive. Probably, cell populations expressing activity in each
tissue and specific activities of both isozymes would help to understand the difference
between mRNA abundance and the activity profile.56

9.2 Pioneering experiments led to fundamental findings


9.2.1 Nutritional studies with isotopes
Womack and Rose57 fed rats with basal amino acid diets lacking glutamic acid, proline,
and arginine, and the rats gained 49 g on average. When they supplied additional
1382_C09.fm Page 143 Tuesday, October 7, 2003 6:23 PM

Chapter nine: The glutamate crossway 143

glutamate, proline, or arginine, rats gained 58, 60, and 78 g, respectively. In another
experiment, rats gained 91 g on an arginine-supplemented basal diet, whereas the addition
of proline and glutamic acid separately and in combination to this basal diet promoted
rats to gain 97, 102, and 106 g, respectively. They concluded that proline and arginine can
be formed from glutamate, and these are interconvertible. Scull and Rose58 fed rats with
arginine-depleted casein hydrolysate diet, and found that two to three times as much
arginine accumulated in the carcass as estimated from dietary arginine intake.
In 1951, the first direct evidence that arginine and proline are formed from glutamate
was obtained.59,60 [14C]glutamate administered to rats by peritoneal injection or orally was
subsequently found as [14C]arginine and [14C]proline in the protein hydrolysate of carcass.
A similar feeding experiment by Stetten and Schoenheimer61 with proline that was doubly
labeled with deuterium and 15N showed that the carbon skeleton of proline was found as
deuterium in the arginine fraction of the body protein. Not only the amidine group of
arginine but also the a- and d-amino groups of ornithine hydrolyzed from arginine con-
tained 15N. Oxidation to glutamate was shown by detecting deuterium and 15N in the
glutamate fraction. Ornithine skeleton was also labeled with deuterium. Stetten62 labeled
a- or d-amino nitrogen of ornithine with 15N, fed it to rats, and found that about 6.5% of
the tissue arginine was derived from the dietary ornithine. a-Amino group contributed
more efficiently to proline and to a lesser extent to glutamic acid. d-Amino group, in
contrast, contributed much to glutamic acid. These results suggest that the initial step in
the ornithine catabolism is the breakdown of ornithine by preferential loss of the d-amino
nitrogen leading to glutamic g-semialdehyde.62 Clutton et al.63 demonstrated by a similar
feeding experiment using deuterated ornithine that 7.5% of arginine in carcass protein
was derived from the deuterated ornithine. Deuterated ornithine was also used by Roloff
et al.64 and found to be converted to glutamic acid and proline in vivo.
These studies altogether proved that glutamate, proline, ornithine, and arginine are
fully interchangeable in living animals.

9.2.2 Perfusion studies of tissues


Windmueller and Spaeth65 elaborated a novel recirculation circuit for studying the small
intestine. Pooled heparinized blood collected from donor rats was pumped into the supe-
rior mesenteric artery after oxygenation. Blood return was collected from the superior
mesenteric vein under venous pressure surveillance. Lymph drains were simultaneously
collected. In early experiments, [14C]glutamine was infused by a separate arterial perfusion
pump, and the venous outflow was serially collected and analyzed to detect single-passage
metabolism. Later, they refined the procedure to detect an even segmental metabolism of
intestine where vascular or luminary perfusion was achieved.66 First of all, 20 to 30% of
plasma glutamine was constantly cleared off from the perfusate in each transit through
the intestines of rats, dogs, cats, hamsters, and monkeys, whereas no other amino acids
were taken up. Carbon of trapped glutamine was recovered as CO2 (57%), citrulline (6%),
proline (5%), alanine (1%), lactate (8%), and citrate (5%) in the venous collections. Besides,
small amounts of aspartate, ornithine, and glutamate were found. Nitrogen of glutamine
was quantitatively distributed in citrulline (34%), alanine (33%), ammonia (23%), and
proline (10%). When glutamine or glutamate was administered intraluminally, 92% of
glutamine administered in the jejunal segments was recovered in the venous output. Of
this glutamine, 37% remained intact; the rest was distributed among similar metabolites
found in the vascular administration, with a similar ratio. With glutamate, 81% was
recovered in the venous blood. However, 97% of recovered glutamate had been metabo-
lized during its translocation, suggesting that the rate-limiting step is GLNase. Thus,
1382_C09.fm Page 144 Tuesday, October 7, 2003 6:23 PM

144 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

glutamine was metabolized similarly by the mucosal cells, regardless of its entrance,
through luminal front or basolateral front.
In other experiments, rat livers were perfused with blood supplemented with graded
levels of ammonia and 21 amino acids to generate maximally 18.8 mmol/min of urea,
which is almost equal to that observed in rats fed a 30% protein diet.67 However, citrulline
release was 36.2 nmol/min, only equivalent to 13% of the rate of citrulline release by the
intestine. Therefore, unless unphysiologically high ornithine and ammonia are loaded
simultaneously, excess citrulline will not accumulate and flood into venous output. Also,
when [14C]citrulline added to the perfusate was recirculated about 40 passes through the
liver, still more than 90% citrulline remained unmetabolized. The residual radioactivity
was in urea. No radioactive arginine was released.
The kidneys extracted about 35% of the citrulline from blood in each pass; this much
of citrulline uptake was equivalent to 83% the rate of intestinal citrulline release. The
kidney released 75% of the citrulline as arginine into the systemic circulation.67 Levillain
et al.68 demonstrated the site of arginine formation along with the nephron. Under stere-
omicroscopic observation, pieces of glomerulus, proximal convoluted tubule, and others
were collected and reacted with [14C]citrulline, arginase, and urease. Arginine production
was counted as [14C]CO2. Arginine synthesis coexisted with the proximal tubules, with
decreasing intensity from the proximal convoluted to the proximal straight tubules, from
122 to 41 fmol/min/mm length. Arginine synthesis increased linearly up to 0.4 mM
citrulline. The proximal straight tubules retained substantial arginase, and this hydrolyzed
40 to 64% arginine formed to urea and ornithine, whereas no hydrolysis occurred in the
proximal convoluted tubules. Therefore, arginine to be returned into the renal vein must
be fabricated in the proximal convoluted tubules. Arginine formed in proximal straight
tubules may contribute to urine concentration after its hydrolysis by arginase to urea.
Localization of ASS and ASL to proximal tubules by immunohistochemical staining of
mouse kidney sections was reported.22 Dhanakoti et al.,69 using similarly prepared kidney
cortical tubules, showed that aspartate that is the second substrate in arginine synthesis
from citrulline can be substituted with glutamate or glutamine just as effectively. Also,
arginine synthesis was proportional to the citrulline concentration in the medium up to
0.5 mM, particularly at around 0.06 mM, a physiological value. Moreover, citrulline infu-
sion from saphenous vein raised plasma citrulline from 62 to 242 mM, while renal uptake
of citrulline increased from 61 to 224 nmol/min/100 g of body weight (BW) and renal
release of arginine from 79 to 265 nmol/min/100 g of BW. A parallel rise of plasma arginine
from 175 to 244 mM was observed. Linear regression of the relationship of citrulline uptake
and arginine output revealed stoichiometric unity. Thus, the plasma citrulline concentra-
tion is the primary factor to determine the rate of arginine production by the kidneys.
Exchange of stable isotopes between citrulline and arginine in the plasma from can-
nulated dogs revealed that the kidney’s contribution for arginine synthesis was about
60%, and the remaining 40% was by extrarenal tissues.70 Also in this species, substantial
amounts of citrulline were derived from the liver as well as the small intestine.

9.3 Overall flow of metabolites and significance in human nutrition


9.3.1 Derivation from animal studies
The significance of the endogenous arginine synthesis in growth and ammonia disposal
has been illustrated by the species differences in the response of animals put on arginine-
depleted status. The typical case is represented by rats and cats, as omnivores and carni-
vores, respectively. Postweaning rats fed on an arginine-deficient diet for 8 days gained
11 g, whereas those on an arginine-replete diet gained 33 g. Citrulline completely replaced
1382_C09.fm Page 145 Tuesday, October 7, 2003 6:23 PM

Chapter nine: The glutamate crossway 145

arginine (35 g), although ornithine failed (13 g).71 Urinary excretion of orotic acid, as a
measure of depleted intermediates of the liver urea cycle, was multiplied 50-fold by
arginine depletion, but was normalized by citrulline or ornithine supplementation. On
the contrary, cats fed on an arginine-depleted diet could not maintain weight and lost 48 g
daily; plasma arginine dropped to 25% of normal.72 However, citrulline supplementation
supported cat’s growth completely, whereas ornithine did not. Plasma arginine was nor-
malized by citrulline feeding but not ornithine.73 Furthermore, some of the cats fed an
arginine-deficient diet acutely developed hyperammonemia and its clinical symptoms. In
such cats, the liver concentration of ornithine was significantly low compared with cats
without hyperammonemia. This suggests that OTC of the hyperammonemic liver was
not sufficiently saturated with endogenous ornithine under the highly catabolic dietary
conditions with arginine exhaustion.74 In fact, PCS activity of total cat intestine is remark-
ably low compared with rats: 16 nmol/min/kg of BW to 308 nmol/min/kg of BW.75
Deprivation of the intestinal citrullinogenesis has been attempted by two different
studies. Hoogenraad et al.76 used d-N-(phosphonacetyl)-L-ornithine (PALO) linked to
glycylglycine to deliver PALO through the cell membrane of intestinal mucosa and to
selectively inhibit OTC. PALO is a transition state analogue and a potent and specific
inhibitor of OTC. When rats were fed on a 30% casein diet treated with arginase to replace
arginine with ornithine, rats gained 15 g for 6 days. However, when rats were given
glyglyPALO in drinking water, the rats lost 15.5 g during the same interval. Interestingly,
supplementation of 1% of either citrulline or arginine in the diet resumed their weight
gain, 9.4 and 5.7 g, respectively. Furthermore, rats fed on normal rat chow, which we may
certainly presume had a sufficient dietary intake of arginine, lost weight when they
received glyglyPALO. Serum arginine reduced to one fourth by glyglyPALO but normal-
ized by addition of 1% citrulline. Serum ammonia was not elevated in any experiment,
which assured that this inhibitor had no effect on liver OTC. These results clearly indicate
that citrulline synthesis by the small intestine has primary importance to suffice arginine
demand for growth, and this fraction will not be fully replaced by dietary arginine.
Another experiment confirmed the indispensability of the small intestine for in vivo
synthesis of arginine.77 Eighty percent of the small intestines of rats was removed, and
the rats were fed by one of four experimental diets: complete (Complete), arginine-free (-
Arg), proline-free (-Pro), and arginine- and proline-free diet (-Arg,Pro). After 4 weeks, rats
fed -Arg or -Arg,Pro lost weight by 28 to 32 g, whereas rats fed Complete gained 80 g. -
Pro resulted in a gain of 58 g. Nitrogen balance was negative in rats fed -Arg and -Arg,Pro.
Urinary orotic acid was high in -Arg and -Arg,Pro but not -Pro. Excretion of nitrate, an
end product of nitric oxide, decreased significantly in -Arg and -Arg,Pro. Concentration
of arginine in the muscle decreased to one third and one fifth in -Arg and -Arg,Pro,
respectively. However, proline was not much affected by any of the diets. Plasma
glutamine increased by twofold in -Arg and -Arg,Pro. These results demonstrate that rats
cannot synthesize arginine from glutamate without the small intestine. Nevertheless,
proline can be synthesized from arginine in such tissues as the liver and kidney even in
the absence of the intestine.

9.3.2 Implication from human clinical studies


Is the kidney an indispensable organ in the conversion of citrulline to arginine? Tizianello
et al.78 showed that citrulline uptake and arginine release by kidneys from patients with
normal renal function were 6.9 and 7.0 mmol/min/1.73 m2, respectively, and those from
patients with renal insufficiency were 2.9 and 2.7 mmol/min/1.73 m2, respectively. Despite
the marked drop in the arginine release, the arterial concentration of arginine was not
lowered in renal insufficiency.78 However, citrulline was raised from 28.7 to 68.6 mM.
1382_C09.fm Page 146 Tuesday, October 7, 2003 6:23 PM

146 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 9.2 Hyperammonemia in a short-bowel patient under essential amino acid supplementation.
On day 0, the patient with thrombosis of superior mesenteric artery had a massive resection of the
small intestine, with a remaining 40 cm left. The patient was maintained by TPN (total parenteral
nutrition) with essential amino acids and glucose to supply 3 g of nitrogen and 2200 kcal daily. Mild
hyperammonemia was noted on day 4. Plasma citrulline, ornithine, and arginine were low, and
orotic acid was high. On day 8, arginine infusion, 6 g daily, was introduced and the plasma ammonia
was corrected to a normal level. Withdrawal of arginine caused a relapse of hyperammonemia.

Studies with uremic animal models in which the kidneys had been resected drastically
reported similar changes. This indicates that accumulated citrulline can be converted to
arginine in other competent tissues, as suggested in Table 9.1.
The effect of an arginine-free diet (-Arg) on humans has been investigated.79 Although
-Arg transiently reduced postprandial plasma arginine, neither hyperammonemia nor
orotic aciduria developed. Castillo et al.,80 using stable isotopes, quantitated de novo a rate
of arginine synthesis in the human body and compared it between arginine-replete and
arginine-free diets for 6 days. They found that the rate (16 mmol/kg/h) did not change in
response to arginine availability. However, plasma arginine and ornithine were low and
citrulline was high on feeding of an arginine-free diet, compared to an arginine-replete
one.80 Therefore, the human intestine appears more efficient than that of rats in making
citrulline and ornithine.
However, strong lines of evidence can be found among clinical reports that suggest
that the small intestine is essential in arginine synthesis in the human body. One is the
observation that a short-bowel (SB) patient who had hypocitrullinemia developed
hypoargininemia, hyperammonemia, and orotic acidemia when the patient was main-
tained on essential amino acid hyperalimentation that lacked in arginine.81–83 Arginine
supplementation relieved the biochemical changes (Figure 9.2). On the contrary, when SB
patients were maintained on an oral therapeutic diet, which contains arginine, tailored
for them, they showed almost normal values for plasma arginine, ornithine, and proline,
whereas glutamine was high and citrulline was low. Neither hyperammonemia nor orotic
aciduria was detected.84 Heird et al.85 reported hyperammonemia in three infants receiving
both essential and nonessential amino acids. However, these infants had jejunal atresia or
1382_C09.fm Page 147 Tuesday, October 7, 2003 6:23 PM

Chapter nine: The glutamate crossway 147

gastroschisis, suggesting severe impairment of intestinal citrullinogenesis. Hyperammone-


mia disappeared by arginine supplementation.85
Patients of urea cycle enzyme deficiencies, CPS, OTC, ASS, or ASL, developed hyper-
glutaminemia and hyperammonemia accompanied by hypoargininemia immediately after
supplementation of arginine, with which their basal diets on low-protein content were
fortified, was discontinued.86 Concomitant decrease in plasma and urinary urea suggested
that dietary arginine was a major origin of urea production in those patients. Therefore,
they lacked not only CPS, OTC, ASS, or ASL of the liver urea cycle, but also corresponding
extrahepatic enzymes that make up the arginine synthetic route. Moreover, the fact that
plasma ornithine did not change or somewhat increased signifies the integrity of OAT
and PCS of the small intestine in those patients. In addition, when patients of CPS, OTC,
or ASS deficiencies received liver transplantation in order to cure hyperammonemic intox-
ication, hypocitrullinemia or hypercitrullinemia persisted, although plasma ammonia and
orotic aciduria were normalized.87 This observation further emphasizes that extrahepatic
CPS and OTC are essential for the citrulline synthesis as precursors of releasable arginine,
and ASS in utilization of citrulline to form argininosuccinate.
In normal humans, plasma arginine increases promptly after oral citrulline load.88 The
major portion of circulating ornithine is a product of arginine by ARGase, because of the
low plasma ornithine levels in ARGase deficiency89 and because arginine ingestion elevates
plasma ornithine almost linearly but does not affect citrulline.90 The systemic release of
citrulline from the intestine evidently determines the plasma levels of citrulline. Crenn
et al.91 surveyed over 57 SB patients to find any correlations among residual length of
intestine, plasma citrulline levels, digestive absorption of proteins and lipids, differences
in surgical procedures, and radiographic findings of the remnant intestines. They found91
complete correlations between citrulline levels, and the lengths and surface areas of resid-
ual intestines. They proposed a cutoff value of 20 mmol/l citrulline to judge between
permanent and transient inability of residual functional enterocytes.
The first patients of PCS deficiency carried a missense mutation in the g-glutamyl-
kinase domain and showed progressive neurodegeneration, joint laxity, skin hyperelastic-
ity, and cataracts with low plasma arginine, citrulline, ornithine, and proline, and para-
doxical hyperammonemia (postprandial alleviation).92 These metabolic derangements
appear mainly to reflect the role of intestinal PCS (the short isoform of PCS) to supply
P5C end products into circulation. However, the peripheral symptoms may suggest the
importance of the long isoform of PCS in specific cells to play a vital role, not known yet.
In the meantime, the importance of ATP availability in the intestinal mitochondria for the
synthesis of citrulline (as substrates for CPS and PCS) was suggested in a clinical record
reporting patients whose mitochondrial oligomycin-sensitive ATPase activity was mark-
edly reduced.93
P5C itself is an aminoaldehyde and a reactive compound. P5C interacts with pyridoxal
phosphate (PLP), a coenzyme form of vitamin B6, to form adducts at physiological tem-
perature and pH.94 This reaction leads to deactivation of PLP and may have clinical
significance in provoking so far unexplained seizures in hyperprolinemia II (PCDH defi-
ciency) patients. The authors also note that PLP forms adducts with physiological inter-
mediates such as pyruvate, oxaloacetate, and acetoacetate. A survey of direct chemical
interactions between intermediates of different enzymological pathways and their effects,
and elucidation of roles of a given enzyme on tissue basis (the global role) and cell basis
(the role in a cellular microenvironment) in a human metabolism will open a new research
area from the glutamate crossway.
1382_C09.fm Page 148 Tuesday, October 7, 2003 6:23 PM

148 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

References
1. Rose, W.C., The nutritive significance of the amino acids, Physiol. Rev., 18, 109–136, 1938.
2. Eagle, H., Amino acid metabolism in mammalian cell cultures, Science, 130, 432–437, 1959.
3. Yu, Y., Terada, K., Nagasaki, A., Takiguchi, M., and Mori, M., Preparation of recombinant
argininosuccinate synthetase and argininosuccinate lyase: expression of the enzymes in rat
tissues, J. Biochem. (Tokyo), 117, 952–957, 1995.
4. Morris, S.M., Jr., Bhamidipati, D., and Kepka-Lenhart, D., Human type II arginase: sequence
analysis and tissue-specific expression, Gene, 193, 157–161, 1997.
5. Wakabayashi, Y., Henslee, J.G., and Jones, M.E., Pyrroline-5-carboxylate synthesis from
glutamate by rat intestinal mucosa: subcellular localization and temperature stability, J. Biol.
Chem., 258, 3873–3882, 1983.
6. Forte-Mcrobbie, C.M. and Pietruszko, R., Purification and characterization of human liver
“high Km” aldehyde dehydrogenase and its identification as glutamic gamma-semialdehyde
dehydrogenase, J. Biol. Chem., 261, 2154–2163, 1986.
7. Ryall, J., Nguyen, M., Bendayan, M., and Shore, G.C., Expression of nuclear genes encoding
the urea cycle enzymes, carbamoyl-phosphate synthetase I and ornithine carbamoyl trans-
ferase, in rat liver and intestinal mucosa, Eur. J. Biochem., 152, 287–292, 1985.
8. Yeh, G.C., Roth, E.F., Jr., Phang, J.M., Harris, S.C., Nagel, R.L., and Rinaldi, A., The effect of
pyrroline-5-carboxylic acid on nucleotide metabolism in erythrocytes from normal and glu-
cose-6-phosphate dehydrogenase-deficient subjects, J. Biol. Chem., 259, 5454–5458, 1984.
9. Strecker, H.J., Purification and properties of rat liver ornithine d-transaminase, J. Biol. Chem.,
240, 1225–1230, 1965.
10. McGivan, J.D., Bradford, N.M., and Beavis, A.D., Factors influencing the activity of ornithine
aminotransferase in isolated rat liver mitochondria, Biochem. J., 162, 147–156, 1977.
11. Herzfeld, A. and Raper, S., Enzymes of ornithine metabolism in adult and developing rat
intestine, Biochim. Biophys. Acta, 428, 600–610, 1976.
12. Sanada, Y., Suemori, I., and Katunuma, N., Properties of ornithine aminotransferase from
rat liver, kidney and small intestine, Biochim. Biophys. Acta, 220, 42–50, 1970.
13. Alonso, E. and Rubio, V., Participation of ornithine aminotransferase in the synthesis and
catabolism of ornithine in mice: studies using gabaculine and arginine deprivation, Biochem.
J., 259, 131–138, 1989.
14. Ross, G., Dunn, D., and Jones, M.E., Ornithine synthesis from glutamate in rat intestinal
mucosa homogenates: evidence for the reduction of glutamate to gamma-glutamyl semi-
aldehyde, Biochem. Biophys. Res. Commun., 85, 140–147, 1978.
15. Yamada, E. and Wakabayashi, Y., Development of pyrroline-5-carboxylate synthase and
N-acetylglutamate synthase and their changes in lactation and aging, Arch. Biochem. Biophys.,
291, 15–23, 1991.
16. Wu, G.Y., Meininger, C.J., Kelly, K., Watford, M., and Morris, S.M., Jr. A cortisol surge
mediates the enhanced expression of pig intestinal pyrroline-5-carboxylate synthase during
weaning, J. Nutr., 130, 1914–1919, 2000.
17. Pinkus, L.M. and Windmueller, H.G., Phosphate-dependent glutaminase of small intestine:
localization and role in intestinal glutamine metabolism, Arch. Biochem. Biophys., 182, 506–517,
1977.
18. Wang, T., Lawler, A.M., Steel, G., Sipila, I., Milam, A.H., and Valle, D., Mice lacking ornithine-
d-amino-transferase have paradoxical neonatal hypoornithinaemia and retinal degeneration,
Nat. Genet., 11, 185–190, 1995.
19. Hurwitz, R. and Kretchmer, N., Development of arginine-synthesizing enzymes in mouse
intestine, Am. J. Physiol., 251, G103–G110, 1986.
20. Ryall, J.C., Quantz, M.A., and Shore, G.C., Rat liver and intestinal mucosa differ in the
developmental pattern and hormonal regulation of carbamoyl-phosphate synthetase I and
ornithine carbamoyl transferase gene expression, Eur. J. Biochem., 156, 453–458, 1986.
21. Shenoy, V., Roig, J.C., Kubilis, P., and Neu, J., Characterization of glutaminase in the devel-
oping rat small intestine, J. Nutr., 126 (Suppl.), 1121S–1130S, 1996.
1382_C09.fm Page 149 Wednesday, October 8, 2003 1:28 PM

Chapter nine: The glutamate crossway 149

22. Morris, S.M., Jr., Sweeney, W.E., Jr., Kepka, D.M., O’Brien, W.E., and Avner, E.D., Localization
of arginine biosynthetic enzymes in renal proximal tubules and abundance of mRNA during
development, Pediatr. Res., 29, 151–154, 1991.
23. De Jonge, W.J., Dingemanse, M.A., De Boer, P.A., Lamers, W.H., and Moorman, A.F., Argi-
nine-metabolizing enzymes in the developing rat small intestine, Pediatr. Res., 43, 442–451,
1998.
24. Blachier, F., M’Rabet-Touil, H., Posho, L., Darcy-Vrillon, B., and Duée, P.-H., Intestinal argi-
nine metabolism during development: evidence for de novo synthesis of L-arginine in
newborn pig enterocytes, Eur. J. Biochem., 216, 109–117, 1993.
25. Wu, G., Borbolla, A.G., and Knabe, D.A., The uptake of glutamine and release of arginine,
citrulline and proline by the small intestine of developing pigs, J. Nutr., 124, 2437–2444, 1994.
26. Wu, G.Y. and Knabe, D.A., Arginine synthesis in enterocytes of neonatal pigs, Am. J. Physiol.
Regul. Integr. Comp. Physiol., 269, R621–R629, 1995.
27. De Jonge, W.J., Kwikkers, K.L., Te Velde, A.A., Van Deventer, S.J.H., Nolte, M.A., Mebius,
R.E., Ruijter, J.M., Lamers, M.C., and Lamers, W.H., Arginine deficiency affects early B cell
maturation and lymphoid organ development in transgenic mice, J. Clin. Invest., 110,
1539–1548, 2002.
28. Wraight, C., Lingelbach, K., and Hoogenraad, N., Comparison of ornithine transcarbamylase
from rat liver and intestine: evidence for differential regulation of enzyme levels, Eur. J.
Biochem., 153, 239–242, 1985.
29. Wraight, C. and Hoogenraad, N., Dietary regulation of ornithine transcarbamylase mRNA
in liver and small intestine, Aust. J. Biol. Sci., 41, 435–440, 1988.
30. Tillman, J.B., Dhahbi, J.M., Mote, P.L., Walford, R.L., and Spindler, S.R., Dietary calorie
restriction in mice induces carbamyl phosphate synthetase I gene transcription tissue spe-
cifically, J. Biol. Chem., 271, 3500–3506, 1996.
31. Herzfeld, A. and Knox, W.E., The properties, developmental formation, and estrogen induc-
tion of ornithine aminotransferase in rat tissues, J. Biol. Chem., 243, 3327–3332, 1968.
32. Lyons, R.T. and Pitot, H.C., Hormonal regulation of ornithine aminotransferase biosynthesis
in rat liver and kidney, Arch. Biochem. Biophys., 180, 472–479, 1977.
33. Rogers, Q.R., Freedland, R.A., and Symmons, R.A., In vivo synthesis and utilization of
arginine in the rat, Am. J. Physiol., 223, 236–240, 1972.
34. Morris, S.M., Jr., Moncman, C.L., Holub, J.S., and Hod, Y., Nutritional and hormonal regu-
lation of mRNA abundance for arginine biosynthetic enzymes in kidney, Arch. Biochem.
Biophys., 273, 230–237, 1989.
35. Schimke, R.T., Enzymes of arginine metabolism in mammalian cell culture, J. Biol. Chem.,
239, 136–145, 1964.
36. Morris, S.M., Jr., Moncman, C.L., Rand, K.D., Dizikes, G.J., Cederbaum, S.D., and O’Brien,
W.E., Regulation of mRNA levels for five urea cycle enzymes in rat liver by diet, cyclic AMP,
and glucocorticoids, Arch. Biochem. Biophys., 256, 343–353, 1987.
37. Herzfeld, A. and Raper, S.M., Effects of cortisol or starvation on the activities of four enzymes
in small intestine and liver of the rat during development, J. Dev. Physiol., 1, 315–327, 1979.
38. Wakabayashi, Y., Yamada, R., and Iwashima, A., Temperature- and time-dependent inacti-
vation of pyrroline-5-carboxylate synthase: suggestive evidence for an allosteric regulation
of the enzyme, Arch. Biochem. Biophys., 238, 469–475, 1985.
39. Wu, G.Y., An important role for pentose cycle in the synthesis of citrulline and proline from
glutamine in porcine enterocytes, Arch. Biochem. Biophys., 336, 224–230, 1996.
40. Kowaloff, E.M., Granger, A.S., and Phang, J.M., Glucocorticoid control of hepatic proline
oxidase, Metabolism, 26, 893–901, 1977.
41. Kawabata, Y., Katunuma, N., and Sanada, Y., Characteristics of proline oxidase in rat tissues,
J. Biochem. (Tokyo), 88, 281–283, 1980.
42. Schrock, H. and Goldstein, L., Interorgan relationships for glutamine metabolism in normal
and acidotic rats, Am. J. Physiol., 240, E519–E525, 1981.
43. Max, S.R., Mill, J., Mearow, K., Konagaya, M., Konagaya, Y., Thomas, J.W., Banner, C., and
Vitkovic, L., Dexamethasone regulates glutamine synthetase expression in rat skeletal mus-
cles, Am. J. Physiol., 255, E397–E403, 1988.
1382_C09.fm Page 150 Tuesday, October 7, 2003 6:23 PM

150 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

44. Falduto, M.T., Young, A.P., and Hickson, R.C., Exercise inhibits glucocorticoid-induced
glutamine synthetase expression in red skeletal muscles, Am. J. Physiol. Cell Physiol., 262,
C214–C220, 1992.
45. Murakami, T., Takiguchi, M., Inomoto, T., Yamamura, K., and Mori, M., Tissue- and devel-
opmental stage-specific expression of the rat ornithine carbamoyltransferase gene in trans-
genic mice, Dev. Genet., 10, 393–401, 1989.
46. Nishiyori, A., Tashiro, H., Kimura, A., Akagi, K., Yamamura, K., Mori, M., and Takiguchi,
M., Determination of tissue specificity of the enhancer by combinatorial operation of tissue-
enriched transcription factors: both HNF-4 and C/EBPb are required for liver-specific activity
of the ornithine transcarbamylase enhancer, J. Biol. Chem., 269, 1323–1331, 1994.
47. Kimura, A., Nishiyori, A., Murakami, T., Tsukamoto, T., Hata, S., Osumi, T., Okamura, R.,
Mori, M., and Takiguchi, M., Chicken ovalbumin upstream promoter-transcription factor
(COUP-TF) represses transcription from the promoter of the gene for ornithine transcarbam-
ylase in a manner antagonistic to hepatocyte nuclear factor-4 (HNF-4), J. Biol. Chem., 268,
11125–11133, 1993.
48. Takiguchi, M. and Mori, M., Transcriptional regulation of genes for ornithine cycle enzymes,
Biochem. J., 312, 649–659, 1995.
49. Inoue, Y., Hayhurst, G.P., Inoue, J., Mori, M., and Gonzalez, F.J., Defective ureagenesis in
mice carrying a liver-specific disruption of hepatocyte nuclear factor 4a (HNF4a): HNF4a
regulates ornithine transcarbamylase in vivo, J. Biol. Chem., 277, 25257–25265, 2002.
50. Christoffels, V.M., Van den Hoff, M.J.B., Lamers, M.C., Van Roon, M.A., de Boer, P.A.J.,
Moorman, A.F.M., and Lamers, W.H., The upstream regulatory region of the carbamoyl-
phosphate synthetase I gene controls its tissue-specific, developmental, and hormonal reg-
ulation in vivo, J. Biol. Chem., 271, 31243–31250, 1996.
51. Christoffels, V.M., Van den Hoff, M.J.B., Moorman, A.F.M., and Lamers, W.H., The far-
upstream enhancer of the carbamoyl-phosphate synthetase I gene is responsible for the tissue
specificity and hormone inducibility of its expression, J. Biol. Chem., 270, 24932–24940, 1995.
52. Van Beers, E.H., Rings, E.H.H.M., Posthuma, G., Dingemanse, M.A., Taminiau, J.A.M.J.,
Heymans, H.S.A., Einerhand, A.W.C., Buller, H.A., and Dekker, J., Intestinal carbamoyl
phosphate synthase I in human and rat: expression during development shows species
differences and mosaic expression in duodenum of both species, J. Histochem. Cytochem., 46,
231–240, 1998.
53. Pendleton, L.C., Goodwin, B.L., Flam, B.R., Solomonson, L.P., and Eichler, D.C., Endothelial
argininosuccinate synthase mRNA 5'-untranslated region diversity: infrastructure for tissue-
specific expression, J. Biol. Chem., 277, 25363–25369, 2002.
54. Flam, B.R., Hartmann, P.J., Harrell-Booth, M., Solomonson, L.P., and Eichler, D.C., Caveolar
localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with
endothelial nitric oxide synthase, Nitric Oxide, 5, 187–197, 2001.
55. Hu, C.A., Lin, W.W., Obie, C., and Valle, D., Molecular enzymology of mammalian Delta1-
pyrroline-5-carboxylate synthase: alternative splice donor utilization generates isoforms with
different sensitivity to ornithine inhibition, J. Biol. Chem., 274, 6754–6762, 1999.
56. Wakabayashi, Y., Yamada, E., Hasegawa, T., and Yamada, R., Enzymological evidence for
the indispensability of small intestine in the synthesis of arginine from glutamate. I. Pyrro-
line-5-carboxylate synthase, Arch. Biochem. Biophys., 291, 1–8, 1991.
57. Womack, M. and Rose, W.C., The role of proline, hydroxyproline, and glutamic acid in
growth, J. Biol. Chem., 171, 37–50, 947.
58. Scull, C.W. and Rose, W.C., Arginine metabolism. I. The relation of the arginine content of
the diet to the increments in tissue arginine during growth, J. Biol. Chem., 89, 109–123, 1930.
59. Sallach, H.J., Koeppe, R.E., and Rose, W.C., The in vivo conversion of glutamic acid into
proline and arginine, J. Am. Chem. Soc., 73, 4500, 1951.
60. Depocas, F. and Bouthillier, L.P., Synthèse de l’acide glutamique marqué dans le groupe
carboxylique gamma avec du carbone radioactif: etude du métabolisme de cet acide aminé
chez le rat, Rev. Can. Biol., 10, 289, 1951.
61. Stetten, M.R. and Schoenheimer, R., The metabolism of l(–)-proline studied with the aid of
deuterium and isotopic nitrogen, J. Biol. Chem., 153, 113–132, 1944.
1382_C09.fm Page 151 Tuesday, October 7, 2003 6:23 PM

Chapter nine: The glutamate crossway 151

62. Stetten, M.R., Mechanism of the conversion of ornithine into proline and glutamic acid in
vivo, J. Biol. Chem., 189, 499–507, 1951.
63. Clutton, R.F., Schoenheimer, R., and Rittenberg, D., Studies in protein metabolism. XII. The
conversion of ornithine into arginine in the mouse, J. Biol. Chem., 132, 227–231, 1940.
64. Roloff, M., Ratner, S., and Schoenheimer, R., The biological conversion of ornithine into
proline and glutamic acid, J. Biol. Chem., 136, 561–562, 1941.
65. Windmueller, H.G. and Spaeth, A.E., Uptake and metabolism of plasma glutamine by the
small intestine, J. Biol. Chem., 249, 5070–5079, 1974.
66. Windmueller, H.G. and Spaeth, A.E., Intestinal metabolism of glutamine and glutamate from
the lumen as compared to glutamine from blood, Arch. Biochem. Biophys., 171, 662–672, 1975.
67. Windmueller, H.G. and Spaeth, A.E., Source and fate of circulating citrulline, Am. J. Physiol,
241, E473–E480, 1981.
68. Levillain, O., Hus-Citharel, A., Morel, F., and Bankir, L., Localization of arginine synthesis
along rat nephron, Am. J. Physiol., 259, F916–F923, 1990.
69. Dhanakoti, S.N., Brosnan, J.T., Herzberg, G.R., and Brosnan, M.E., Renal arginine synthesis:
studies in vitro and in vivo, Am. J. Physiol., 259, E437–E442, 1990.
70. Yu, Y.M., Burke, J.F., Tompkins, R.G., Martin, R., and Young, V.R., Quantitative aspects of
interorgan relationships among arginine and citrulline metabolism, Am. J. Physiol., 271,
E1098–E1109, 1996.
71. Milner, J.A. and Visek, W.J., Urinary metabolites characteristic of urea-cycle amino acid
deficiency, Metabolism, 24, 643–651, 1975.
72. Rogers, Q.R. and Morris, J.G., Essentiality of amino acids for the growing kitten, J. Nutr.,
109, 718–723, 1979.
73. Morris, J.G., Rogers, Q.R., Winterrowd, D.L., and Kamikawa, E.M., The utilization of orni-
thine and citrulline by the growing kitten, J. Nutr., 109, 724–729, 1979.
74. Stewart, P.M., Batshaw, M., Valle, D., and Walser, M., Effects of arginine-free meals on
ureagenesis in cats, Am. J. Physiol., 241, E310–E315, 1981.
75. Rogers, Q.R. and Phang, J.M., Deficiency of pyrroline-5-carboxylate synthase in the intestinal
mucosa of the cat, J. Nutr., 115, 146–150, 1985.
76. Hoogenraad, N., Totino, N., Elmer, H., Wraight, C., Alewood, P., and Johns, R.B., Inhibition
of intestinal citrulline synthesis causes severe growth retardation in rats, Am. J. Physiol., 249,
G792–G799, 1985.
77. Wakabayashi, Y. and Yamada, E., Arginine but not proline changes to be an essential amino
acid in the rat with massive resection of the small intestine, Clin. Nutr., 11S, 84, 1992.
78. Tizianello, A., De Ferrari, G., Garibotto, G., Gurreri, G., and Robaudo, C., Renal metabolism
of amino acids and ammonia in subjects with normal renal function and in patients with
chronic renal insufficiency, J. Clin. Invest., 65, 1162–1173, 1980.
79. Carey, G.P., Kime, Z., Rogers, Q.R., Morris, J.G., Hargrove, D., Buffington, C.A., and Brusilow,
S.W., An arginine-deficient diet in humans does not evoke hyperammonemia or orotic
aciduria, J. Nutr., 117, 1734–1739, 1987.
80. Castillo, L., Ajami, A., Branch, S., Chapman, T.E., Yu, Y.-M., Burke, J.F., and Young, V.R.,
Plasma arginine kinetics in adult man: response to an arginine-free diet, Metabolism, 43,
114–122, 1994.
81. Yamada, E., Wakabayashi, Y., Saito, A., Yoda, K., Tanaka, Y., and Miyazaki, M., Hyper-
ammonaemia caused by essential amino acid supplements in patient with short bowel,
Lancet, 341, 1542–1543, 1993.
82. Yokoyama, K., Ogura, Y., Kawabata, M., Hinoshita, F., Suzuki, Y., Hara, S., Yamada, A.,
Mimura, N., Nakayama, M., Kawaguchi, Y., and Sakai, O., Hyperammonemia in a patient
with short bowel syndrome and chronic: renal failure, Nephron, 72, 693–695, 1996.
83. Kapila, S., Saba, M., Lin, C.-H., and Bawle, E.V., Arginine deficiency-induced hyperammone-
mia in a home total parenteral nutrition-dependent patient: a case report, J. Parenter. Enteral
Nutr., 25, 286–288, 2003.
84. Pita, A.M., Wakabayashi, Y., Fernandez-Bustos, M.A., Virgili, N., Riudor, E., Soler, J., and
Farriol, M., Plasma urea-cycle-related amino acids, ammonium levels, and urinary orotic
acid excretion in short-bowel patients managed with an oral diet, Clin. Nutr., 22, 93–98, 2003.
1382_C09.fm Page 152 Tuesday, October 7, 2003 6:23 PM

152 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

85. Heird, W.C., Nicholson, J.F., Driscoll, J.M., Schullinger, J.N., and Winters, R.W., Hyperam-
monemia resulting from intravenous alimentation using a mixture of synthetic L-amino
acids: a preliminary report, J. Pediatr., 81, 162–165, 1972.
86. Brusilow, S.W., Argininine, an indispensable amino acid for patients with inborn errors of
urea synthesis, J. Clin. Invest., 74, 2144–2148, 1984.
87. Rabier, D., Narcy, C., Bardet, J., Parvy, P., Saudubray, J.M., and Kamoun, P., Arginine remains
an essential amino acid after liver transplantation in urea cycle enzyme deficiencies, J. Inherit.
Metab. Dis., 14, 277–280, 1991.
88. Rajantie, J., Simell, O., and Perheentupa, J., Oral administration of urea cycle intermediates
in lysinuric protein intolerance: effect on plasma and urinary arginine and ornithine, Metab-
olism, 32, 49–51, 1983.
89. Adriaenssens, K., Karcher, D., Marescau, B., Van Broeckhoven, C., Lowenthal, A., and Ter-
heggen, H.C., Hyperargininemia: the rat as a model for the human disease and the compar-
ative response to enzyme replacement therapy with free arginase and arginase-loaded
erythrocytes in vivo, Int. J. Biochem., 16, 779–786, 1984.
90. Kamoun, P., Rabier, D., Bardet, J., and Parvy, P., Citrulline concentrations in human plasma
after arginine load, Clin. Chem., 37, 1287–1287, 1991.
91. Crenn, P., Coudray-Lucas, C., Thuillier, F., Cynober, L., and Messing, B., Postabsorptive
plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal
failure in humans, Gastroenterology, 119, 1496–1505, 2000.
92. Baumgartner, M.R., Hu, C.A.A., Almashanu, S., Steel, G., Obie, C., Aral, B., Rabier, D.,
Kamoun, P., Saudubray, J.M., and Valle, D., Hyperammonemia with reduced ornithine,
citrulline, arginine and proline: a new inborn error caused by a mutation in the gene encoding
Delta1-pyrroline-5-carboxylate synthase, Hum. Mol. Genet., 9, 2853–2858, 2000.
93. Parfait, B., De Lonlay, P., Von Kleist-Retzow, J.C., Cormier-Daire, V., Chretien, D., Rtig, A.,
Rabier, D., Saudubray, J.M., Rustin, P., and Munnich, A., The neurogenic weakness, ataxia
and retinitis pigmentosa (NARP) syndrome mtDNA mutation (T8993G) triggers muscle
ATPase deficiency and hypocitrullinaemia, Eur. J. Pediatr., 158, 55–58, 1999.
94. Farrant, R.D., Walker, V., Mills, G.A., Mellor, J.M., and Langley, G.J., Pyridoxal phosphate
de-activation by pyrroline-5-carboxylic acid: increased risk of vitamin B6 deficiency and
seizures in hyperprolinemia type II, J. Biol. Chem., 276, 15107–15116, 2001.
95. De Almeida, A.F., Curi, R., Newsholme, P., and Newsholme, E.A., Maximal activities of key
enzymes of glutaminolysis, glycolysis, Krebs cycle and pentose-phosphate pathway of sev-
eral tissues in mature and aged rats, Int. J. Biochem., 21, 937–940, 1989.
96. Ardawi, M.S.M. and Newsholme, E.A., Intracellular localization and properties of phos-
phate-dependent glutaminase in rat mesenteric lymph nodes, Biochem. J., 217, 289–296, 1984.
97. Chader, G. J., Hormonal effects on the neural retina. I. Glutamine synthetase development
in the retina and liver of the normal and triiodothyronine-treated rat, Arch. Biochem. Biophys.,
144, 657–662, 1971.
98. Jones, M.E., Anderson, A.D., Anderson, C., and Hodes, S., Citrulline synthesis in rat tissues,
Arch. Biochem. Biophys., 95, 499–507, 1961.
99. Ratner, S., A radiochemical assay for argininosuccinate synthetase with [U-14C]aspartate,
Anal. Biochem., 135, 479–488, 1983.
100. Ratner, S. and Murakami-Murofushi, K., A new radiochemical assay for argininosuccinase
with purified [14C]argininosuccinate, Anal. Biochem., 106, 134–147, 1980.
101. Wakabayashi, Y., Iwashima, A., Yamada, E., and Yamada, R., Enzymological evidence for the
indispensability of small intestine in the synthesis of arginine from glutamate. II. N-acetyl-
glutamate synthase, Arch. Biochem. Biophys., 291, 9–14, 1991.
102. Herzfeld, A., Mezl, V.A., and Knox, W.E., Enzymes metabolizing d1-pyrroline-5-carboxylate
in rat tissues, Biochem. J., 166, 95–103, 1977.
103. Herzfeld, A. and Raper, S.M., The heterogeneity of arginases in rat tissues, Biochem. J., 153,
469–478, 1976.
1382_C10.fm Page 153 Tuesday, October 7, 2003 6:27 PM

chapter ten

Arginine metabolism in mammals


Guoyao Wu
Texas A&M University
Sidney M. Morris, Jr.
University of Pittsburgh School of Medicine

Contents
Introduction..................................................................................................................................153
10.1 Arginine synthesis .............................................................................................................155
10.1.1 Arginine as a semiessential amino acid for most mammals.........................155
10.1.2 The intestinal-renal axis for endogenous arginine synthesis ........................155
10.1.3 The citrulline–NO cycle .......................................................................................156
10.2 Arginine catabolism ..........................................................................................................156
10.2.1 Catabolism via arginases .....................................................................................157
10.2.2 Catabolism via arginine:glycine amidinotransferase......................................158
10.2.3 Catabolism via NOS .............................................................................................159
10.2.4 Catabolism via arginine decarboxylase.............................................................159
10.3 Metabolism of methylarginines.......................................................................................160
10.4 Concluding remarks and perspectives ..........................................................................160
Acknowledgments ......................................................................................................................161
References .....................................................................................................................................161

Introduction
L-Arginine (2-amino-5-guanidinovaleric acid) is an amino acid with remarkable metabolic
and regulatory versatility. In 1988, it was identified as the physiological precursor for the
synthesis of nitric oxide (NO) in animal cells (Table 10.1).1,2 The discovery of NO synthesis
has stimulated an enormous interest in arginine metabolism over the past decade. Thus,
much effort has been directed to explore nutritional or therapeutic roles of arginine to
treat many human diseases that are associated with a relative or absolute deficiency of
arginine or with a reduced bioavailability of NO3–5 (see Chapter 35). However, it should
be borne in mind that other aspects of arginine metabolism in addition to NO synthesis
play very important physiological roles. These include the synthesis of arginine itself, as
well as the catabolism of arginine to produce compounds such as proline, polyamines
(putrescine, spermidine, and spermine), creatine, agmatine, and glutamate (Figure 10.1).

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 153
1382_C10.fm Page 154 Tuesday, October 7, 2003 6:27 PM

154 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 10.1 Enzymes Involved in the Synthesis and Catabolism of Arginine, Ornithine,
and Citrulline
Amino Acid Produced by: Used as Substrate by:
Arginine Argininosuccinate lyase Arginase
NOS
Arginine:glycine
amidinotransferase
Arginine decarboxylase
Ornithine Arginase Ornithine decarboxylase
Ornithine aminotransferase Ornithine aminotransferase
Arginine:glycine Ornithine carbamyltransferase
amidinotransferase
Citrulline Ornithine carbamyltransferase Argininosuccinate synthetase
NOS
Dimethylarginine
dimethylaminohydrolase

Note: Because the reaction catalyzed by ornithine aminotransferase can be reversible under physiologic
conditions, ornithine may be either a substrate or product of this reaction, depending on the specific
metabolic conditions.

Arginine

fumarate fumarate
aspartate aspartate

Citrulline-NO Urea Cycle


Cycle
-
NH3 + HCO3
1
NO + Citrulline Arginine Ornithine
2 4
glycine
urea
3 Proline
CO2
ornithine
7
Creatine Agmatine Ornithine P5C

5 6
Glutamate
urea CO2

Putrescine Polyamines

Figure 10.1 Overview of mammalian arginine metabolism. Not all enzymes depicted are expressed
in all cell types. For the sake of clarity, not all reactants or cofactors are shown. 1, NOS; 2, argin-
ine:glycine amidinotransferase; 3, arginine decarboxylase; 4, arginase; 5, agmatinase; 6, ornithine
decarboxylase; 7, ornithine aminotransferase; 8, arginine transporter; P5C, D1-pyrroline-5-carboxy-
late. The dashed line indicates that recycling of citrulline is variable and not quantitative.
1382_C10.fm Page 155 Tuesday, October 7, 2003 6:27 PM

Chapter ten: Arginine metabolism in mammals 155

Therefore, the impact of arginine metabolism extends to virtually every cellular and organ
function in the body. The major objective of this chapter is to present an overview of
mammalian arginine metabolism and the biochemical basis for the clinical applications
of arginine. For more detailed coverage of arginine metabolism and its regulation, refer
to recent reviews.6–10

10.1 Arginine synthesis


10.1.1 Arginine as a semiessential amino acid for most mammals
Owing to its endogenous synthesis in most mammals (including humans, rats, and pigs),
arginine is classified as a nutritionally semiessential amino acid, depending on develop-
mental stage and metabolic state.3 On the basis of nitrogen balance, arginine is considered
as a nonessential amino acid for healthy adult mammals7 (see Chapter 27), with the
exception of strict carnivores.11 However, endogenous arginine synthesis may not be
sufficient to meet metabolic needs in growing infants3 and other neonates (e.g., piglets12
and young rats13), in adults under highly catabolic conditions (e.g., sepsis, burn injury,
and trauma),14,15 and in adults with massive small bowel resection16 (see Chapter 9). In
these conditions, arginine supplementation can become essential for health (see Chapter
35). Moreover, following liver transplantation, hepatic arginase is released into the circu-
lation and can result in a large decline in plasma arginine levels.17–20 This condition also
may require arginine supplementation for optimal recovery.

10.1.2 The intestinal-renal axis for endogenous arginine synthesis


Although arginine is formed in the liver via the urea cycle, there is no net synthesis of
arginine by this organ. Once formed, arginine is immediately hydrolyzed to produce
urea and ornithine. The latter is recycled in the urea cycle as the carrier molecule that
collects waste nitrogen atoms and bicarbonate (see Chapter 7). Instead, the majority of
whole-body arginine synthesis in adults is performed in a metabolic collaboration by
the small intestine and kidneys in what has been termed the intestinal-renal axis.7
Studies over the past 25 years have established that absorptive epithelial cells of the
small intestine (enterocytes) in mammals play the major role in whole-body synthesis of
citrulline, the precursor of arginine.7 Glutamine, glutamate, and proline in enteral diet, as
well as glutamine in arterial blood, are substrates for the intestinal synthesis of citrulline.21
Intestinal catabolism of glutamine via phosphate-dependent glutaminase, pyrroline-5-
carboxylate (P5C) synthase, and ornithine aminotransferase (OAT) yields ornithine, which
is converted to citrulline by carbamylphosphate synthase I and ornithine carbamyltrans-
ferase.7 All of these enzymes are located in mitochondria of enterocytes. Proline oxidase,
another mitochondrial enzyme, oxidizes proline to form P5C, which is converted into
citrulline via OAT, carbamylphosphate synthase I, and ornithine carbamyltransferase.7
The intestine-derived citrulline is released into the circulation and taken up by extrahepatic
tissues (primarily kidneys) for arginine synthesis. (Because it synthesizes N-acetyl-
glutamate, an essential allosteric activator of carbamylphosphate synthase-I,7 the activity
of N-acetylglutamate synthase may regulate the rate of intestinal synthesis of citrulline
and arginine. However, experiments are needed to fully test this hypothesis.)
Declines in plasma concentrations of citrulline have been used as a marker for reduced
small intestinal mass, intestinal injury, and rejection of small bowel transplants.22–24
Changes in plasma citrulline concentrations also can be reflective of changes in intestinal
citrulline synthesis as a function of diet or development.12,25 The crucial role of the small
intestine in endogenous arginine synthesis is dramatically illustrated by the arginine
1382_C10.fm Page 156 Tuesday, October 7, 2003 6:27 PM

156 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

deficiencies, which can result from an inherited deficiency of P5C synthase in humans,26,27
the naturally very low activity of P5C synthase in the intestine of cats,28 a limited supply
of enteral proline in neonatal pigs,29 or resection of the small bowel.16 Dietary arginine is
essential in these circumstances.
With regard to arginine synthesis, enterocytes of neonates differ from those of adults
in two respects. First, unlike the case in adults, most of the citrulline synthesized in
enterocytes of neonates is converted locally to arginine because of relatively high argini-
nosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) activities, coupled with
relatively little intestinal arginase activity.30,31 As ASS expression declines and arginase
expression increases in enterocytes around the time of weaning, enterocytes release most
of the synthesized citrulline rather than converting it into arginine.32,33 Second, catabolism
of proline in neonatal enterocytes represents an important source of P5C for ornithine and
citrulline synthesis.34 Endogenous synthesis of arginine is of enormous nutritional and
physiological importance because arginine is remarkably deficient in the milk of most
mammals, including humans, cows, pigs, and rats,35,36 and because arginine requirements
by young mammals are particularly high.37 For example, we have estimated that endog-
enous synthesis of arginine must provide at least 60% of arginine for the 7-day-old sucking
piglet.3 Consistent with this notion is the finding that an inhibition of intestinal citrulline
synthesis for 12 h decreases plasma concentrations of citrulline and arginine by 52 and
76%, respectively, in neonatal pigs.25
In both neonatal and adult mammals, citrulline released by the small intestine is not
taken up by the liver but is utilized for arginine synthesis by ASS and ASL in extrahepatic
tissues, with the proximal tubules of the kidneys being the major site.7,38 In healthy adult
rats, rates of renal arginine synthesis are determined by the availability of citrulline rather
than by renal capacity for arginine synthesis.39 Because the uptake of arginine by the liver
also is low,40 intestinally derived citrulline or arginine is equally effective as a source of
arginine for the whole body.

10.1.3 The citrulline–NO cycle


Arginine also is formed from citrulline in a pathway known as the citrulline–NO cycle41,42
or the arginine–citrulline cycle43,44 (Figure 10.1). This pathway, which is comprised of the
enzymes ASS and ASL, does not represent de novo synthesis of arginine but is instead a
recycling pathway whereby citrulline produced by NO synthase (NOS) is converted back
into arginine. ASS and ASL are present at low levels in virtually all nonhepatic cells and
are induced when inducible NOS (iNOS) is expressed.41,42 However, the efficiency of this
recycling pathway is by no means quanititative, as evidenced by the net production of
citrulline by NO-producing cells.7 The quantitative contribution of this pathway to argi-
nine production in vivo is unknown.

10.2 Arginine catabolism


Arginine transport by the plasma membrane is the first step of arginine utilization by
animal cells. The most important mechanism for arginine uptake by many cell types is
system y+, a high-affinity, Na+-independent transporter of the basic amino acids — argi-
nine, lysine, and ornithine.40 However, other transporters, such as b0,+, B0,+, and y+L, also
transport arginine (see Chapter 4), and cells may express more than one type of arginine
transporter. Arginine transport can be highly regulated by a variety of stimuli.40,45 Once
inside the cell, arginine can serve as a substrate for only four enzymes in mammals in
addition to arginyl-tRNA synthetase: the NOS isozymes, the arginase isozymes,
arginine:glycine amidinotransferase, and arginine decarboxylase (Figure 10.1). Owing to
1382_C10.fm Page 157 Tuesday, October 7, 2003 6:27 PM

Chapter ten: Arginine metabolism in mammals 157

ARGININE

6
2 3
1 ARGININE X Ornithine Putrescine Polyamines

urea CO2 P5C Proline


[NG-OH-Ar gini ne]

ORNT1 ORNT1 ?
NO +
Citrulline 4 5
ARGININE X Ornithine P5C Glutamate

αKG Glu
Cytosol urea Mitochondrion

Figure 10.2 Metabolic roles and subcellular localization of the arginases and related enzymes. Not
all enzymes depicted are expressed in all cell types. For purposes of clarity, not all reactants or
cofactors are shown. 1, NOS; 2, arginase I; 3, ornithine decarboxylase; 4, arginase II; 5, ornithine
aminotransferase; 6, arginine transporter; ORNT1, mitochondrial transporter of ornithine/citrulline
and of arginine; P5C, D1-pyrroline-5-carboxylate. NG-OH-arginine, an intermediate in NO synthesis,
is an endogenous inhibitor of the arginases. (Modified from Figure 10.5 of Morris, S.M., Jr., Regu-
lation of arginine availability and its impact on NO synthesis, in Nitric Oxide: Biology and Pathobiology,
Ignarro, L.J., Ed., Academic Press, San Diego, 2000, pp. 187–197. Reprinted by permission of Aca-
demic Press.)

their impact on multiple cellular processes, changes in activity or expression of the NOS
and arginase isozymes probably represent the most important regulated changes in argi-
nine metabolism (Figure 10.2).
Changes in plasma concentrations of arginine, ornithine, or citrulline are often used
as markers for activities of specific arginine metabolic enzymes.17–27 However, it is not
always appreciated that each of these amino acids can be produced or catabolized by
multiple enzymes. The enzymes involved in metabolism of these key amino acids are
therefore listed in Table 10.1 as an aid to investigators in this field.

10.2.1 Catabolism via arginases


There are two distinct isozymes of mammalian arginase (types I and II), which are encoded
by separate genes and differ in molecular and immunological properties, tissue distribu-
tion, subcellular localization, and regulation of expression.46 Both isozymes catalyze the
hydrolysis of arginine to urea and ornithine. In healthy adults, type I arginase, a cytosolic
enzyme, is highly expressed in periportal hepatocytes as a component of the urea cycle
and, to a limited extent, in a few other tissues and cells, such as brain,47 small intestine,48
and red blood cells (only in primates).49 However, a variety of cytokines and other inflam-
matory stimuli can induce expression of type I arginase in many cell types.41,50 Type II
arginase, a mitochondrial enzyme, is expressed at modest to low levels in many extra-
hepatic tissues. In healthy adults, the highest expression of type II arginase is found in
the kidney, prostate, testis, small intestine, and lactating mammary gland,51–53 but its
expression also can be induced by inflammatory stimuli such as bacterial lipopolysaccha-
ride and by cyclic AMP.51,54,55 Type II arginase appears to play a significant role in arginine
catabolism and homeostasis, as plasma concentrations of arginine are doubled in mice
deficient in this enzyme.56 These results indicate that rates of endogenous arginine
1382_C10.fm Page 158 Tuesday, October 7, 2003 6:27 PM

158 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

synthesis do not decrease sufficiently, if at all, to compensate for the reduced arginase
activity to restore normal plasma levels of arginine. This notion is thus consistent with
the finding that arginine catabolism is more important than arginine synthesis in main-
taining arginine homeostasis in adults.57 Similarly, overexpression of arginase I in the small
intestine of transgenic mice resulted in markedly reduced plasma arginine concentra-
tions,58,59 again demonstrating that endogenous arginine synthesis cannot compensate for
large changes in arginine catabolism. These transgenic mice also exhibited decreased
growth of the suckling neonate and impaired development of skeletal muscle and lym-
phoid organs.58,59
The different subcellular localization of the arginase isozymes may provide a mech-
anism for regulating the subsequent metabolic fate of ornithine (Figure 10.2). Ornithine
can be utilized for the formation of proline, polyamines, glutamate, and glutamine.7
Formation of these products from arginine is of great nutritional and physiological impor-
tance for diverse functions such as lactation, growth, development, issue remodeling, and
responses to a wide range of hormones and signaling molecules.42,60–63 Much of the orni-
thine produced by arginase is converted to P5C by OAT, as indicated by the occurrence
of hyperornithinemia in adult mammals (e.g., humans and mice) when OAT is defi-
cient.64,65 Owing to the production of ornithine, arginase can be limiting for polyamine
synthesis in several cell types, including vascular smooth muscle cells,66,67 endothelial
cells,68,69 and activated macrophages.70 Arginase is present in calf serum70,71 and thus may
generate sufficient ornithine to support the proliferation of cultured cells that lack this
enzyme. Conversely, elevated arginase activity in some tumor cells may contribute to their
high proliferation rate.72 Remarkably, arginase-dependent production of polyamines also
may be essential for regeneration of injured neurons.73
The arginases can inhibit both inducible74–76 and constitutive68,77–79 NO synthesis in
mammalian cells by regulating the availability of the common substrate arginine. Inter-
estingly, recent work has indicated that high-level expression of arginase I in astrocytes
can apparently deplete arginine to such an extent that subsequent induction of iNOS
expression also is inhibited (R.R. Ratan, personal communication). It remains to be deter-
mined whether this observation will hold for other cell types. Depletion of arginine by
arginase can also have other consequences. For example, elevated arginase activity in
activated macrophages can deplete extracellular arginine sufficiently to result in reduced
expression of the zeta chain of the T cell receptor in cocultured T cells (A. Ochoa, personal
communication). This may contribute to immune cell dysfunction in patients with arginine
deficiency. In some cases, increased expression of the arginases in macrophages may be
beneficial to invading pathogens via effects on the synthesis of NO or polyamines.80–84
The precise roles of the individual arginase isozymes in most cell types in vivo remain
unknown, but investigations of this exciting area are increasing. The recent development
of genetic knockout mouse models of arginase deficiencies56,85 will facilitate efforts to
define the physiological roles of these important enzymes.

10.2.2 Catabolism via arginine:glycine amidinotransferase


Creatine synthesis represents another major use of arginine.7 This pathway is initiated by
arginine:glycine amidinotransferase, which transfers the guanidino group from arginine
to glycine to form guanidinoacetate and ornithine. This enzyme is present predominantly
in the renal tubules, pancreas, and, to a much lesser extent, liver and other organs. The
kidneys are the principal site of guanidinoacetate formation.86 Guanidinoacetate is methyl-
ated by guanidinoacetate N-methyltransferase, which is located primarily in the liver,
pancreas, and, to a much lesser extent, kidneys to form creatine. Circulating creatine is
actively taken up by skeletal muscle and nerve where it is phosphorylated and eventually
1382_C10.fm Page 159 Tuesday, October 7, 2003 6:27 PM

Chapter ten: Arginine metabolism in mammals 159

undergoes nonenzymatic, irreversible dehydration to yield creatinine. The latter is


excreted by the kidneys. Thus, creatine homeostasis primarily involves the kidney, liver,
and skeletal muscle. Approximately 2.3 g of arginine per day is required to maintain
creatine homeostasis in adult humans.7
The methylation of guanidinoacetate to form creatine consumes more methyl groups
than all other methylation reactions combined.87 Thus, creatine synthesis from arginine
plays an important role in regulating the availability of the methyl group donor for other
methylation reactions, such as the synthesis of methionine from homocysteine, an inde-
pendent risk factor for cardiovascular disease.88 The importance of creatine biosynthesis
is graphically demonstrated by the finding that a deficiency of guanidinoacetate N-methyl-
transferase in humans causes a severe creatine deficiency and developmental abnormali-
ties in muscle and brain during early infancy.89 Furthermore, an arginine deficiency in
mice decreases creatine synthesis and results in abnormal neuromotor behavior.58

10.2.3 Catabolism via NOS


As this topic is covered in detail in Chapter 14, only a few points will be noted here. NO
is formed from L-arginine by three NOS isoforms: nNOS or NOS1 (originally identified
as constitutive in neuronal tissue), iNOS or NOS2 (originally identified as being inducible
by cytokines in activated macrophages and liver), and eNOS or NOS3 (originally identified
as constitutive in vascular endothelial cells).90 A quantitatively small amount of NO is
produced by nNOS or eNOS in animal cells,30,68,91–93 whereas much larger amounts of NO
are generated by iNOS in almost all cell types following its induction by inflammatory
cytokines and lipopolysaccharide.70,91,94–96
Because arginine is the only physiologic nitrogen-containing substrate for NOS, pro-
cesses that regulate arginine availability can play an important role in regulating NO
synthesis.42 In fact, a major conundrum in this field is known as the arginine paradox.97
This is based on the observation that cellular rates of NO synthesis are a function of
extracellular arginine concentration, despite the fact that intracellular levels of arginine
should be sufficient to fully saturate the NOS enzymes, based on their Km values for
arginine.98 Thus, recent studies have shown that arginine transporters91,95 and the arginases
(see Section 10.2.1) can play important roles in regulating NO synthesis. Because deficien-
cies or excesses of NO production can lead to numerous cellular and organ dysfunctions,99
elucidating the roles that dietary arginine and arginine metabolic enzymes play in regulat-
ing NO synthesis is important for developing strategies to maintain health and treat disease.
NOS activity also can have an impact on arginase activity via production of
NG-hydroxy-L-arginine (Figure 10.2), a potent endogenous inhibitor of the arginases.100,101
NG-hydroxy-L-arginine, a relatively stable intermediate in NO synthesis, can be released
from NOS and accumulate in sufficient concentration to inhibit arginase activity in cul-
tured cells.96,102 However, the impact of this compound on arginase activity in vivo remains
to be determined.

10.2.4 Catabolism via arginine decarboxylase


Arginine decarboxylase, a mitochondrial enzyme that produces agmatine, is the most
recent addition to the family of mammalian arginine metabolic enzymes. Relatively little
is known about this enzyme, which has not been purified or cloned from any vertebrate
species. However, arginine decarboxylase activity has been reported for rat aorta, brain,
liver, kidney, stomach, and intestine.103 Agmatine is of considerable interest because of its
effects as a cell signaling molecule.104–106 However, the physiologic roles of endogenously
synthesized agmatine have yet to be established.
1382_C10.fm Page 160 Tuesday, October 7, 2003 6:27 PM

160 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Agmatine is hydrolyzed by agmatinase, also a mitochondrial enzyme,107 to produce


urea and putrescine, thus representing an alternate pathway for polyamine synthesis in
animal cells (Figure 10.1). The first cDNAs for vertebrate agmatinase were recently
cloned,108,109 allowing investigators to determine that agmatinase is expressed to varying
degrees in several human cell types, with highest expression in liver and kidney.108,109 Its
expression in human hepatocytes can be induced by hepatitis B virus,108 possibly suggest-
ing a role for agmatinase in the pathogenesis of viral hepatitis.

10.3 Metabolism of methylarginines


Because the methylarginines NG-monomethyl-L-arginine (NMMA) and NGNG-dimethyl-
L-arginine (asymmetrical dimethylarginine (ADMA)) are competitive inhibitors of all NOS
isozymes, there has been growing interest in mammalian metabolism of this family of
arginine derivatives.5,109–111 Free arginine is not a substrate for methylation; arginine resi-
dues in proteins are methylated by a family of protein arginine N-methyltransferases
(PRMT)112 to form NMMA, ADMA, or NGNNG-dimethyl-L-arginine (symmetrical dimethyl-
arginine (SDMA)). Methylation of arginine residues in proteins appears to play roles in
regulating intra- or intermolecular interactions of proteins, protein sorting, gene transcrip-
tion, and cell signaling.112 All PRMT identified to date can monomethylate arginine resi-
dues in proteins. However, further methylation of NMMA to form ADMA and SDMA is
catalyzed by type I PRMT and type II PRMT, respectively. All of the arginine methylation
reactions involve the modification of guanidino nitrogen atoms and require S-adenosyl-
methionine. When intracellular proteins are degraded by proteases and peptidases, free
methylarginines (NMMA, ADMA, and SDMA) are released.
Free NMMA and ADMA produced in the body are hydrolyzed by dimethylarginine
dimethylaminohydrolase (DDAH) to form citrulline and methylamines.113 Two DDAH
isozymes are expressed, and DDAH activity is widespread in mammalian tissues and
cells, including heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, and
endothelial cells.114 Interestingly, the crystal structure of a Pseudomonas DDAH revealed
similarities to arginine:glycine amidinotransferase and arginine deiminase.115 Homo-
cysteine recently was shown to inhibit DDAH in a cell-free system, suggesting that this
may represent a mechanism whereby hyperhomocysteinemia results in elevated plasma
levels of ADMA, which in turn inhibits NO production in the cardiovascular system.116
Concentrations of free NMMA, ADMA, and SDMA in plasma are low in healthy
subjects (0.5–1 mM)117,118 but can be elevated in patients with various cardiovascular and
other disorders, such as diabetes, renal failure, hypercholesterolemia, atherosclerosis,
schizophrenia, and multiple sclerosis,110,111 suggesting a role for dimethylarginines in these
diseases. Interestingly, endogenous NMMA and ADMA have recently been shown to
inhibit neuronal NO production and prevent NO-mediated excitotoxic injury in cultured
neurons.119 Although SDMA itself is not an inhibitor of NOS, it is a competitive inhibitor
of arginine transport by cells, as are the other methylarginines.40 Therefore, elevated
concentrations of SDMA under certain pathological conditions may indirectly lead to
reduced NO synthesis.

10.4 Concluding remarks and perspectives


Recent studies have shown that arginine displays remarkable metabolic and regulatory
versatility in mammals. Therefore, the beneficial or deleterious effects of arginine
metabolism critically depend on the relative activities of specific arginine-catabolizing
enzymes; thus, precise regulation of these enzymes in vivo has important implications for
health and disease. Much of the information about the role of arginases in regulating NO,
1382_C10.fm Page 161 Tuesday, October 7, 2003 6:27 PM

Chapter ten: Arginine metabolism in mammals 161

polyamine, proline, and glutamate synthesis have been generated from in vitro studies
and should be verified in vivo. In addition, the recent cloning of cDNA for mouse N-acetyl-
glutamate synthase120 will provide a powerful tool to define the regulatory role of this
enzyme in intestinal citrulline synthesis. Furthermore, future studies to fully explain the
arginine paradox97 will not only provide new knowledge about the regulation of mam-
malian NO synthesis but will also have important implications for the clinical application
of arginine.4 Finally, arginine and citrulline were recently found to be unusually abundant
in porcine allantoic fluid (e.g., 4 to 6 mM arginine on day 40 of gestation)121,122 and in
ovine allantoic fluid (e.g., 9.7 mM citrulline on day 60 of gestation)119; thus, the roles of
arginine or citrulline in conceptus development should be elucidated. We predict that
arginine or citrulline will provide an effective nutritional or pharmacotherapeutic treat-
ment for many human diseases. We also anticipate that exciting new roles for arginine
in regulating cell and tissue function in health and disease will be discovered in the
coming years.

Acknowledgments
Work in our laboratories is supported, in part, by grants from the U.S. Department of
Agriculture, American Heart Association, and Juvenile Diabetes Research Foundation; by
a Hatch project from the Texas Agricultural Experiment Station; by a Texas A&M Univer-
sity Faculty Fellowship (to G.W.); and by grants from the National Institutes of Health (to
S.M.M.). We thank Frances Mutscher for office support.

References
1. Palmer, R.M.J., Ashton, D., and Moncada, S., Vascular endothelial cells synthesize nitric oxide
from L-arginine, Nature, 333, 664–666, 1988.
2. Hibbs, J.B., Jr., Taintor, R.R., Vavrin, Z., and Rachlin, E.M., Nitric oxide: a cytotoxic activated
macrophage effector molecule, Biochem. Biophys. Res. Commun., 157, 87–94, 1988.
3. Wu, G., Meininger, C.J., Knabe, D.A., Bazer, F.W., and Rhoads, J.M., Arginine nutrition in
development, health and disease, Curr. Opin. Clin. Nutr. Metab. Care, 3, 59–66, 2000.
4. Wu, G. and Meininger, C.J., Arginine nutrition and cardiovascular function, J. Nutr., 130,
2626–2629, 2000.
5. Cooke, J.P., Mont-Reynaud, R., Tsao, P.S., and Maxwell, A.J., Nitric oxide and vascular
disease, in Nitric Oxide: Biology and Pathology, Ignarro, L.J., Ed., Academic Press, New York,
2000, pp. 759–783.
6. Cynober, L., Le Boucher, J., and Vasson, M.-P., Arginine metabolism in mammals, J. Nutr.
Biochem., 6, 402–413, 1995.
7. Wu, G. and Morris, S.M., Jr., Arginine metabolism: nitric oxide and beyond, Biochem. J., 336,
1–17, 1998.
8. Morris, S.M., Jr., Regulation of enzymes of the urea cycle and arginine metabolism, Annu.
Rev. Nutr., 22, 87–105, 2002.
9. Takiguchi, M. and Mori, M., Transcriptional regulation of genes for ornithine cycle enzymes,
Biochem. J., 312, 649–659, 1995.
10. Boger, R.H. and Bode-Boger, S.M., The clinical pharmacology of L-arginine, Annu. Rev.
Pharmacol. Toxicol., 41, 79–99, 2001.
11. Morris, J.G., Nutritional and metabolic responses to arginine deficiency in carnivores, J. Nutr.,
115, 524–531, 1985.
12. Flynn, N.E., Knabe, D.A., and Wu, G., Postnatal changes of plasma amino acids in suckling
pigs, J. Anim. Sci., 78, 2369–2375, 2000.
1382_C10.fm Page 162 Tuesday, October 7, 2003 6:27 PM

162 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

13. Wu, G., Flynn, N.E., Flynn, S.P., Jolly, C.A., and Davis, P.K., Dietary protein or arginine
deficiency impairs constitutive and inducible nitric oxide synthesis by young rats, J. Nutr.,
129, 1347–1354, 1999.
14. Beaumier, L., Castillo, L., Yu, Y.M., Ajami, A.M., and Young, V.R., Arginine: new and exciting
developments for an “old” amino acid, Biomed. Environ. Sci., 9, 296–315, 1996.
15. Hallemeesch, M.M., Lamers, W.H., and Deutz, N.E., Reduced arginine availability and nitric
oxide production, Clin. Nutr., 21, 273–279, 2002.
16. Wakabayashi, Y., The glutamate crossway, in Amino Acid Metabolism and Therapy in Health
and Nutritional Disease, Cynober, L.A., Ed., CRC Press, Boca Raton, 1995, pp. 89–98.
17. Roth, E., Steininger, R., Winkler, S., Langle, F., Grunberger, T., Fugger, R., and Muhlbacher,
F., L-Arginine deficiency after liver transplantation as an effect of arginase efflux from the
graft: influence on nitric oxide metabolism, Transplantation, 57, 665–669, 1994.
18. Langle, F., Steininger, R., Roth, R., Winkler, S., Andel, H., Acimovic, S., Fugger, R., and
Muhlbacher, F., L-arginine deficiency and hemodynamic changes as a result of arginase efflux
following orthotopic liver transplantation, Transplant Proc., 27, 2872–2873, 1995.
19. Ikemoto, M., Tsunekawa, S., Tanaka, K., Tanaka, A., Yamaoka, Y., Ozawa, K., Fukuda, Y.,
Moriyasu, F., Totani, M., Kasai, Y., Mori, T., and Ueda, K., Liver-type arginase in serum
during and after liver transplantation: a novel index in monitoring conditions of the liver
graft and its clinical significance, Clin. Chim. Acta, 271, 11–23, 1998.
20. Yagnik, G.P., Takahashi, Y., Tsoulfas, G., Reid, K., Murase, N., and Geller, D.A., Blockade of
the L-arginine/NO synthase pathway worsens hepatic apoptosis and liver transplant pres-
ervation injury, Hepatology, 36, 573–581, 2002.
21. Wu, G., Intestinal mucosal amino acid catabolism, J. Nutr., 128, 1249–1252, 1998.
22. Crenn, P., Coudray-Lucas, C., Thuillier, F., Cynober, L., and Messing, B., Postabsorptive
plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal
failure in humans, Gastroenterology, 119, 1496–1505, 2000.
23. Pappas, P.A., Saudubray, J.M., Tzakis, A.G., Rabier, D., Carreno, M.R., Gomez-Marin, O.,
et al., Serum citrulline as a marker of acute cellular rejection for intestinal transplantation,
Transplant Proc., 34, 915–917, 2002.
24. Wu, G., Becker, R.M., Bose, C.L., and Rhoads, J.M., Serum amino acid concentrations in
preterm infants, J. Pediatr., 139, 334–337, 2001.
25. Flynn, N.E. and Wu, G., An important role for endogenous synthesis of arginine in main-
taining arginine homeostasis in neonatal pigs, Am. J. Physiol., 271, R1149–R1155, 1996.
26. Kamoun, P., Aral, B., and Saudubray, J.M., A new inherited metabolic disease: pyrroline-5-
carboxylate synthetase deficiency, Bull. Acad. Natl. Med. (Paris), 182, 131–139, 1998.
27. Baumgartner, M.R., Hu, C.A.A., Almashanu, S., Steel, G., Obie, C., Aral, B., Rabier, D.,
Kamoun, P., Saudubray, J.-M., and Valle, D., Hyperammonemia with reduced ornithine,
citrulline, arginine and proline: a new inborn error caused by a mutation in the gene encoding
D1-pyrroline-5-carboxylate synthase, Hum. Mol. Genet., 9, 2853–2858, 2000.
28. Rogers, Q.R. and Phang, J.M., Deficiency of pyrroline-5-carboxylate synthase in the intestinal
mucosa of the cat, J. Nutr., 115, 146–150, 1985.
29. Brunton, J.A., Bertolo, R.F.P., Pencharz, P.B., and Ball, R.O., Proline ameliorates arginine
deficiency during enteral but not parenteral feeding in neonatal pigs, Am. J. Physiol., 277,
E223–E231, 1999.
30. Wu, G. and Knabe, D.A., Arginine synthesis in enterocytes of neonatal pigs, Am. J. Physiol.,
269, R621–R629, 1995.
31. Wu, G., Knabe, D.A., Flynn, N.E., Yan, W., and Flynn, S.P., Arginine degradation in devel-
oping porcine enterocytes, Am. J. Physiol., 271, G913–G917, 1996.
32. Wu, G., Knabe, D.A., and Flynn, N.E., Synthesis of citrulline from glutamine in pig entero-
cytes, Biochem. J., 299, 115–121, 1994.
33. Wu, G., Urea synthesis in enterocytes of developing pigs, Biochem. J., 312, 717–723, 1995.
34. Wu, G., Synthesis of citrulline and arginine from proline in enterocytes of postnatal pigs,
Am. J. Physiol., 272, G1382–G1390, 1997.
35. Wu, G. and Knabe, D.A., Free and protein-bound amino acids in sow’s colostrum and milk,
J. Nutr., 124, 415–424, 1994.
1382_C10.fm Page 163 Tuesday, October 7, 2003 6:27 PM

Chapter ten: Arginine metabolism in mammals 163

36. Davis, T.A., Nguyen, H.V., Garcia-Bravo, R., Florotto, M.L., Jackson, E.M., Lewis, D.S., Lee,
D.R., and Reeds, P.J., Amino acid composition of human milk is not unique, J. Nutr., 124,
1126–1132, 1994.
37. Visek, W.J., Arginine needs, physiological state and usual diets: a reevaluation, J. Nutr., 116,
36–46, 1986.
38. Morris, S.M., Jr., Sweeney, W.E., Kepka, D.M., O’Brien, W.E. and Avner, E.D., Localization
of arginine biosynthetic enzymes in renal proximal tubules and abundance of mRNAs during
development, Pediatr. Res., 29, 151–154, 1991.
39. Dhanakoti, S.N., Brosnan, J.T., Herzberg, G.R., and Brosnan, M.E., Renal arginine synthesis:
studies in vitro and in vivo, Am. J. Physiol., 259, E437–E442, 1990.
40. Closs, E.I. and Mann, G.E., Membrane transport of arginine and cationic amino acid analogs,
in Nitric Oxide: Biology and Pathology, Ignarro, L.J., Ed., Academic Press, New York, 2000, pp.
225–241.
41. Morris, S.M., Jr., Arginine synthesis, metabolism, and transport: regulators of nitric oxide
synthesis, in Cellular and Molecular Biology of Nitric Oxide, Laskin, J.D. and Laskin, D.L., Eds.,
Marcel Dekker, New York, 1999, pp. 57–85.
42. Morris, S.M., Jr., Regulation of arginine availability and its impact on NO synthesis, in Nitric
Oxide: Biology and Pathobiology, Ignarro, L.J., Ed., Academic Press, San Diego, 2000,
pp. 187–197.
43. Wu, G. and Brosnan, J.T., Macrophages can convert citrulline into arginine, Biochem. J., 281,
45–48, 1992.
44. Hecker, M., Sessa, W.C., Harris, H.J., Anggard, E.E., and Vane, J.R., The metabolism of
L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor:
cultured endothelial cells recycle L-citrulline to L-arginine, Proc. Natl. Acad. Sci. U.S.A., 87,
8612–8616, 1990.
45. MacLeod, C.L. and Kakuda, D.K., Regulation of CAT: cationic amino acid transporter gene
expression, Amino Acids, 11, 171–191, 1996.
46. Jenkinson, C.P., Grody, W.W., and Cederbaum, S.D., Comparative properties of arginases,
Comp. Biochem. Physiol., 114B, 107–132, 1996.
47. Yu, H., Iyer, R.K., Kern, R.M., Rodriguez, W.I., Grody, W.W., and Cederbaum, S.D., Expression
of arginase isozymes in mouse brain, J. Neurosci. Res., 66, 406–422, 2001.
48. Flynn, N.E., Meininger, C.J., Kelly, K., Ing, N.H., Morris, S.M., Jr., and Wu, G., Glucocorticoids
mediate the enhanced expression of intestinal type II arginase and argininosuccinate lyase
in postweaning pigs, J. Nutr., 129, 799–803, 1999.
49. Kim, P.S., Iyer, R.K., Lu, K.V., Yu, H., Karimi, A., Kern, R.M., Tai, D.K., Cederbaum, S.D., and
Grody, W.W., Expression of the liver form of arginase in erythrocytes, Mol. Genet. Metab., 76,
100–110, 2002.
50. Mori, M. and Gotoh, T., Relationship between arginase activity and nitric oxide production,
in Nitric Oxide: Biology and Pathology, Ignarro, L.J., Ed., Academic Press, San Diego, 2000,
pp. 199–208.
51. Gotoh, T., Sonoki, T., Nagasaki, A., Terada, K., Takiguchi, M., and Mori, M., Molecular cloning
of cDNA for nonhepatic mitochondrial arginase (arginase II) and comparison of its induction
with nitric oxide synthase in a murine macrophage-like cell line, FEBS Lett., 395, 119–122,
1996.
52. Vockley, J.G., Jenkinson, C.P., Shukula, H., Kern, R.M., Grody, W.W., and Cederbaum, S.C.,
Cloning and characterization of the human type II arginase gene, Genomics, 38, 118–123, 1996.
53. Morris, S.M., Jr., Bhamidipati, D., and Kepka-Lenhart, D., Human type II arginase: sequence
analysis and tissue-specific expression, Gene, 193, 157–161, 1997.
54. Wang, W.W., Jenkinson, C.P., Griscavage, J.M., Kern, R.M., Arabolos, N.S., Byrns, R.E.,
Cederbaum, S.D., and Ignarro, L.J., Co-induction of arginase and nitric oxide synthase in
murine macrophages activated by lipopolysaccharide, Biochem. Biophys. Res. Commun., 210,
1009–1016, 1995.
55. Morris, S.M., Jr., Kepka-Lenhart, D., and Chen, L.C., Differential regulation of arginases and
inducible nitric oxide synthase in murine macrophage cells, Am. J. Physiol., 275, E740–E747,
1998.
1382_C10.fm Page 164 Tuesday, October 7, 2003 6:27 PM

164 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

56. Shi, O., Morris, S.M., Zoghbi, H., Porter, C.W., and O’Brien, W.E., Generation of a mouse
model for arginase II deficiency by targeted disruption of the arginase II gene, Mol. Cell.
Biol., 21, 811–813, 2001.
57. Castillo, L., Chapman, T.E., Sanchez, M., Yu, Y.M., Burke, J.F., Ajami, A.M., Vogt, J., and
Young, V.R., Plasma arginine and citrulline kinetics in adults given adequate and arginine-
free diets, Proc. Natl. Acad. Sci. U.S.A., 90, 7749–7753, 1993.
58. de Jonge, W.J., Marescau, B., D’Hooge, R., De Deyn, P.P., Hallemeesch, M.M., Deutz, N.E.P.,
Ruijter, J.M., and Lamers, W.H., Overexpression of arginase alters circulating and tissue
amino acids and guanidino compounds and affects neuromotor behavior in mice, J. Nutr.,
131, 2732–2740, 2001.
59. de Jonge, W.J., Hallemeesch, M.M., Kwikkers, K.L., Ruijter, J.M., de Gier-de Vries, C.,
van Roon, M.A., Meijer, A.J., Marescau, B., De Deyn, P.P., Deutz, N.E.P., and Lamers, W.H.,
Overexpression of arginase I in enterocytes of transgenic mice elicits a selective arginine
deficiency and affects skin, muscle, and lymphoid development, Am. J. Clin. Nutr., 76,
128–140, 2002.
60. O’Quinn, P.R., Knabe, D.A., and Wu, G., Arginine catabolism in lactating porcine mammary
tissue, J. Anim. Sci., 80, 467–474, 2002.
61. Durante, W., Regulation of L-arginine transport and metabolism in vascular smooth muscle
cells, Cell Biochem. Biophys., 35, 19–34, 2001.
62. Wu, G., Flynn, N.E., Knabe, D.A., and Jaeger, L.A., A cortisol surge mediates the enhanced
polyamine synthesis in porcine enterocytes during weaning, Am. J. Physiol., 279, R554–R559,
2000.
63. Wu, G., Flynn, N.E., and Knabe, D.A., Enhanced intestinal synthesis of polyamines from
proline in cortisol-treated pigs, Am. J. Physiol., 279, E395–E402, 2000.
64. Wang, T., Lawler, A.M., Steel, G., Sipila, I., Milam, A.H., and Valle, D., Mice lacking ornithine-
d-aminotransferase have paradoxical neonatal hypoornithinemia and retinal degeneration,
Nat. Genet., 11, 185–190, 1995.
65. Valle, D. and Simell, O., The hyperornithinemias, in The Metabolic and Molecular Bases of
Inherited Disease, Scriver, C.R., Beaudet, A.L., Sly, W.S., Valle, D., Childs, B., Kinzler, K.W.,
and Vogelstein, B., Eds., McGraw-Hill, New York, 2001, pp. 1857–1895.
66. Ignarro, L.J., Buga, G.M., Wei, L.H., Bauer, P.M., Wu, G., and del Soldato, P., Role of the
arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation,
Proc. Natl. Acad. Sci. U.S.A., 97, 4202–4208, 2001.
67. Wei, L.H., Wu, G., Morris, S.M., Jr., and Ignarro, L.J., Elevated arginase I expression in rat
aortic smooth muscle cells increases cell proliferation, Proc. Natl. Acad. Sci. U.S.A., 98,
9260–9264, 2001.
68. Li, H., Meininger, C.J., Hawker, J.R., Jr., Haynes, T.E., Kepka-Lenhart, D., Mistry, S.K., Morris,
S.M., Jr., and Wu, G., Regulatory role of arginase I and II in nitric oxide, polyamine, and
proline syntheses in endothelial cells, Am. J. Physiol., 280, E75–E82, 2001.
69. Li, H., Meininger, C.J., Hawker, J.R., Jr., Kelly, K.A., Morris, S.M., Jr., and Wu G., Activities
of arginase I and II are limiting for endothelial cell proliferation, Am. J. Physiol., 282, R64–R69,
2002.
70. Kepka-Lenhart, D., Mistry, S.K., Wu, G., and Morris, S.M., Jr., Arginase I: a limiting factor
for nitric oxide and polyamine synthesis by activated macrophages? Am. J. Physiol., 279,
R2237–R2242, 2000.
71. Holtta, E. and Pohjanpelto, P., Polyamine dependence of Chinese hamster ovary cells in
serum-free culture is due to deficient arginase activity, Biochim. Biophys. Acta, 721, 321–327,
1982.
72. Singh, R., Pervin, S., Karimi, A., Cederbaum, S., and Chaudhuri, G., Arginase activity in
human breast cancer cell lines: NG-hydroxy-L-arginine selectively inhibits cell proliferation
and induces apoptosis in MDA-MB-468 cells, Cancer Res., 60, 3305–3312, 2000.
73. Cai, D., Deng, K., Mellado, W., Lee, J., Ratan, R.R., and Filben, M.T., Arginase I and
polyamines are downstream from cyclic AMP in the pathway that overcomes inhibition of
axonal regeneration by MAG and myelin, Neuron, 35, 711–719, 2002.
1382_C10.fm Page 165 Tuesday, October 7, 2003 6:27 PM

Chapter ten: Arginine metabolism in mammals 165

74. Gotoh, T. and Mori, M., Arginase II downregulates nitric oxide (NO) production and prevents
NO-mediated apoptosis in murine macrophage-derived RAW 264.7 cells, J. Cell Biol., 144,
427–434, 1999.
75. Tenu, J.-P., Lepoivre, M., Moali, C., Brollo, M., Mansuy, D., and Boucher, J.-L., Effects of the
new arginase inhibitor N_-hydroxy-nor-L-arginine on NO synthase activity in murine mac-
rophages, Nitric Oxide, 3, 427–438, 1999.
76. Rutschman, R., Lang, R., Hesse, M., Ihle, J.N., Wynn, T.A., and Murray, P.J., Cutting edge:
Stat6-dependent substrate depletion regulates nitric oxide production, J. Immunol., 166,
2173–2177, 2001.
77. Baggio, R., Emig, F.A., Christianson, D.W., Ash, D.E., Chakder, S., and Rattan, S., Biochemical
and functional profile of a newly developed potent and isozyme-selective arginase inhibitor,
J. Pharmacol. Exp. Ther., 290, 1409–1416, 1999.
78. Cox, J.D., Kim, N.N., Traish, A.M., and Christianson, D.W., Arginase-boronic acid complex
highlights a physiological role in erectile function, Nat. Struct. Biol., 6, 1043–1047, 1999.
79. Kim, N.N., Cox, J.D., Baggio, R.F., Emig, F.A., Mistry, S.K., Harper, S.L., Speicher, D.W.,
Morris, S.M., Jr., Ash, D.E., Traish, A., and Christianson, D.W., Probing erectile function: S-(2-
boronoethyl)-L-cysteine binds to arginase as a transition state analogue and enhances smooth
muscle relaxation in human penile corpus cavernosum, Biochemistry, 40, 2678–2688, 2001.
80. Gobert, A.P., Daulouede, S., Lepoivre, M., Boucher, J.L., Bouteille, B., Buguet, A., Cespuglio,
R., Veyret, B., and Vincendeau, P., L-Arginine availability modulates local nitric oxide pro-
duction and parasite killing in experimental trypanosomiasis, Infect. Immun., 68, 4653–4657,
2000.
81. Gobert, A.P., McGee, D.J., Akhtar, M., Mendz, G.L., Newton, J.C., Cheng, Y., Mobley, H.L.,
and Wilson, K.T., Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic
cells: a strategy for bacterial survival, Proc. Natl. Acad. Sci. U.S.A., 98, 13844–13849, 2001.
82. Gobert, A.P., Cheng, Y., Wang, J.Y., Boucher, J.L., Iyer, R.K., Cederbaum, S.D., Casero, R.A.,
Jr., Newton, J.C., and Wilson, K.T., Helicobacter pylori induces macrophage apoptosis by
activation of arginase II, J. Immunol., 168, 4692–4700, 2002.
83. Abdallahi, O.M., Bensalem, H., Augier, R., Diagana, M., De Reggi, M., and Gharib, B.,
Arginase expression in peritoneal macrophages and increase in circulating polyamine levels
in mice infected with Schistosoma mansoni, Cell. Mol. Life Sci., 58, 1350–1357, 2001.
84. Iniesta, V., Carlos Gomez-Nieto, L., Molano, I., Mohedano, A., Carcelen, J., Miron, C., Alonso,
C., and Corraliza, I., Arginase I induction in macrophages, triggered by Th2-type cytokines,
supports the growth of intracellular Leishmania parasites, Parasite Immunol., 24, 113–118,
2002.
85. Iyer, R.K., Yoo, R.K., Kern, R.M., Rozengurt, N., Tsoa, R., O’Brien, W.E., Yu, H., Grody, W.W.,
and Cederbaum, S.D., Mouse model for human arginase deficiency, Mol. Cell. Biol., 22,
4491–4498, 2002.
86. Wyss, M. and Kaddurah-Daouk, R., Creatine and creatinine metabolism, Physiol. Rev., 80,
1107–1213, 2000.
87. Stead, L.M., Au, K.P., Jacobs, R.L., Brosnan, M.E., and Brosnan, J.T., Methylation demand
and homocysteine metabolism: effects of dietary provision of creatine and guanidinoacetate,
Am. J. Physiol., 281, E1095–E1100, 2001.
88. Wu, G. and Meininger, C.J., Regulation of nitric oxide synthesis by dietary factors, Annu.
Rev. Nutr., 22, 61–86, 2002.
89. Stockler, S., Isbrandt, D., Hanefeld, F., Schmidt, B., and von Figura, K., Guanidinoacetate
methyltransferase deficiency: the first inborn error of creatine metabolism in man, Am. J.
Hum. Genet., 58, 914–922, 1996.
90. Alderton, W.K., Cooper, C.E., and Knowles, R.G., Nitric oxide synthases: structure, function
and inhibition, Biochem. J., 357, 593–615, 2001.
91. Stevens, B.R., Kakuda, D.K., Yu, K., Waters, M., Vo, C.B., and Raizada, M.K., Induced nitric
oxide synthesis is dependent on induced alternatively spliced CAT-2 encoding L-arginine
transport in brain astrocytes, J. Biol. Chem., 271, 24017–24022, 1996.
1382_C10.fm Page 166 Tuesday, October 7, 2003 6:27 PM

166 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

92. Wu, G., Haynes, T.E., Li, H., Yan, W., and Meininger, C.J., Glutamine metabolism to glu-
cosamine is necessary for glutamine inhibition of endothelial nitric oxide synthesis, Biochem.
J., 353, 245–252, 2001.
93. Meininger, C.J., Kelly, K.A., Li, H., Haynes, T.E., and Wu, G., Glucosamine inhibits inducible
nitric oxide synthesis, Biochem. Biophys. Res. Commun., 279, 234–239, 2000.
94. Meininger, C.J. and Wu, G., L-Glutamine inhibits nitric oxide synthesis in bovine venular
endothelial cells, J. Pharmacol. Exp. Ther., 281, 448–453, 1997.
95. Nicholson, B., Manner, C.K., Kleeman, J., and MacLeod, C.L., Sustained nitric oxide produc-
tion in macrophages requires the arginine transporter CAT2, J. Biol. Chem., 276, 15881–15885,
2001.
96. Buga, G.M., Singh, R., Pervin, S., Rogers, N.E., Schmitz, D.A., Jenkinson, C.P., Cederbaum,
S.D., and Ignarro, L.J., Arginase activity in endothelial cells: inhibition by NG-hydroxy-
arginine during high-output nitric oxide production, Am. J. Physiol., 271, H1988–H1998, 1996.
97. Kurz, S. and Harrison, D.G., Insulin and the arginine paradox, J. Clin. Invest., 99, 369–370,
1997.
98. Stuehr, D.J., Structure-function aspects in the nitric oxide synthases, Annu. Rev. Pharmacol.
Toxicol., 37, 339–359, 1997.
99. Ignarro, L.J., Cirino, G., Casini, A., and Napoli, C., Nitric oxide as a signaling molecule in
the vascular system: an overview, J. Cardiovasc. Pharmacol., 34, 879–886, 1999.
100. Boucher, J.L., Custot, J., Vadon, S., Delaforge, M., Lepoivre, M., Tenu, J.P., Yapo, A., and
Mansuy, D., NG-hydroxy-L-arginine, an intermediate in the L-arginine to nitric oxide pathway,
is a strong inhibitor of liver and macrophage arginase, Biochem. Biophys. Res. Commun., 203,
1614–1621, 1994.
101. Daghigh, F., Fukuto, J.M., and Ash, D.E., Inhibition of rat liver arginase by an intermediate
in NO biosynthesis, NG-hydroxy-L-arginine: implications for the regulation of nitric oxide
biosynthesis by arginase, Biochem. Biophys. Res. Commun., 202, 174–180, 1994.
102. Hecker, M., Nematollahi, H., Hey, C., Busse, R., and Racke, K., Inhibition of arginase by NG-
hydroxy-L-arginine in alveolar macrophages: implications for the utilization of L-arginine for
nitric oxide synthesis, FEBS Lett., 359, 251–254, 1995.
103. Regunathan, S. and Reis, D.J., Characterization of arginine decarboxylase in rat brain and
liver: distinction from ornithine decarboxylase, J. Neurochem., 74, 2201–2208, 2000.
104. Blantz, R.C., Satriano, J., Gabbai, F., and Kelly, C., Biological effects of arginine metabolites,
Acta Physiol. Scand., 168, 21–25, 2000.
105. Reis, D.J. and Regunathan, S., Is agmatine a novel neurotransmitter in brain? Trends Phar-
macol. Sci., 21, 187–193, 2000.
106. Raasch, W., Schafer, U., Chun, J., and Dominiak, P., Biological significance of agmatine, an
endogenous ligand at imidazoline binding sites, Br. J. Pharmacol., 133, 755–780, 2001.
107. Sastre, M., Regunathan, S., Galea, E., and Reis, D.J., Agmatinase activity in rat brain: a
metabolic pathway for the degradation of agmatine, J. Neurochem., 67, 1761–1765, 1996.
108. Mistry, S.K., Burwell, T.J., Chambers, R.M., Rudolph-Owen, L., Spaltmann, F., Cook, W.J.,
and Morris, S.M., Jr., Cloning of human agmatinase: an alternate path for polyamine syn-
thesis induced in liver by hepatitis B virus, Am. J. Physiol., 282, G375–G381, 2002.
109. Iyer, R.K., Kim, H.K., Tsoa, R.W., Grody, W.W., and Cederbaum, S.D., Cloning and charac-
terization of human agmatinase, Mol. Genet. Metab., 75, 209–218, 2002.
110. Vallance, P., The asymmetrical dimethylarginine/dimethylarginine dimethylaminohydrolase
pathway in the regulation of nitric oxide generation, Clin. Sci., 100, 159–160, 2001.
111. Boger, R.H. and Bode-Boger, S.M., Asymmetric dimethylarginine, derangements of the endo-
thelial nitric oxide synthase pathway, and cardiovascular diseases, Semin. Thromb. Hemost.,
26, 539–45, 2000.
112. McBride, A.E. and Silver, P.A., State of the arg:protein methylation at arginine comes of age,
Cell, 106, 5–8, 2001.
113. Ogawa, T., Kimoto, M., and Sasaoka, K., Purification and properties of a new enzyme, NG,NG-
dimethylarginine dimethylaminohydrolase, from rat kidney, J. Biol. Chem., 264, 10205–10209,
1989.
1382_C10.fm Page 167 Tuesday, October 7, 2003 6:27 PM

Chapter ten: Arginine metabolism in mammals 167

114. Leiper, J.M., Maria, J.S., Chubb, A., MacAllister, R.J., Charles, I.G., Whitley, G.S.J., and
Vallance P., Identification of two human dimethylarginine dimethylaminohydrolases with
distinct tissue distributions and homology with microbial arginine deiminases, Biochem. J.,
343, 209–214, 1999.
115. Murray-Rust, J., Leiper, J., McAlister, M., Phelan, J., Tilley, S., Santa Maria, J., Vallance, P.,
and McDonald, N., Structural insights into the hydrolysis of cellular nitric oxide synthase
inhibitors by dimethylarginine dimethylaminohydrolase, Nat. Struct. Biol., 8, 679–683, 2001.
116. Stuhlinger, M.C., Tsao, P.S., Her, J.H., Kimoto, M., Balint, R.F., and Cooke, J.P., Homocysteine
impairs the nitric oxide synthase pathway: role of asymmetric dimethylarginine, Circulation,
104, 2569–2575, 2001.
117. Teerlink, T., Nijveldt, R.J., de Jong, S., and van Leeuwen, P.A.M., Determination of arginine,
asymmetric dimethylarginine, and symmetric dimethylarginine in human plasma and other
biological samples by high-performance liquid chromatography, Anal. Biochem., 303, 131–137,
2002.
118. Vishwanathan, K., Tackett, R.L., Stewart, J.T., and Bartlett, M.G., Determination of arginine
and methylated arginines in human plasma by liquid chromatography-tandem mass spec-
trometry, J. Chromatogr. B, 748, 157–166, 2000.
119. Cardounel, A.J. and Zweier, J.L., Endogenous methyl-arginines regulate neuronal nitric oxide
synthase and prevent excitotoxic injury, J. Biol. Chem., 277, 33995–34002, 2002.
120. Caldovic, L., Morizono, H., Yu, X.L., Thompson, M., Shi, D.S., Gallegos, R., et al., Identifica-
tion, cloning and expression of the mouse N-acetylglutamate synthase gene, Biochem. J., 364,
825–831, 2002.
121. Wu, G., Bazer, F.W., Tuo, W., and Flynn, S.P., Unusual abundance of arginine and ornithine
in porcine allantoic fluid, Biol. Reprod., 54, 1261–1265, 1996.
122. Wu, G., Pond, W.G., Ott, T., and Bazer, F.W., Maternal dietary protein deficiency decreases
amino acid concentrations in fetal plasma and allantoic fluid of pigs, J. Nutr., 128, 894–902,
1998.
123. Kwon, H., Wu, G., Bazer, F.W., and Spencer T.E., Developmental changes of amino acids in
ovine uterine and fetal fluids during pregnancy, Biol. Reprod., 66 (Suppl. 1), 186–187, 2002.
1382_C10.fm Page 168 Tuesday, October 7, 2003 6:27 PM
1382_C11.fm Page 169 Tuesday, October 7, 2003 6:29 PM

chapter eleven

Glutamine metabolism
Rudolf Oehler
University of Vienna
Erich Roth
University of Vienna

Contents
Introduction..................................................................................................................................169
11.1 Biochemical reactions and their regulation...................................................................170
11.1.1 Gln synthesis .........................................................................................................170
11.1.1.1 Glutamine synthetase reaction ............................................................171
11.1.1.2 Gln transport through cell membranes..............................................171
11.1.1.3 Gln is a carrier of nitrogen and carbon..............................................172
11.1.2 Gln breakdown......................................................................................................173
11.1.2.1 Glutaminase reaction.............................................................................173
11.1.2.2 Gln utilization.........................................................................................173
11.1.2.3 Gln is a precursor of a variety of molecules .....................................174
11.1.3 Gln interorgan exchanges....................................................................................174
11.2 Effects of Gln starvation ...................................................................................................175
11.2.1 Energy metabolism ...............................................................................................176
11.2.2 Protein synthesis rate ...........................................................................................177
11.2.3 Cell protective mechanisms ................................................................................177
11.2.4 Apoptosis ...............................................................................................................178
11.2.5 Osmosignaling.......................................................................................................179
11.2.6 Immune response..................................................................................................179
Acknowledgments ......................................................................................................................180
References .....................................................................................................................................180

Introduction
The pioneer of biochemical research, Sir Hans Krebs, once stated that “most amino acids
have multiple functions, but glutamine appears to be the most versatile.” The present
chapter describes the special biochemical properties of glutamine (Gln), which are respon-
sible for the versatility of this amino acid. It explains how Gln is involved in intracellular
metabolism and in interorgan nitrogen transport. It delineates how Gln is used as energetic

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 169
1382_C11.fm Page 170 Tuesday, October 7, 2003 6:29 PM

170 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

fuel by several cell types, which implies that these cells suffer from Gln starvation during
catabolic states. In addition, the chapter summarizes recent findings on the effect of Gln
starvation on function, stress response, and apoptosis of Gln-utilizing cells and gives an
overview of the current knowledge on the molecular mechanisms of Gln sensing.

11.1 Biochemical reactions and their regulation


11.1.1 Gln synthesis
Gln is a polar amino acid that is uncharged at pH 7.0. It contains five C-atoms and bears
a 5-carboxamide in its R-group, which can be readily deaminated for nitrogen release (see
Figure 11.1). The C5 structure makes Gln compatible with the C5-dicarboxylic acids of the
tricarboxylic acid (TCA) cycle and their metabolites. Therefore, Gln plays a central role in
nitrogen as well as carbohydrate metabolism.

alanine
pyruvate
lactate purines
GSH pyrimidines
GABA glucosamines

NH4+
ATP GLN- ADP+Pi
AA αKA synthetase
TCA- TA
cycle αKG Glu Gln
NH4+ Glutaminase H2O

NADH GLU-DH NAD+


NH4+ CO2
ATP
CO2 + H2O
ATP urea-
cycle

expressed in some tissues


expressed in all tissues

urea

Figure 11.1 Metabolic functions of Gln. Gln is formed by the combination of the TCA cycle inter-
mediate a-ketoglutarate (a-KG) with two nitrogens in a two-step reaction. In the first reaction a-
KG is converted to glutamate (Glu) by the activity of transaminases (TA). In this reaction the a-
amino group is enzymatically transferred from an amino acid (AA) to the a-carbon atom of a-KG,
resulting in the corresponding a-keto acid analog of the incoming amino acid and Glu. Glu is then
transformed into Gln by the combination with an ammonium ion catalyzed by the ubiquitous
expressed energy-dependent glutamine synthetase (GS). In Gln catabolism, Gln is converted to Glu
through hydrolysis catalyzed by the enzyme glutaminase, which is expressed only in Gln-utilizing
cells. Glu can then be transformed to a-KG by either transamination or glutamate dehydrogenase
(GLU-DH). The free ammonia resulting from the glutaminase reaction and from the GLU-DH
reaction is then either introduced into the urea cycle (in liver) or released from the tissue (in kidney).
As a-KG, the carbon backbone of Gln enters the TCA cycle. Once in the TCA cycle, the Gln-derived
carbons can either be oxidized to CO2 and water resulting in ATP formation or be introduced in the
synthesis of lactate, pyruvate, or alanine. Gln is also used in the synthesis of purines, pyrimidines
and glucosamines. Glu is a substrate for synthesis glutathione (GSH) and g-aminobutyrate (GABA).
1382_C11.fm Page 171 Tuesday, October 7, 2003 6:29 PM

Chapter eleven: Glutamine metabolism 171

11.1.1.1 Glutamine synthetase reaction


Gln is formed from glutamate, which itself is readily convertible to a-ketoglutarate,
feeding the TCA cycle (Figure 11.1). For Gln formation, glutamate is enzymatically
combined with an ammonium ion (NH4+) by the action of glutamine synthetase.
Glutamine synthetase is an enzyme that can be found in numerous tissues. It promotes
the reaction

ATP + NH4+ + glutamate Æ ADP + Pi + Gln + H+

In the first step of this reaction, the enzyme-bound high-energy intermediate glutamyl
5-phosphate is formed, an acyl phosphate resulting from the phosphorylation of the
5-carbocyl group of glutamate by ATP. Then in the second step, the glutamyl 5-phos-
phate combines with an ammonium ion in the active site to form Gln and release
phosphate. This energy-dependent reaction is irreversible and tightly regulated. It was
found that the activity of glutamine synthetase is subject to regulation by over 40
metabolites.1 Some of these serve as substrates or allosteric effectors, including Gln,
glutamate, ammonia, ATP, and a-ketoglutarate. Thus, glutamine synthetase is regulated
by its substrates and products. The Gln formation rate in skeletal muscle increases in
response to enhanced Gln withdrawal from the plasma by Gln-utilizing cells under
stress conditions. In contrast, the Gln formation rate decreases when plasma Gln is
restored in patients by parenteral Gln administration (see Chapter 36 for details). This
indicates that glutamine synthetase activity in skeletal muscle is regulated to maintain
plasma Gln at a defined level. Substitution of a-ketoglutarate can also help to spare
muscle Gln pools in these patients (see Chapter 37 for details), demonstrating that
synthesis of Gln in skeletal muscle can keep pace with Gln consumption if ample
precursors are provided.

11.1.1.2 Gln transport through cell membranes


The Gln formed by glutamine synthetase is a water-soluble compound that can readily
pass through cell membranes, whereas the precursor glutamate, which bears a net charge,
needs facilitation by specific transport systems.2 However, Gln does not freely cross the
plasma membrane. The cytoplasmatic Gln level is far above its transmembrane equilib-
rium distribution, and the transport of Gln is highly regulated. Several Gln-transporting
systems have been described in the past few years that can be divided into Na+-dependent
and Na+-independent systems. The former utilizes the potential energy present in the
transmembrane Na+ electrochemical gradient, maintained largely by the Na+/K+-ATPase,
to drive the uptake of amino acids against their concentration gradients. Na+-dependent
Gln transporters include systems ASC, N, A, B0,+, and y+L, whereas Na+-independent
transporters include systems L and b0,+.3 All of these transporters mediate the movement
of several other amino acids, to varying degrees. System ASC was originally named for
three of its preferred substrates (alanine, serine, cysteine) to distinguish it from system A
activity. It takes up Gln with high affinity and transports a wide panel of other zwitterionic
amino acids such as serine, threonine, cysteine, alanine, and asparagine, as well as
bulky/branched-chain amino acids (leucine, valine, methionine) to a lesser degree. The
catalytic mechanism of ASC involves a Na+-dependent exchange of intracellular and
extracellular amino acids. This transporter can therefore mediate either Gln uptake or
release. Net Gln movement through this transporter differs from cell type to cell type. The
ASC system is expressed in the liver, kidney, skeletal muscle, intestinal epithelia, lung,
placenta, and pancreas. In contrast, system N has a rather narrow substrate specificity of
1382_C11.fm Page 172 Tuesday, October 7, 2003 6:29 PM

172 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Gln, histidine, and asparagine only — all substrates containing nitrogen in their side chain.
It is expressed in liver and skeletal muscle, in which it represents a potentially rate-limiting
step in metabolism. Hepatic and skeletal muscle system N activities exhibit reciprocal
regulation during catabolic states in which system N-mediated Gln uptake is predictably
enhanced in the liver and decreased in skeletal muscle. Gln transport mediated by system
N is electrogenic, with a proposed two Na+ ions and amino acid transported inward,
coupled to the efflux of one H+. The more ubiquitously expressed system A was found to
mediate Gln uptake only after prolonged periods of amino acid starvation. These data
show that Gln uptake and exchange are mediated by a number of transporters that show
a tissue-specific expression pattern. In addition, because of the wide range of intracellular
substrate levels and transmembrane electrical potentials, the affinity of each system for
Gln varies significantly between the different cell types even at similar expression levels.
Today it is not yet clear how the different Gln transporter systems are regulated, and
further studies are needed to understand the influence of these systems on the interorgan
Gln flux.

11.1.1.3 Gln is a carrier of nitrogen and carbon


The interconversion of charged glutamate to uncharged Gln includes the toxic compound
ammonia. The formed Gln is nontoxic and, as described above, membrane permeable.
Thus, Gln formation is suited to detoxify ammonia and to remove it from the cell. In fact,
approximately one-third of all amino acid-derived nitrogen transported by blood is in the
form of Gln.4 Its concentration in blood (approximately 650 mmol/l of plasma) exceeds
that of all other amino acids by far. More than 60 to 80% of the plasma flux of Gln is
provided by de novo synthesis in the postabsorptive state, whereas the contribution by the
diet plays only a minor role.5 De novo synthesis is inhibited only at very high levels of
Gln uptake (125 mg/kg*h) leading to a doubling of the normal plasma concentration. Gln
serves as a vehicle for transportation of ammonia in a nontoxic form from peripheral
tissues to visceral organs where it can be excreted as ammonium by kidneys or converted
to urea by the liver. Gln is the major source of both nitrogen used in hepatic ureagenesis
and nitrogen excreted in urine. Perfusion of isolated rat livers with Gln was found to
stimulate urea output.6 This indicates that Gln regulates the ammonia detoxification pro-
cesses in the liver.
Ammonia derives from degradation of nitrogen-containing compounds. A main
source of ammonia is amino acid catabolism in the course of protein breakdown. The 20
amino acid catabolic pathways converge to form only five carbohydrate products (pyru-
vate, acetyl-CoA, a-ketoglutarate, fumarate, and oxaloacetate), all of which enter the TCA
cycle. Thus, the carbohydrate backbone of all amino acids can be converted to any TCA
cycle intermediate. The amino group of the degraded amino acids is commonly transferred
to a-ketocarboxylic acids by transamination. A number of aminotransferases transfer the
amino group to the TCA cycle intermediate a-ketoglutarate to form glutamate. a-Keto-
glutarate is also converted to glutamate by combination with ammonia via the mitochon-
drial glutamate dehydrogenase. Glutamate itself is then combined with a second nitrogen
in the course of Gln formation via glutamine synthetase (see Figure 11.1). Thus, Gln can
potentially incorporate nitrogen as well as the carbohydrate skeleton from all 20 amino
acids and should therefore be a suitable end product of protein breakdown. In fact, amino
acids, mainly branched-chain amino acids, are the major source of nitrogen and carbon
for Gln synthesis by skeletal muscle. Net synthesis of Gln also occurs in the lungs, adipose
tissue, brain, and, under certain conditions, liver. The release of Gln from these tissues
serves to transport nitrogen and carbon to Gln-utilizing cells.
1382_C11.fm Page 173 Tuesday, October 7, 2003 6:29 PM

Chapter eleven: Glutamine metabolism 173

11.1.2 Gln breakdown


11.1.2.1 Glutaminase reaction
Gln catabolic pathway results in the same intermediates as seen in Gln synthesis, but with
other enzymes involved. Gln is converted to glutamate and ammonia by the mitochondrial
enzyme glutaminase in the nonreversible reaction

Gln + H2O Æ glutamate + NH4+

Glutaminase activity was found only in certain tissues, including liver, kidney, entero-
cytes, and immune cells. The liver has evolved an elegant mechanism for Gln metabolism
and concerted ammonia detoxification. In the periportal region, ammonia and glutamate
are being generated from incoming Gln by the hepatic isoform of the enzyme glutaminase,
while at the same time high incoming concentrations of ammonia from the splanchic bed
are utilized for urea synthesis (see Chapter 7 for more details). In the perivenous portion
of the liver, high levels of Gln synthetase convert glutamate and excess ammonia into
Gln, effectively reducing the ammonia concentrations in hepatic venous blood to nontoxic
levels. With this intraorgan Gln cycle, the liver is able to effectively detoxify incoming
blood from ammonia and control output of Gln. In contrast, the kidney counteracts
acidosis utilizing the kidney isoform of glutaminase to generate ammonia from Gln. The
kidney disposes of excess nitrogen by consuming Gln and excreting the ammonia
produced.

11.1.2.2 Gln utilization


In the healthy individual, the major site of Gln catabolism is the small intestine, where
Gln is the principal respiratory fuel of enterocytes. In addition, a high rate of Gln utilization
was also found in all lymphoid tissues examined, including lymph nodes, spleen, thymus,
and Peyer’s patches, in neutrophils, in monocytes, and in macrophages. The catabolism
of Gln in the intestine results in the production of CO2, alanine, pyruvate, and lactate from
the carbon skeleton and of ammonia and alanine from the carboxamide and amino groups.
Complete oxidation of Gln via glutamate, a-ketoglutarate, and the TCA cycle results in
27 ATP equivalents, which is one of the highest rates of all nonessential amino acids.
Under physiologic conditions, Gln oxidation can account for as much as one third of the
ATP production in cultured enterocytes, lymphocytes, and monocytes. The rate of Gln
utilization of human neutrophils was recently found to depend on the extracellular con-
centration of the other major energy substrate, glucose.7 Decreasing the extracellular
concentration of glucose from 5.5 to 1 or 0 mM increased the apparent Vmax of Gln
utilization by 70 and 134%, respectively. This suggests that Gln metabolism may be par-
ticularly important as a fuel for neutrophils in situations where the blood glucose concen-
tration is low. The rate of glucose utilization, however, is independent of the extracellular
Gln concentration. As shown in isolated human monocytes, reduction of extracellular Gln
below the physiological plasma concentration leads to a decreased intracellular ATP level
in the presence of 11 mM glucose.8 This suggests that although both glucose and Gln are
used by leukocytes for synthesis of common metabolic intermediates, glucose cannot
replace Gln completely. Not all utilized Gln is used for ATP production. For example,
HeLa and CHO cells each convert approximately 30% of catabolized Gln carbons to CO2
and 15% to lactate, while approximately 20% of carbons are incorporated into macromol-
ecules. The total amount of cellular energy derived from Gln depends on the extent of
oxidation and the rate of Gln utilization. These factors in turn depend largely on the
absolute amounts and relative proportions of Gln and glucose available, as well as the
1382_C11.fm Page 174 Tuesday, October 7, 2003 6:29 PM

174 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

type and proliferative state of the cell. For example, lymphocytes derive a large portion
of their energy from Gln oxidation. This portion increases strongly after activation with
RNA or mitogenic antibodies directed against CD3, which stimulate lymphocytes to
proliferate. This proliferation is highly dependent on the Gln concentration in culture
medium.

11.1.2.3 Gln is a precursor of a variety of molecules


Gln is used as an amino donor in a variety of reactions. For example, it is directly involved
in purine nucleotide synthesis. The amide groups of three Gln molecules can contribute
to each purine molecule that is synthesized. Similarly, formation of guanosine monophos-
phate requires nitrogen from Gln. In addition, pyrimidine nucleotides are synthesized by
a sequence of reactions that involve Gln and aspartate. Purines and pyrimidines are
substrates for DNA synthesis. The increased DNA synthesis in mitogen-stimulated lym-
phocytes is therefore believed to be the reason for the increased Gln utilization by these
cells.
Gln is the donor of nitrogen for synthesis of amino sugars such as glucosamine
6-phosphate, galactosamine, and N-acetylgalactosamine. Glucosamine is further metabo-
lized to synthesize N-acetylglucosamine and sialic acids. Amino sugars are found in both
glycoproteins and proteoglycans. The Gln product glutamate is part of the tripeptide
glutathione (GSH), in which its 5-carboxyl group forms the peptide linkage to the 1-amino
group of cysteine (GSH synthesis and function are described further below). In the central
nervous system, glutamate is an important excitatory neurotransmitter, and it also serves
as the precursor for g-aminobutyric acid (GABA), an important inhibitory neurotransmit-
ter. Nerve endings and neurons may also use Gln for synthesis of glutamate (and hence
GABA) because neurons have high glutaminase activity. Some of the glutamate and GABA
released by neurons are taken up by glial cells, which in turn have high glutamine
synthetase activity used in the resynthesis of Gln.

11.1.3 Gln interorgan exchanges


All these data indicate an extensive exchange of Gln between Gln-producing and Gln-con-
suming tissues. This Gln interorgan exchange is summarized in Figure 11.2. Although the
Gln-forming enzyme glutamine synthase is expressed in most cell types, net synthesis of
Gln occurs predominantly in skeletal muscle and lung. Excess Gln is released into the
blood and transported to Gln-consuming tissues. The Gln-degrading enzyme glutaminase
can only be found in a small number of tissues, such as liver, kidney, enterocytes, and
immune cells. Liver and kidney use Gln for nitrogen extraction and produce urea and
ammonia, respectively. In contrast, gut enterocytes and leukocytes utilize Gln as a main
respiratory fuel for ATP production. Thus, Gln plays an important role in nitrogen as well
as carbohydrate metabolism. The plasma Gln level is determined by the balance of Gln
production and Gln consumption.
About 20 years ago we found a marked muscle Gln depletion in septic patients that
correlated with poor survival.9 Numerous subsequent studies showed that Gln levels in
skeletal muscle as well as in plasma are strongly reduced in sepsis and other catabolic
states, which is associated with a worse prognosis of the disease. During catabolic states,
a combination of increased Gln use by Gln-consuming cells and decreased nutrient uptake
creates a Gln demand that is met primarily by increased Gln efflux from lung and muscle
tissue. Although plasma Gln levels are usually maintained in injured patients, lung and
muscle stores can be rapidly depleted after severe injury or infection. Muscle is a major
producer of Gln during normal and catabolic states. In the normal postabsoptive 200-g
rat, Gln is released from the hindquarter at a rate of approximately 0.4 mmol/min. Given
1382_C11.fm Page 175 Tuesday, October 7, 2003 6:29 PM

Chapter eleven: Glutamine metabolism 175

muscle
protein
break down
lung

plasma
(~ 0,6 mM)
urea
ATP
glucose
Gln
liver gut

NH3 ATP

kidney leukocytes

Figure 11.2 Overview of physiological Gln interorgan exchanges. The plasma Gln level is deter-
mined by the balance of Gln production and Gln consumption. Net synthesis of Gln occurs pre-
dominantly in skeletal muscle and lung, whereas net Gln uptake occurs mainly in liver, kidney, gut,
and leukocytes. Liver and kidney use Gln for nitrogen extraction and produce urea and ammonia,
respectively. In contrast, gut enterocytes and leukocytes utilize Gln as a main respiratory fuel for
ATP production.

that skeletal muscle Gln concentrations are approximately 6 to 8 mmol/l of intracellular


water, even a normal rate of release cannot be sustained for long without de novo synthesis.
During catabolic states the efflux of Gln from muscle increases markedly, and increased
net production rate must eventually compensate for this heightened release. The muscles
maintain a rapid efflux of Gln in part by increasing the rate of proteolysis while decreasing
the rate of protein synthesis. This increases the intracellular pool of amino acids for
production of Gln, which can be derived from all amino acids through their conversion
to glutamate, either directly or through a-ketoglutarate. In critical illness, muscle Gln
stores and Gln supplied by muscle proteolysis do not suffice to meet demand for Gln,
because of either the magnitude of Gln use or depletion of muscle mass. This leads to a
decrease in plasma Gln levels, and Gln-utilizing cells therefore suffer from Gln starvation
under these conditions.

11.2 Effects of Gln starvation


Gln is used as a substrate for ATP formation mainly in gut enterocytes and leukocytes.
Thus, these cells exhibit the highest susceptibility to Gln starvation under catabolic con-
ditions. Both cell types play important roles in the defense against microorganisms: while
enterocytes are essential for integrity of gut mucosa, which prevents translocation of
bacteria from the lumen into the abdominal cavity, leukocytes detect and eliminate invad-
ing microorganisms in the course of immune response. Both capabilities are essential
during critical illness. However, reduced availability of Gln under these conditions has
adverse effects on energy metabolism, protein synthesis, cell protective mechanisms, via-
bility, and function of these Gln-consuming cells (summarized in Figure 11.3).
1382_C11.fm Page 176 Tuesday, October 7, 2003 6:29 PM

176 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

AMPK pathway catabolic pathways ↑


ATP↓ AMPK anabolic pathways ↓
protein synthesis ↓

? Cellular redox state TRX:ASK1 ↓ JNK


GSH↓ redox-sensitive
kinases NF-kB
AP-1
Osmo-signaling
Fas ↑
delayed Hsp70-induction apoptosis
cell volume ↓
Erk ↓
p38 ↓ autophagic-proteolysis ↑
Gln-depletion
Translation p70s6k
mTOR ↓ 4E-BP1 translation ↓
...

glutaminyl-tRNA synthetase
QRS:Gln:ASK1 complex ↓ JNK

Figure 11.3 Cellular effects of Gln starvation. Reduced availability of Gln affects a number of dif-
ferent molecular mechanisms. Gln starvation leads to a reduction of intracellular ATP. Cellular
energy depletion leads to activation of AMPK, which regulates enzyme activity and gene expression.
However, until now there has been no study on the effect of Gln starvation on AMPK activation.
Gln starvation leads to a reduced intracellular glutathione (GSH) level. A decrease in the GSH:GSSG
ratio affects the activity of redox-sensitive kinases. Gln starvation leads to a cell shrinkage, which
affects the osmo-sensitive pathways. In addition, reduced availability of amino acids reduces the
activity of mTOR, which is a central regulatory factor in the control of translation. The available
Gln is also detected by glutaminyl-tRNA synthetase, which acts as a regulatory cofactor in the Fas-
mediated apoptosis pathway. All these mechanisms result in a reduction of anabolic pathways and
an increase in catabolic pathways in order to spare resources. In addition, several of these mecha-
nisms lead to an increased susceptibility of the cell to apoptosis triggers.

11.2.1 Energy metabolism


A major effect of Gln starvation is certainly the reduction of intracellular ATP. ATP is
required, directly or indirectly, for all energy-consuming processes. Energy deprivation
of mammalian cells leads to the activation of the AMP-activated protein kinase (AMPK)
cascade via an increase in the AMP:ATP ratio.10 The AMPK functions as a metabolic
master switch that inhibits anabolic processes and stimulates catabolic processes, thereby
preserving ATP. For example, AMPK inhibits acetyl-CoA-carboxylase (ACC) by phos-
phorylation, activates phosphofructokinase (PFK), and reduces the expression of genes
involved in fatty acid synthesis. It is still unclear whether the AMPK pathway becomes
activated by Gln starvation. To our knowledge, there is only one study to date that
investigated the role of AMPK in a Gln-utilizing cell type. Marsin and coworkers11
showed that hypoxia stimulates glycolysis in activated monocytes by phosphorylation
of PFK by AMPK. Thus, AMPK is involved in the regulation of the energy metabolism
of monocytes, but further studies are needed to characterize the role of AMPK in
monocyte function and the effect of Gln starvation on the AMPK pathway. However,
the sustained ATP decrease in Gln-starving monocytes8 indicates that even if ATP-
preserving countermeasures are activated, they are not sufficient to restore the normal
energy status of these cells.
1382_C11.fm Page 177 Tuesday, October 7, 2003 6:29 PM

Chapter eleven: Glutamine metabolism 177

11.2.2 Protein synthesis rate


Studies employing a variety of cell types and different tissues demonstrate that cells
respond to reduction of amino acid availability by a decrease of overall protein synthesis
rate. This control occurs predominantly at the level of initiation of translation of mRNA
into proteins (see Chapter 16 for more details). Translation initiation is regulated by over
a dozen proteins referred to as eukaryotic initiation factors (eIFs). The phosphorylation
status of these factors determines the efficiency of unwinding of structured mRNA and
the binding of mRNA to the ribosomal complex. It has been shown that slight changes in
the phosphorylation patterns of eIFs can lead to preferential translation of mRNAs con-
taining highly structured 5'-untranslated regions or favor the binding of mRNAs contain-
ing a 5'-terminal oligopyrimidine (TOP) tract.12 Thus, the response of translation initiation
to a change in amino acid availability can be general, affecting the translation of most, if
not all, mRNAs, but it can also be specific, affecting the translation of a single subset of
mRNAs. A major regulating factor of eIF phosphorylation status is the mammalian target
of rapamycin (mTOR).13 The activity and phosphorylation state of mTOR and downstream
effectors are decreased in response to amino acid limitation and stimulated upon their
readdition. For example, administration of leucine promoted activation of mTOR in the
liver of food-deprived rats14 and in Xenopus laevis oocytes.15 However, the nutrient metab-
olites and signaling molecules that act upstream of mTOR are unknown. mTOR was
recently found to be down-regulated by AMPK activation in rat skeletal muscle cells,
which suggests a direct connection to energy metabolism.16 In addition, a recent study in
yeast showed that Gln starvation caused nuclear localization and activation of TOR-
inhibited transcription factors GLN3, RTG1, and RTG3, all of which mediate Gln synthe-
sis.17 These findings suggest that the TOR pathway senses Gln concentrations.

11.2.3 Cell protective mechanisms


In the course of inflammation cells are confronted with a number of cytotoxic mediators,
including endotoxin, cytokines, and reactive oxygen species (ROS). Cells express a group
of proteins that are essential to cellular survival under such stressful conditions, the heat
shock proteins. The 70-kDa heat shock protein (Hsp70) has attracted the most interest
because it is highly inducible in leukocytes.18,19 Preemptive Hsp70 induction protects cells
against cytotoxic mediators and reduces organ dysfunction and mortality in animal mod-
els of sepsis.20 Under conditions with low plasma Gln, such as polytrauma, severe sepsis,
and acute respiratory distress syndrome, Hsp70 expression was found to be impaired in
granulocytes,21 monocytes,22 lymphocytes,23 and intestinal epithelial cells.24,25 We have
shown in vitro in isolated lymphocytes that Hsp70 induction depends on the availability
of Gln in a dose-dependent manner.26 While Hsp70 can be induced at the physiological
Gln concentration to nearly maximum levels, it shows a reduced inducibility (–47%) at
pathological Gln levels (0.2 mM). This indicates that Gln-starving cells are unable to
express normal amounts of Hsp70. Since plasma Gln depletion occurs during systemic
inflammation, the impaired Hsp70 expression under these conditions is likely to have
deleterious effects on the survival and function of leukocytes and may contribute to the
immunosuppression observed in these patients.
Many researchers have found that Hsp70 expression can be induced in a variety of
cell types and healthy animals by administration of high-dose Gln (5 to 20 mM or 5 mg/kg,
respectively).27,28 The elevated Hsp70 level protects cells and animals from subsequent
stress situations such as heat shock,25 sepsis,28 and cold ischemia-reperfusion injury.29 The
increase in Hsp70 is probably induced by a stress response of the cell to the nonphysio-
logically high Gln concentration rather than being related to a starvation effect.
1382_C11.fm Page 178 Tuesday, October 7, 2003 6:29 PM

178 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

While Hsp70 protects cells against oxidative stress by repair or removal of damaged
proteins, the second major protection factor of mammalian cells, the antioxidant glu-
tathione (GSH), reacts directly with ROS in order to prevent oxidative damage. Gln (via
glutamate) is one of the three precursor amino acids of GSH (L-g-glutamyl-L-cysteinyl-
glycine). A significant correlation between reduced Gln supply and diminished intra-
cellular GSH was observed in cultured peripheral blood mononuclear cells,30 activated
T-cells,31 Peyer’s patches,32 and critically ill patients.33 These data clearly show that leu-
kocytes are unable to maintain their GSH levels when Gln is depleted.
GSH is one of the three redox couples that determines the antioxidative capacity of a
cell: nicotinamide adenine dinucleotide phosphate (NADPH/NADP+), thioredoxin
(TRXred/TRXox), and glutathione (GSH/GSSG). Although glutathione is (due to its high
concentration) the most important determinant of the cellular redox state, the GSH/GSSG
ratio is clearly affected by the redox ratios of the other couples. Under physiological
conditions, GSH levels are 10- to 100-fold higher than GSSG levels. Gln starvation-induced
reduction of GSH content is likely to show a strong effect on the cellular redox state.
Shifting the redox state toward oxidizing conditions affects the activation of several sig-
naling molecules, including protein kinase B, protein phosphatases 1 and 2A, calcineurin,
nuclear factor (NF)-kB, c-Jun N-terminal kinase (JNK), apoptosis signal-regulated kinase
1 (ASK1), and p38 mitogen-activated protein kinase (MAPK).34,35 These mediators are
named redox-sensitive kinases. Oxidizing conditions activate pathways that reduce pro-
liferation and cytokine production and increase apoptosis.36–38

11.2.4 Apoptosis
Apoptosis can be induced by a range of environmental, physical, or chemical stresses.
Recent studies have established that the survival-promoting effects of Hsp70 can be partly
attributed to the suppression of apoptosis.39 Thus, the reduced Hsp70 expression in Gln-
starving cells, together with their impaired antioxidative capacity, is likely to make them
more sensitive to induction of apoptosis. We showed in premonocytic U937 cells that the
number of apoptotic cells increased only marginally after Gln starvation for 4 h. However,
Gln starvation renders these cells more susceptible to specific apoptosis triggers.40 The
number of apoptotic cells was significantly higher (50 to 60%) in Gln-starving cells when
exposed to Fas-ligand or heat shock. No effect of Gln starvation was found on UV irra-
diation-induced apoptosis. Hence, Gln starvation sensitizes cells to apoptosis induction,
probably by an impaired stress response. However, the increased susceptibility depends
on the apoptosis trigger.
Apoptosis is initiated by two principal pathways. The intrinsic pathway (stimulated,
for example, by tumor necrosis factor-a (TNF-a)) involves mitochondria, whereas the
extrinsic pathway is directly activated by the ligation of death receptors such as Fas. Binding
of Fas-ligand or agonistic antibodies to Fas receptor initiates the activation of the caspase
cascade and promotes JNK activation through interaction with ASK1, a member of the
mitogen-activated protein kinase kinase kinase (MAPKKK) family.41 These activation steps
finally lead to apoptosis. Studies in myelocytic HL-60 cells showed an increase in the Fas-
mediated apoptosis pathway by Gln starvation.42 Gln starvation for 12 h induced a reduc-
tion of cell volume by about 40%. This shrinkage promoted a ligand-independent activation
of the Fas-mediated apoptosis pathway. Accordingly, cell shrinkage did not induce apop-
tosis in CD95 receptor-negative lymphoma L1210 cells. Replacement of glutamine with
surrogate compatible osmolytes counteracted cell volume decrement and protected the
CD95-expressing cells from apoptosis. A similar increase in Fas-mediated apoptosis was
observed in Gln-starving HeLa cells.43 Fas-ligation activated ASK1 and JNK in Gln-starving
cells but not in normal cells. The authors found a Gln-dependent association of ASK1 with
1382_C11.fm Page 179 Tuesday, October 7, 2003 6:29 PM

Chapter eleven: Glutamine metabolism 179

glutaminyl-tRNA synthetase (Gln-RS). The ASK1 activity was inhibited by the interaction
with Gln-RS. This antiapoptotic function of Gln-RS was weakened during Gln starvation.
Thus, Gln directly interferes with the signal transduction pathway of Fas-mediated apop-
tosis. Isolated human neutrophils react differently to Gln starvation.7 When exposed to Fas-
ligand, neutrophil apoptosis is increased by a reduction in the extracellular concentration
of glucose but is unaffected by Gln concentration.
A crucial step in the apoptotic mechanism of TNF-a is the perturbation of mitochon-
drial functions leading to the formation of ROS. Thus, the cytotoxicity of TNF-a depends
on the antioxidative capacity of the cell, as well as on the mitochondrial activity. In mouse
fibrosarcoma L929cells, Gln is utilized as a major energy source and drives mitochondrial
ATP formation, while glucose is mainly converted to lactate through glycolysis. Gln
starvation desensitizes these cells to TNF-a cytotoxicity, while lack of glucose in the
medium does not alter the TNF-a response.44 Thus, Gln starvation protects these cells
from TNF-a cytotoxicity by reducing mitochondrial activity and thereby preventing ROS
formation. A similar relation of Gln metabolism and TNF-a-induced cytotoxicity was
described in Ehrlich ascites tumor-bearing mice.45 Feeding mice with a Gln-enriched diet
led to a high rate of Gln oxidation, which promoted a selective depletion of mitochondrial
GSH by about 58%. The increase in ROS production by TNF-a further depleted mitochon-
drial GSH and finally induced apoptosis. Taken together, these data clearly show that Gln
starvation affects cellular susceptibility to apoptosis in dependence of the cell type and of
the apoptosis trigger.

11.2.5 Osmosignaling
In recent years, it has become increasingly evident that cell volume represents a major
determinant of cellular function in a variety of cell types. In mammalian cells, hydration
may change dynamically in response to hormones, ethanol, aniso-osmotic environments,
and oxidative stress, or by cumulative substrate uptake. Gln has been shown to increase
hepatocyte hydration due to the cumulative uptake of Gln into the cells, which activates
osmosignaling pathways involving MAPK.46 Gln-induced cell swelling also activates
extracellular signal-regulated kinases (ERK) and p38 (MAPK). A similar relation of Gln
to osmosignaling pathways is described also in other cell types, including myelocytic
HL-60 cells42 and adipocytes.47 Therefore, it seems that the osmoregulatory effect of Gln
is not restricted to Gln-consuming cells (more details on osmosignaling are given in
Chapters 16 through 18).

11.2.6 Immune response


In addition to their impaired proliferative responsiveness to mitogens, Gln-starving T-lym-
phocytes show remarkable changes in their function. It was found that the expression of
the cell surface activation markers CD25, CD45RO, and CD71 and the production of
interferon-g and TNF-a are dependent on the concentrations of Gln. Gln starvation arrests
the cells in the G0/G1 phase and reduces lymphokine-activated killer cell activity. The
growth arrest can be reversed by addition of Gln by a cAMP-inhibitable and Raf-inde-
pendent activation of the JNK MAPK.48
In contrast to the rapidly proliferating lymphocytes, macrophages are terminally
differentiated cells that have lost their ability to divide. Gln starvation reduces the expres-
sion of human leukocyte antigen HLA-DR on monocyte-derived macrophages, and
decreases tetanus toxoid-induced antigen presentation. In addition, low Gln levels down-
regulate the expression of intercellular adhesion molecule-1 (ICAM-1/CD54), Fc receptor
for immunoglobulin (Ig) G (Fc gamma RI/CD64), and complement receptors type 3 (CR3;
1382_C11.fm Page 180 Tuesday, October 7, 2003 6:29 PM

180 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

CD11b/CD18) and type 4 (CR4; CD11c/CD18). These reductions are associated with a
decreased phagocytosis of IgG-sensitized ox erythrocytes or opsonized Escherichia coli.
Monocyte expression of CD14, CD71, and Fc gamma RIII/CD16 and the capacity to
phagocytose latex beads are not affected by Gln starvation.49 Gln has an important effect
on the maturation of myelomonocytic U937 cells along the monocytic pathway. Gln star-
vation reduced DNA synthesis and enhanced the development of vacuoles.
Direct evidence for the influence of Gln starvation on gut immunology was shown in
a mouse model. Parenteral feeding of mice with Gln-free solutions increased translocation
of bacteria and toxins from the gut into the circulation, caused intestinal immunoglobulin
(Ig) A deficiency, and reduced lymphocyte yield in the gut-associated lymphoid tissue.
The addition of Gln to the feeding solution improved the outcome of endotoxin-challenged
animals and normalized intestinal IgA levels and lymphocyte numbers in the PP, the
lamina propria, and the intraepithelial layer (see Chapter 42 for more details). We could
show in recent in vivo studies that restoring plasma Gln levels by Gln supplementation
in patients undergoing elective surgery is associated with a diminished decrease in HLA-
DR50 and with an increased TNF-a production in response to ex vivo lipopolysaccharide
(LPS) stimulation.40 Thus, maintaining plasma Gln levels seems not only to be essential
for the leukocyte energy status but also for their function and is therefore important for
the inflammatory response.

Acknowledgments
We thank Christine Brostjan, Maja Munk Eliasen, Andreas Spittler, Maria Zellner, and
Susanne Oehler for helpful discussions and careful reading of the manuscript.

References
1. Stadtman, E.R., The story of glutamine synthetase regulation, J. Biol. Chem., 276, 44357–44364,
2001.
2. Danbolt, N.C., Glutamate uptake, Prog. Neurobiol., 65, 1–105, 2001.
3. Bode, B.P., Recent molecular advances in mammalian glutamine transport, J. Nutr., 131
(Suppl.), 2475S–2485S, 2001 (discussion, 2486S–2487S).
4. Elia, M., Folmer, P., Schlatmann, A., Goren, A., and Austin, S., Amino acid metabolism in
muscle and in the whole body of man before and after ingestion of a single mixed meal,
Am. J. Clin. Nutr., 49, 1203–1210, 1989.
5. Obled, C., Papet, I., and Breuille, D., Metabolic bases of amino acid requirements in acute
diseases, Curr. Opin. Clin. Nutr. Metab. Care, 5, 189–197, 2002.
6. Pastor, C.M., Morris, S.M., Jr., and Billiar, T.R., Sources of arginine for induced nitric oxide
synthesis in the isolated perfused liver, Am. J. Physiol., 269, G861–G866, 1995.
7. Healy, D.A., Watson, R.W., and Newsholme, P., Glucose, but not glutamine, protects against
spontaneous and anti-Fas antibody-induced apoptosis in human neutrophils, Clin. Sci.
(Lond.), 103, 179–189, 2002.
8. Spittler, A., Winkler, S., Götzinger, P., Oehler, R., Willheim, M., Tempfer, C., Weigel, G., Függer,
R., Boltz Nitulescu, G., and Roth, E., Influence of glutamine on the phenotype and function
of human monocytes, Blood, 86, 1564–1569, 1995.
9. Roth, E., Funovics, J., Mühlbacher, F., Schemper, M., Mautitz, W., Sporn, P., and Fritsch, A.,
Metabolic disorders in severe abdominal sepsis: glutamine deficiency in skeletal muscle,
Clin. Nutr., 1, 25, 1982.
10. Hardie, D.G. and Hawley, S.A., AMP-activated protein kinase: the energy charge hypothesis
revisited, Bioessays, 23, 1112–1119, 2001.
11. Marsin, A.S., Bouzin, C., Bertrand, L., and Hue, L., The stimulation of glycolysis by hypoxia
in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phos-
phofructo-2-kinase, J. Biol. Chem., 277, 30778–30783, 2002.
1382_C11.fm Page 181 Tuesday, October 7, 2003 6:29 PM

Chapter eleven: Glutamine metabolism 181

12. Shah, O.J., Anthony, J.C., Kimball, S.R., and Jefferson, L.S., 4E-BP1 and S6K1: translational
integration sites for nutritional and hormonal information in muscle, Am. J. Physiol. Endo-
crinol. Metab., 279, E715–E729, 2000.
13. Rohde, J., Heitman, J., and Cardenas, M.E., The TOR kinases link nutrient sensing to cell
growth, J. Biol. Chem., 276, 9583–9586, 2001.
14. Jefferson, L.S. and Kimball, S.R., Amino acid regulation of gene expression, J. Nutr., 131
(Suppl.), 2460S–2466S, 2001 (discussion, 2486S–2847S).
15. Christie, G.R., Hajduch, E., Hundal, H.S., Proud, C.G., and Taylor, P.M., Intracellular sensing
of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase in a target of rapamycin-
dependent manner, J. Biol. Chem., 277, 9952–9957, 2002.
16. Bolster, D.R., Crozier, S.J., Kimball, S.R., and Jefferson, L.S., AMP-activated protein kinase
suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian
target of rapamycin (mTOR) signaling, J. Biol. Chem., 277, 23977–23980, 2002.
17. Crespo, J.L., Powers, T., Fowler, B., and Hall, M.N., The TOR-controlled transcription acti-
vators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine,
Proc. Natl. Acad. Sci. U.S.A, 99, 6784–6789, 2002.
18. Oehler, R., Pusch, E., Zellner, M., Dungel, P., Hergovich, N., Homoncik, M., Eliasen, M.,
Brabec, M., and Roth, E., Cell type specific variations in the induction of Hsp70 in human
leukocytes by fever-like whole body hyperthermia, Cell Stress Chaperones, 6, 306–315, 2001.
19. Polla, B.S., Bachelet, M., Elia, G., and Santoro, G.M., Stress proteins in inflammation, Ann.
N.Y. Acad. Sci., 851, 75–85, 1998.
20. Bruemmer-Smith, S., Stüber, F., and Schroeder, S., Protective functions of intracellular heat-
shock protein (HSP) 70-expression in patients with severe sepsis, Intensive Care Med., 27,
1835–1841, 2001.
21. Weingartmann, G., Oehler, R., Derkits, S., Oismuller, C., Fugger, R., and Roth, E., HSP70
expression in granulocytes and lymphocytes of patients with polytrauma: comparison with
plasma glutamine, Clin. Nutr., 18, 121–124, 1999.
22. Durand, P., Bachelet, M., Brunet, F., Richard, M.J., Dhainaut, J.F., Dall’Ava, J., and Polla, B.S.,
Inducibility of the 70 kD heat shock protein in peripheral blood monocytes is decreased in
human acute respiratory distress syndrome and recovers over time, Am. J. Respir. Crit. Care
Med., 161, 286–292, 2000.
23. Schroeder, S., Lindemann, C., Hoeft, A., Putensen, C., Decker, D., von Ruecker, A., and Stuber,
F., Impaired inducibility of heat shock protein 70 in peripheral blood lymphocytes of patients
with severe sepsis, Crit. Care Med., 27, 1080–1084, 1999.
24. Fukudo, S., Abe, K., Hongo, M., Utsumi, A., and Itoyama, Y., Brain-gut induction of heat
shock protein (HSP) 70 mRNA by psychophysiological stress in rats, Brain Res., 757, 146–148,
1997.
25. Wischmeyer, P.E., Musch, M.W., Madonna, M.B., Thisted, R., and Chang, E.B., Glutamine
protects intestinal epithelial cells: role of inducible HSP70, Am. J. Physiol., 272, G879–G884,
1997.
26. Oehler, R., Pusch, E., Dungel, P., Zellner, M., Munk-Eliasen, M., and Roth, E., Reduced
expression of the major inducible 70 kDa heat shock protein in glutamine starved leukocytes,
Br. J. Nutr., 87 (Suppl. 1), S17–S21, 2002.
27. Wischmeyer, P.E., Glutamine and heat shock protein expression, Nutrition, 18, 225–228, 2002.
28. Wischmeyer, P.E., Kahana, M., Wolfson, R., Ren, H., Musch, M.M., and Chang, E.B.,
Glutamine induces heat shock protein and protects against endotoxin shock in the rat, J. Appl.
Physiol., 90, 2403–2410, 2001.
29. Kojima, R., Tamaki, T., Kawamura, A., Konoeda, Y., Tanaka, M., Katori, M., Yokota, N.,
Hayashi, T., Takahashi, Y., and Kakita, A., Expression of heat shock proteins induced by L(+)-
glutamine injection and survival of hypothermically stored heart grafts, Transplant Proc., 30,
3746–3747, 1998.
30. Roth, E., Oehler, R., Manhart, N., Exner, R., Wessner, B., Strasser, E., and Spittler, A., Regu-
lative potential of glutamine: relation to glutathione metabolism, Nutrition, 18, 217–221, 2002.
1382_C11.fm Page 182 Tuesday, October 7, 2003 6:29 PM

182 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

31. Chang, W.K., Yang, K.D., Chuang, H., Jan, J.T., and Shaio, M.F., Glutamine protects activated
human T cells from apoptosis by up-regulating glutathione and Bcl-2 levels, Clin. Immunol.,
104, 151–160, 2002.
32. Manhart, N., Vierlinger, K., Spittler, A., Bergmeister, H., Sautner, T., and Roth, E., Oral feeding
with glutamine prevents lymphocyte and glutathione depletion of Peyer’s patches in endot-
oxemic mice, Ann. Surg., 234, 92–97, 2001.
33. Wernerman, J., Luo, J.L., and Hammarqvist, F., Glutathione status in critically-ill patients:
possibility of modulation by antioxidants, Proc. Nutr. Soc., 58, 677–680, 1999.
34. Filomeni, G., Rotilio, G., and Ciriolo, M.R., Cell signalling and the glutathione redox system,
Biochem. Pharmacol., 64, 1057–1064, 2002.
35. Filomeni, G., Rotilio, G., and Ciriolo, M.R., Glutathione disulfide induces apoptosis in U937
cells by a redox-mediated p38 MAP kinase pathway, FASEB J., 17, 64–66, 2003.
36. Mates, J.M., Perez-Gomez, C., Nunez de Castro, I., Asenjo, M., and Marquez, J., Glutamine
and its relationship with intracellular redox status, oxidative stress and cell prolifera-
tion/death, Int. J. Biochem. Cell Biol., 34, 439–458, 2002.
37. Thannickal, V.J. and Fanburg, B.L., Reactive oxygen species in cell signaling, Am. J. Physiol.
Lung Cell Mol. Physiol., 279, L1005–L1028, 2000.
38. Utsugi, M., Dobashi, K., Koga, Y., Shimizu, Y., Ishizuka, T., Iizuka, K., Hamuro, J., Nakazawa,
T., and Mori, M., Glutathione redox regulates lipopolysaccharide-induced IL-12 production
through p38 mitogen-activated protein kinase activation in human monocytes: role of glu-
tathione redox in IFN-gamma priming of IL-12 production, J. Leukoc. Biol., 71, 339–347, 2002.
39. Beere, H.M. and Green, D.R., Stress management: heat shock protein-70 and the regulation
of apoptosis, Trends Cell Biol., 11, 6–10, 2001.
40. Exner, R., Tamandl, D., Goetzinger, P., Mittlboeck, M., Fuegger, R., Sautner, T., Spittler, A.,
and Roth E., Perioperative Gly-Gln infusion diminishes the surgery induced period of im-
munosuppression: accelerated restoration of the LPS stimulated TNF-alpha response, Ann.
Surg., 237, 110–115, 2003.
41. Wajant, H., The Fas signaling pathway: more than a paradigm, Science, 296, 1635–1636, 2002.
42. Fumarola, C., Zerbini, A., and Guidotti, G.G., Glutamine deprivation-mediated cell shrinkage
induces ligand-independent CD95 receptor signaling and apoptosis, Cell Death Differ., 8,
1004–1013, 2001.
43. Ko, Y.G., Kim, E.Y., Kim, T., Park, H., Park, H.S., Choi, E.J., and Kim, S., Glutamine-dependent
antiapoptotic interaction of human glutaminyl-tRNA synthetase with apoptosis signal-reg-
ulating kinase 1, J. Biol. Chem., 276, 6030–6036, 2001.
44. Goossens, V., Grooten, J., and Fiers, W., The oxidative metabolism of glutamine: a modulator
of reactive oxygen intermediate-mediated cytotoxicity of tumor necrosis factor in L929 fib-
rosarcoma cells, J. Biol. Chem., 271, 192–196, 1996.
45. Obrador, E., Carretero, J., Esteve, J.M., Pellicer, J.A., Pascual, A., Petschen, I., and Estrela,
J.M., Glutamine potentiates TNF-alpha-induced tumor cytotoxicity, Free Radic. Biol. Med., 31,
642–650, 2001.
46. Haussinger, D., Graf, D., and Weiergraber, O.H., Glutamine and cell signaling in liver, J. Nutr.,
131 (Suppl.), 2509S–2514S, 2001 (discussion, 2523S–2524S).
47. Ritchie, J.W., Baird, F.E., Christie, G.R., Stewart, A., Low, S.Y., Hundal, H.S., and Taylor, P.M.,
Mechanisms of glutamine transport in rat adipocytes and acute regulation by cell swelling,
Cell Physiol. Biochem., 11, 259–270, 2001.
48. Rhoads, J.M., Argenzio, R.A., Chen, W., Graves, L.M., Licato, L.L., Blikslager, A.T., Smith, J.,
Gatzy, J., and Brenner, D.A., Glutamine metabolism stimulates intestinal cell MAPKs by a
cAMP-inhibitable, Raf-independent mechanism, Gastroenterology, 118, 90–100, 2000.
49. Spittler, A., Oehler, R., Goetzinger, P., Holzer, S., Reissner, C.M., Leutmezer, F., Rath, V., Wrba,
F., Fuegger, R., Boltz Nitulescu, G., and Roth, E., Low glutamine concentrations induce
phenotypical and functional differentiation of U937 myelomonocytic cells, J. Nutr., 127,
2151–2157, 1997.
50. Spittler, A., Sautner, T., Gornikiewicz, A., Manhart, N., Oehler, R., Bergmann, M., Fugger, R.,
and Roth, E., Postoperative glycyl-glutamine infusion reduces immunosuppression: partial
prevention of the surgery induced decrease in HLA-DR expression on monocytes, Clin. Nutr.,
20, 37–42, 2001.
1382_C12.fm Page 183 Tuesday, October 7, 2003 6:31 PM

section B

Control of and by amino acids


1382_C12.fm Page 184 Tuesday, October 7, 2003 6:31 PM
1382_C12.fm Page 185 Tuesday, October 7, 2003 6:31 PM

chapter twelve

Insulin and the regulation


of amino acid catabolism
and protein turnover
Jean-Pascal De Bandt
Université Paris 5

Contents
Introduction..................................................................................................................................185
12.1 The physiological role of insulin ....................................................................................186
12.1.1 Mechanisms of insulin action .............................................................................186
12.1.2 Insulin and the regulation of metabolism ........................................................187
12.1.3 Effect on gene transcription ................................................................................188
12.2 Insulin and amino acid catabolism ................................................................................188
12.3 Insulin and protein turnover ...........................................................................................190
12.3.1 Control of protein metabolism at the cellular level........................................190
12.3.1.1 Regulation of protein catabolism ........................................................190
12.3.1.2 Regulation of protein synthesis ...........................................................191
12.3.2 Insulin and the control of protein metabolism in vivo in humans...............193
12.3.2.1 Regulation of protein catabolism ........................................................193
12.3.2.2 Regulation of protein synthesis ...........................................................194
12.4 Conclusion ..........................................................................................................................195
References .....................................................................................................................................195

Introduction
Insulin is a key hormone, the various effects of which have long been known. The main
metabolic effects of insulin include the regulation of glycemia, energy metabolism, protein
accretion, and growth. We all know from our student textbooks that insulin promotes
glucose uptake and storage by various cell populations, and amino acid transport and
utilization for protein synthesis. But even though we have made tremendous strides in
understanding the mechanisms of insulin action, some areas remain uncharted, including
certain aspects of the regulation of amino acid catabolism and protein turnover.

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 185
1382_C12.fm Page 186 Tuesday, October 7, 2003 6:31 PM

186 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

This knowledge gap has many causes: studies of insulin action in situations of acute
insulin withdrawal or of insulin resistance, while yielding interesting data, may have
underestimated compensatory mechanisms; extrapolation of data from experimental stud-
ies to human beings is sometimes difficult; organ specificity of insulin action, for example,
according to the composition of muscle fibers, is only now being explored. The issue is
further complicated by the fact that both insulin and amino acids exert regulatory action
on glucose and amino acid metabolism and on protein turnover via interfering signaling
pathways (see Chapters 16 and 18 for details).
From a teleological point of view, the importance of these interactions that hamper
our understanding was only to be expected. Insulin is one of the main hormones of energy
storage and homeostasis and of protein accretion. Storage and utilization of amino acids,
and even more so of essential amino acid, as an energy source, is not cost-effective. For
example, we are all well aware of the importance of peripheral amino acid mobilization
during the metabolic response to stress; however, when sustained during prolonged stress
situations, this has adverse consequences in terms of malnutrition and increased morbidity.
The study of the kinetics and dose–response relationship of amino acid effect on protein
metabolism has shown that “the mechanisms of control of muscle protein synthesis contain
a physiological device that limits the amount of protein that can be stored in muscle.”1
Thus, when the protein pool is at its highest, amino acid excess must be dealt with.
According to amino acid supply levels, amino acid availability therefore ought to act to
control the anabolic response to insulin. For example, at high amino acid supply levels,
peak amino acid utilization for protein synthesis can be reached and excess amino acid
then has to be degraded; this requires a relative decrease in the insulin effect on the former
process and a relative insulin resistance in order to promote amino acid oxidation at the
expense of glucose utilization. Conversely, if amino acid supply levels are low, the pro-
moting effect of insulin on protein synthesis has to be blunted.

12.1 The physiological role of insulin


12.1.1 Mechanisms of insulin action
After binding of insulin, insulin receptors become tyrosine-phosphorylated through an
autophosphorylation reaction. The receptor then phosphorylates a number of intracellular
substrates such as the insulin receptor substrate (IRS)-1 and IRS-2 and Shc, initiating
various signaling pathways (Figure 12.1). However, many insulin responses are mediated
through IRS-1 and IRS-2. These proteins are characterized by the presence of an NH2-
terminal pleckstrin homology (PH) domain, which can mediate interaction with phospho-
lipids, and a phosphotyrosine-binding domain, which allows binding to the insulin recep-
tor.2,3 IRS proteins couple insulin receptors to the phosphatidylinositol 3-kinase (PI3-
kinase) and extracellular signal-regulated kinase (ERK) cascades:

• PI3-kinase belongs to a family of enzymes that phosphorylate phosphoinositides.


Among PI3-kinases, class IA enzymes consist of a p110 catalytic subunit and a p85
regulatory subunit. P85 presents two SH2 domains (SH2 for Src-homology-2) en-
abling binding to phosphorylated tyrosine residues. Insulin specifically activates
PI3-kinase type IA, the products of which include phosphatidylinositol-3,4-bisphos-
phate (PtdIns(3,4)P2) and phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3).
These phospholipids activate various signaling kinases, including the mammalian
target of rapamycin (mTOR), alternate protein kinase C isoforms, and phosphoi-
nositide-dependent kinase (PDK). PDK phosphorylates and activates protein ki-
nase B (PKB)/akt, which in turn can phosphorylate several substrates, including
1382_C12.fm Page 187 Tuesday, October 7, 2003 6:31 PM

Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 187

insulin

Insulin receptor

GLUT4 translocation Cbl

CAP
Shc IRS1
Grb2 PI3K
ras--GDP
SOS
p85 p110
PtdIns(4)P,
ras--GTP PtdIns(4,5)P2
raf

PtdIns(3,4)P2,
MEKK
PtdIns(3,4,5)P3
MAPKinase
cascade MEK PDK1
aPKC
PKB/Akt

ERK
mTOR Forkhead
GSK3
transcription factors

p70s6k

Gene expression, Glucose metabolism,


Cell growth, Glycogen/lipid/protein synthesis
Differentiation Specific gene expression

Figure 12.1 Insulin signaling cascade. The insulin receptor undergoes autophosphorylation and
catalyzes the phosphorylation of cellular proteins such as IRS, Shc, or Cbl. These proteins then
interact with SH2 domain-containing molecules. Activation of phosphatidylinositol-3-OH kinase
(PI3K) results in the generation of phosphatidylinositol-3,4-diphosphate (PtdIns(3,4)P2) and phos-
phatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3). PtdIns(3,4)P2 and PtdIns(3,4,5)P3 activate a va-
riety of downstream signaling kinases, including phosphoinositide-dependent kinase 1 (PDK1),
protein kinase B (PKB/Akt), alternate protein kinases C (aPKC: PKC z and l), and the mammalian
target of rapamycin (mTOR). Tyrosine phosphorylation of Shc proteins, which in turn interact with
the adapter protein Grb2, recruits the Son-of-sevenless (SOS) exchange protein for activation of Ras.
Ras stimulates a serine kinase cascade (MAPkinase) through stepwise activation of Raf, MEKK (MEK
kinases), MEK (MAP/ERK kinases), and ERK (extracellular signal-regulated kinases), leading to the
phosphorylation of transcription factors. (Adapted from Saltiel, A.R. and Kahn, C.R., Nature, 414,
799, 2001; White, M.F., Am. J. Physiol., 283, E413, 2002.)

glycogen synthase kinase 3 (GSK-3) and mTOR. This pathway seems to be involved
in the control of various biological processes such as glucose transport and me-
tabolism, protein synthesis, and cell survival.2,3
• ERK cascade activation involves the GRB-2/SOS complex, which can bind activat-
ed Shc and IRS proteins, and in turn interacts with Ras, thus yielding the active
form of Ras (Ras-GTP). Ras association with Raf-1 enables the activation of the
latter and initiation of the MEK/ERK signaling cascade. These mitogen-activated
protein kinases (MAPKs) have a wide range of potential substrates, including
transcription factors and other protein kinases.2,3

12.1.2 Insulin and the regulation of metabolism


Insulin is a multifunctional anabolic hormone. While on a short-term basis it regulates
protein and energy balance, it also stimulates cell growth and differentiation.2
Insulin plays a prominent role in the moment-to-moment adaptation of protein and
energy metabolism to nutrient supply. It increases glucose uptake in muscle and adipose
tissue and inhibits hepatic glucose production. It also promotes the storage of nutrient in
1382_C12.fm Page 188 Tuesday, October 7, 2003 6:31 PM

188 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

adipose tissue, liver, and muscle by stimulating lipogenesis, glycogenesis, and, at least in
vitro, protein synthesis and by inhibiting lipolysis, glycogenolysis, and protein breakdown.
The role of insulin in promoting growth is an evolving research field. The role of
distinct forms of PKB/akt has been emphasized: mice lacking akt2 are insulin resistant
and undergo a diabetes-like syndrome, and the absence of akt1 is associated with growth
stunting.4 The role of insulin is complicated by the fact that insulin, insulin-like growth
factor-1 (IGF-I), growth hormone, and thyroid hormones are interdependent, with complex
interrelationships in growth and aging.5 Moreover, while the physiological roles of insulin
and insulin-like growth factors are distinct — these hormones acting, respectively, on fuel
uptake and metabolism and on cell growth and differentiation — ligand interaction with
the insulin receptor and IGF-I receptor activates very similar signaling pathways, so that
it may also result in very similar biological effects.3 It has even been suggested that in vivo
specificity of insulin and IGF is in part related to the level of receptor expression in target
tissues with high levels of insulin receptors and low levels of IGF-I receptors in hepato-
cytes, adipocytes, and skeletal muscle and the opposite in fibroblasts and various undif-
ferentiated progenitor cells.3

12.1.3 Effect on gene transcription


Insulin can affect the transcription of different genes via two different mechanisms. First,
via ERK and MAPK activation, insulin can activate genes involved in cell growth.6 Second,
insulin acts via PKB-mediated forkhead transcription factors. Forkhead transcription fac-
tors are normally located in the nucleus where they activate transcription of genes, includ-
ing phosphoenol pyruvate carboxykinase and tyrosine aminotransferase. PKB activation
by insulin leads to the phosphorylation of these transcription factors and to the inactivation
of these genes.6 Cis-acting elements or insulin response elements (IREs) that mediate the
action of insulin on gene transcription mediate the insulin-dependent transcriptional
inhibition of several hepatic genes such as those encoding phosphoenolpyruvate carboxy-
kinase (PEPCK), insulin-like growth factor-binding protein 1 (IGFBP-1), tyrosine amino-
transferase, apolipoprotein CIII, and glucose-6-phosphatase.6 The other consensus IREs
are the Ets motif and the serum response element, which mediate stimulatory effects of
insulin on several genes.6 For example, insulin has been demonstrated to increase IGFBP-3
gene transcription7 or cytoskeletal gamma-actin gene.8

12.2 Insulin and amino acid catabolism


Overall, insulin favors amino acid uptake by various organs. It modulates the activity of
several amino acid transport systems such as systems A, ASC, CAT, N, and Nm.9–12 System
A is a key target of hormonal regulation. It has been shown that insulin up-regulates
system A by various mechanisms according to the tissue: it activates system A in a gene
transcription-dependent manner in hepatocytes, and through a rapid mechanism in mus-
cle.9 This effect is independent of protein synthesis and electrochemical gradient of sodium.
Surprisingly, system A activity is also up-regulated in situations of insulin deficiency or
insulin resistance. This is eventually related to another mechanism of system A regulation
called adaptive regulation, i.e., the derepression of system A in the absence of amino acids.9
In skeletal muscle cells, insulin induces plasma membrane abundance of SAT2, one
of the cloned members of the system A transporter family. This occurs in a PI3-kinase-
dependent manner,13 as demonstrated by its inhibition by the PI3-kinase inhibitor, wort-
mannin, but does not involve mTOR or MAPK pathways.14 Hyde et al.13 demonstrated
that this increase results from increased recruitment of the transporter from an endosomal
compartment to the cell membrane. Notably, these authors also observed that SAT2 was
1382_C12.fm Page 189 Tuesday, October 7, 2003 6:31 PM

Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 189

present in a specific population of endosomal vesicles, from which the glucose transporter
GLUT4 was excluded.13
In adipocytes, insulin stimulated glutamine transport via the ASCT2 members of the
ASC transporter. This effect may involve both the ERK/MAPK pathway and the insulin-
induced modifications of ion transport through the cell membrane.15
Concerning amino acid oxidation, it has been largely demonstrated in the liver that
the regulation of the urea cycle depends on substrate availability and hormones.12 Beliveau
Carey et al.16 demonstrated that substrate availability exerts moment-to-moment control
over the velocity of urea synthesis. However, while insulin increases amino acid transport
to the liver as stated above, it preserves amino acids from catabolism, as suggested, for
example, by the absence of any increase in 15N-urea from 15N-glutamine in isolated rat
hepatocytes compared with the stimulating effect of glucagon.17
Among amino acid-degrading enzymes only phenylalanine hydroxylase and the
branched-chain alpha-keto acid dehydrogenase (BCKAD) complex are regulated by phos-
phorylation/dephosphorylation mechanisms and are thus potential targets of insulin
action. Most other amino acid-degrading enzymes show adaptive induction in conditions
of increased gluconeogenesis.18
Phenylalanine hydroxylase, the major regulatory step of the phenylalanine degrada-
tion pathway, catalyzes the conversion of phenylalanine to tyrosine. Insulin has been
shown to antagonize the glucagon-stimulated phosphorylation of phenylalanine hydrox-
ylase in hepatocytes.19 This may be relevant to the potential use of phenylalanine as a
gluconeogenic precursor. Induction of diabetes in rats is associated with a significant
increase in the abundance of both phenylalanine hydroxylase protein and phenylalanine
hydroxylase-specific mRNA.20
Given the importance of branched-chain amino acids (BCAAs) and particularly of
leucine in the control of protein turnover, their metabolism and regulation by insulin have
been the focus of much research. BCAAs form the bulk of the amino acids released by the
splanchnic bed after a meal. The first two enzymes of the catabolic pathway of BCAAs are
BCAA-aminotransferase (BCAT) and BCKAD. In humans, transamination and oxidative
capacities are highest (66% of total capacities) in skeletal muscle.21 This is notably different
from rats in which, besides a considerably (nearly 10-fold) higher total oxidative capacity,
oxidation is mainly hepatic (60%). Hormonal regulation of BCAAT expression has not been
demonstrated except in the mammary gland.22 However, in type I diabetic patients, Nair
et al.23 have observed that insulin treatment decreases leucine transamination. BCKAD
complex represents the key step of BCAA catabolism and is subject to tight regulation.
BCKAD is subject to end-product inhibition, but the most important mechanism is its
regulation by phosphorylation/dephosphorylation. Inactivating phosphorylation is per-
formed by a BCKAD kinase and dephosphorylation by a BCKAD phosphatase, which act
on the E1 component of the enzyme complex.18 Little is known about the regulation of the
phosphatase. Evidence of BCKAD regulation by insulin is essentially indirect, deduced
either from in vivo experiments or from studies in the diabetic situation. In diabetic animals,
Lombardo et al.24 observed an increase in the hepatic BCKAD activity related to an increase
in its protein mass and decreased mass of its associated kinase. These alterations appear
to occur posttranscriptionally, since diabetes had no effect on the gene expressions of
BCKAD subunits or BCKAD kinase. On the other hand, the protein expression of BCKAD
kinase has been shown to be downregulated posttranscriptionally in the skeletal muscle
but upregulated pretranscriptionally in the cardiac muscle.25 In type I diabetic patients,
insulin withdrawal increases and insulin administration decreases leucine oxidation,23,26–29
while in streptozotocin diabetic rats, an increase in nitrogen exchange between leucine and
alanine has been demonstrated together with an increase in total alanine release by mus-
cle.30 In perfused rat hindquarter, insulin decreased leucine oxidation.31 Insulin and leucine
1382_C12.fm Page 190 Tuesday, October 7, 2003 6:31 PM

190 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

both stimulate BCKAD activity, promoting dephosphorylation in the adipose tissue


enzyme.32 However, in normal fasted adults 13C-leucine oxidation measured after a 12-h
fast33 was insensitive to increased insulin with leucine clamped at a low concentration, and
oxidation increased with the physiological increases in leucine concentration after a 4-day
fast, while insulin decreased.34 This suggests that in the whole body the overriding regu-
latory influence on BCKAD is substrate availability. At increasing protein supply but
constant energy level eliciting insulin responses,35,36 leucine oxidation ranged from inhibi-
tion at the lowest to marked stimulation at the highest protein intakes, consistent with the
changes in plasma leucine. It would thus appear that there is an equilibrium between the
effect of insulin on BCKAD and protein feeding-related induction of the enzyme when the
BCAA supply is from the diet and disposal is a priority.

12.3 Insulin and protein turnover


12.3.1 Control of protein metabolism at the cellular level
12.3.1.1 Regulation of protein catabolism
Pioneering studies, in particular by Mortimore’s group,37 demonstrated that autophagic
protein degradation, which is largely responsible for liver protein degradation during
fasting, is inhibited by amino acid and insulin (see also Chapter 16). Interestingly, these
authors indicate that insulin failed to inhibit protein breakdown in the absence of amino
acids or in the presence of maximally suppressive amino acids, while it virtually sup-
pressed the zonal loss of effectiveness of regulatory amino acids at amino acid levels
matching those in the portal vein in fed rats (Figure 12.2).37

400
Valine release (nmol/min,100g rat)

350

300

250

200

150

100

50

0
0 2 4 6 8 10
Multiples of plasma amino acid concentration

Figure 12.2 Insulin and amino acid control of hepatic proteolysis. Isolated rat livers were perfused
with either regulatory amino acids (leucine, tyrosine, glutamine, proline, methionine, histidine,
tryptophan) with () or without (●) insulin or a complete plasma amino acid mixture () at multiples
of their concentration in normal plasma. Protein degradation was evaluated by the measurement
of valine release. (Adapted from Mortimore, G.E. et al., Diabetes Metab. Rev., 5, 49, 1989.)
1382_C12.fm Page 191 Tuesday, October 7, 2003 6:31 PM

Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 191

Blommaart et al.38 showed, in isolated hepatocytes, that insulin and hypo-osmotic


conditions similarly enhance the sensitivity of autophagic proteolysis to inhibition by
amino acids. While hepatic cell swelling has been demonstrated to be an important
regulator of liver metabolism and protein turnover, insulin stimulates Na+-H+ exchange,
Na+-K+-2 Cl– cotransport, and the Na+-K+-ATPase in the liver, leading to intracellular K+,
Na+, and Cl– accumulation and thus cell swelling39 (see Chapter 16 for more details). In
parallel, insulin through its known stimulatory effect on amino transport via the Na+-
dependent system A may further enhance amino-induced cell swelling. As both cell
swelling and insulin activate PI3-kinase, a possible action via PDK1 and the mTOR path-
way may provide a common ground for these effects. Notably, distinct classes of PI3-kinase
are involved in the signaling pathways that control macroautophagy. Class IA enzymes,
which are stimulated by insulin, inhibit macroautophagy, whereas class III enzymes trigger
autophagic sequestration.40 It should also be mentioned that Petiot et al.40 did not observe
any involvement of PKB/akt in the control of these processes. On the other hand, phos-
phorylation of ribosomal protein S6 has been shown to be inhibitory for autophagy in rat
hepatocytes; both hypo-osmolarity and insulin increased phosphorylation of S6 at low
amino acid concentrations.38
An inhibitory effect of insulin on ATP and ubiquitin-dependent protein degradation
has also been demonstrated. In vitro, in rat L6 myotubes, insulin lowered levels of 14-kDa
ubiquitin-conjugating enzyme E2(14k) mRNA.41 Bennett et al.42 showed that insulin inhib-
ited protein (lysozyme) degradation by the proteasome in reticulocyte extracts by more
than 90%. This effect was also observed in HepG2 cells.42 Mitch et al.43 observed in acutely
diabetic rats that insulin reversed muscle proteolysis and that this was associated with a
normalization of the mRNAs encoding the ubiquitine–proteasome system component.
However, the mechanisms of the inhibition of proteolysis by insulin may vary according
to the fiber composition of the muscle studied.44 In a study of the influence of a euglycemic
hyperinsulinemic clamp on proteolysis measured in vitro, Larbaud et al.44 observed that
the antiproteolytic effect of insulin was suppressed by inhibitors of the lysosomal path-
ways in the slow-twitch red-fiber soleus, but by a proteasome inhibitor in the fast-twitch
mixed-fiber extensor digitorum longus.

12.3.1.2 Regulation of protein synthesis


Insulin regulates protein synthesis in mammalian cells. Besides its effect on gene expres-
sion, insulin acts at the level of mRNA translation. In the adrenalectomized streptozotocin
diabetic rat, global translation rate is inhibited by 40% even with in vivo infusions of
glucose and amino acids, and this inhibition is entirely restored by insulin infusion at
physiological levels.45 Furthermore, the increased insulin secretion within the first 20 min
of refeeding in a 4-day fasted rat is causally associated with increased skeletal muscle
protein synthesis.46 However, in vivo studies indicate that insulin effects on protein syn-
thesis may be tissue specific. Boirie et al.47 have studied, in miniature swine, fractional
synthesis rates (FSR) of mitochondrial and cytoplasmic proteins in liver, heart, and skeletal
muscle, and myosin heavy chain in muscle. Insulin infusion increased the FSR of muscle
mitochondrial proteins but had no effect on FSR of myosin. In the heart, insulin only
increased FSR of cytoplasmic protein. In liver, insulin stimulated neither mitochondrial
nor cytoplasmic protein FSR.
Only the steps of the peptide chain initiation process (Figure 12.3) affected by insulin
will be addressed here (see Chapters 16 and 18 for more details on this process). Insulin
induces a global increase in the rate of translation but also a marked increase in the
translation of specific mRNAs such as ribosomal proteins and elongation factor.48 It acts
on several steps in the translation process:
192

insulin PKB/Akt - GSK3

eIF2B eIF2B--P

GDP GTP

eIF2 eIF2 eIF2


GTP GDP GDP
Met-tRNAi
eIF4E eIF4A
eIF4G
60S
eIF3 eIF2 eIF3
eIF2 GTP 40S
1382_C12.fm Page 192 Tuesday, October 7, 2003 6:31 PM

40S eIF3
GTP Met-tRNAi
40S m7GTP-mRNA
Met-tRNAi
40S
43S preinitiation complex
80S eIF3 80S initiation complex
m7GTP-mRNA
60S
eIF4E eIF4A
eIF4G ELONGATION
60S eIF4F

eIF4E eIF4A
eIF4E
eIF4G
eIF4E-BP1
+
insulin PKB/Akt mTOR eIF4E-BP1 -P

Figure 12.3 Initiation of protein synthesis in mammalian cells. Regulation by insulin. Dissociation of the 80S ribosome into its components, 40S and 60S,
requires the eukaryotic initiation factor (eIF) 3. The binding of the initiator Met-tRNA to the 40S subunit is mediated by eIF2 (only when it is complexed
with GTP). At the following stage, eIF4F (complex of eIF4E, eIF4G, and eIF4A) binds to mRNA (m7GTP-mRNA), and the eIF4F-mRNA complex then
binds to the 43S preinitiation complex. The last stage is the addition of the 60S subunit to form the 80S initiation complex, which can enter elongation.
This stage involves the hydrolysis of the GTP bound to eIF2 and the release of eIF2-GDP along with the other eIFs. Insulin acts via PI3-kinase/PKB and
phosphorylation/inactivation of GSK-3, and enables eIF2B to promote GTP exchange on, and thus activation of, eIF2. On the other hand, insulin favors
the dissociation of eIF4E from its binding protein, eIF4E-BP1, enabling its association with the other proteins of the eIF4F complex and the subsequent
interaction of this complex with the 43S preinitiation complex. (Adapted from Proud, C.G. and Denton, R.M., Biochem. J., 328, 329, 1997; Kimball, S.R.
et al., J. Appl. Physiol., 93, 1168, 2002.)
Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition
1382_C12.fm Page 193 Tuesday, October 7, 2003 6:31 PM

Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 193

• Regulation of eukaryotic initiation factor (eIF) 2 activity: eIF2 exists as an inactive


eIF2-GDP and an active eIF2-GTP complex, eIF2B promoting the exchange of GDP
by GTP on eIF2. eIF2B is inhibited by its phosphorylation by GSK-3. Insulin acts
via PI3-kinase and PKB/akt, which phosphorylates and inactivates GSK-3, thus
alleviating its inhibitory effect on eIF2B and leading to enhanced nucleotide ex-
change on eIF2. Increased availability of active eILF2-GTP thus promotes transla-
tion initiation at the stage of the 43S preinitiation complex.48
• Regulation of the eIF4F complex: Insulin favors the dissociation of eIF4E from
E-BP1, enabling its association with the other proteins of the eIF4F complex and
the subsequent interaction of this complex with the 43S preinitiation complex.48
This may occur via PI3-kinase and PKB/akt in the insulin signaling cascade and
modulation of the phosphorylation state of eIF4E-binding protein 1 (eIF4E-BP1)
and ribosomal protein S6 kinase (p70S6K). However, amino acids are required for
the maintenance of the insulin effect on eIF4E-BP1 and p70S6K 49 (see Chapters 16
and 18 for more details).
• Regulation of eEF2: Translation elongation requires eukaryotic elongation factors
(eEFs). eEF2, which mediates ribosomal translocation, is phosphorylated/inacti-
vated by eEF2-kinase, which is regulated by both MAPK and the mTOR signaling
pathway.50 Insulin decreases the activity of eEF2-kinase.48 This effect is influenced
by amino acids.51
• Activation of p70S6K by insulin leading to the phosphorylation of the ribosomal
protein S6 has been suggested to play an important role in the regulation of specific
mRNAs encoding for ribosomal and elongation factors, the so-called 5'-terminal
oligopyrimidine (TOP) mRNAs.52

12.3.2 Insulin and the control of protein metabolism in vivo in humans


Thus from in vitro or cells-in-culture studies it seems reasonable to assume that insulin
exerts a marked inhibitory effect on proteolysis and a stimulatory one on synthesis. While
the in vivo antiproteolytic effect of insulin has been repeatedly demonstrated, the anabolic
effect of insulin on protein synthesis is largely debated.
The main difficulty in interpreting whole-body studies arises from the combination
of direct and indirect actions of insulin and of the associated changes in amino acid
concentrations. The direct effect of amino acids on protein synthesis and catabolism and
the hypoaminoacidemia induced by insulin administration can largely account for the
discrepancies in insulin effects.53 Given that the role of insulin is to preserve amino acids
for protein synthesis, insulin-induced or insulin-independent changes in amino acid avail-
ability may affect the response of protein metabolism because of its sensitivity to amino
acid concentration.

12.3.2.1 Regulation of protein catabolism


In humans, the rate of whole-body protein degradation falls in response to physiological
increases in plasma insulin.54 Conversely, whole-body protein degradation is increased in
insulin-withdrawn type 1 diabetic patients.23,28,29 When the fall in plasma amino acid is
prevented either by amino acid perfusion or by an oral protein supply, an inhibition of
proteolysis by insulin is observed in type I diabetes.27,55 Of note, Biolo et al.53 observed
that whole-body proteolysis was suppressed during meal ingestion in type I diabetic
patients despite insulin withdrawal; this finding suggests that the influences of amino
acids and insulin on proteolysis are separate and additive. In normal subjects, insulin-
induced inhibition of proteolysis is enhanced by higher levels of amino acids.34
1382_C12.fm Page 194 Tuesday, October 7, 2003 6:31 PM

194 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

The tissue location of insulin action on proteolysis is a subject of debate. While several
investigations indicate that insulin is most effective on skeletal muscles,56–58 this preference
has not been consistently observed.59,60 In a study in type I diabetic patients, Nair et al.23
observed that insulin also causes an inhibition of protein breakdown in splanchnic tissues.

12.3.2.2 Regulation of protein synthesis


On a whole-body basis, the anabolic effect of insulin on protein synthesis has not been
unequivocally demonstrated.28,61,62 On the contrary, some studies have even suggested an
insulin-induced reduction in protein synthesis.23,26 In diabetic patients, some studies indi-
cate that both protein breakdown and synthesis increase during insulin deprivation; net
protein loss may thus be due to a greater increase in catabolism.63
While limitations of method may be responsible, as suggested by Biolo et al.,64 who
observed a stimulating effect of insulin using arteriovenous catheterization and muscle
biopsy approaches, one explanation could be the sensitivity of protein synthesis to amino
acid availability:

• A study using varying intakes of protein eliciting similar insulin levels indicates
that the response of protein synthesis depends on plasma leucine levels.35 Volpi et
al.,65 measuring whole-body protein kinetics in healthy volunteers during intra-
gastric infusion of water, glucose–lipid–amino acid meal, or isocaloric glucose–lip-
id meal, showed that dietary amino acids account for approximately 90% of post-
prandial protein anabolism and insulin for only 10%. When amino acid levels are
clamped, increasing insulin up to levels inducing maximal suppression of proteol-
ysis did not influence whole-body protein synthesis.34,57 However, during an insu-
lin–glucose clamp with amino acid infusion, Bennet et al.61 observed in type I
diabetic patients that protein synthesis was unaltered by insulin, but that there
was a concomitant decrease in the intramuscular concentrations of a number of
amino acids, including leucine. This suggests that the decrease in amino acid
availability has hampered the effect of insulin on protein synthesis. In another
study in healthy subjects, the same authors62 reported that insulin, given with a
sufficient amount of amino acids, may stimulate leg and whole-body protein
synthesis.
• It has been suggested that the relative sensitivity of protein synthesis and proteol-
ysis to insulin and amino acids may differ between rats and humans; this might
thus also account for the lack of stimulating effect of insulin in humans.66 In
humans, the inhibition of proteolysis could be the major response to insulin; a
reduction in intracellular amino acid concentrations and specifically of leucine
would thus inhibit protein synthesis.
• This question is further complicated by possible pathology-related or amino acid-
induced alterations in the sensitivity to insulin action. Deficient protein turnover
sensitivity to insulin action has been described both in type I and type II diabe-
tes.27,62,67,68 On the other hand, while the stimulatory effect of specific amino acids
such as leucine and arginine on insulin secretion has been largely demonstrated
(see Chapter 20 for details), several in vivo studies have pointed to a deterioration
of tissue response to insulin action in various situations, including high-protein
diet. Infusion of amino acids during a euglycemic hyperinsulinemic clamp in
normal fasted volunteers decreases whole-body glucose oxidation, nonoxidative
glucose disposal, forearm glucose disposal, and responsiveness of hepatic glucose
production to insulin.69 Patti et al.70 have shown that this occurs via decreased
insulin-stimulated tyrosine phosphorylation of IRS-1 and a marked inhibition of
insulin-stimulated PI3-kinase. They suggested that amino acids act as specific
1382_C12.fm Page 195 Tuesday, October 7, 2003 6:31 PM

Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 195

positive signals for maintenance of protein stores, while inhibiting other actions
of insulin on glucose metabolism. Tremblay and Marette71 further demonstrated
in L6 myotubes that amino acids speed up the time-dependent deactivation of IRS-
1-associated PI3-kinase via a rapamycin-sensitive increase in serine/threonine
phosphorylation of IRS-1 and decreased binding of PI3-kinase to IRS-1.

Thus, while protein synthesis is clearly regulated by amino acid levels, the exact
contribution of insulin remains to be clearly delineated.

12.4 Conclusion
While we gradually map the pathways of insulin action on amino acid metabolism, new
knowledge concerning the interference of nutrients comes and blurs the picture. Although
insulin is demonstrably a major contributor to the regulation of amino acid and protein
metabolism, the precise delineation of the pathways involved, along with the possible
species-to-species and tissue-to-tissue particularities, remains to be established. Building
on initial studies, which indicated that insulin acts via PI3-kinase while numerous medi-
ators also act on this pathway, we now know that a specific class of PI3-kinase is induced
by insulin. The possibility of subtypes of other intermediates of insulin signaling cannot
be ruled out, while the unraveling of the complex world of transcription factors will
probably yield interesting findings. For example, peroxisome-proliferator-activated recep-
tor (PPAR) a activators promote BCAA oxidation by increasing expression of components
of the BCKAD complex and decreasing expression of the BCKAD kinase,72 while PPARg
agonist potentiates insulin-stimulated Akt phosphorylation in adipose, muscle, and liver
tissues.73 The effects of exercise on muscle insulin signaling and action are also of great
interest in the context of the present “diabesity” pandemic. Muscle insulin action on
glucose transport, glycogen synthesis, and amino acid transport has been demonstrated
even after a single bout of exercise.74 Moreover, some data suggest that insulin in combi-
nation with prior contractions induces a stimulation of protein synthesis at physiological
concentrations of amino acids.75

References
1. Rennie, M.J., Bohé, J., and Wolfe, R.R., Latency, duration and dose response relationships of
amino acid effects on human muscle protein synthesis, J. Nutr., 132, 3225S, 2002.
2. Saltiel, A.R. and Kahn, C.R., Insulin signalling and the regulation of glucose and lipid
metabolism, Nature, 414, 799, 2001.
3. Siddle, K., Urso, B., Niesler, C.A., Cope, D.L., Molina, L., Surinya, K.H., and Soos, M.A.,
Specificity in ligand binding and intracellular signalling by insulin and insulin-like growth
factor receptors, Biochem. Soc. Trans., 29, 513, 2001.
4. Glass, D.J., Signalling pathways that mediate skeletal muscle hypertrophy and atrophy, Nat.
Cell Biol., 5, 87, 2003.
5. Tatar, M., Bartke, A., and Antebi, A., The endocrine regulation of aging by insulin-like signals,
Science, 299, 1346, 2003.
6. White, M.F., IRS proteins and the common path to diabetes, Am. J. Physiol., 283, E413, 2002.
7. Phillips, L.S., Pao, C.I., and Villafuerte, B.C., Molecular regulation of insulin-like growth
factor-I and its principal binding protein, IGFBP-3, Prog. Nucleic Acid Res. Mol. Biol., 60, 195,
1998.
8. Messina, J.L., Regulation of gamma-actin gene expression by insulin, J. Cell Physiol., 160, 287,
1994.
9. Palacin, M., Estevez, R., Bertran, J., and Zorzano, A., Molecular biology of mammalian plasma
membrane amino acid transporters, Physiol. Rev., 78, 969, 1998.
1382_C12.fm Page 196 Wednesday, October 8, 2003 1:29 PM

196 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

10. Low, S.Y., Taylor, P.M., and Rennie, M.J., Responses of glutamine transport in cultured rat
skeletal muscle to osmotically induced changes in cell volume, J. Physiol., 492, 877, 1996.
11. Hundal, H.S., Rennie, M.J., and Watt, P.W., Characteristics of L-glutamine transport in per-
fused rat skeletal muscle, J. Physiol., 393, 283, 1987.
12. Meijer, A.J., Lamers, W.H., and Chamuleau, R.A., Nitrogen metabolism and ornithine cycle
function, Physiol. Rev., 70, 701, 1990.
13. Hyde, R., Peyrollier, K., and Hundal, H.S., Insulin promotes the cell surface recruitment of
the SAT2/ATA2 system A amino acid transporter from an endosomal compartment in
skeletal muscle cells, J. Biol. Chem., 277, 13628, 2002.
14. McDowell, H.E., Eyers, P.A., and Hundal, H.S., Regulation of system A amino acid transport
in L6 rat skeletal muscle cells by insulin, chemical and hyperthermic stress, FEBS Lett., 441,
15, 1998.
15. Ritchie, J.W., Baird, F.E., Christie, G.R., Stewart, A., Low, S.Y., Hundal, H.S., and Taylor, P.M.,
Mechanisms of glutamine transport in rat adipocytes and acute regulation by cell swelling,
Cell Physiol. Biochem., 11, 259, 2001.
16. Beliveau Carey, G., Cheung, C.W., Cohen, N.S., Brusilow, S., and Raijman, L., Regulation of
urea and citrulline synthesis under physiological conditions, Biochem. J., 292, 241, 1993.
17. Nissim, I., Yudkoff, M., and Brosnan, J.T., Regulation of [15N]urea synthesis from
[5-15N]glutamine: role of pH, hormones, and pyruvate, J. Biol. Chem., 271, 31234, 1996.
18. Harper, A.E., Miller, R.H., and Block, K.P., Branched-chain amino acid metabolism, Annu.
Rev. Nutr., 4, 409, 1984.
19. Fisher, M.J., Dickson, A.J., and Pogson, C.I., The role of insulin in the modulation of glucagon-
dependent control of phenylalanine hydroxylation in isolated liver cells, Biochem. J., 242, 655,
1987.
20. Green, A.K., McDowall, I.L., Richardson, S.C., and Fisher, M.J., The effect of vanadate upon
the expression of phenylalanine hydroxylase in streptozotocin-diabetic rat liver, Biochim.
Biophys. Acta, 1180, 21, 1992.
21. Suryawan, A., Hawes, J.W., Harris, R.A., Shimomura, Y., Jenkins, A.E., and Hutson, S.M., A
molecular model of human branched-chain amino acid metabolism, Am. J. Clin. Nutr., 68,
72, 1998.
22. Tovar, A.R., Becerril, E., Hernandez-Pando, R., Lopez, G., Suryawan, A., Desantiago, S.,
Hutson, S.M., and Torres, N., Localization and expression of BCAT during pregnancy and
lactation in the rat mammary gland, Am. J. Physiol., 280, E480, 2001.
23. Nair, K.S., Ford, G.C., Ekberg, K., Fernqvist-Forbes, E., and Wahren, J., Protein dynamics in
whole body and in splanchnic and leg tissues in type I diabetic patients, J. Clin. Invest., 95,
2926, 1995.
24. Lombardo, Y.B., Thamotharan, M., Bawani, S.Z., Paul, H.S., and Adibi, S.A., Posttranscrip-
tional alterations in protein masses of hepatic branched-chain keto acid dehydrogenase and
its associated kinase in diabetes, Proc. Assoc. Am. Physicians, 110, 40, 1998.
25. Lombardo, Y.B., Serdikoff, C., Thamotharan, M., Paul, H.S., and Adibi, S.A., Inverse altera-
tions of BCKA dehydrogenase activity in cardiac and skeletal muscles of diabetic rats, Am.
J. Physiol., 277, E685, 1999.
26. Luzi, L., Castellino, P., Simonson, D.C., Petrides, A.S., and DeFronzo, R.A., Leucine metab-
olism in IDDM: role of insulin and substrate availability, Diabetes, 39, 38, 1990.
27. Tessari, P., Pehling, G., Nissen, S.L., Gerich, J.E., Service, F.J., Rizza, R.A., and Haymond,
M.W., Regulation of whole-body leucine metabolism with insulin during mixed-meal ab-
sorption in normal and diabetic humans, Diabetes, 37, 512, 1988.
28. Pacy, P.J., Nair, K.S., Ford, C., and Halliday, D., Failure of insulin infusion to stimulate
fractional muscle protein synthesis in type I diabetic patients: anabolic effect of insulin and
decreased proteolysis, Diabetes, 38, 618, 1989.
29. Umpleby, A.M., Boroujerdi, M.A., Brown, P.M., Carson, E.R., and Sonksen, P.H., The effect
of metabolic control on leucine metabolism in type 1 (insulin-dependent) diabetic patients,
Diabetologia, 29, 131, 1986.
1382_C12.fm Page 197 Tuesday, October 7, 2003 6:31 PM

Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 197

30. Meynial-Denis, D., Chavaroux, A., Foucat, L., Mignon, M., Prugnaud, J., Bayle, G., Renou,
J.P., and Arnal, M., Contribution of proteolysis and de novo synthesis to alanine production
in diabetic rat skeletal muscle: a 15N/1H nuclear magnetic resonance study, Diabetologia, 40,
1159, 1997.
31. Hutson, S.M., Cree, T.C., and Harper, A.E., Regulation of leucine and alpha-ketoisocaproate
metabolism in skeletal muscle, J. Biol. Chem., 253, 8126, 1978.
32. Frick, G.P. and Goodman, H.M., Insulin regulation of the activity and phosphorylation of
branched-chain 2-oxo acid dehydrogenase in adipose tissue, Biochem. J., 258, 229, 1989.
33. Hutson, S.M., Zapalowski, C., Cree, T.C., and Harper, A.E., Regulation of leucine and alpha-
ketoisocaproic acid metabolism in skeletal muscle: effects of starvation and insulin, J. Biol.
Chem., 255, 2418, 1980.
34. Frexes-Steed, M., Lacy, D.B., Collins, J., and Abumrad, N.N., Role of leucine and other amino
acids in regulating protein metabolism in vivo, Am. J. Physiol., 262, E925, 1992.
35. Pacy, P.J., Price, G.M., Halliday, D., Quevedo, M.R., and Millward, D.J., Nitrogen homeostasis
in man: the diurnal responses of protein synthesis and degradation and amino acid oxidation
to diets with increasing protein intakes, Clin. Sci., 86, 103, 1994.
36. McHardy, K.C., McNurlan, M.A., Milne, E., Calder, A.G., Fearns, L.M., Broom, J., and Garlick,
P.J., The effect of insulin suppression on postprandial nutrient metabolism: studies with
infusion of somatostatin and insulin, Eur. J. Clin. Nutr., 45, 515, 1991.
37. Mortimore, G.E., Pösö, A.R., and Lardeux, B.R., Mechanism and regulation of protein deg-
radation in liver, Diabetes Metab. Rev., 5, 49, 1989.
38. Blommaart, E.F., Luiken, J.J., Blommaart, P.J., van Woerkom, G.M., and Meijer, A.J., Phos-
phorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes,
J. Biol. Chem., 270, 2320, 1995.
39. Häussinger, D., Lang, F., and Gerok, W., Regulation of cell function by the cellular hydration
state, Am. J. Physiol., 267, E343, 1994.
40. Petiot, A., Ogier-Denis, E., Blommaart, E.F., Meijer, A.J., and Codogno, P., Distinct classes of
phosphatidylinositol 3'-kinases are involved in signalling pathways that control macro-
autophagy in HT-29cells, J. Biol. Chem., 275, 992, 2000.
41. Wing, S.S. and Banville, D., 14-kDa ubiquitin-conjugating enzyme: structure of the rat gene
and regulation upon fasting and by insulin, Am. J. Physiol., 267, E39, 1994.
42. Bennett, R.G., Hamel, F.G., and Duckworth, W.C., Insulin inhibits the ubiquitin-dependent
degrading activity of the 26S proteasome, Endocrinology, 141, 2508, 2000.
43. Mitch, W.E., Bailey, J.L., Wang, X., Jurkovitz, C., Newby, D., and Price, S.R., Evaluation of
signals activating ubiquitin-proteasome proteolysis in a model of muscle wasting, Am. J.
Physiol., 276, C1132, 1999.
44. Larbaud, D., Balage, M., Taillandier, D., Combaret, L., Grizard, J., and Attaix, D., Differential
regulation of the lysosomal, Ca2+-dependent and ubiquitin/proteasome-dependent prote-
olytic pathways in fast-twitch and slow-twitch rat muscle following hyperinsulinaemia, Clin.
Sci., 101, 551, 2001.
45. Odedra, B.R., Dalal, S.S., and Millward, D.J., Muscle protein synthesis in the streptozotocin-
diabetic rat: a possible role for corticosterone in the insensitivity to insulin infusion in vivo,
Biochem. J., 202, 363, 1982.
46. Millward, D.J., Odedra, B., and Bates, P.C., The role of insulin, corticosterone and other
factors in the acute recovery of muscle protein synthesis on refeeding food-deprived rats,
Biochem. J., 216, 583, 1983.
47. Boirie, Y., Short, K.R., Ahlman, B., Charlton, M., and Nair, K.S., Tissue-specific regulation of
mitochondrial and cytoplasmic protein synthesis rates by insulin, Diabetes, 50, 2652, 2001.
48. Proud, C.G. and Denton, R.M., Molecular mechanisms for the control of translation by
insulin, Biochem. J., 328, 329, 1997.
49. Hara, K., Yonezawa, K., Weng, Q.P., Kozlowski, M.T., Belham, C., and Avruch, J., Amino acid
sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector
mechanism, J. Biol. Chem., 273, 14484, 1998.
50. Browne, G.J. and Proud, C.G., Regulation of peptide-chain elongation in mammalian cells,
Eur. J. Biochem., 269, 5360, 2002.
1382_C12.fm Page 198 Tuesday, October 7, 2003 6:31 PM

198 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

51. Campbell, L.E., Wang, X., and Proud, C.G., Nutrients differentially regulate multiple trans-
lation factors and their control by insulin, Biochem. J., 344, 433, 1999.
52. Proud, C.G., Wang, X., Patel, J.V., Campbell, L.E., Kleijn, M., Li, W., and Browne, G.J.,
Interplay between insulin and nutrients in the regulation of translation factors, Biochem. Soc.
Trans., 29, 541, 2001.
53. Biolo, G., Inchiostro, S., Tiengo, A., and Tessari, P., Regulation of postprandial whole-body
proteolysis in insulin-deprived IDDM, Diabetes, 44, 203, 1995.
54. Liu, Z. and Barrett, E.J., Human protein metabolism: its measurement and regulation, Am.
J. Physiol., 283, E1105, 2002.
55. Inchiostro, S., Biolo, G., Bruttomesso, D., Fongher, C., Sabadin, L., Carlini, M., Duner, E.,
Tiengo, A., and Tessari, P., Effects of insulin and amino acid infusion on leucine and pheny-
lalanine kinetics in type 1 diabetes, Am. J. Physiol., 262, E203, 1992.
56. Möller-Loswick, A.C., Zachrisson, H., Hyltander, A., Körner, U., Matthews, D.E., and Lund-
holm, K., Insulin selectively attenuates breakdown of nonmyofibrillar proteins in peripheral
tissues of normal men, Am. J. Physiol., 266, E645, 1994.
57. Gelfand, R.A. and Barrett, E.J., Effect of physiologic hyperinsulinemia on skeletal muscle
protein synthesis and breakdown in man, J. Clin. Invest., 80, 1, 1987.
58. Heslin, M.J., Newman, E., Wolf, R.F., Pisters, P.W., and Brennan, M.F., Effect of hyperinsuline-
mia on whole body and skeletal muscle leucine carbon kinetics in humans, Am. J. Physiol.,
262, E911, 1992.
59. Arfvidsson, B., Zachrisson, H., Möller-Loswick, A.C., Hyltander, A., Sandström, R., and
Lundholm, K., Effect of systemic hyperinsulinemia on amino acid flux across human legs
in postabsorptive state, Am. J. Physiol., 260, E46, 1991.
60. Tessari, P., Biolo, G., Inchiostro, S., Sacca, L., Nosadini, R., Boscarato, M.T., Trevisan, R.,
De Kreutzenberg, S.V., and Tiengo, A., Effects of insulin on whole body and forearm leucine
and KIC metabolism in type 1 diabetes, Am. J. Physiol., 259, E96, 1990.
61. Bennet, W.M., Connacher, A.A., Smith, K., Jung, R.T., and Rennie, M.J., Inability to stimulate
skeletal muscle or whole body protein synthesis in type 1 (insulin-dependent) diabetic
patients by insulin-plus-glucose during amino acid infusion: studies of incorporation and
turnover of tracer L-[1-13C]leucine, Diabetologia, 33, 43, 1990.
62. Bennet, W.M., Connacher, A.A., Jung, R.T., Stehle, P., and Rennie, M.J., Effects of insulin and
amino acids on leg protein turnover in IDDM patients, Diabetes, 40, 499, 1991.
63. Charlton, M. and Nair, K.S., Protein metabolism in insulin-dependent diabetes mellitus,
J. Nutr., 128, 323S, 1998.
64. Biolo, G., Declan Fleming, R.Y., and Wolfe, R.R., Physiologic hyperinsulinemia stimulates
protein synthesis and enhances transport of selected amino acids in human skeletal muscle,
J. Clin. Invest., 95, 811, 1995.
65. Volpi, E., Lucidi, P., Cruciani, G., Monacchia, F., Reboldi, G., Brunetti, P., Bolli, G.B., and
De Feo, P., Contribution of amino acids and insulin to protein anabolism during meal ab-
sorption, Diabetes, 45, 1245, 1996.
66. Millward, J., Insulin and the regulation of amino acid catabolism and protein turnover, in
Amino Acid Metabolism and Therapy in Health and Nutritional Disease, Cynober, L., Ed., CRC
Press, Boca Raton, FL, 1995, p. 127.
67. Gougeon, R., Marliss, E.B., Jones, P.J., Pencharz, P.B., and Morais, J.A., Effect of exogenous
insulin on protein metabolism with differing nonprotein energy intakes in type 2 diabetes
mellitus, Int. J. Obes. Relat. Metab. Disord., 22, 250, 1998.
68. Tessari, P., Nosadini, R., Trevisan, R., De Kreutzenberg, S.V., Inchiostro, S., Duner, E., Biolo,
G., Marescotti, M.C., Tiengo, A., and Crepaldi, G., Defective suppression by insulin of leucine-
carbon appearance and oxidation in type 1, insulin-dependent diabetes mellitus: evidence
for insulin resistance involving glucose and amino acid metabolism, J. Clin. Invest., 77, 1797,
1986.
69. Krebs, M., Krssak, M., Bernroider, E., Anderwald, C., Brehm, A., Meyerspeer, M., Nowotny,
P., Roth, E., Waldhausl, W., and Roden, M., Mechanism of amino acid-induced skeletal muscle
insulin resistance in humans, Diabetes, 51, 599, 2002.
1382_C12.fm Page 199 Tuesday, October 7, 2003 6:31 PM

Chapter twelve: Insulin and the regulation of amino acid catabolism and protein turnover 199

70. Patti, M.E., Brambilla, E., Luzi, L., Landaker, E.J., and Kahn, C.R., Bidirectional modulation
of insulin action by amino acids, J. Clin. Invest., 101, 1519, 1998.
71. Tremblay, F. and Marette, A., Amino acid and insulin signaling via the mTOR/p70 S6 kinase
pathway: a negative feedback mechanism leading to insulin resistance in skeletal muscle
cells, J. Biol. Chem., 276, 38052, 2001.
72. Kobayashi, R., Murakami, T., Obayashi, M., Nakai, N., Jaskiewicz, J., Fujiwara, Y., Shimomura,
Y., and Harris, R.A., Clofibric acid stimulates branched-chain amino acid catabolism by three
mechanisms, Arch. Biochem. Biophys., 407, 231, 2002.
73. Jiang, G., Dallas-Yang, Q., Li, Z., Szalkowski, D., Liu, F., Shen, X., Wu, M., Zhou, G., Doebber,
T., Berger, J., Moller, D.E., and Zhang, B.B., Potentiation of insulin signaling in tissues of
Zucker obese rats after acute and long-term treatment with PPARgamma agonists, Diabetes,
51, 2412, 2002.
74. Wojtaszewski, J.F., Nielsen, J.N., and Richter, E.A., Effect of acute exercise on insulin signaling
and action in humans, J. Appl. Physiol., 93, 384, 2002.
75. Kimball, S.R., Farrell, P.A., and Jefferson, L.S., Role of insulin in translational control of
protein synthesis in skeletal muscle by amino acids or exercise, J. Appl. Physiol., 93, 1168, 2002.
1382_C12.fm Page 200 Tuesday, October 7, 2003 6:31 PM
1382_C13.fm Page 201 Tuesday, October 7, 2003 6:33 PM

chapter thirteen

Control of amino acid metabolism


by counterregulatory hormones
Jan Wernerman
Karolinska Institutet

Contents
Introduction..................................................................................................................................201
13.1 Plasma concentration of amino acids.............................................................................202
13.2 Muscle free amino acids...................................................................................................204
13.3 Protein metabolism............................................................................................................204
13.4 Conclusion and perspective.............................................................................................206
References .....................................................................................................................................207

Introduction
The metabolic alterations seen following trauma were early recognized to be attributable
to increased levels of stress hormones, predominantly adrenaline, cortisol, and glucagon.1,2
In animal experiments a hypoglycemia model was used that, to some extent, produced
alterations, preferable in carbohydrate metabolism, which had similarities to what was
seen following trauma.3 The hypoglycemic event also created elevations of adrenaline,
cortisol, and glucagon, and thereby these hormones were given the name counterregula-
tory hormones. In particular, glucagon has also been used in clinical contexts to counteract
hypoglycemia. In carbohydrate metabolism the counterregulatory hormones induce a state
of hyperglycemia, enhanced endogenous glucose production as well as enhanced gluco-
neogenesis. The obvious links between carbohydrate and amino acid metabolism soon
led to a series of experiments where the hypoglycemic event was exchanged by an infusion
of the three most important hormones: adrenaline, cortisol, and glucagon.4 The triple
hormone cocktail soon became a standardized experimental setup that could be used in
experimental animals or in healthy volunteers to study metabolic events associated with
trauma and sepsis in a standardized manner.
In carbohydrate metabolism it was quite obvious that the three counterregulatory
hormones had a synergistic effect upon, for example, plasma glucose levels, compared to
when the singular hormones were given individually.5 For both cortisol and glucagon,

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 201
1382_C13.fm Page 202 Tuesday, October 7, 2003 6:33 PM

202 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

metabolic effects were seen at a much lower plasma level than when these hormones were
given isolated. The mechanisms for these actions are perhaps still not fully understood,
but cortisol may act on the adrenergic receptors to down-regulate them and also to sustain
the effects for longer periods of time. In general, cortisol induces net protein breakdown
and hyperaminoacidemia.6 The regulation of glucagon effects is less well understood, but
glucagon facilitates the uptake of substrates into the liver, preferably by system A.7 In high
concentrations, glucagon causes hypoaminoacidemia. In general terms, stress hormones
increase the whole-body metabolic rate.8,9 When it comes to the effects of the counterreg-
ulatory hormones upon amino acid metabolism and protein metabolism, interest was
initially focused upon alanine as one of the important precursors for gluconeogenesis. In
parallel to the effects upon plasma glucose, the effects on plasma alanine concentration
are mainly confined to the time interval when the plasma concentrations of the counter-
regulatory hormones are elevated.10 This singular finding clearly indicates that the hor-
mone cocktail infused only partially mimics the actual events in conjunction with trauma
and sepsis. Also, concerning glutamine kinetics and the exchange of alanine across the
leg, there are distinct differences between a triple hormone infusion and catabolic
patients.10–12

13.1 Plasma concentration of amino acids


When looking upon the effects of hormones on the plasma amino acid concentrations,
there are two major things to consider: the direction of changes and the time pattern.
Sometimes the direction of changes is not consistent over time, which makes this compli-
cated but also fascinating when trying to understand the underlying mechanisms. The
major part of the effects elicited by a triple hormone combination is produced by adren-
aline alone,10,13 and is therefore abolished by a simultaneous beta-blockade.13 During the
infusion of counterregulatory hormones for up to 6 h, there is a general pattern in which
the concentrations of most of the essential amino acids decrease.10,14,15 The only exception
is alanine, which starts to increase after a short period of stress hormone infusion.10 When
the infusion of counterregulatory hormone stops, this is the end of the story, and the
plasma amino acid concentrations go back to normal.10 However, there are a number of
exceptions. Glutamine, for example, decreases under the influence of counterregulatory
hormones, but afterwards, glutamate exhibits an overshot above the basal value before
coming back to normal (Figure 13.1). Another pattern is shown by the branched-chain
amino acids (leucine, valine, and isoleucine), which initially go down but later increase
to be significantly elevated in plasma 24 h after the end of the infusion. The increased
plasma concentrations of branched-chain amino acids are also seen on the third day after
surgical trauma.16 However, also following elective surgery the biphasic pattern is seen
with a decrease of the plasma concentrations of branched-chained amino acids at 12 h
postoperatively, coming back to normal 24 h following the operation.17 Among the other
essential amino acids, the aromatic amino acids show high plasma levels at 24 h; this is
marginally significant, but on the 3rd and even 10th days postoperatively, values are
high.16,17 In general, plasma amino acid patterns following elective surgical trauma in
otherwise healthy individuals show increased amino acid concentrations in plasma during
a 2-week period following surgery. It is therefore obvious that the counterregulatory
hormones as well as the surgical trauma initiate changes over time that are much longer
than those during which the plasma levels of counterregulatory hormones are elevated.
During prolonged periods of stress, as for intensive care patients, plasma amino acid levels
are normal or close to normal for most amino acids.18,19 The exception is glutamine, which
holds a low plasma concentration, also related to outcome.20 On the other hand, the levels
1382_C13.fm Page 203 Tuesday, October 7, 2003 6:33 PM

Chapter thirteen: Control of amino acid metabolism by counterregulatory hormones 203

700
*
600
plasma conc . (µ mo l/L)
500

400
** *
300
**

200
**
100
** glutamine
BCAA
st ress hormon e infusi on

0
0 6 12 18 24

time (h)

Figure 13.1 Concentrations of glutamine and the sum of branched-chain amino acids (BCAA) in
plasma of healthy volunteers during and after a combined stress hormone infusion of adrenaline,
cortisol, and glucagon. * and ** denote significantly different from basal values, p < 0.05 and p < 0.01,
respectively. (Data from Wernerman, J. et al, Clin. Nutr., 4, 207–216, 1985; Shamoon, H. et al., Diabetes,
29, 875–881, 1980; Wernerman, J. et al., Clin. Physiol., 13, 309–319, 1993.)

of aromatic amino acids, in particular phenylalanine, are usually higher than normal.19 In
experiments with a prolonged infusion of counterregulatory hormones over several days,
slight increases in the plasma levels of alanine and phenylalanine were the only changes
seen.21 As for the exchange of amino acids across muscle tissue, a prolonged stress hormone
infusion does not elicit any changes, compared to the basal situation.21 In burn patients
there is a considerable efflux of alanine,12 while the efflux of glutamine is sometimes
reported elevated but in other studies not found different from what is seen in healthy
individuals.12,22,23
For glutamine a decrease in plasma concentration is seen in response to adrenaline
alone. This slight decrease is accompanied by increases in the rate of appearance as well
as the rate of disappearance.24,25 The rate of appearance corresponds to an increased release
from skeletal muscle, while the rate of disappearance corresponds to an increase in renal
gluconeogenesis.24 In response to physiological concentrations of cortisol or glucagon, the
plasma concentration of glutamine, as well as the rates of appearance and disappearance,
is not altered.26 For alanine the plasma concentration increases slowly in conjunction with
a triple hormone infusion. In parallel, the efflux from the leg is only marginally increased
after 60 min of stress hormone infusion but later on is unaltered, compared to basal.10,12
This is in contrast to the marked increase in alanine concentration in skeletal muscle
described below, which is seen accompanying adrenaline alone as well as a triple hormone
combination.10 The increase of essential amino acids, in particular the branched-chain
amino acids and the aromatic amino acids seen in response to counterregulating hormones
as well as following trauma and sepsis, is still obscure. However, a relation to an increase
in protein degradation has been suggested.27
1382_C13.fm Page 204 Tuesday, October 7, 2003 6:33 PM

204 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

13.2 Muscle free amino acids


In general, tissue free amino acids are believed to stay in balance with plasma concentra-
tion. However, this is an oversimplification, and many times plasma is even a poor
reflection of what is happening inside the cells. As the tissue free amino acid pool is the
precursor pool for protein synthesis as well as the substrate pool for oxidative processes,
changes in the tissue free amino acid pool are perhaps a better reflection of metabolic
events. It must be emphasized that the intracellular free amino acid concentrations vary
considerably between different tissues. Following an infusion of the counterregulatory
hormones, the changes come much slower, compared to plasma.10,14
In muscle, glutamine and alanine are the two amino acids responding already within
an hour, glutamine going down, while alanine increases. Supposedly this reflects an
enhanced export of gluconeogenetic precursors from muscle. The branched-chain amino
acids respond with a biphasic pattern similar to that seen in plasma (Figure 13.2).
Glutamine concentration, on the other hand, shows a steady decrease in muscle during
the 24 h after a 6-h triple hormone infusion without any signs of restitution.15 Following
elective surgery, a similar pattern is seen over the first 3 or 4 days.16,17 After that, a gradual
restitution back to normal is seen up to the 7th or 10th day (Figure 13.3). Following severe
septic events, glutamine is shown to be the last amino acid to be restored back to normal
concentration in muscle.16,28 This slow restoration stands in contrast to the rapid restoration
of intracellular free glutamine depletion that occurs during refeeding after short-term
starvation.29 The regulating mechanisms for glutamine depletion as well as repletion are
not well understood. Muscle glutamine concentration drops rapidly in septic patients in
the ICU, most often down to 25% of normal levels.18,19 In general, nonessential amino acids
stay rather stable in plasma concentration during counterregulatory hormone infusion,
but in muscle, several nonessential amino acids such as glutamine, alanine, and glutamate
exhibit distinct changes of concentrations. Glutamate, which holds a comparatively high
concentration in muscle, compared to the low concentration in plasma, shows a rapid
decrease during hormone infusion and a gradual normalization over the next 18 h. Muscle
alanine concentration increases during the counterregulatory hormone infusion, but is
rapidly normalized after that. The majority of these experiments are performed in healthy
volunteers in the basal state. Simultaneous food intake given as intravenous nutrition
abolishes these changes.30 The metabolic handling of the nutrients, on the other hand, is
only marginally affected by elevated concentrations of stress hormones, at least in terms
of amino acids.31,32 During a prolonged stress hormone infusion, however, intracellular
glutamine concentration in muscle decreases despite food intake.21

13.3 Protein metabolism


As amino acids are the brick stones and currency of protein metabolism, amino acid
metabolism and protein metabolism are closely related. It was early shown that an infusion
of counterregulatory hormones induces a negative whole-body nitrogen balance in healthy
volunteers.33,34 This is, of course, a reflection of what also happens after a surgical trauma.
On the tissue level, muscle protein synthesis as reflected by the concentration and size
distribution of ribosomes decreases following a triple hormone infusion.35 The same hap-
pens during infusions of adrenaline or cortisol only, but the combination gives a more
profound decrease. Also, an in vitro assay of incorporation of a labeled amino acid into
polypeptide chains in a cellular fraction of isolated ribosomes shows a decrease in response
to stress hormones.36 The effects on skeletal muscle concern both synthesis and degrada-
tion of muscle proteins.37 In quantitative terms, the muscle protein synthesis rate is only
marginally decreased, by 15%, at the end of a 6-h stress hormone infusion.38 However,
1382_C13.fm Page 205 Tuesday, October 7, 2003 6:33 PM

Chapter thirteen: Control of amino acid metabolism by counterregulatory hormones 205

15 .75

muscle conc. (mmol/k g)


**
10 0.50 *

***

*
5 0.25

**
glutamine
BCAA
st ress hor mone i nfusi on

0
0 6 12 18 24
time (h)
Figure 13.2 Concentrations of glutamine and the sum of branched-chain amino acids (BCAA) in
muscle of healthy volunteers during and after a combined stress hormone infusion of adrenaline,
cortisol, and glucagon. The left scale on the y-axis applies for glutamine, while the right scale applies
for the sum of branched-chain amino acids. *, **, and *** denote significantly different from basal
values, p < 0.05, p < 0.01, and p < 0.001, respectively. (Data from Wernerman, J. et al, Clin. Nutr., 4,
207–216, 1985; Wernerman, J. et al., Clin. Physiol., 13, 309–319, 1993.)

after normalization of plasma hormonal levels 18 h later and then continuing decrease of
the muscle protein synthesis rate, a drop of 30% is seen, compared to the preinfusion
levels. This has also been investigated in circulating lymphocytes, which also reveal a 40
to 60% decrease of in vivo protein synthesis following a 6-h triple hormone infusion.38,39
This goes together with an increase in the count of circulating leukocytes but a decrease
in the circulating numbers of lymphocytes. All subfractions of lymphocytes show a similar
decrease as the total numbers, except for the natural killer cells, which respond with an
increased fraction. Cortisol alone elicits none of these changes.40 For the circulating lym-
phocytes, the time pattern is different from that of the skeletal muscle. The decrease in
protein synthesis rate seen at the end of the 6-h stress hormone infusion is totally abolished
18 h later, when the decrease seen in the muscle protein synthesis rate is even more
pronounced.38 This also has some resemblance to the response seen following elective
surgery, when a depression of the muscle protein synthesis rate occurs in the early post-
operative period and is not reversed on the third postoperative day regardless of nutrition
support.41 For protein synthesis in lymphocytes, the postoperative time course has not
been fully characterized, but 24 h postoperatively, an enhanced protein synthesis rate in
circulating lymphocytes is seen regardless of postoperative nutrition or a preoperative
glucose load.42 Elevated stress hormone concentrations also influence the synthesis rate
of albumin, which is unaffected at the end of a 6-h infusion.38 Subsequently, at 18 h
postinfusion a 15% increase in albumin synthesis rate is seen. This is in accord with the
increased albumin synthesis rate seen 24 h postoperatively and in ICU patients.43–45 How-
ever, the possibility of an early decrease in albumin and total liver protein synthesis rate
1382_C13.fm Page 206 Tuesday, October 7, 2003 6:33 PM

206 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

16 1.00

**
muscle conc. (mmol/k g) ** *
12 0.75 *

**
* ***
8 0.50

4 0.25

glutamine
elective BCAA
su rgery

0
0 24 48 72 96 120 144 168 192 216 240

time (h)
Figure 13.3 Concentrations of glutamine and the sum of branched-chain amino acids (BCAA) in
muscle of metabolically healthy patients undergoing elective abdominal surgery of medium size.
The left scale on the y-axis applies for glutamine, while the right scale applies for the sum of
branched-chain amino acids. *, **, and *** denote significantly different from basal values, p < 0.05,
p < 0.01, p < 0.001, respectively. (Data from Petersson, B. et al., Br. J. Surg., 79, 212–216, 1992; Essén,
P. et al., Clin. Physiol., 12, 163–177, 1992.)

in response to stress hormones is suggested by the findings of a sharp decrease in total


liver protein synthesis rate as well as albumin synthesis rate already during a laparoscopic
surgical procedure.46 Presently, however, there are no data concerning albumin synthesis
during the early phase of a stress hormone infusion.

13.4 Conclusion and perspective


Amino acid metabolism is regulated to a great extent by hormones, which also means that
protein metabolism is influenced.47 In conjunction with trauma and sepsis, the stress
hormones are of particular importance, and it is their combined effect that works on amino
acid and protein metabolism. In addition to the mainly catabolic hormones discussed here,
hormones with anabolic or partly anabolic actions, such as insulin, growth hormone,
insulin-like growth factor I, and sex steroids, are also active and make the final picture
even more complicated. As emphasized above, it is the direction of changes and the time
pattern of changes that are important. Furthermore, as indicated above, individual tissues
may respond quite differently to the same hormonal challenge. Although a lot of work
was done on basic physiology 30 to 40 years ago, there are still many questions that need
to be answered, especially when several hormones appear simultaneously. New tech-
niques, in particular the use of stable isotopes, also provide the possibility of reassessing
old dogmas, which sometimes appear to be oversimplifications of the underlying mech-
anisms.
1382_C13.fm Page 207 Tuesday, October 7, 2003 6:33 PM

Chapter thirteen: Control of amino acid metabolism by counterregulatory hormones 207

References
1. Alberti, K.G.M.M., Batstone, G.F., and Foster, K.J., Relative role of various hormones in
mediating the metabolic response to injury, J. Parenter. Enteral Nutr., 4, 141–145, 1980.
2. Bessey, P.Q. and Lowe, K.A., Early hormonal changes affect the catabolic response to trauma,
Ann. Surg., 218, 476–489, 1993.
3. Garber, A.J., Cryer, P.E., Santiago, J.V., Haymond, M.W., Pagliara, A.S., and Kipnis, D.M.,
The role of adrenergic mechanisms in the substrate and hormonal response to insulin-
induced hypoglycemia in man, J. Clin. Invest., 58, 7–15, 1976.
4. Gelfand, R.A., Matthews, D.E., Bier, D.M., and Sherwin, R.S., Role of counterregulatory
hormones in the catabolic response to stress, J. Clin. Invest., 74, 2238–2248, 1984.
5. Shamoon, H., Hendler, R., and Sherwin, R.S., Synergistic interactions among antiinsulin
hormones in the pathogenesis of stress hyperglycemia in humans, J. Clin. Endocrinol. Metab.,
52, 1235–1241, 1981.
6. Cynober, L.A., Plasma amino acid levels with a note on membrane transport: characteristics,
regulation, and metabolic significance, Nutrition, 18, 761–766, 2002.
7. Boden, G., Rezvani, I., and Owen, O.E., Effects of glucagon on plasma amino acids, J. Clin.
Invest., 73, 87, 1984.
8. Brillon, D.J., Zheng, B., Campbell, R.G., and Matthews, D.E., Effect of cortisol on energy
expenditure and amino acid metabolism in humans, Am. J. Physiol., 268, E501–E513, 1994.
9. Ratheiser, K.M., Brillon, D.J., Campbell, R.G., and Matthews, D.E., Epinephrine produces a
prolonged elevation in metabolic rate in humans, Am. J. Clin. Nutr., 68, 1046–1052, 1998.
10. Wernerman, J., Brandt, R., Strandell, T., Allgén, L.-G., and Vinnars, E., The effect of stress
hormones on the interorgan flux of amino acids and the concentration of free amino acids
in skeletal muscle, Clin. Nutr., 4, 207–216, 1985.
11. Gore, D.C. and Jahoor, F., Glutamine kinetics in burn patients: comparison with hormonally
induced stress in volunteers, Arch. Surg., 129, 1318–1323, 1994.
12. Brown, J.A., Gore, D.C., and Jahoor, F., Catabolic hormones alone fail to reproduce the stress-
induced efflux of amino acids, Arch. Surg., 129, 819–824, 1994.
13. Shamoon, H., Jacob, R., and Sherwin, R.S., Epinephrine-induced hypoaminoacidemia in
normal and diabetic human subjects: effect of beta blockade, Diabetes, 29, 875–881, 1980.
14. Wernerman, J., Hammarqvist, F., Botta, D., and Vinnars, E., Stress hormones alter the pattern
of free amino acids in human skeletal muscle, Clin. Physiol., 13, 309–319, 1993.
15. Hammarqvist, F., Ejesson, B., and Wernerman, J., Stress hormones initiate prolonged changes
in the muscle amino acid pattern, Clin. Physiol., 21, 44–50, 2001.
16. Petersson, B., Vinnars, E., Waller, S.-O., and Wernerman, J., Long-term changes in muscle
free amino acid levels after elective abdominal surgery, Br. J. Surg., 79, 212–216, 1992.
17. Essén, P., Wernerman, J., Sonnenfeld, T., Thunell, S., and Vinnars, E., Free amino acids in
plasma and muscle during 24 hours post-operatively: a descriptive study, Clin. Physiol., 12,
163–177, 1992.
18. Roth, E., Funovics, J., Mühlbacher, M., Schemper, M., Mauritz, W., Sporn, P., and Fritsch, A.,
Metabolic disorders in severe abdominal sepsis: glutamine defiency in skeletal muscle, Clin.
Nutr., 1, 25–42, 1982.
19. Gamrin, L., Essén, P., Forsberg, A.-M., Hultman, E., and Wernerman, J., A descriptive study
of skeletal metabolism in critically ill patients: free amino acids, protein, energy-rich phos-
phates, nucleic acids, fat, water and electrolytes, Crit. Care Med., 24, 575–583, 1996.
20. Oudemans-van Straaten, H.M., Bosman, R.J., Treskes, M., van der Spoel, H.J., and Zandstra,
D.F., Plasma glutamine depletion and patient outcome in acute ICU admissions, Intensive
Care Med., 27, 84–90, 2001.
21. Bessey, P.Q., Jiang, Z.M., Johnson, D.J., Smith, R.J., and Wilmore, D.W., Posttraumatic skeletal
muscle proteolysis: the role of the hormonal environment, World J. Surg., 13, 465–470, 1989.
22. Clowes, G.H., Jr., Randall, H.T., and Cha, C.J., Amino acid and energy metabolism in septic
and traumatized patients, J. Parenter. Enteral Nutr., 4, 195–205, 1980.
1382_C13.fm Page 208 Tuesday, October 7, 2003 6:33 PM

208 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

23. Vesali, R.F., Klaude, M., Rooyackers, O.E., Tjäder, I.I., Barle, H., and Wernerman, J., Longi-
tudinal pattern of glutamine/glutamate balance across the leg in long-stay intensive care
unit patients, Clin. Nutr., 21, 505–514, 2002.
24. Gerich, J.E., Meyer, C., and Stumvoll, M.W., Hormonal control of renal and systemic
glutamine metabolism, J. Nutr., 130 (Suppl.), 995S–1001S, 2000.
25. Ratheiser, K.M., Pesola, G.R., Campbell, R.G., and Matthews, D.E., Epinephrine transiently
increases amino acid disappearance to lower amino acid levels in humans, J. Parenter. Enteral
Nutr., 23, 279–287, 1999.
26. Battezzati, A., Simonson, D.C., Luzi, L., and Matthews, D.E., Glucagon increases glutamine
uptake without affecting glutamine release in humans, Metabolism, 47, 713–723, 1998.
27. Fürst, P., Regulation of intracellular metabolism of amino acids, in Nutrition in Cancer and
Trauma Sepsis, Bozetti, F. and Dionigi, R., Eds., Karger, Basel, Switzerland, 1985, pp. 21–53.
28. Askanazi, J., Carpentier, Y.A., Michelsen, C.B., Elwyn, D.H., Furst, P., Kantrowitz, L.R.,
Gump, F.E., and Kinney, J.M., Muscle and plasma amino acids following injury: influence
of intercurrent infection, Ann. Surg., 192, 78–85, 1980.
29. Andersson, K., Luo, J.L., Hammarqvist, F., and Wernerman, J., The effect of fasting on muscle
glutathione levels, Clin. Nutr., 13 (Special Suppl.), O.12, 1994.
30. Hammarqvist, F., von der Decken, A., Vinnars, E., and Wernerman, J., Stress hormone and
amino acid infusion in healthy volunteers: short-term effects on protein synthesis and amino
acid metabolism in skeletal muscle, Metabolism, 43, 1158–1163, 1994.
31. Fong, Y.M., Albert, J.D., Tracey, K., Hesse, D.G., Calvano, S., Matthews, D.E., and Lowry,
S.F., The influence of substrate background on the acute metabolic response to epinephrine
and cortisol, J. Trauma, 31, 1467–1476, 1991.
32. Schiefermeier, M., Ratheiser, K.M., Zauner, C., Roth, E., Eichler, H.G., and Matthews, D.E.,
Epinephrine does not impair utilization of exogenous amino acids in humans, Am. J. Clin.
Nutr., 65, 1765–1773, 1997.
33. Watters, J.M., Bessey, P.Q., Dinarello, C.A., Wolff, S.M., and Wilmore, D.W., Both inflamma-
tory and endocrine mediators stimulate host responses to sepsis, Arch. Surg., 121, 179–190,
1986.
34. Wilmore, D.W., Are the metabolic alterations associated with critical illness related to the
hormonal environment? Clin. Nutr., 5, 9–19, 1986.
35. Wernerman, J., Botta, D., Hammarqvist, F., Thunell, S., von der Decken, A., and Vinnars, E.,
Stress hormones given to healthy volunteers alter the concentration and configuration of
ribosomes in skeletal muscle, reflecting changes in protein synthesis, Clin. Sci., 77, 611–616,
1989.
36. Wernerman, J., von der Decken, A., Hammarqvist, F., Botta, D., and Vinnars, E., Incorporation
of 14C-leucine into human skeletal muscle protein in a cell-free system as a measure of
protein synthesis: influence of stress hormones, J. Clin. Biochem. Nutr., 9, 269–278, 1990.
37. Gore, D.C., Jahoor, F., Wolfe, R.R., and Herndon, D.N., Acute response of human muscle
protein to catabolic hormones, Ann. Surg., 218, 679–684, 1993.
38. McNurlan, M.A., Sandgren, A., Hunter, P., Essén, P., Garlick, P.J., and Wernerman, J., Protein
synthesis rates of skeletal muscle, lymphocytes, and albumin with stress hormone infusion
in healthy man, Metabolism, 45 1388–1394, 1996.
39. Januszkiewicz, A., Essén, P., McNurlan, M., Ringdén, O., Wernerman, J., and Garlick, P.,
Protein synthesis of circulating human T lymphocytes does not respond to a cortisol chal-
lenge within 24 hours, Acta Anaesth. Scand., 44, 202–209, 2000.
40. Januszkiewicz, A., Essén, P., McNurlan, M., Ringdén, O., Garlick, P., and Wernerman, J., A
combined stress hormone infusion decreases in vivo protein synthesis in human T lym-
phocytes in healthy volunteers, Metabolism, 50, 1308–1314, 2001.
41. Essén, P., McNurlan, M.A., Sonnenfeld, T., Milne, E., Vinnars, E., Wernerman, J., and Garlick,
P., Muscle protein synthesis after operation: effects of intravenous nutrition, Eur. J. Surg.,
159, 195–200, 1993.
42. Essén, P., McNurlan, M.A., Thorell, A., Tjäder, I., Caso, G., Andersson, S., Wernerman, J.,
and Garlick, P.J., Determination of protein synthesis in lymphocytes in vivo after surgery,
Clin. Sci., 91, 99–106, 1996.
1382_C13.fm Page 209 Tuesday, October 7, 2003 6:33 PM

Chapter thirteen: Control of amino acid metabolism by counterregulatory hormones 209

43. van Acker, B., Hulsewé, K.W.E., Wagenmakers, A.J.M., Deutz, N.E.P., von Meyenfeldt, M.F.,
and Soeters, P.B., Effect of surgery on albumin synthesis rate in humans, Clin. Nutr., 17
(Suppl. 1), 14, 1998.
44. Mansoor, O., Cayol, M., Gachon, P., Boirie, Y., Schoeffler, P., Obled, C., and Beaufrer, B.,
Albumin and fibrinogen syntheses increase while muscle protein synthesis decreases in head-
injured patients, Am. J. Physiol., 273, E898–E902, 1997.
45. Essén, P., McNurlan, M.A., Gamrin, L., Garlick, P.J., and Wernerman, J., Tissue protein
synthesis rate in critically ill patients, Crit. Care Med., 26, 92–100, 1998.
46. Barle, H., Nyberg, B., Ramel, S., Essén, P., McNurlan, M., Wernerman, J., and Garlick, P.,
Inhibition of liver protein synthesis during laparoscopic surgery, Am. J. Physiol., 277,
E591–E596, 1999.
47. Miers, W.R. and Barrett, E.J., The role of insulin and other hormones in the regulation of
amino acid and protein metabolism in humans, J. Basic Clin. Physiol. Pharmacol., 9, 235–253,
1998.
1382_C13.fm Page 210 Tuesday, October 7, 2003 6:33 PM
1382_C14.fm Page 211 Tuesday, October 7, 2003 6:34 PM

chapter fourteen

Nitric oxide
Eric J. Mahoney
Rhode Island Hospital
Jorge E. Albina
Rhode Island Hospital

Contents
14.1 The biology of nitric oxide...............................................................................................211
14.1.1 The nitric oxide synthases...................................................................................212
14.1.1.1 nNOS (NOS I).........................................................................................214
14.1.1.2 iNOS (NOS II).........................................................................................214
14.1.1.3 eNOS (NOS III) ......................................................................................215
14.1.2 Nitric oxide: mechanisms of action ...................................................................215
14.2 Nitric oxide: physiology and pathophysiology in mammalian cells .......................217
14.2.1 Nitric oxide and the vasculature........................................................................217
14.2.2 Nitric oxide, wound repair, infection, and septic shock ................................220
14.2.3 Nitric oxide and apoptosis ..................................................................................223
14.2.4 Nitric oxide and cancer........................................................................................224
14.2.5 Nitric oxide and cellular energy metabolism...................................................226
14.2.6 Nitric oxide and the nervous system ................................................................226
14.3 Conclusion ..........................................................................................................................228
Acknowledgments ......................................................................................................................228
References .....................................................................................................................................228

14.1 The biology of nitric oxide


Nitric oxide (NO) and its biochemistry have had an almost unprecedented effect on the
way in which cellular interactions are understood. In historical perspective, the chemical
properties of nitric oxide were described by Joseph Priestley1 in 1772 after exposing various
metals to nitric acid. More recently, nitric oxide was allocated to the fields of food pres-
ervation in the curing of meat as well as an environmental pollutant from combustion
engines. However, upon its discovery as an effector molecule in mammalian physiology,2–6
nitric oxide was catapulted to a position of extreme importance. Its functions have been
shown to include signal transduction, cytotoxicity, vascular tone, and inflammation, to

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 211
1382_C14.fm Page 212 Tuesday, October 7, 2003 6:34 PM

212 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

name only a few. This recognition was demonstrated by it being named “molecule of the
year 1992” in Science7 and culminated in Drs. Furchgott, Murad, and Ignarro being
awarded the Nobel Prize for Medicine in 1998 for the discovery and subsequent physio-
logic characterization of nitric oxide.
The reader is advised that the information presented herein has been obtained from
various animal and cell models. Like many other physiologically relevant molecules, there
can be dramatic differences in function across species and even across organ systems
within the same species. Further complicating this picture, the actions of nitric oxide can
change according to additional factors such as NO concentration, redox status of the target
cell, or even which reactive nitrogen species is active given the specific milieu. Although
the term NO is used for simplicity, what NO synthase (NOS)-containing cells contribute
to their environment is proposed to be a mixture of NO, NO2, N2O3, NO2–, NO3–, nitro-
samines, nonprotein nitrosothiols, and S-nitrosylated proteins.8 This chapter will attempt
to provide a cogent view of this highly protean molecule and its role in a biological system.

14.1.1 The nitric oxide synthases


The nitric oxide synthases are a family of three isoforms9 that catalyze the conversion of
L-arginine to nitric oxide and citrulline.

L - arginine æ ææÆ nitric oxide + citrulline


NOS

Based on initial description, these three isoforms were divided into two groups:
constitutive, containing nNOS (NOS I) and eNOS (NOS III), and inducible (iNOS, NOS II).
However, as will be discussed, it is now realized that the expression of nNOS and eNOS
can be induced under certain physiological conditions. Similarly, iNOS may function
constitutively under other conditions.
The three isoforms do share the same domain structure and catalytic mechanism.
Specifically, the overall amino acid sequence homology for the three human NOS isoforms
is approximately 55%, with particularly strong sequence conservation noted in regions
involved in catalysis.10 Across species, the homology between equivalent isoforms aver-
ages 90 ± 6%.9
Each of the NOS isomers is a homodimer made up of monomers containing a reductase
domain and an oxygenase domain, with the active site being an iron protoporphyrin IX
(heme) within the oxygenase domain. Each domain has absolute cofactor requirements:
the C-terminal reductase domain requires NADPH, FAD, and FMN; the N-terminal oxy-
genase domain requires heme, tetrahydrobiopterin, and L-arginine. Connecting these two
domains is a calmodulin consensus-binding domain.11 Along with these requirements,
each NOS is functionally active only in dimeric form because the active site requires two
hemes.12 In order to dimerize, each monomer must incorporate the heme moiety, and
dimerization is promoted and stabilized by the binding of tetrahydrobiopterin and L-argi-
nine.13,14 Further stabilizing dimerization, each isoform has been shown to contain zinc,15
whose binding site is at the dimeric interface.16 The binding of zinc has been shown to
function structurally rather than catalytically and is important for optimum enzymatic
activity.15
The flow of electrons within NOS has been established, from NADPH to FAD to
FMN to heme.17 In the absence of calmodulin, a calcium-dependent protein, FMN is
unable to pass along the electrons due to the quaternary structure of the protein.11 This
halts any further reduction of FAD by NADPH.18 Once calmodulin binds, it causes a
1382_C14.fm Page 213 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 213

NADPH FAD FMN


H4B Fe ARG

H4B Fe ARG

CaM CaM

H4B Fe ARG

CaM H4B Fe ARG

FMN FAD NADPH

Figure 14.1 Calmodulin-induced electron transfer in NOS heterodimer. In the absence of calmodulin,
the quaternary structure of NOS is such that electron transfer cannot occur between the reductase
domain and the heme moiety. Upon calmodulin binding, structural alterations allow electron trans-
fer from FMN to the heme moiety located on the adjacent subunit. H4B, tetrahydrobiopterin; ARG,
arginine; CaM, calmodulin. (Adapted from Panda, K. et al., J. Biol. Chem., 276, 23349, 2001. With
permission.)

conformation change in the reductase domain that properly aligns adjacent reductase
and oxygenase domains,11 and allows the transfer of electrons from FMN in the reductase
domain to the oxygenase domain18,19 (see Figure 14.1). Upon acceptance of the electrons
from FMN, heme binds to and activates oxygen and catalyzes the stepwise oxygenation
of L-arginine.20 Along with this, observations of other investigators have demonstrated
that electron transfer from tetrahydrobiopterin is another absolute requirement for L-argi-
nine oxidation.18
All three NOS isoforms have comparable specific activities, about 1 mM/min/mg of
protein at 37˚C.21 With their functional requirements the same, the main differences
between the three isoforms are their regulatory features. Most notably, in the constitutive
group, essential calmodulin binding and subsequent activation are calcium dependent
and reversible.8 Given such dependence on transient intracellular calcium fluxes, the
activity of the constitutive NOSs will be measured in seconds to minutes.22 Contrary to
this, iNOS (NOS II) binds calmodulin irreversibly and is calcium independent. Upon
stimulation, there is a delay of 6 to 8 h before iNOS-derived NO production to allow for
transcription and de novo protein synthesis. Once produced, however, iNOS will be active
for hours to days and produce more that 1000-fold larger quantities than either constitutive
NOS.23
1382_C14.fm Page 214 Tuesday, October 7, 2003 6:34 PM

214 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

14.1.1.1 nNOS (NOS I)


Although originally isolated in nerves, nNOS has since been found in skeletal muscle,
neutrophils, pancreatic islets, and endometrium, as well as respiratory and gastrointestinal
epithelia.9 Through its regulation by transient increases in intracellular calcium concen-
tration, nNOS participates in signal transduction.24 Additionally, nNOS has been shown
to be influenced by the brain protein termed protein inhibitor of neuronal NOS (PIN). It
has been demonstrated that PIN inactivates nNOS by destabilizing the nNOS dimer.25
In the brain, nNOS-derived NO is believed to be involved in several forms of synaptic
plasticity26 and neuronal development.27 nNOS-derived NO has been implicated as a
retrograde messenger from the postsynaptic to the presynaptic neurons to elicit long-term
potentiation,28,29 a process believed to be important in memory and learning. (Refer to
Section 14.2.6, “Nitric Oxide and the Nervous System.”)
In skeletal muscle, nNOS is associated with the sarcolemma through interactions with
the dystrophin complex.30,31 This is via a specialized PDZ domain at the N-terminus of
nNOS that binds with PDZ repeats on a1-syntrophin, a binding partner of dystrophin.
This PDZ domain is a sequence of approximately 100 amino acids that is found on a
diverse group of cytoskeletal proteins and enzymes.32

14.1.1.2 iNOS (NOS II)


The inducible nitric oxide synthase isoform was originally described in inflammatory cells
upon lipopolysaccharide (LPS) stimulation. However, most, if not all, cells can be stimu-
lated to express iNOS under appropriate conditions. In humans, iNOS expression has
been documented in hepatocytes, chondrocytes, adenocarcinoma cells, keratinocytes, res-
piratory epithelia, and macrophages.22 iNOS may even function constitutively in some
cells under certain physiological conditions,33 notably the large airway in humans.22 In
monocyte-derived cells, LPS and interferon-g are strong inducers of iNOS. In these cells,
other cytokines such as tumor necrosis factor-a (TNF-a) and interleukin (IL)-1b have
minimal effects. In contrast, cells such as hepatocytes and myocytes are more sensitive to
iNOS induction by IL-1b and TNF-a.23 It must be noted that dramatic species variability
in ease and conditions of iNOS expression does exist. For example, rat macrophages will
produce iNOS in vitro without additional stimulation. Murine and human macrophages,
however, will require cytokine and endotoxin to express iNOS. Murine cells will express
iNOS in response to LPS and stimulatory cytokines such as interferon-g, IL-1b, IL-6, and
TNF-a; however, most human cells require a combination of cytokines for iNOS expres-
sion, which include interferon-g, IL-1b, and TNF-a.34
In stark contrast to the constitutive isoforms, iNOS (NOS II) is calcium independent
and binds calmodulin irreversibly. Therefore, the regulation of iNOS is at the level of
transcription and substrate availability rather than intracellular calcium concentration. In
connection to this, it has been proposed that a physiological role of intracellular arginase
in macrophages could be to reduce the availability of L-arginine to iNOS.35

NOS
L - arginine æ æææ Æ nitric oxide + citrulline
æarginase
æææÆ ornithine

Moreover, it has been shown that certain TH2 cytokines, specifically IL-4 and IL-10,
down-regulate NO production by suppressing the induction of iNOS and by enhancing
arginase expression.36 The increased arginase activity would presumably reduce the intra-
cellular L-arginine concentration that is available to iNOS. Other investigators37 have
challenged this on several fronts. First, iNOS has a large kinetic advantage over arginase,
1382_C14.fm Page 215 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 215

with iNOS having a micromolar Km for L-arginine vs. a millimolar Km of arginase.38


Furthermore, in rat peritoneal macrophages stimulated with interferon-g and LPS, it has
been shown that most of the L-arginine consumption is preferentially through iNOS.39
The iNOS gene promoter region is regulated by multiple transcription factors, includ-
ing nuclear factor (NF)-kB,22 AP-1, NF-IL6,40 interferon regulatory factor-1 (IRF-1),41,42 and
hypoxia-inducible factor-1 (HIF-1).43 In rat mesangial cells, NF-kB binding to the iNOS
promoter was shown to be absolutely essential for iNOS expression induced by IL-1b.44
Cytokine-induced expression of iNOS is abrogated by inhibitors of NF-kB.45 Additional
regulation of iNOS expression is demonstrated by the fact that murine macrophages
deficient in IRF-1 show markedly reduced iNOS expression.42,46 Even in an experimental
model of wound infection, interferon-g knockout mice demonstrate attenuated iNOS
expression. 47 Similarly, cAMP-responsive element binding protein (CREB) and
CAAT/enhancer-binding protein (C/ERB) are involved in cAMP induction of iNOS
expression.44
Additional modes of regulation of iNOS have also been discovered. In human mac-
rophages, iNOS expression is associated with the cross-linking of CD69, an NK cell family
signal-transducing receptor.48 In rat smooth muscle cells, transforming growth factor-b
inhibits iNOS gene transcription49 and can decrease iNOS message stability, reduce iNOS
mRNA translation, and increase the degradation of iNOS protein.50 Moreover, NO itself
has been shown to affect iNOS expression. NO is able to inhibit iNOS dimerization via
limiting the availability of heme by directly inhibiting heme synthesis and by inhibiting
insertion of heme onto the iNOS monomer.51 Also, NO has a negative feedback loop on
the regulation of NF-kB.45 The ultimate degradation of the iNOS protein itself is via
proteosome digestion.52

14.1.1.3 eNOS (NOS III)


Although originally isolated from the endothelium, this constitutive NOS has also been
found in platelets, cardiac myocytes, and the hippocampus.53 Stimuli that have been shown
to elicit eNOS-derived NO include shear stress, bradykinin, ADP, and hypoxia. Hypoxia
has also been shown to down-regulate eNOS expression in pulmonary epithelia cells.54,55
Thus, in underventilated areas of lung, vasoconstriction will occur, improving the venti-
lation-to-perfusion matching.
eNOS has been found to be localized to the plasmalemmal caveolae.56 In the resting
state, eNOS is acylated to the caveolin protein; however, upon increased intracellular
calcium concentration due to cellular stimulation, calcium-bound calmodulin is able to
competitively displace caveolin. This binding of calmodulin to eNOS results in the afore-
mentioned conformational change in eNOS quaternary structure that allows electron flow
and nitric oxide production.57 With a return of intracellular calcium to normal levels, eNOS
will disassociate with calmodulin and reassociate with caveolin.
Inflammatory mediators also have been shown to regulate eNOS expression. Tumor
necrosis factor-a has been demonstrated to down-regulate eNOS mRNA in bovine aortic
endothelial cells and human vein endothelial cells,58–60 as well as to destabilize eNOS
mRNA in rat vascular smooth muscle cells.9

14.1.2 Nitric oxide: mechanisms of action


Lancaster61 has demonstrated that given the large diffusion ratio of nitric oxide, NO will
be able to remain at significant concentrations at relatively far distances from the NO-
producing cell. Taking this and the half-life of NO into account, he submitted that the in
vivo effects of NO are mainly paracrine in nature. Moreover, given the lipophilic nature
of NO, cell membranes will not pose a boundary to the molecule. Therefore, in simplest
1382_C14.fm Page 216 Tuesday, October 7, 2003 6:34 PM

216 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

terms, NO produced by one cell penetrates the membranes of another cell and regulates
the activity of that target cell. The actual influence that NO exerts in a biologic system is
a function of its capacity to react with a wide array of molecules depending on the redox
state, local concentration, and availability of molecular targets. It has been described that
different concentrations or rates of NO production may influence certain reactions: at
lower concentrations, direct effects of NO would predominate, such as cGMP activation;
however, at high concentrations, such as iNOS-derived NO, indirect effects such as nitro-
sation, nitration, and oxidation will prevail.62 With such a wide array of resultant path-
ways, multiple mechanisms of actions are set in motion, even within a single cell.
At low concentrations, the NO-mediated stimulation of guanlylate cyclase, with result-
ant formation of cGMP, is considered the main pathway of NO activity.63,64 The resultant
increase in intracellular cGMP will alter the activity of cGMP-regulated ion channels,
cGMP-regulated phosphodiesterases, and cGMP-dependent protein kinases. Specific reg-
ulatory roles attributed to NO-cGMP interaction include (but are not limited to) neural
modulation, regulation of vascular tone, inhibition of platelet aggregation, leuko-
cyte–endothelial interaction, and inhibition of protein synthesis.65,66 It has been suggested
that this latter function is due to phosphorylation of eukaryotic initiation factor-2a.67
With regard to intracellular proteins, NO and related molecules can covalently modify
cysteine residues in proteins and therefore regulate cellular activity.68,69 NO has been
shown to affect all three mitogen-activated protein kinase (MAPK) cascades (i.e.,
SAP/JNK, p38, ERK/MAPK)70 with either activation or inhibition of the target enzyme.
Furthermore, the Janus kinases (JAK) have been shown to be inhibited by NO.71 In addi-
tion, NO has been shown to both increase72 and suppress73 protein kinase C activity.
Specifically, protein kinase C has critical thiol residues that influence its kinase activity.
S-nitrosylation of these thiols reversibly decreases the phosphotransferase activity of pro-
tein kinase C.73
Although multiple sites on a target protein might be susceptible to attack by NO and
related species, nitrosation occurs preferentially either at transition metals69 or thiols74 that
regulate protein function. The hierarchical order of attack preference seems to be heme
moieties,75 with further preferences as thiols > tyrosine > amines.74 Such molecular selec-
tivity is a result of radical biochemistry and thermodynamic law. As described by
Jaeschke76 for O2– but with direct applicability to NO and other free radicals, NO initiates
a radical chain reaction. Each reaction is spontaneous, and the energy gain from each
consecutive molecular bond has to be larger than the dissociation energy of the old bond
according to thermodynamic laws. Thus, each reaction results in the formation of increas-
ingly stable radicals and focuses the next reaction on preferred, susceptible targets.
Overall, nitrosylation has been shown to regulate a diverse array of enzymatic activ-
ities, including tissue-type plasminogen activator (t-PA),77 cathepsin B,68 glyceraldehyde-
3-phosphate dehydrogenase,78 K+ ion channels of vascular smooth muscle,79 and NMDA
glutamate receptors.80 S-nitrosylation of target proteins has been shown to be a mechanism
by which cells express antimicrobial properties.81 The efficacy of this trait is dependent
on the target’s intracellular thiol pool,81 which can absorb NO, thus diminishing its cyto-
toxic effect. This has specifically been shown to be true in Salmonella typhimurium,81 which
is resistant to NO attack. Furthermore, utilizing S-nitrosylation, NO has also been shown
to activate p21, a nonheme enzyme that is known to play a central role in converting
extracellular signals into intracellular responses,82 notably in mitogen-stimulated T
cells.83,84
Higher concentrations of NO can increase the target cells’ labile iron pool.69 This is
via NO attacking iron-regulatory protein (IRP), also known as aconitase, which binds to
iron-responsive element (IRE), a protein that regulates the expression of several genes
involved in iron uptake, storage, and utilization, including ferritin and the transferrin
1382_C14.fm Page 217 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 217

receptor. Ultimately, the action of NO causes repression of ferritin mRNA translocation,


along with stabilization of transferrin receptor mRNA against degradation,85 which results
in a net increase in target cell iron pool and alterations in metabolic pathways under iron
control.
Furthermore, NO can alter redox-sensitive transcription factors, such as activator
protein-1 (AP-1), and NF-kB.86 AP-1 is important in the control of cell proliferation and
differentiation, and NO has been shown to both inhibit87 and increase88,89 AP-1 binding to
DNA. NF-kB, on the other hand, is a transcription factor that regulates many genes
involved in the inflammatory response, including TNF-a, IL-2, IL-8, intercellular adhesion
molecule (ICAM)-1, VCAM-1, and iNOS.90 Its direct interaction with NO has been dem-
onstrated to be due to NO attack on the cysteine residue involved in the nuclear translo-
cation and DNA binding of NF-kB.91 This can significantly alter the capacity of NF-kB to
bind to DNA.92 However, the indirect effects of NO on NF-kB can vary depending on cell
type, stimulus, NO species, NO concentration, and redox status of the cell.57 NO and NO-
related species have been shown to activate NF-kB in human peripheral blood mononu-
clear cells53 and RAW 264.7 cells.93 Conversely, NO has been shown to inhibit NF-kB
activation in hepatocytes94 and in RAW 264.7 cells, the latter being interpreted as feedback
inhibition of NO on NF-kB.93 In this context, NO was shown to stabilize the NF-kB inhibitor
IkB-a in endothelial cells.95
At even higher concentrations of NO, more closely associated with NO’s cytostatic
and cytotoxic properties, NO can inhibit other key enzymes, including ribonucleotide
reductase, the rate-limiting step in DNA replication,96,97 and complexes I and II of mito-
chondrial electron transport chain.98 In these high NO environments, NO can directly
interfere with DNA and cause strand breaks when the intracellular thiol pool has been
depleted99,100 (see Table 14.1 for listing of NO-regulated gene products).
Cells, however, have developed mechanisms to defend against nitrosative stress. For
example, glutathione can react with NO to form GSNO. This acts as an NO sink and
confers protection against attack by NO.74 A cell can then utilize the NO against oxidative
attack via slow release from the protein. Rauhala et al.101 demonstrated that GSNO is
approximately 100-fold more potent than GSH in suppressing iron-induced generation of
hydroxyl ions and that it suppresses the peroxidation of lipids. In addition, in rat hepa-
tocytes stimulated with TNF-a, NO was shown to protect the cells against TNF-a-induced
apoptosis by inducing heat shock protein-70 expression.102
Certain bacteria, such as Escherichia coli, have developed mechanisms to sense oxida-
tive and nitrosative stress and react to them, namely, through OxyR and SoxRS. OxyR is
a nonmetal protein that is activated by hydrogen peroxide and S-nitrosothiol. Once acti-
vated by the stressor, OxyR up-regulates genes that can protect the organism,103 such as
catalase.104 SoxRS is a multigene system that is initiated under nitrosative stress due to
interaction with its Fe–S cluster.105 This induces transcription of resistance genes such as
manganese superoxide dismutase and glucose-6-phosphate dehydrogenase.106

14.2 Nitric oxide: physiology and pathophysiology


in mammalian cells
14.2.1 Nitric oxide and the vasculature
As stated in the Section 14.1.1, “The Nitric Oxide Synthases,” eNOS-derived NO is a major
regulator of vascular tone and blood pressure. In response to shear stress and agonists
such as bradykinin and ADP, the vascular endothelium produces NO, which in turn
rapidly diffuses to the vascular smooth muscle and causes relaxation through a
1382_C14.fm Page 218 Wednesday, October 8, 2003 1:32 PM

218 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 14.1 Index of NO-Regulated Genes and Gene Products

Protective Mediators Matrix and Matrix Metalloproteinases


Heme-oxygenase-1 MMP-9
Superoxide dismutase TIMP-1
DNA-protein kinase t-PA
Heat shock protein-70 PAI-I
Collagen
Pro-Inflammatory Mediators Fibronectin
INOS Laminin
Cyclooxygenase 2 SPARC
Phospholipase A2
Growth Factors
Cytokines and Chemokines VEGF
Interleukin-8 FLT-1
MIP-2 Erythropoietin
MIP-1a Adrenomedulin
MCP-1 TGF-b
M-CSF
TNF-a

Adhesion Molecules
ICAM-1
VCAM-1
E-Selectin
P-Selectin

MIP = macrophage inflammatory protein; FLT = fms-like tyrosine kinase.


Source: Pfeilschifter, J. et al., Eur. J. Physiol., 442, 479, 2001. Reprinted with permission.

cGMP-mediated dephosphorylation of the myosin light chains,107 as well as via nitrosy-


lation of a thiol-containing domain in a calcium-dependent potassium channel (KCA++).79
Multiple pieces of evidence supporting NO regulation of blood pressure have been
presented. First, eNOS knockout mice are found to have profound hypertension.108–110
Second, administration of the eNOS inhibitor N-nitro-L-arginine to wild-type animals
promotes vasoconstriction.111 Moreover, eNOS-derived NO has been demonstrated to be
critically important in maintaining the normal low vascular resistance in the lung, and
eNOS knockout mice have pulmonary hypertension and lack the normal vasodilatory
response to acetylcholine exposure.109
The vasodilatory properties of NO have been exploited therapeutically. Nitrates,
including nitroglycerin and isosorbide dinitrate, are cornerstone therapy in the treatment
of coronary artery disease and myocardial ischemia. Via NO-mediated vasodilation of
both venous and arterial vessels, these medications are able to decrease preload (left
ventricular end-diastolic pressure) and afterload (peripheral vascular resistance) as well
as dilate the coronary arteries. This leads to an overall improvement in the oxygen
supply–demand ratio and decreased myocardial oxygen consumption.107 More recently,
the presence of NO-sensitive vasculature has been taken advantage of in the treatment of
erectile dysfunction (ED). NO was shown to mediate penile erection by relaxation of the
corpus cavernosum,112,113 leading to tumescence. Sildenafil citrate (Viagra‚*) is a selective
inhibitor of cGMP-specific phosphodiesterase type 5 (PDE5). When sexual stimulation

* Registered trademark of Pfizer, Inc., New York, NY.


1382_C14.fm Page 219 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 219

causes local release of NO, inhibition of PDE5 by sildenafil promotes increased levels of
cGMP in the corpus cavernosum, leading to smooth muscle relaxation, increased blood
flow, and erection.114
Special mention must be made about other physiologically relevant nitric oxide func-
tions in vasomotor regulation. Bronchial epithelial and endothelial cells use NO to match
ventilation and perfusion.115 Clinically, inhaled nitric oxide therapy has been shown to
improve gas exchange and pulmonary hemodynamics in patients with acute respiratory
distress syndrome (ARDS) and children with primary pulmonary hypertension.116,117 NO
therapy is effective because the inhaled nitric oxide goes only to those areas of the lung
that are ventilated, producing vasodilation of vascular smooth muscle cells and a redis-
tribution of blood flow to ventilated regions, with an overall improvement in ventilation-
perfusion matching and oxygenation.118 Also, in the kidney, macula densa tubular epithe-
lial cells employ NO to dilate afferent arterioles in order to increase glomerular filtration.119
The role of NO in endothelial function is much more diverse than just vasomotor
regulation. Endothelial-derived NO is important in preventing leukocyte adhesion to the
endothelium under noninflammatory conditions.120 Actually, both nNOS and eNOS were
shown to be involved in the regulation of leukocyte–endothelial interactions under non-
inflammatory conditions.121 Utilizing intravital microscopy of ileal mesenteric microcircu-
lation, investigators were able to demonstrate that baseline rolling of neutrophils was
significantly increased in both nNOS and eNOS knockout mice. Also, firm leukocyte
adhesion was greater in the nNOS and eNOS knockout animals. The increased rolling and
adherence of neutrophils was shown to be due to an up-regulation of P-selectin on the
vessels of the nNOS and eNOS knockout animals.121 NO has been shown to inhibit the
expression of P-selectin as well as other endothelial cell adhesion molecules, including
VCAM-1 and ICAM-1.122,123 It has been proposed that tonic endogenous NO release inhibits
P-selectin, ICAM-1, and VCAM-1 expression on the endothelium. This latter effect is not
cGMP dependent, but rather is associated with inhibition of NF-kB.122 Experimental inhi-
bition of endogenous eNOS-derived NO by L-NMA results in activation of NF-kB, which
suggests further that endogenous NO may tonically inhibit NF-kB within the endothelial
cells during baseline, noninflammatory conditions.95 This effect can be overridden during
inflammation, however, because it was demonstrated that constitutive NO production
was insufficient to inhibit TNF-a-induced activation of NF-kB.124 In this situation, a higher
concentration of NO, such as that resulting from iNOS induction, was predicted to be
required to suppress cytokine-activated NF-kB. (For further discussion see Section 14.2.2.)
The constitutively expressed NO has long been recognized to inhibit platelet adhesion
and aggregation.64,125 In fact, as early as 1987, sodium nitroprusside, a potent NO donor
used therapeutically for blood pressure control, was shown to prevent platelet adhesion
to the endothelium.126 During platelet activation, agonists such as ADP, thrombin, and
thromboxane stimulate calcium mobilization and entry into the platelet. An NO-mediated
cGMP-dependent protein kinase within the platelet acts to inhibit receptor agonist-evoked
calcium mobilization from intracellular sources127 and inhibit cytoskeletal reorganiza-
tion.128 In vivo, NO has been shown to inhibit platelet aggregation synergistically with
prostacyclin.129
Besides functioning on the surface of the endothelium, NO also influences the per-
meability of the endothelial barrier.130 In the exteriorized ileal mesentery model, Lefer et
al.121 described that eNOS and nNOS knockout mice had increased neutrophil transmi-
gration and accumulation in the peritoneum after thioglycollate injection when compared
to the wild type. Several years prior to this, Kubes and Granger,130 analyzing feline
mesenteric lymph vessels under baseline, noninflammatory conditions, demonstrated a
fivefold increase in lymph flow and lymph protein flux after infusion of a NOS inhibitor.
Since this effect occurred without any change in the capillary hydrostatic pressure, it was
1382_C14.fm Page 220 Tuesday, October 7, 2003 6:34 PM

220 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

concluded that NO minimizes the loss of fluid and protein out of the vasculature by
tonically preventing microvascular permeability. Then, utilizing an anti-CD11/CD18 anti-
body to prevent leukocyte adherence, these authors went on to show that leukocyte
adhesion is responsible for a significant portion of the NOS inhibitor increase in fluid and
protein leakage. Therefore, NO maintains an intact endothelial barrier by reducing
microvascular permeability and by inhibiting leukocyte adherence.130 The dysregulation
of endothelial NO production has been implicated in the development of atherosclerotic
plaques131 as well as the impaired vascular responsiveness characteristic of atherosclerotic
vessels.132,133 Oxidized low-density lipoproteins (LDLs) are able to inflict an oxidative stress
on the endothelium and decrease the level of NO.134 This has been supported by findings
that demonstrate oxidized LDLs scavenge NO directly131 as well as down-regulate eNOS
expression.134,135

14.2.2 Nitric oxide, wound repair, infection, and septic shock


In healing wounds, iNOS protein has been localized to keratinocytes, inflammatory cells,
fibroblasts, sebaceous glandular epithelium, muscle, and the endothelium. It is found not
only in rodent and murine wounds but also in human burns,136 chronic venous leg ulcers,137
skin abscesses,138 and granulomata.139
In mice, Yamasaki et al.140 reported overall delayed wound healing in iNOS knockout
mice that had undergone cutaneous excisional wounds, as well as in wild-type mice
treated with an iNOS inhibitor. These investigators were able to demonstrate normaliza-
tion of wound healing times after local adenoviral transfer of an iNOS expression vector.
In direct contrast to these findings, Most et al.,141 utilizing a murine incisional wound
model, failed to find any difference between wild-type and iNOS knockout mice in either
tensile strength or collagen content. Experiments from this laboratory have been per-
formed to explain this discrepancy.47 The data support a role for bacterial colonization in
the excisional wound model used by Yamasaki et al.140 that stimulated NO production,
since bacterial products are known to induce iNOS expression in inflammatory cells.139
However, in the incisional model employed by Most et al.,141 the wounds remain sterile,
and iNOS-derived NO from inflammatory cells does not appear to be required for sterile
wound repair.
Positive iNOS protein staining by immunohistochemical analysis is detectable in the
epithelium, sebaceous glands, and skeletal muscle of murine incisional and excisional
cutaneous wounds. In the epithelium, it has been shown that there is a large induction of
iNOS expression in keratinocytes during the inflammatory phase of wound healing.142 In
vitro and in vivo studies have shown that both exogenously and endogenously derived
NO is a potent enhancer of cytokine-induced vascular endothelial growth factor
(VEGF)–mRNA expression within keratinocytes.143 Also in keratinocytes, NO has been
shown to promote proliferation and reepithelialization.144 Additionally, NO is produced
at the skin surface, where it has been shown to be bactericidal.145
The role of nitric oxide regulation of fibroblasts is less clear. Analysis of skin wound
fibroblasts demonstrated that these cells express iNOS protein.146 NO has been shown to
suppress matrix production147 and fibroblast proliferation, and to increase cell apoptosis.148
In addition, in anastamotic wound tissue, investigators showed a correlation between
markedly increased NO production and impaired collagen synthesis that resulted in
decreased intestinal anastamotic bursting pressure.149
Other investigators, however, have challenged these results and have submitted data
showing greater collagen synthesis in the presence of NO. These researchers have dem-
onstrated enhanced fibroblast collagen synthesis by approximately 75 and 87% in the
presence of 100 and 400 mM SNAP, an NO donor, respectively.150 Furthermore, in rats that
1382_C14.fm Page 221 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 221

underwent colonic anastamosis, continuous IV infusion of s-methylisothiourea, reportedly


a selective iNOS inhibitor, resulted in a significantly reduced anastamotic bursting strength
compared to control animals.151
As stated in Section 14.1.1.3, endothelial-derived NO is important in preventing leu-
kocyte adhesion to the endothelium.122 During inflammation, however, constitutive NO
production is insufficient to inhibit TNF-a-induced activation of NF-kB within the endo-
thelium.124 In this situation, a higher concentration of NO, such as iNOS derived, at the
site of inflammation may be required to suppress cytokine-activated NF-kB. This is sup-
ported with multiple data from Granger and Kubes152–154 that showed NO attenuation of
leukocyte adhesion during inflammatory conditions and stimuli such as reactive oxygen
metabolites, ischemia-reperfusion, and oxidized low-density lipoproteins. Similarly, the
cytokine-induced expressions of M-CSF and IL-6 by the endothelium are inhibited by NO
through inhibition of NF-kB.95 Also, endothelial-derived IL-8 has been shown to be both
inhibited122 and induced155 by NO.
At this juncture, the influence of nitric oxide derived from inflammatory cells at the
site of injury deserves special mention. As stated previously, marked species variability
influences which stimuli are necessary to induce iNOS expression. In the rat macrophage,
culturing alone is enough to induce iNOS expression. This is contrasted to murine mac-
rophages, which require cytokines and LPS for their stimulation in vitro. In vivo, the sterility
of the wound will influence iNOS protein expression; in our experience, bacterial coloni-
zation and bacterial products were required to induce iNOS production in murine dermal
wounds.47 The inflammatory cells from which iNOS-derived NO originates appears to be
both the macrophage and the neutrophil. In rodent sterile cutaneous wounds, iNOS
expression occurs during the initial 48 to 72 h of injury, and the macrophages contribute
the bulk of iNOS-derived NO.156
The expression of iNOS-derived NO in human inflammatory cells has been much
more controversial. The inducibility of iNOS mRNA and the identification of iNOS protein
have been firmly established by several researchers,157,158 and both human neutrophils and
macrophages have been demonstrated to express iNOS at sites of inflammatory foci such
as urinary tract infections159 and skin lesions in patients with leishmaniasis.160 However,
consistent evidence of NO production by identification of NO degradation products has
been much more challenging. The final production of iNOS-derived NO in humans
appears to be highly restricted compared to rat and rodent models.
As reported by Weinberg and colleagues,161 human peripheral blood monocytes har-
vested from a large cohort of donors (23 to 74 depending on culture conditions) mostly
failed to produce NO2–/NO3– during 3- to 4-day cultures with a variety of iNOS inducers,
including cytokines and growth factors (e.g., IFN-g, TNF-a, IL-1, IL-2, IL-4, IL-6, GM-CSF),
as well as LPS, Leishmania monocytogenes, Candida albicans, Staphylococous epidermidis, Myco-
bacterium avium complex, and M. tuberculosis. However, they were able to demonstrate a
modest but statistically significant increase in NO2–/NO3– production from peritoneal
macrophages that were incubated with LPS ± IFN-g, whereas the peripheral blood mono-
cytes did not.
Subsequently, members of that laboratory tested freshly isolated monocytes from eight
healthy male volunteers for iNOS expression and NO production in response to IFN-a2b
treatment.162 Their research demonstrated iNOS mRNA induction, positive iNOS immuno-
blotting, and a dose-dependent increase in NO2–/NO3– production in response to IFN-a2b.
Moreover, in separate experiments,163 these researchers showed that untreated peripheral
blood mononuclear cells from 25 patients suffering from rheumatoid arthritis (RA) had a
twofold greater NOS activity at baseline in culture than normal controls. In response to
IFN-g ± LPS, RA mononuclear cells had a significantly increased NOS activity over baseline
compared to NO response to treatment in controls.
1382_C14.fm Page 222 Tuesday, October 7, 2003 6:34 PM

222 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Similarly, Hickman-Davis and colleagues164 showed that exposure of alveolar macro-


phages from normal volunteers and from lung transplant recipients to IFN-g failed to
affect NO2–/NO3– production. However, coincubation of the alveolar macrophages from
the transplant recipients with Klebsiella resulted in a significant increase in nitrite/nitrate
production.
Furthermore, Nicholson and colleagues165 demonstrated that 65% of alveolar macro-
phages from 11 patients with untreated, culture-positive tuberculosis were positive for
iNOS antigen, and Nozaki and colleagues166 showed increased production of iNOS in
BCG-inoculated alveolar macrophages in patients with pulmonary fibrosis. Most dramat-
ically, Anstey and colleagues167 found high fasting urinary and plasma nitrate levels and
leukocytes’ iNOS antigen in healthy controls and asymptomatic malaria patients in an
endemic area.
In an extensive review of over 100 articles regarding NO production and NOS II
expression in human mononuclear cells, Weinberg168 concluded that human monocytes
and macrophages are able to produce NOS II and NO but in lower levels than murine
cells when stimulated in parallel. The exceptions are monocytes and tissue macrophages
isolated from ill patients suffering from malaria, tuberculosis, or rheumatoid arthritis, as
shown above, which express higher levels of NOS II and NO than normal control mono-
cytes.
The most widely accepted role for nitric oxide and its related species during infectious
diseases is as a weapon against the invading bacteria. In vitro, NO has been shown to be
directly toxic to M. tuberculosis,169 Staphylococcus aureus,170 and Leishmania major.171 However,
different bacteria may be more susceptible to one but resistant to another nitric oxide
species. For example, NO is toxic to S. aureus170,172 and Leishmania major173 but is not directly
toxic to E. coli174 or S. typhimurium.175 The latter two require NO to interact with hydrogen
peroxide to form peroxynitrite for bactericidal activity.176 In all, NO has been shown to
exert antimicrobial activity against viruses, intracellular and extracellular bacteria, para-
sites, and fungi.177
As a cytotoxic effector molecule, NO should not be equated with superoxide. Ding
et al.178 demonstrated that the synthesis of NO and O2– are independently regulated and
that the stimuli necessary for their production may be different. O2– production is rapid,
occurring in seconds to minutes after appropriate stimulation,179 whereas NO production
will require several hours and will require de novo protein synthesis.9,180,181 Moreover,
through in vitro studies utilizing S. aureus, it has been demonstrated that NO and O2– have
different temporal killing profiles. Whereas superoxide exposure was able to kill the
bacterium within 2 to 5 h, exposure to the NO donor SNAP failed to do so. Rather, the
prolonged exposure to SNAP caused a dose-related delayed killing over 24 h.170 The
authors surmised that NO causes stabilized microbial killing over a prolonged period of
exposure.
Stenger et al.182 treated immunocompetent mice that had resolved leishmania infection
with the iNOS inhibitor more than 100 days after the mice had received the parasitic
inoculation. This treatment led to reactivation of the infection disease with a 10,000- to
100,000-fold increase of the parasite burden in the cutaneous and lymphoid tissue. This
suggested that a continuous production of iNOS-derived NO is required in order to
maintain control of this organism.
Furthermore, NO may also act as a paracrine signal messenger to the endothelium
and inflammatory cells. As stated previously, NO acting upon the endothelium inhibits
neutrophil rolling and adherence. After systemic administration of LPS, iNOS-derived NO
was shown to abrogate neutrophil migration to an injury site.183 In this regard, NO has
been shown to induce TNF-a production in LPS-simulated neutrophils184 as well as inhibit
neutrophil superoxide production.185 In the macrophage, NO has been shown to activate
1382_C14.fm Page 223 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 223

NF-kB (in contrast to its action on endothelial NF-kB).53,93 Furthermore, T cell activation
has been shown to occur by S-nitrosylation of p21ras. In the granuloma, which is comprised
largely of macrophages, T cells, and fibroblasts, NO may function to coordinate the
simultaneous activities and location of each cell type in order to achieve the appropriate
architecture, extracellular matrix, and isolation of the offending agent.186
Systemically, nitric oxide also influences the overall homeostasis of the host during
severe infection and endotoxemia. Endotoxemia is known to induce the expression of
NOS not only in the professional inflammatory cells but also in the gastrointestinal tract,187
hepatocytes,188,189 spleen,187 skeletal muscle,190 cardiac myocytes,191 vascular smooth mus-
cle,192 and endothelial cells.58 Given this widespread distribution of NO production during
endotoxemia, and with the knowledge of the role of NO in vasomotor tone and blood
pressure regulation, it is easy to envision nitric oxide playing a pivotal role in the hypoten-
sion characteristic of septic shock.
Ochoa et al.193 demonstrated elevations in blood NO2–/NO3– concentrations in septic
patients and showed that increased levels of these metabolites in the blood correlated with
increased endotoxin concentration in the plasma. At the same time, it was demonstrated
that hypotension after endotoxemic challenge could be attenuated with the use of the
NOS inhibitor L-NMMA in a rodent model of endotoxemia.194 Other investigators195 have
been able to reproduce this finding and to translate it to the clinical setting. In the latter
studies, septic patients who were treated with NOS inhibitors had improved mean arterial
pressures and an increase in the systemic vascular resistances.196,197 Unfortunately, the
mortalities in each study remained high (60 and 50%, respectively), but the cohort size of
each was too small for conclusive judgment regarding treatment.

14.2.3 Nitric oxide and apoptosis


Apoptosis is the orderly, energy-dependent process by which the cell fragments its own
DNA with specific endonucleases, contracts the chromatin, and dies. Cell necrosis, on the
other hand, is characterized by random fragmentation of DNA, nuclear swelling, and
lysis. NO and its related species have been shown to be influential in both modes of cell
demise. Of course, as with other functions of NO, the ultimate effect will depend on the
cellular milieu, including the redox status of the cell, as well as which specific cell type it
is acting upon. The sensitivity to NO varies dramatically from one cell type to another,
with death occurring as a result of either necrosis or apoptosis.198 In this regard, NO has
been shown to have conflicting effects on cell apoptosis. Without a doubt, NO can mediate
DNA damage100 and inhibit protein and nucleic acid synthesis97 with resultant target cell
death. Evidence to support this has been demonstrated in vitro, where exogenous NO
application induces apoptosis in macrophages.199 IL-1b has been shown to induce apop-
totic cell death in pancreatic beta cells by stimulating NO production.200,201
The mechanism involved in NO-induced apoptosis has been shown to involve p53,
the caspase family of enzymes, and bcl-2. p53 is a tumor suppressor gene that has also
been shown to be part of the DNA damage repair pathway. Activation of p53 after DNA
damage results in p53 binding to DNA and transcribing genes involved in cell cycle
inhibition, such as p21. p21 is itself an inhibitor of the cell cycle-dependent kinases and
results in G1 arrest.202 The caspases are a family of proteases involved in apoptosis whose
catalytic site utilizes a cysteine moiety.102 These proteases are normally present within a
cell as inactive proenzymes that, when cleaved to their active forms, initiate a cascade
that culminates in apoptosis. And finally, bcl-2, which is in fact a family of genes that
encode antiapoptotic proteins, is able to prevent cell death by inhibiting the activation of
the caspases.
1382_C14.fm Page 224 Tuesday, October 7, 2003 6:34 PM

224 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Evidence for NO-induced involvement in each step of this pathway has been shown.63
In RAW 264.7 macrophages, activation of iNOS resulted in p53 accumulation, which
preceded DNA fragmentation. The degree of p53 accumulation was shown to correlate
with frequency of apoptosis, and iNOS inhibition resulted in a down-regulation of p53
and diminished apoptotic features.203
In human leukemic cells204 and rat mesangial cells,205 NO donors have been shown to
activate caspases. Moreover, cytokine-induced caspase activation is blocked by iNOS
inhibitors,206 thereby attesting to their interrelationship. Similarly, pharmacologic inhibi-
tion of caspases blocks NO-mediated apoptosis.207 Moreover, in bcl-2-transfected animals,
NO-mediated caspase activation was attenuated.206
In keeping with the duality of NO, NO has been shown to inhibit apoptosis in several
cell types depending on physiological conditions. This effect has been shown in rat hepa-
tocytes,102 human B cells,208 mouse splenocytes,209 human eosinophils,210 rat ovarian folli-
cles,211 rat cardiac myocytes,212 and human umbilical vein endothelial cells,213 as well as
others. In hepatocytes, NO can prevent apoptosis by indirect inhibition of caspase-3-like
protease activation via a cGMP-dependent mechanism, as well as by direct inhibition of
caspase-3-like protease activity through S-nitrosylation.102 Kim et al.102 further showed that
low-dose NO can protect hepatocytes against apoptosis through NO-mediated up-regu-
lation of heat shock protein-70.
In connection to this, it has been shown that NO-mediated protection against apoptosis
is inducible. For example, it has been shown in vitro that stimulation of macrophages with
a low, nontoxic dose of NO or equivalent cytokine stimulation can protect the cells against
a subsequent high dose of NO that would otherwise cause apoptosis.214 Some investigators
have proposed that it is cyclooxygenase-2 induction during the pretreatment period that
confers protection to the macrophage against apoptosis.215 In addition, macrophages that
overexpress cyclooxygenase-2 have demonstrated resistance to NO-stimulated apopto-
sis.215 Others have stated that this phenomenon is due to NF-kB activation, because block-
ing NF-kB removed the protection of NO pretreatment and restored p53 accumulation
and DNA fragmentation after high-dose NO exposure.63 A third proposed mechanism,
seen in splenocytes, is an NO-induced cGMP-dependent increase in bcl-2.209

14.2.4 Nitric oxide and cancer


Similar to its attack upon nonneoplastic cells, NO has also been shown to have tumoricidal
activity. In the P815 mastocytoma cell line, coculture of tumor cells with activated macro-
phages or direct exposure of tumor cells to NO gas or the NO donor SNP resulted in NO-
dependent DNA fragmentation within the tumor cell, consistent with apoptosis.216 Mac-
rophage-derived NO has also been shown to induce regression of UV-induced murine
cancers.217 Thus, the host appears to be able to direct inducible nitric oxide synthase-
derived NO against neoplastic growth.
Multiple researchers have attempted to piece together the mechanism by which this
occurs in hopes of finding applicability in cancer treatment. Hung et al.218 found that
interferon-g is absolutely required for iNOS-derived NO production and subsequent anti-
tumor activity. More interestingly, Hung et al.218 and others219 demonstrated that macro-
phages themselves do not possess the intrinsic capacity to direct the attack against the
specific tumor cells. Instead, this tumor specificity is based on CD4+ T cell activation by
antigen presenting cells (APCs), which in turn recruit and activate effector tumoricidal
macrophages. This tumoricidal activity is via superoxide and NO.
In the human breast cancer cell line MDA-MB-231, sustained exposure to the NO
donor DETA-NONOate induced cytostasis and ultimately apoptosis of the tumor cells.220
This cytostasis occurred during the G1 phase of the cell cycle and was associated with the
1382_C14.fm Page 225 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 225

down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein.


Cyclin D1 is a member of the G1 cyclins, which regulate progression of a cell through the
G1 phase of the cell cycle. Exposure of these tumor cells to exogenous NO led to a decrease
in cyclin D1 due to the attenuation of cyclin D1 synthesis; thus, the direct protein synthesis
inhibitory property of NO was responsible.
Both murine and human tumor cells, like all normal nonneoplastic cells, can produce
NO either constitutively or in response to induction by exogenous stimuli.221–223 It is not
only the exogenously derived NO that may affect tumor activity. Researchers have been
able to demonstrate in vivo tumor regression of hepatic metastasis of murine sarcoma
tumors after systemically up-regulating the iNOS expression within the tumor itself.224
Others have shown in the murine K-1735 melanoma cell line, after successful transfection
of an enzymatically active iNOS gene, results in DNA fragmentation and apoptosis that
directly correlated with endogenous NO production.225 These researchers also had been
able to demonstrate that, in addition to inducing apoptosis, iNOS gene transfection
abolished the ability of the tumor cells to metastasize. This suggests that the endogenous
expression of iNOS may play a particularly important role in conferring metastatic
potential to tumor cells. In this K-1735 melanoma cell line, it was further demonstrated
that metastatic cells do not exhibit appropriately high levels of iNOS expression after
incubation with different cytokines or LPS, whereas nonmetastatic cells did.226,227 Even
in human colon cancer, tumor cells isolated from metastatic foci exhibit lower NO
activity than tumor cells from the primary tumor.223 Thus, metastatic cells appear to lose
expression of iNOS, and according to Dong et al.,228 “[this] loss of iNOS expression and
hence resistance to induction of apoptosis may contribute to the survival of metastatic
cells in the circulation.”
Unfortunately, along with a decrease in endogenous iNOS expression, tumor cells
may develop other means to exploit or circumvent NO-mediated tumor surveillance.
Tumor cells, such as in the human glioblastoma and hepatocellular carcinoma cell lines,
produce constitutive levels of endogenous NO, which leads to increased VEGF expres-
sion,229 thereby promoting neovascularization for the tumor. Tumor cells may also
respond to nitrosative stress by increasing cellular defenses. It has been shown that
exposure of tumor cells to exogenous NO leads to an up-regulation of the large catalytic
subunit of the DNA-dependent protein kinase (DNA-PKcs). Since DNA-PKcs is essential
for repair of double-stranded DNA breaks, this would confer protection to the tumor
cells against nitrosative attack.230 Tumor cells expressing bcl-2 may escape macrophage-
derived, NO-induced apoptosis. This was dramatically demonstrated in the NO-sensi-
tive P815 mastocytoma cell line, which became NO resistant after stable transfection
with the bcl-2 gene.231 bcl-2 overexpression is known to be a frequent finding among
carcinomas, lymphomas, and leukemias.232
Perhaps most dramatically, tumor cells may actively inhibit macrophage NO produc-
tion. Tumor-infiltrating macrophages have been shown to have impaired iNOS function,233
and peritoneal macrophages from tumor-bearing mice have reduced NO production.234,235
In kinetic studies of peritoneal macrophages from mammary tumor-bearing mice,
DiNapoli et al.236 demonstrated that iNOS hyporesponsiveness became more pronounced
as the malignancy progressed. It was concluded from these studies that a tumor-derived
product, specifically phosphatidyl serine, caused a concentration-dependent inhibition of
iNOS mRNA transcription within the macrophages. The decreased iNOS mRNA tran-
scription ultimately resulted in a diminished capacity of macrophages to lyse the target
tumor cells, even upon appropriate stimulation. This was further shown to be iNOS
specific, because other cell functions such as superoxide production, mitochondrial dehy-
drogenase activity, and protein synthesis were not affected.
1382_C14.fm Page 226 Tuesday, October 7, 2003 6:34 PM

226 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

14.2.5 Nitric oxide and cellular energy metabolism


As stated previously, NADPH supplies reducing equivalents to NOS for the oxidation of
L-arginine to NO and citrulline. NADPH, in turn, is produced through the hexose–mono-
phosphate shunt. Macrophages have been shown to have enhanced glucose utilization
through this shunt during activation, as well as enhanced overall glucose uptake, glyco-
lytic, and tricarboxylic acid cycle activity.78 The increase in glycolytic flux of activated
macrophages has been shown to be NO dependent.237 Moreover, activated macrophages
have inhibited mitochondrial respiration, localized to inhibition of complexes I and II of
the electron transport chain.98 Activated macrophages have also been shown to inhibit
aconitase, a citric acid cycle enzyme with a catalytically active iron–sulfur cluster, in target
tumor cells via NO.98,238 By blocking oxidative metabolism and respiration, NO forces the
target cell to rely on glycolysis as the predominant energy-generating pathway.239 An
example of this increased glucose reliance, NO production has been shown to increase
expression of the Glut 1 glucose transporter in rat L6 skeletal muscle cells.240 This NO-
mediated increase in glucose transport was also shown in human peripheral blood mono-
nuclear cells in vitro after exposure to NO donors.53
Furthermore, in vitro studies have shown NO-mediated alterations of glyceraldehyde-
3-phosphate dehydrogenase, a glycolytic enzyme.78 The NO attack on glyceraldehyde-3-
phosphate dehydrogenase was demonstrated to induce the acyl phosphatase activity of
the enzyme, which uncouples ATP production241 (see Figure 14.2). Subsequently, the spe-
cific mechanism of this alteration was described by Mohr et al.,242,243 depicting S-nitrosyla-
tion of the enzyme’s thiol group, which initiates subsequent covalent modification by
NAD.

14.2.6 Nitric oxide and the nervous system


As stated in Section 14.1.1.1, NO has been identified as a modulator of nerve cell growth
and synaptic plasticity, and as a neurotransmitter. Certain NO-responsive targets in the
neuron have been identified, including GAP-43, a growth cone constituent, and SNAP-25,
a synaptic protein involved in axonal growth and synaptogenesis.244 By modulation of
these neuronal proteins, NO is able to regulate neuronal process outgrowth and
remodeling.
Long-term potentiation is the process by which stimulation of certain central nervous
system synapses produces a pronounced and prolonged potentiation of the excitatory
postsynaptic potential and increases the probability that the postsynaptic neuron will fire
with subsequent stimulation. This process is believed to be important in memory and
learning. Long-term potentiation in vivo requires a presynaptic increase in transmitter
release that is believed to be triggered through retrograde stimulation by a neurotrans-
mitter released from the postsynaptic neuron.245,246 This retrograde messenger results in
an overall strengthening of the synaptic connection. Multiple investigators have proposed
NO as the retrograde messenger.28,29,247 Support for this hypothesis comes from the finding
that long-term potentiation is significantly reduced in mouse brains exposed to the NOS
inhibitor N-nitro-L-arginine.227 From which isoform the NO is elicited is controversial, since
long-term potentiation was significantly reduced only in mice that were double knockouts
for nNOS and eNOS.227
Rats that were exposed to carbon monoxide prenatally were found to have selective
impairment of long-term potentiation that correlated with decreased nNOS in the hippoc-
ampus.248 Carbon monoxide is known to bind the heme in NOS and inhibit enzyme
activity.249 Moreover, in an animal model of lead poisoning, rats that were exposed to lead
prenatally were found to have decreased nNOS activity compared to controls.250 From
1382_C14.fm Page 227 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 227

Figure 14.2 Impact of NO on glucose metabolism and energy in macrophages. Glucose metabolism
(top left panel): main graphic reports glycolytic flux and insert graphic shows glucose oxidation by
resident rat peritoneal macrophage in culture. Culture media included 6 mM L-arginine ((–)ARG in
the figure) or 1 mM L-arginine ((+)ARG in the figure). Addition of N-MMA (0.5 mM) to high
L-arginine cultures restored all measurements to those obtained in (–)-L-arginine cultures (not
shown). Theoretical ATP yields (top right panel) were calculated from glucose metabolism data
based on the known ATP yield of glucose oxidation or its metabolism through glycolysis. ATP
content of cells cultured as described above by the end of a 24-h culture is shown in the lower left
panel. Data in the lower right panel, in turn, show the incorporation of [32P] into [32P]-ATP during
the last 4 h of the 24-h culture.241

these findings, it was proposed that the cognitive defects associated with lead poisoning
may be attributable to NO-mediated long-term potentiation and neuronal plasticity.
Of course, in excess, the toxic properties of NO will predominate. In mice that
underwent middle cerebral artery occlusion with resultant cerebral ischemia, a dramatic
rise in NO concentration was found.251 This overproduction of NO ultimately causes
neurotoxicity.252 In nNOS knockout mice, there is a reduction in ischemia after middle
cerebral artery occlusion.253
Other important characteristics of NO in the nervous system that must not be over-
looked include cGMP-dependent modulation of vascular tone and cerebral blood flow,254
as well as NO as a potent antioxidant that can protect neurons from oxidative stress.255
Furthermore, the autonomic nervous system employs nNOS-derived NO as a nonadren-
ergic, noncholinergic neurotransmitter that mediates the actions of autonomic motor neu-
rons on smooth muscle.30 This includes both vascular smooth muscle cells and nonvascular
smooth muscle cells, such as those within enteric sphincters, where NO may elicit inhib-
itory responses. This latter property is exemplified in vivo by nNOS knockout mice, which
suffer from hypertrophic pyloric stenosis.26
1382_C14.fm Page 228 Tuesday, October 7, 2003 6:34 PM

228 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

14.3 Conclusion
As stated in Section 14.1, the discovery of nitric oxide and the nitric oxide synthases has
had a significant impact on the understanding of cellular physiology. With its roles in
signal transduction, vascular tone, and innate immunity, to name only a few, nitric oxide
plays a pivotal role in every organ system studied to date.
Currently, there are over 49,000 articles and multiple textbooks dedicated to nitric
oxide. With ongoing research, the information presented herein will quickly become dated.
But with advancements in the understanding of the physiological and pathophysiological
effects of NO, novel clinical applications of pharmaceutical therapy will undoubtedly
follow.

Acknowledgments
This work was supported by the National Institute of General Medical Sciences Grant
GM-42859, funds allocated by Rhode Island Hospital to the Division of Surgical Research,
Department of Surgery, and by the Armand D. Versaci Research Scholar in Surgical
Sciences Award made to Eric J. Mahoney, M.D., which was generously provided by the
Carter Family Charitable Trust. The authors are also grateful to Jill Rose for her invaluable
assistance.

References
1. Moncada, S., Foreword, in Methods in Nitric Oxide Research, Feelisch, M. and Stamler, J.S.,
Eds., John Wiley & Sons, Chichester, England, 1996, p. xvii.
2. Furchgott, R.F. and Zawadzki, J.V., The obligatory role of endothelial cells in the relaxation
of arterial smooth muscle by acetylcholine, Nature, 288, 373, 1980.
3. Ignarro, L.J., Lippton, H., Edwards, J.C., et al., Mechanism of vascular smooth muscle relax-
ation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement
of S-nitrosothiols as active intermediates, J. Pharmacol. Exp. Ther., 218, 739, 1981.
4. Ignarro, L.J., Edwards, J.C., Gruetter, D.Y., et al., Possible involvement of S-nitrosothiols in
the activation of guanylate cyclase by nitroso compounds, FEBS Lett., 110, 275, 1980.
5. Mellion, B.T., Ignarro, L.J., Ohlstein, E.H., et al., Evidence for the inhibitory role of guanosine
3',5'-monophosphate in ADP-induced human platelet aggregation in the presence of nitric
oxide and related vasodilators, Blood, 57, 946, 1981.
6. Stuehr, D.J. and Marletta, M.A., Mammalian nitrate biosynthesis: mouse macrophages pro-
duce nitrite and nitrate in response to Escherichia coli lipopolysaccharide, Proc. Natl. Acad.
Sci. U.S.A., 82, 7738, 1985.
7. Koshland, D.E., Jr., The molecule of the year, Science, 258, 1861, 1992.
8. Nathan, C., Nitric oxide as a secretory product of mammalian cells, FASEB J., 6, 3051, 1992.
9. Nathan, C. and Xie, Q.W., Regulation of biosynthesis of nitric oxide, J. Biol. Chem., 269, 13725,
1994.
10. Michel, T., Xie, Q.W., and Nathan, C., Molecular biological analysis of nitric oxide synthases,
in Methods in Nitric Oxide Research, Feelisch, M. and Stamler, J.S., Eds., John Wiley & Sons,
Chichester, England, 1996, p. 161.
11. Panda, K., Ghosh, S., and Stuehr, D.J., Calmodulin activates intersubunit electron transfer
in the neuronal nitric-oxide synthase dimer, J. Biol. Chem., 276, 23349, 2001.
12. Xie, Q.W., Leung, M., Fuortes, M., et al., Complementation analysis of mutants of nitric oxide
synthase reveals that the active site requires two hemes, Proc. Natl. Acad. Sci. U.S.A., 93, 4891,
1996.
13. Baek, K.J., Thiel, B.A., Lucas, S., et al., Macrophage nitric oxide synthase subunits: purifica-
tion, characterization, and role of prosthetic groups and substrate in regulating their asso-
ciation into a dimeric enzyme, J. Biol. Chem., 268, 21120, 1993.
1382_C14.fm Page 229 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 229

14. Tzeng, E., Billiar, T.R., Robbins, P.D., et al., Expression of human inducible nitric oxide
synthase in a tetrahydrobiopterin (H4B)-deficient cell line: H4B promotes assembly of en-
zyme subunits into an active dimer, Proc. Natl. Acad. Sci. U.S.A., 92, 11771, 1995.
15. Miller, R.T., Martasek, P., Raman, C.S., et al., Zinc content of Escherichia coli-expressed
constitutive isoforms of nitric-oxide synthase: enzymatic activity and effect of pterin, J. Biol.
Chem., 274, 14537, 1999.
16. Rodriguez-Crespo, I., Nishida, C.R., Knudsen, G.M., et al., Mutation of the five conserved
histidines in the endothelial nitric-oxide synthase hemoprotein domain: no evidence for a
non-heme metal requirement for catalysis, J. Biol. Chem., 274, 21617, 1999.
17. Adak, S., Ghosh, S., Abu-Soud, H.M., et al., Role of reductase domain cluster 1 acidic residues
in neuronal nitric-oxide synthase: characterization of the FMN-FREE enzyme, J. Biol. Chem.,
274, 22313, 1999.
18. Bec, N., Gorren, A.C., Voelker, C., et al., Reaction of neuronal nitric-oxide synthase with
oxygen at low temperature: evidence for reductive activation of the oxy-ferrous complex by
tetrahydrobiopterin, J. Biol. Chem., 273, 13502, 1998.
19. Abu-Soud, H.M. and Stuehr, D.J., Nitric oxide synthases reveal a role for calmodulin in
controlling electron transfer, Proc. Natl. Acad. Sci. U.S.A., 90, 10769, 1993.
20. Stuehr, D.J., Structure-function aspects in the nitric oxide synthases, Annu. Rev. Pharmacol.
Toxicol., 37, 339, 1997.
21. Feldman, P.L., Griffith, O.W., and Stuehr, D.J., The surprising life of nitric oxide, Chem. Eng.
News, 26, 1993.
22. Nathan, C. and Xie, Q.W., Nitric oxide synthases: roles, tolls, and controls, Cell, 78, 915, 1994.
23. Beck, K.F., Eberhardt, W., Frank, S., et al., Inducible NO synthase: role in cellular signalling,
J. Exp. Biol., 202, 645, 1999.
24. Garthwaite, J. and Boulton, C.L., Nitric oxide signaling in the central nervous system, Annu.
Rev. Physiol., 57, 683, 1995.
25. Jaffrey, S.R. and Snyder, S.H., PIN: an associated protein inhibitor of neuronal nitric oxide
synthase, Science, 274, 774, 1996.
26. Huang, P.L., Dawson, T.M., Bredt, D.S., et al., Targeted disruption of the neuronal nitric oxide
synthase gene, Cell, 75, 1273, 1993.
27. Schmidt, H.H. and Walter, U., NO at work, Cell, 78, 919, 1994.
28. Bohme, G.A., Bon, C., Stutzmann, J.M., et al., Possible involvement of nitric oxide in long-
term potentiation, Eur. J. Pharmacol., 199, 379, 1991.
29. Schuman, E.M. and Madison, D.V., A requirement for the intercellular messenger nitric oxide
in long-term potentiation, Science, 254, 1503, 1991.
30. Brenman, J.E., Chao, D.S., Gee, S.H., et al., Interaction of nitric oxide synthase with the
postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains, Cell,
84, 757, 1996.
31. Brenman, J.E., Chao, D.S., Xia, H., et al., Nitric oxide synthase complexed with dystrophin
and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy, Cell, 82, 743,
1995.
32. Cho, K.O., Hunt, C.A., and Kennedy, M.B., The rat brain postsynaptic density fraction
contains a homolog of the Drosophila discs-large tumor suppressor protein, Neuron, 9, 929,
1992.
33. Guo, F.H., De Raeve, H.R., Rice, T.W., et al., Continuous nitric oxide synthesis by inducible
nitric oxide synthase in normal human airway epithelium in vivo, Proc. Natl. Acad. Sci. U.S.A.,
92, 7809, 1995.
34. Geller, D.A. and Billiar, T.R., Molecular biology of nitric oxide synthases, Cancer Metastasis
Rev., 17, 7, 1998.
35. Morris, S.M., Jr., Arginine synthesis, metabolism, and transport: regulators of nitric oxide
synthesis, in Cellular and Molecular Biology of Nitric Oxide, Laskin, J.D. and Laskin, D.L., Eds.,
Marcel Dekker, New York, 1999, p. 57.
36. Modolell, M., Corraliza, I.M., Link, F., et al., Reciprocal regulation of the nitric oxide syn-
thase/arginase balance in mouse bone marrow-derived macrophages by TH1 and TH2
cytokines, Eur. J. Immunol., 25, 1101, 1995.
1382_C14.fm Page 230 Tuesday, October 7, 2003 6:34 PM

230 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

37. Louis, C.A., Mody, V., Henry, W.L., Jr., et al., Regulation of arginase isoforms I and II by IL-4
in cultured murine peritoneal macrophages, Am. J. Physiol., 276, R237, 1999.
38. Morris, S.M., Jr., Kepka-Lenhart, D., and Chen, L.C., Differential regulation of arginases and
inducible nitric oxide synthase in murine macrophage cells, Am. J. Physiol., 275, E740, 1998.
39. Albina, J.E., Henry, W.L., Jr., Mastrofrancesco, B., et al., Macrophage activation by culture in
an anoxic environment, J. Immunol., 155, 4391, 1995.
40. Lowenstein, C.J., Alley, E.W., Raval, P., et al., Macrophage nitric oxide synthase gene: two
upstream regions mediate induction by interferon gamma and lipopolysaccharide, Proc. Natl.
Acad. Sci. U.S.A., 90, 9730, 1993.
41. Xie, Q.W., Whisnant, R., and Nathan, C., Promoter of the mouse gene encoding calcium-
independent nitric oxide synthase confers inducibility by interferon gamma and bacterial
lipopolysaccharide, J. Exp. Med., 177, 1779, 1993.
42. Kamijo, R., Harada, H., Matsuyama, T., et al., Requirement for transcription factor IRF-1 in
NO synthase induction in macrophages, Science, 263, 1612, 1994.
43. Semenza, G.L., Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1,
Annu. Rev. Cell Dev. Biol., 15, 551, 1999.
44. Eberhardt, W., Pluss, C., Hummel, R., et al., Molecular mechanisms of inducible nitric oxide
synthase gene expression by IL-1beta and cAMP in rat mesangial cells, J. Immunol., 160, 4961,
1998.
45. Pfeilschifter, J., Eberhardt, W., Hummel, R., et al., Therapeutic strategies for the inhibition
of inducible nitric oxide synthase: potential for a novel class of anti-inflammatory agents,
Cell Biol. Int., 20, 51, 1996.
46. Shiraishi, A., Dudler, J., and Lotz, M., The role of IFN regulatory factor-1 in synovitis and
nitric oxide production, J. Immunol., 159, 3549, 1997.
47. Mahoney, E., Reichner, J., Robinson Bostom, L., et al., Bacterial colonization and the expres-
sion of inducible nitric oxide synthase (iNOS) in murine wounds, Am. J. Pathol., 161, 2143,
2002.
48. De Maria, R., Cifone, M.G., Trotta, R., et al., Triggering of human monocyte activation through
CD69, a member of the natural killer cell gene complex family of signal transducing receptors,
J. Exp. Med., 180, 1999, 1994.
49. Perrella, M.A., Yoshizumi, M., Fen, Z., et al., Transforming growth factor-beta 1, but not
dexamethasone, down-regulates nitric-oxide synthase mRNA after its induction by interleu-
kin-1 beta in rat smooth muscle cells, J. Biol. Chem., 269, 14595, 1994.
50. Vodovotz, Y., Bogdan, C., Paik, J., et al., Mechanisms of suppression of macrophage nitric
oxide release by transforming growth factor beta, J. Exp. Med., 178, 605, 1993.
51. Albakri, Q.A. and Stuehr, D.J., Intracellular assembly of inducible NO synthase is limited
by nitric oxide-mediated changes in heme insertion and availability, J. Biol. Chem., 271, 5414,
1996.
52. Musial, A. and Eissa, N.T., Inducible nitric-oxide synthase is regulated by the proteasome
degradation pathway, J. Biol. Chem., 276, 24268, 2001.
53. Lander, H.M., Sehajpal, P., Levine, D.M., et al., Activation of human peripheral blood mono-
nuclear cells by nitric oxide-generating compounds, J. Immunol., 150, 1509, 1993.
54. Ziesche, R., Petkov, V., Williams, J., et al., Lipopolysaccharide and interleukin 1 augment the
effects of hypoxia and inflammation in human pulmonary arterial tissue, Proc. Natl. Acad.
Sci. U.S.A., 93, 12478, 1996.
55. Liao, J.K., Zulueta, J.J., Yu, F.S., et al., Regulation of bovine endothelial constitutive nitric
oxide synthase by oxygen, J. Clin. Invest., 96, 2661, 1995.
56. Shaul, P.W., Smart, E.J., Robinson, L.J., et al., Acylation targets endothelial nitric-oxide syn-
thase to plasmalemmal caveolae, J. Biol. Chem., 271, 6518, 1996.
57. Michel, T. and Feron, O., Nitric oxide synthases: which, where, how, and why? J. Clin. Invest.,
100, 2146, 1997.
58. Lamas, S., Michel, T., Brenner, B.M., et al., Nitric oxide synthesis in endothelial cells: evidence
for a pathway inducible by TNF-alpha, Am. J. Physiol., 261, C634, 1991.
59. Nishida, K., Harrison, D.G., Navas, J.P., et al., Molecular cloning and characterization of the
constitutive bovine aortic endothelial cell nitric oxide synthase, J. Clin. Invest., 90, 2092, 1992.
1382_C14.fm Page 231 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 231

60. Lamas, S., Marsden, P.A., Li, G.K., et al., Endothelial nitric oxide synthase: molecular cloning
and characterization of a distinct constitutive enzyme isoform, Proc. Natl. Acad. Sci. U.S.A.,
89, 6348, 1992.
61. Lancaster, J.R., Jr., Simulation of the diffusion and reaction of endogenously produced nitric
oxide, Proc. Natl. Acad. Sci. U.S.A., 91, 8137, 1994.
62. Grisham, M.B., Jourd’Heuil, D., and Wink, D.A., Nitric oxide. I. Physiological chemistry of
nitric oxide and its metabolites: implications in inflammation, Am. J. Physiol., 276, G315, 1999.
63. Brune, B., von Knethen, A., and Sandau, K.B., Nitric oxide and its role in apoptosis, Eur. J.
Pharmacol., 351, 261, 1998.
64. Ignarro, L.J., Biosynthesis and metabolism of endothelium-derived nitric oxide, Annu. Rev.
Pharmacol. Toxicol., 30, 535, 1990.
65. Curran, R.D., Ferrari, F.K., Kispert, P.H., et al., Nitric oxide and nitric oxide-generating
compounds inhibit hepatocyte protein synthesis, FASEB J., 5, 2085, 1991.
66. Kolpakov, V., Gordon, D., and Kulik, T.J., Nitric oxide-generating compounds inhibit total
protein and collagen synthesis in cultured vascular smooth muscle cells, Circ. Res., 76, 305,
1995.
67. Kim, Y.M., Son, K., Hong, S.J., et al., Inhibition of protein synthesis by nitric oxide correlates
with cytostatic activity: nitric oxide induces phosphorylation of initiation factor eIF-2 alpha,
Mol. Med., 4, 179, 1998.
68. Stamler, J.S., Simon, D.I., Osborne, J.A., et al., S-nitrosylation of proteins with nitric oxide:
synthesis and characterization of biologically active compounds, Proc. Natl. Acad. Sci. U.S.A.,
89, 444, 1992.
69. Stamler, J.S., Redox signaling: nitrosylation and related target interactions of nitric oxide,
Cell, 78, 931, 1994.
70. Lander, H.M., Jacovina, A.T., Davis, R.J., et al., Differential activation of mitogen-activated
protein kinases by nitric oxide-related species, J. Biol. Chem., 271, 19705, 1996.
71. Duhe, R.J., Evans, G.A., Erwin, R.A., et al., Nitric oxide and thiol redox regulation of Janus
kinase activity, Proc. Natl. Acad. Sci. U.S.A., 95, 126, 1998.
72. Klann, E., Roberson, E.D., Knapp, L.T., et al., A role for superoxide in protein kinase C
activation and induction of long-term potentiation, J. Biol. Chem., 273, 4516, 1998.
73. Gopalakrishna, R., Chen, Z.H., and Gundimeda, U., Nitric oxide and nitric oxide-generating
agents induce a reversible inactivation of protein kinase C activity and phorbol ester binding,
J. Biol. Chem., 268, 27180, 1993.
74. Simon, D.I., Mullins, M.E., Jia, L., et al., Polynitrosylated proteins: characterization, bioac-
tivity, and functional consequences, Proc. Natl. Acad. Sci. U.S.A., 93, 4736, 1996.
75. Wink, D.A., Cook, J.A., Kim, S.Y., et al., Superoxide modulates the oxidation and nitrosation
of thiols by nitric oxide-derived reactive intermediates: chemical aspects involved in the
balance between oxidative and nitrosative stress, J. Biol. Chem., 272, 11147, 1997.
76. Jaeschke, H., Mechanisms of oxidant stress-induced acute tissue injury, Proc. Soc. Exp. Biol.
Med., 209, 104, 1995.
77. Stamler, J.S., Simon, D.I., Jaraki, O., et al., S-nitrosylation of tissue-type plasminogen activator
confers vasodilatory and antiplatelet properties on the enzyme, Proc. Natl. Acad. Sci. U.S.A.,
89, 8087, 1992.
78. Mateo, R.B., Reichner, J.S., Mastrofrancesco, B., et al., Impact of nitric oxide on macrophage
glucose metabolism and glyceraldehyde-3-phosphate dehydrogenase activity, Am. J. Physiol.,
268, C669, 1995.
79. Bolotina, V.M., Najibi, S., Palacino, J.J., et al., Nitric oxide directly activates calcium-depen-
dent potassium channels in vascular smooth muscle, Nature, 368, 850, 1994.
80. Lipton, S.A., Choi, Y.B., Pan, Z.H., et al., A redox-based mechanism for the neuroprotective
and neurodestructive effects of nitric oxide and related nitroso-compounds, Nature, 364, 626,
1993.
81. De Groote, M.A., Testerman, T., Xu, Y., et al., Homocysteine antagonism of nitric oxide-
related cytostasis in Salmonella typhimurium, Science, 272, 414, 1996.
82. Lander, H.M., Ogiste, J.S., Teng, K.K., et al., p21ras as a common signaling target of reactive
free radicals and cellular redox stress, J. Biol. Chem., 270, 21195, 1995.
1382_C14.fm Page 232 Tuesday, October 7, 2003 6:34 PM

232 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

83. Downward, J., Graves, J.D., Warne, P.H., et al., Stimulation of p21ras upon T-cell activation,
Nature, 346, 719, 1990.
84. Graves, J.D., Downward, J., Rayter, S., et al., CD2 antigen mediated activation of the guanine
nucleotide binding proteins p21ras in human T lymphocytes, J. Immunol., 146, 3709, 1991.
85. Pantopoulos, K. and Hentze, M.W., Nitric oxide signaling to iron-regulatory protein: direct
control of ferritin mRNA translation and transferrin receptor mRNA stability in transfected
fibroblasts, Proc. Natl. Acad. Sci. U.S.A., 92, 1267, 1995.
86. Sen, C.K. and Packer, L., Antioxidant and redox regulation of gene transcription, FASEB J.,
10, 709, 1996.
87. Tabuchi, A., Sano, K., Oh, E., et al., Modulation of AP-1 activity by nitric oxide (NO) in vitro:
NO-mediated modulation of AP-1, FEBS Lett., 351, 123, 1994.
88. Felley-Bosco, E., Ambs, S., Lowenstein, C.J., et al., Constitutive expression of inducible nitric
oxide synthase in human bronchial epithelial cells induces c-fos and stimulates the cGMP
pathway, Am. J. Respir. Cell Mol. Biol., 11, 159, 1994.
89. Pilz, R.B., Suhasini, M., Idriss, S., et al., Nitric oxide and cGMP analogs activate transcription
from AP-1-responsive promoters in mammalian cells, FASEB J., 9, 552, 1995.
90. Siebenlist, U., Franzoso, G., and Brown, K., Structure, regulation and function of NF-kappa
B, Annu. Rev. Cell Biol., 10, 405, 1994.
91. Matthews, J.R., Botting, C.H., Panico, M., et al., Inhibition of NF-kappaB DNA binding by
nitric oxide, Nucleic Acids Res., 24, 2236, 1996.
92. DelaTorre, A., Schroeder, R.A., and Kuo, P.C., Alteration of NF-kappa B p50 DNA binding
kinetics by S-nitrosylation, Biochem. Biophys. Res. Commun., 238, 703, 1997.
93. Chen, F., Kuhn, D.C., Sun, S.C., et al., Dependence and reversal of nitric oxide production
on NF-kappa B in silica and lipopolysaccharide-induced macrophages, Biochem. Biophys. Res.
Commun., 214, 839, 1995.
94. Taylor, B.S., Kim, Y.M., Wang, Q., et al., Nitric oxide down-regulates hepatocyte-inducible
nitric oxide synthase gene expression, Arch. Surg., 132, 1177, 1997.
95. Peng, H.B., Libby, P., and Liao, J.K., Induction and stabilization of I kappa B alpha by nitric
oxide mediates inhibition of NF-kappa B, J. Biol. Chem., 270, 14214, 1995.
96. Kwon, N.S., Stuehr, D.J., and Nathan, C.F., Inhibition of tumor cell ribonucleotide reductase
by macrophage-derived nitric oxide, J. Exp. Med., 174, 761, 1991.
97. Lepoivre, M., Chenais, B., Yapo, A., et al., Alterations of ribonucleotide reductase activity
following induction of the nitrite-generating pathway in adenocarcinoma cells, J. Biol. Chem.,
265, 14143, 1990.
98. Drapier, J.C. and Hibbs, J.B., Jr., Differentiation of murine macrophages to express nonspecific
cytotoxicity for tumor cells results in L-arginine-dependent inhibition of mitochondrial iron-
sulfur enzymes in the macrophage effector cells, J. Immunol., 140, 2829, 1988.
99. Fehsel, K., Kroncke, K.D., Meyer, K.L., et al., Nitric oxide induces apoptosis in mouse
thymocytes, J. Immunol., 155, 2858, 1995.
100. Wink, D.A., Kasprzak, K.S., Maragos, C.M., et al., DNA deaminating ability and genotoxicity
of nitric oxide and its progenitors, Science, 254, 1001, 1991.
101. Rauhala, P., Lin, A.M., and Chiueh, C.C., Neuroprotection by S-nitrosoglutathione of brain
dopamine neurons from oxidative stress, FASEB J., 12, 165, 1998.
102. Kim, Y.M., de Vera, M.E., Watkins, S.C., et al., Nitric oxide protects cultured rat hepatocytes
from tumor necrosis factor-alpha-induced apoptosis by inducing heat shock protein 70
expression, J. Biol. Chem., 272, 1402, 1997.
103. Hausladen, A., Privalle, C.T., Keng, T., et al., Nitrosative stress: activation of the transcription
factor OxyR, Cell, 86, 719, 1996.
104. Loewen, P., Probing the structure of catalase HPII of Escherichia coli: a review, Gene, 179, 39,
1996.
105. Hidalgo, E. and Demple, B., An iron-sulfur center essential for transcriptional activation by
the redox-sensing SoxR protein, EMBO J., 13, 138, 1994.
106. Nunoshiba, T., deRojas-Walker, T., Wishnok, J.S., et al., Activation by nitric oxide of an
oxidative-stress response that defends Escherichia coli against activated macrophages, Proc.
Natl. Acad. Sci. U.S.A., 90, 9993, 1993.
1382_C14.fm Page 233 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 233

107. P.D.R. Nitrostat®, in Physicians’ Desk Reference, 56th ed., Healthcare, T., Ed., Medical Econom-
ics Co., Inc., Montvale, NJ, 2002, p. 2658.
108. Shesely, E.G., Maeda, N., Kim, H.S., et al., Elevated blood pressures in mice lacking endo-
thelial nitric oxide synthase, Proc. Natl. Acad. Sci. U.S.A., 93, 13176, 1996.
109. Steudel, W., Ichinose, F., Huang, P.L., et al., Pulmonary vasoconstriction and hypertension
in mice with targeted disruption of the endothelial nitric oxide synthase (NOS 3) gene, Circ.
Res., 81, 34, 1997.
110. Huang, P.L., Huang, Z., Mashimo, H., et al., Hypertension in mice lacking the gene for
endothelial nitric oxide synthase, Nature, 377, 239, 1995.
111. Kubes, P., Suzuki, M., and Granger, D.N., Nitric oxide: an endogenous modulator of leuko-
cyte adhesion, Proc. Natl. Acad. Sci. U.S.A., 88, 4651, 1991.
112. Ignarro, L.J., Bush, P.A., Buga, G.M., et al., Nitric oxide and cyclic GMP formation upon
electrical field stimulation cause relaxation of corpus cavernosum smooth muscle, Biochem.
Biophys. Res. Commun., 170, 843, 1990.
113. Rajfer, J., Aronson, W.J., Bush, P.A., et al., Nitric oxide as a mediator of relaxation of the
corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission,
N. Engl. J. Med., 326, 90, 1992.
114. P.D.R. Viagra®, in Physician’s Desk Reference, 56th ed., Healthcare, T., Ed., Medical Economics
Co., Inc., Montvale, NJ, 2002, p. 2732.
115. Gaston, B., Drazen, J.M., Loscalzo, J., et al., The biology of nitrogen oxides in the airways,
Am. J. Respir. Crit. Care Med., 149, 538, 1994.
116. Rossaint, R., Gerlach, H., Schmidt-Ruhnke, H., et al., Efficacy of inhaled nitric oxide in
patients with severe ARDS, Chest, 107, 1107, 1995.
117. Abman, S.H., Griebel, J.L., Parker, D.K., et al., Acute effects of inhaled nitric oxide in children
with severe hypoxemic respiratory failure, J. Pediatr., 124, 881, 1994.
118. Rossaint, R., Falke, K.J., Lopez, F., et al., Inhaled nitric oxide for the adult respiratory distress
syndrome, N. Engl. J. Med., 328, 399, 1993.
119. Wilcox, C.S., Welch, W.J., Murad, F., et al., Nitric oxide synthase in macula densa regulates
glomerular capillary pressure, Proc. Natl. Acad. Sci. U.S.A., 89, 11993, 1992.
120. Gaboury, J., Woodman, R.C., Granger, D.N., et al., Nitric oxide prevents leukocyte adherence:
role of superoxide, Am. J. Physiol., 265, H862, 1993.
121. Lefer, D.J., Jones, S.P., Girod, W.G., et al., Leukocyte-endothelial cell interactions in nitric
oxide synthase-deficient mice, Am. J. Physiol., 276, H1943, 1999.
122. De Catarina, R., Libby, P., Peng, H.B., et al., Nitric oxide decreases cytokine-induced endo-
thelial activation: nitric oxide selectively reduces endothelial expression of adhesion mole-
cules and proinflammatory cytokines, J. Clin. Invest., 96, 60, 1995.
123. Gauthier, T.W., Davenpeck, K.L., and Lefer, A.M., Nitric oxide attenuates leukocyte-endo-
thelial interaction via P-selectin in splanchnic ischemia-reperfusion, Am. J. Physiol., 267, G562,
1994.
124. Xie, Q.W., Kashiwabara, Y., and Nathan, C., Role of transcription factor NF-kappa B/Rel in
induction of nitric oxide synthase, J. Biol. Chem., 269, 4705, 1994.
125. Moncada, S., Palmer, R.M., and Higgs, E.A., Nitric oxide: physiology, pathophysiology, and
pharmacology, Pharmacol. Rev., 43, 109, 1991.
126. Radomski, M.W., Palmer, R.M., and Moncada, S., Endogenous nitric oxide inhibits human
platelet adhesion to vascular endothelium, Lancet, 2, 1057, 1987.
127. Geiger, J., Nolte, C., and Walter, U., Regulation of calcium mobilization and entry in human
platelets by endothelium-derived factors, Am. J. Physiol., 267, C236, 1994.
128. Horstrup, K., Jablonka, B., Honig-Liedl, P., et al., Phosphorylation of focal adhesion vaso-
dilator-stimulated phosphoprotein at Ser157 in intact human platelets correlates with fibrin-
ogen receptor inhibition, Eur. J. Biochem., 225, 21, 1994.
129. Radomski, M.W., Palmer, R.M., and Moncada, S., The anti-aggregating properties of vascular
endothelium: interactions between prostacyclin and nitric oxide, Br. J. Pharmacol., 92, 639,
1987.
130. Kubes, P. and Granger, D.N., Nitric oxide modulates microvascular permeability, Am. J.
Physiol., 262, H611, 1992.
1382_C14.fm Page 234 Tuesday, October 7, 2003 6:34 PM

234 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

131. Chin, J.H., Azhar, S., and Hoffman, B.B., Inactivation of endothelial derived relaxing factor
by oxidized lipoproteins, J. Clin. Invest., 89, 10, 1992.
132. Bossaller, C., Habib, G.B., Yamamoto, H., et al., Impaired muscarinic endothelium-dependent
relaxation and cyclic guanosine 5'-monophosphate formation in atherosclerotic human cor-
onary artery and rabbit aorta, J. Clin. Invest., 79, 170, 1987.
133. Tanner, F.C., Noll, G., Boulanger, C.M., et al., Oxidized low density lipoproteins inhibit
relaxations of porcine coronary arteries: role of scavenger receptor and endothelium-derived
nitric oxide, Circulation, 83, 2012, 1991.
134. Aikawa, M., Sugiyama, S., Hill, C.C., et al., Lipid lowering reduces oxidative stress and
endothelial cell activation in rabbit atheroma, Circulation, 106, 1390, 2002.
135. Rodriguez, J.A., Grau, A., Eguinoa, E., et al., Dietary supplementation with vitamins C and
E prevents downregulation of endothelial NOS expression in hypercholesterolemia in vivo
and in vitro, Atherosclerosis, 165, 33, 2002.
136. Paulsen, S.M., Wurster, S.H., and Nanney, L.B., Expression of inducible nitric oxide synthase
in human burn wounds, Wound Repair Regen., 6, 142, 1998.
137. Abd-El-Aleem, S.A., Ferguson, M.W., Appleton, I., et al., Expression of nitric oxide synthase
isoforms and arginase in normal human skin and chronic venous leg ulcers, J. Pathol., 191,
434, 2000.
138. Annane, D., Sanquer, S., Sebille, V., et al., Compartmentalised inducible nitric-oxide synthase
activity in septic shock, Lancet, 355, 1143, 2000.
139. Facchetti, F., Vermi, W., Fiorentini, S., et al., Expression of inducible nitric oxide synthase in
human granulomas and histiocytic reactions, Am. J. Pathol., 154, 145, 1999.
140. Yamasaki, K., Edington, H.D., McClosky, C., et al., Reversal of impaired wound repair in
iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer, J. Clin. Invest., 101,
967, 1998.
141. Most, D., Efron, D.T., Shi, H.P., Tantry, U.S., and Barbul, A., Characterization of incisional
wound healing in inducible nitric oxide synthase knockout mice, Surgery, 132(5), 866–876,
2002.
142. Frank, S., Madlener, M., Pfeilschifter, J., et al., Induction of inducible nitric oxide synthase
and its corresponding tetrahydrobiopterin-cofactor-synthesizing enzyme GTP-cyclohydro-
lase I during cutaneous wound repair, J. Invest. Dermatol., 111, 1058, 1998.
143. Frank, S., Stallmeyer, B., Kampfer, H., et al., Nitric oxide triggers enhanced induction of
vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during
cutaneous wound repair, FASEB J., 13, 2002, 1999.
144. Stallmeyer, B., Kampfer, H., Kolb, N., et al., The function of nitric oxide in wound repair:
inhibition of inducible nitric oxide-synthase severely impairs wound reepithelialization,
J. Invest. Dermatol., 113, 1090, 1999.
145. Weller, R., Pattullo, S., Smith, L., et al., Nitric oxide is generated on the skin surface by
reduction of sweat nitrate, J. Invest. Dermatol., 107, 327, 1996.
146. Witte, M.B., Barbul, A., Schick, M.A., et al., Upregulation of arginase expression in wound-
derived fibroblasts, J. Surg. Res., 105, 35, 2002.
147. Efron, D.T., Most, D., and Barbul, A., Role of nitric oxide in wound healing, Curr. Opin. Clin.
Nutr. Metab. Care, 3, 197, 2000.
148. Albina, J.E. and Henry, W.L., Jr., TGF-b prevents loss of fibroblast viability resulting from
the induction of nitric oxide synthase (NOS) by IFN-g and endotoxin (ENDO) but reduces
proliferation, FASEB J., Part I, 6, A1077, 1992.
149. Thornton, F.J., Ahrendt, G.M., Schaffer, M.R., et al., Sepsis impairs anastomotic collagen gene
expression and synthesis: a possible role for nitric oxide, J. Surg. Res., 69, 81, 1997.
150. Witte, M.B., Thornton, F.J., Efron, D.T., et al., Enhancement of fibroblast collagen synthesis
by nitric oxide, Nitric Oxide, 4, 572, 2000.
151. Efron, D.T., Thornton, F.J., Steulten, C., et al., Expression and function of inducible nitric
oxide synthase during rat colon anastomotic healing, J. Gastrointest. Surg., 3, 592, 1999.
152. Granger, D.N., Cell adhesion and migration. II. Leukocyte-endothelial cell adhesion in the
digestive system, Am. J. Physiol., 273, G982, 1997.
1382_C14.fm Page 235 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 235

153. Granger, D.N. and Kubes, P., The microcirculation and inflammation: modulation of leuko-
cyte-endothelial cell adhesion, J. Leukoc. Biol., 55, 662, 1994.
154. Granger, D.N. and Kubes, P., Nitric oxide as antiinflammatory agent, Meth. Enzymol., 269,
434, 1996.
155. Villarete, L.H. and Remick, D.G., Nitric oxide regulation of IL-8 expression in human endo-
thelial cells, Biochem. Biophys. Res. Commun., 211, 671, 1995.
156. Reichner, J.S., Meszaros, A.J., Louis, C.A., et al., Molecular and metabolic evidence for the
restricted expression of inducible nitric oxide synthase in healing wounds, Am. J. Pathol.,
154, 1097, 1999.
157. Weinberg, R.S., Thomson, J.C., Lao, R., et al., Stem cell factor amplifies newborn and sickle
erythropoiesis in liquid cultures, Blood, 81, 2591, 1993.
158. Reiling, N., Ulmer, A.J., Duchrow, M., et al., Nitric oxide synthase: mRNA expression of
different isoforms in human monocytes/macrophages, Eur. J. Immunol., 24, 1941, 1994.
159. Wheeler, M.A., Smith, S.D., Garcia-Cardena, G., et al., Bacterial infection induces nitric oxide
synthase in human neutrophils, J. Clin. Invest., 99, 110, 1997.
160. Qadoumi, M., Becker, I., Donhauser, N., et al., Expression of inducible nitric oxide synthase
in skin lesions of patients with American cutaneous leishmaniasis, Infect. Immun., 70, 4638,
2002.
161. Weinberg, J.B., Misukonis, M.A., Shami, P.J., et al., Human mononuclear phagocyte inducible
nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric
oxide production by blood monocytes and peritoneal macrophages, Blood, 86, 1184, 1995.
162. Sharara, A.I., Perkins, D.J., Misukonis, M.A., et al., Interferon (IFN)-alpha activation of
human blood mononuclear cells in vitro and in vivo for nitric oxide synthase (NOS) type 2
mRNA and protein expression: possible relationship of induced NOS2 to the anti-hepatitis
C effects of IFN-alpha in vivo, J. Exp. Med., 186, 1495, 1997.
163. St. Clair, E.W., Wilkinson, W.E., Lang, T., et al., Increased expression of blood mononuclear
cell nitric oxide synthase type 2 in rheumatoid arthritis patients, J. Exp. Med., 184, 1173, 1996.
164. Hickman-Davis, J.M., O’Reilly, P., Davis, I.C., et al., Killing of Klebsiella pneumoniae by human
alveolar macrophages, Am. J. Physiol., 282, L944, 2002.
165. Nicholson, S., Bonecini-Almeida Mda, G., Lapa e Silva, J.R., et al., Inducible nitric oxide
synthase in pulmonary alveolar macrophages from patients with tuberculosis, J. Exp. Med.,
183, 2293, 1996.
166. Nozaki, Y., Hasegawa, Y., Ichiyama, S., et al., Mechanism of nitric oxide-dependent killing
of Mycobacterium bovis BCG in human alveolar macrophages, Infect. Immun., 65, 3644, 1997.
167. Anstey, N.M., Weinberg, J.B., Hassanali, M.Y., et al., Nitric oxide in Tanzanian children with
malaria: inverse relationship between malaria severity and nitric oxide production/nitric
oxide synthase type 2 expression, J. Exp. Med., 184, 557, 1996.
168. Weinberg, J.B., Nitric oxide production and nitric oxide synthase type 2 expression by human
mononuclear phagocytes: a review, Mol. Med., 4, 557, 1998.
169. Long, R., Light, B., and Talbot, J.A., Mycobacteriocidal action of exogenous nitric oxide,
Antimicrob. Agents Chemother., 43, 403, 1999.
170. Kaplan, S.S., Lancaster, J.R., Jr., Basford, R.E., et al., Effect of nitric oxide on staphylococcal
killing and interactive effect with superoxide, Infect. Immun., 64, 69, 1996.
171. Vouldoukis, I., Riveros-Moreno, V., Dugas, B., et al., The killing of Leishmania major by human
macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23
surface antigen, Proc. Natl. Acad. Sci. U.S.A., 92, 7804, 1995.
172. Hoehn, T., Huebner, J., Paboura, E., et al., Effect of therapeutic concentrations of nitric oxide
on bacterial growth in vitro, Crit. Care Med., 26, 1857, 1998.
173. Assreuy, J., Cunha, F.Q., Epperlein, M., et al., Production of nitric oxide and superoxide by
activated macrophages and killing of Leishmania major, Eur. J. Immunol., 24, 672, 1994.
174. Pacelli, R., Wink, D.A., Cook, J.A., et al., Nitric oxide potentiates hydrogen peroxide-induced
killing of Escherichia coli, J. Exp. Med., 182, 1469, 1995.
175. De Groote, M.A., Granger, D., Xu, Y., et al., Genetic and redox determinants of nitric oxide
cytotoxicity in a Salmonella typhimurium model, Proc. Natl. Acad. Sci. U.S.A., 92, 6399, 1995.
1382_C14.fm Page 236 Tuesday, October 7, 2003 6:34 PM

236 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

176. Brunelli, L., Crow, J.P., and Beckman, J.S., The comparative toxicity of nitric oxide and
peroxynitrite to Escherichia coli, Arch. Biochem. Biophys., 316, 327, 1995.
177. De Groote, M.A. and Fang, F.C., NO inhibitions: antimicrobial properties of nitric oxide, Clin.
Infect. Dis., 21, S162, 1995.
178. Ding, A.H., Nathan, C.F., and Stuehr, D.J., Release of reactive nitrogen intermediates and
reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating
cytokines and evidence for independent production, J. Immunol., 141, 2407, 1988.
179. Mulsch, A., NO synthases: mechanism of activation, identity of NOX and expression in
human cells, Res. Immunol., 142, 561, 1991.
180. Stuehr, D.J. and Marletta, M.A., Induction of nitrite/nitrate synthesis in murine macrophages
by BCG infectin, lymphokines, or interferon-gamma, J. Immunol., 139, 518, 1987.
181. Granger, D.L., Hibbs, J.B., Jr., Perfect, J.R., et al., Metabolic fate of L-arginine in relation to
microbiostatic capability of murine macrophages, J. Clin. Invest., 85, 264, 1990.
182. Stenger, S., Donhauser, N., Thuring, H., et al., Reactivation of latent leishmaniasis by inhi-
bition of inducible nitric oxide synthase, J. Exp. Med., 183, 1501, 1996.
183. Tavares-Murta, B.M., Machado, J.S., Ferreira, S.H., et al., Nitric oxide mediates the inhibition
of neutrophil migration induced by systemic administration of LPS, Inflammation, 25, 247,
2001.
184. Van Dervort, A.L., Yan, L., Madara, P.J., et al., Nitric oxide regulates endotoxin-induced TNF-
alpha production by human neutrophils, J. Immunol., 152, 4102, 1994.
185. Clancy, R.M., Leszczynska-Piziak, J., and Abramson, S.B., Nitric oxide, an endothelial cell
relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the
NADPH oxidase, J. Clin. Invest., 90, 1116, 1992.
186. Krueger, M.R., Tames, D.R., and Mariano, M., Expression of NO-synthase in cells of foreign-
body and BCG-induced granulomata in mice: influence of L-NAME on the evolution of the
lesion, Immunology, 95, 278, 1998.
187. Salter, M., Knowles, R.G., and Moncada, S., Widespread tissue distribution, species distri-
bution and changes in activity of Ca(2+)-dependent and Ca(2+)-independent nitric oxide
synthases, FEBS Lett., 291, 145, 1991.
188. Curran, R.D., Billiar, T.R., Stuehr, D.J., et al., Multiple cytokines are required to induce
hepatocyte nitric oxide production and inhibit total protein synthesis, Ann. Surg., 212, 462,
1990.
189. Billiar, T.R., Curran, R.D., Stuehr, D.J., et al., Inducible cytosolic enzyme activity for the
production of nitrogen oxides from L-arginine in hepatocytes, Biochem. Biophys. Res. Com-
mun., 168, 1034, 1990.
190. Garcia, Y.R., May, J.J., Green, A.M., et al., Acetylcholine receptor-reactive antibody induces
nitric oxide production by a rat skeletal muscle cell line: influence of cytokine environment,
J. Neuroimmunol., 120, 103, 2001.
191. Ziolo, M.T., Katoh, H., and Bers, D.M., Expression of inducible nitric oxide synthase depresses
beta-adrenergic-stimulated calcium release from the sarcoplasmic reticulum in intact ven-
tricular myocytes, Circulation, 104, 2961, 2001.
192. Fleming, I., Gray, G.A., Schott, C., et al., Inducible but not constitutive production of nitric
oxide by vascular smooth muscle cells, Eur. J. Pharmacol., 200, 375, 1991.
193. Ochoa, J.B., Udekwu, A.O., Billiar, T.R., et al., Nitrogen oxide levels in patients after trauma
and during sepsis, Ann. Surg., 214, 621, 1991.
194. Thiemermann, C. and Vane, J., Inhibition of nitric oxide synthesis reduces the hypotension
induced by bacterial lipopolysaccharides in the rat in vivo, Eur. J. Pharmacol., 182, 591, 1990.
195. Meyer, J., Traber, L.D., Nelson, S., et al., Reversal of hyperdynamic response to continuous
endotoxin administration by inhibition of NO synthesis, J. Appl. Physiol., 73, 324, 1992.
196. Avontuur, J.A., Tutein Nolthenius, R.P., Buijk, S.L., et al., Effect of L-NAME, an inhibitor of
nitric oxide synthesis, on cardiopulmonary function in human septic shock, Chest, 113, 1640,
1998.
197. Broccard, A., Hurni, J.M., Eckert, P., et al., Tissue oxygenation and hemodynamic response
to NO synthase inhibition in septic shock, Shock, 14, 35, 2000.
1382_C14.fm Page 237 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 237

198. Bonfoco, E., Krainc, D., Ankarcrona, M., et al., Apoptosis and necrosis: two distinct events
induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric ox-
ide/superoxide in cortical cell cultures, Proc. Natl. Acad. Sci. U.S.A., 92, 7162, 1995.
199. Sandau, K., Pfeilschifter, J., and Brune, B., The balance between nitric oxide and superoxide
determines apoptotic and necrotic death of rat mesangial cells, J. Immunol., 158, 4938, 1997.
200. Ankarcrona, M., Dypbukt, J.M., Brune, B., et al., Interleukin-1 beta-induced nitric oxide
production activates apoptosis in pancreatic RINm5F cells, Exp. Cell Res., 213, 172, 1994.
201. Kaneto, H., Fujii, J., Seo, H.G., et al., Apoptotic cell death death triggered by nitric oxide in
pancreatic beta-cells, Diabetes, 44, 733, 1995.
202. Liebermann, D.A., Hoffman, B., and Steinman, R.A., Molecular controls of growth arrest and
apoptosis: p53-dependent and independent pathways, Oncogene, 11, 199, 1995.
203. Messmer, U.K. and Brune, B., Nitric oxide-induced apoptosis: p53-dependent and p53-
independent signalling pathways, Biochem. J., 319, 299, 1996.
204. Yabuki, M., Kariya, S., Inai, Y., et al., Molecular mechanisms of apoptosis in HL-60 cells
induced by a nitric oxide-releasing compound, Free Radic. Res., 27, 325, 1997.
205. Sandau, K., Pfeilschifter, J., and Brune, B., Nitrosative and oxidative stress induced heme
oxygenase-1 accumulation in rat mesangial cells, Eur. J. Pharmacol., 342, 77, 1998.
206. Messmer, U.K., Reimer, D.M., Reed, J.C., et al., Nitric oxide induced poly(ADP-ribose) poly-
merase cleavage in RAW 264.7 macrophage apoptosis is blocked by Bcl-2, FEBS Lett., 384,
162, 1996.
207. Brockhaus, F. and Brune, B., U937 apoptotic cell death by nitric oxide: Bcl-2 downregulation
and caspase activation, Exp. Cell Res., 238, 33, 1998.
208. Mannick, J.B., Asano, K., Izumi, K., et al., Nitric oxide produced by human B lymphocytes
inhibits apoptosis and Epstein-Barr virus reactivation, Cell, 79, 1137, 1994.
209. Genaro, A.M., Hortelano, S., Alvarez, A., et al., Splenic B lymphocyte programmed cell death
is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels,
J. Clin. Invest., 95, 1884, 1995.
210. Beauvais, F., Michel, L., and Dubertret, L., The nitric oxide donors, azide and hydroxylamine,
inhibit the programmed cell death of cytokine-deprived human eosinophils, FEBS Lett., 361,
229, 1995.
211. Chun, S.Y., Eisenhauer, K.M., Kubo, M., et al., Interleukin-1 beta suppresses apoptosis in rat
ovarian follicles by increasing nitric oxide production, Endocrinology, 136, 3120, 1995.
212. Cheng, W., Li, B., Kajstura, J., et al., Stretch-induced programmed myocyte cell death, J. Clin.
Invest., 96, 2247, 1995.
213. Dimmeler, S., Haendeler, J., Nehls, M., et al., Suppression of apoptosis by nitric oxide via
inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein
(CPP)-32-like proteases, J. Exp. Med., 185, 601, 1997.
214. Brune, B., Golkel, C., and von Knethen, A., Cytokine and low-level nitric oxide prestimulation
block p53 accumulation and apoptosis of RAW 264.7 macrophages, Biochem. Biophys. Res.
Commun., 229, 396, 1996.
215. von Knethen, A. and Brune, B., Cyclooxygenase-2: an essential regulator of NO-mediated
apoptosis, FASEB J., 11, 887, 1997.
216. Cui, S., Reichner, J.S., Mateo, R.B., et al., Activated murine macrophages induce apoptosis
in tumor cells through nitric oxide-dependent or -independent mechanisms, Cancer Res., 54,
2462, 1994.
217. Yim, C.Y., Batian, N.R., Smith, J.C., et al., Macrophage nitric oxide synthesis delays progres-
sion of ultraviolet light-induced murine skin cancers, Cancer Res., 53, 5507, 1993.
218. Hung, K., Hayashi, R., Lafond-Walker, A., et al., The central role of CD4(+) T cells in the
antitumor immune response, J. Exp. Med., 188, 2357, 1998.
219. DiNapoli, M.R., Calderon, C.L., and Lopez, D.M., The altered tumoricidal capacity of macro-
phages isolated from tumor-bearing mice is related to reduced expression of the inducible
nitric oxide synthase gene, J. Exp. Med., 183, 1323, 1996.
220. Pervin, S., Singh, R., and Chaudhuri, G., Nitric oxide-induced cytostasis and cell cycle arrest
of a human breast cancer cell line (MDA-MB-231): potential role of cyclin D1, Proc. Natl.
Acad. Sci. U.S.A., 98, 3583, 2001.
1382_C14.fm Page 238 Tuesday, October 7, 2003 6:34 PM

238 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

221. Forstermann, U., Gorsky, L.D., Pollock, J.S., et al., Hormone-induced biosynthesis of endo-
thelium-derived relaxing factor/nitric oxide-like material in N1E-115 neuroblastoma cells
requires calcium and calmodulin, Mol. Pharmacol., 38, 7, 1990.
222. Radomski, M.W., Jenkins, D.C., Holmes, L., et al., Human colorectal adenocarcinoma cells:
differential nitric oxide synthesis determines their ability to aggregate platelets, Cancer Res.,
51, 6073, 1991.
223. Fast, D.J., Lynch, R.C., and Leu, R.W., Nitric oxide production by tumor targets in response
to TNF: paradoxical correlation with susceptibility to TNF-mediated cytotoxicity without
direct involvement in the cytotoxic mechanism, J. Leukoc. Biol., 52, 255, 1992.
224. Xie, K., Huang, S., Dong, Z., et al., Direct correlation between expression of endogenous
inducible nitric oxide synthase and regression of M5076 reticulum cell sarcoma hepatic
metastases in mice treated with liposomes containing lipopeptide CGP 31362, Cancer Res.,
55, 3123, 1995.
225. Xie, K., Huang, S., Dong, Z., et al., Transfection with the inducible nitric oxide synthase gene
suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells,
J. Exp. Med., 181, 1333, 1995.
226. Son, H., Hawkins, R.D., Martin, K., et al., Long-term potentiation is reduced in mice that are
doubly mutant in endothelial and neuronal nitric oxide synthase, Cell, 87, 1015, 1996.
227. Xie, K., Dong, Z., and Fidler, I.J., Activation of nitric oxide synthase gene for inhibition of
cancer metastasis, J. Leukoc. Biol., 59, 797, 1996.
228. Dong, Z., Staroselsky, A.H., Qi, X., et al., Inverse correlation between expression of inducible
nitric oxide synthase activity and production of metastasis in K-1735 murine melanoma cells,
Cancer Res., 54, 789, 1994.
229. Chin, K., Kurashima, Y., Ogura, T., et al., Induction of vascular endothelial growth factor by
nitric oxide in human glioblastoma and hepatocellular carcinoma cells, Oncogene, 15, 437,
1997.
230. Xu, W., Liu, L., Smith, G.C., et al., Nitric oxide upregulates expression of DNA-PKcs to
protect cells from DNA-damaging anti-tumor agents, Nat. Cell Biol., 2, 339, 2000.
231. Albina, J.E., Martin, B.-A., Henry, W.L., Jr., et al., B cell lymphoma-2 transfected P815 cells
resist reactive nitrogen intermediate-mediated macrophage-dependent cytotoxicity, J. Immu-
nol., 157, 279, 1996.
232. Torigoe, T., Millan, J.A., Takayama, S., et al., Bcl-2 inhibits T-cell-mediated cytolysis of a
leukemia cell line, Cancer Res., 54, 4851, 1994.
233. Calderon, C., Huang, Z.H., Gage, D.A., et al., Isolation of a nitric oxide inhibitor from
mammary tumor cells and its characterization as phosphatidyl serine, J. Exp. Med., 180, 945,
1994.
234. Mills, C.D., Shearer, J., Evans, R., et al., Macrophage arginine metabolism and the inhibition
or stimulation of cancer, J. Immunol., 149, 2709, 1992.
235. Wiggington, J.M., Kuhns, D.B., Back, T.C., et al., Interleukin 12 primes macrophages for nitric
oxide production in vivo and restores depressed nitric oxide production by macrophages
from tumor-bearing mice: implications for the antitumor activity of interleukin 12 and/or
interleukin 2, Cancer Res., 56, 1131, 1996.
236. DiNapoli, M.R., Calderon, C.L., and Lopez, D.M., Phosphatidyl serine is involved in the
reduced rate of transcription of the inducible nitric oxide synthase gene in macrophages
from tumor-bearing mice, J. Immunol., 158, 1810, 1997.
237. Albina, J.E. and Mastrofrancesco, B., Modulation of glucose metabolism in macrophages by
products of nitric oxide synthase, Am. J. Physiol., 264, C1594, 1993.
238. Drapier, J.C. and Hibbs, J.B., Jr., Murine cytotoxic activated macrophages inhibit aconitase
in tumor cells: inhibition involves the iron-sulfur prosthetic group and is reversible, J. Clin.
Invest., 78, 790, 1986.
239. Hibbs, J.B., Jr., Taintor, R.R., Vavrin, Z., et al., Synthesis of nitric oxide from a terminal
guanidino nitrogen atom of L-arginine: a molecular mechanism regulating cellular prolifer-
ation that targets intracellular iron, in Nitric Oxide from L-Arginine: A Bioregulatory System,
Moncada, S. and Higgs, E.A., Eds., Elsevier Science Publishers B.V., Amsterdam, 1990, p. 189.
1382_C14.fm Page 239 Tuesday, October 7, 2003 6:34 PM

Chapter fourteen: Nitric oxide 239

240. Bedard, S., Marcotte, B., and Marette, A., Cytokines modulate glucose transport in skeletal
muscle by inducing the expression of inducible nitric oxide synthase, Biochem. J., 325, 487,
1997.
241. Albina, J.E., Mastrofrancesco, B., and Reichner, J.S., NO induces acyl phosphatase activity
in glyceraldehyde-3-phosphate dehydrogenase (GAPDH): a mechanism for uncoupling gly-
colysis from ATP synthesis in cells producing NO, Acta Physiol. Scand., 167 (Suppl. 645), 177,
1999.
242. Mohr, S., Stamler, J.S., and Brune, B., Mechanism of covalent modification of glyceraldehyde-
3-phosphate dehydrogenase at its active site thiol by nitric oxide, peroxynitrite and related
nitrosating agents, FEBS Lett., 348, 223, 1994.
243. Mohr, S., Stamler, J.S., and Brune, B., Posttranslational modification of glyceraldehyde-3-
phosphate dehydrogenase by S-nitrosylation and subsequent NADH attachment, J. Biol.
Chem., 271, 4209, 1996.
244. Hess, D.T., Patterson, S.I., Smith, D.S., et al., Neuronal growth cone collapse and inhibition
of protein fatty acylation by nitric oxide, Nature, 366, 562, 1993.
245. Bliss, T.V. and Collingridge, G.L., A synaptic model of memory: long-term potentiation in
the hippocampus, Nature, 361, 31, 1993.
246. Hawkins, R.D., Kandel, E.R., and Siegelbaum, S.A., Learning to modulate transmitter release:
themes and variations in synaptic plasticity, Annu. Rev. Neurosci., 16, 625, 1993.
247. O’Dell, T.J., Hawkins, R.D., Kandel, E.R., et al., Tests of the roles of two diffusible substances
in long-term potentiation: evidence for nitric oxide as a possible early retrograde messenger,
Proc. Natl. Acad. Sci. U.S.A., 88, 11285, 1991.
248. Mereu, G., Cammalleri, M., Fa, M., et al., Prenatal exposure to a low concentration of carbon
monoxide disrupts hippocampal long-term potentiation in rat offspring, J. Pharmacol. Exp.
Ther., 294, 728, 2000.
249. White, K.A. and Marletta, M.A., Nitric oxide synthase is a cytochrome P-450 type hemopro-
tein, Biochemistry, 31, 6627, 1992.
250. Chetty, C.S., Reddy, G.R., Murthy, K.S., et al., Perinatal lead exposure alters the expression
of neuronal nitric oxide synthase in rat brain, Int. J. Toxicol., 20, 113, 2001.
251. Malinski, T., Bailey, F., Zhang, Z.G., et al., Nitric oxide measured by a porphyrinic micro-
sensor in rat brain after transient middle cerebral artery occlusion, J. Cereb. Blood Flow Metab.,
13, 355, 1993.
252. Dawson, T.M., Dawson, V.L., and Snyder, S.H., A novel neuronal messenger molecule in
brain: the free radical, nitric oxide, Ann. Neurol., 32, 297, 1992.
253. Huang, Z., Huang, P.L., Panahian, N., et al., Effects of cerebral ischemia in mice deficient in
neuronal nitric oxide synthase, Science, 265, 1883, 1994.
254. Kobari, M., Fukuuchi, Y., Tomita, M., et al., Role of nitric oxide in regulation of cerebral
microvascular tone and autoregulation of cerebral blood flow in cats, Brain Res., 667, 255,
1994.
255. Mohanakumar, K.P., Hanbauer, I., and Chiueh, C.C., Neuroprotection by nitric oxide against
hydroxyl radical-induced nigral neurotoxicity, J. Chem. Neuroanat., 14, 195, 1998.
256. Pfeilschifter, J., Eberhardt, W., and Beck, K.F., Regulation of gene expression by nitric oxide,
Eur. J. Physiol., 442, 479, 2001.
1382_C14.fm Page 240 Tuesday, October 7, 2003 6:34 PM
1382_C15.fm Page 241 Tuesday, October 7, 2003 6:38 PM

chapter fifteen

Control of amino acid metabolism


by lipids, ketone bodies, and
glucose substrates
Yves Boirie
Centre de Recherche en Nutrition Humaine
Stéphane Walrand
Centre de Recherche en Nutrition Humaine
Bernard Beaufrère*
Centre de Recherche en Nutrition Humaine

Contents
Introduction..................................................................................................................................241
15.1 Sparing actions of glucose and fat on protein metabolism........................................242
15.2 Mechanisms of nitrogen sparing by glucose and fat ..................................................243
15.2.1 Glucose ...................................................................................................................243
15.2.2 Lipids ......................................................................................................................244
15.3 Effects of the fatty acid chain length..............................................................................245
15.3.1 Medium-chain triglycerides ................................................................................245
15.3.2 Structured MCT.....................................................................................................245
15.3.3 Other lipids ............................................................................................................246
15.4 Nitrogen-sparing effects of ketone bodies ....................................................................246
15.5 Conclusion ..........................................................................................................................247
References .....................................................................................................................................247

Introduction
Protein turnover and energy metabolism are closely related in all species and in numerous
physiological and pathological conditions. Protein synthesis and, to a lesser extent, protein
breakdown are energy-requiring processes, and the strong relationship between energy
and protein metabolism has been the subject of extensive reviews.1–4 Schematically, for a

* In memoriam.

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 241
1382_C15.fm Page 242 Tuesday, October 7, 2003 6:38 PM

242 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

given nitrogen intake, a limited energy supply decreases nitrogen balance, while an excess
of energy above the maintenance level increases it, at least within certain limits. Therefore,
it can be said that any energy source is nitrogen sparing with a rough proportionality
between the level of energy intake and nitrogen sparing. However, it is still controversial
whether this effect depends on the source of energy administered, i.e., carbohydrates
(CHO) or fat. Moreover, the mechanisms for any sparing effect of nonprotein energy
substrates are still unknown. Therefore, this review will focus on the effects of glucose
and various fat sources, including medium-chain triglycerides and ketone bodies, on
amino acid and protein kinetics. This issue is of importance for the underlying mechanisms
of relative protein sparing observed during prolonged fasting and for the choice of fat
and glucose intakes in order to optimize postprandial protein synthesis.

15.1 Sparing actions of glucose and fat on protein metabolism


According to earlier studies by Cathcart5 and Munro,6 administration of CHO during
fasting in man reduces nitrogen excretion, while substituting CHO by an isocaloric amount
of fat induces a transient rise of nitrogen excretion, thus further decreasing nitrogen
balance. This specific effect of CHO was later confirmed by Richardson et al.7 in a study
comparing two isocaloric diets, rich in either fat (fat:CHO ratio = 1:1) or CHO (CHO:fat
ratio = 2:1). The nitrogen balance was higher during the high CHO diet, but this was true
only for the subjects who were probably below their energy (and possibly protein) require-
ments, as indicated by a weight loss during the experiment. Actually, for the subjects
receiving normal energy intakes, there was no difference between the two diets, with even
a trend toward a higher protein-sparing effect of the high-fat diet. Vazquez et al.8 also
demonstrated in short-term-fasting obese subjects (1 week) that low amounts of CHO
reduced nitrogen excretion and leucine oxidation (which was increased by fasting), while
fat had no effect. In a further study,9 the same group reported similar findings in obese
subjects receiving for 4 weeks a very low caloric diet, which was either ketogenic (i.e.,
low in CHO) or nonketogenic (i.e., higher in CHO), this latter diet inducing a higher
nitrogen balance. Finally, this was also found in rats receiving an adequate energy and a
low-protein intake by total parenteral nutrition.10 Taken as a whole, these data indicate
that CHO have a better nitrogen-sparing effect than fat, particularly under circumstances
of limited energy supply.
This classic view may be challenged on various grounds. First, during fasting in obese
patients, there is an inverse relationship between the urinary nitrogen excretion (expressed
in many studies as the P ratio, i.e., fasting urinary nitrogen loss/fasting metabolic rate)
and the level of adiposity, both in humans and in animal models, suggesting a protein-
sparing effect of lipids (see reviews in Elia11 and Henry12). Second, a study by McCargar
et al.13 was unable to reproduce the results of Richardson et al.7 In healthy subjects receiving
75% of their maintenance energy intake, nitrogen balances were identical whether the
energy was provided mostly by fat or CHO (CHO:fat ratio = 2:1 or 1:1), and at maintenance
energy intake, the protein-sparing action of the fat diet was higher than that of CHO.
There is no clear explanation for the different results obtained in these two otherwise
similar studies. Results similar to those of McCargar et al.13 were also reported in rats.14
After a single meal of variable fat and CHO content, Flatt et al.15 observed similar nitrogen
excretion, but the meals were not isocaloric. Third, this problem was also extensively
evaluated during total parenteral nutrition in adults and children. Basically, all these
studies compared the effects of isocaloric amounts of glucose and lipids — given as long-
chain triglycerides (LCT) — administered at or above maintenance levels, on protein
metabolism. In a classic study, Jeejeebhoy et al.16 compared the effects of the so-called
glucose system (i.e., glucose alone) and lipid system (i.e., 83% of LCT) in malnourished
1382_C15.fm Page 243 Tuesday, October 7, 2003 6:38 PM

Chapter fifteen: Control of amino acid metabolism 243

patients to an equivalent nitrogen-sparing effect of these two substrates. Numerous similar


data were obtained in adults17–20 and children21,22 using various fat:CHO ratios. The only
study23 showing a better efficiency (i.e., lower nitrogen excretion) of glucose suffers from
various methodological problems: the energy intake was variable and the nitrogen bal-
ances were of very short duration. Many of the trials were conducted in patients presenting
evolutive infections or an inflammatory state, these two factors potentially resulting in
profound modifications of protein metabolism, which may mask a specific effect of glucose
or lipid. In addition, the glucose intake was often quite high, particularly in the studies
performed in newborns,21,22,24 and often above the maximal oxidative capacity for glucose.
Consequently, fat oxidation was certainly low and a possible specific effect of fat on protein
metabolism was unlikely to appear in these studies. Finally, more recent studies25–27 dem-
onstrated that a fat–glucose regimen spares more nitrogen than glucose alone. Of particular
interest is a study conducted in well-nourished nonseptic children under long-term total
parenteral nutrition (TPN),27 showing a better nitrogen balance, a lower leucine oxidation,
and a lower protein breakdown with a fat–glucose mixture than with glucose alone,
glucose being administered at a rather low level.
In summary, it is difficult to draw definitive conclusions from the available data and
further studies are warranted on this matter. According to most studies in man, it would
seem that at energy intakes at or above maintenance levels, fat and CHO exert a nitrogen-
sparing effect that is at least equivalent and possibly better for fat. On the contrary, at low
energy levels, glucose could be more efficient than fat, despite discrepancies in results.7,13

15.2 Mechanisms of nitrogen sparing by glucose and fat


15.2.1 Glucose
The effects of glucose on amino acid metabolism are difficult to assess since intravenous
glucose or oral CHO administration result in numerous metabolic and hormonal modifi-
cations. Undoubtedly, a glucose infusion results in a decreased whole-body protein break-
down,28 but also induces an increased insulin secretion, a potent anabolic hormone. The
mechanism of insulin action on protein metabolism in vivo is beyond the scope of this
review, but there is general agreement that insulin reduces protein breakdown and amino
acid oxidation, while its stimulating effect on protein synthesis remains debated in adults.3
Physiopathological models such as insulin-dependent diabetes29 do not clarify the situa-
tion since, in this case, hyperglycemia is associated with profound hypoinsulinemia result-
ing in increased protein breakdown and amino acid oxidation. A study30 in which the
effects of hyperglycemia and insulin were separated by means of a hyperglycemic clamp
at different insulin levels has indicated that whole-body protein breakdown was unaf-
fected by plasma glucose level, suggesting an absence of effect of glucose per se on protein
breakdown. Amino acid oxidation (and estimates of whole-body protein synthesis) was
not measured in this study. In a recent work, Ebeling et al.31 have reported that branched-
chain amino acid concentrations were increased in plasma and muscles after CHO deple-
tion induced by exercise as a sign of increased protein breakdown. This work raises the
question about the relationship between glycogen stores or intramuscular glucose avail-
ability and amino acid utilization for oxidation or protein synthesis. The effect of a con-
tinuous infusion of amino acids with or without a concomitant administration of glucose
and long-chain triglycerides on the amino acid balance across the leg and the arm has
also been examined in healthy humans.32 Amino acid balance was negative in the fasted
state but reached a positive balance during amino acid infusion. Surprisingly, this effect
was not dependent on any contribution from glucose and lipids. These authors postulated
1382_C15.fm Page 244 Tuesday, October 7, 2003 6:38 PM

244 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

that only extracellular amino acid levels may determine amino acid balance across tissues
independently of nonprotein calories.32
By contrast, in vitro studies demonstrate an effect of glucose alone on protein metab-
olism. Glucose stimulated protein synthesis in incubated rat skeletal muscle.33 When
glucose was removed from the medium, leucine oxidation was accelerated, suggesting a
substrate competition at the mitochondrial level between glucose and amino acids.34
Glucose also had an inhibitory effect on protein degradation measured as the tyrosine
release in the medium.35 It is, however, difficult to compare in vitro with in vivo data,
particularly with respect to the amount of energy available for the tissues, and in summary,
the effect of glucose on protein sparing in vivo is certainly mostly mediated by insulin.
An emerging aspect of the glucose-sparing effect on protein metabolism is related to
its action in the digestive tract. Indeed, when CHO are administered concomitantly in a
meal, exogenous amino acids entering the body will be differently distributed. A recent
study from Mariotti et al.36 has indicated that sucrose addition reduced early deamination
of dietary nitrogen and reduced endogenous nitrogen oxidation 4 h after meal intake.
Thus, the metabolic fate of dietary nitrogen in the postprandial period is modulated by
CHO. Other recent observations from our group have used a different approach to analyze
the postprandial protein anabolism from intrinsically dietary milk proteins added to
maltodextrine.37 Preliminary results indicated an effect of CHO on the absorption rate of
proteins, which is an important determinant of postprandial protein balance in humans.38,39
Therefore, metabolic but also digestive actions of glucose on protein utilization are to be
considered.

15.2.2 Lipids
By contrast with glucose, lipids are unlikely to act through a hormonal modification.
Although high free fatty acids (FA) levels (>3 mM) obtained by infusing LCT and heparin
resulted in moderate increase in plasma insulin,40 most studies in humans and animals
did not show any significant hormonal modifications.41–43 Therefore, a direct effect of free
fatty acids on protein metabolism should be considered. An elegant study by Tessari et
al.42 demonstrated an inverse relationship between leucine oxidation and free fatty acids
levels (and therefore free fatty acid oxidation) in dogs. In humans, a moderate increase of
free fatty acids (~1 mM) also resulted in a 20% reduction of leucine oxidation.41
The mechanism of the FA sparing was mostly studied for the branched amino acid
oxidation. Long-chain FA inhibit the branched-chain keto acid dehydrogenase (BCKADH)
for some,44,45 if not all,34,46 authors. It is probably not the FA themselves that regulate
BCKADH but the products of their b-oxidation, possibly through the generation of a high
NADH2/NAD+ ratio.47 This is achieved during a parenteral infusion of lipids, resulting
in high FA levels. During oral intake, plasma FA are low (due to the simultaneous intake
of CHO and to hyperinsulinemia), but FA oxidation is still increased,13 although this was
not reported by all authors.14 A switch from a high-fat to a low-fat diet in adult rats led
to a lower branched-chain amino acid dehydrogenase activity, revealing a reduction in
branched-chain amino acid degradation.48 This regulation of amino acid oxidation by FA
oxidation is part of the general concept of substrate competition for mitochondrial oxida-
tion, initially suggested by Krebs49 and then by Randle et al.50 (for glucose and FA). The
effects of the level of saturation of FA are largely unexplored, and those of the FA chain
length will be discussed below.
A last possible mechanism of action for FA is a direct effect on protein synthesis or
breakdown. Inhibition of protein breakdown by high FA levels was reported in dogs,42
but data in humans are conflicting.41,51 Conversely, lipolysis inhibition that induced low
FA concentrations stimulated muscle protein breakdown and reduced muscle protein
1382_C15.fm Page 245 Tuesday, October 7, 2003 6:38 PM

Chapter fifteen: Control of amino acid metabolism 245

synthesis in dogs.52 The interpretation of these changes is difficult, and protein synthesis
changes might be due only to a decreased amino acid availability resulting from their
increased oxidation. As it has been previously suggested for glucose when it was added
to a protein meal, preliminary results indicate that adding lipids to a protein meal may
have a direct action through proteolysis modulation and an indirect effect by changing
postprandial protein absorption from the digestive tract.37

15.3 Effects of the fatty acid chain length


Only long-chain FA are normally present in the organism in significant amounts. However,
LCT infusions used in parenteral nutrition can induce side effects such as excessive fat
deposition and alterations of the reticuloendothelial system.53 Therefore, alternative
sources have been proposed, medium-chain triglycerides (MCT) being the most widely
used. Their specific effects on protein metabolism will be discussed below, while other
characteristics have been the subject of a review.54

15.3.1 Medium-chain triglycerides


MCT are made of glycerol and medium-chain FA (8 to 10 carbons). They are more com-
pletely and more rapidly oxidized55 than LCT, and therefore potentially more nitrogen
sparing according to the hypothesis described above (Section 15.2.1).
Numerous clinical studies have compared nitrogen balances in patients suffering from
various diseases who were infused with LCT or physical mixtures of MCT and LCT (MCT
cannot be infused alone, due to a potential neurologic toxicity and to the essential FA
requirements). Results of these trials are conflicting, showing either a better balance with
MCT56–60 or no difference at all60–65; some of the discrepanies in these results were obtained
by the same authors. Similar opposite results were obtained in animal models, nitrogen
balances being better,66 equal,67,68 or worse69 with MCT than with LCT. Therefore, from a
clinical point of view, it is likely that the nitrogen-sparing effect of MCT is at least equiv-
alent to that of LCT. By contrast, in vivo amino acid kinetics and in vitro studies demonstrate
a deleterious action of MCT on branched-chain amino acid oxidation. This was first
demonstrated in dogs70 and then confirmed in normal humans: the decreased leucine
oxidation observed with LCT was annihilated by the addition of MCT to LCT (both
triglyceride intakes being isocaloric).41 Additionaly, the influence of the chain length of
FA on whole-body leucine kinetic in healthy subjects has been recently reported.51 Whole-
body protein synthesis and breakdown decreased during MCT administration, whereas
LCT decreased leucine oxidation. This protein-sparing effect of LCT appeared to be dis-
sociated from FA effects on glucose metabolism; both MCT and LCT diminished insulin
ability to increase glucose disappearance and to decrease glucose production.51 In vitro
results also demonstrated that octanoate (C8 FA) activates BCKADH34,45,71,72 by a direct
action on the BCKADH kinase73 (octanoate inhibits the kinase, thus keeping BCKADH
under its active-dephosphorylated form). In summary, it is difficult to fully reconcile
experimental models and clinical studies results. A deleterious effect of medium-chain FA
on distinct amino acid oxidative pathways is likely, but this effect would be minimized
in a clinical setting due to the overall superior oxidation of the MCT.

15.3.2 Structured MCT


These compounds are made of both long-chain and medium-chain FA, carried by the same
glycerol. The various FA can be linked on the glycerol molecule either at random or on a
defined position, depending on the mode of synthesis. Although the FA acid content of
1382_C15.fm Page 246 Tuesday, October 7, 2003 6:38 PM

246 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

structured MCT and physical mixture of MCT and LCT are identical, FA derived from
these two compounds seem to have different intravascular metabolisms. Studies from the
group of Blackburn reported dramatic improvements of various parameters of protein
metabolism (e.g., nitrogen balance and leucine kinetics) in rats receiving structured MCT
either orally74 or intravenously.75,76 One study available in healthy man showed no differ-
ence in nitrogen balances,77 whereas structured MCT administered parenterally were as
efficient as physical LCT and MCT mixture on nitrogen balance in patients scheduled for
abdominal surgery.78

15.3.3 Other lipids


Various other substrates were proposed, such as odd-numbered MCT (trinonanoïn)79 or
short-chain triglycerides. Triacetin has been demonstrated to have the same effect as LCT
on leucine kinetics in dogs, warranting further studies in man.80
In summary, numerous alternative fat sources might be proposed in the near future
for total parenteral nutrition. Some of those might be of interest with respect to nitrogen
sparing, but also in combination with other metabolic actions like eicosapentenoic acid
having specific action on protein breakdown.81 Other aspects of caring for stressed patients
is to improve their immunity, which may involve specific fatty acid derivatives.82

15.4 Nitrogen-sparing effects of ketone bodies


During prolonged fasting, ketone bodies (KB) replace glucose as a primary metabolic fuel
for brain, thus resulting in a decreased need for glucose and in a decreased hepatic glucose
production. Fewer precursors are therefore needed for gluconeogenesis, and particularly
fewer amino acids. This would result in a decreased muscle proteolysis, thus explaining
the relative protein sparing observed during prolonged fasting, which is a crucial mech-
anism for survival. Thus, it was very tempting to speculate that KB were the metabolic
signal for the reduction of muscle protein breakdown. As a matter of fact, Sherwin et al.83
demonstrated that an infusion of DL-b hydroxybutyrate of sodium (Na DL-bOH) in fasting
obese subjects reduced nitrogen excretion and also plasma alanine levels. The nitrogen-
sparing effect of KB was further confirmed in obese patients.84 Nair et al.85 studied leucine
kinetics in volunteers receiving Na DL-bOHB and demonstrated a decreased leucine oxi-
dation and an increase in both whole-body and muscle protein synthesis.
The results of Sherwin et al.83 were challenged by further in vivo studies. In particular,
Fery et al.86 showed that alcalosis induced by Na DL-bOHB was responsible for the reduced
alaninemia, since it was reproduced by a sodium bicarbonate infusion. The actual effect
of KB by themselves were, on the contrary, to raise alaninemia. Miles et al.87 also reported
that KB had no effect on the leucine carbon rate of appearance (whole-body protein
breakdown) and confirmed the results of Fery et al. Finally, we41 infused normal subjects
with a mixture of acid and sodic form of D-bOHB, inducing no change of plasma pH, and
found no effect on leucine oxidation and fluxes. The discrepancy between our results and
those of Nair et al.85 could be explained by the different doses infused (which was lower
in our study), by the racemic form of bOHB used (the D form being the only physiological
circulating KB), and by methodological issues. Finally, the use of ketogenic low-caloric
diets in the treatment of obesity has not proved efficient, rather resulting in a higher
nitrogen excretion.8 This latter situation is, however, quite different from the experimental
infusion of KB during which FA are low, while they are high during a ketogenic diet. With
respect to in vitro studies, the data are also controversial showing34,88 or not46,89 a substrate
competition between KB and amino acids.
1382_C15.fm Page 247 Tuesday, October 7, 2003 6:38 PM

Chapter fifteen: Control of amino acid metabolism 247

Thus, from the currently available data, it seems unlikely that KB are the primary
metabolic signal for the reduction of protein breakdown during fasting. They might act
indirectly, either through their stimulating but moderate effect on insulin secretion90 or
through their alcalinizing effect when infused as their sodic form, thus correcting the
fasting acidosis.

15.5 Conclusion
Although the control of amino acid metabolism by energy substrates has been extensively
studied, the specific effects of glucose, lipids, and ketone bodies are not completely clear,
due to the interactions with hormonal changes and with the pathophysiological situations
that were studied (level of protein and energy intake, underlying diseases). During TPN,
lipids probably exert a specific nitrogen-sparing effect and may help to reduce the poorly
tolerated high glucose intakes; the type of lipid to be used remains controversial. However,
new aspects of protein metabolism with respect to digestion–absorption modulating action
of glucose and lipids or other components like immunity influenced by nutrients are now
to be considered together in short-term as in long-term physiological studies beyond
nitrogen balance.

References
1. Pellett, P.L. and Young, V.R., The effects of different levels of energy intake on protein
metabolism and of different levels of protein intake on energy metabolism: a statistical
evaluation from the published literature, in Protein Energy Interactions, Scrimshaw, N.S. and
Schürch, B., Eds., I.D.E.C.G., Waterville Valley, NH, 1991, pp. 81–136.
2. Jequier, E., Effect of different levels of carbohydrate, fat and protein intake on protein
metabolism and thermogenesis, in Protein Energy Interactions, Scrimshaw, N.S. and Schürch,
B., Eds., I.D.E.C.G., Waterville Valley, NH, 1991, pp. 123–138.
3. Young, V.R., Yu, Y.M., and Fukagawa, N.K., Energy and protein turnover, in Energy Metab-
olism: Tissue Determinants and Cellular Corollaries, Kinney, J.M. and Tucker, H.N., Eds., Raven
Press, New York, 1991, pp. 439–466.
4. Young, V.R., Yu, Y.M., and Fukagawa, N.K., Whole body energy and nitrogen (protein)
relationships, in Energy Metabolism: Tissue Determinants and Cellular Corollaries, Kinney, J.M.
and Tucker, H.N., Eds., Raven Press, New York, 1991, pp. 139–161.
5. Cathcart, E.P., The influence of carbohydrates and fats on protein metabolism, J. Physiol., 39,
311–330, 1990.
6. Munro, H.N., General aspects of the regulation of protein metabolism by diet and by hor-
mones, in Mammalian Protein Metabolism, Munro, H.N. and Allison, J.B., Eds., Academic Press,
New York, 1964, pp. 381–482.
7. Richardson, D.P., Wayler, A.H., Scrimshaw, N.S., and Young, V.R. Quantitative effect of an
isoenergetic exchange of fat for carbohydrate on dietary protein utilization in healthy young
men, Am. J. Clin. Nutr., 32, 2217–2226, 1979.
8. Vazquez, J.A., Morse, E.L., and Adibi, S.A., Effect of dietary fat, carbohydrate, and protein
on branched-chain amino acid catabolism during caloric restriction, J. Clin. Invest., 76,
737–743, 1985.
9. Vazquez, J.A. and Adibi, S.A., Protein sparing during treatment of obesity: ketogenic versus
nonketogenic very low calorie diet, Metabolism, 41, 406–414, 1992.
10. Vazquez, J.A., Paul, H.S., and Adibi, S.A., Regulation of leucine catabolism by caloric sources:
role of glucose and lipid in nitrogen sparing during nitrogen deprivation, J. Clin. Invest., 82,
1606–1613, 1988.
11. Elia, M., Effect of starvation and very low calorie diets on protein-energy interrelationships
in lean and obese subjects, in Protein Energy Interactions, Scrimshaw, N.S. and Schürch, B.,
Eds., I.D.E.C.G., Waterville Valley, NH, 1991, pp. 249–283.
1382_C15.fm Page 248 Tuesday, October 7, 2003 6:38 PM

248 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

12. Henry, C.J.K., Quantitative relationships between protein and energy metabolism: influence
of body composition, in Protein Energy Interactions, Scrimshaw, N.S. and Schürch, B., Eds.,
I.D.E.C.G., Waterville Valley, NH, 1991, pp. 191–200.
13. McCargar, L.J., Clandinin, M.T., Belcastro, A.N., and Walker, K., Dietary carbohydrate-to-fat
ratio: influence on whole-body nitrogen retention, substrate utilization, and hormone re-
sponse in healthy male subjects, Am. J. Clin. Nutr., 49, 1169–1178, 1989.
14. Hartsook, E.W., Hershberger, T.V., and Nee, J.C.M., Effects of dietary protein content and
ratio of fat to carbohydrate calories on energy metabolism and body composition of growing
rats, J. Nutr., 103, 167–178, 1973.
15. Flatt, J.P., Ravussin, E., Acheson, K.J., and Jequier, E., Effects of dietary fat on postprandial
substrate oxidation and on carbohydrate and fat balances, J. Clin. Invest., 76, 1019–1024, 1985.
16. Jeejeebhoy, K.N., Anderson, G.H., Nakooda, A.F., Greenberg, G.R., Sanderson, I., and Marliss,
E.B., Metabolic studies in total parenteral nutrition with lipid in man, J. Clin. Invest., 57,
125–136, 1976.
17. Bark, S., Holm, I., Hakansson, I., and Wretlind, A., Nitrogen sparing effect of fat emulsion
compared with glucose in the postoperative period, Acta Chir. Scand., 142, 423–427, 1976.
18. Baker, J.P., Detsky, A.S., Stewart, S., Whitwell, J., Marliss, E.B., and Jeejeebhoy, K.N., Ran-
domized trial of total parenteral nutrition in critically ill patients: metabolic effects of varying
glucose:lipid ratios as the energy source, Gastroenterology, 87, 53–59, 1984.
19. De Chalain, T.M.B., Michell, W.L., O’Keefe, S.J., and Ogden, J.M., The effect of fuel source
on amino acid metabolism in critically ill patients, J. Surg. Res., 52, 167–176, 1992.
20. Smith, R.G., Mackie, W., Kohlhardt, S., and Kee, A.J., The effect on protein and amino acid
metabolism of an intravenous nutrition regimen providing seventy percent of nonprotein
calories as lipid, Surgery, 111, 12–20, 1992.
21. Pineault, M., Chessex, P., Bisaillon, S., and Brisson, G., Total parenteral nutrition in the
newborn: impact of the quality of infused energy on nitrogen metabolism, Am. J. Clin. Nutr.,
47, 298–304, 1988.
22. Pencharz, P., Beesley, J., Sauer, P., Van Aerde, J., Canagarayar, U., Renner, J., McVet, M.,
Wesson, D., and Swyer, P., Total-body protein turnover in parenterally fed neonates: effects
of energy source studied by using 15N glycine and 1-13C leucine, Am. J. Clin. Nutr., 50,
395–400, 1989.
23. Long, J.M., Wilmore, D.W., Mason, A.D., and Pruitt, B.A., Effect of carbohydrate and fat
intake on nitrogen excretion during total intravenous feeding, Ann. Surg., 185, 417–422, 1977.
24. Van Aerde, J.E.E., Sauer, P.J.J., Pencharz, P.B., Smith, J.M., and Swyer, P.R., Effect of replacing
glucose with lipid on the energy metabolism of newborn infants, Clin. Sci., 76, 581–588, 1989.
25. MacFie, J., Smith, R.C., and Hill, G.L., Glucose or fat as a nonprotein energy source? A
controlled trial in gastroenterological patients requiring intravenous nutrition, Gastroenter-
ology, 80, 103–107, 1981.
26. Nose, O., Tipton, J.R., Ament, M.E., and Yabuuchi, H., Effect of the energy source on changes
in energy expenditure, respiratory quotient and nitrogen balance during total parenteral
nutrition in children, Ped. Res., 21, 538–541, 1987.
27. Bresson, J.L., Bader, B., Rocchiccioli, F., Mariotti, A., Ricour, C., Sachs, C., and Rey, J., Protein
metabolism kinetics and energy substrate utilization in infants fed parenteral solutions with
different glucose fat ratios, Am. J. Clin. Nutr., 54, 370–376, 1991.
28. Robert, J.J., Bier, D., Schoeller, D., Wolfe, R., Matthews, D.E., Munro, H.N., and Young, V.R.,
Effects of intravenous glucose on whole body leucine dynamics, studied with 1-13C leucine
in healthy young and elderly adults, J. Gerontol., 39, 673–681, 1984.
29. Umpleby, A.M., Boroujerdi, M.A., Brown, P.M., Carson, E.R., and Sonksen, P.H., The effect
of metabolic control on leucine metabolism in type 1 (insulin-dependent) diabetic patients,
Diabetologia, 29, 131–141, 1986.
30. Heiling, V.J., Campbell, P.J., Gottesman, I.S., Tsalikian, E., Beaufrère, B., Gerich, J.E., and
Haymond, M.W., Differential effects of hyperglycemia and hyperinsulinemia on leucine rate
of appearance in normal humans, J. Clin. Endocrinol. Metab., 76, 203–206, 1993.
1382_C15.fm Page 249 Tuesday, October 7, 2003 6:38 PM

Chapter fifteen: Control of amino acid metabolism 249

31. Ebeling, P., Tuominen, J.A., Laipio, M.L., Virtanen, M.A., Koivisto, E., and Koivisto, V.A.,
Carbohydrate depletion has profound effects on the muscle amino acid and glucose metab-
olism during hyperinsulinaemia, Diabetes Obes. Metab., 3, 113–120, 2001.
32. Svanberg, E., Moller-Loswick, A.C., Matthews, D.E., Korner, U., Andersson, M., and
Lundholm, K., The role of glucose, long-chain triglycerides and amino acids for promotion
of amino acid balance across peripheral tissues in man, Clin. Physiol., 19, 311–320, 1999.
33. Hedden, M.P. and Buse, M.G., Effects of glucose, pyruvate, lactate and amino acids on muscle
protein synthesis, Am. J. Physiol., 242, E184–E192, 1982.
34. Buse, M.G., Biggers, J.F., Friderici, K.H., and Buse, J.F., Oxidation of branched chain amino
acids by isolated hearts and diaphragms of the rat: the effect of fatty acids, glucose and
pyruvate respiration, J. Biol. Chem., 247, 8085–8096, 1972.
35. Goldberg, A.L., Tischler, M., De Martino, G., and Griffin, G., Hormonal regulation of protein
degradation and synthesis in skeletal muscle, Fed. Proc., 39, 31–36, 1980.
36. Mariotti, F., Mahe, S., Luengo, C., Benamouzig, R., and Tome, D., Postprandial modulation
of dietary and whole-body nitrogen utilization by carbohydrates in humans, Am. J. Clin.
Nutr., 72, 954–962, 2000.
37. Dangin, M., Boirie, Y., Guillet, C., and Beaufrère, B., Influence of the protein digestion rate
on protein turnover in young and elderly subjects, J. Nutr., 132, 3228–3233, 2002.
38. Boirie, Y., Dangin, M., Gachon, P., Vasson, M.P., Maubois, J.L., and Beaufrère, B., Slow and
fast dietary proteins differently modulate postprandial protein accretion, Proc. Natl. Acad.
Sci. U.S.A., 94, 14930–14935, 1997.
39. Dangin, M., Boirie, Y., Garcia-Rodenas, C., Gachon, P., Fauquant, J., Callier, P., Ballevre, O.,
and Beaufrère, B., The digestion rate of protein is an independent regulating factor of
postprandial protein retention, Am. J. Physiol., 280, E340–E348, 2001.
40. Ferrannini, E., Barrett, E.J., Bevilacqua, S., Jacob, R., Walesky, M., Sherwin, R.S., and Defronzo,
R.A., Effect on free fatty acids on blood amino acid levels in humans, Am. J. Physiol., 250,
E686–E694, 1986.
41. Beaufrère, B., Chassard, D., Broussolle, C., Riou, J.P., and Beylot, M., Effects of D-b-hydroxy-
butyrate, long and medium chain triglycerides on leucine metabolism in man, Am. J. Physiol.,
262, E268–E274, 1992.
42. Tessari, P., Nissen, S.L., Miles, J.M., and Haymond, M.W., Inverse relationship of leucine flux
and oxidation to free fatty acids availability in vivo, J. Clin. Invest., 77, 575–581, 1986.
43. Caroll, K.F. and Nestel, P.F., Effect of long chain triglycerides on human insulin secretion,
Diabetes, 21, 923–929, 1972.
44. Buffington, C.K., De Buysere, M.J., and Olson, M.J., Studies on the regulation of the branched
chain a-ketoacid dehydrogenase in the perfused rat heart, J. Biol. Chem., 254, 10453–10458,
1979.
45. Buxton, D.B., Varron, L.L., Taylor, M.K., and Olson, M.S., Regulatory effects of fatty acids
on decarboxylation of leucine and 4-methyl-2-oxopentanoate in the perfused rat heart, Bio-
chem. J., 221, 593–599, 1984.
46. Odessey, R. and Goldberg, A.L., Oxidation of leucine by rat skeletal muscle, Am. J. Physiol.,
223, 1376–1388, 1972.
47. Buse, M.G., Herlong, H.F., and Weigand, D.A., The effects of diabetes, insulin and the redox
potential on leucine metabolism by isolated rat hemidiaphragm, Endocrinology, 98, 1166–1175,
1976.
48. Brooks, S.P. and Lampi, B.J., Time course of enzyme changes after a switch from a high-fat
to a low-fat diet, Comp. Biochem. Physiol. B Biochem. Mol. Biol., 118, 359–365, 1997.
49. Krebs, H.A., Metabolism of amino acids. III. Deamination of amino acids, Biochem. J., 29,
1620–1628, 1935.
50. Randle, P.J., Garland, P.B., Hales, C.N., and Newsholme, E.A., The glucose fatty acid cycle:
its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus, Lancet, i,
785–789, 1963.
51. Keller, U., Turkalj, I., Laager, R., Bloesch, D., and Bilz, S., Effects of medium- and long-chain
fatty acids on whole body leucine and glucose kinetics in man, Metabolism, 51, 754–760, 2002.
1382_C15.fm Page 250 Tuesday, October 7, 2003 6:38 PM

250 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

52. Lowell, B.B. and Goodman, M.N., Protein sparing in skeletal muscle during prolonged
starvation: dependence on lipid fuel availability, Diabetes, 36, 14–19, 1987.
53. Carpentier, Y. and Thonnart, N., Lipids in clinical nutrition, in Lipids in Modern Nutrition,
Horisberger, M. and Bracco, U., Eds., Raven, New York, 1987, pp. 147–155.
54. Bach, A.C., Frey, A., and Lutz, O., Clinical and experimental effects of medium chain tri-
glyceride based fat emulsions: a review, Clin. Nutr., 8, 223–235, 1989.
55. Johnson, R.C., Young, S.K., Cotter, R., Lin, L., and Rowe, W.B., Medium chain triglyceride
lipid emulsion: metabolism and tissue distribution, Am. J. Clin. Nutr., 52, 502–508, 1990.
56. Czarnetzki, H.D., Schweder, R., Helmki, U., Teichert, H.M., and Nagel, R., MCT and nitrogen
metabolism, in Nutrition in Clinical Practice, Harting, N., Dietze, G., Weiner, R., and Fürst, P.,
Eds., Karger, Basel, Switzerland, 1989, pp. 83–89.
57. Dawes, R.F.H., Royle, G.T., Dennison, A.R., Crowe, P.J., and Ball, M., Metabolic studies of a
lipid emulsion containing medium chain triglycerides in perioperative and total parenteral
nutrition infusions, World. J. Surg., 10, 38–46, 1986.
58. Dennison, A.R., Ball, M., Crowe, P.J., White, K., Hands, L., Watkins, R.M., and Kettlewell,
M., The metabolic consequences of infusing emulsions containing medium chain triglycer-
ides for parenteral nutrition: a comparative study with conventional lipid, Ann. Rev. Coll.
Surg. Engl., 68, 111–121, 1986.
59. Dennison, A.R., Ball, M., Hands, L.J., Corwe, P.J., Watkins, E.M., and Kettlewell, M., Total
parenteral nutrition using conventional and medium chain triglycerides: effect on liver
function tests, complement and nitrogen balance, J. Parenter. Enteral Nutr., 12, 15–19, 1988.
60. Ball, M.J., Parenteral nutrition in the critically ill: use of a medium chain triglyceride emul-
sion, Intensive Care Med., 19, 89–95, 1993.
61. Clarke, P.J., Ball, M.J., Hands, L.J., Dennison, A.R., Turnbridge, A., White, K., and Kettlewell,
M.G.W., Use of a lipid containing medium chain triglyceride in patients receiving TPN: a
randomized prospective trial, Br. J. Surg., 74, 701–704, 1987.
62. Bach, A.C., Guiraud, M., Gibault, J.P., Schirardin, H., Frey, A., and Bouletreau, P., Medium
chain triglycerides in septic patients on total parenteral nutrition, Clin. Nutr., 7, 157–163, 1988.
63. Calon, B., Pottecher, T., Frey, A., Ravanello, J., Otteni, J.C., and Bach, A.C., Long chain versus
medium and long chain triglyceride based fat emulsion in parenteral nutrition of severe
head trauma patients, Infusiontherapie, 17, 246–248, 1990.
64. Crowe, P.J., Dennison, A.R., and Royle, G.T., A new intravenous emulsion containing medi-
um chain triglyceride: studies of its metabolic effects in the perioperative period compared
with a conventional long chain triglyceride emulsion, J. Parenter. Enteral Nutr., 9, 720–724,
1985.
65. Jiang, Z.M., Zang, S.Y., Wang, X.R., Yang, N.F., Zhu, Y., and Wilmore, D., A comparison of
medium-chain and long-chain triglycerides in surgical patients, Ann. Surg., 217, 175–184,
1993.
66. Wasa, Y., Ogashi, S., Iwada, M., Mizobuchi, S., and Tamiya, T., The effect of medium chain
triglycerides (MCT) emulsion on protein metabolism in burned rats, Clin. Nutr., 7, 42, 1988
(abstract).
67. Grancher, D., Jean Blain, C., Frey, A., Schirardin, H., and Bach, A.C., Studies on the tolerance
of medium chain triglycerides in dogs, J. Parenter. Enteral Nutr., 11, 280–286, 1987.
68. Fried, R.C., Mullen, J.L., Blackburn, G.L., Buzby, G.P., Georgieff, M., and Stein, T.P., Effects
of nonglucose substrates (xylitol, medium chain triglycerides, long chain triglycerides) and
carnitine on nitrogen metabolism in stressed rats, J. Parenter. Enteral Nutr., 14, 134–138, 1990.
69. Stein, T.P., Fried, R.C., Torosian, M.H., Leskiw, M.J., Schluter, M.D., Settle, R.G., and Buzby,
G.P., Comparison of glucose, LCT and LCT plus MCT as calorie source for parenterally
nourished septic rats, Am. J. Physiol., 250, E312–E318, 1986.
70. Rodriguez, N.R., Schwenk, W.F., Beaufrère, B., Miles, J.M., and Haymonds, M.W., Trioctanoin
infusion increases in vivo leucine oxidation: a lesson in isotope modelling, Am. J. Physiol.,
251, E343–E348, 1986.
71. Spydevold, O. and Hokland, B., Oxidation of branched chain amino acids in skeletal muscle
and liver of rat: effects of octanoate and energy state, Biochem. Biophys. Acta, 676, 279–288,
1981.
1382_C15.fm Page 251 Tuesday, October 7, 2003 6:38 PM

Chapter fifteen: Control of amino acid metabolism 251

72. Wagenmakers, A.J.M. and Veerkamp, J.H., Interaction of octanoate with branched chain
2-oxoacid oxidation in rat and human muscle in vitro, Int. J. Biochem., 16, 977–984, 1984.
73. Paxton, R. and Harris, R.A., Regulation of branched chain a-ketoacid dehydrogenase kinase,
Arch. Biochem. Biophys., 231, 48, 1984.
74. De Michele, J.J., Karlstad, M.D., Babayan, V.K., Istfan, N., Blackburn, G.L., and Bistrian, B.R.,
Enhanced skeletal muscle and liver protein synthesis with structured lipids in enterally fed
burned rats, Metabolism, 37, 787, 1988.
75. Mok, K.T., Maiz, A., Yamazaki, K., Sobrado, J., Babayan, V.K., Moldawer, L.L., Bistrian, B.R.,
and Blackburn, G.L., Structured medium chain and long chain triglyceride emulsions are
superior to physical mixtures in sparing body protein in the burned rats, Metabolism, 33,
910–915, 1984.
76. Maiz, A., Yamazaki, K., Sobrado, J., Babayan, V.K., Moldawer, L.L., Bistrian, B.R., and Black-
burn, G.L., Protein metabolism during total parenteral nutrition (TPN) in injured rats using
medium chain triglycerides, Metabolism, 33, 901–909, 1984.
77. Sandstrom, S., Hyltander, A., Korner, U., and Lundholm, K., Structured triglycerides to
postoperative patients: a safety and tolerance study, J. Parenter. Enteral Nutr., 17, 153–157,
1993.
78. Chambrier, C., Guiraud, M., Gibault, J.P., Labrosse, H., and Bouletreau, P., Medium- and
long-chain triacylglycerols in postoperative patients: structured lipids versus a physical
mixture, Nutrition, 15, 274–277, 1999.
79. Linseisen, J. and Wolfram, G., Odd-numbered medium-chain triglycerides (trinonanoin) in
total parenteral nutrition: effects on parameters of fat metabolism in rabbits, J. Parenter. Enteral
Nutr., 17, 522–528, 1993.
80. Bailey, J.W., Miles, J.M., and Haymond, M.W., Effect of parenteral administration of short-
chain triglycerides on leucine metabolism, Am. J. Clin. Nutr., 1993, 58, 912–916.
81. Whitehouse, A.S. and Tisdale, M.J., Downregulation of ubiquitin-dependent proteolysis by
eicosapentaenoic acid in acute starvation, Biochem. Biophys. Res. Commun., 285, 598–602, 2001.
82. Calder, P.C. and Grimble, R.F., Polyunsaturated fatty acids, inflammation and immunity, Eur.
J. Clin. Nutr., 56, 14–19, 2002.
83. Sherwin, R.S., Hendler, R.G., and Felig, P., Effect of ketone infusions on amino acid and
nitrogen metabolism in man, J. Clin. Invest., 55, 1382–1390, 1975.
84. Pawan, G.L.S. and Semple S.J.G., Effect of 3-hydroxybutyrate in obese subjects on very low
energy diets and during therapeutic starvation, Lancet, i, 15–17, 1986.
85. Nair, K.S., Welle, S.L., Halliday, D., and Campbell, R.G., Effect of b-hydroxybutyrate on
whole-body leucine kinetics and fractional mixed skeletal muscle protein synthesis in hu-
mans, J. Clin. Invest., 82, 198–205, 1988.
86. Fery, F. and Balasse, E.O., Differential effects of sodium acetoacetate and acetoacetic acid
infusion on alanine and glutamine metabolism in man, J. Clin. Invest., 66, 323–331, 1980.
87. Miles, J.M., Nissen, S.L., Rizza, R.A., Gerich, J.E., and Haymond, M.W., Failure of infused
b-hydroxybutyrate to decrease proteolysis in man, Diabetes, 32, 197–205, 1983.
88. Wu, G. and Thompson, J.R., Ketone bodies inhibit leucine degradation in chick skeletal
muscle, Int. J. Biochem., 19, 937–943, 1987.
89. Paul, H.S. and Adibi, S.A., Leucine oxidation in diabetes and starvation: effects of ketone
bodies on branched chain amino acid oxidation in vitro, Metabolism, 27, 185–200, 1978.
90. Miles, J.M., Haymond, M.H., and Gerich, J.E., Suppression of glucose production and stim-
ulation of insulin secretion by physiological concentrations of ketone bodies in man, J. Clin.
Endocrinol. Metab., 52, 34–37, 1981.
1382_C15.fm Page 252 Tuesday, October 7, 2003 6:38 PM
1382_C16.fm Page 253 Tuesday, October 7, 2003 6:40 PM

chapter sixteen

Amino acid signaling and the


control of protein metabolism
Alfred J. Meijer and Peter F. Dubbelhuis
Academic Medical Center, Amsterdam

Contents
Introduction..................................................................................................................................253
16.1 Amino acid-dependent signaling and the control of autophagic protein
degradation.........................................................................................................................254
16.2 Amino acid-dependent signaling and the control of protein synthesis...................258
16.2.1 Amino acids and p70S6 kinase activation........................................................258
16.2.2 Amino acid stimulation of 4E-BP1 phosphorylation......................................258
16.2.3 Amino acid stimulation of eEF2 kinase ............................................................259
16.2.4 Amino acid stimulation of eIF2a .......................................................................259
16.2.5 Participation of PI 3-kinase and protein kinase B in amino
acid-dependent signaling? Amino acid/insulin synergy ..............................259
16.2.6 Amino acids and the activation of mTOR........................................................261
16.2.7 Amino acids and protein phosphatases............................................................261
16.2.8 Negative feedback by amino acid signaling on insulin signaling ...............262
16.2.9 mTOR-dependent signaling is antagonized by AMP kinase ........................262
16.2.10 Amino acid signaling in pancreatic b-cells ......................................................263
16.2.11 Amino acid signaling in vivo .............................................................................264
16.3 Mechanisms ........................................................................................................................264
16.4 Conclusions.........................................................................................................................266
References .....................................................................................................................................266

Introduction
Although amino acids are important as substrates for many metabolic pathways such as
protein synthesis, gluconeogenesis, and synthesis of urea and other N-containing com-
pounds, they can also serve as regulators of metabolism. For example, in the liver, the
central amino acid catabolite, glutamate, promotes the activity of the ornithine cycle via
synthesis of N-acetylglutamate, the essential activator of carbamoyl-phosphate synthase
(cf. Chapter 7). Glutamate and aspartate are essential components of the malate/aspartate

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 253
1382_C16.fm Page 254 Tuesday, October 7, 2003 6:40 PM

254 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

shuttle responsible for the mitochondrial oxidation of cytosolic NADH during aerobic
glycolysis in many tissues and during ethanol oxidation in the liver, and they kinetically
control the rate of these processes.1 Another pathway that is controlled (i.e., inhibited) by
amino acids is the autophago-lysosomal pathway of protein degradation,2,3 a good exam-
ple of feedback inhibition.
A new element was added to mechanisms involved in the control of metabolism by
amino acids after the discovery that cell swelling, associated with the Na+-dependent
influx of some amino acids into cells or with the intracellular accumulation of nonpermeant
amino acid catabolites, such as glutamate and aspartate, exerts anabolic and anticatabolic
effects. For instance, amino acid-induced cell swelling stimulates glucose-driven glycogen
synthesis,4 lipogenesis,5 and protein synthesis,6 and it inhibits catabolic pathways such as
glycogenolysis7 and protein degradation.8,9 These phenomena are reminiscent of those
occurring in microorganisms in which changes in the synthesis or degradation of macro-
molecules are used to counteract changes in extra- and intracellular osmolarity.10
Work initiated by our laboratory and now confirmed by many others has revealed
the existence of an amino acid-stimulated signal transduction pathway that shares com-
ponents and acts in concert with a signaling pathway that is also stimulated by insulin.
It is this pathway that simultaneously controls (autophagic) protein degradation and
protein synthesis, but in opposite directions. This chapter summarizes the major findings
in this field.

16.1 Amino acid-dependent signaling and the control of autophagic


protein degradation
During macroautophagy, portions of the cytoplasm, which sometimes even contain entire
organelles, are surrounded by a sequestering double membrane. The newly formed initial
autophagosomes mature stepwise into degradative autophagosomes: they acquire lyso-
somal membrane proteins by fusion with vesicles deficient in hydrolytic enzymes. This
is followed by acidification and fusion with existing lysosomes and ultimately leads to
the degradation of the sequestered macromolecular compounds (see Blommaart et al.11
for a review). Although the autophagosomes constitute only a small percentage of the
total cell volume (maximally about 1 to 2%), their turnover is high, with a half-life of
about 8 min in hepatocytes.11 In the liver of rats and mice in vivo, macroautophagy is
responsible for a net loss of 20 to 25% of liver cell protein after 24 h fasting during which
the concentrations of circulating insulin and amino acids fall.2 The origin of the seques-
tering membrane is still under debate. It may be formed by invaginations of the ribosome-
free parts of the endoplasmic reticulum, post-Golgi membranes and Golgi membranes, or
by a combination of these.3,11
Autophagic protein degradation, which occurs in almost all cell types, is inhibited by
amino acids and insulin and is stimulated by glucagon in the liver.2,11 In the liver, insulin
and glucagon only exert their effects on autophagy at intermediate amino acid concentra-
tions but not at either high or very low amino acid concentrations, when autophagy
proceeds at minimal or maximal rates, respectively.2,11 As will be discussed below, these
effects parallel those on signal transduction. Control of autophagic protein degradation
by amino acids and hormones occurs at the level of sequestration, i.e., the first step of the
autophagic pathway. At moderate concentrations (1 mM), leucine also increases the intra-
lysosomal pH; this effect could not have been caused by accumulation of ammonia (a
known acidotropic agent) because under the conditions studied leucine was not
catabolized.12
1382_C16.fm Page 255 Tuesday, October 7, 2003 6:40 PM

Chapter sixteen: Amino acid signaling and the control of protein metabolism 255

From a survey of the literature regarding the specificity of amino acid inhibition of
autophagy in liver, we concluded that leucine, phenylanine, and tyrosine, in combination
with a few other amino acids, such as alanine, glutamine, and proline, were most
effective.11 As indicated earlier, the plasma membrane transport of alanine, glutamine, and
proline (in contrast to that of leucine, phenylalanine, and tyrosine) is Na+ coupled, and
their intracellular accumulation, combined with the intracellular accumulation of imper-
meant glutamate and aspartate formed by their catabolism, results in increased cell vol-
ume. A combination of low concentrations of leucine, phenylalanine, and tyrosine and
hypo-osmotically induced cell swelling (without intracellular accumulation of glutamate
and aspartate) was as effective in inhibiting autophagy as a complete mixture of amino
acids.13,14 It must be pointed out that leucine and the aromatic amino acids do not cause
cell swelling.5 In muscle, too, leucine and glutamine have been known for a long time as
potent inhibitors of proteolysis.15–18 Taken together, the data suggest that it is the combi-
nation of leucine, phenylalanine, and tyrosine, and cell swelling that results in maximal
inhibition of autophagy. Earlier studies, initiated by Hallbrucker and colleagues,8 and
extended by our laboratory,9 also stressed the importance of cell swelling in the control of
autophagy. As discussed below, the specificity of amino acids with regard to their ability
to inhibit autophagy closely parallels their effect on signal transduction.
Indications that protein phosphorylation was involved in the control of autophagy
were provided by experiments with isolated hepatocytes, which showed strong inhibition
of the process by inhibitors of tyrosine kinases and of protein phosphatases19–21; however,
the possibility was not taken into consideration that amino acids might influence protein
phosphorylation. The first indications that amino acids affect signal transduction came
from experiments carried out in our own laboratory, in which we showed that a complete
physiological mixture of amino acids at concentrations similar to those present in the
portal vein of fed rats strongly and rapidly (t 1/2 , 10 min) stimulated phosphorylation of
ribosomal protein S6 with no effect on its rate of dephosphorylation,13,14 when they were
added to isolated hepatocytes. S6 is a component of the 40S subunit, has five phosphory-
lation sites, and was long known as one of the end points in insulin signaling. It is generally
assumed that S6 phosphorylation is required for translation of the terminal oligopyrimi-
dine (TOP) tract family of mRNA molecules that contain an oligopyrimidine tract
upstream of their transcription initiation site and encode proteins participating in mRNA
translation,22 although this view has recently been questioned.23 The increase in S6 phos-
phorylation caused by amino acid addition could be completely prevented by rapamycin,
which indicated that the serine/threonine protein kinase mTOR (mammalian target of
rapamycin) was located upstream of S6 in the signaling process.14 Interestingly, when
insulin was added in the absence of amino acids, this did not affect S6 phosphorylation;
however, insulin potentiated the effect of low but not high amino acid concentrations. On
the other hand, glucagon inhibited the effect of amino acids on S6 phosphorylation at low
but not high amino acid concentrations. Hypo-osmotically induced cell swelling mimicked
the effect of insulin in that it had no effect by itself but promoted the effect of low but not
high amino acid concentrations. A combination of physiological concentrations of leucine,
phenylalanine, and tyrosine, together with either insulin or hypo-osmotically induced cell
swelling, stimulated S6 phosphorylation maximally.14 All these effects on S6 phosphory-
lation were independent of protein synthesis because they were observed in the presence
of cycloheximide, and they were similar to the effects of amino acids, cell swelling, insulin,
and glucagon on autophagic proteolysis, as discussed above. Indeed, under a variety of
conditions a linear relationship was observed between the degree of S6 phosphorylation
and the percentage of inhibition of autophagic proteolysis. Furthermore, inhibition of
amino acid-stimulated S6 phosphorylation by rapamycin partially albeit not completely
reversed the inhibition of autophagic proteolysis by amino acids. The fact that inhibition
1382_C16.fm Page 256 Tuesday, October 7, 2003 6:40 PM

256 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

was not completely prevented by rapamycin may be explained by the ability of leucine
to inhibit not only autophagic sequestration but also the lysosomal proton pump,12 as
discussed above. Stimulation of autophagy by rapamycin was also observed in interphase
embryonic stem cells24 and in yeast.25–27 By contrast, in myotubes, inhibition of autophagic
proteolysis by leucine occurs by a mechanism that appears to be independent of mTOR.28
Possibly, in this cell type, inhibition of the lysosomal proton pump by leucine (see above)
may have been largely responsible for the inhibition of proteolysis.
Krause et al.29 recently showed that in hepatocytes the combination of leucine with
either glutamine-induced cell swelling or insulin also maximally stimulated phosphoryla-
tion and activation of p70S6 kinase, the enzyme responsible for phosphorylation of S6. In
the perfused liver, a combination of glutamine and leucine (together with tyrosine) was
also effective in stimulating p70S6 kinase and S6 phosphorylation.30 These data are there-
fore consistent with our data on the effects of leucine in combination with either hypo-
osmotically induced cell swelling or insulin on the phosphorylation of S6 (which reflects
the p70S6 kinase activity in situ), as discussed earlier. We were recently able to demonstrate
that phosphorylation of p70S6 kinase was also greatly stimulated when hepatocytes were
exposed to low concentrations of leucine in combination with lactate and NH3. The
combination of lactate and NH3 allows intracellular glutamate and aspartate to accumulate
to high concentrations31 and also results in an increased cell volume (P.F. Dubbelhuis and
A.J. Meijer, unpublished data).
On the basis of all these data, we therefore tentatively conclude that the minimal
requirement for S6 phosphorylation is the presence of leucine and an increase in cell
volume, whether induced by hypo-osmolarity of the extracellular environment or by
intracellular amino acid accumulation. Control experiments carried out in our laboratory
(data not shown) indicated that cell swelling does not affect leucine transport across the
plasma membrane.
Because amino acids and insulin acted in synergy with regard to both mTOR-depen-
dent S6 phoshorylation and inhibition of autophagic proteolysis, and because interruption
of signaling with rapamycin increased autophagy, we also considered the possibility that
phosphatidylinositol 3-kinase (PI 3-kinase), an important component of insulin signaling
upstream of mTOR, was involved both in amino acid signaling and in the control of
autophagy. Amino acid-induced S6 phosphorylation could be prevented by the PI 3-kinase
inhibitors wortmannin and LY294002,32 suggesting that, indeed, PI 3-kinase contributes
to amino acid signaling (see Section 16.2 for further discussion of this issue). However, in
contrast to that caused by rapamycin, interruption of amino acid signaling by wortmannin
or LY294004 did not stimulate but rather inhibited autophagy. This unexpected result led
us to the hypothesis that phosphatidylinositol 3-phosphate (PI(3)P), produced by consti-
tutively active PI 3-kinase class III, may be essential for autophagy and that phosphati-
dylinositol 3,4-bisphosphate (PI(3,4)P2) and phosphatidylinositol 3,4,5-trisphosphate
(PI(3,4,5)P3), produced by insulin-stimulated PI 3-kinase class I, inhibit the process.11,32
Because wortmannin and LY294002 are not specific and inhibit both classes of PI 3-kinase,
this would explain our results. The hypothesis that PI(3)P might be necessary for autoph-
agy was based on the situation in yeast, in which autophagy is also very active under
nutrition-poor conditions. Yeast contains Vps34, a homologue of mammalian class III PI
3-kinase, which also produces PI(3)P,33,34 but does not contain the class I enzyme and
cannot produce PI(3,4)P2 and PI(3,4,5)P3.
Our hypothesis was confirmed by experiments with HT-29 cells, in which the level
of phosphatidylinositol phospholipids within the cells was manipulated either by addition
of interleukin (IL)-13, by feeding the cells synthetic lipids, or by transfection experiments
resulting in altered PI 3-kinase class III activity.35 Likewise, manipulation of PI(3,4)P2 and
PI(3,4,5)P3 by modulation of the activity of the tumor suppressor PTEN (phosphatase and
1382_C16.fm Page 257 Tuesday, October 7, 2003 6:40 PM

Chapter sixteen: Amino acid signaling and the control of protein metabolism 257

tensin homologue deleted from chromosome 10), a lipid phosphatase that specifically
dephosphorylates the 3-position of PI(3,4)P2 and PI(3,4,5)P3,36 resulted in predictable
changes in autophagy, with high rates of autophagy by overexpression of PTEN and low
rates by inactivation of the gene.37 There is now also ample evidence in support of the
participation of PI(3)P in autophagy in yeast.38–40 In Saccaromyces cerevisiae, Vps34 together
with its adapter protein Vps15 forms a complex with Apg6 and Apg14, two other proteins
that are part of the machinery that controls autophagy in this organism.38 In mammalian
cells, the tumor suppressor protein Beclin I, which is associated with the antiapoptotic
protein Bcl2 in the mitochondrial outer membrane and the endoplasmic reticulum mem-
brane, is the functional homologue of the yeast Apg6 protein41; Beclin I also forms a
complex with PI 3-kinase and its p150 adapter protein.42
Most rewarding was our discovery that 3-methyladenine, the classical inhibitor of
autophagic sequestration with an unknown mechanism,43 turned out to be a PI 3-kinase
inhibitor.32,35 Recent data have shown that inhibitors of cAMP phosphodiesterase, such as
caffeine and theophylline, are also inhibitors of PI 3-kinase,44 so that, in retrospect, the
previously reported anomalous inhibitory effects of these compounds on autophagy45 now
find their explanation.
Experiments in nature have also provided evidence in support of the importance of
the phosphatidylinositol phospholipids in the control of autophagy in vivo. One example
is X-linked myotubular myopathy, characterized by severe neonatal hypotonia and gen-
eralized muscle weakness, which is caused by a mutation in myotubularin, a PI(3)P
phosphatase that removes phosphate from the 3-position of PI(3)P, but not from PI(3,4)P2
and PI(3,4,5)P3.36,46 A mutation in this gene results in high levels of PI(3)P and accumulation
of autophagic vacuoles.47 A second example is PTEN, already discussed above, which is
mutated in a large number of human cancers, including glioblastoma and prostate cancer,
in addition to a wide range of advanced malignancies, such as endometrial, breast, lung,
kidney, bladder, testis, and head and neck cancers, and melanoma and lymphoma.36,48
Likewise, PTEN +/– mice developed hyperplastic changes in prostate, skin, liver, colon,
endometrium, thyroid, and thymus, or developed T-cell lymphoma and teratocarcinoma.48
There is no doubt that in addition to increased protein synthesis (cf. Section 16.2), defective
autophagy contributes to tumor cell growth in all these cases. It is surprising that this
possibility was not considered at all in recent discussions on the role of PTEN in tumor
cell growth.49
Notwithstanding the fact that both wortmannin and LY294002 were able to prevent
amino acid-stimulated S6 phosphorylation (see above), the issue of whether amino acids
are able to activate PI 3-kinase class I is still controversial: direct attempts to show such
activation either have failed29,50–52 or were successful, albeit that activation was only tran-
sient.53,54 In hepatocytes and in intestinal cells, PI 3-kinase was also activated by hypo-
osmotically induced cell swelling.53,55,56 It has been suggested that the PI 3-kinase inhibitors
are not specific and that they also inhibit mTOR.50,51 In our opinion, however, this is not
likely for two reasons: first, much higher concentrations of wortmannin were required to
inhibit mTOR activity in vitro than to inhibit PI 3-kinase activity in vitro.32,57 Second, in
cells overexpressing protein kinase C-delta, PI 3-kinase-independent phosphorylation of
4E-BP1, a downstream target of mTOR (see Section 16.2), was increased; this increase in
4E-BP1 phosphorylation was rapamycin sensitive but wortmannin insensitive.58 This is
difficult to understand if mTOR were inhibited by wortmannin (see Section 16.2 for further
discussion of the involvement of PI 3-kinase in amino acid signaling).
Amino acids do not activate protein kinase B (see Section 16.2). Yet expression of
constitutively active protein kinase B or expression of a dominant-negative mutant form
of the kinase respectively inhibited or activated autophagy in HT-29 cells37; this indicates
that protein kinase B, like PI 3-kinase, is involved in the control of autophagy.
1382_C16.fm Page 258 Tuesday, October 7, 2003 6:40 PM

258 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Apart from the fact that amino acids can influence autophagy via the mTOR
pathway, they may also affect autophagy via extracellular signal-regulated (Erk 1/2)
mitogen-activated protein (MAP) kinase. In HT-29 cells, amino acid depletion resulted in
the phosphorylation and activation of MAP kinase, which in turn phosphorylated
Ga-interacting protein (GAIP). This resulted in stimulation of the GTP-ase activity of the
a-subunit of the trimeric Gai3 protein, leading to activation of autophagy.59 In perfused liver,
amino acid-induced cell swelling resulted in activation of p38 MAP kinase and inhibition
of autophagy.60 Clearly, in addition to the mTOR pathway, members of the MAP kinase
family are also part of the mechanism involved in the control of autophagy by amino acids,
although their precise role may be different in different experimental models.59 It is unlikely
that mTOR-dependent signaling is influenced by the MAP kinase pathway.61
Although autophagy occurs in almost all cell types, its contribution to overall pro-
teolysis is cell type dependent.11 The major extralysosomal proteolytic process is the
ubiquitin–proteasome pathway.62 Although insulin can inhibit this process,63 it is not
known whether amino acids and mTOR signaling can also inhibit it.

16.2 Amino acid-dependent signaling and the control of protein


synthesis
16.2.1 Amino acids and p70S6 kinase activation
Since our initial observations in hepatocytes, amino acid-dependent signaling and its
interaction with insulin signaling have been confirmed (or rediscovered) in many cell
types. These include muscle cells, adipocytes, hepatoma cells, CHO cells, and pancreatic
b-cells.50,51,61,64–66 Apart from the fact that amino acid-stimulated phosphorylation of p70S6
kinase and S6 was rapamycin sensitive, the involvement of mTOR in the amino acid
response was also supported by other experiments. For instance, in human rhabdomyo-
sarcoma Rh30 cells harboring a rapamycin-resistant mutant of mTOR, amino acids stim-
ulated p70S6 kinase activity in a rapamycin-insensitive manner.67 In CHO-IR cells, a
rapamycin-resistant mutant of p70S6 kinase could be phosphorylated at Thr 412 (critical
for enzyme activity) in the presence of insulin in a wortmannin-sensitive manner, irre-
spective of the presence of amino acids.50
As in hepatocytes, in many other cell types amino acids and insulin were found to
act in synergy.50,51,66,68,69 Insulin alone did not induce p70S6 kinase activation; in cases where
it did stimulate on its own, this could be ascribed to amino acids produced by autophagy.52
Also, as in hepatocytes, leucine proved to be the most effective among the various amino
acids in other cell types.50,51,64,66,70–73 The data also showed that leucine alone could not
completely mimic the effect of a mixture of all amino acids. Apparently, other amino acids
act in concert with leucine to elicit full activation of p70S6 kinase. As discussed in Section
16.1, a possible explanation is that amino acids, which are transported together with Na+,
increase cell volume and can potentiate the effect of leucine.

16.2.2 Amino acid stimulation of 4E-BP1 phosphorylation


Another substrate of mTOR, apart from p70S6 kinase, is 4E-BP1 (also known as
PHAS-1).74,75 4E-BP1 has several phosphorylation sites that are targets of different protein
kinases, and phosphorylation of the protein results in dissociation of the eIF4E.4E-BP1
complex; eIF4E then becomes available for initiation of cap-dependent mRNA transla-
tion.76 Like p70S6 kinase, in various cell types phosphorylation of 4E-BP1 was greatly
stimulated by amino acids in a rapamycin-sensitive manner,50–52,64–67,77 with leucine again
1382_C16.fm Page 259 Tuesday, October 7, 2003 6:40 PM

Chapter sixteen: Amino acid signaling and the control of protein metabolism 259

being the most effective.50,65,66 Insulin was not required for this effect. In the absence of
amino acids, insulin was unable to stimulate 4E-BP1 phosphorylation.50,77

16.2.3 Amino acid stimulation of eEF2 kinase


Eukaryotic elongation factor 2 (eEF2), which mediates the translocation step of elongation,
becomes inactive when it is phosphorylated at Thr56. Phosphorylation at this position is
controlled by eEF2 kinase. This kinase becomes inhibited when phosphorylated at Ser366
by p70S6 kinase, so that the same factors (e.g., amino acids) that control p70S6 kinase
activity also control eEF2 kinase phosphorylation, and thus the activity of eEF2.78 eEF2
kinase can also be phosphorylated by p90RSK, in which case eEF2 phosphorylation is
insensitive to rapamycin but sensitive to inhibitors of the MEK/Erk signaling pathway.78

16.2.4 Amino acid stimulation of eIF2a


Another factor controlling protein synthesis is the eukaryotic initiation factor eIF2. In the
absence of amino acids, this factor becomes inactivated when Ser51 of the a-subunit of
eIF2 is phosphorylated. In the presence of amino acids, when Ser51 is dephosphorylated,
eIF2 becomes active and recruits charged initiator tRNA to the 40S ribosomal subunit.79
In the activation process by amino acid-induced dephosphorylation, leucine in particular
is effective.70
In yeast, a single kinase, GCN2 (general control nondepressible), is responsible for
eIF2a phosphorylation. This kinase is activated by uncharged tRNAs because its C-ter-
minus structurally resembles histidyl-tRNA synthetase and other aminoacyl-tRNA syn-
thetases.80 This is then followed by increased synthesis of the transcription factor GCN4,
which in turn is responsible for increased transcription of a large number of genes involved
in amino acid synthesis and other metabolic processes needed under these conditions,
including genes encoding proteins required for autophagy.80,81 Although this has been
disputed, uncharged tRNA may be a sensor of amino acid starvation (see Section 16.3).
In this context, it is of importance to note that eIF2a kinase is not only important in the
regulation of protein synthesis but is also essential in starvation-induced autophagy in
both yeast and mammalian cells.82

16.2.5 Participation of PI 3-kinase and protein kinase B in amino acid-dependent


signaling? Amino acid/insulin synergy
A simple explanation for the synergy between insulin and amino acids in stimulating
mTOR downstream targets would be that insulin promotes amino acid transport across
the plasma membrane. This is not very likely, however, because stimulation of protein
synthesis in muscle by insulin was accompanied by decreased rather than increased
intracellular amino acid concentrations.61
As discussed earlier, in the absence of amino acids, insulin alone did not affect phos-
phorylation of mTOR downstream targets. However, insulin alone did stimulate the
activity of PI 3-kinase and protein kinase B,29,50–52,64,68,83 which are both signaling compo-
nents located upstream of mTOR.76 Stimulation of phosphorylation of p70S6 kinase, of S6,
and of 4E-BP1 by amino acids alone in the absence of insulin could be prevented by
inhibitors of PI 3-kinase.32,50,51,61,64,65,71 This suggests that PI 3-kinase is located upstream of
p70S6 kinase in the signaling pathway. As already discussed in Section 16.1, it is not
entirely clear whether amino acids are able to stimulate PI 3-kinase activity. There is general
agreement, however, that amino acids do not affect protein kinase B activity.29,50,51,54,64,67,68
1382_C16.fm Page 260 Tuesday, October 7, 2003 6:40 PM

260 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 16.1 Amino acids as regulators of protein metabolism. The scheme is a composition of data
discussed in this chapter. IR, insulin receptor; IRS, insulin receptor substrate; PI3K, phosphatidyli-
nositol 3-kinase; PI3P, phosphatidylinositol 3-phosphate; PI45P2, phosphatidylinositol 4,5-bisphos-
phate; PI345P3, phosphatidylinositol 3,4,5-trisphosphate; PDK1, phosphoinositide-dependent kinase 1;
PKB, protein kinase B; AA, amino acids; RVD, regulatory volume decrease; GS, glycogen synthase;
ACC, acetyl-CoA carboxylase; mTOR, mammalian target of rapamycin; PA, phosphatidylic acid;
AMPK, AMP-activated protein kinase; pHlys, intralysosomal pH; PP2A, protein phosphatase 2A;
GAPP, glutamate-activated protein phosphatase; 4E-BP1, eukaryotic protein translation initiation
factor 4E-binding protein-1; eIF2a, eukaryotic initiation factor 2a; eIF2aK, eukaryotic initiation factor
2a kinase; eEF2, eukaryotic elongation factor 2; eEF2K, eukaryotic elongation factor 2 kinase; AA-
tRNA, aminoacyl-tRNA. For the purpose of clarity, the mTOR-associated proteins, raptor and TSC1
and TSC2, have not been drawn.

Because amino acids may not directly activate PI 3-kinase, it is possible that PI 3-kinase
is on a pathway parallel to that of amino acids, and that the activation of both PI 3-kinase
(by insulin) and mTOR or another kinase (by amino acids) is required for full activation
of p70S6 kinase.76,78 It has been suggested that the phosphatidylinositol lipids are required
for membrane anchoring of one or more kinases, the activity of which is regulated by
amino acids.84 Another possibility arose from studies showing that amino acid-dependent
p70S6 kinase activation was abrogated in PDK1 –/– cells; however, amino acids were still
able to increase phosphorylation of 4E-BP1 in a rapamycin-sensitive manner. This indi-
cated that activation of mTOR function in the presence of amino acids was maintained in
these PDK1-deficient cells.78 Using p70S6 kinase phospho-specific antibodies, Wang et al.78
concluded that activation of p70S6 kinase requires two separate inputs: one through PDK1,
which results in phosphorylation of Thr229, and another through mTOR (PI 3-kinase and
PDK1 independent), resulting in phosphorylation of Thr389. (Note: Thr229 and Thr389
are equivalent to Thr252 and Thr412 of the long-splice variant of p70S6 kinase.) (Figure
16.1; cf. also Section 16.3.)
In order to account for the ability of high concentrations of amino acids to activate
p70S6 kinase in the absence of insulin by a mechanism that is sensitive to wortmannin or
LY294002, however, one has to assume that either basal activity of PI 3-kinase or only a
1382_C16.fm Page 261 Tuesday, October 7, 2003 6:40 PM

Chapter sixteen: Amino acid signaling and the control of protein metabolism 261

slight stimulation of PI 3-kinase by amino acids (or possibly inhibition of PTEN) may be
sufficient for Thr389 phosphorylation in p70S6 kinase.
In our opinion, Figure 16.1 satisfactorily accounts for the synergy between amino acids
and insulin with regard to p70S6 kinase activation. Cell swelling may mimic the effect of
insulin on PI 3-kinase,53,55 so that the effect of high concentrations of amino acids on p70S6
kinase consists of two components: one due to amino acids that increase cell volume,
resulting in PI 3-kinase and PDK1 activation, and another component (e.g., leucine),
required for mTOR activation (see also next paragraph). This would explain why high
concentrations of amino acids are able to activate p70S6 kinase in the absence of insulin.

16.2.6 Amino acids and the activation of mTOR


The mechanism by which amino acids activate mTOR is still unclear, and results are
controversial. In vitro, mTOR kinase activity toward 4E-BP1, with mTOR being immuno-
precipitated from either rapamycin-treated or amino acid-depleted CHO-IR or PC12 cells,
was not different from mTOR immunoprecipitated from control cells.50,85 This suggests
that mTOR activity changes were lost during isolation and may perhaps not be due to
phosphorylation of mTOR, but rather to an allosteric effect on mTOR itself. Other studies,
however, did show stable changes in mTOR activity. For instance, mTOR isolated from
amino acid-stimulated Jurkat cells could phosphorylate the protein phosphatase PP2A
in vitro.86 Addition of amino acids to HEK293 cells in the absence of insulin increased
phosphorylation of Ser2448 of mTOR; moreover, in vitro, mTOR could be phosphorylated
by protein kinase B, but only when mTOR was immunopurified from cells incubated with
amino acids.87 Changes in Ser2448 phosphorylation also ran in parallel with mTOR-
dependent signaling and rates of protein synthesis in rat skeletal muscle in vivo.88 Although
these experiments do show that mTOR can undergo stable changes in phosphorylation
in response to amino acid addition, it is not always clear whether Ser2448 phosphorylation
is essential for mTOR activity.76,83

16.2.7 Amino acids and protein phosphatases


It has been suggested that amino acids act as inhibitors of a protein phosphatase. In yeast,
for example, the rapamycin-sensitive TOR proteins affect PP2A activity, by modulating
the association of PP2A with the Tap42 protein. In the presence of nutrients Tap42 becomes
phosphorylated and associates with PP2A, which then becomes inhibited, while nutrient
deprivation or rapamycin addition reverses these events.76
The involvement of PP2A in amino acid signaling in mammalian cells is controversial.
Thus, in brain cells and in Jurkat cells, p70S6 kinase appeared to be tightly associated with
protein phosphatase 2A.86,89 In cells carrying the p70S6 kinase mutant that is resistant to
rapamycin and to amino acid depletion, the association with PP2A was lost.86 Moreover,
in some cell types, phosphorylation and activation of p70S6 kinase, S6, and 4E-BP1 could
be induced by PP2A inhibitors in the presence of rapamycin or in the absence of amino
acids.84,86,90 Similarly, dexamethasone-induced dephosphorylation of p70S6 kinase and of
4E-BP1 in L6 myoblasts in the presence of amino acids can be corrected by PP2A inhibi-
tors.91 In other cell types, however, PP2A inhibitors had no effect.50,89 In rat hepatocytes,
the PP2A inhibitor calyculin, but not okadaic acid, induced rapamycin-insensitive hyper-
phosphorylation of p70S6 kinase, which was additive to the rapamycin-sensitive phos-
phorylation induced by amino acid addition.92 However, the calyculin-induced hyperphos-
phorylation did not affect p70S6 kinase activity. Phosphatase inhibitors also did not affect
amino acid-induced phosphorylation of S6.14 According to Krause et al.,93 there are two
protein phosphatases in amino acid signaling, one protein phosphatase located upstream
1382_C16.fm Page 262 Tuesday, October 7, 2003 6:40 PM

262 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

of mTOR, which is perhaps activated by glutamate (and activates both glycogen synthase
and acetyl-CoA carboxylase) and a second protein phosphatase that dephosphorylates
p70S6 kinase and becomes inhibited when mTOR is activated (cf. Figure 16.1). The exist-
ence of two protein phosphatases would explain discrepancies in results obtained with
protein phosphatase inhibitors because the outcome of such experiments would have
depended on whether the protein phosphatase inhibitor had been added before or after
exposure of the cells to amino acids.

16.2.8 Negative feedback by amino acid signaling on insulin signaling


Although insulin and amino acids synergize with regard to their effects on mTOR-medi-
ated signaling, there are now also several reports showing that in muscle cells, adipocytes,
and hepatoma cells (but not in hepatocytes29), amino acids cause a time-dependent down-
regulation of insulin-mediated activation of PI 3-kinase, protein kinase B, and glucose
transport in a rapamycin-sensitive fashion.51,69,94–96 The presence of amino acids resulted
in increased ser/thr phosphorylation of IRS-1 and in decreased binding of the p85 regu-
latory subunit of PI 3-kinase to IRS-1, followed by increased, presumably proteasomal,
degradation of IRS-1.69,94 It has been proposed that this mechanism may underlie dimin-
ished glucose tolerance during high-protein feeding.51,69 Consumption of fish protein may
be favorable in this regard because of the relatively low plasma levels of leucine, tyrosine,
and some other amino acids under these conditions.69
Apparently, and paradoxically, amino acids are required for insulin-mediated activa-
tion of mTOR and its downstream targets, but they inhibit the initial part of the insulin
signaling pathway. The paradox lies in the fact that PI 3-kinase activity is essential for
amino acid-induced activation of mTOR and its downstream targets (see above). Down-
regulation of PI 3-kinase activation by amino acids would be counterproductive, therefore,
and would eventually lead to diminished protein synthesis and increased autophagic
protein breakdown. This is highly unlikely. Possibly, part of the activation of PI 3-kinase
by insulin proceeds independently of IRS-1. There is, indeed, evidence that the pathway
via IRS-2 may escape feedback inhibition by amino acids.94 This residual, IRS-2-mediated
activation of PI 3-kinase would in that case be sufficient for amino acid-induced activation
of mTOR and its downstream targets. mTOR may thus be considered to be a metabolic
switch that integrates both nutrition-mediated and insulin-mediated signals.94 Amino
acids would then simultaneously decrease transport and utilization of glucose by insulin-
sensitive tissues, and at the same time increase protein synthesis and decrease autophagic
proteolysis, thus contributing to stimulation of cell growth. The fact that an increase in
cAMP, a catabolic signal, decreases mTOR activity97 further supports a role of mTOR as
a nutritional sensor and nicely accounts for the glucagon/insulin antagonism we previ-
ously observed with regard to S6 phosphorylation in hepatocytes.14

16.2.9 mTOR-dependent signaling is antagonized by AMP kinase


In a recent study with HEK293 cells, yet another function of mTOR was proposed in that
this protein kinase may act as a sensor of not only amino acids but also intracellular ATP.98
It was noted that in contrast to several other protein kinases, the Km of ATP for mTOR
was high, about 1 mM, and within the physiological range of ATP concentrations. More-
over, by inhibiting either glycolytic or mitochondrial ATP production, a correlation was
found between the intracellular ATP concentration and the degree of phosphorylation of
p70S6 kinase or 4E-BP1, as indicators of mTOR activity in situ.98 Because inhibition of ATP
production also increases intracellular AMP levels via adenylate kinase, we considered
that AMP kinase may also contribute to mTOR inhibition when energy production is
1382_C16.fm Page 263 Tuesday, October 7, 2003 6:40 PM

Chapter sixteen: Amino acid signaling and the control of protein metabolism 263

compromised. Indeed, we and others found that under various conditions, activation of
AMP kinase strongly inhibited amino acid-dependent signaling88,92,93,99,100 and protein syn-
thesis.88,99,101 Activation of AMP kinase inhibited protein synthesis even more than rapamy-
cin, suggesting that AMP kinase inhibits protein synthesis not only through inhibition of
mTOR-mediated signaling but also at other steps controlling the protein-synthesizing
machinery.99 AMP kinase can also phosphorylate (and activate) eEF2 kinase independently
of mTOR.101,102
The association of mTOR with the mitochondrial outer membrane103 is of interest
because adenylate kinase is located in the mitochondrial intermembrane space. mTOR is
thus in a perfect position to sense changes in the AMP/ATP ratio.99
It has been reported that glucose helps to stimulate mTOR-dependent signaling in
CHO cells.104 Although this possibility could not have been considered at the time, in
retrospect, this may have been due to a decrease in AMP kinase activity. Likewise, the
activation by hypoxia of the endoplasmic reticulum protein kinase PERK,105 a mammalian
eIF2a kinase, which results in inhibition of protein synthesis, may have been caused by
AMP kinase action.
In view of the inhibitory effect of AMP kinase activation on protein synthesis via
interference with mTOR-dependent signaling, one would expect stimulation of autophagy.
Indeed, in yeast, the AMP kinase homologue snf1p is required for autophagy.106 However,
in hepatocytes, activation of AMP kinase results in inhibition of autophagy.107 Apparently,
in hepatocytes, AMP kinase, in addition to its ability to inhibit mTOR signaling, also
inactivates other protein components involved in the regulation of autophagy.

16.2.10 Amino acid signaling in pancreatic b-cells


Amino acid signaling also occurs in pancreatic b-cells and constitutes a fascinating feed-
back loop in the regulation of whole-body nitrogen metabolism. In these cells, too, amino
acids stimulated p70S6 kinase and 4E-BP1 phosphorylation in a rapamycin-sensitive and
wortmannin-sensitive fashion. Strikingly, b-cells from p70S6 kinase-deficient mice under-
produced insulin.108 As in other cells, insulin alone, whether produced by the b-cells
themselves (after glucose addition) or added externally, was not effective unless amino
acids were also present.66,77 Among the various amino acids, leucine was again most
effective.66,73 In addition to the ability of amino acids to promote b-cell proliferation via
increased signaling, it has been proposed that cytosolic glutamate in b-cells can directly
stimulate exocytosis of insulin presumably by causing swelling of the insulin-containing
granules.109Although attractive, this idea was refuted by data showing that with glutamine
present, the intracellular glutamate concentration was extremely high, yet insulin release
remained low.110 Interestingly, glutamine and leucine (in the absence of glucose) acted
synergistically, and in the presence of these two amino acids alone, insulin production
was as high as observed in the presence of glucose alone. According to the traditional
view, allosteric activation of glutamate dehydrogenase by leucine provides a-oxoglutarate
for the citric acid cycle.110 The combination of glutamine plus leucine was also particularly
effective in stimulating p70S6 kinase phosphorylation.73,111 Experiments with transaminase
inhibitors and with leucine analogues indicated that both the metabolism of leucine and
its ability to stimulate glutamate dehydrogenase were required to stimulate p70S6 kinase
phosphorylation.73,111 Inhibition of the mitochondrial respiratory chain, when glycolysis
was the only source of ATP production, eliminated the ability of glutamine plus leucine
to stimulate p70S6 kinase. It was concluded that the same mitochondrial events that
generate signals for leucine-stimulated exocytosis of insulin are required to activate the
amino acid signaling pathway, and that activation of protein synthesis by amino acid
signaling contributes to enhanced b-cell function.73 Although overall ATP levels did not
1382_C16.fm Page 264 Tuesday, October 7, 2003 6:40 PM

264 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

change, it is likely that AMP, and thus AMP kinase activity, was increased. If, as discussed
above, AMP kinase inhibits mTOR-dependent signaling, a mechanistic explanation is
established for the coupling between mitochondrial function and insulin release, in addi-
tion to the increase in cytosolic Ca++ concentration following closure of the plasma mem-
brane K+ATP channel by a high cytosolic ATP/ADP ratio.111 It is possible that part of the
stimulation of insulin production by glucose is caused by a decrease in the activity of
AMP kinase.112,113 Similarly, proper functioning of the malate–aspartate shuttle in
b-cells114,115 may be required to keep AMP kinase activity low.

16.2.11 Amino acid signaling in vivo


Although studies on amino acid-dependent signal transduction have mainly been carried
out in isolated cells, there is now ample evidence that amino acid signaling also plays an
important role in vivo. For example, the protein anabolic response after a protein meal in
man, rats, and mice occurred in the absence of changes in insulin concentration and was
accompanied by increased phosphorylation of 4E-BP1 and p70S6 kinase in muscle and
liver.61,116,117 In the rat, inhibition of insulin production by diazoxide eliminated the effect
of amino acids, suggesting that, as in isolated cells, insulin and amino acids are also both
required in vivo to induce a positive nitrogen balance.117 Likewise, in man, leucine and
insulin synergized with respect to their ability to stimulate p70S6 kinase phosphorylation
in muscle, while insulin but not leucine increased protein kinase B phosphorylation.118
Other data also suggested that in vivo the leucine-induced enhancement of protein syn-
thesis and the phosphorylation states of 4E-BP1 and p70S6 kinase are facilitated by
increases in serum insulin.119

16.3 Mechanisms
From all these studies, the picture emerges that amino acids somehow directly activate
mTOR activity. Whether PI 3-kinase is necessary for this activation is not known; however,
for stimulation of phosphorylation of downstream targets of mTOR (e.g., p70S6 kinase),
PI 3-kinase activity is required. Very recent studies have shed further light on the mech-
anism by which mTOR-mediated signaling may be controlled. mTOR-dependent signaling
in normal cells appears to be restrained by the tumor suppressor proteins TSC1 (hamartin)
and TSC2 (tuberin). Mutations in TSC1 and TSC2 result in tuberous sclerosis complex
(TSC), with development of benign tumors in various organs.120 The loss of function of
these proteins results in constitutive activation of p70S6 kinase/4E-BP1.121–123 There is still
some debate as to whether TSC1 and TSC2 inhibit mTOR121 or whether they directly affect
the activity of p70S6 kinase/4E-BP1.122 Interestingly, in normal cells, the function of TSC1
and TSC2 is suppressed by activation of PI 3-kinase (and possibly also protein kinase
B).121,122 This mechanism is in agreement with the notion, discussed in Section 16.2, that
PI 3-kinase and amino acid-dependent signaling are, indeed, on parallel pathways leading
to activation of mTOR or mTOR downstream targets. It also nicely explains why amino
acid-dependent signaling is sensitive to inhibition by PI 3-kinase inhibitors (Figure 16.1).
Moreover, it explains, at least in part, the synergy between amino acids and insulin.
The question still to be answered is the mechanism by which amino acids can activate
mTOR. Apart from the possibility that amino acids may stimulate a protein kinase acting
on mTOR as substrate, an attractive mechanism is also that amino acids, indeed, inhibit
a protein phosphatase. The simplest mechanism would be if mTOR in mammalian cells,
in analogy with yeast, would be a direct substrate for PP2A, although this remains to be
proven.87 Association of PP2A with mTOR downstream targets is also possible (cf. Figure
16.1). The recently discovered raptor, a protein that is associated with mTOR and controls
1382_C16.fm Page 265 Tuesday, October 7, 2003 6:40 PM

Chapter sixteen: Amino acid signaling and the control of protein metabolism 265

mTOR kinase activity, may also be part of the amino acid-sensing mechanism.124,125 Alter-
natively, amino acids may affect the activity of TSC1-2. At present, information on this
issue is not available.
Whatever the nature of the protein(s) that are activated (protein kinases, raptor) or
inhibited (protein phosphatases, TSC1-2) by amino acids, the amino acids can do so by a
direct, allosteric effect on these proteins. However, it is also possible that the plasma
membrane contains a specific amino acid receptor. The existence of a receptor was pro-
posed on the basis of the specific binding to the hepatocyte plasma membrane of Leu8-
Map, a small cell-impermeant globular peptide with eight leucine residues on the outside
of the molecule; moreover, the peptide effectively inhibited autophagy, and replacement
of the leucine residues by isoleucine rendered the peptide inactive.126 However, Leu8-Map
did not affect amino acid signaling,72,127 and its effect on autophagy could be ascribed to
the degradation of the peptide to free leucine.127
Evidence that amino acids may not act via a surface receptor was provided by the
demonstration that inhibition of plasma amino acid transport inhibited the activation of
p70S6 kinase.67 Likewise, in Xenopus laevis oocytes, leucine-induced signaling could only
be observed after overexpression of the L-leucine transport protein.128 These data clearly
indicate that the direct target for amino acids must be located intracellularly.
An as yet hypothetical mechanism is one in which the cell responds to changes in the
charging of tRNAs. This hypothesis is based on data in yeast (see Section 16.2) showing
that upon amino acid starvation, free, uncharged tRNA strongly binds to the protein kinase
GCN2, which then becomes activated and phosphorylates eIF2a. Whether free tRNA,
indeed, controls amino acid signaling in mammalian cells is controversial. Thus, in one
study with T-lymphoblastoid Jurkat cells, inhibition of amino acid–tRNA synthetase with
amino acid alcohols did prevent amino acid-induced activation of p70S6 kinase.67 This
could not be confirmed, however, in studies with freshly isolated rat adipocytes72 or with
CHO cells.104 In HEK293 cells, amino acid deprivation did not affect amino acid–tRNA
levels,98 which suggested that intracellular amino acid pools rather than the degree of
amino acid–tRNA charging controls amino acid signaling. Although these differences in
results may be ascribed to a difference in experimental systems, it is not likely that the
amino acid-sensing mechanism would be cell type dependent, especially because of the
similarity in amino acid specificity of amino acid signaling in the various cell types. Further
studies are clearly required to resolve this issue.
However, if tRNA is always fully charged with amino acids,98 the conclusion must be
that protein synthesis is never substrate limited, even under amino acid-deprived condi-
tions, and that the rate of protein synthesis is determined by the amino acid concentration
dependence of amino acid signaling only.
If, on the other hand, tRNA acts as an amino sensor (like in yeast) and tRNA charging
determines amino acid-dependent signaling, a possible mechanism underlying the ability
of cell swelling to potentiate this process can be provided. Thus, during regulatory volume
decrease when intracellular chloride falls, amino acid–tRNA synthetases may become
activated because chloride ions inhibit these enzymes, in analogy with the situation in
certain bacteria (cf. Figure 16.1).127
Recently, it was shown that in addition to amino acids, the mitogenic second messen-
ger phosphatidylic acid was also able to activate mTOR-mediated signaling, but only
when amino acids were present in sufficient amounts.129 This indicates that phosphatidylic
acid governs signaling in parallel to amino acids,130 similar to PI 3-kinase and amino acids,
as discussed in Section 16.2 (cf. Figure 16.1). Whether amino acids are able to affect
phosphatidylic acid concentrations (or vice versa) is not known.
Previously, we postulated that amino acid signaling, ultimately leading to S6 phospho-
rylation, provides an efficient mechanism by which both autophagic protein degradation
1382_C16.fm Page 266 Tuesday, October 7, 2003 6:40 PM

266 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

and protein synthesis could be oppositely controlled (cf. Introduction). We also proposed
a mechanism by which S6 phosphorylation may contribute to the reciprocal control of
protein synthesis and degradation. We proposed that S6 phosphorylation may promote
binding of ribosomes to the endoplasmic reticulum (ER) and enhance ER-linked protein
synthesis.14 In this context, it is of importance that in hepatocytes, synthesis of export
protein, but not of housekeeping protein, declines after amino acid deprivation.131 Ribo-
some binding to the ER would reduce the availability of ribosome-free regions of the ER,
which may be the source of the autophagosomal membrane.11 Thus, a common mechanism
would stimulate ER-linked protein synthesis while at the same time inhibiting proteolysis.
Removal of ribosomes by autophagy132 is thus prevented. We still think such a mechanism
is possible. As discussed in Section 16.1, activation of PI 3-kinase class I also simultaneously
stimulates protein synthesis and inhibits autophagic protein degradation. This provides
a second mechanism for the opposite control of protein synthesis and degradation.

16.4 Conclusions
As discussed in this review, there is now overwhelming evidence that amino acids are
important as signaling molecules, with insulin-like actions with regard to protein synthesis
and (autophagic) protein degradation. The ability of amino acids to stimulate signaling
in b-cells and insulin production further adds to their protein anabolic properties. It is
fascinating that the same signaling pathway appears to control both protein synthesis and
degradation. It must be pointed out that this pathway is different from the pathway of
glutamate signaling via glutamate receptors in the central nervous system and other
tissues. These receptors gate cation channels, and their activation causes depolarization
and an increase in cytosolic Ca++133; they also interact with PI 3-kinase.134 In cerebellar
Purkinje cells, the glutamate receptors interact with Beclin, and a role has been proposed
in the regulation of autophagy.135 Whether the glutamate receptor signaling pathway can
interact with the amino acid signaling pathway discussed in this chapter has not been
studied so far. This is an intriguing possibility, although the interaction of Ca++ with the
insulin–amino acid-dependent signaling pathway is complex.136,137
In amino acid-dependent signaling, mTOR occupies a central role as both a sensor of
intracellular amino acid concentrations and, via AMP kinase, a sensor of the cellular energy
state. It may be speculated that mTOR also senses amino acid-induced increases in cell
volume (or in connection with this, perhaps the intracellular chloride concentration),
although at present there is no evidence yet to support this. The role of mTOR in the
control of autophagic protein degradation, first proposed by us,14 is now generally
accepted, as indicated by a number of recent reviews.138–140 The importance of (amino acid-
dependent) mTOR-mediated signaling in cancer becomes more and more evident.76 Inter-
ventions used to combat cancer growth that interfere with amino acid-dependent signal-
ing, such as treatment with rapamycin and rapamycin analogues,141 not only inhibit
protein synthesis but also, at the same time, accelerate (autophagic) protein degradation,
and thus act as a two-edged sword.

References
1. Meijer, A.J. and van Dam, K., The metabolic significance of anion transport in mitochondria,
Biochim. Biophys. Acta, 346, 213–244, 1974.
2. Mortimore, G.E., Pösö, A.R., and Lardeux, B.R., Mechanism and regulation of protein deg-
radation in liver, Diabetes Metab. Rev., 5, 49–70, 1989.
3. Seglen, P.O. and Bohley, P., Autophagy and other vacuolar protein degradation mechanisms,
Experientia, 48, 158–172, 1992.
1382_C16.fm Page 267 Tuesday, October 7, 2003 6:40 PM

Chapter sixteen: Amino acid signaling and the control of protein metabolism 267

4. Baquet, A., Hue, L., Meijer, A.J., van Woerkom, G.M., and Plomp, P.J.A.M., Swelling of rat
hepatocytes stimulates glycogen synthesis, J. Biol. Chem., 265, 955–959, 1990.
5. Baquet, A., Maisin, L., and Hue, L., Swelling of rat hepatocytes activates acetyl-CoA car-
boxylase in parallel to glycogen synthase, Biochem. J., 278, 887–890, 1991.
6. Stoll, B., Gerok, W., Lang, F., and Häussinger, D., Liver cell volume and protein synthesis,
Biochem. J., 287, 217–222, 1992.
7. Lang F., Stehle, T., and Häussinger, D., Water, K+, H+, lactate and glucose fluxes during cell
volume regulation in perfused rat liver, Pflugers Arch., 413, 209–216, 1989.
8. Hallbrucker, C., vom Dahl, S., Lang, F., and Häussinger, D., Control of hepatic proteolysis
by amino acids: the role of cell volume, Eur. J. Biochem., 197, 717–724, 1991.
9. Meijer, A.J., Gustafson, L.A., Luiken, J.J.F.P., Blommaart, P.J., Caro, L.H.P., van Woerkom,
G.M., Spronk, C., and Boon, L., Cell swelling and the sensitivity of autophagic proteolysis
to inhibition by amino acids in isolated rat hepatocytes, Eur. J. Biochem., 215, 449–454, 1993.
10. Csonka, L.N., Physiological and genetic responses of bacteria to osmotic stress, Microbiol.
Rev., 53, 121–147, 1989.
11. Blommaart, E.F.C., Luiken, J.J.F.P., and Meijer, A.J., Autophagic proteolysis: control and
specificity, Histochem. J., 29, 365–385, 1997.
12. Luiken, J.J.F.P., Aerts, J.M.F.G., and Meijer, A.J., The role of the intralysosomal pH in the
control of autophagic proteolytic flux in rat hepatocytes, Eur. J. Biochem., 235, 564–573, 1996.
13. Luiken, J.J.F.P., Blommaart, E.F.C., Boon, L., van Woerkom, G.M., and Meijer, A.J., Cell
swelling and the control of autophagic proteolysis in hepatocytes: involvement of phospho-
rylation of ribosomal protein S6? Biochem. Soc. Trans., 22, 508–511, 1994.
14. Blommaart E.F.C., Luiken, J.J.F.P., Blommaart, P.J., van Woerkom, G.M., and Meijer, A.J.,
Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepato-
cytes, J. Biol. Chem., 270, 2320–2326, 1995.
15. Buse, M.G. and Reid, S.S., Leucine: a possible regulator of protein turnover in muscle, J. Clin.
Invest., 56, 1250–1261, 1975.
16. Fulks, R.M., Li, J.B., and Goldberg, A.L., Effects of insulin, glucose, and amino acids on
protein turnover in rat diaphragm, J. Biol. Chem., 250, 290–298, 1975.
17. Li, J.B. and Jefferson, L.S., Influence of amino acid availability on protein turnover in perfused
skeletal muscle, Biochim. Biophys. Acta, 544, 351–359, 1978.
18. Rennie, M.J., Hundal, H.S., Babij, P., MacLennan, P., Taylor, P.M., Watt, P.W., Jepson, M.M.,
and Millward, D.J., Characteristics of a glutamine carrier in skeletal muscle have important
consequences for nitrogen loss in injury, infection, and chronic disease, Lancet, ii, 1008–1012,
1986.
19. Holen, I., Gordon, P.B., and Seglen, P.O., Protein kinase-dependent effects of okadaic acid
on hepatocytic autophagy and cytoskeletal integrity, Biochem. J., 284, 633–636, 1992.
20. Holen, I., Gordon, P.B., and Seglen, P.O., Inhibition of hepatocytic autophagy by okadaic
acid and other protein phosphatase inhibitors, Eur. J. Biochem., 215, 113–122, 1993.
21. Holen, I., Strømhaug, P.E., Gordon, P.B., Fengsrud, M., Berg, T.O., and Seglen, P.O., Inhibition
of autophagy and multiple steps in asialoglycoprotein endocytosis by inhibitors of tyrosine
protein kinases (tyrphostins), J. Biol. Chem., 270, 12823–12831, 1995.
22. Dufner, A. and Thomas, G., Ribosomal S6 kinase signaling and the control of translation,
Exp. Cell Res., 253, 100–109, 1999.
23. Stolovich, M., Tang, H., Hornstein, E., Levy, G., Cohen, R., Bae, S.S., Birnbaum, M.J., and
Meyuhas, O., Transduction of growth or mitogenic signals into translational activation of
TOP mRNAs is fully reliant on the phosphatidylinositol 3-kinase-mediated pathway but
requires neither S6K1 nor rpS6 phosphorylation, Mol. Cell Biol., 22, 8101–8113, 2002.
24. Eskelinen, E.L., Prescott, A.R., Cooper, J., Brachmann, S.M., Wang, L., Tang, X., Backer, J.M.,
and Lucocq, J.M., Inhibition of autophagy in mitotic animal cells, Traffic, 3, 878–893, 2002.
25. Noda, T. and Ohsumi, Y., Tor, a phosphatidylinositol kinase homologue, controls autophagy
in yeast, J. Biol. Chem., 273, 3963–3966, 1998.
26. Kamada, Y., Funakoshi, T., Shintani, T., Nagano, K., Ohsumi, M., and Ohsumi, Y., Tor-
mediated induction of autophagy via an Apg1 protein kinase complex, J. Cell Biol., 150,
1507–1513, 2000.
1382_C16.fm Page 268 Tuesday, October 7, 2003 6:40 PM

268 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

27. Cruz, M.C., Goldstein, A.L., Blankenship, J., Del Poeta, M., Perfect, J.R., McCusker, J.H.,
Bennani, Y.L., Cardenas, M.E., and Heitman, J., Rapamycin and less immunosuppressive
analogs are toxic to Candida albicans and Cryptococcus neoformans via FKBP12-dependent
inhibition of TOR, Antimicrob. Agents Chemother., 45, 3162–3170, 2001.
28. Mordier, S., Deval, C., Bechet, D., Tassa, A., and Ferrara, M., Leucine limitation induces
autophagy and activation of lysosome-dependent proteolysis in C2C12 myotubes through
a mammalian target of rapamycin-independent signaling pathway, J. Biol. Chem., 275,
29900–29906, 2000.
29. Krause, U., Bertrand, L., Maisin, L., Rosa, M., and Hue, L., Signalling pathways and combi-
natory effects of insulin and amino acids in isolated rat hepatocytes, Eur. J. Biochem., 269,
3742–3750, 2002.
30. Shah, O.J., Antonetti, D.A., Kimball, S.R., and Jefferson, L.S., Leucine, glutamine, and tyrosine
reciprocally modulate the translation initiation factors eIF4F and eIF2B in perfused rat liver,
J. Biol. Chem., 274, 36168–36175, 1999.
31. Meijer, A.J., Gimpel, J.A., Deleeuw, G., Tischler, M.E., Tager, J.M., and Williamson, J.R.,
Interrelationships between gluconeogenesis and ureogenesis in isolated hepatocytes, J. Biol.
Chem., 253, 2308–2320, 1978.
32. Blommaart, E.F.C., Krause, U., Schellens, J.P.M., Vreeling-Sindelárová, H., and Meijer, A.J.,
The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy
in isolated rat hepatocytes, Eur. J. Biochem., 243, 240–246, 1997.
33. Schu, P.V., Takegawa, K., Fry, M.J., Stack, J.H., Waterfield, M.D., and Emr, S.D., Phosphati-
dylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting, Science, 260,
88–91, 1993.
34. Stack, J.H. and Emr, S.D., Vps34p required for yeast vacuolar protein sorting is a multiple
specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3-ki-
nase activities, J. Biol. Chem., 269, 31552–31562, 1994.
35. Petiot, A., Ogier-Denis, E., Blommaart, E.F.C., Meijer, A.J., and Codogno, P., Distinct classes
of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macro-
autophagy in HT-29 cells, J. Biol. Chem., 275, 992–998, 2000.
36. Maehama, T., Taylor, G.S., and Dixon, J.E., PTEN and myotubularin: novel phosphoinositide
phosphatases, Annu. Rev. Biochem., 70, 247–279, 2001.
37. Arico, S., Petiot, A., Bauvy, C., Dubbelhuis, P.F., Meijer, A.J., Codogno, P., and Ogier-Denis
E., The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the
phosphatidylinositol 3-kinase/protein kinase B pathway, J. Biol. Chem., 276, 35243–35246,
2001.
38. Kihara, A., Noda, T., Ishihara, N., and Ohsumi, Y., Two distinct Vps34 phosphatidylinositol
3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces
cerevisiae, J. Cell Biol., 52, 519–530, 2001.
39. Simonsen, A., Wurmser, A.E., Emr, S.D., and Stenmark, H., The role of phosphoinositides in
membrane transport, Curr. Opin. Cell. Biol., 13, 485–492, 2001.
40. Wurmser, A.E. and Emr, S.D., Novel PtdIns(3)P-binding protein Etf1 functions as an effector
of the Vps34 PtdIns 3-kinase in autophagy, J. Cell Biol., 158, 761–772, 2002.
41. Liang, X.H., Jackson, S., Seaman, M., Brown, K., Kempkes, B., Hibshoosh, H., and Levine,
B., Induction of autophagy and inhibition of tumorigenesis by beclin 1, Nature, 402, 672–676,
1999.
42. Kihara, A., Kabeya, Y., Ohsumi, Y., and Yoshimori, T., Beclin-phosphatidylinositol 3-kinase
complex functions at the trans-Golgi network, EMBO Rep., 2, 330–335, 2001.
43. Seglen, P.O. and Gordon, P.B., 3-Methyladenine: specific inhibitor of autophagic/lysosomal
protein degradation in isolated rat hepatocytes, Proc. Natl. Acad. Sci. U.S.A., 79, 1889–1892,
1982.
44. Foukas, L.C., Daniele, N., Ktori, C., Anderson, K.E., Jensen, J., and Shepherd, P.R., Direct
effects of caffeine and theophylline on p110 delta and other phosphoinositide 3-kinases:
differential effects on lipid kinase and protein kinase activities, J. Biol. Chem., 277,
37124–37130, 2002.
1382_C16.fm Page 269 Tuesday, October 7, 2003 6:40 PM

Chapter sixteen: Amino acid signaling and the control of protein metabolism 269

45. Holen, I., Gordon, P.B., Strømhaug, P.E., and Seglen, P.O., Role of cAMP in the regulation of
hepatocytic autophagy, Eur. J. Biochem., 236, 163–170, 1996.
46. Blondeau, F., Laporte, J., Bodin, S., Superti-Furga, G., Payrastre, B., and Mandel, J.L., Myo-
tubularin, a phosphatase deficient in myotubular myopathy, acts on phosphatidylinositol
3-kinase and phosphatidylinositol 3-phosphate pathway, Hum. Mol. Genet., 9, 2223–2229,
2000.
47. Walker, D.M., Urbe, S., Dove, S.K., Tenza, D., Raposo, G., and Clague, M.J., Characterization
of MTMR3: an inositol lipid 3-phosphatase with novel substrate specificity, Curr. Biol., 11,
1600–1605, 2001.
48. Tamura, M., Gu, J., Tran, H., and Yamada, K.M., PTEN gene and integrin signaling in cancer,
J. Natl. Cancer Inst., 91, 1820–1828, 1999.
49. Leslie, N.R. and Downes, C.P., PTEN: The down side of PI 3-kinase signalling, Cell. Signal.,
14, 285–295, 2002.
50. Hara, K., Yonezawa, K., Weng, Q.P., Kozlowski, M.T., Belham, C., and Avruch, J., Amino acid
sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector
mechanism, J. Biol. Chem., 273, 14484–14494, 1998.
51. Patti, M.E., Brambilla, E., Luzi, L., Landaker, E.J., and Kahn, C.R., Bidirectional modulation
of insulin action by amino acids, J. Clin. Invest., 101, 1519–1529, 1998.
52. Shigemitsu, K., Tsujishita, Y., Hara, K., Nanahoshi, M., Avruch, J., and Yonezawa K., Regu-
lation of translational effectors by amino acid and mammalian target of rapamycin signaling
pathways: possible involvement of autophagy in cultured hepatoma cells, J. Biol. Chem., 274,
1058–1065, 1999.
53. Krause, U., Rider, M.H., and Hue, L., Protein kinase signaling pathway triggered by cell
swelling and involved in the activation of glycogen synthase and acetyl-CoA carboxylase in
isolated rat hepatocytes, J. Biol. Chem., 271, 16668–16673, 1996.
54. Peyrollier, K., Hajduch, E., Blair, A.S., Hyde, R., and Hundal, H.S., L-leucine availability
regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 ac-
tivity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin
(mTOR) pathway in the L-leucine-induced up-regulation of system A amino acid transport,
Biochem. J., 350, 361–368, 2000.
55. Webster, C.R., Blanch, C.J., Phillips, J., and Anwer, M.S., Cell swelling-induced translocation
of rat liver Na(+)/taurocholate cotransport polypeptide is mediated via the phosphoinositide
3-kinase signaling pathway, J. Biol. Chem., 275, 29754–29760, 2000.
56. Tilly, B.C., Edixhoven, M.J., Tertoolen, L.G., Morii, N., Saitoh, Y., Narumiya, S., and de Jonge,
H.R., Activation of the osmo-sensitive chloride conductance involves P21rho and is accom-
panied by a transient reorganization of the F-actin cytoskeleton, Mol. Biol. Cell, 7, 1419–1427,
1996.
57. Brunn, G.J., Williams, J., Sabers, C., Wiederrecht, G., Lawrence, J.C., Jr., and Abraham, R.T.,
Direct inhibition of the signaling functions of the mammalian target of rapamycin by the
phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, EMBO J., 15, 5256–5267,
1996.
58. Kumar, V., Pandey, P., Sabatini, D., Kumar, M., Majumder, P.K., Bharti, A., Carmichael, G.,
Kufe, D., and Kharbanda, S., Functional interaction between RAFT1/FRAP/mTOR and
protein kinase cdelta in the regulation of cap-dependent initiation of translation, EMBO J.,
19, 1087–1097, 2000.
59. Ogier-Denis, E., Pattingre, S., El Benna, J., and Codogno, P., Erk1/2-dependent phosphor-
ylation of Galpha-interacting protein stimulates its GTPase accelerating activity and auto-
phagy in human colon cancer cells, J. Biol. Chem., 275, 39090–39095, 2000.
60. Häussinger, D., Schliess, F., Dombrowski, F., and Vom Dahl, S., Involvement of p38MAPK
in the regulation of proteolysis by liver cell hydration, Gastroenterology, 116, 921–935, 1999.
61. Shah, O.J., Anthony, J.C., Kimball, S.R., and Jefferson, L.S., 4E-BP1 and S6K1: translational
integration sites for nutritional and hormonal information in muscle, Am. J. Physiol., 279,
E715–E729, 2000.
62. Hershko, A. and Ciechanover, A., The ubiquitin system, Annu. Rev. Biochem., 67, 425–479,
1998.
1382_C16.fm Page 270 Tuesday, October 7, 2003 6:40 PM

270 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

63. Bennett, R.G., Hamel, F.G., and Duckworth, W.C., Insulin inhibits the ubiquitin-dependent
degrading activity of the 26S proteasome, Endocrinology, 141, 2508–2517, 2000.
64. Wang, X., Campbell, L.E., Miller, C.M., and Proud, C.G., Amino acid availability regulates
p70 S6 kinase and multiple translation factors, Biochem. J., 334, 261–267, 1998.
65. Fox, H.L., Kimball, S.R., Jefferson, L.S., and Lynch, C.J., Amino acids stimulate phosphor-
ylation of p70S6k and organization of rat adipocytes into multicellular clusters, Am. J. Physiol.,
274, C206–C213, 1998.
66. Xu, G., Kwon, G., Marshall, C.A., Lin, T.A., Lawrence, J.C., Jr., and McDaniel, M.L., Branched-
chain amino acids are essential in the regulation of PHAS-I and p70 S6 kinase by pancreatic
beta-cells: a possible role in protein translation and mitogenic signaling, J. Biol. Chem., 273,
28178–28184, 1998.
67. Iiboshi, Y., Papst, P.J., Kawasome, H., Hosoi, H., Abraham, R.T., Houghton, P.J., and Terada,
N., Amino acid-dependent control of p70(s6k): involvement of tRNA aminoacylation in the
regulation, J. Biol. Chem., 274, 1092–1099, 1999.
68. Campbell, L.E., Wang, X., and Proud, C.G., Nutrients differentially regulate multiple trans-
lation factors and their control by insulin, Biochem. J., 344, 433–441, 1999.
69. Tremblay, F. and Marette, A., Amino acid and insulin signaling via the mTOR/p70 S6 kinase
pathway: a negative feedback mechanism leading to insulin resistance in skeletal muscle
cells, J. Biol. Chem., 276, 38052–38060, 2001.
70. Kimball, S.R., Horetsky, R.L., and Jefferson, L.S., Implication of eIF2B rather than eIF4E in
the regulation of global protein synthesis by amino acids in L6 myoblasts, J. Biol. Chem., 273,
30945–30953, 1998.
71. Shigemitsu, K., Tsujishita, Y., Miyake, H., Hidayat, S., Tanaka, N., Hara, K., and Yonezawa,
K., Structural requirement of leucine for activation of p70 S6 kinase, FEBS Lett., 447, 303–306,
1999.
72. Lynch, C.J., Fox, H.L., Vary, T.C., Jefferson, L.S., and Kimball, S.R., Regulation of amino acid-
sensitive TOR signaling by leucine analogues in adipocytes, J. Cell Biochem., 77, 234–251, 2000.
73. Xu, G., Kwon, G., Cruz, W.S., Marshall, C.A., and McDaniel, M.L., Metabolic regulation by
leucine of translation initiation through the mTOR-signaling pathway by pancreatic beta-
cells, Diabetes, 50, 353–360, 2001.
74. Brunn, G.J., Hudson, C.C., Sekulic, A., Williams, J.M., Hosoi, H., Houghton, P.J., Lawrence,
J.C., Jr., and Abraham, R.T., Phosphorylation of the translational repressor PHAS-I by the
mammalian target of rapamycin, Science, 277, 99–101, 1997.
75. Burnett, P.E., Barrow, R.K., Cohen, N.A., Snyder, S.H., and Sabatini, D.M., RAFT1 phospho-
rylation of the translational regulators p70 S6 kinase and 4E-BP1, Proc. Natl. Acad. Sci. U.S.A.,
95, 1432–1437, 1998.
76. Gingras, A.C., Raught, B., and Sonenberg, N., Regulation of translation initiation by
FRAP/mTOR, Genes Dev., 15, 807–826, 2001.
77. Xu, G., Marshall, C.A., Lin, T.A., Kwon, G., Munivenkatappa, R.B., Hill, J.R., Lawrence, J.C.,
Jr., and McDaniel, M.L., Insulin mediates glucose-stimulated phosphorylation of PHAS-I by
pancreatic beta cells: an insulin-receptor mechanism for autoregulation of protein synthesis
by translation, J. Biol. Chem., 273, 4485–4491, 1998.
78. Wang, X., Li, W., Williams, M., Terada, N., Alessi, D.R., and Proud, C.G., Regulation of
elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase, EMBO J., 20, 4370–4379, 2001.
79. Dever, T.E., Translation initiation: adept at adapting, Trends Biochem. Sci., 24, 398–403, 1999.
80. Hinnebusch, A.G., Translational regulation of yeast GCN4: a window on factors that control
initiator-tRNA binding to the ribosome, J. Biol. Chem., 272, 21661–21664, 1997.
81. Natarajan, K., Meyer, M.R., Jackson, B.M., Slade, D., Roberts, C., Hinnebusch, A.G., and
Marton, M.J., Transcriptional profiling shows that Gcn4p is a master regulator of gene
expression during amino acid starvation in yeast, Mol. Cell Biol., 21, 4347–4368, 2001.
82. Talloczy, Z., Jiang, W., Virgin, H.W., Leib, D.A., Scheuner, D., Kaufman, R.J., Eskelinen, E.L.,
and Levine, B., Regulation of starvation- and virus-induced autophagy by the eIF2alpha
kinase signaling pathway, Proc. Natl. Acad. Sci. U.S.A., 99, 190–195, 2002.
1382_C16.fm Page 271 Tuesday, October 7, 2003 6:40 PM

Chapter sixteen: Amino acid signaling and the control of protein metabolism 271

83. Reynolds, T.H., Bodine, S.C., and Lawrence, J.C., Jr., Control of Ser2448 phosphorylation in
the mammalian target of rapamycin by insulin and skeletal muscle load, J. Biol. Chem., 277,
17657–17662, 2002.
84. Tang, H., Hornstein, E., Stolovich, M., Levy, G., Livingstone, M., Templeton, D., Avruch, J.,
and Meyuhas, O., Amino acid-induced translation of TOP mRNAs is fully dependent on
phosphatidylinositol 3-kinase-mediated signaling, is partially inhibited by rapamycin, and
is independent of S6K1 and rpS6 phosphorylation, Mol. Cell Biol., 21, 8671–8683, 2001.
85. Kleijn, M. and Proud, C.G., Glucose and amino acids modulate translation factor activation
by growth factors in PC12 cells, Biochem. J., 347, 399–406, 2000.
86. Peterson, R.T., Desai, B.N., Hardwick, J.S., and Schreiber, S.L., Protein phosphatase 2A
interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycin
associated protein, Proc. Natl. Acad. Sci. U.S.A., 96, 4438–4442, 1999.
87. Navé, B.T., Ouwens, M., Withers, D.J., Alessi, D.R., and Shepherd, P.R., Mammalian target
of rapamycin is a direct target for protein kinase B: identification of a convergence point for
opposing effects of insulin and amino-acid deficiency on protein translation, Biochem. J., 344,
427–431, 1999.
88. Bolster, D.R., Crozier, S.J., Kimball, S.R., and Jefferson, L.S., AMP-activated protein kinase
suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian
target of rapamycin (mTOR) signaling, J. Biol. Chem., 277, 23977–23980, 2002.
89. Westphal, R.S., Coffee, R.L., Jr., Marotta, A., Pelech, S.L., and Wadzinski, B.E., Identification
of kinase-phosphatase signaling modules composed of p70 S6 kinase-protein phosphatase
2A (PP2A) and p21-activated kinase-PP2A, J. Biol. Chem., 274, 687–692, 1999.
90. Parrott, L.A. and Templeton, D.J., Osmotic stress inhibits p70/85 S6 kinase through activation
of a protein phosphatase, J. Biol. Chem., 274, 24731–24736, 1999.
91. Shah, O.J., Kimball, S.R., and Jefferson, L.S., Glucocorticoids abate p70(S6k) and eIF4E func-
tion in L6 skeletal myoblasts, Am. J. Physiol. Endocrinol. Metab., 279, E74–E82, 2000.
92. Dubbelhuis, P.F. and Meijer, A.J., Amino acid-dependent signal transduction, in Cell and
Molecular Responses to Stress, Vol. 3, Sensing, Signaling and Cell Adaptation, Storey, K.B. and
Storey, J.M., Eds., Elsevier, Amsterdam, 2002, pp. 207–219.
93. Krause, U., Bertrand, L., and Hue, L., Control of p70 ribosomal protein S6 kinase and acetyl-
CoA carboxylase by AMP-activated protein kinase and protein phosphatases in isolated
hepatocytes, Eur. J. Biochem., 269, 3751–3759, 2002.
94. Takano, A., Usui, I., Haruta, T., Kawahara, J., Uno, T., Iwata, M., and Kobayashi, M., Mam-
malian target of rapamycin pathway regulates insulin signaling via subcellular redistribution
of insulin receptor substrate 1 and integrates nutritional signals and metabolic signals of
insulin, Mol. Cell Biol., 21, 5050–5062, 2001.
95. Hartley, D. and Cooper, G.M., Role of mTOR in the degradation of IRS-1: regulation of PP2A
activity, J. Cell Biochem., 85, 304–314, 2002.
96. Terruzzi, I., Allibardi, S., Bendinelli, P., Maroni, P., Piccoletti, R., Vesco, F., Samaja, M., and
Luzi, L., Amino acid- and lipid-induced insulin resistance in rat heart: molecular mecha-
nisms, Mol. Cell. Endocrinol., 190, 135–145, 2002.
97. Scott, P.H. and Lawrence, J.C., Jr., Attenuation of mammalian target of rapamycin activity
by increased cAMP in 3T3-L1 adipocytes, J. Biol. Chem., 273, 34496–34501, 1998.
98. Dennis, P.B., Jaeschke, A., Saitoh, M., Fowler, B., Kozma, S.C., and Thomas, G., Mammalian
TOR: a homeostatic ATP sensor, Science, 294, 1102–1105, 2001.
99. Dubbelhuis, P.F. and Meijer, A.J., Hepatic amino acid-dependent signaling is under the
control of AMP-dependent protein kinase, FEBS Lett., 521, 39–42, 2002.
100. Larsen, A.K., Møller, M.T., Blankson, H., Samari, H.R., Holden, L., and Seglen, P.O., Naringin-
sensitive phosphorylation of plectin, a cytoskeletal cross-linking protein, in isolated rat
hepatocytes, J. Biol. Chem., 277, 34826–34835, 2002.
101. Horman, S., Browne, G., Krause, U., Patel, J., Vertommen, D., Bertrand, L., Lavoinne, A.,
Hue, L., Proud, C.G., and Rider, M., Activation of AMP-activated protein kinase leads to the
phosphorylation of elongation factor 2 and an inhibition of protein synthesis, Curr. Biol., 12,
1419–1423, 2002.
1382_C16.fm Page 272 Tuesday, October 7, 2003 6:40 PM

272 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

102. McLeod, L.E. and Proud, C.G., ATP depletion increases phosphorylation of elongation factor
eEF2 in adult cardiomyocytes independently of inhibition of mTOR signalling, FEBS Lett.,
531, 448–452, 2002.
103. Desai, B.N., Myers, B.R., and Schreiber, S.L., FKBP12-rapamycin-associated protein associates
with mitochondria and senses osmotic stress via mitochondrial dysfunction, Proc. Natl. Acad.
Sci. U.S.A., 99, 4319–4324, 2002.
104. Patel, J., Wang, X., and Proud, C.G., Glucose exerts a permissive effect on the regulation of
the initiation factor 4E binding protein 4E-BP1, Biochem. J., 358, 497–503, 2001.
105. Koumenis, C., Naczki, C., Koritzinsky, M., Rastani, S., Diehl, A., Sonenberg, N., Koromilas,
A., and Wouters, B.G., Regulation of protein synthesis by hypoxia via activation of the
endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor
eIF2alpha, Mol. Cell Biol., 22, 7405–7416, 2002.
106. Wang, Z., Wilson, W.A., Fujino, M.A., and Roach, P.J., Antagonistic controls of autophagy
and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase,
and the cyclin-dependent kinase Pho85p, Mol. Cell Biol., 21, 5742–5752, 2001.
107. Samari, H.R. and Seglen, P.O., Inhibition of hepatocytic autophagy by adenosine, amino-
imidazole-4-carboxamide riboside, and N6-mercaptopurine riboside: evidence for involve-
ment of AMP-activated protein kinase, J. Biol. Chem., 273, 23758–23763, 1998.
108. Pende, M., Kozma, S.C., Jaquet, M., Oorschot, V., Burcelin, R., Le Marchand-Brustel, Y.,
Klumperman, J., Thorens, B., and Thomas, G., Hypoinsulinaemia, glucose intolerance and
diminished beta-cell size in S6K1-deficient mice, Nature, 408, 994–997, 2000.
109. Maechler, P. and Wollheim, C.B., Mitochondrial glutamate acts as a messenger in glucose-
induced insulin exocytosis, Nature, 402, 685–689, 1999.
110. MacDonald, M.J. and Fahien, L.A., Glutamate is not a messenger in insulin secretion, J. Biol.
Chem., 275, 34025–34027, 2000.
111. McDaniel, M.L., Marshall, C.A., Pappan, K.L., and Kwon, G., Metabolic and autocrine reg-
ulation of the mammalian target of rapamycin by pancreatic beta-cells, Diabetes, 51,
2877–2885, 2002.
112. Salt, I.P., Johnson, G., Ashcroft, S.J., and Hardie, D.G., AMP-activated protein kinase is
activated by low glucose in cell lines derived from pancreatic beta cells, and may regulate
insulin release, Biochem. J., 335, 533–539, 1998.
113. da Silva Xavier, G., Leclerc, I., Salt, I.P., Doiron, B., Hardie, D.G., Kahn, A., and Rutter, G.A.,
Role of AMP-activated protein kinase in the regulation by glucose of islet beta cell gene
expression, Proc. Natl. Acad. Sci. U.S.A., 97, 4023–4028, 2000.
114. Eto, K., Tsubamoto, Y., Terauchi, Y., Sugiyama, T., Kishimoto, T., Takahashi, N., Yamauchi,
N., Kubota, N., Murayama, S., Aizawa, T., Akanuma, Y., Aizawa, S., Kasai, H., Yazaki, Y.,
and Kadowaki, T., Role of NADH shuttle system in glucose-induced activation of mitochon-
drial metabolism and insulin secretion, Science, 283, 981–985, 1999.
115. Tan, C., Tuch, B.E., Tu, J., and Brown, S.A., Role of NADH shuttles in glucose-induced insulin
secretion from fetal beta-cells, Diabetes, 51, 2989–2996, 2002.
116. Long, W., Saffer, L., Wei, L., and Barrett, E.J., Amino acids regulate skeletal muscle PHAS-I
and p70 S6-kinase phosphorylation independently of insulin, Am. J. Physiol., 279, E301–E306,
2000.
117. Balage, M., Sinaud, S., Prod'homme, M., Dardevet, D., Vary, T.C., Kimball, S.R., Jefferson,
L.S., and Grizard, J., Amino acids and insulin are both required to regulate assembly of the
eIF4E.eIF4G complex in rat skeletal muscle, Am. J. Physiol., 281, E565–E574, 2001.
118. Greiwe, J.S., Kwon, G., McDaniel, M.L., and Semenkovich, C.F., Leucine and insulin activate
p70 S6 kinase through different pathways in human skeletal muscle, Am. J. Physiol., 281,
E466–E471, 2001.
119. Anthony, J.C., Lang, C.H., Crozier, S.J., Anthony, T.G., MacLean, D.A., Kimball, S.R., and
Jefferson, L.S., Contribution of insulin to the translational control of protein synthesis in
skeletal muscle by leucine, Am. J. Physiol., 282, E1092–E1101, 2002.
120. Young, J. and Povey, S., The genetic basis of tuberous sclerosis, Mol. Med. Today, 4, 313–319,
1998.
1382_C16.fm Page 273 Tuesday, October 7, 2003 6:40 PM

Chapter sixteen: Amino acid signaling and the control of protein metabolism 273

121. Inoki, K., Li, Y., Zhu, T., Wu, J., and Guan, K.L., TSC2 is phosphorylated and inhibited by
Akt and suppresses mTOR signalling, Nat. Cell Biol., 4, 648–657, 2002.
122. Jaeschke, A., Hartkamp, J., Saitoh, M., Roworth, W., Nobukuni, T., Hodges, A., Sampson, J.,
Thomas, G., and Lamb, R., Tuberous sclerosis complex tumor suppressor-mediated S6 kinase
inhibition by phosphatidylinositide-3-OH kinase is mTOR independent, J. Cell Biol., 159,
217–224, 2002.
123. Kenerson, H.L., Aicher, L.D., True, L.D., and Yeung, R.S., Activated mammalian target of
rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors, Cancer
Res., 62, 5645–5650, 2002.
124. Kim, D.H., Sarbassov, D.D., Ali, S.M., King, J.E., Latek, R.R., Erdjument-Bromage, H., Tempst,
P., and Sabatini, D.M., mTOR interacts with raptor to form a nutrient-sensitive complex that
signals to the cell growth machinery, Cell, 110, 163–175, 2002.
125. Hara, K., Maruki, Y., Long, X., Yoshino, K., Oshiro, N., Hidayat, S., Tokunaga, C., Avruch,
J., and Yonezawa, K., Raptor, a binding partner of target of rapamycin (TOR), mediates TOR
action, Cell, 110, 177–189, 2002.
126. Miotto, G., Venerando, R., Marin, O., Siliprandi, N., and Mortimore, G.E., Inhibition of
macroautophagy and proteolysis in the isolated rat hepatocyte by a nontransportable deriv-
ative of the multiple antigen peptide Leu8-Lys4-Lys2-Lys-beta Ala, J. Biol. Chem., 269,
25348–25353, 1994.
127. van Sluijters, D.A., Dubbelhuis, P.F., Blommaart, E.F.C., and Meijer, A.J., Amino-acid-depen-
dent signal transduction, Biochem. J., 351, 545–550, 2000.
128. Christie, G.R., Hajduch, E., Hundal, H.S., Proud, C.G., and Taylor, P.M., Intracellular sensing
of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase in a target of rapamycin-
dependent manner, J. Biol. Chem., 277, 9952–9957, 2002.
129. Fang, Y., Vilella-Bach, M., Bachmann, R., Flanigan, A., and Chen, J., Phosphatidic acid-
mediated mitogenic activation of mTOR signaling, Science, 294, 1942–1945, 2001.
130. Chen, J. and Fang, Y., A novel pathway regulating the mammalian target of rapamycin
(mTOR) signaling, Biochem. Pharmacol., 64, 1071–1077, 2002.
131. Tanaka, K. and Ichihara, A., Different effect of amino acid deprivation on syntheses of intra-
and extracellular proteins in rat hepatocytes in primary culture, J. Biochem. (Tokyo), 94,
1339–1348, 1983.
132. Lardeux, B.R. and Mortimore, G.E., Amino acid and hormonal control of macromolecular
turnover in perfused rat liver: evidence for selective autophagy, J. Biol. Chem., 262,
14514–14519, 1987.
133. Nedergaard, M., Takano, T., and Hansen, A.J., Beyond the role of glutamate as a neurotrans-
mitter, Nat. Rev. Neurosci., 3, 748–755, 2002.
134. Perkinton, M.S., Ip, J.K., Wood, G.L., Crossthwaite, A.J., and Williams, R.J., Phosphatidy-
linositol 3-kinase is a central mediator of NMDA receptor signalling to MAP kinase (Erk1/2),
Akt/PKB and CREB in striatal neurones, J. Neurochem., 80, 239–254, 2002.
135. Yue, Z., Horton, A., Bravin, M., DeJager, P.L., Selimi, F., and Heintz, N., A novel protein
complex linking the delta 2 glutamate receptor and autophagy: implications for neurode-
generation in lurcher mice, Neuron, 35, 921–933, 2002.
136. Worrall, D.S. and Olefsky, J.M., The effects of intracellular calcium depletion on insulin
signaling in 3T3-L1 adipocytes, Mol. Endocrinol., 16, 378–389, 2002.
137. Hannan, K.M., Thomas, G., and Pearson, R.B., Activation of S6K1 requires an initial calcium-
dependent priming event involving formation of a high molecular weight signalling com-
plex, Biochem. J., 370, 469–477, 2003.
138. Dennis, P.B., Fumagalli, S., and Thomas, G., Target of rapamycin (TOR): balancing the
opposing forces of protein synthesis and degradation, Curr. Opin. Genet. Dev., 9, 49–54, 1999.
139. Schmelzle, T. and Hall, M.N., TOR, a central controller of cell growth, Cell, 103, 253–262. 2000.
140. Raught, B., Gingras, A.C., and Sonenberg, N., The target of rapamycin (TOR) proteins, Proc.
Natl. Acad. Sci. U.S.A., 98, 7037–7044, 2001.
141. Hidalgo, M. and Rowinsky, E.K., The rapamycin-sensitive signal transduction pathway as
a target for cancer therapy, Oncogene, 19, 6680–6686, 2000.
1382_C16.fm Page 274 Tuesday, October 7, 2003 6:40 PM
1382_C17.fm Page 275 Tuesday, October 7, 2003 6:43 PM

chapter seventeen

The role of amino acids


in the control of proteolysis
Stephan vom Dahl
Heinrich Heine University
Dieter Häussinger
Heinrich Heine University

Contents
Abbreviations...............................................................................................................................275
Introduction..................................................................................................................................276
17.1 Assessment of autophagic proteolysis and sequestration in rat liver......................276
17.2 Regulation of proteolysis by amino acids .....................................................................277
17.3 Mechanisms of amino acid-induced proteolysis regulation ......................................278
17.3.1 Sites of amino acid-dependent inhibition of proteolysis ...............................278
17.3.2 Cell hydration and proteolysis regulation........................................................279
17.3.3 Signaling in hydration-dependent regulation of autophagic proteolysis ...281
17.3.4 Cell hydration-independent signaling in proteolysis regulation .................282
17.4 Clinical relevance of amino acid-dependent proteolysis regulation ........................284
Acknowledgments ......................................................................................................................284
References .....................................................................................................................................284

Abbreviations
AV autophagic vacuole
Erk extracellular signal-regulated kinase
FAK focal adhesion kinase
mTOR mammalian target of rapamycin
p38MAPK p38 mitogen-activated protein kinase
p70S6K p70 ribosomal S6 protein kinase
PI 3-kinase phosphoinositide 3-kinase
RVD regulatory volume decrease

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 275
1382_C17.fm Page 276 Tuesday, October 7, 2003 6:43 PM

276 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Introduction
Protein degradation in animal cells involves lysosomal and nonlysosomal proteolysis, the
latter comprising ubiquitin-dependent proteolysis and the action of a variety of other
cytosolic proteases. Substrate selectivity may exist for the different ways of cellular pro-
teolysis: for example, short-lived proteins are preferentially degraded by nonlysosomal
proteolysis. During amino acid starvation in liver, the most important pathway is lysos-
omal proteolysis involving the formation of autophagic vacuoles.
The process of autophagic proteolysis can be divided into several steps:

• Sequestration
• Acidification
• Fusion
• Digestion

During the first step (sequestration), a membrane envelops a region of cytoplasm into
a closed vacuole, forming the early autophagosome, which is considered a distinct
organelle and is morphologically characterized by a double-layer sequestrational mem-
brane. The half-life of an autophagosome is about 8 min.1 Then an acidic intraorganellar
pH within the autophagosome is generated by a proton pump (acidification2). This organelle
is termed late autophagosome. The next step is fusion of the autophagosome with the
primary lysosome. With this formation of a secondary lysosome, the process of digestion
is activated. After digestion, a residual body is visualized by electron microscopy.
Autophagic and endocytotic pathways can meet and intermix their contents in lysosomal
and prelysosomal compartments.
Owing to the heterogeneity of autophagic vacuoles (macroautophagy), the term
microautophagy has been coined. The existence of microautophagy is suggested by the fact
that the complete disappearance of autophagic vacuoles (AV), as it occurs in the presence
of supraphysiological amino acid concentrations, was accompanied by only 70% inhibition
of proteolysis, with degradable protein still being demonstrable in lysosomal fractions.3
It is only macroautophagic proteolysis, in the following specified as autophagic proteol-
ysis, and not the other ways of cellular proteolysis, that is controlled by amino acids. This
chapter deals with macroautophagic proteolysis only and will focus on liver tissue and
cell hydration changes, either by changes of ambient osmolarity, by amino acids, or by
hormones. For comprehensive work on other pathways of proteolysis and cell hydration-
independent amino acid signaling, the reader is referred to the comprehensive work of
Doherty and Mayer,4 Blommaart et al.,5 and van Sluijters et al.6

17.1 Assessment of autophagic proteolysis and sequestration


in rat liver
Hepatic proteolysis has been studied in the isolated perfused rat liver, in isolated hepa-
tocytes, either being perifused or in suspension, or in primary hepatocyte cultures. As
branched-chain amino acids are not catabolized in liver, the steady-state release of leucine,
valine, or isoleucine reflects the rate of hepatic protein breakdown, provided conditions
are met that allow correction for the reutilization of these amino acids for protein synthesis
or minimization of this latter process.
In vivo prelabeling of hepatic proteins by i.p. injection of either [14C]-valine or [3H]-leu-
cine 16 h prior to the perfusion experiment or to isolation of hepatocytes allows the
monitoring of proteolysis even in the presence of high concentrations of branched-chain
amino acids. The label released from the liver under these conditions is almost exclusively
1382_C17.fm Page 277 Tuesday, October 7, 2003 6:43 PM

Chapter seventeen: The role of amino acids in the control of proteolysis 277

derived from the labeled proteins. The reutilization of labeled amino acids for protein
synthesis can be minimized by using a nonrecirculating, open system for hepatocyte
isolation/liver perfusion or by addition of unlabeled valine/leucine at a concentration of
0.1 to 1 mmol/l, respectively.
The efficacy of this approach is based on the assumption that no significant channeling
of proteolysis-derived amino acids into protein synthesis exists. The extent of protein
synthesis can independently be assessed by bolus injection of labeled valine to the perfu-
sate.7 Using these prelabeling techniques, it has to be kept in mind that due to different
half-lives of proteins, lasting from minutes to days, inhomogeneous labeling of these
proteins will necessarily result.
In general, open experimental systems, such as nonrecirculating liver perfusion or
hepatocyte perifusion, are preferable for proteolysis studies, because they allow the main-
taining of constant substrate concentrations and prevent the accumulation of metabolites,
which interfere with proteolysis itself, e.g., ammonia.8 Cycloheximide, which at a concen-
tration of 5 mmol/l inhibits protein synthesis by 95%, may also be employed to prevent
branched-chain amino acid reutilization; however, this inhibitor affects proteolysis itself,
depending on the experimental system used. Whereas in perifused hepatocytes cyclohex-
imide was reported not to interfere with proteolysis,9,10 it abolishes the insulin sensitivity
of proteolysis in primary cultures of hepatocytes and, after transient stimulation, inhibits
proteolysis in perfused rat liver.11 Advantages of the perfused rat liver for studying pro-
teolysis are the integrity of the cytoskeleton and cell polarity and maintained cell–cell
interactions. The integrity of hepatic architecture in this model is of relevance, since key
roles of microtubules and integrin-based cellular signal transduction for proteolysis reg-
ulation were shown recently.11–13 Functionally, the various steps of proteolysis can be
studied by analyzing the accumulation of electro-injected [14C]-lactose in autophagic vac-
uoles.14,15 Lysosomal proteolysis requires an acidic intraorganellar pH of about 5, which
is generated by vacuolar H+-ATPases.2 Acidotropic agents, i.e., weak bases such as meth-
ylamine, chloroquine, or ammonia, accumulate in this compartment and inhibit proteol-
ysis due to vacuolar alkalinization and lysosomal swelling, which impair fusion and
lysosomal protease activation.
Morphologically, AV are defined as bits of cytoplasm sequestered from the remaining
cytoplasm by one or two membranes. The morphology of autophagic vacuoles has been
described in detail elsewhere.1 The fractional volume of autophagic vacuoles that is
defined as the volume of autophagic vacuoles per volume of liver cell cytoplasm (Vav/Vc)
can be calculated by morphometric methods and averages, in the absence of amino acids,
to be about 0.5% of the whole cytoplasm of rat liver.16,17

17.2 Regulation of proteolysis by amino acids


In perfused liver or isolated hepatocytes, the rate of protein degradation may become as
high as 4 to 5% of cytosolic protein per hour. About 70% of this proteolytic rate can be
ascribed to macroautophagy, i.e., autophagic proteolysis. This process is regulated by
insulin, glucagon, amino acids, and cell hydration.3,5,7,18–20 Amino acid withdrawal from
perfused rat liver increases autophagic proteolysis dramatically and instantaneously,19 and
after a short lag period, proteolysis rises to a sustained maximal rate. Reinstitution of
amino acids at four- to fivefold physiological concentrations almost completely suppresses
this autophagic proteolytic response.21,22
Amino acid control of proteolysis is observed not only in isolated perfused rat liver
but also in isolated or cultivated rat hepatocytes.9,23,24 However, quantitative differences
exist regarding the antiproteolytic effects exerted by specific amino acids or amino acid
mixtures, depending on the experimental system used. In perfused rat liver, the amino
1382_C17.fm Page 278 Tuesday, October 7, 2003 6:43 PM

278 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

acids with highest antiproteolytic capacity were leucine, phenylalanine, tyrosine,


glutamine, proline, histidine, and tryptophane (so-called regulatory amino acids), whereas
other amino acids were almost ineffective (nonregulatory amino acids). When the regu-
latory amino acids were tested as a mixture, they suppressed proteolysis below 0.5-fold
and above 3-fold, their respective physiological concentrations, whereas at the 2- to 3-fold
physiological level, the proteolysis inhibition almost disappeared.
This concentration-dependent loss of efficacy was shown for most regulatory amino
acids25,26 and can be avoided by alanine, which itself was only slightly effective, suggesting
a permissive function for this amino acid.3 Similar conclusions were deduced from exper-
iments with isolated perifused hepatocytes from starved rats. Here, both alanine and
leucine had little effect on proteolysis; however, when added together, these amino acids
exhibited a strong antiproteolytic effect resembling that of a complete amino acid mixture.9
A detailed review of amino acid requirements for proteolysis inhibition in rat liver is given
in Mortimore and Pöso.27 In isolated rat hepatocytes, the strongest inhibition was found
with glutamine and asparagine, followed by leucine, aromatic amino acids, and histidine.23
Serine, glycine, and alanine were largely ineffective in isolated rat hepatocytes, but inhi-
bition of proteolysis by these amino acids was reported in perfused rat liver.28
Thus, although the data from many laboratories consistently show an inhibition of
proteolysis by amino acids, there is variability with respect to the magnitude of the
antiproteolytic effect, the nutritional state of the animals, the experimental system used,
and the concentrations of the employed amino acids. Even more complex is the emerging
image, when combinations of different amino acids are used. Further, the permissive action
of alanine can be mimicked by pyruvate or octanoate, suggestive of a regulatory role of
alanine in linking energy needs and proteolysis.

17.3 Mechanisms of amino acid-induced proteolysis regulation


The mechanisms involved in amino acid-induced regulation of proteolysis in rat liver are
only incompletely understood. Recent work has brought substantial progress: new prin-
ciples of regulation, e.g., cell hydration as an independent regulation principle, have been
identified and new potential signaling pathways in autophagic proteolysis, i.e., the phos-
phoinositide 3-kinase (PI 3-kinase) way and the p38MAPK pathway, have been characterized.

17.3.1 Sites of amino acid-dependent inhibition of proteolysis


Morphometric studies revealed that amino acid deprivation leads to an instant formation
of autophagic vacuoles (sequestration) in rat liver, which ceases upon amino acid rein-
stallation.29 Studies with isolated hepatocytes loaded with [14C]-sucrose by electroperme-
abilization showed that amino acids inhibit the sequestrational step, with histidine being
most effective.20 Besides sequestration, other steps are also involved. For example, aspar-
agine, like vinblastine, impedes fusion of autophagosomes with primary lysosomes.30
Further, ammonia derived from the breakdown of amino acids may inhibit proteolysis
due to inhibition of acidification and fusion.31 However, this phenomenon may only play
a role at unphysiologically high amino acid concentrations.21
Hepatic proteolysis requires energy and is sensitive to changes of intracellular ATP.14,15
Sequestration, acidification, and fusion were all found to be energy dependent, with
sequestration being the step most sensitive to small changes in intracellular ATP. Amino
acids, however, were shown to inhibit proteolysis without affecting cellular ATP levels,14
suggesting that amino acids do not exert their regulatory role in protein degradation via
changes of intracellular ATP.
1382_C17.fm Page 279 Tuesday, October 7, 2003 6:43 PM

Chapter seventeen: The role of amino acids in the control of proteolysis 279

Like amino acids, insulin inhibits autophagic sequestration in isolated hepatocytes,


but only in the presence of amino acids.30 Glucagon, on the other hand, stimulated pro-
teolysis, but only in the presence of amino acids. In perfused rat liver, however, inhibition
or stimulation of proteolysis by insulin or glucagon does not require the presence of amino
acids (except 0.1 mmol/l leucine).32–34
Studies on the antiproteolytic effects of leucine and glutamine, which are considered
the most potent regulatory amino acids, suggested that their albeit unidentified site of
recognition may be close to the plasma membrane.25 Similar conclusions were obtained
for phenylalanine.26 The structural requirements of leucine and its analogues for proteol-
ysis inhibition have recently been characterized.25

17.3.2 Cell hydration and proteolysis regulation


It was recognized since the late 1980s that the cellular hydration state can change within
minutes under the influence of hormones and nutrients (reviewed in the work of
Häussinger and others35–37). Many amino acids are taken up by liver via concentrative,
Na+-dependent transport systems in the plasma membrane (e.g., glutamine via system N
and glycine and alanine via system A). These transporters can build up intra- or extracel-
lular concentration gradients of >20. The accumulation of Na+ and amino acids within the
cell leads to osmotic hepatocyte swelling and induces a volume regulatory K+ efflux from
the cell.38–40 This regulatory volume decrease (RVD) does not completely restore original
cell volume but prevents excessive cell swelling. The cells remain in a slightly swollen
state as they face increased amino acid concentrations in the extracellular environment.
It was pointed out that amino acid-induced cell swelling occurs already at concentra-
tions in the physiological concentration range. Glutamine-induced cell swelling is half
maximal at concentrations of about 0.7 mmol/l, i.e., a physiological portal concentration,
and is maximal at 2 mmol/l. Thus, physiological fluctuations of portal amino acid con-
centrations are probably accompanied by parallel alterations of liver cell volume in vivo.
A possible link between amino acid-induced cell swelling and proteolysis inhibition came
from the observation that hypo-osmotic cell swelling in perfused rat liver inhibited pro-
teolysis.41 As an example, the antiproteolytic effects of glutamine and glycine can fully be
explained by amino acid-dependent cell swelling (Figure 17.1): the proteolysis inhibition
by these amino acids can quantitatively be mimicked fully by hypo-osmotic cell swelling
when induced to the same extent as by the combination of these two amino acids.28,33,41
The threefold higher antiproteolytic capacity of glycine in livers from starved rats than in
livers from fed rats is explained by the threefold higher swelling of glycine during star-
vation,28,42 due to adaptive up-regulation of system A under these conditions.43 When
individual amino acids are tested for their swelling potency in the fed and starved states,
glutamine appears to be most effective to increase cell hydration in liver.28,42 This may, in
part, explain why the protein anabolic effects of this amino acid received special attention
in the past.44 However, not all amino acids exert their antiproteolytic effects via changes
of cell hydration, but they do activate specific signaling mechanisms, as discussed below.
Cell hydration changes are of essential importance for the actions of the hormones
insulin and glucagon on proteolysis. These hormones are potent and rapidly acting mod-
ulators of cell hydration.34,45–47 In liver, insulin stimulates Na+/H+ exchange and Na-K-2
Cl cotransport and the Na+/K+-ATPase. The concerted action of these transporters leads
to cellular accumulation of potassium, sodium, chloride, and, consequently, cell swelling.
Recently, insulin-stimulated Na-K-2 Cl cotransport has been characterized in liver.48
In contrast, glucagon leads to a depletion of intracellular K+ via activation of Ba2+-
and quinidine-sensitive K+ channels. Accordingly, glucagon shrinks the cells. The antipro-
teolytic action of insulin can be quantitatively mimicked by hypo-osmotic cell swelling to
1382_C17.fm Page 280 Tuesday, October 7, 2003 6:43 PM

280 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

40

30

20

10

-20 -10 10 20
% change of cell water
-10

-20
hypotonic
glutamine
-30 glycine
alanine
glutamine + glycine
-40 insulin
insulin/bumetanide
ethanol
acetaldehyde
ethanol/bumetanide
ethanol/methylpyrazole
glucagon/hypotonic taurocholate
glucagon/insulin glycerol
cAMP/hypotonic IGF-1
hypertonic/insulin BaCl2
cAMP+vasopressin glucagon + insulin
insulin + cAMP glucagon + hypotonic

Figure 17.1 Relationship between cell hydration and proteolysis control in perfused rat liver. Cell
volume in perfused rat liver was determined as intracellular water space according to vom Dahl et
al.96 and averaged — in livers from fed rats — 569 ± 7 ml/g (n = 234). Proteolysis was assessed as
[3H]-leucine release into effluent perfusate from amino acid-free perfused livers of fed rats, which
had been prelabeled by i.p. injection of [3H]-leucine 16 h prior to the perfusion experiment.41
Autophagic proteolysis is already maximally stimulated in the absence of amino acids or hormones.
Proteolysis-stimulating effects of hyperosmolarity, glucagon, or cAMP become apparent when pro-
teolysis is preinhibited by either hypo-osmolarity, insulin, or amino acids. (Modified according to
Häussinger et al.33 and vom Dahl and Häussinger 42.)

the same extent as insulin. Hyperosmotic shrinkage reverses both insulin-induced cell
swelling and the antiproteolytic effect of the hormone, and abolition of insulin-induced
cell swelling by inhibitors of Na+/H+ exchange (amiloride), Na-K-2 Cl cotransport (bumet-
anide, furosemide), and the Na+/K+-ATPase (ouabain) also prevents insulin-induced pro-
teolysis inhibition.32,34
Half-maximal cell swelling or shrinkage by insulin or glucagon is already found at
physiological portal hormone concentrations in vivo.47 Accordingly, physiological fluctu-
ations of portal insulin and glucagon concentrations may modify liver cell volume also
in vivo. As shown in Figure 17.1, there is a linear relationship between proteolytic activity
and cell hydration: an increase of cell hydration is followed by a decrease of proteolysis,
whereas cell shrinkage stimulates proteolysis.33 In livers from fed rats, 1% increase of cell
hydration is accompanied by a 2% decrease of proteolysis on average.42 This relationship
1382_C17.fm Page 281 Tuesday, October 7, 2003 6:43 PM

Chapter seventeen: The role of amino acids in the control of proteolysis 281

is maintained, regardless of whether cell volume is increased by hypo-osmolarity, amino


acids, ethanol, acetaldehyde,49 Ba2+, insulin, taurocholic acid, glycerol, or bumetanide42
(Figure 17.1) or decreased by glucagon, cAMP, or vasopressin.47 In summary, alterations
of the cellular hydration state do act as a second messenger of nutrients and hormones
(reviewed in Häussinger and others35–37,50,51).
An important element in amino acid-dependent proteolysis regulation is the cytoskel-
eton. Cell swelling is known to increase mRNA levels for b-actin and tubulin, to increase
actin polymerization, and to stabilize microtubules.52,53 Further, the decrease of autophago-
somes and the antiproteolytic action of hypo-osmolarity or glutamine/glycine are largely
abolished in the presence of colchicine.11,12 Microtubular integrity is a prerequisite for
proteolysis regulation by cell volume. The microtubular integrity is disturbed following
the preparation of isolated hepatocytes, but not in the hepatocyte in the in situ perfused
liver. This might explain why proteolysis is very sensitive to hypo-osmotic cell swelling
in perfused rat liver but not in freshly isolated hepatocytes.10,54 However, an inhibition of
proteolysis upon hypo-osmotic cell swelling was also demonstrated in isolated hepato-
cytes, when amino acids were present.10 It could be speculated that amino acids accelerate
the reorganization of cytoskeletal structures in hepatocytes and thereby help in the resto-
ration of the sensitivity of proteolysis to cell volume changes. In line with this, glutamine
was found to increase actin polymerization and to stabilize microtubules in rat liver.52,53
The mechanisms of how cell swelling could relate to proteolysis regulation have at
least partially been elucidated. Swelling impairs the acidification of intracellular vacuolar
compartments, such as endocytotic vesicles,55–57 as do lysosomotropic agents, e.g., NH4+,
and it might be conceivable that the prevention of vacuolar acidification adds partially to
the mechanisms of swelling-induced proteolysis inhibition.58 Nevertheless, the time course
and inhibitor profile of swelling-induced alkalinization of intracellular vesicles make a
dominant role of this mechanism less likely.55–57

17.3.3 Signaling in hydration-dependent regulation of autophagic proteolysis


Substantial progress has been made in identifying signal transduction pathways linking
cell volume changes to alterations in proteolysis.51 Proteolysis inhibition by cell swelling
strongly depends on activation of the p38MAPK in perfused rat liver.17 Specific inhibition
of the p38MAPK abolishes the antiproteolytic effects and the decrease of AV exerted by hypo-
osmolarity and glutamine but is without effect on cell swelling under these conditions.17
It has been shown that destruction of microtubules by colchicine does not prevent swell-
ing-induced cell volume changes and p38MAPK activation, suggestive of a location of
p38MAPK upstream from sequestrational sites and the putative microtubular element in the
swelling proteolysis signaling cascade.
Whereas in bacteria, plants, and fungi, two-component histidine kinases were identi-
fied to be involved in sensing of and subsequent adaptation to adverse osmotic condi-
tions,59 the mechanisms of “osmosensing” in mammalian cells are far from being under-
stood. Integrins are candidates to be involved in “mechanotransduction,” i.e., the
conversion of a mechanical stimulus into covalent modifications of signaling components.
Integrins are heterodimers, with each subunit having a single transmembrane domain.
They establish cell adhesion to the extracellular matrix and bind inside the cell to cyto-
plasmic proteins, which in turn interact with different signal transduction components
and the cytoskeleton60–70 (for a review, see Aplin et al.71). In normal liver, the most important
integrins are a1b1, a5b1, and a9b1.72–74
Recent results from our laboratory show that integrins could play a role in sensing
hepatocyte swelling induced by hypo-osmolarity or amino acid accumulation in perfused
rat liver, i.e., an intact organ model authentically preserving hepatocyte polarity and
1382_C17.fm Page 282 Tuesday, October 7, 2003 6:43 PM

282 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 17.2 Antiproteolytic effect of glutamine and its abolition by the integrin antagonistic peptide
GRGDSP, but not the inactive analogue GRGESP (A), and lack of effect of integrin antagonistic
peptide GRGDSP on the antiproteolytic effect of phenylalanine (B). Livers from fed rats were used
and proteolysis was assessed as described in Figure 17.1. In the presence of either GRGDSP (10
mmol/l, D) or GRGESP (10 mmol/l, ), glutamine (2 mmol/l) was infused for 30 min (A). Phenyla-
lanine (2 mmol/l) (B) was present from 130 to 160 min, and either GRGDSP (10 mmol/l, ‡ ) or
GRGESP (10 mmol/l, ®) was present since 100 min perfusion time. Results are from three experi-
ments for each condition; data are shown as means ± SEM.

three-dimensional anchoring to the extracellular matrix. Based on experiments with the


fibronectin-derived hexapeptide GRGDSP75 and the Src inhibitor PP-2,76 a role of integrin-
mediated activation of Src-type kinases as a trigger of p38MAPK and extracellular signal-
regulated kinase (Erk)-1/Erk-2 activation by hepatocyte swelling has been shown. An
important link between membrane-located integrin-dependent osmosensing and subse-
quent activation of p38MAPK is the Src kinases. Activation of Src is necessary for proteolysis
regulation by cell hydration. In line with this, the swelling-induced proteolysis inhibition
by glutamine is blunted by the RGD peptide (Figure 17.2A). Proteolysis inhibition by
phenylalanine, which does not involve cell swelling and p38MAPK 17, is insensitive to the
RGD peptide and PP-2 (Figure 17.2B), indicating that the inhibitors do not generally
interfere with the regulation of autophagic proteolysis and specifically impair the swell-
ing-related signal transduction toward proteolysis.

17.3.4 Cell hydration-independent signaling in proteolysis regulation


Not all amino acids exert their antiproteolytic effects via an increase of cell hydration, e.g.,
leucine, phenylalanine, and asparagine do not lead to changes of cell hydration in liver,17
and here other mechanisms seem to be involved.
Phenylalanine action on proteolysis resides on the activation of mammalian target of
rapamycin (mTOR) and p70S6K kinase,5,77,78 a signaling cascade that can clearly be differ-
entiated from the swelling-related antiproteolytic signaling cascade6 and does not seem
to be involved into cell hydration-dependent signaling.12,13,17
In freshly isolated suspended hepatocytes hypo-osmolarity activates PI 3-kinase,
leading to increased glycogen and fatty acid synthesis79,80 and taurocholate uptake.81
Further, hypo-osmolarity sensitizes these cells to proteolyis inhibition by amino acids,10
which depends on ribosomal S6 phosphorylation in a rapamycin-sensitive manner.77 This
suggests that multiple osmosensing mechanisms exist in hepatocytes, which could be
1382_C17.fm Page 283 Tuesday, October 7, 2003 6:43 PM

Chapter seventeen: The role of amino acids in the control of proteolysis 283

Non-swelling Swelling amino


amino acids acids

integrins GRGDSP
Wort- PI 3-
mannin kinase
Src PP-2

Rapamycin mTOR p38MAPK SB 203 580

p70S6 kinase microtubules colchicine

S6-P

segregation

proteolysis

Figure 17.3 Hypothetical scheme of proteolysis regulation by amino acids in rat liver. Swelling-
dependent and non-swelling-dependent ways of proteolysis regulation by amino acids, as outlined
in vom Dahl et al.13 (right panel) and van Sluijters et al.6 (left panel). S6, ribosomal protein S6; p70S6K,
70-kDa S6 kinase.

differentially linked to intracellular signaling pathways. The exact interaction between


the PI-3 kinase-dependent proteolysis regulation pathway6 and cell hydration-dependent
proteolysis regulation mechanisms13 is unclear, but inhibitor experiments suggest — up
to now — that the two pathways are distinctly separated from each other, but do converge
at the level of formation of autophagic vacuoles, i.e., sequestration.
A current working hypothesis as to the role of amino acid-dependent signaling with
respect to autophagic proteolysis is outlined in Figure 17.3. Integrins sense hepatocyte
swelling by amino acids, leading to activation of Src-type kinases, which in turn mediate
activation of Erk-1/Erk-2 and p38MAPK. Impairment of integrin–matrix interaction and
inhibition of Src-type kinases, but not disruption of the actin cytoskeleton, prevents the
p38MAPK-dependent inhibition of autophagy due to cell swelling and the regulatory
volume decrease triggered by hypo-osmolarity and/or swelling amino acids like glycine
or glutamine. Thus, integrins may act as cell volume sensors, at least in response to
hepatocyte swelling. Probably, as in bacteria, plants, and fungi,59 multiple osmosensing
mechanisms exist also in mammalian cells, and future work will unravel their relative
contributions.
1382_C17.fm Page 284 Tuesday, October 7, 2003 6:43 PM

284 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

17.4 Clinical relevance of amino acid-dependent proteolysis


regulation
Hypo-osmotic and hormone- and amino acid-induced cell swelling not only inhibits
proteolysis in liver, but can simultaneously stimulate protein synthesis.82 Interestingly,
mRNA turnover is also regulated by amino acids83,84 and cell hydration in parallel to
protein turnover.85 These findings may shed a new light on the understanding of protein
catabolic states and the known anabolic effects of insulin and amino acids.
Data from Calmus et al.86 suggested a negative correlation between the amount of
proteolysis and the viability of the graft in human liver transplantation, and the anti-
proteolytic effect of the standard University of Wisconsin solution has been characterized
in vitro.87,88 Here, the inverse relationship between proteolysis activity and cell volume
was confirmed in a rat model of cold preservation injury.87,89
In view of these data from liver, it is quite conceivable that in skeletal muscle, protein
turnover may also be controlled by cellular hydration. Protein turnover in skeletal muscle
is regulated in a complex way, but glutamine seems to play an important role.90 In skeletal
muscle, glutamine is transported via a Na+-dependent carrier (system N), which builds
up remarkably high intra- and extracellular glutamine concentration gradients. A rela-
tionship between the intramuscular glutamine concentration and protein turnover has
been described by Rennie et al.91 Protein catabolic states, such as sepsis, are characterized
by a marked lowering of intracellular glutamine concentrations (reviewed in Roth et al.44).
These largely empirical observations may find their explanation in cell volume changes
resulting from changed intra- and extracellular glutamine concentration gradients. Indeed,
irrespective of the nature of the underlying disease, a close correlation between the cellular
hydration state of muscle and whole-body nitrogen balance has been shown.92 Here, it
was hypothesized that cellular shrinkage in liver and skeletal muscle triggers the protein
catabolic states that accompany various diseases. In critically ill patients, e.g., burn
patients,93 proteolysis is accelerated, and the stimulation of proteolysis is preceded by
muscle cell dehydration.94 In healthy humans, induction of hyperosmolar conditions by
salt loading induced an acceleration of protein degradation, whereas generation of hypo-
osmotic plasma conditions by water loading induced an inhibition of proteolysis, as
measured by standard [13C]-leucine techniques.95 Therefore, it is well conceivable that the
physician already interferes with cell hydration by trying to counteract protein catabolism
by the infusion of amino acids.

Acknowledgments
This work was supported by SFB 575, “Experimentelle Hepatologie.”

References
1. Pfeifer, U., Inhibition by insulin of the formation of autophagic vacuoles in rat liver: a
morphometric approach to the kinetics of intracellular degradation by autophagy, J. Cell
Biol., 78, 152, 1978.
2. Tager, J.M., Aerts, J.M.F.G., Oude-Elferink, R.J.A., Groen, A.K., Meijer, A.J., Gordon, P.D., and
Seglen, P.O., pH regulation of intracellular membrane flow, in pH Homeostasis, Häussinger,
D., Ed., Academic Press, London, 1988, p. 123.
3. Mortimore, G.E. and Pösö, R.A., Intracellular protein catabolism and its control during
nutrient deprivation and supply, Annu. Rev. Nutr., 7, 539, 1987.
4. Doherty, F.J. and Mayer, R.J., Intracellular Protein Degradation, Rickwood, D., Ed., IRL Press,
Oxford, 1995, p. 1.
1382_C17.fm Page 285 Tuesday, October 7, 2003 6:43 PM

Chapter seventeen: The role of amino acids in the control of proteolysis 285

5. Blommaart, E.F.C., Luiken, J.J.F.P., and Meijer, A.M., Autophagic proteolysis: control and
specificity, Histochem. J., 29, 365, 1997.
6. van Sluijters, D.A., Dubbelhuis, P.F., Blommaart, E.F., and Meijer, A.J., Amino-acid-dependent
signal transduction, Biochem. J., 351 (Pt. 3), 545, 2000.
7. Mortimore, G.E. and Mondon, C.E., Inhibition by insulin of valine turnover in liver, J. Biol.
Chem., 245, 2375, 1969.
8. Seglen, P.O., Inhibitor of protein degradation formed during incubation of isolated rat hepa-
tocytes in a cell culture medium: its identification as ammonia, Exp. Cell Res., 107, 207, 1977.
9. Leverve, X.M., Caro, L.H.P., Plomp, P.J.A.M., and Meijer, A.J., Control of proteolysis in
perifused rat hepatocytes, FEBS Lett., 219, 455, 1987.
10. Meijer, A.J., Gustafson, L.A., Luiken, J.J.F.P., Blommaart, P.J.E., Caro, L.H.P., Woerkom,
G.M.V., Spronk, C., and Boon, L., Cell swelling and the sensitivity of autophagic proteolysis
to inhibition by amino acids in isolated rat hepatocytes, Eur. J. Biochem., 215, 449, 1993.
11. vom Dahl, S., Stoll, B., Gerok, W., and Häussinger, D., Inhibition of proteolysis by cell swelling
in liver requires intact microtubular structures, Biochem. J., 308, 529, 1995.
12. vom Dahl, S., Dombrowski, F., Schliess, F., Pfeifer, U., and Häussinger, D., Cell hydration
controls autophagosome formation in rat liver in a microtubule-dependent way downstream
from p38MAPK activation, Biochem. J., 354, 31, 2001.
13. vom Dahl, S., Schliess, F., Reissmann, R., Goerg, B., Weiergräber, O., Kacalton, M., Dom-
browski, F., and Häussinger, D., Involvement of integrins into osmo-sensing and signaling
towards autophagic proteolysis in rat liver, J. Biol. Chem., 278, 27088, 2003.
14. Plomp, P.J.A.M., Wolvetang, E.J., Groen, A.K., Meijer, A.J., Gordon, P.B., and Seglen, P.O.,
Energy dependence of autophagic protein degradation in isolated rat hepatocytes, Eur. J.
Biochem., 164, 197, 1987.
15. Plomp, P.J.A.M., Gordon, P.B., Meijer, A.J., Hoyvik, H., and Seglen, P.O., Energy dependence
of different steps in the autophagic-lysosomal pathway, J. Biol. Chem., 264, 6699, 1989.
16. Pfeifer, U., Application of test substances to the surface of rat liver in situ: opposite effects
of insulin and isoproterenol on cellular autophagy, Lab. Invest., 50, 348, 1984.
17. Häussinger, D., Schliess, F., Dombrowski, F., and vom Dahl, S., Involvement of p38MAPK in
the regulation of proteolysis by liver cell hydration, Gastroenterology, 116, 921, 1999.
18. Häussinger, D., Regulation and functional significance of liver cell volume, Prog. Liver Dis.,
14, 29, 1996.
19. Mortimore, G.E. and Schworer, C.M., Induction of autophagy by amino-acid deprivation in
perfused rat liver, Nature, 270, 174, 1977.
20. Seglen, P.O. and Gordon, P.B., Amino acid control of autophagic sequestration and protein
degradation in rat hepatocytes, J. Cell Biol., 99, 435, 1984.
21. Pöso, A.R., Schwörer, C.M., and Mortimore, G.E., Acceleration of proteolysis in perfused rat
liver by deletion of glucogenic amino acids: regulatory role of glutamine, Biochem. Biophys.
Res. Commun., 107, 1433, 1982.
22. Pösö, R., Wert, J.J., and Mortimore, G.E., Multifunctional control by amino acids of depriva-
tion-induced proteolysis in liver, J. Biol. Chem., 257, 12114, 1982.
23. Seglen, P.O., Gordon, P.B., and Poli, A., Amino acid inhibition of the autophagic/lysosomal
pathway of protein degradation in isolated rat hepatocytes, Biochim. Biophys. Acta, 630, 103,
1980.
24. Sommercorn, J.M. and Swick, R.W., Protein degradation in primary monolayer cultures of
adult rat hepatocytes: further evidence for the regulation of protein degradation by amino
acids, J. Biol. Chem., 256, 4816, 1981.
25. Miotto, G., Venerando, R., Khurann, K.K., Siliprandi, N., and Mortimore, G.E., Control of
hepatic proteolysis by leucine and isovaleryl-L-carnitine through a common locus: evidence
for a possible mechanism of recognition at the plasma membrane, J. Biol. Chem., 267, 22060,
1992.
26. Kadowaki, M., Pösö, A.R., and Mortimore, G.E., Parallel control of hepatic proteolysis by
phenylalanine and phenylpyruvate through independent inhibitory sites at the plasma mem-
brane, J. Biol. Chem., 267, 22060, 1992.
1382_C17.fm Page 286 Tuesday, October 7, 2003 6:43 PM

286 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

27. Mortimore, G.E and Pöso, A.R., The lysosomal pathway of intracellular proteolysis in liver:
regulation by amino acids, Adv. Enzyme Regul., 25, 257, 1986.
28. Hallbrucker, C., vom Dahl, S., Lang, F., and Häussinger, D., Control of hepatic proteolysis
by amino acids, Eur. J. Biochem., 197, 717, 1991.
29. Schworer, C.M., Shiffer, K.A., and Mortimore, G.E., Quantitative relationship between au-
tophagy and proteolysis during graded amino acid deprivation in perfused rat liver, J. Biol.
Chem., 256, 7652, 1981.
30. Seglen, P.O., Kovacs, A.L., and Gordon, P.B., Autophagic protein degradation in hepatocytes,
in Regulation of Hepatic Function, Grunnet, N. and Quistorff, B., Eds., Munksgaard, Copen-
hagen, 1991, p. 358.
31. Seglen, P.O., Grinde, B., and Solheim, A.S., Inhibition of the lysosomal pathway of protein
degradation in isolated rat hepatocytes by ammonia, methylamine, chloroquine and leupep-
tin, Eur. J. Biochem., 95, 215, 1979.
32. Hallbrucker, C., vom Dahl, S., Lang, F., Gerok, W., and Häussinger, D., Inhibition of hepatic
proteolysis by insulin: role of hormone-induced alterations of cellular K+ balance, Eur. J.
Biochem., 199, 467, 1991.
33. Häussinger, D., Hallbrucker, C., vom Dahl, S., Decker, S., Schweizer, U., Lang, F., and Gerok,
W., Cell volume is a major determinant of proteolysis control in liver, FEBS Lett., 283, 70, 1991.
34. vom Dahl, S., Hallbrucker, C., Lang, F., Gerok, W., and Häussinger, D., Regulation of liver
cell volume and proteolysis by glucagon and insulin, Biochem. J., 278, 771, 1991.
35. Häussinger, D., The role of cellular hydration in the regulation of cell function, Biochem. J.,
313, 697, 1996.
36. Häussinger, D. and Lang, F., Regulation of cell function by the cellular hydration state, Am.
J. Physiol., 267, 343, 1994.
37. Lang, F., Busch, G.L., Ritter, M., Völkl, H., Waldegger, S., Gulbins, E., and Häussinger, D.,
Functional significance of cell volume regulatory mechanisms, Physiol. Rev., 78, 247, 1998.
38. Bakker-Grunwald, T., Potassium permeability and volume control in isolated rat hepatocytes,
Biochim. Biophys. Acta, 731, 239, 1983.
39. Kristensen, L.O. and Folke, M., Volume-regulatory K+ efflux during concentrative uptake of
alanine in isolated rat hepatocytes, Biochem. J., 221, 265, 1984.
40. Häussinger, D., Stehle, T., and Lang, F., Volume regulation in liver: further characterization
by inhibitors and ionic substitutions, Hepatology, 11, 243, 1990.
41. Häussinger, D., Hallbrucker, C., vom Dahl, S., Lang, F., and Gerok, W., Cell swelling inhibits
proteolysis in perfused rat liver, Biochem. J., 272, 239, 1990.
42. vom Dahl, S. and Häussinger, D., The role of the nutritional state in the control of proteolysis
by the cellular hydration state in the perfused rat liver, J. Nutr., 126, 395, 1996.
43. Hayes, M.R. and McGivan, J.D., Differential effects of starvation on alanine and glutamine
transport in isolated rat hepatocytes, Biochem. J., 204, 365, 1982.
44. Roth, E., Karner, J., and Ollenschläger, G., Glutamine: an anabolic effector? J. Parenter. Enteral
Nutr., 130S, 24, 1990.
45. Hallbrucker, C., vom Dahl, S., Lang, F., Gerok, W., and Häussinger, D., Modification of liver
cell volume by insulin and glucagon, Pflügers Arch., 418, 519, 1991.
46. Häussinger, D. and Lang, F., Cell volume and hormone action, Trends Pharmacol. Sci., 13, 371,
1992.
47. vom Dahl, S., Hallbrucker, C., Lang, F., Gerok, W., and Häussinger, D., Regulation of cell
volume in the perfused rat liver by hormones, Biochem. J., 280, 105, 1991.
48. Schliess, F., Schäfer, C., vom Dahl, S., Fischer, R., Lordnejad, M.R., and Häussinger, D.,
Hepatic expression and regulation of the Na+/K+/2Cl– cotransporter NKCC1 in hepatocytes
and HuH-7 hepatoma cells, Arch. Biochem. Biophys., 401, 187, 2002.
49. vom Dahl, S. and Häussinger, D., Bumetanide-sensitive cell swelling mediates the inhibitory
effect of ethanol on proteolysis in rat liver, Gastroenterology, 114, 1046, 1998.
50. Häussinger, D., Osmoregulation of liver cell function: signalling, osmolytes and cell hetero-
geneity, Contrib. Nephrol., 123, 185, 1998.
51. Häussinger, D. and Schliess, F., Osmotic induction of signaling cascades: role in regulation
of cell function, Biochem. Biophys. Res. Commun., 255, 551, 1999.
1382_C17.fm Page 287 Tuesday, October 7, 2003 6:43 PM

Chapter seventeen: The role of amino acids in the control of proteolysis 287

52. Theodoropoulos, P.A., Strournaras, C., Stoll, B., Markogiannakis, E., Lang, F., Gravanis, A.,
and Häussinger, D., Hepatocyte swelling leads to rapid decrease of the total G-/total actin
ratio and increases actin mRNA levels, FEBS Lett., 311, 241, 1992.
53. Häussinger, D., Stoll, B., vom Dahl, S., Theodoropoulos, P.A., Markogiannakis, E., Gravanis,
A., Lang, F., and Stournaras, C., Microtubule stabilization and induction of tubulin mRNA
by cell swelling in isolated rat hepatocytes, Biochem. Cell Biol., 72, 12, 1994.
54. Meijer, A.J., Inhibition of autophagic proteolysis by cell swelling in hepatocytes, Biochem. J.,
312, 987, 1995.
55. Schreiber, R., Stoll, B., Lang, F., and Häussinger, D., Effects of anisoosmolarity and hydrop-
eroxides on intracellular pH in isolated rat hepatocytes as assessed by (2',7')-bis(carboxyeth-
yl)-5(6)-carboxyfluorescein and fluorescein isothiocyanate-dextran fluorescence, Biochem. J.,
303, 113, 1994.
56. Schreiber, R. and Häussinger, D., Characterization of the swelling-induced alkalinization of
endocytotic vesicles in fluorescein isothiocyanate-dextran loaded rat hepatocytes, Biochem.
J., 309, 19, 1995.
57. Schreiber, R., Zhang, F., and Häussinger, D., Regulation of vesicular pH in liver macrophages
and parenchymal cells by ammonia and anisotonicity as assessed by fluorescein isothiocy-
anate dextran fluorescence, Biochem. J., 315, 385, 1996.
58. Luiken, J.J.F.P., Aerts, J.M.F.G., and Meijer, A.J., The role of the intralysosomal pH in the
control of autophagic proteolytic flux in rat hepatocytes, Eur. J. Biochem., 235, 564, 1996.
59. Loomis, W.F., Shaulsky, G., and Wang, N., Histidine kinases in signal transduction pathways
of eukaryotic cells, J. Cell Sci., 110, 1141, 1997.
60. Aikawa, R., Nagai, T., Kudoh, S., Zou, Y., Tanaka, M., Tamura, M., Akazawa, H., Takano, H.,
Nagai, R., and Komuro, I., Integrins play a critical role in mechanical stress-induced p38
MAPK activation, Hypertension, 39, 233, 2002.
61. Chen, K.D., Li, Y.S., Kim, M., Li, S., Yuan, S., Chien, S., and Shyy, J.Y., Mechanotransduction
in response to shear stress: roles of receptor tyrosine kinases, integrins, and Shc, J. Biol. Chem.,
274, 18393, 1999.
62. Clark, E.A. and Brugge, J.S., Integrins and signal transduction pathways: the road taken,
Science, 268, 233, 1995.
63. Jaeschke, H., Cellular adhesion molecules: regulation and functional significance in the
pathogenesis of liver diseases, Am. J. Physiol., 273, G602, 1997.
64. Li, S., Kim, M., Hu, Y.L., Jalali, S., Schlaepfer, D.D., Hunter, T., Chien, S., and Shyy, J.Y., Fluid
shear stress activation of focal adhesion kinase: linking to mitogen-activated protein kinases,
J. Biol. Chem., 272, 30455, 1997.
65. Low, S.Y., Rennie, M.J., and Taylor, P.M., Involvement of integrins and the cytoskeleton in
modulation of skeletal muscle glycogen synthesis by changes in cell volume, FEBS Lett., 417,
101, 1997.
66. Ruwhof, C. and van der Laarse, A., Mechanical stress-induced cardiac hypertrophy: mech-
anisms and signal transduction pathways, Cardiovasc. Res., 47, 23, 2000.
67. Schlaepfer, D.D. and Hunter, T., Signal transduction from the extracellular matrix: a role for
the focal adhesion protein-tyrosine kinase FAK, Cell Struct. Funct., 21, 445, 1996.
68. Schoenwaelder, S.M. and Burridge, K., Bidirectional signaling between the cytoskeleton and
integrins, Curr. Opin. Cell Biol., 11, 274, 1999.
69. Ueki, K., Mimura, T., Nakamoto, T., Sasaki, T., Aizawa, S., Hirai, H., Yano, S., Naruse, T.,
and Nojima, Y., Integrin-mediated signal transduction in cells lacking focal adhesion kinase
p125FAK, FEBS Lett., 432, 197, 1998.
70. Zhou, X., Li, J., and Kucik, D.F., The microtubule cytoskeleton participates in control of beta2
integrin avidity, J. Biol. Chem., 276, 44762, 2001.
71. Aplin, A.E., Howe, A., Alahari, S.K., and Juliano, R.L., Signal transduction and signal mod-
ulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell
adhesion molecules, and selectins, Pharmacol. Rev., 50, 197, 1998.
72. Carloni, V., Mazzocca, A., Pantaleo, P., Cordella, C., Laffi, G., and Gentilini, P., The integrin,
a6b1, is necessary for the matrix-dependent activation of FAK and MAP kinase and the
migration of human hepatocarcinoma cells, Hepatology, 34, 42, 2001.
1382_C17.fm Page 288 Tuesday, October 7, 2003 6:43 PM

288 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

73. Hsu, S.L., Cheng, C., and Shi, Y.R., Proteolysis of integrin alpha5 and beta1 subunits involved
in retinoic acid-induced apoptosis in human hepatoma Hep3B cells, Cancer Lett., 167, 193,
2001.
74. Torimura, T., Ueno, T., Kin, M., Harada, R., Nakamura, T., Kawaguchi, T., Harada, M.,
Kumashiro, R., Watanabe, H., Avraham, R., and Sata, M., Autocrine motility factor enhances
hepatoma cell invasion across the basement membrane through activation of beta1 integrins,
Hepatology, 34, 62, 2001.
75. Chen, B.M. and Grinnell, A.D., Integrins and modulation of transmitter release from motor
nerve terminals by stretch, Science, 269, 1578, 1995.
76. Hanke, J.H., Gardner, J.P., Dow, R.L., Changelian, P.S., Brissette, W.H., Weringer, E.J., Pollok,
B.A., and Connelly, P.A., Discovery of a novel, potent, and Src family-selective tyrosine kinase
inhibitor: study of Lck- and FynT-dependent T cell activation, J. Biol. Chem., 271, 695, 1996.
77. Blommaart, E.F., Luiken, J.J., Blommaart, P.J., van Woerkom, G.M., and Meijer, A.J., Phos-
phorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes,
J. Biol. Chem., 270, 2320, 1995.
78. Blommaart, E.F., Krause, U., Schellens, J.P., Vreeling-Sindelarova, H., and Meijer, A.J., The
phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in
isolated rat hepatocytes, Eur. J. Biochem., 243, 240, 1997.
79. Krause, U., Rider, M.H., and Hue, L., Protein kinase signaling pathway triggered by cell
swelling and involved in the activation of glycogen synthase and acetyl-CoA carboxylase in
isolated rat hepatocytes, J. Biol. Chem., 271, 16668, 1996.
80. Meijer, A.J., Baquet, A., Gustafson, L., van Woerkom, G.M., and Hue, L., Mechanism of
activation of liver glycogen synthase by swelling, J. Biol. Chem., 267, 5823, 1992.
81. Webster, C.R., Blanch, C.J., Phillips, J., and Anwer, M.S., Cell swelling-induced translocation
of rat liver Na(+)/taurocholate cotransport polypeptide is mediated via the phosphoinositide
3-kinase signaling pathway, J. Biol. Chem., 275, 29754, 2000.
82. Stoll, B., Gerok, W., Lang, F., and Häussinger, D., Liver cell volume and protein synthesis,
Biochem. J., 287, 217, 1992.
83. Balavoine, S., Feldmann, G., and Lardeux, B., Rates of RNA degradation in isolated rat
hepatocytes: effects of amino acids and inhibitors of lysosomal function, Eur. J. Biochem., 189,
617, 1987.
84. Lardeux, B.R. and Mortimore, G.E., Amino acid and hormonal control of macromolecular
turnover in perfused rat liver, J. Biol. Chem., 262, 14514, 1987.
85. Newsome, W.P., Warskulat, U., Noe, B., Wettstein, M., Stoll, B., Gerok, W., and Häussinger,
D., Modulation of phosphoenolpyruvate carboxy kinase mRNA levels by the hepatocellular
hydration state, Biochem. J., 304, 555, 1994.
86. Calmus, Y., Cynober, L., Dousset, B., Lim, S.K., Soubrane, O., Conti, F., Houssin, D., and
Giboudeau, J., Evidence for the detrimental role of proteolysis during liver preservation in
humans, Gastroenterology, 108, 1510, 1995.
87. Neveux, N., De Bandt, J.P., Charrueau, C., Savier, E., Chaumeil, J.C., Hannoun, L., Giboudeau,
J., and Cynober, L.A., Deletion of hydroxyethylstarch from University of Wisconsin solution
induces cell shrinkage and proteolysis during and after cold storage of rat liver, Hepatology,
25, 678, 1997.
88. Charrueau, C., Savier, E., Blonde-Cynober, F., Coudray-Lucas, C., Poupon, R., Giboudeau,
J., Chaumeil, J.C., Hannoun, L., and Cynober, L., Effect of two storage solutions on proteolysis
in isolated rat liver cells, Int. J. Pharmaceut., 170, 257, 1998.
89. Neveux, N., De Bandt, J.P., Fattal, E., Hannoun, L., Poupon, R., Chaumeil, J.C., Delattre, J.,
and Cynober, L.A., Cold preservation injury in rat liver: effect of liposomally-entrapped
adenosine triphosphate, J. Hepatol., 33, 68, 2000.
90. Sudgen, P.H. and Fuller, S.J., Regulation of protein turnover in skeletal and cardiac muscle,
Biochem. J., 273, 21, 1991.
91. Rennie, M.J., Hundal, H.S., Babij, P., MacLennan, P., Taylor, P.M., Watt, P.W., Jepson, M.M.,
and Millward, D.J., Characteristics of a glutamine carrier in skeletal muscle may have im-
portant consequences for nitrogen loss in injury, infection and chronic disease, Lancet, 2,
1008, 1986.
1382_C17.fm Page 289 Tuesday, October 7, 2003 6:43 PM

Chapter seventeen: The role of amino acids in the control of proteolysis 289

92. Häussinger, D., Roth, E., Lang, F., and Gerok, W., Cellular hydration state: an important
determinant of protein catabolism in health and disease, Lancet, 341, 1340, 1993.
93. Cynober, L., Amino acid metabolism in thermal burns, J. Parenter. Enteral Nutr., 13, 196, 1989.
94. Finn, P.J., Plank, L.D., Clark, M.A., Connolly, A.B., and Hill, G.L., Progressive cellular dehy-
dration and proteolysis in critically ill patients, Lancet, 347, 654, 1996.
95. Berneis, K., Ninnis, R., Häussinger, D., and Keller, U., Effects of hyper- and hypoosmolality
on whole body protein and glucose balance, Am. J. Physiol., 276, E188, 1999.
96. vom Dahl, S., Hallbrucker, C., Lang, F., Gerok, W., and Häussinger, D., A non-invasive
technique for cell volume determination in perfused rat liver, Biol. Chem. Hoppe-Seyler, 372,
411, 1991.
1382_C17.fm Page 290 Tuesday, October 7, 2003 6:43 PM
1382_C18.fm Page 291 Tuesday, October 7, 2003 6:46 PM

chapter eighteen

Anabolic effects and signaling


pathways triggered by amino acids
in the liver
Louis Hue
Institute of Cellular Pathology and Université Catholique de Louvain
Luc Bertrand
Institute of Cellular Pathology and Université Catholique de Louvain

Contents
Introduction..................................................................................................................................291
18.1 Stimulation of glycogen synthesis and lipogenesis by glutamine ............................292
18.2 Comparison of the effects of glutamine and insulin...................................................294
18.3 Leucine, a connection between amino acids and insulin ...........................................294
18.4 Amino acids and protein synthesis ................................................................................294
18.5 Signaling pathways triggered by amino acids .............................................................297
18.5.1 PtdIns-3-K ..............................................................................................................297
18.5.2 Protein phosphatases and GAPP .......................................................................297
18.5.3 AMPK .....................................................................................................................298
18.5.4 Interaction between leucine, glutamine, and insulin .....................................299
18.6 Conclusions.........................................................................................................................299
Acknowledgments ......................................................................................................................299
References .....................................................................................................................................299

Introduction
Nutrients have been known for a long time to control metabolism. Their metabolic effects
were usually considered to be indirect and mediated by hormones, e.g., the effect of
glucose resulting from the stimulation of insulin secretion. However, in recent years,
experimental evidence accumulated, especially in models of isolated cells or organs, to
demonstrate that nutrients can affect metabolism by themselves. Nutrients, such as glu-
cose, fatty acids, and certain amino acids, can modulate gene expression via transcription
factors binding to response elements in the promoter region of certain genes. Besides these

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 291
1382_C18.fm Page 292 Tuesday, October 7, 2003 6:46 PM

292 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

long-term effects, nutrients can also exert short-term metabolic effects. For example, glu-
cose stimulates glycogen synthesis by controlling the (de)phosphorylation and activation
states of glycogen phosphorylase and glycogen synthase (GS). Similarly, the short-term
metabolic effects of amino acids are numerous and well documented. We describe in this
review the short-term effects of amino acids that do not result from changes in gene
expression but involve complex phosphorylation cascades.

18.1 Stimulation of glycogen synthesis and lipogenesis by glutamine


Glycogen synthesis is controlled by the availability of substrates and by the hormonal and
dietary status of the organism. In perfused livers or preparations of isolated hepatocytes,
glucose is a poor substrate for glycogen synthesis, and the addition of insulin is of little
help.1,2 In these preparations, glycogen synthesis is enhanced by several amino acids, such
as glutamine, alanine, asparagine, and proline.3,4 Besides their effects on glycogen synthe-
sis, glutamine, proline, and, to a lesser extent, alanine also stimulate lipogenesis and inhibit
ketogenesis.4,5 Glycogen synthesis and lipogenesis are mainly controlled by the activity of
GS and acetyl-coenzyme A carboxylase (ACC), respectively. Both enzymes are intercon-
vertible by (de)phosphorylation, the active form being dephosphorylated. They possess
multiple phosphorylation sites, which are phosphorylated following a hierarchic order.
Phosphorylation of the primary sites allows for the phosphorylation of the secondary sites
by different protein kinases, eventually leading to the inactivation of the enzymes.
Several detailed studies have been performed to elucidate the mechanism responsible
for the stimulation of glycogen synthesis by amino acids in hepatocytes.6 A breakthrough
was achieved when a stimulation of glycogen synthesis was found with amino isobutyric
acid, a nonmetabolizable amino acid analogue, which is transported in a Na+-dependent
manner, like glutamine.7 This led to the hypothesis that cell swelling and the ionic mod-
ifications resulting from the Na+-dependent entry of amino acid could stimulate glycogen
synthesis. Indeed, a single and direct relationship between an increase in cell volume and
stimulation of glycogen synthesis was observed in hepatocytes incubated with various
amino acids.8 Furthermore, prevention of swelling by hyperosmotic media blocked the
amino acid-induced activation of GS. In addition, cell swelling induced by hypo-osmotic
media, even in the absence of amino acids, could also activate GS. A similar relationship
between changes in cell volume and activity of ACC was also reported.9 This suggested
the involvement of a common regulatory mechanism triggered by cell swelling.
Swollen cells respond to a hypo-osmotic stress by an intricate mechanism of regulatory
volume decrease, which aims at restoring the initial cell volume. This mechanism leads
to an electrogenic K+ efflux followed by Cl–, which are permeant ions distributed across
the plasma membrane according to its potential.10 A fall in the intracellular concentration
of KCl is indeed observed in swollen hepatocytes. These ionic changes have consequences
on the activity of the protein phosphatases that activate GS and ACC. For example, in
vitro measurements have demonstrated that normal intracellular concentrations (above 50
mM) of Cl– inhibit glycogen synthase activation and, to a lesser extent, ACC activation,
whereas concentrations found in swollen cells had no detectable inhibitory effect.11 On
the other hand, glutamate, whose concentration can increase to values up to 20 to 25 mM
in hepatocytes incubated with glutamine, allosterically stimulates ACC and greatly stim-
ulates its activation by a type 2A protein phosphatase, the glutamate-activated protein
phosphatase (GAPP) (Figure 18.1).12,13 GAPP can dephosphorylate a synthetic peptide
containing Ser79, the inactivating phosphorylation site in ACC. This Ser is phosphorylated
by the AMP-activated protein kinase (AMPK). AMPK is well conserved in eukaryotes and
acts as an energy/nutrient sensor in cells.14 It is activated by an increased AMP:ATP ratio,
as occurs during oxygen deprivation or various cellular stresses. Once activated, AMPK
1382_C18.fm Page 293 Tuesday, October 7, 2003 6:46 PM

Chapter eighteen: Anabolic effects of amino acids in the liver 293

Figure 18.1 Signaling pathways involved in the regulation of ACC activity by insulin, amino acids,
and AMP-activated protein kinase in liver. Left panel: During oxygen deprivation, the AMP:ATP
ratio increases. The change in nucleotide concentrations induces AMPK phosphorylation and acti-
vation. AMPK is able to phosphorylate (on Ser79) and inactivate liver ACC, and thus inhibit fatty
acids synthesis. Right panel: Insulin and nutrients, like glutamine or leucine, activate ACC and so
stimulate fatty acids synthesis by different pathways. Glutamine leads to the accumulation of
glutamate, which stimulates the protein phosphatase GAPP, which in turn activates ACC. The
mechanism involved in ACC activation by GAPP is not fully understood. GAPP could dephospho-
rylate inhibiting sites (other than Ser79, because its phosphorylation state is not affected by amino
acids treatment68). Insulin by itself is not able to activate ACC, even if its signaling pathway down
to PKB is working. However, leucine alone or in combination with insulin is able to induce ACC
activation by a still unknown mechanism. Finally, leucine also increases the glutamine-induced ACC
activation. This observation is explained by the fact that leucine stimulates glutaminase and so
increases glutamate concentration. Calyculin A (CA), an inhibitor of protein phosphatase, prevents
ACC activation by amino acids.

stimulates ATP-producing pathways and inhibits energy-requiring processes, such as


glycogen synthesis, lipogenesis, and protein synthesis. AMPK phosphorylates and inac-
tivates ACC and glycogen synthase; it also leads to the inactivation of protein kinase and
factors involved in the protein synthesis machinery. Surprisingly, the activation of ACC
that occurs in hepatocytes incubated with glutamine does not correspond to a decreased
phosphorylation state of Ser79in ACC, although it is mediated by a protein phosphatase.15
The mechanism involved in this amino acid-induced activation of ACC is discussed below.
In addition to regulation by (de)phosphorylation, translocation and compartmentation
of key regulatory enzymes should be taken into account when considering the control of
glycogen metabolism in the liver. Indeed, stimulation of glycogen synthesis by glucose
promotes the translocation of GS from the cytosol to the actin-reach area of the cell cortex
close to the plasma membrane.16,17 Glucose 6-phosphate mediates this process and is
therefore a key signal for both the activation and translocation of GS.18 Glucokinase also
undergoes translocation. In resting cells, i.e., in the presence of a low, though physiological,
concentration of glucose, glucokinase is sequestered by the glucokinase regulatory protein
in the nucleus. When glucose concentration increases, glucokinase leaves the nucleus to
the cytosol.19,20 This compartmentation of glucokinase increases the sensitivity of glycogen
synthesis to small changes in glucokinase activity. The question in point here is to know
if and how the stimulation of glycogen synthesis by glutamine superimposes to these to-
and-fro movements of GS between various subcellular compartments. However, the stim-
ulatory effect of glutamine on glycogen synthesis cannot be explained by a change in
glucose 6-phosphate concentration, which indeed remains unaffected in glutamine-treated
1382_C18.fm Page 294 Tuesday, October 7, 2003 6:46 PM

294 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

hepatocytes.4,21 It is, however, tempting to speculate that glutamate could interfere in one
way or another with the subcellular distribution of GS and glucokinase.
Translocation of ACC from an active cytosolic pool to an inactive mitochondrial pool
upon fasting has been reported.22,23 This observation has not been confirmed24 and is
difficult to interpret because of the citrate-induced polymerization of ACC into macro-
molecular complexes.25–28 Nevertheless, some interaction between ACC and the cytoskel-
eton could occur.29 In addition, a cytosolic 75-kDa protein co-purifies with ACC and could
participate in the control of ACC activity.30 The possibility that this ACC-associated protein
is involved in ACC activation by amino acids is certainly worth considering.

18.2 Comparison of the effects of glutamine and insulin


The anabolic effects of glutamine and other Na+-cotransported amino acids resemble those
of insulin, which is indeed known for its stimulation of glycogen synthesis in skeletal
muscle and lipogenesis in adipose tissue.31 However, in short-term experiments with
hepatocytes in suspension or perfused livers, we repeatedly failed to obtain any evidence
in favor of such an effect of insulin, added alone or in combination with glutamine.15
Moreover, the activation of GS and ACC by glutamine persists in hepatocytes from strep-
tozotocin-diabetic rats.15 Taken together, these data demonstrate that the effect of
glutamine is independent of insulin and is not mediated by the insulin signaling pathway
(Figure 18.1).
The lack of insulin effect on GS and ACC cannot be attributed to a defect in signal
transduction. First, insulin is able to antagonize the effects of submaximal concentrations
of hormones, such as glucagon, that act via cyclic AMP.32,33 Second, we and others con-
firmed that in isolated hepatocytes, the insulin signaling pathway is functional, from the
insulin receptor down to phosphatidylinositol-3-kinase (PtdIns-3-K).15,34 PtdIns-3-K is a
lipid kinase responsible for the synthesis of phosphatidylinositol-3,4,5-trisphosphate; it
mediates most if not all the metabolic effects of insulin through the activation of protein
kinase B or Akt (PKB).35 Therefore, the lack of GS and ACC activation by insulin in
hepatocytes results from the inhibition of a step downstream of PtdIns-3-K and PKB, and
this inhibition cannot be relieved by incubation of hepatocytes with glutamine.

18.3 Leucine, a connection between amino acids and insulin


Leucine, a branched-chain amino acid that is poorly metabolized by the liver and that
does not induce swelling, activates ACC9 but not GS8,15 (Table 18.1, Figure 18.1). However,
ACC activation by leucine differed from the effect of glutamine. The maximal effect of
leucine was observed within 10 min, whereas it required about 45 to 60 min with
glutamine, and the overall effect of glutamine on ACC activation was larger than that of
leucine. Moreover, the effects of glutamine and leucine on ACC activation were additive,
whereas leucine antagonized the activation of GS by glutamine. Interestingly, the activa-
tion of ACC by leucine was enhanced by insulin, in contrast to the effect of glutamine
(Table 18.1). These data suggest that glutamine and leucine act by different mechanisms
and that leucine exerts a permissive effect on insulin action.15

18.4 Amino acids and protein synthesis


The regulation of the protein synthesis machinery involves (de)phosphorylation of various
translation factors and ribosomal proteins as evidenced by numerous experimental studies
carried out in several tissues and cell types36–41 (see also Chapter 7).
1382_C18.fm Page 295 Tuesday, October 7, 2003 6:46 PM

Chapter eighteen: Anabolic effects of amino acids in the liver 295

Table 18.1 Effects of Glutamine (Gln), Leucine (Leu), and Insulin (Ins), Alone or in
Combination, on GS, ACC, and p70S6K Activity in Hepatocytes

Maximal activation of:


Treatment GS ACC P70S6K
(fold activation relative to the control value)
Control 1 1 1
Insulin 1 1 1
Glutamine 2.5 3 20–40
Leucine 1 2 10–20
Gln + Leu 1.5 6 100
Ins + Gln 2.5 3 20–40
Ins + Leu 1 3 30

Note: Insulin alone had no effect. Maximal activation of ACC and p70S6K by leucine or leucine
and insulin was at 10 min. The effects of glutamine, alone or together with insulin or
leucine, were maximal at 60 min.
Source: From Krause, U. et al., Eur. J. Biochem., 269, 3742, 2002.

The protein kinase p70S6K plays an important role in the control of protein synthesis
in response to hormones, mitogens, and nutrients.42–44 Once activated, p70S6K phospho-
rylates the S6 ribosomal protein present in the 40S ribosomal subunit. It is involved in the
translation of mRNAs that contain oligopyrimidine sequences upstream of their transcrip-
tion initiation site and are members of the terminal oligopyrimidine (TOP) family of RNA.
The proteins encoded by these mRNAs are ribosomal proteins and proteins involved in
the translation machinery. p70S6K activation involves a complex sequence of multiple
hierarchical phosphorylations by several protein kinases.45–50 One of these is the mamma-
lian target of rapamycin (mTOR), which phosphorylates Thr389in p70S6K (Figure 18.2).
Phosphorylation of Thr389 correlates with p70S6K activity. The prior phosphorylation of
Thr389 by mTOR seems to be required for the phosphorylation of Thr229 by the 3-phos-
phoinositide-dependent protein kinase-1 (PDK1), a constitutively active protein kinase.
mTOR plays a central role in the control of protein synthesis by nutrients and energy.
It has an unusually high constant of affinity (Km) for ATP (about 1 mM) and has been
proposed to act as an ATP sensor of the cell.51 mTOR is a protein kinase whose active
center shares some common structural features with lipid kinases, such as PtdIns-3-K, and
which can be inhibited by wortmannin.52 It is also specifically inhibited by the immuno-
suppressant rapamycin.53,54 The mechanism of activation of mTOR is complex and not
fully understood (Figure 18.2). mTOR is inhibited by the hamartin (or tuberous sclerosis
complex 1 (TSC1))–tuberin (or TSC2) complex.55–58 Phosphorylation of tuberin by PKB
circumvents this inhibition, thereby explaining the effect of insulin. MTOR phosphorylates
directly p70S6K, but it may also phosphorylate and thereby inactivate a protein phos-
phatase that in turn inactivates p70S6K (Figure 18.2).47,49
Amino acids are direct precursors of protein synthesis, and hence their availability
controls protein turnover. Besides their mass effect, certain amino acids exert a control on
protein metabolism and have been shown to inhibit autophagy in the liver. Blommaart et
al. were the first to demonstrate that certain amino acids stimulate the phosphorylation
of the ribosomal protein S6 in isolated hepatocytes.59,60 The stimulation of S6 phosphory-
lation and the inhibition of autophagy were equally sensitive to the same amino acids,
among which leucine, tyrosine, and phenylalanine were the most effective. Their effects
were blocked by rapamycin, indicating that the mTOR-dependent activation of p70S6K
was responsible for S6 phosphorylation. Remarkably, insulin alone had no effect in isolated
1382_C18.fm Page 296 Tuesday, October 7, 2003 6:46 PM

296 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 18.2 Signaling pathways involved in the regulation of p70S6K activity by insulin, amino
acids, and AMP-activated protein kinase in liver. Right panel: Insulin, glutamine, or leucine activate
p70S6K and stimulate protein synthesis by different pathways, which all converge on mTOR. mTOR
activates p70S6K both by direct phosphorylation and by inhibition of protein phosphatase (PPase).
After phosphorylation by mTOR, p70S6K is further phosphorylated and fully activated by PDK-1.
p70S6K activation by glutamine involves GAPP. The link between GAPP and mTOR could be the
TSC1–TSC2 complex. From studies in other cell types, this complex is known to inhibit mTOR
activity. The inhibition can be relieved after phosphorylation of TSC2. We propose that glutamate
activates mTOR via GAPP, which could lead to TSC1–TSC2 complex (de)phosphorylation and
separation. Insulin by itself is not able to activate p70S6K. However, leucine alone or in combination
with insulin is able to induce p70S6K activation. The signaling pathways involved in these insu-
lin/leucine effects are still unknown. We speculate that they all converge on the TSC1–TSC2 complex.
Finally, leucine also increases the glutamine-induced p70S6K activation by increasing glutamate
concentration. Calyculin A (CA) has a dual effect on p70S6K activity, indicating that two phospha-
tases are involved, one upstream of mTOR, which leads to p70S6K activation, and one downstream
of mTOR, which inactivates p70S6K. Left panel: During oxygen deprivation, AMPK is able to induce
the dephosphorylation and inactivation of p70S6K, and thus the inhibition of protein synthesis. The
targets of AMPK could be the TSC1–TSC2 complex or mTOR.

hepatocytes unless low concentrations of amino acids were added. These initial observa-
tions were later confirmed by many other groups in various cell types.47,61–67 These studies,
including ours,15,68 demonstrated that incubation of cells with leucine enhanced the phos-
phorylation and activation state of p70S6K synergistically with insulin (Table 18.1) and in
a rapamycin-sensitive manner.
In addition, our recent work on isolated hepatocytes showed that glutamine leads to
a sustained activation of p70S6K and further enhanced the effects of leucine on p70S6K
(Table 18.1). However, glutamine did not act in synergism with insulin. Taken together,
these results indicate that, as already shown for GS and ACC activation, the signal trans-
duction leading to p70S6K activation by leucine differs from that of glutamine, both being
different from the insulin signaling pathway. Rapamycin was found to inhibit p70S6K
activation by leucine or glutamine alone, or leucine together with glutamine or insulin,
underscoring the fact that mTOR is a common target for these amino acids and insulin.15
1382_C18.fm Page 297 Tuesday, October 7, 2003 6:46 PM

Chapter eighteen: Anabolic effects of amino acids in the liver 297

Wortmannin was also found to inhibit p70S6K activation by glutamine,69 leucine,70 or


hypo-osmotic media,69 although this effect might be related to the fact that wortmannin
has been reported to inhibit mTOR52 or other lipid kinases (see below).

18.5 Signaling pathways triggered by amino acids


Several attempts have been made in the past to elucidate the signaling pathways triggered
by amino acids. The purpose of this section is to highlight the findings that represent a
significant advance in our understanding of the metabolic effects of amino acids.

18.5.1 PtdIns-3-K
It is now clear that the signaling pathway mediating the response to glutamine and leucine
differs from the insulin signaling pathway (Figure 18.1 and Figure 18.2). However, the
first steps of the insulin signaling cascade from the insulin receptor down to PtdIns-3-K
and PKB, which are indeed activated by insulin, are not affected by amino acids in the
liver.15 The increase in total PtdIns-3-K activity by glutamine and leucine, which we and
others have reported, did not correspond to an increased PtdIns-3-K activity associated
with IRS-1.15,69,70 Moreover, in other cell types, it has been shown that amino acids could
antagonize, at least in part, the activation of PtdIns-3-K induced by insulin.65,71
What then is the mechanism by which wortmannin inhibits the metabolic effects of
glutamine in liver? Besides PtdIns-3-K, other wortmannin-sensitive lipid kinases could be
involved. PtdIns-3-K and 3-phosphatidylinositol-5-kinase could work in concert and pro-
duce phosphatidylinositol-3,5-bisphosphate, whose concentration increases in cells sub-
mitted to a hypo-osmotic shock.72 Whether glutamine increases phosphatidylinositol-3,5-
bisphosphate in liver and what the downstream targets of this phospholipid are remain
to be established.

18.5.2 Protein phosphatases and GAPP


Our recent work has demonstrated that the activation of ACC and p70S6K by amino acids
is prevented by inhibitors of type 1 and 2A protein phosphatases and by activation of
AMPK.68 These results suggest that the known mechanisms of activation of ACC and
p70S6K should be reinterpreted (Figure 18.1 and Figure 18.2). ACC activation by amino
acids is blocked by protein phosphatase inhibitors but does not involve dephosphorylation
of Ser-79. We suggest that the protein phosphatase involved is GAPP, which is likely to
be activated by the amino acid-induced accumulation of glutamate. We speculate that
GAPP dephosphorylates inactivating sites other than Ser79.
In contrast with ACC, the active form of p70S6K is (multi)phosphorylated (Figure
18.2). Therefore, GAPP cannot be directly involved, although it could participate in the
activation cascade. p70S6K is activated by several protein kinases, among which mTOR
plays a crucial role. p70S6K activation by amino acids corresponds to an increased phos-
phorylation of Thr389 by mTOR. We expect that Thr229 by PDK1 also participates in
p70S6K activation by amino acids. In support of the intervention of an inhibiting protein
phosphatase downstream of mTOR is our observation68 that the amino acid-induced
activation of p70S6K is reinforced when inhibitors of protein phosphatase are added after
preincubation with amino acids. The mTOR-sensitive protein phosphatase would then be
inhibited, thus reinforcing the direct effect of mTOR on Thr389 phosphorylation. Moreover,
another protein phosphatase should also be involved in the activation of p70S6K by amino
acids. Indeed, preincubation of hepatocytes with protein phosphatase inhibitors prevents
the activation and phosphorylation of p70S6K by amino acids.68 This indicates that a
1382_C18.fm Page 298 Tuesday, October 7, 2003 6:46 PM

298 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

protein phosphatase upstream of mTOR should be involved. We suggest that this protein
phosphatase is GAPP, which could lead to the parallel activation of ACC and p70S6K by
amino acids. We have to speculate that GAPP activates mTOR through an indirect mech-
anism, which leads to the (de)phosphorylation of a site in TSC2 that promotes the inter-
action with TSC1 and hence the inhibition of mTOR. Taken together, these data support
the hypothesis that two phosphatases are involved, one upstream of mTOR, which leads
to ACC and p70S6K activation, and one downstream of mTOR, which inactivates p70S6K.

18.5.3 AMPK
The three anabolic pathways in this case, glycogen synthesis, lipogenesis, and protein
synthesis, share some common regulatory features. They are energy and nutrient sensitive.
When the energy supply is limited, as during hypoxia or metabolic stress, these pathways
are turned off. The fall in ATP concentration, which occurs under these conditions, is not
the only mechanism involved. Activation of AMPK by the increase in the intracellular
AMP:ATP ratio leads to a rapid and efficient inhibition of these three anabolic pathways.
Therefore, AMPK is another player in the game. It is indeed able to phosphorylate and
inactivate liver GS, although the exact site phosphorylated by AMPK is not known. AMPK
is also able to inactivate ACC by phosphorylation of Ser79. This overrules and antagonizes
ACC activation by amino acids.68 An additional inactivation of GAPP by AMPK has not
been ruled out. On the other hand, AMPK activation abrogates the amino acid-induced
activation of p70S6K, indicating that AMPK is involved in the regulation of p70S6K.68,73,74
However, AMPK does not act directly on p70S6K. Indeed, AMPK is unable to phospho-
rylate and inactivate p70S6K in vitro, and more importantly, the inactivation of p70S6K
by AMPK results from a decreased phosphorylation of Thr389, the site phosphorylated
by mTOR.68 AMPK could phosphorylate and inactivate mTOR. However, the fact that
p70S6K inactivation by AMPK is slower than the inactivation by rapamycin in intact cells
advocates against a direct action of AMPK on mTOR. We suggest that AMPK could
phosphorylate TSC2 on a site that promotes mTOR inhibition by the inhibitory TSC1–TSC2
complex. This site phosphorylated by AMPK could be dephosphorylated by GAPP.
Recently, a protein–protein interaction between AMPK and mTOR has been proposed.75
The putative intervention of TSC1–TSC2 complex in this interaction remains to be estab-
lished.
The indirect inactivation of p70S6K is but one mechanism by which AMPK inhibits
protein synthesis. Recent work from our group has demonstrated that AMPK activation
results in the phosphorylation and inactivation of eEF2, a factor controlling the elongation
step. Here again, AMPK does not directly phosphorylate and inactivate eEF2. The inhibi-
tion involves phosphorylation and activation by AMPK of eEF2 kinase, the upstream
protein kinase.76
There is no doubt that AMPK activation leads to the inhibition of the basal as well as
the amino acid-stimulated rate of glycogen, lipid, and protein synthesis. One may thus
wonder whether an inhibition of AMPK could mediate the stimulation of the anabolic
pathways by amino acids. AMPK would then act as a master switch for nutrients. The
experimental evidence so far obtained does not support this hypothesis. In hepatocytes
incubated with amino acids under normoxic conditions, the activity of AMPK is barely
detectable. Moreover, amino acids do not antagonize the dose-dependent activation of
AMPK that is observed in hepatocytes incubated with a stimulator of AMPK.68 An amino
acid-induced inactivation of AMPK is thus ruled out.
1382_C18.fm Page 299 Tuesday, October 7, 2003 6:46 PM

Chapter eighteen: Anabolic effects of amino acids in the liver 299

18.5.4 Interaction between leucine, glutamine, and insulin


By contrast with glutamine, leucine transport into liver cells does not depend on Na+, and
so does not cause swelling and GAPP activation. Liver metabolism of leucine is very
limited because this organ contains very little transaminase acting on branched-chain
amino acids. However, in liver, leucine stimulates glutaminase, thereby increasing the
intracellular concentration of glutamate when glutamine is present. The resulting stimu-
lation of GAPP by glutamate could explain the synergism between leucine and glutamine
for ACC activation.
The activation of p70S6K induced by leucine results from hyperphosphorylation of
this enzyme in a rapamycin-sensitive manner, as is the case for glutamine. Insulin further
activates p70S6K in hepatocytes incubated with leucine but not with glutamine. Moreover,
glutamine and leucine synergistically and maximally activate p70S6K. All these observa-
tions confirm that the control by glutamine, leucine, and insulin differs.

18.6 Conclusions
The experimental evidence reviewed in this chapter indicates that amino acids and insulin
exert combinatory effects that are mediated by different, but linked, signaling pathways.
Indeed, work from several groups, including ours, shows that leucine exerts a permissive
effect on insulin signaling to both p70S6K and ACC, although the signaling pathways
used to modulate these two targets are different. Moreover, the fact that activation of
p70S6K by glutamine, leucine, and insulin is rapamycin sensitive indicates that they all
converge on mTOR or its regulator, namely, the TSC1/TSC2. In addition, a common
activating protein phosphatase is probably involved in the activation of both ACC and
p70S6K by amino acids. Finally, AMPK is able to prevent p70S6K as well as ACC activation
by glutamine, leucine, and insulin. Therefore, the mTOR–p70S6K axis and ACC appear
as central targets for the control of protein synthesis and lipogenesis, respectively. By
contrast, GS seems to be exclusively activated by glutamine, whereas leucine acts as a
negative modulator on this activation.
Several questions remain unanswered. Whether there is a specific leucine sensor77 and
which upstream elements leading to ACC and p70S6K activation mediate the effects of
leucine remain to be elucidated. Similarly, the target of AMPK in the regulation pathway
of p70S6K activity is still unknown. Finally, the elucidation of the mechanism responsible
for the insulin resistance that is observed in liver, as well as the permissive effect of leucine
on insulin action, remains an important challenge for the future.

Acknowledgments
The work carried out in the authors’ laboratory was supported by grants from the Belgian
Fund for Medical Scientific Research, the European Union (FP5), and the Federal and
Regional Authorities of Belgium.

References
1. Hue, L., Bontemps, F., and Hers, H., The effects of glucose and of potassium ions on the
interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in
isolated rat liver preparations, Biochem. J., 152, 105, 1975.
2. Stalmans, W. and van de Werve, G., Regulation of glycogen metabolism by insulin, in Short-
Term Regulation of Liver Metabolism, Hue, L. and van de Werve, G., Eds., Elsevier, Amsterdam,
1981, p. 119.
1382_C18.fm Page 300 Tuesday, October 7, 2003 6:46 PM

300 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

3. Katz, J., Golden, S., and Wals, P.A., Stimulation of hepatic glycogen synthesis by amino acids,
Proc. Natl. Acad. Sci. U.S.A., 73, 3433, 1976.
4. Lavoinne, A., Baquet, A., and Hue, L., Stimulation of glycogen synthesis and lipogenesis by
glutamine in isolated rat hepatocytes, Biochem. J., 248, 429, 1987.
5. Baquet, A., Lavoinne, A., and Hue, L., Comparison of the effects of various amino acids on
glycogen synthesis, lipogenesis and ketogenesis in isolated rat hepatocytes, Biochem. J., 273,
57, 1991.
6. Hue, L., Gaussin, V., and Krause, U., Anabolic response to cell swelling in the liver, in
Contributions of Physiology to the Understanding of Diabetes, Zahnd, G.R. and Wollheim, C.B.,
Eds, Springer-Verlag, Berlin, 1997, p. 10.
7. Rognstad, R., Effects of amino acids analogs and amino acid mixtures on glycogen synthesis
in rat hepatocytes, Biochem. Arch., 2, 185, 1986.
8. Baquet, A. et al., Swelling of rat hepatocytes stimulates glycogen synthesis, J. Biol. Chem.,
265, 955, 1990.
9. Baquet, A., Maisin, L., and Hue, L., Swelling of rat hepatocytes activates acetyl-CoA carbox-
ylase in parallel to glycogen synthase, Biochem. J., 278, 887, 1991.
10. Hoffmann, E.K. and Simonsen, L.O., Membrane mechanisms in volume and pH regulation
in vertebrate cells, Physiol. Rev., 69, 315, 1989.
11. Meijer, A.J. et al., Mechanism of activation of liver glycogen synthase by swelling, J. Biol.
Chem., 267, 5823, 1992.
12. Baquet, A. et al., Mechanism of activation of liver acetyl-CoA carboxylase by cell swelling,
Eur. J. Biochem., 217, 1083, 1993.
13. Gaussin, V. et al., Activation of hepatic acetyl-CoA carboxylase by glutamate and Mg2+ is
mediated by protein phosphatase-2A, Biochem. J., 316, 217, 1996.
14. Hardie, D.G. and Hawley, S.A., AMP-activated protein kinase: the energy charge hypothesis
revisited, Bioessays, 23, 1112, 2001.
15. Krause, U. et al., Signalling pathways and combinatory effects of insulin and amino acids
in isolated rat hepatocytes, Eur. J. Biochem., 269, 3742, 2002.
16. Fernandez-Novell, J.M. et al., Glucose induces the translocation and the aggregation of
glycogen synthase in rat hepatocytes, Biochem. J., 281, 443, 1992.
17. Fernandez-Novell, J.M. et al., Glucose induces the translocation of glycogen synthase to the
cell cortex in rat hepatocytes, Biochem. J., 321, 227, 1997.
18. Fernandez-Novell, J.M. et al., Role of glucose 6-phosphate in the translocation of glycogen
synthase in rat hepatocytes, Biochem. J., 288, 497, 1992.
19. Agius, L. et al., Evidence for a role of glucose-induced translocation of glucokinase in the
control of hepatic glycogen synthesis, J. Biol. Chem., 271, 30479, 1996.
20. de la Iglesia, N. et al., Glucokinase regulatory protein is essential for the proper subcellular
localisation of liver glucokinase, FEBS Lett., 456, 332, 1999.
21. Carabaza, A. et al., Role of AMP on the activation of glycogen synthase and phosphorylase
by adenosine, fructose, and glutamine in rat hepatocytes, J. Biol. Chem., 265, 2724, 1990.
22. Allred, J.B. and Roman-Lopez, C.R., Enzymatically inactive forms of acetyl-CoA carboxylase
in rat liver mitochondria, Biochem. J., 251, 881, 1988.
23. Allred, J.B. et al., Mitochondrial storage forms of acetyl CoA carboxylase: mobilization/ac-
tivation accounts for increased activity of the enzyme in liver of genetically obese Zucker
rats, J. Nutr., 119, 478, 1989.
24. Moir, A.M. and Zammit, V.A., Changes in the properties of cytosolic acetyl-CoA carboxylase
studied in cold-clamped liver samples from fed, starved and starved-refed rats, Biochem. J.,
272, 511, 1990.
25. Beaty, N.B. and Lane, M.D., Kinetics of activation of acetyl-CoA carboxylase by citrate:
relationship to the rate of polymerization of the enzyme, J. Biol. Chem., 258, 13043, 1983.
26. Beaty, N.B. and Lane, M.D., Kinetics of citrate-induced activation and polymerization of
chick liver acetyl-CoA carboxylase, Ann. N.Y. Acad. Sci., 447, 23, 1985.
27. Thampy, K.G. and Wakil, S.J., Regulation of acetyl-coenzyme A carboxylase. II. Effect of
fasting and refeeding on the activity, phosphate content, and aggregation state of the enzyme,
J. Biol. Chem., 263, 6454, 1988.
1382_C18.fm Page 301 Tuesday, October 7, 2003 6:46 PM

Chapter eighteen: Anabolic effects of amino acids in the liver 301

28. Mabrouk, G.M. et al., Acute hormonal control of acetyl-CoA carboxylase: the roles of insulin,
glucagon, and epinephrine, J. Biol. Chem., 265, 6330, 1990.
29. Geelen, M.J. et al., Studies on the intracellular localization of acetyl-CoA carboxylase, Biochem.
Biophys. Res. Commun., 233, 253, 1997.
30. Quayle, K.A., Denton, R.M., and Brownsey, R.W., Evidence for a protein regulator from rat
liver which activates acetyl-CoA carboxylase, Biochem. J., 292, 75, 1993.
31. Saltiel, A.R. and Kahn, C.R., Insulin signalling and the regulation of glucose and lipid
metabolism, Nature, 414, 799, 2001.
32. Loten, E.G. et al., Stimulation of a low Km phosphodiesterase from liver by insulin and
glucagon, J. Biol. Chem., 253, 746, 1978.
33. Exton, J.H., Mechanisms of hormonal regulation of hepatic glucose metabolism, Diabetes
Metab. Rev., 3, 163, 1987.
34. Dubbelhuis, P.F. et al., Inhibition of autophagic proteolysis by inhibitors of phosphoinositide
3-kinase can interfere with the regulation of glycogen synthesis in isolated hepatocytes,
Biochem. J., 368, 827, 2002.
35. Vanhaesebroeck, B. and Alessi, D.R., The PI3K-PDK1 connection: more than just a road to
PKB, Biochem. J., 346, 561, 2000.
36. Shah, O.J. et al., 4E-BP1 and S6K1: translational integration sites for nutritional and hormonal
information in muscle, Am. J. Physiol. Endocrinol. Metab., 279, E715, 2000.
37. van Sluijters, D.A. et al., Amino-acid-dependent signal transduction, Biochem. J., 351, 545,
2000.
38. Gingras, A.C., Raught, B., and Sonenberg, N., Regulation of translation initiation by
FRAP/mTOR, Genes Dev., 15, 807, 2001.
39. Proud, C.G., Regulation of mammalian translation factors by nutrients, Eur. J. Biochem., 269,
5338, 2002.
40. Browne, G.J. and Proud, C.G., Regulation of peptide-chain elongation in mammalian cells,
Eur. J. Biochem., 269, 5360, 2002.
41. Proud, C.G. et al., Interplay between insulin and nutrients in the regulation of translation
factors, Biochem. Soc. Trans., 29, 541, 2001.
42. Pullen, N. and Thomas, G., The modular phosphorylation and activation of p70s6k, FEBS
Lett., 410, 78, 1997.
43. Dufner, A. and Thomas, G., Ribosomal S6 kinase signaling and the control of translation,
Exp. Cell. Res., 253, 100, 1999.
44. Martin, K.A. and Blenis, J., Coordinate regulation of translation by the PI 3-kinase and mTOR
pathways, Adv. Cancer Res., 86, 1, 2002.
45. Burnett, P.E. et al., RAFT1 phosphorylation of the translational regulators p70 S6 kinase and
4E-BP1, Proc. Natl. Acad. Sci. U.S.A., 95, 1432, 1998.
46. Pullen, N. et al., Phosphorylation and activation of p70s6k by PDK1, Science, 279, 707, 1998.
47. Hara, K. et al., Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1
through a common effector mechanism, J. Biol. Chem., 273, 14484, 1998.
48. Dufner, A. et al., Protein kinase B localization and activation differentially affect S6 kinase
1 activity and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylation,
Mol. Cell. Biol., 19, 4525, 1999.
49. Peterson, R.T. et al., Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is
activated by inhibition of FKBP12-rapamycin associated protein, Proc. Natl. Acad. Sci. U.S.A.,
96, 4438, 1999.
50. Weng, Q.P. et al., Regulation of the p70 S6 kinase by phosphorylation in vivo: analysis using
site-specific anti-phosphopeptide antibodies, J. Biol. Chem., 273, 16621, 1998.
51. Dennis, P.B. et al., Mammalian TOR: a homeostatic ATP sensor, Science, 294, 1102, 2001.
52. Brunn, G.J. et al., Direct inhibition of the signaling functions of the mammalian target of
rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, EMBO
J., 15, 5256, 1996.
53. Chung, J. et al., Rapamycin-FKBP specifically blocks growth-dependent activation of and
signaling by the 70 kd S6 protein kinases, Cell, 69, 1227, 1992.
1382_C18.fm Page 302 Tuesday, October 7, 2003 6:46 PM

302 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

54. Price, D.J. et al., Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase, Science,
257, 973, 1992.
55. Gao, X. et al., Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling, Nat.
Cell. Biol., 4, 699, 2002.
56. McManus, E.J. and Alessi, D.R., TSC1-TSC2: a complex tale of PKB-mediated S6K regulation,
Nat. Cell. Biol., 4, E214, 2002.
57. Potter, C.J., Pedraza, L.G., and Xu, T., Akt regulates growth by directly phosphorylating Tsc2,
Nat. Cell. Biol., 4, 658, 2002.
58. Tee, A.R. et al., Tuberous sclerosis complex-1 and -2 gene products function together to inhibit
mammalian target of rapamycin (mTOR)-mediated downstream signaling, Proc. Natl. Acad.
Sci. U.S.A., 99, 13571, 2002.
59. Blommaart, E.F. et al., Phosphorylation of ribosomal protein S6 is inhibitory for autophagy
in isolated rat hepatocytes, J. Biol. Chem., 270, 2320, 1995.
60. Blommaart, E.F., Luiken, J.J., and Meijer, A.J., Autophagic proteolysis: control and specificity,
Histochem. J., 29, 365, 1997.
61. Campbell, L.E., Wang, X., and Proud, C.G., Nutrients differentially regulate multiple trans-
lation factors and their control by insulin, Biochem. J., 344, 433, 1999.
62. Fox, H.L. et al., Amino acids stimulate phosphorylation of p70S6k and organization of rat
adipocytes into multicellular clusters, Am. J. Physiol., 274, C206, 1998.
63. Iiboshi, Y. et al., Amino acid-dependent control of p70(s6k): involvement of tRNA amino-
acylation in the regulation, J. Biol. Chem., 274, 1092, 1999.
64. Kimball, S.R., Horetsky, R.L., and Jefferson, L.S., Implication of eIF2B rather than eIF4E in
the regulation of global protein synthesis by amino acids in L6 myoblasts, J. Biol. Chem., 273,
30945, 1998.
65. Patti, M.E. et al., Bidirectional modulation of insulin action by amino acids, J. Clin. Invest.,
101, 1519, 1998.
66. Wang, X. et al., Amino acid availability regulates p70 S6 kinase and multiple translation
factors, Biochem. J., 334, 261, 1998.
67. Xu, G. et al., Branched-chain amino acids are essential in the regulation of PHAS-I and p70
S6 kinase by pancreatic beta-cells: a possible role in protein translation and mitogenic sig-
naling, J. Biol. Chem., 273, 28178, 1998.
68. Krause, U., Bertrand, L., and Hue, L., Control of p70 ribosomal protein S6 kinase and
acetylCoA carboxylase activity by AMP-activated protein kinase and protein phosphatases
in isolated rat hepatocytes, Eur. J. Biochem., 269, 3751, 2002.
69. Krause, U., Rider, M.H., and Hue, L., Protein kinase signaling pathway triggered by cell
swelling and involved in the activation of glycogen synthase and acetyl-CoA carboxylase in
isolated rat hepatocytes, J. Biol. Chem., 271, 16668, 1996.
70. Peyrollier, K. et al., L-Leucine availability regulates phosphatidylinositol 3-kinase, p70 S6
kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involve-
ment of the mammalian target of rapamycin (mTOR) pathway in the L-leucine-induced up-
regulation of system A amino acid transport, Biochem. J., 350, 361, 2000.
71. Tremblay, F. and Marette, A., Amino acid and insulin signaling via the mTOR/p70 S6 kinase
pathway: a negative feedback mechanism leading to insulin resistance in skeletal muscle
cells, J. Biol. Chem., 276, 38052, 2001.
72. Dove, S.K. et al., Osmotic stress activates phosphatidylinositol-3,5-bisphosphate synthesis,
Nature, 390, 187, 1997.
73. Dubbelhuis, P.F. and Meijer, A.J., Hepatic amino acid-dependent signaling is under the
control of AMP-dependent protein kinase, FEBS Lett., 521, 39, 2002.
74. Bolster, D.R. et al., AMP-activated protein kinase suppresses protein synthesis in rat skeletal
muscle through down-regulated mammalian target of rapamycin (mTOR) signaling, J. Biol.
Chem., 277, 23977, 2002.
75. Kimura, N. et al., A possible linkage between AMP-activated protein kinase (AMPK) and
mammalian target of rapamycin (mTOR) signalling pathway, Genes Cells, 8, 65, 2003.
1382_C18.fm Page 303 Tuesday, October 7, 2003 6:46 PM

Chapter eighteen: Anabolic effects of amino acids in the liver 303

76. Horman, S. et al., Activation of AMP-activated protein kinase leads to the phosphorylation
of elongation factor 2 and an inhibition of protein synthesis, Curr. Biol., 12, 1419, 2002.
77. Christie, G.R. et al., Intracellular sensing of amino acids in Xenopus laevis oocytes stimulates
p70 S6 kinase in a target of rapamycin-dependent manner, J. Biol. Chem., 277, 9952, 2002.
1382_C18.fm Page 304 Tuesday, October 7, 2003 6:46 PM
1382_C19.fm Page 305 Tuesday, October 7, 2003 6:48 PM

chapter nineteen

Amino acids and immune function


Philip C. Calder
University of Southampton
Parveen Yaqoob
The University of Reading

Contents
Introduction..................................................................................................................................306
19.1 The immune system..........................................................................................................306
19.1.1 Components of the immune system..................................................................306
19.1.2 Innate immunity ...................................................................................................307
19.1.3 Acquired immunity ..............................................................................................307
19.2 Specific amino acids, immune function, and infection ...............................................309
19.2.1 Arginine..................................................................................................................309
19.2.1.1 Metabolic and physiological functions of arginine of relevance
to immune function ...............................................................................309
19.2.1.2 Arginine, immune function, and infection ........................................309
19.2.1.2.1 Cell culture studies .............................................................309
19.2.1.2.2 Animal feeding studies ......................................................310
19.2.1.2.3 Arginine and immune function: studies in healthy
subjects ..................................................................................311
19.2.1.2.4 Arginine, immune function, and infection: human
clinical studies......................................................................311
19.2.2 Glutamine...............................................................................................................312
19.2.2.1 Metabolic functions of glutamine of relevance to immune
function....................................................................................................312
19.2.2.2 Glutamine, immune cell function, and infection: cell culture
and animal feeding studies ..................................................................312
19.2.2.2.1 Cell culture studies .............................................................312
19.2.2.2.2 Animal feeding studies ......................................................312
19.2.2.3 Glutamine, immune function, and infection: human clinical
studies ......................................................................................................313
19.2.3 Sulfur amino acids................................................................................................314
19.2.3.1 Metabolic functions of sulfur amino acids of relevance
to immune function ...............................................................................314
19.2.3.2 Cysteine, glutathione, and immune function....................................315

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 305
1382_C19.fm Page 306 Tuesday, October 7, 2003 6:48 PM

306 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

19.2.3.2.1 Glutathione status in infection and catabolic stress......315


19.2.3.2.2 Cysteine, glutathione, and immune function: cell
culture studies......................................................................315
19.2.3.2.3 Glutathione and immune function: studies in healthy
humans..................................................................................315
19.2.3.2.4 Glutathione and immune function: human clinical
studies ...................................................................................315
19.2.3.3 Taurine and immune function .............................................................315
19.3 Conclusion ..........................................................................................................................316
References .....................................................................................................................................316

Introduction
The immune system exists to protect the host from pathogenic invaders and from other
noxious insults. Upon infection there is a marked increase in demand for substrates by
the immune system; these substrates will provide energy and the precursors for the
synthesis of new cells, effector molecules (e.g., antibodies, cytokines, acute phase proteins),
and protective molecules (e.g., glutathione). The physiology and biochemistry of an
infected individual is fundamentally changed in a way that will ensure that the immune
system receives nutrients from within the body. Muscle protein is catabolized to provide
amino acids for synthesizing new cells, proteins, and peptides for the immune response.
Furthermore, amino acids are converted to glucose, a preferred fuel, together with
glutamine, for the immune system. An increase in urinary nitrogen and sulfur excretion
occurs as a result of this catabolic process. The extent of this process is highlighted by the
significant increase in urinary nitrogen excretion from 9 g/day in mild infection to 20 to
30 g/day following major burns or severe traumatic injury.1 The loss of nitrogen from the
body of an adult during a bacterial infection may be equivalent to 60 g of tissue protein,
and in a period of persistent malarial infection, equivalent to over 500 g of protein. Despite
the mobilization of muscle proteins, the plasma concentrations of some amino acids, such
as glutamine, fall during infections and catabolic stress,2–6 presumably because demands
exceed supply. Since certain amino acids have been demonstrated to play a role in sup-
porting an efficient immune response and since catabolic states are associated with
increased susceptibility to infections, a case has been made for providing an exogenous
supply of these amino acids or their precursors in certain clincal settings. This chapter
describes the effect of altered amino acid supply on the ability of the immune system to
respond efficiently when challenged and on the subsequent ability of the host to deal with
infectious agents. The chapter begins with a description of the immune system and its
components and how they respond in an integrated manner when challenged. A more
full description of the immune response may be found in any immunology textbook. The
influence of some amino acids on immune function has been examined in great detail,
and there is a strong metabolic and clinical rationale behind this research. It is these amino
acids (arginine, glutamine, the sulfur amino acids) that this chapter deals with in detail.

19.1 The immune system


19.1.1 Components of the immune system
The immune system acts to protect the host from infectious agents that exist in the
environment (bacteria, viruses, fungi, parasites) and from other noxious insults. The
immune system has two functional divisions: the innate (or natural) and the acquired
1382_C19.fm Page 307 Tuesday, October 7, 2003 6:48 PM

Chapter nineteen: Amino acids and immune function 307

Figure 19.1 The interrelationship between the innate and acquired immune responses.

(also termed specific or adaptive). Both components of immunity involve various blood-
borne factors and cells. All cells of the immune system originate in bone marrow. They
are found circulating in the bloodstream, organized into lymphoid organs such as the
thymus, spleen, and lymph nodes, or dispersed in other locations around the body.

19.1.2 Innate immunity


Innate immunity is the first line of defense against infectious agents. It is present before
exposure to pathogens and is concerned with preventing the entry of infectious agents
into the body and, if they do enter, with their rapid elimination. The innate immune system
includes physical barriers, soluble factors, and phagocytic cells. Innate immunity has no
memory and is therefore not influenced by prior exposure to an organism. Phagocytic
cells express surface receptors specific for bacterial antigens. Binding of antigen to the
receptors triggers phagocytosis and subsequent destruction of the pathogenic microorgan-
ism by complement or by toxic chemicals, such as superoxide radicals and hydrogen
peroxide. Natural killer cells also possess surface receptors and destroy pathogens by
release of cytotoxic proteins. In this way, innate immunity provides a first line of defense
against invading pathogens. However, an immune response often requires the coordinated
actions of both innate immunity and the more powerful and flexible acquired immunity
(Figure 19.1).

19.1.3 Acquired immunity


The acquired immune response involves lymphocytes. It is highly specific, since each
lymphocyte carries surface receptors for a single antigen. The acquired immune response
becomes effective over several days after the initial activation, but it also persists for some
1382_C19.fm Page 308 Tuesday, October 7, 2003 6:48 PM

308 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

time after the removal of the initiating antigen. This persistence gives rise to immunolog-
ical memory, which is the basis for a stronger, more effective immune response upon
reexposure to an antigen (i.e., reinfection with the same pathogen).
B lymphocytes are characterized by their ability to produce antibodies (immuno-
globulins (Ig)), which are specific for an individual antigen. Antibodies work in several
ways to combat invading pathogens. They can neutralize microorganisms by binding to
them and preventing their attachment to host cells, and they can activate complement
proteins in plasma, which in turn promote the destruction of bacteria by phagocytes.
Immunity involving antibodies (humoral immunity) deals with extracellular pathogens.
However, some pathogens, particularly viruses, but also some bacteria, infect individuals
by entering cells. These pathogens will escape humoral immunity and are instead dealt
with by cell-mediated immunity, which is conferred by T lymphocytes. T lymphocytes
express antigen-specific T cell receptors on their surface. However, unlike B lymphocytes,
they are only able to recognize antigens that are presented to them on a cell surface; this
is the distinguishing feature between humoral and cell-mediated immunity. Therefore,
infection of a cell by an intracellular pathogen is signaled to T lymphocytes by cell surface
expression of peptide fragments derived from the pathogen. These fragments are trans-
ported to the surface of the infected cell and expressed there in conjunction with proteins
termed major histocompatibility complex (MHC); in humans, MHC is termed human
leukocyte antigen (HLA). It is the combination of the pathogen-derived peptide fragment
bound to MHC that is recognized by T lymphocytes. Intracellular pathogens stimulate
cytotoxic T lymphocytes to destroy the infected cell, while extracellular pathogens stim-
ulate a helper T cell-mediated response. In delayed type hypersensitivity (DTH), antigen-
activated CD4+ T lymphocytes (helper T cells) secrete cytokines, which have several effects,
including recruitment of neutrophils and monocytes from the blood to the site of antigen
challenge and activation of monocytes in order to effect elimination of the antigen.
Communication within the acquired immune system and between the innate and
acquired systems is brought about by direct cell-to-cell contact involving adhesion mole-
cules and by the production of chemical messengers, which send signals from one cell to
another (Figure 19.1). Chief among these chemical messengers are proteins called cyto-
kines, which can act to regulate the activity of the cell that produced the cytokine or of
other cells. Each cytokine can have multiple activities on different cell types. Cytokines
act by binding to specific receptors on the cell surface and thereby induce changes in
growth, development, or activity of the target cell. Tumor necrosis factor (TNF)-a, inter-
leukin (IL)-1, and IL-6 are among the most important cytokines produced by monocytes
and macrophages. These cytokines activate neutrophils, monocytes, and macrophages to
initiate bacterial and tumor cell killing, increase adhesion molecule expression on the
surface of neutrophils and endothelial cells, stimulate T and B lymphocyte proliferation,
and initiate the production of other proinflammatory cytokines. Thus, TNF, IL-1, and IL-6
are mediators of both natural and acquired immunity and are an important link between
them (Figure 19.1). In addition, these cytokines mediate the systemic effects of inflamma-
tion such as fever, weight loss, and acute phase protein synthesis in the liver. Inflammation
is the body’s immediate response to infection or injury and is an integral part of the innate
immune response. Thus, production of appropriate amounts of TNF, IL-1, and IL-6 is
important in response to infection. However, inappropriate production or overproduction
can be dangerous, and these cytokines, particularly TNF, are implicated in causing some
of the pathological responses that occur in acute and chronic inflammatory conditions.
Helper T lymphocytes can be subdivided into two broad categories according to the
pattern of cytokines they produce. Th1 cells produce IL-2 and interferon (IFN)-g, which
activate macrophages, natural killer cells, and cytotoxic T lymphocytes, and are the
principal effectors of cell-mediated immunity against viruses and bacteria. Th2 cells
1382_C19.fm Page 309 Tuesday, October 7, 2003 6:48 PM

Chapter nineteen: Amino acids and immune function 309

produce IL-4, which stimulates IgE production, IL-5, an eosinophil activating factor, and
IL-10, which together with IL-4 suppresses cell-mediated immunity. Th2 cells are respon-
sible for defense against helminthic parasites, which is due to IgE-mediated activation of
mast cells and basophils.

19.2 Specific amino acids, immune function, and infection


19.2.1 Arginine
19.2.1.1 Metabolic and physiological functions of arginine of relevance to immune
function
Arginine is an intermediate in many metabolic pathways and is an important substrate
for the synthesis of proteins, of the polyamines required for DNA, RNA, and protein
synthesis, and of the potent gaseous mediator nitric oxide (see Chapter 10). The inducible
form of nitric oxide synthase (iNOS) is of most relevance to the immune system. iNOS
expression, and hence nitric oxide production, is induced in monocytes and macrophages
in response to a variety of stimuli, particularly the Th1 cytokine IFN-g and the Gram-
negative bacterial wall component endotoxin (lipopolysaccharide (LPS)). Nitric oxide is a
regulator of many immune functions (see Chapter 14), and inhibition of its production
increases the host susceptibility to viral, bacterial, fungal, protozoal, and helminthic infec-
tions. Thus, nitric oxide (and hence the availability of the substrate for its synthesis,
arginine) appears to be essential for effective host defense.
An alternative pathway of arginine metabolism is via arginase, which converts argi-
nine to ornithine. Arginase activity is increased in macrophages by stimulation with LPS
and with T cell-derived cytokines7–10 and is increased by mitogenic stimulation of lym-
phocytes.11 One potential fate of the ornithine produced by arginase is in the synthesis of
the polyamines (putrescine, spermidine, spermine). Polyamines are required for mainte-
nance of cell viability, and their levels increase during cell growth, differentiation, and
proliferation. They act to facilitate DNA, RNA, and protein synthesis. Inhibition of
polyamine synthesis by inhibiting ornithine decarboxylase leads to a reduction in cell
viability, cell cycle arrest, and inhibition of cell differentiation.
Arginine is also a secretagogue for several hormones, including prolactin, growth
hormone, and insulin-like growth factor-1 (IGF-1), that can influence immune function.12–15
Prolactin induces maturation of dendritic cells, by increasing the expression of the antigen-
presenting MHC class II molecules and costimulatory molecules. Prolactin can also stim-
ulate the release of Th1 cytokines by T lymphocytes. Growth hormone can potentiate the
cytokine responses of human T cells, improve the antigen-presenting capability of den-
dritic cells, and increase the numbers of hematopoietic progenitor cells in the bone marrow.
IGF-1 plays an important role in the maturation of lymphocytes in the bone marrow and
in their function in the periphery. In rodents, IGF-1 can restore age-related thymic invo-
lution and increase lymphocyte number and activity. In addition, the thymotropic effects
of growth hormone appear to be mediated through IGF-1.

19.2.1.2 Arginine, immune function, and infection


19.2.1.2.1 Cell culture studies. Arginine is required for optimal proliferation of lym-
phocytes in vitro: diminished mitogenic responses of rodent and human T lymphocytes
are seen in arginine-free conditions16,17 (Figure 19.2) or in conditions in which arginine use
is inhibited.18 Although this effect may be due to decreased protein and polyamine
synthesis in the absence of arginine, some workers have demonstrated that normal pro-
liferative responses are observed when nitric oxide donors such as sodium nitroprusside
1382_C19.fm Page 310 Tuesday, October 7, 2003 6:48 PM

310 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 19.2 The influence of arginine and glutamine on T lymphocyte proliferation. Rat lymphocytes
were incubated under standard conditions in four different sets of conditions: (1) in the absence of
glutamine but with increasing concentrations of arginine, (2) in the absence of arginine but with
increasing concentrations of glutamine, (3) in the presence of 1 mM arginine and increasing con-
centrations of glutamine, and (4) in the presence of 2 mM glutamine and increasing concentrations
of arginine. Lymphocyte proliferation in response to concanavalin A was determined as [3H]thymi-
dine incorporation (cpm/well) over the final 18 h of a 66-h culture period. (Data are from Calder,
P.C., Proc. Nutr. Soc., 54, 123A, 1995.)

are provided to cultures lacking arginine.19 This indicates that arginine promotes T lym-
phocyte proliferative responses through generation of nitric oxide. High concentrations
of arginine have also been shown to increase monocyte and natural killer cell cytotoxicity
in vitro.20,21

19.2.1.2.2 Animal feeding studies. Studies conducted many years ago showed that
supplemental dietary arginine reduced trauma-induced thymic involution, lessened
weight loss, improved wound healing, and prolonged survival in injured rats (see Barbul22
for references). Furthermore, adding arginine to the diet of normal healthy mice or rats
increased thymic weight and the number of thymic lymphocytes (see Barbul22). Increased
dietary arginine enhanced the DTH response in normal mice and in tumor-bearing or
septic animals.23–25 The improved DTH response was found to correlate with increased
T cell proliferation in response to stimulation by mitogens or tumor antigens.24,25 In addi-
tion, there was an increase in specific T cell cytotoxicity, IL-2 production, and IL-2 receptor
expression on T lymphocytes and in macrophage-mediated and natural killer cell cyto-
toxicity.24,25 Rejection of implanted tumors in mice was associated with elevated levels of
nitric oxide production and iNOS expression in peritoneal macrophages and with a reduc-
tion in arginase activity,26 suggesting a role for nitric oxide in the antitumor effects of
arginine. However, whether the effect of nitric oxide is directly on tumor cells or via
improved immune function (or both of these) is not clear. In a burn model in guinea pigs,
Saito et al.27 showed an increase in DTH and in survival in arginine-supplemented animals
compared with controls. Madden et al.28 demonstrated a similar survival advantage of
1382_C19.fm Page 311 Tuesday, October 7, 2003 6:48 PM

Chapter nineteen: Amino acids and immune function 311

supplemental dietary arginine in animals subjected to lethal bacterial peritonitis. Gianotti


et al.29 also found improved survival in arginine-fed mice following cecal ligation and
puncture or gavage with Escherichia coli and burn injury. They also reported a reduction
in bacterial translocation and increased bacteriacidal activity of host phagocytes in the
arginine-supplemented animals. The survival advantage of arginine was eliminated by
the concomitant administration of a NOS inhibitor,29 again suggesting that nitric oxide is
involved. A number of animal studies indicate that ornithine a-ketoglutarate modulates
immune function in various models of burns, sepsis, tumor bearing, and catabolic stress,
and that this action is related to arginine generation; these studies are addressed elsewhere
(Chapter 37).

19.2.1.2.3 Arginine and immune function: studies in healthy subjects. In healthy human
subjects, arginine supplementation (30 g/day) increased blood T lymphocyte proliferation
in response to mitogens and decreased CD8+ cell numbers, while not affecting total lym-
phocyte or CD4+ cell numbers.30,31

19.2.1.2.4 Arginine, immune function, and infection: human clinical studies. Daly et al.32
studied the immunologic effects of supplemental dietary arginine in patients undergoing
major operations for gastrointestinal malignancy. Patients received enteral arginine
(25 g/day) or isonitrogenous glycine (43 g/day) for 7 days postoperatively. Arginine
supplementation was associated with an increased number of circulating CD4+ cells and
an enhanced response of peripheral blood lymphocytes to mitogens by day 7. Although,
only the arginine-supplemented group achieved a positive nitrogen balance (by day 6),
there was no difference in clinical outcome between the two groups.
A large number of studies incorporating arginine into enteral formulae also containing
other so-called immunonutrients have been conducted in intensive care and surgical
patients. The majority of these trials have used the commercially available product
IMPACT®, which contains arginine, nucleotides, and n-3 fatty acids. Meta-analyses of
controlled, randomized studies using IMPACT or similar immunonutrient mixes identified
significant reductions in infections and in length of hospital stay; in general, these effects
are more pronounced in surgical than critically ill patients.33–35 Despite these apparent
benefits of immunonutrition, none of the three meta-analyses identified a significant effect
of immunonutrition on mortality either across all trials considered or within surgical or
critically ill patients.
A number of the studies of enteral nutrition involving arginine reported immune
or inflammatory outcomes (see Calder36 for references). A number of studies have
reported circulating lymphocyte numbers and subsets and circulating immunoglobulin
concentrations, and most report little difference in these compared with the control
group. Some studies have reported aspects of immune function such as phagocytosis,
respiratory burst, lymphocyte proliferation, human leukocyte antigen-DR expression on
monocytes, and cytokine production; several of these studies report some significant
improvements in these functions in patients receiving immunonutrition compared with
the control group. Although these observations fit with the effects of arginine that might
be predicted based upon studies in cell culture, animals, and healthy humans, and could
be used as evidence of the efficacy of arginine in trauma and postsurgery settings, the
complex nature of the formulae prevents such a clear interpretation. The effects could
be due to any one of the specified nutrients (i.e., arginine, RNA, n-3 PUFA) or to the
combination of these nutrients.
1382_C19.fm Page 312 Tuesday, October 7, 2003 6:48 PM

312 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

19.2.2 Glutamine
19.2.2.1 Metabolic functions of glutamine of relevance to immune function
The immune system is considered to be an important user of glutamine supplied from
the bloodstream (see Wilmore and Shabert,37 Calder and Yaqoob,38 and Calder and
Newsholme39 for reviews). In immune cells glutamine acts as an important energy source,
a nitrogen donor for the synthesis of purines and pyrimidines, the building blocks of RNA
and DNA, a substrate for protein synthesis, and a precursor to glutamate, which is
incorporated into the antioxidant glutathione. The activity of phosphate-dependent
glutaminase is high in lymphoid organs and in lymphocytes, macrophages, and neutro-
phils. Glutaminase activity increases in the popliteal lymph node in response to an immu-
nological challenge. Consistent with the high activity of glutaminase, glutamine is utilized
at a high rate by cultured lymphocytes, macrophages, and neutrophils. Mitogenic stimu-
lation of lymphocytes increases both glutaminase activity and the rate of glutamine utili-
zation. Glutamine utilization by macrophages was increased by BCG activation in vivo or
by bacterial LPS stimulation in vitro.40 It has been calculated that glutamine can contribute
up to 35% of the energy requirement of immune cells in culture.41
Glutathione is a tripeptide antioxidant composed of glutamate, cysteine, and glycine.
Glutathione concentrations in the liver, lung, small intestine, and immune cells fall in
response to infection, inflammatory stimuli, and trauma (see Chapter 39 for more details).
The fall in hepatic glutathione concentration and in the export of glutathione from the rat
liver can be prevented by oral provision of glutamine.42,43 Glutamine-enriched parenteral
nutrition elevated plasma glutathione concentration in rats44 and promoted the release of
glutathione from the rat gut into the bloodstream.45 The immunologic effects of glutathione
are dealt with later (see Section 19.2.3.2). Although glutamine is able to preserve glu-
tathione concentrations in the liver, gut, kidney, and bloodstream, it is not clear whether
it also preserves glutathione concentrations within immune cells. However, incubation of
human blood mononuclear cells with increasing concentrations of glutamine resulted in
higher intracellular glutathione concentrations in both CD4+ and CD8+ cells.46 Thus, one
means by which glutamine might exert immunological effects is through maintenance of
glutathione status. This requires further investigation.

19.2.2.2 Glutamine, immune cell function, and infection: cell culture and animal
feeding studies
19.2.2.2.1 Cell culture studies. Several specific immunomodulatory actions of
glutamine in vitro have been reported (see Wilmore and Shabert,37 Calder and Yaqoob,38
and Calder and Newsholme39 for reviews). Increasing the availability of glutamine in
culture has been shown to enhance T lymphocyte proliferation (Figure 19.2), IL-2, and
IFN-g production by lymphocytes; IL-2 receptor expression by lymphocytes; B lymphocyte
differentiation into antibody-producing cells; phagocytosis by human monocytes and
murine macrophages; MHC class II expression and antigen presentation by human mono-
cytes; IL-1 and TNF-a production by rodent macrophages; and IL-6 and IL-8 production
by human monocytes; and to improve the defective antimicrobial activities of blood
neutrophils taken from patients with burns or postsurgery.

19.2.2.2.2 Animal feeding studies. Animal studies have reported that enrichment of
the diet with glutamine increases ex vivo T lymphocyte proliferation.47–49 A glutamine-
enriched diet also increased the proportion of CD4+ lymphocytes in the spleen and
increased the proportion of mitogen-stimulated lymphocytes bearing the IL-2 receptor.49
Feeding mice a glutamine-enriched diet increased production of TNF-a, IL-1b, and IL-6
1382_C19.fm Page 313 Tuesday, October 7, 2003 6:48 PM

Chapter nineteen: Amino acids and immune function 313

by LPS-stimulated macrophages50 and increased IL-2 and IFN-g but not IL-4 production
by mitogen-stimulated spleen lymphocytes.49
Glutamine-supplemented parenteral nutrition improved survival in rats following
cecal ligation and puncture.51 Likewise, intravenous glutamine improved the survival of
rats following the administration of live E. coli.52,53 Suzuki et al.54 fed mice for 10 days on
diets containing casein or casein supplemented with 20 or 40 g of glutamine/kg and then
innoculated them intravenously with live Staphylococcus aureus. Over the following 20
days, during which the mice were maintained on the same diets they had been fed prior
to infection, 80% of the control animals died, while mortality was 60% in the 20 g of
glutamine/kg group and 30% in the 40 g of glutamine/kg group. Another study showed
that inclusion of glutamine in parenteral nutrition decreased mortality to intratracheally
innoculated Pseudomonas.55 These studies did not measure indices of immune function.
Yoo et al.48 reported that proliferation of blood lymphocytes from E. coli-infected piglets
was significantly higher if the piglets consumed a diet containing glutamine compared
with a diet that did not contain glutamine. Shewchuk et al.47 reported that mitogen-
stimulated proliferation of spleen lymphocytes taken from tumor-bearing rats fed a diet
containing an increased amount of glutamine was greater than that of those taken from
rats fed a standard casein-containing diet. Furthermore, infusion of alanyl-glutamine into
tumor-bearing rats increased the in vitro phagocytic capacity of alveolar macrophages,56
while infusion into septic rats increased in vitro proliferation of mitogen-stimulated blood
lymphocytes.57 These studies indicate that provision of glutamine either parenterally or
enterally increases the function of various immune cells and that this might account for
the enhanced resistance to infection observed in other studies. A number of animal studies
indicate that ornithine a-ketoglutarate modulates immune function in various models of
burns, sepsis, tumor bearing, and catabolic stress, and that this action is related in part to
glutamine generation; these studies are addressed elsewhere (Chapter 37).
A series of studies has examined the influence of glutamine on the gut-associated and
respiratory lymphoid systems in mice undergoing various challenges. Feeding rats a
glutamine-free diet for 7 days resulted in decreased mucosal wet weight and a decreased
number of intraepithelial lymphocytes,58 suggesting that glutamine is required for main-
tenance of the gut-associated immune system. Parenteral glutamine or alanyl-glutamine
maintained the lymphocyte yield from Peyer’s patches and intestinal integrity in mice
given an intranasal inoculation of influenza virus.59,60 Enteral glutamine increased total
cellularity of Peyer’s patches (and spleen) in LPS-treated mice61; this effect was mainly
due to an increase in T cell number. In another study, inclusion of glutamine in parenteral
nutrition improved the concentration of SIgA in the intestinal lumen and improved intes-
tine IL-4 and IL-10 concentrations62 (see Chapter 42 for more details).

19.2.2.3 Glutamine, immune function, and infection: human clinical studies


In man, plasma and muscle glutamine levels are lowered (by up to 50%) by sepsis, major
injury, and burns, and following surgery.2–6 Low plasma glutamine concentration at admis-
sion of patients to intensive care was associated with higher severity of illness and higher
mortality.63 The lowered plasma glutamine concentrations that occur are most likely the
result of demand for glutamine exceeding the supply, and thus glutamine should be
considered a conditionally essential amino acid during catabolic stress.64 Lowered plasma
glutamine may contribute, at least in part, to the immunosuppression that accompanies
catabolic stress states. Thus, there has been significant interest in provision of glutamine
to patients following surgery, radiation treatment, or bone marrow transplantation, or
suffering from injury, sepsis, or burns. Both parenteral and enteral routes of administration
have been investigated (see also Chapters 34, 36, and 38).
1382_C19.fm Page 314 Tuesday, October 7, 2003 6:48 PM

314 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

The provision of glutamine intravenously to patients following bone marrow trans-


plantation resulted in a lower level of infection and a shorter stay in the hospital than for
patients receiving glutamine-free parenteral nutrition.65 A later report showed that
glutamine treatment resulted in greater numbers of total lymphocytes, T lymphocytes,
and CD4+ lymphocytes (but not B lymphocytes or natural killer cells) in the bloodstream
after the patients were discharged.66 The authors suggested that glutamine specifically
enhances T lymphocyte numbers and that this might be responsible for the diminished
infection rate observed. Very low birth weight babies who received a glutamine-enriched
premature feeding formula had a much lower rate of sepsis than babies who received a
standard formula.67 In a study of patients in intensive care, glutamine provision decreased
mortality compared with standard parenteral nutrition and changed the pattern of mor-
tality.68 In a more recent study, in which patients received enteral glutamine vs. standard
enteral feed within 48 h of the trauma, there was a significant reduction in the 15-day
incidence of pneumonia, bacteremia, and severe sepsis in the glutamine group, although
this was not associated with reduced mortality.69 None of these studies reported immu-
nological outcomes of the treatments. However, another study of patients in intensive care
reported that enteral glutamine increased the blood CD4:CD8 ratio.70 Parenteral adminis-
tration of glutamine into patients after colorectal surgery increased mitogen-stimulated
proliferation of blood lymphocytes,71 suggesting that glutamine does improve T lympho-
cyte function in patients at risk of sepsis; glutamine did not affect ex vivo TNF-a or IL-6
production. In another study, postoperative patients who received alanyl-glutamine
parenterally had increased blood lymphocyte numbers.72 More recently, infusion of a
parenteral mixture containing glycyl-glutamine for 48 h after major abdominal surgery
resulted in better maintenance of the HLA-DR expression on circulating monocytes than
in control patients who received a standard parenteral mixture.73 Patients with esophageal
cancer being treated with radiochemotherapy had higher blood lymphocyte counts and
better lymphocyte proliferative responses if they consumed glutamine (30 g/day) for 28
days.74 These studies indicate that glutamine is able to maintain lymphocyte numbers and
(some) immune cell responses in patients normally at risk of immunosuppression and
infection. In addition to a direct immunological effect, glutamine, even provided parenter-
ally, improves gut barrier function in patients at risk of infection.75 This would have the
benefit of decreasing the translocation of bacteria from the gut and so eliminating a key
source of infection.

19.2.3 Sulfur amino acids


19.2.3.1 Metabolic functions of sulfur amino acids of relevance to immune function
Methionine and cysteine play roles as precursors for the synthesis of proteins. Metal-
lothionein, the major zinc transport protein, and many acute phase proteins contain high
proportions of sulfur amino acids. Methionine is involved in polyamine synthesis. Cys-
teine is the rate-limiting substrate for the synthesis of the antioxidant glutathione. Cysteine
can also be converted to taurine and inorganic sulfate; taurine is a putative antioxidant
and cell membrane stabilizer, and is the predominant free nitrogenous compound in
immune cells.
During the response to infection and injury the urinary excretion of sulfur increases,
but to a lesser extent than that of nitrogen, suggesting that sulfur amino acids are prefer-
entially retained and so “spared” from catabolism. Urinary sulfate losses during HIV
infection in humans were equivalent to 10 g of cysteine per day,76 in contrast to losses of
approximately 3 g/day for healthy individuals on a Western diet. Since cysteine is the
precursor for both sulfate and glutathione, this finding may be linked with the decline in
tissue glutathione pools that has been observed in HIV infection.77,78
1382_C19.fm Page 315 Tuesday, October 7, 2003 6:48 PM

Chapter nineteen: Amino acids and immune function 315

19.2.3.2 Cysteine, glutathione, and immune function


19.2.3.2.1 Glutathione status in infection and catabolic stress. Infection of mice with
the influenza virus resulted in a significant decrease in the glutathione content of the
blood.79 Substantial decreases in glutathione concentrations in blood and lung epithelial
lining fluid were noted in asymptomatic HIV infection in humans.77 The glutathione
content of blood and skeletal muscle in patients undergoing elective abdominal surgery
fell within 24 h of the operation.80

19.2.3.2.2 Cysteine, glutathione, and immune function: cell culture studies. Culture of
human lymphocytes in the absence of cysteine markedly decreases their ability to prolif-
erate in response to mitogenic stimulation.16 Depletion of glutathione in cultured human
lymphocytes diminished cytotoxic T cell activity.81 Glutathione depletion was associated
with diminished IFN-g but not IL-2 or IL-4 production by antigen-stimulated murine
lymph node cells in culture.82 This effect was mediated by antigen-presenting cells, and
the authors suggest that glutathione acts via inducing IL-12 production by these cells to
alter the Th1/Th2 balance in favor of a Th1 response. In another study, a rise in the
glutathione content of lymphocytes from adult humans was accompanied by an increase
in lymphocyte proliferation and IL-2 production in response to mitogen.83 These studies
suggest that glutathione promotes a range of cell-mediated immune responses.

19.2.3.2.3 Glutathione and immune function: studies in healthy humans. Depletion of


glutathione through an exercise regimen decreased the number of CD4+ cells by 30% in
a subset of individuals.84 Treatment with the cysteine precursor N-acetylcysteine (NAC)
(400 mg/day for 4 weeks) prevented the exercise-induced fall in intracellular glutathione
concentrations and increased the number of CD4+ cells by 25%.84 Immune cell functions
were not investigated in this study.

19.2.3.2.4 Glutathione and immune function: human clinical studies. Administration


of 600 mg of NAC/day to patients with HIV infection for 7 months increased the number
of lymphocytes in the circulation and enhanced ex vivo lymphocyte proliferation in
response to tetanus toxin.76 There was also a decrease in plasma IL-6 concentration.76 This
study indicates that while glutathione improves cell-mediated immune function, it acts
to diminish the production of inflammatory cytokines, which can be damaging to the host
if produced in excess amounts. This suggestion is supported by the inverse relationship
between glutathione concentration in monocytes from patients with cirrhosis and the
ability of those monocytes to produce IL-1, IL-8, and TNF-a.85 Treatment of the cirrhotic
patients with the glutathione precursor oxothiazalidine-4-carboxylate increased monocyte
glutathione content and decreased IL-1, IL-8, and TNF-a production.85
NAC infusion into patients with sepsis increased blood glutathione concentration,
decreased plasma concentrations of IL-8 and soluble TNF receptors, improved respiratory
function, and shortened the number of days needed in intensive care.86,87 While not affect-
ing mortality rates, NAC shortened hospital length of stay. Studies on asymptomatic HIV
patients have shown that NAC can raise blood glutathione concentrations and increase
natural killer cell activity and T lymphocyte proliferation in response to mitogens or
tetanus toxin.76,88 Furthermore, studies have shown that survival time was improved in
HIV patients who maintained high concentrations of glutathione in CD4+ T lymphocytes.89

19.2.3.3 Taurine and immune function


Taurine is a sulfonated b-amino acid derived from methionine and cysteine metabolism;
it is not a component of proteins (see also Chapter 44). In humans, plasma taurine
1382_C19.fm Page 316 Tuesday, October 7, 2003 6:48 PM

316 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

concentrations are decreased by trauma and sepsis.90,91 Taurine is present in high concen-
trations in most tissues and particularly in cells of the immune system: taurine contributes
50% of the free amino acid pool within lymphocytes and is the most abundant free
nitrogenous compound therein. The role of taurine within lymphocytes is not well known
(see Redmond et al.92 for a review). Cats fed a taurine-deficient diet have atrophy of the
lymph nodes and spleen, a decrease in circulating lymphocyte numbers, and impaired
respiratory burst by phagocytes.93 These effects are reversed by taurine supplementation
of the diet. Administration of taurine to mice prevented the decline in T cell number that
occurs with aging and enhanced the proliferative responses of T lymphocytes from both
young and old mice94; the effect was more marked in cells from old than young animals.
In neutrophils taurine appears to play a role in maintaining phagocytic capacity and
microbicidal action through interaction with myeloperoxidase, an enzyme involved in
respiratory burst (see Redmond et al.92). Taurinechloramine is formed by complexing of
taurine with hypochlorous acid (HOCl) produced by myeloperoxidase. Hypochlorous
acid, although toxic to bacteria, causes damage to host tissues, and it has been proposed
that the formation of taurinechloramine is a mechanism to protect the host from this
damage. However, taurinechloramine is bacteriacidal in its own right, and so it is proposed
that it represents a bacteriacidal molecule that confers the advantage of decreasing
hypochlorous acid-induced host tissue damage (see Redmond et al.92). Although taurine
appears not to affect mediator production by macrophages, taurinechloramine decreases
nitric oxide, superoxide, PGE 2, TNF-a, and IL-6 production by leukocytes in culture (see
Seabra et al.95 and references therein). It has been proposed that taurine may offer a
therapeutic approach for treatment of acute inflammatory events.96

19.3 Conclusion
Cell culture and animal studies indicate that certain amino acids, most notably arginine,
glutamine, and cysteine, are required for efficient function of cells of the immune system.
Poorly nourished humans display impaired immune responses, which predispose to
increased morbidity and mortality. Patients in the catabolic state exhibit impaired or
deranged immune responses, show altered profiles of amino acids in the bloodstream,
and are at risk of infections and subsequent complications. Improved amino acid supply
to such patients is believed to be a suitable therapeutic approach. The most studied amino
acids in this context are arginine and glutamine, although cysteine has also been studied.
Animal models and clinical trials provide evidence of immune improvements with argi-
nine and glutamine, and these are associated with improved outcome. There is a need to
identify the most suitable mix of amino acids for use in different patient groups who are
at risk of impaired immune function.

References
1. Wilmore, D.W., Alterations in protein, carbohydrate and fat metabolism in injured and septic
patients, J. Am. Coll. Nutr., 2, 3, 1983.
2. Askanazi, J. et al., Muscle and plasma amino acids following injury: influence of intercurrent
infection, Ann. Surg., 192, 78, 1980.
3. Milewski, P.J. et al., Intracellular free amino acids in undernourished patients with and
without sepsis, Clin. Sci., 62, 83, 1982.
4. Stinnett, J.D. et al., Plasma and skeletal muscle amino acids following severe burn injury in
patients and experimental animals, Ann. Surg., 195, 75, 1982.
5. Parry-Billings, M. et al., Does glutamine contribute to immunosuppression after major burns?
Lancet, 336, 523, 1990.
1382_C19.fm Page 317 Tuesday, October 7, 2003 6:48 PM

Chapter nineteen: Amino acids and immune function 317

6. Parry-Billings, M. et al., Effects of major and minor surgery on plasma glutamine and
cytokine levels. Arch. Surg., 127, 1237, 1992.
7. Munder, M. et al., Th1/Th2-regulated expression of arginase isoforms in murine macro-
phages and dendritic cells, J. Immunol., 163, 3771, 1999.
8. Chang, C.I. et al., The involvement of tyrosine kinases, cyclic AMP/protein kinase A, and
p38 mitogen-activated protein kinase in IL-13-mediated arginase I induction in macrophages:
its implications in IL-13-inhibited nitric oxide production, J. Immunol., 165, 2134, 2000.
9. Corraliza, I.M. et al., Arginase induction by suppressors of nitric oxide synthesis (IL-4, IL-10
and PGE2) in murine bone-marrow-derived macrophages, Biochem. Biophys. Res. Commun.,
206, 667, 1995.
10. Sonoki, T. et al., Coinduction of nitric-oxide synthase and arginase I in cultured rat peritoneal
macrophages and rat tissues in vivo by lipopolysaccharide, J. Biol. Chem., 272, 3689, 1997.
11. Klein, D. and Morris, D.R., Increased arginase activity during lymphocyte mitogenesis, Biochem.
Biophys. Res. Commun., 81, 199, 1978.
12. Matera, L. et al., Prolactin in autoimmunity and antitumor defence, J. Neuroimmunol., 109,
47, 2000.
13. Murphy, W.J. and Longo, D.L., Growth hormone as an immunomodulating therapeutic
agent, Immunol. Today, 21, 211, 2000.
14. Clark, R. et al., Insulin-like growth factor-1 stimulation of lymphopoiesis, J. Clin. Invest., 92,
540, 1993.
15. Hinton, P.S. et al., Insulin-like growth factor-I enhances immune response in dexamethasone-
treated or surgically stressed rats maintained with total parenteral nutrition, J. Parenter.
Enteral Nutr., 19, 444, 1995.
16. Chuang, J.C., Yu, C.L., and Wang, S.R., Modulation of lymphocyte proliferation by amino acids,
Clin. Exp. Immunol., 81, 173, 1990.
17. Calder, P.C., Requirement for both glutamine and arginine by proliferating lymphocytes, Proc.
Nutr. Soc., 54, 123A, 1995.
18. Christie, G.S. et al., The effects of an arginine antagonist on stimulated human lymphocytes in
culture, Pathology, 3, 139, 1971.
19. Efron, D.T. et al., Nitric oxide generation from L-arginine is required for optimal human
peripheral blood lymphocyte DNA synthesis, Surgery, 110, 327, 1991.
20. Moriguchi, S. et al., Functional changes in human lymphocytes and monocytes after in vitro
incubation with arginine, Nutr. Res., 7, 719, 1987.
21. Park, K.G.M. et al., Stimulation of lymphocyte natural cytotoxicity by L-arginine, Lancet, 337,
645, 1991.
22. Barbul, A., Arginine and immune function, Nutrition, 6, 53, 1990.
23. Reynolds, J.V., Thom, A.K., Zhang, S.M., Ziegler, M.M., Naji, A., and Daly, J.M., Arginine,
protein malnutrition and cancer, J. Surg. Res., 45, 513, 1988.
24. Reynolds, J.V. et al., Immunological effects of arginine in tumor-bearing and non-tumor-
bearing hosts, Ann. Surg., 211, 202, 1990.
25. Lewis, B. and Langkamp-Henken, B., Arginine enhances in vivo immune responses in young,
adult and aged mice, J. Nutr., 130, 1827, 2000.
26. Mills, C.D. et al., Macrophage arginine metabolism and the inhibition or stimulation of
cancer, J. Immunol., 149, 2709, 1992.
27. Saito, H. et al., Metabolic and immune effects of dietary arginine supplementation after burn,
Arch. Surg., 122, 784, 1987.
28. Madden, H.P. et al., Stimulation of T cell immunity by arginine enhances survival in peri-
tonitis, J. Surg. Res., 44, 658, 1988.
29. Gianotti, L. et al., Arginine-supplemented diets improve survival in gut-derived sepsis and
peritonitis by modulating bacterial clearance: the role of nitric oxide, Ann. Surg., 217, 644,
1993.
30. Barbul, A. et al., Arginine stimulates lymphocyte immune response in healthy human beings,
Surgery, 90, 244, 1981.
31. Barbul, A. et al., Arginine enhances wound healing and lymphocyte immune responses in
humans, Surgery, 108, 331, 1990.
1382_C19.fm Page 318 Tuesday, October 7, 2003 6:48 PM

318 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

32. Daly, J.M. et al., Immune and metabolic effects of arginine in the surgical patient, Ann. Surg.,
208, 512, 1988.
33. Beale, R.J., Bryg, D.J., and Bihari, D.J., Immunonutrition in the critically ill: a systematic
review of clinical outcome, Crit. Care Med., 27, 2799, 1999.
34. Heys, S.D. et al., Enteral nutritional supplementation with key nutrients in patients with
critical illness and cancer: a meta-analysis of randomized controlled clinical trials, Ann. Surg.,
229, 467, 1999.
35. Heyland, D.K. et al., Should immunonutrition become routine in critically ill patients? A
systematic review of the evidence, J. Am. Med. Assoc., 286, 944, 2001.
36. Calder, P.C., Lipids and the critically ill patient, in Nutrition and Critical Care, Cynober, L.
and Moore, F.A., Eds., Karger, Basel, Switzerland, 2003, in press.
37. Wilmore, D. and Shabert, J.K., Role of glutamine in immunologic responses, Nutrition, 14,
618, 1998.
38. Calder, P.C. and Yaqoob, P., Glutamine and the immune system, Amino Acids, 17, 227, 1999.
39. Calder, P.C. and Newsholme, P., Glutamine and the immune system, in Nutrition and Immune
Function, Calder, P.C., Field, C.J., and Gill, H.S., Eds., CABI, New York, 2002, p. 109.
40. Murphy, C.J. and Newsholme, P., The importance of glutamine metabolism in murine macro-
phages and human monocytes to L-arginine biosynthesis and rates of nitrite or urea produc-
tion, Clin. Sci., 95, 397, 1998.
41. Spolarics, Z. et al., Glutamine and fatty acid oxidation are the main sources of energy in
Kupffer and endothelial cells, Am. J. Physiol., 261, G185, 1991.
42. Hong, R.W. et al., Glutamine preserves liver glutathione after lethal hepatic injury, Ann.
Surg., 215, 114, 1992.
43. Welbourne, T.C., King, A.B., and Horton, K., Enteral glutamine supports hepatic glutathione
efflux during inflammation, J. Nutr. Biochem., 4, 236, 1993.
44. Denno, R. et al., Glutamine-enriched total parenteral nutrition enhances plasma glutathione
in the resting state, J. Surg. Res., 61, 35, 1996.
45. Cao, Y. et al., Glutamine enhances gut glutathione production, J. Parenter. Enteral Nutr., 22,
224, 1998.
46. Chang, W.K., Yang, K.D., and Shaio, M.F., Lymphocyte proliferation modulated by glutamine:
involved in the redox reaction, Clin. Exp. Immunol., 117, 482, 1999.
47. Shewchuk, L.D., Baracos, V.E., and Field, C.J., Dietary L-glutamine supplementation reduces
growth of the Morris Hepatoma 7777 in exercise-trained and sendentary rats, J. Nutr., 127,
158, 1997.
48. Yoo, S.S., Field, C.J., and McBurney, M.I., Glutamine supplementation maintains intramus-
cular glutamine concentrations and normalizes lymphocyte function in infected early
weaned pigs, J. Nutr., 127, 2253, 1997.
49. Kew, S. et al., Dietary glutamine enhances murine T-lymphocyte responsiveness, J. Nutr.,
129, 1524, 1999.
50. Wells, S.M. et al., Dietary glutamine enhances cytokine production by murine macrophages,
Nutrition, 15, 881, 1999.
51. Ardawi, M.S.M., Effect of glutamine-enriched total parenteral nutrition on septic rats, Clin.
Sci., 81, 215, 1991.
52. Inoue, Y., Grant, J.P., and Snyder, P.J., Effect of glutamine-supplemented intravenous nutrition
on survival after Escherichia coli-induced peritonitis, J. Parenter. Enteral Nutr., 17, 41, 1993.
53. Naka, S. et al., Alanyl-glutamine-supplemented total parenteral nutrition improves survival
and protein metabolism in rat protracted bacterial peritonitis model, J. Parenter. Enteral Nutr.,
20, 417, 1996.
54. Suzuki, I. et al., Effect of a glutamine-supplemented diet in response to methicellin-resistant
Staphylococcus aureus infection in mice, J. Nutr. Sci. Vitaminol., 39, 405, 1993.
55. DeWitt, R.C. et al., Glutamine-enriched total parenteral nutrition preserves respiratory im-
munity and improves survival to Pseudomonas pneumonia, J. Surg. Res., 84, 13, 1999.
56. Kweon, M.N. et al., Effect of alanyl-glutamine-enriched infusion on tumour growth and
cellular immune function in rats, Amino Acids, 1, 7, 1991.
1382_C19.fm Page 319 Tuesday, October 7, 2003 6:48 PM

Chapter nineteen: Amino acids and immune function 319

57. Yoshida, S. et al., Effect of glutamine supplementation on lymphocyte function in septic rats,
J. Parenter. Enteral Nutr., 16, 30S, 1992.
58. Horvath, K. et al., Isocaloric glutamine-free diet and the morphology and function of rat
small intestine, J. Parenter. Enteral Nutr., 20, 128, 1996.
59. Li, J. et al., Effect of glutamine-enriched TPN on small intestine gut-associated lymphoid
tissue (GALT) and upper respiratory tract immunity, Surgery, 121, 542, 1997.
60. Li, J. et al., Glycyl-L-glutamine-enriched total parenteral nutrition maintains small intestine
gut-associated lymphoid tissue and upper respiratory tract immunity, J. Parenter. Enteral
Nutr., 22, 31, 1998.
61. Manhart, N. et al., Influence of enteral diets supplemented with key nutrients on lymphocyte
subpopulations in Peyer’s patches of endotoxin-boostered mice, Clin. Nutr., 19, 265, 2000.
62. Kudsk, K.A. et al., Glutamine-enriched total parenteral nutrition maintains intestinal inter-
leukin-4 and mucosal immunoglobulin A levels, J. Parenter. Enteral Nutr., 24, 270, 2000.
63. Oudemans-van Straaten, H.M. et al., Plasma glutamine depletion and patient outcome in
acute ICU admissions, Intensive Care Med., 27, 84, 2001.
64. Lacey, J.M. and Wilmore, D.W., Is glutamine a conditionally essential amino acid? Nutr. Rev.,
48, 297, 1990.
65. Ziegler, T.R. et al., Clinical and metabolic efficacy of glutamine-supplemented parenteral
nutrition following bone marrow transplantation: a double-blinded, randomized, controlled
trial, Ann. Int. Med., 116, 821, 1992.
66. Ziegler, T.R. et al., Effects of glutamine supplementation on circulating lymphocytes after
bone marrow transplantation: a pilot study, Am. J. Med. Sci., 315, 4, 1998.
67. Neu, J. et al., Enteral glutamine supplementation for very low birthweight infants decreases
morbidity, J. Pediatr., 131, 691, 1997.
68. Griffiths, R.D., Jones, C., and Palmer, T.E.A., Six-month outcome of critically ill patients given
glutamine-supplemented parenteral nutrition, Nutrition, 13, 295, 1997.
69. Houdijk, A.P.J. et al., Randomised trial of glutamine-enriched parenteral nutrition on infec-
tious morbidity in patients with multiple trauma, Lancet, 352, 772, 1998.
70. Jensen, G.L. et al., A double blind, prospective, randomized study of glutamine-enriched
compared with standard peptide-based feeding in critically ill patients, Am. J. Clin. Nutr.,
64, 615, 1996.
71. O’Riordain, M. et al., Glutamine supplemented parenteral nutrition enhances T-lymphocyte
response in surgical patients undergoing colorectal resection, Ann. Surg., 220, 212, 1994.
72. Morlion, B.J. et al., Total parenteral nutrition with glutamine dipeptide after major abdominal
surgery: a randomized, double-blind, controlled study, Ann. Surg., 227, 302, 1998.
73. Spittler, A. et al., Postoperative glycyl-glutamine infusion reduces immunosuppression: par-
tial prevention of the surgery induced decrease in HLA-DR expression on monocytes, Clin.
Nutr., 20, 37, 2001.
74. Yoshida, S. et al., Effects of glutamine supplements and radiochemotherapy on systemic
immune and gut barrier function in patients with advanced esophageal cancer, Ann. Surg.,
227, 485, 1998.
75. van der Hulst, R.R.W. et al., Glutamine and the preservation of gut integrity, Lancet, 341,
1363, 1993.
76. Breitkreutz, R. et al., Improvement of immune functions in HIV infection by sulfur supple-
mentation: two randomized trials, J. Mol. Med., 78, 55, 2000.
77. Staal, F.J.T., Ela, S.W., and Roederer, M., Glutathione deficiency in human immunodeficiency
virus infection, Lancet, i, 909, 1992.
78. De Rosa, S.C. et al., N-acetylcysteine replenishes glutathione in HIV infection, Eur. J. Clin.
Invest., 30, 915, 2000.
79. Hennett, T., Peterhans, E., and Stocker, R., Alterations in antioxidant defences in lung and
liver of mice infected with influenza A virus, J. Gen. Virol., 73, 39, 1992.
80. Luo, J.L. et al., Skeletal muscle glutathione after surgical trauma, Ann. Surg., 223, 420, 1996.
81. Droge, W. et al., Functions of glutathione and glutathione disulfide immunology and immu-
nopathology, FASEB J., 8, 1131, 1994.
1382_C19.fm Page 320 Tuesday, October 7, 2003 6:48 PM

320 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

82. Peterson, J.D. et al., Glutathione levels in antigen-presenting cells modulate Th1 versus Th2
response patterns, Proc. Natl. Acad. Sci. U.S.A., 95, 3071, 1998.
83. Wu, D. et al., In vitro glutathione supplementation enhances interleukin-2 production and
mitogenic response of peripheral blood mononuclear cells from young and old subjects,
J. Nutr., 124, 655, 1994.
84. Kinscherf, R. et al., Effect of glutathione depletion and oral N-acetyl-cysteine treatment on
CD4+ and CD8+ cells, FASEB J., 8, 448, 1994.
85. Pena, L.R., Hill, D.B., and McClain, C.J., Treatment with glutathione precursor decreases
cytokine activity, J. Parenter. Enteral Nutr., 23, 1, 1999.
86. Spapen, H. et al., Does N-acetyl cysteine influence the cytokine response during early human
septic shock? Chest, 113, 1616, 1998.
87. Bernard, G.R. et al., A trial of antioxidants N-acetylcysteine and procysteine in ARDS, Chest,
112, 164, 1997.
88. Simon, G., Moog, C., and Obert, G., Effects of glutathione precursors on human immuno-
deficiency virus replication, Chem. Biol. Interact., 91, 217, 1994.
89. Herzenberg, L.A. et al., Glutathione deficiency is associated with impaired survival in HIV
disease, Proc. Natl. Acad. Sci. U.S.A., 94, 1967, 1997.
90. Vente, J.P. et al., Plasma amino acid profiles in sepsis and stress, Ann. Surg., 209, 57, 1989.
91. Paauw, J.D. and Davis, A.T., Taurine concentrations in serum of critically injured patients
and age- and sex-matched healthy control subjects, Am. J. Clin. Nutr., 52, 657, 1990.
92. Redmond, H.P. et al., Immunonutrition: the role of taurine, Nutrition, 14, 599, 1998.
93. Schuller-Levis, G. et al., Immunologic consequences of taurine deficiency in cats, J. Leukoc.
Biol., 47, 321, 1990.
94. Negoro, S. and Hara, H., The effect of taurine on the age-related decline of the immune
response in mice: the restorative effect on the T cell proliferative response to costimulation
with ionomycin and phorbol myristate acetate, Adv. Exp. Biol. Med., 315, 229, 1992.
95. Seabra, V., Stachlewitz, R.F., and Thurman, R.G., Taurine blunts LPS-induced increases in
intracellular calcium and TNF-a production by Kupffer cells, J. Leukoc. Biol., 64, 615, 1998.
96. Stapelton, P.P., Redmond, H.P., and Bouchier-Hayes, D., Taurine and inflammation: a new
approach to an old problem? J. Leukoc. Biol., 61, 231, 1997.
1382_C20.fm Page 321 Tuesday, October 7, 2003 6:50 PM

chapter twenty

Amino acid-mediated insulin


secretion
Willy J. Malaisse
Brussels Free University

Contents
Introduction..................................................................................................................................321
20.1 Branched-chain amino acids...........................................................................................322
20.2 Monoaminodicarboxylic aliphatic amino acids and their derivatives....................323
20.2.1 L-glutamine ...........................................................................................................324
20.2.2 L-asparagine ..........................................................................................................324
20.3 Cationic amino acids........................................................................................................325
20.3.1 L-arginine and L-ornithine ..................................................................................325
20.3.2 L-lysine...................................................................................................................328
20.4 Aromatic amino acids......................................................................................................328
20.5 Heterocyclic amino acids ................................................................................................329
20.5.1 L-histidine..............................................................................................................329
20.5.2 L-tryptophane .......................................................................................................330
20.5.3 L-proline.................................................................................................................330
20.6 L-alanine .............................................................................................................................330
20.7 Glycine, L-serine, and L-threonine .................................................................................331
20.8 Sulfur-containing amino acids .......................................................................................332
20.9 a-Aminoisobutyric acid...................................................................................................332
20.10 Taurine................................................................................................................................332
20.11 Concluding remarks.........................................................................................................333
Acknowledgments ......................................................................................................................333
References .....................................................................................................................................333

Introduction
The knowledge that several amino acids are able to stimulate insulin secretion from the
pancreatic islet B-cells raises several questions.
First, the mechanisms by which amino acids are identified by the endocrine pancreas
as insulinotropic agents was and, to a certain extent, remains an open question. In essence,

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 321
1382_C20.fm Page 322 Tuesday, October 7, 2003 6:50 PM

322 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

in this respect, the situation is comparable to that concerning the mode of action of other
insulinotropic nutrients, especially D-glucose. In the latter case, a debate had long opposed
the defenders of the so-called receptor and metabolic hypotheses. The first hypothesis
postulates that D-glucose itself acting upon a stereospecific receptor acts as a signal in the
process of glucose-stimulated insulin release. According to the second hypothesis, D-glu-
cose owes its insulinotropic potential to its capacity to act in the islet cells as a nutrient
augmenting, for instance, the rate of ATP generation.
Second, the physiological relevance of the insulinotropic action of amino acids may
be questioned. One of the approaches used to answer such a question consists in exploring
the effects upon insulin release and other functional variables of a mixture of circulating
amino acids, all tested at their physiological concentration in isolated pancreatic islets.
According to a recent study,1 under these experimental conditions, the mixture of amino
acids augments by about 50% the rate of insulin release evoked in the islets by D-glucose,
the hexose also being used at a concentration close to that found in the extracellular fluid
in the fed state (i.e., about 8.3 mM in fed rats).
Third, the possible modulation of the insulinotropic action of amino acids by envi-
ronmental factors (e.g., starvation or aging) or its perturbation in disease states (e.g., type
2 diabetes mellitus) cannot be ignored. Along the same line of thinking, the question could
be raised whether advantage might be taken of the insulinotropic action of amino acids,
as well as some selected analogs or derivatives, to either design provocative tests for
assessment of the functional capacity of the endocrine pancreas or develop novel insulino-
tropic tools for the treatment of type 2 diabetes.2
Although the above list should not be considered exhaustive, it is largely sufficient
to justify current interest in the process of amino acid-mediated insulin secretion. This is
indeed the very topic of the present chapter. In dealing with such an issue, an attempt
will here be made to review essential knowledge on the insulinotropic action of amino
acids.

20.1 Branched-chain amino acids


L-leucine, L-isoleucine, and L-valine are branched-chain monoaminomonocarboxylic ali-
phatic amino acids. They are potent insulin secretagogues. Already in 1963, Floyd et al.3
provided evidence that stimulation of insulin release represents the cause of leucine-
induced hypoglycemia in man.
The mechanism by which L-leucine stimulates insulin secretion was extensively
reviewed in 1981,4 1984,5 and 1986.6 In this period, the elucidation of L-leucine mode of
action in the B-cell was based mainly on two contributions. First, it was established that
the insulinotropic action of branched-chain 2-keto acids, e.g., 2-ketoisocaproate, is causally
related to their capacity to act as nutrients in islet cells.7,8 Second, it was shown that certain
amino acids, like L-leucine and its nonmetabolized analog b(-)2-amino-bicyclo[2,2,1]hep-
tane-2-carboxylic acid (BCH) are potent activators of glutamate dehydrogenase.9 As a
matter of fact, there was a tight correlation between the capacity of different amino acids
to activate glutamate dehydrogenase in islet homogenates and the ability of L-glutamine
to augment insulin release in the presence of each of these amino acids,9 with the following
hierarchy: L-leucine = BCH > L-norvaline > L-isoleucine. The amino acids glycine, L-serine,
L-valine, L-norleucine, and L-lysine failed to activate glutamate dehydrogenase, and
L-glutamine failed to augment insulin output recorded in their presence.9
The results recorded with BCH in intact islets thus provided a key finding in support
of the fuel concept for insulin release. In intact rat islets, BCH increased the islet content
or output of NH4+, 2-ketoglutarate, malate, pyruvate, and alanine. BCH caused a dose-
related increase in 14CO2 output from islets prelabeled with L-[U-14C]glutamine. BCH
1382_C20.fm Page 323 Tuesday, October 7, 2003 6:50 PM

Chapter twenty: Amino acid-mediated insulin secretion 323

increased the islet content of ATP and stimulated both 45Ca net uptake and insulin release.
The capacity of seven distinct amino acids to activate glutamate dehydrogenase tightly
correlated with their ability to augment 14CO2 output from islets prelabeled with
[U-14C]glutamine and to stimulate insulin release in the presence of L-glutamine. The
activation of glutamate dehydrogenase by BCH may thus account for the insulin-releasing
capacity of the leucine analog.10
In further work, it was observed that L-glutamine causes a dose-related enhancement
of insulin release evoked, in rat pancreatic islets, by BCH. In the islets exposed to
L-glutamine, BCH decreased the deamidation of glutamine, but stimulated the oxidative
deamination of glutamate, increased the rate of generation and islet content of 2-ketoglut-
arate, and augmented the oxidation of L-[U-14C]glutamine. BCH antagonized the sparing
action of L-glutamine upon the oxidation of endogenous fatty acids. The stimulation of
insulin release by the association of L-glutamine and BCH was commensurate with the
estimated increase in O2 consumption and coincided with an increase in the islet
NADPH/NADP+ ratio, net uptake of 45Ca, and cyclic AMP concentration. It was concluded
that insulin release evoked by these amino acids is causally linked to an increase in
catabolic fluxes, the secretagogues acting in the islet cells as a fuel (L-glutamine) or enzyme
activator (BCH).11
As far as other branched-chain amino acids are concerned, the situation can be sum-
marized as follows. In the absence of any other exogenous nutrient, only L-leucine and
L-norvaline augment insulin release from isolated rat pancreatic islets, while L-isoleucine,
L-valine, and L-norleucine failed to do so. In the presence of L-glutamine (10 mM), L-leucine,
L-norvaline, and L-isoleucine significantly increase insulin output, while L-valine and
L-norleucine still fail to do so. L-norleucine, however, augments insulin secretion in the
presence of L-leucine or D-glucose, while L-valine fails to do so. The negative results
obtained with L-valine do not mean that this amino acid is not metabolized in islet cells.
As a matter of fact, L-valine, like L-norvaline, L-isoleucine, and L-norleucine, inhibits insulin
release evoked by 2-ketoisocaproate in the rat islets, as a result of the transamination
between the 2-keto acid and these amino acids.12
Taken as a whole, the above considerations emphasize the view that sufficient endog-
enous amino acids are available in the B-cells of isolated islets to stimulate insulin release,
provided that their rate of transamination is enhanced, as the result of glutamate dehy-
drogenase activation. Likewise, the capacity of 3-phenylpyruvate to stimulate insulin
release is attributable to the catabolism of endogenous amino acids in the islet cells, acting
as partners in transaminase reactions leading to the conversion of 3-phenylpyruvate into
L-phenylalanine.13,14 Furthermore, additional work indicated that any differences between
exogenous L-leucine and L-norleucine, in terms of their respective insulinotropic capacity,
reflect the actual availability of a 2-keto acid partner rather than differences in the intrinsic
properties of the relevant amino acid aminotransferase.15

20.2 Monoaminodicarboxylic aliphatic amino acids and their


derivatives
L-glutamic acid and L-aspartic acid and their derivatives, L-glutamine and L-asparagine,
have been the object of extensive studies in rat pancreatic islets. Interest in these amino
acids stems not only from the observation that they considerably augment the insulin
secretory response to L-leucine and other activators of glutamate dehydrogenase, but also
from the view that they may cover in vivo part of the basal energy expenditure of islet cells.
At variance with L-glutamine and L-asparagine, L-glutamate and L-aspartate fail to
affect significantly the secretory and cationic response of rat islets to L-leucine.16,17
1382_C20.fm Page 324 Tuesday, October 7, 2003 6:50 PM

324 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Incidentally, it was claimed that glutamate acts as an intracellular messenger that


couples, in the process of glucose-stimulated insulin secretion, glucose metabolism to
insulin release.18 This was soon invalidated.19 Likewise, a recent work on the possible
modulation of glucose-stimulated secretion by drugs supposed to act at the intervention
of metabotropic glutamate and GABAB receptors is poorly contributive, as it was con-
ducted in tumoral islet cells exposed to increasing concentrations of D-glucose, and yet
no information was provided on the control value for insulin output recorded in the sole
presence of the hexose.20

20.2.1 L-glutamine

The metabolism of L-glutamine (1.0 to 10.0 mM) in isolated rat islets and its modulation
by environmental factors were first examined in detail from 1980 to 1982.21,22 L-glutamine
is rapidly taken up and metabolized in pancreatic islets. The rate of L-glutamine deami-
dation largely exceeds the rate of glutamate conversion to g-aminobutyrate and a-keto-
glutarate. The latter conversion occurs in part by oxidative deamination and in part by
transamination reactions coupled with the conversion of 2-keto acids (pyruvate, oxaloa-
cetate), themselves derived from the metabolism of glutamine to their corresponding
amino acids (alanine, aspartate). An important fraction of malate formed from a-keto-
glutarate leaves the Krebs cycle and is converted to pyruvate, this process being apparently
associated with the induction of a more reduced state in cytosolic redox couples.
L-glutamine abolishes the oxidation of endogenous fatty acids and stimulates lipogenesis.
A sparing action of L-glutamine upon the utilization of endogenous nutrients is docu-
mented by the fact that the glutamine-induced increase in O2 consumption is much lower
than expected from the rate of 14CO2 output from islets exposed to L-[U-14C]glutamine.
L-glutamine, although decreasing K+ conductance, fails to stimulate insulin release both
in the absence and presence of D-glucose. It is proposed that L-glutamine represents a
major fuel for pancreatic islets under physiological conditions.21
The production of 14CO2 from L-[U-14C]glutamine, which reflected the generation of
ATP through the metabolism of exogenous glutamine, appeared to be regulated by the
redox state of nicotinamide nucleotides and the ATP content of the islet cells. The influence
of environmental factors on glutamine oxidation was examined in order to identify ATP-
requiring processes. Glutamine oxidation was decreased in the absence of extracellular
Ca2+, under conditions aiming at inhibition of the (Na+ + K+)-dependent ATPase and,
provided that glucose was present in the incubation medium, by cycloheximide. These
findings were interpreted to suggest that the handling of Ca2+ by the islet cells, the active
transport of univalent cations, and the biosynthesis of proinsulin represent three major
ATP-consuming processes in this fuel sensor organ.22
Under normal environmental conditions and in the absence of any other exogenous
nutrient, L-glutamine is well oxidized but, as already mentioned, fails to stimulate insulin
release. However, a marked stimulation of insulin release by L-glutamine, without alter-
ation in its oxidation rate, occurs when the intracellular pH of the islet cells is decreased
or when theophylline is added to the incubation medium.23
L-glutamine augments the islet production of g-aminobutyrate, which can be further
oxidized.21 The basal g-aminobutyrate content is high in islets.

20.2.2 L- asparagine

The secretory and oxidative responses of rat pancreatic islets to L-asparagine,17 its metab-
olism,24 and the metabolic interaction between L-asparagine and L-leucine in these islets25
were examined in the cited references. Briefly, the following information was obtained.
1382_C20.fm Page 325 Tuesday, October 7, 2003 6:50 PM

Chapter twenty: Amino acid-mediated insulin secretion 325

L-asparagine (2 to 10 mM) failed to affect insulin release in the absence of any


other exogenous nutrient or the presence of D-glucose, but caused a concentration-
related and progressive enhancement of insulin release evoked by L-leucine, BCH, or
2-ketoisocaproate.
The deamidation of L-asparagine and the conversion of aspartate to oxalacetate, by
transamination, may occur in both the cytosol and mitochondria. Oxalacetate is then
converted to pyruvate in part via phosphoenolpyruvate and in part via malate. The latter
modality, by consuming NADH and generating NADPH, may lead to changes in the redox
state of the cytosolic NADH/NAD+ and NADPH/NADP+ couples. Such changes may in
turn account, in part at least, for the capacity of L-asparagine to augment insulin release
induced by certain nutrient secretagogues.
L-leucine inhibited the uptake and deamidation of L-asparagine, but failed to exert
any obvious primary effect upon the further catabolism of aspartate derived from exog-
enous asparagine. L-asparagine augmented the oxidation of L-leucine, an effect possibly
attributable to activation of 2-ketoisocaproate dehydrogenase. The association of L-aspar-
agine and L-leucine exerted a sparing action on the utilization of endogenous amino acids,
so that the integrated rate of nutrient oxidation was virtually identical in the sole presence
of L-leucine and simultaneous presence of L-asparagine and L-leucine. It is proposed that
the enhancing action of L-asparagine upon insulin release evoked by L-leucine is attribut-
able to an increased generation rate of cytosolic NADPH rather than any increase in
nutrients oxidation.

20.3 Cationic amino acids


The basic diaminomonocarboxylic aliphatic acids L-lysine and L-arginine are established
as protein constituents. Such is not the case for the basic diaminomonocarboxylic amino
acid L-ornithine. Yet, these three cationic amino acids apparently share, to a certain extent,
a common mode of action as insulinotropic agents.

20.3.1 L- arginine and L-ornithine


L-arginine is a potent insulin secretagogue and is often used as a tool to assess the secretory
capacity of the endocrine pancreas.
L-arginine and L-ornithine are metabolized in rat pancreatic islets. Three sites of the
catabolism of these amino acids deserve attention, namely, the conversion of L-arginine
to L-ornithine and urea, the generation of putrescine and polyamines from L-ornithine,
and its conversion to L-glutamate.26
The rate of urea formation in pancreatic islets exposed to L-arginine yielded the highest
metabolic flow encountered so far in the study of islet metabolism.
Islet homogenates displayed ornithine decarboxylase activity. In intact islets exposed
to either L-arginine or L-ornithine, the steady-state L-ornithine intracellular content was
sufficient to ensure virtual saturation of ornithine decarboxylase, even if allowance is made
for the subcellular distribution of the amino acid. The generation of amines from L-orni-
thine led to a more rapid isotopic equilibration of the intracellular pool of putrescine than
that of spermidine or spermine.
Islet homogenates also displayed ornithine–glutamate transaminase activity. The rate
of L-ornithine transamination was lower than that found with other amino acids. Thus,
relative to the transamination rate of L-aspartate, that of L-alanine, L-leucine, and L-orni-
thine amounted to 22, 19, and 2%, respectively.
In light of this information, at least five modalities have been considered to account
for the insulinotropic action of L-arginine.
1382_C20.fm Page 326 Tuesday, October 7, 2003 6:50 PM

326 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

First, within the framework of the current fuel concept for the insulinotropic action
of nutrient secretagogues, the question was raised whether the catabolism of L-arginine
may generate sufficient ATP to account for its insulinotropic capacity. Despite the fact
that the steady-state content of L-ornithine in intact islet cells exposed to the latter amino
acid was sufficient to virtually saturate ornithine-glutamate transaminase, the rate of
conversion of L-ornithine to L-glutamate was 20 times lower than the maximal velocity
of ornithine-glutamate transaminase in cell homogenates. This finding is consistent with
the knowledge that in islet cells, the rate of amino acid transamination is largely depend-
ent on the availability of the 2-keto acid partner. The channeling of L-ornithine to
L-glutamate in intact islet cells was documented by a series of converging observations,
including the identification of L-glutamate itself, the cycloheximide-sensitive incorpora-
tion of radioactive amino acids such as L-glutamate or L-proline into the protein of cells
exposed to 14C-labeled ornithine, the presence of a component of L-[1-14C]ornithine oxi-
dation resistant to D,L-a-difluoromethyl ornithine (DFMO), and the production of 14CO2
from either L-[U-14C]arginine or L-[U-14C]ornithine in excess of what could be accounted
for by the generation of putrescine and polyamines. Thus, a contributive role for the
production of ATP linked with the generation of NADH in the reactions catalyzed by
glutamaldehyde dehydrogenase and glutamate dehydrogenase or in the further catabo-
lism of 2-ketoglutarate in the Krebs cycle should not be ignored. For instance, in islets
exposed to 10.0 mM L-[U-14C]arginine, the DFMO-resistant production of 14CO2 amounted
to 47.2 ± 6.6 pmol/islet per 120 min, a value comparable to that found at a noninsulino-
tropic hexose concentration (approximately 2.0 mM) in islets exposed to D-[U-14C]glucose.
Nevertheless, this first hypothesis concerning the insulinotropic action of L-arginine and
L-ornithine, namely, that they act as precursors of metabolized nutrients, meets with
several objections. First, it is known from previous studies that L-arginine fails to evoke
some characteristic features otherwise encountered in the process of nutrient-stimulated
insulin release. Thus, at variance with nutrient secretagogues, L-arginine does not inhibit
86Rb outflow from islets perifused in the absence of exogenous nutrient27 and fails to

provoke a phosphate flush in islets prelabeled with [32P]orthophosphate.28 Second,


L-arginine, unlike nutrient secretagogues,29,30 fails to activate phospholipase C, in fair
agreement with a prior observation based on the labeling of inositol-containing phos-
pholipids in islets exposed to [32P]orthophosphate.31 Third, L-arginine and L-ornithine
stimulate not only insulin release but also glucagon and somatostatin secretion, whereas
several nutrient secretagogues exert opposite effects upon the release of insulin and
glucagon, respectively.32,33 L-arginine and L-ornithine impair, modestly but significantly,
the utilization and oxidation of D-glucose by islet cells. Such a situation, which is remi-
niscent of that found in islets either exposed to exogenous L-glutamine or rendered
alkalotic by incubation in an alkaline medium, suggests that the oxidation of L-glutamate
generated from L-arginine or L-ornithine may be counterbalanced by a decrease in
D-glucose oxidation. Expressed in terms of CO2 formation, the latter two phenomena are
indeed of the same magnitude, and hence, their concomitant occurrence could result in
an unchanged overall rate of ATP generation.
Second, it was proposed that the insulinotropic action of L-arginine may be related to
the generation of nitric oxide (NO). This seems most unlikely, however, since L-ornithine
mimics, in virtually all respects, the effect of L-arginine upon islet function. Incidentally,
although rat pancreatic islets are apparently devoid of ornithine transcarbamylase activity
and fail to produce urea when incubated in the absence or presence of L-glutamine,34 a
citrulline–nitric oxide cycle was proposed to play a role in the response of adult human
or rodent pancreatic islets to cytokines.35 Cytokines indeed modestly increase arginino-
succinate synthetase activity in both human and rat islets. It was proposed, therefore, that
the citrulline–NO cycle may be important for the regulation of NO production, e.g., during
1382_C20.fm Page 327 Tuesday, October 7, 2003 6:50 PM

Chapter twenty: Amino acid-mediated insulin secretion 327

insulitis in early insulin-dependent diabetes mellitus, considering that L-arginine avail-


ability may be limiting for NO production and that argininosuccinate synthetase may be
the rate-limiting enzyme in such a cycle.35
Third, an increase in either ornithine or putrescine content of cells exposed to
L-arginine or L-ornithine could play a role in the secretory response to these amino acids
by providing, for instance, a substrate for the reaction catalyzed by islet transglutami-
nase.36
Fourth, it would be unwise, in our opinion, to rule out a change in the cytosolic
concentration of spermidine and spermine with consequent changes in such variables as
the mitochondrial transport of Ca2+ 37 or the activity of a polyamine-sensitive protein
kinase identified in islet homogenates.38 Indeed, although L-arginine and L-ornithine failed
to affect significantly the total content in polyamines, their cytosolic generation might
lead to a localized increase in polyamine concentration, the bulk of preformed polyamines
being apparently sequestered by cellular organelles.39 The inhibitor of ornithine decar-
boxylase DFMO was used to investigate a possible role for the de novo generation of
polyamines in the process of arginine- or ornithine-induced insulin release. The inhibitor
of ornithine decarboxylase failed to affect the oxidation and insulinotropic action of
D-glucose. It only caused a partial decrease in the secretory response to either L-arginine
or L-ornithine. In this respect, the results collected in individual experiments appeared
rather variable, but the factors responsible for such variability were not identified. Taken
as a whole, the experimental data suggest that the generation of putrescine and
polyamines may, at the most, play a limited role in the secretory response to L-arginine
or L-ornithine. This view is compatible with the finding that the nonmetabolized amino
acid 2-aminoisobutyrate, which markedly increases the activity of ornithine decarboxy-
lase in islets as in other tissues, barely augments the release of insulin evoked by L-arginine
or L-ornithine.40
Last, it was proposed that the uptake of cationic amino acids by the B-cells may
lead to membrane depolarization.41,42 In order to assess the possible role of L-arginine
accumulation in islet cells as a determinant of its insulinotropic action, the uptake of
L-arginine and other cationic amino acids (L-ornithine, L-homoarginine, D,L-a-methylor-
nithine, D,L-a-difluoromethylornithine) by rat pancreatic islets was compared to the ionic
and secretory responses of the islets to the same amino acids.43 A tight correlation was
found between the net uptake of these amino acids and their capacity to stimulate 86Rb
efflux, 45Ca uptake and efflux, and insulin release. In the latter respect, there was little
difference between metabolized and nonmetabolized amino acids. Thus, although
L-homoarginine and 4-amino-1-guanylpiperidine-4-carboxylic acid failed to act as a
substrate for either arginase or amino acid aminotransferase in islet homogenates, they
both stimulated 86Rb efflux, 45Ca uptake and efflux, and insulin secretion in intact islets.
These findings are compatible with the view that the accumulation of these positively
charged amino acids in islet cells represents an essential determinant of their secretory
action. Hence, the release of insulin evoked by these amino acids could be due to
depolarization of the plasma membrane with subsequent gating of voltage-sensitive
Ca2+ channels or to some other biophysical effect, as suggested by the persistence of a
sizeable secretory response to L-arginine or L-ornithine in islets perifused at a high
concentration of extracellular K+ (50 mM).
Finally, the salt ornithine a-ketoglutarate was recently reported to stimulate insulin
secretion.44
In conclusion, the stimulation of insulin release by L-arginine or L-ornithine appears
mainly attributable to the biophysical effects of these cationic amino acids, e.g., upon cell
membrane polarization.
1382_C20.fm Page 328 Tuesday, October 7, 2003 6:50 PM

328 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

20.3.2 L- lysine

L-lysine, an essential amino acid in humans, is a cationic amino acid with positively
charged side chains. Its insulinotropic action either in vitro in pieces of rat pancreatic
tissue45 or in vivo in human subjects46 was already documented from 1966 to 1968.
L-lysine is taken up by islets by a saturable transport mechanism. L-[U-14C]lysine is
oxidized in a time- and concentration-related manner (2 to 10 mM) and augments NH4+
production in rat islets. These two effects are commensurate assuming full degradation
of the amino acid. L-lysine (10.0 mM) exerts little effect upon the oxidation of endogenous
nutrients in islets prelabeled with [U-14C]palmitate or L-[U-14C]glutamine. L-lysine aug-
ments both the ATP content and ATP/ADP ratio. The latter effect is less marked, however,
than that caused by 7.0 mM D-glucose.47
L-lysine (10 mM) augments 86Rb efflux from prelabeled islets. This effect is only
transient, however, in the absence but not presence of D-glucose. In the presence of the
hexose, the increase in 86Rb outflow is only partially decreased in the absence of extra-
cellular Ca2+. At variance with other cationic amino acids (L-arginine, L-homoarginine,
L-ornithine), L-lysine augments, like D-glucose, the net uptake of 86Rb over a 60-min
incubation, whether in the absence or presence of D-glucose (7.0 mM).
L-lysine (10 mM) increases 45Ca efflux from prelabeled islets, this effect being more
pronounced in the presence than absence of D-glucose. In both cases, L-lysine also increases
45Ca net uptake over a 60-min incubation.47

L-lysine, when tested at a 10.0 mM concentration, only causes a minor increase in


insulin output from islets deprived of any other exogenous nutrient. Its insulinotropic
action is more pronounced, however, at a 20 mM concentration. At a low concentration
of D-glucose (2.8 mM), L-lysine (10.0 mM) considerably increases insulin release, provided
that theophylline (1.4 mM) is also present in the incubation medium. In the absence of
the phosphodiesterase inhibitor, but at a higher concentration of D-glucose (7.0 mM), a
rapid, marked, and sustained increase in insulin output is also recorded in response to
10.0 mM L-lysine. Under these conditions, the secretory response to L-lysine is much less
severely affected by intracellular acidification than that evoked by either L-arginine or
L-homoarginine (also 10.0 mM). Moreover, at variance with such cationic amino acids as
L-ornithine and L-arginine, L-lysine augments insulin release evoked by Ba2+ (2.0 mM) and
theophylline (1.4 mM) in the nominal absence of extracellular Ca2+.47
Taken as a whole, these findings indicate that the functional response of the pancreatic
islet B-cells to L-lysine involves not only a biophysical mechanism similar to that respon-
sible for the insulinotropic action of L-homoarginine, but also a significant, albeit modest,
metabolic component, which reflects the capacity of L-lysine to act as a fuel in islet cells.47

20.4 Aromatic amino acids


L-phenylalanine is oxidized to L-tyrosine in the reaction catalyzed by phenylalanine
hydroxylase. L-tyrosine is further metabolized, with the ultimate formation of fumarate
and acetoacetate. Little information is available on the insulinotropic action of the two
aromatic amino acids. L-phenylalanine and L-tyrosine, although they were incorporated
at respective concentrations of 80 and 95 mM in the recent study dealing with the mixture
of circulating amino acids, tested at their physiological concentrations.1 L-phenylalanine
stimulates insulin secretion in man,46 in rat pancreatic pieces exposed to 5.6 mM
D-glucose,45 and in cultured fetal rat pancreas.48 L-phenylalanine (10 mM) fails to stimulate
insulin release from rat islets incubated in the absence of any other exogenous nutrient
or in the presence of L-glutamine (10 mM). It augments insulin output in the presence of
8.3 mM D-glucose, but this stimulatory effect fades out at a higher concentration of the
1382_C20.fm Page 329 Tuesday, October 7, 2003 6:50 PM

Chapter twenty: Amino acid-mediated insulin secretion 329

sugar.13 These results were considered in the framework of an extensive study on the
insulinotropic action of 3-phenylpyruvate.13,14

20.5 Heterocyclic amino acids


20.5.1 L- histidine

Only limited information is available on the insulinotropic efficiency of L-histidine. In


healthy subjects, the infusion of L-histidine (0.2 mol) failed to increase plasma insulin
concentration.49 Likewise, in incubated pieces of rabbit pancreas,50 in isolated rat islets,51
or in the perfused eel pancreas,52 L-histidine (2.0 to 10.0 mM) failed to stimulate insulin
release in the presence of 2.7 to 16.7 mM D-glucose. In one study, however, L-histidine (8.3
mM) was found to increase insulin secretion from pieces of rabbit pancreas incubated in
the presence of 8.3 mM D-glucose.53 A modest increase in insulin output also was observed
in pieces of rat pancreas exposed to L-histidine (10.0 mM) in the presence of 5.6 mM
D-glucose.45 The results of a further study54 clearly indicate that at the same high concen-
tration (10.0 mM), L-histidine provokes a rapid and sustained, albeit slowly decreasing,
increment in insulin output from rat islets exposed to either D-glucose (7.0 or 8.3 mM) or
L-leucine (10.0 mM).
Metabolic data clearly indicate that the insulinotropic action of L-histidine cannot be
ascribed to any favorable effect of the amino acid upon islet respiration.54 It also seems
unlikely that L-histidine stimulates insulin release through the generation of histamine.
According to prior publications,51,55 histamine (0.1 to 1.0 mM) fails to significantly affect
insulin release from islets exposed to D-glucose (2.8 to 16.7 mM). No generation of
14C-labeled histamine from L-[U-14C]histidine was detected in rat islets, and the production

of 14CO2 from L-[U-14C]histidine was suppressed by mitochondrial poisons.54


Several features of the cationic and secretory response in islet cells to L-histidine
suggest that this amino acid stimulates insulin release by a mechanism similar to that
involved in the functional response to other cationic amino acids. L-histidine increased
86Rb outflow from prelabeled islets exposed to D-glucose (7.0 mM). More importantly, the

L-histidine-induced increase in 86Rb outflow persisted in the absence of extracellular Ca2+.


It coincided, at normal extracellular Ca2+ concentration, with a stimulation of 45Ca outflow,
the latter process being suppressed in the absence of extracellular Ca2+. L-histidine also
stimulated 45Ca net uptake while failing to stimulate insulin release in glucose-deprived
islets. These findings are compatible with the view that L-histidine, like other cationic
amino acids, causes the gating of voltage-sensitive K+ and Ca2+ channels, presumably as
the result of a primary depolarization of the plasma membrane. There were, nevertheless,
two obvious differences between the functional response to L-histidine and to other cat-
ionic amino acids. First, L-histidine (10.0 mM) augmented, over a 90-min incubation, the
release of insulin evoked by L-leucine (10.0 mM), whereas L-arginine, L-ornithine, and
L-lysine failed to do so. It should be realized, however, that in islets exposed to L-leucine,
an alteration in intracellular pH is thought to interfere with the normal coupling between
metabolic and secretory events.56 Because L-histidine is the sole amino acid with significant
buffering capacity at a close-to-physiological pH, its accumulation in the islet cells might
conceivably counteract the unfavorable effect of L-leucine upon cellular pH. Second, when
tested at a concentration of 10.0 mM, L-histidine provoked cationic and secretory responses
that were much less marked, and apparently more rapidly evanescent, than those evoked
by L-arginine, L-ornithine, or L-homoarginine.54
At first glance, the fact that L-histidine, which carries a weakly basic imidazolium
function, is a weaker insulin secretagogue than either L-arginine or L-ornithine appears
compatible with the role currently ascribed to the cellular accumulation of these positively
1382_C20.fm Page 330 Tuesday, October 7, 2003 6:50 PM

330 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

charged amino acids in their insulinotropic action. If the cytosolic pH of islet cells is not
lower than 6.7, the ratio of ionized to nonionized side chains of L-histidine would not
exceed 2:10. Hence, relative to the situation found with L-arginine or L-ornithine, less than
17% of the molecules of L-histidine would carry a positive charge on their side chain.
Although the absolute value for the net uptake of L-[U-14C]histidine was similar to that
previously found with other cationic amino acids, the number of ionized molecules of
L-histidine present in the islet cells exposed to a 10 mM concentration of the amino acid
would then be below the threshold of concentration required for stimulation of insulin
release at increasing concentrations of other cationic amino acids, such as L-arginine,
L-ornithine, or L-homoarginine.43 Yet, at this concentration, L-histidine provoked a sizeable
stimulation of insulin release. Therefore, these findings might well argue against the
concept that the insulinotropic action of cationic amino acids is solely attributable to their
accumulation as positively charged molecules inside the islet B-cell. The finding that
L-arginine stimulates insulin release from electrically permeated islets exposed to a sub-
stimulatory Ca2+ concentration (50 mM) also conflicts with the latter concept.57

20.5.2 L- tryptophane

L-tryptophane is usually the least abundant of the amino acids in the diet and is not a
major substrate for the generation of high-energy phosphates. Nevertheless, when rested
at a 10 mM concentration, it augments insulin release in pieces of rat pancreatic tissue
incubated in the presence of 5.6 mM D-glucose.45 In this respect, its insulinotropic action
relative to that of other amino acids appears comparable in man and rat.46 L-tryptophane
(5 mM) had no insulinotropic effect on rabbit pancreas pieces, however.50

20.5.3 L- proline

L-proline (265 mM) was incorporated in the mixture of amino acids tested at their physi-
ological concentrations.1 It may be transformed to L-glutamate. Its possible role as an
insulinotropic agent was considered in the framework of the catabolic fate of L-arginine
or L-ornithine in rat pancreatic islets, with emphasis on the cycloheximide-sensitive incor-
poration of radioactivity into tricarboxylic acid (TCA)-precipitable material in islets
exposed to 14C-labeled L-ornithine.26 L-proline induced only a weak insulin release from
incubated pieces of rabbit pancreas.58

20.6 L- alanine
L-alanine, one of the nonessential amino acids, is electrically neutral.
Previous studies concerning the fate and insulinotropic action of L-alanine in pancre-
atic islet cells have documented that (1) L-[U-14C]alanine uptake by islets from obese-
hyperglycemic mice is concentration (1.0 to 25.0 mM) and time (10 to 120 min) related
with, at equilibrium, an apparent distribution ratio close to 11.2, the uptake of L-[U-14C]
alanine being little affected by either D-glucose (17.0 mM) or L-leucine (20.0 mM)55; (2) L-[U-
14C]alanine (5.0 mM) is oxidized by the same islets, albeit to a lesser extent than L-[U-
14C]leucine tested at the same concentration60; and (3) L-alanine (5.0 mM) has no substantial

effects on basal or glucose-stimulated insulin release in the islets from the obese-hyperg-
lycemic animals.61,62 In cultured fetal rat pancreas, L-alanine also failed to stimulate insulin
release when present alone or together with D-glucose in the incubation medium and was
only effective in the presence of caffeine.63
In vivo, a modest increase in plasma insulin concentration was observed in fetal and
neonatal lambs during infusion of L-alanine, with an increase in plasma L-alanine
1382_C20.fm Page 331 Tuesday, October 7, 2003 6:50 PM

Chapter twenty: Amino acid-mediated insulin secretion 331

concentration of about 0.50 to 0.75 mM.64 In newborn infants, however, the intravenous
injection of L-alanine (1.7 mmol/kg of body weight), failed to affect significantly plasma
insulin concentration.65
More recent experiments suggest that the insulinotropic effect of L-alanine may be
attributable to Na+ cotransport. Thus, Dunne et al.66 first observed that in RINm5F cells,
L-alanine (2 to 10 mM) caused cell depolarization, associated with a net inward membrane
current and leading to the generation of Ca2+ spike potentials and an increase in [Ca2+]i,
these effects of the A-type amino acid being suppressed in the absence of external Na+ or
by exchange of the L-form of the amino acid with its D-stereoisomer. McClenaghan et al.67
then reported that at an elevated level of D-glucose, L-alanine promptly induced transient
insulin release from perifused islet cells from ob/ob mice, such a secretory response being
again effectively suppressed by removal of Na+. Last, McClenaghan et al.68 observed that
in BRIN-BD11 cells, the insulinotropic action of L-alanine was again abolished in the
absence of Na+, being also enhanced by ouabain and suppressed in the absence of extra-
cellular Ca2+.
In a recent study conducted in islets prepared from normal adult rats, L-alanine was
found (1) to inhibit pyruvate kinase in islet homogenates, (2) not to affect the oxidation
of endogenous fatty acids in islets prelabeled with [U-14C]palmitate, (3) to stimulate 45Ca
uptake in islets deprived of any other exogenous nutrient, and (4) to augment insulin
release evoked by either 2-ketoisocaproate or L-leucine, while failing to affect significantly
glucose- or glyceraldehyde-induced insulin secretion.69 The oxidation of L-[U-14C]alanine
was unaffected by D-glucose but inhibited by L-leucine. Inversely, L-alanine decreased the
oxidation of D-[U-14C]glucose but failed to affect L-[-14C]leucine oxidation. It was concluded
that the occurrence of a positive insulinotropic action of L-alanine is restricted to selected
experimental conditions, the secretory data being compatible with the view that stimula-
tion of insulin secretion by the tested nutrients reflects, as a rule, their capacity to augment
ATP generation in the islet B-cells. However, the possible role of Na+ cotransport in the
secretory response to L-alanine cannot be ignored.
In the study just mentioned, the results recorded in the presence of D-glucose were
considered as follows. In the presence of increasing concentrations of D-glucose, L-alanine
(5.0 to 20.0 mM) failed to augment significantly insulin secretion. Such a situation may be
accounted for by the fact that the oxidation of exogenous L-[U-14C]alanine was compen-
sated by an alanine-induced decrease in D-[U-14C]glucose oxidation. The latter phenome-
non might itself be attributable to both a decrease in pyruvate kinase activity and com-
petition between alanine-derived and glucose-derived pyruvate in terms of the further
oxidation of this 2-keto acid. It was indeed previously documented that pyruvate gener-
ated by other enzymatic reaction(s) than that catalyzed by pyruvate kinase is less efficiently
converted to L-lactate than pyruvate generated from phospho-enol-pyruvate.70–72 The same
comments may apply to the situation found in the presence of D-glyceraldehyde, in which
case L-alanine again failed to augment insulin output.
This interpretation is consistent with the finding that in the sole presence of D-glucose
(8.3 mM), L-alanine (0.56 mM) fails to affect significantly insulin release, while the omission
of L-alanine augments insulin output from rat islets as evoked, in the presence of D-glucose,
by a full amino acid mixture containing 22 amino acids together with taurine, all tested
at their physiological concentrations.1

20.7 Glycine, L- serine, and L- threonine


Glycine (10 mM) stimulates insulin release from pieces of rat pancreas incubated in the
presence of 5.6 mM D-glucose.45 Glycine also augments insulin output from the perfused
rat pancreas,73 but not from isolated mouse islets.74
1382_C20.fm Page 332 Tuesday, October 7, 2003 6:50 PM

332 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

The monoaminomonocarboxylic aliphatic amino acids L-serine and L-threonine have


both hydroxyl groups in addition to the ammonium group on their carbon chains and are
converted, at the intervention of specific dehydratases, to ammonium and, respectively,
pyruvate and 2-ketobutyrate. They were used at respective concentrations of 670 and
290 mM in the already mentioned mixture of 22 amino acids. Although L-threonine caused
insulin release in vivo in man,46 it was ineffective in vitro.50

20.8 Sulfur-containing amino acids


The sulfur-containing monoaminomonocarboxylic aliphatic amino acids L-methionine and
L-cysteine are established protein constituents. They were used at respective concentrations
of 55 and 30 mM in the mixture of 22 amino acids examined for its insulinotropic action
under close-to-physiological conditions.1 L-methionine is a weak insulinotropic agent
whether in vivo in man46 or in vitro in rabbit50 or rat45 pancreatic tissue.

20.9 a-Aminoisobutyric acid


In early work, a-aminoisobutyric acid was found to stimulate insulin release in fetal rat
pancreas,63 but not in isolated mouse islets.62 Intraperitoneal injection of a-aminoisobutyric
acid in rats 210 min before sacrifice or preincubation of rat islets with a-aminoisobutyric
acid (10 mM) increases the activity of ornithine decarboxylase in the islets.40 This procedure
was used, therefore, to evaluate the possible role of polyamine generation in the secretory
response of the B-cell to such cationic amino acids as L-arginine and L-ornithine. Although
a-aminoisobutyric acid exerted little or no direct effect upon insulin release evoked by
various secretagogues, including L-arginine and L-ornithine, in rat islets, pretreatment with
a-aminoisobutyric acid, whether in vivo or in vitro, slightly increased the secretory response
to L-arginine and L-ornithine (10 mM each) in islets incubated in the presence of 5.6 mM
D-glucose. These findings suggest that the de novo generation of polyamines only plays a
restricted role in the secretory response of islet cells to L-arginine or L-ornithine.40

20.10 Taurine
Recent work75 has drawn attention to the presence of an abnormally low concentration of
taurine in the plasma of rats undergoing protein malnutrition and the possible participa-
tion of this anomaly in the perturbation of pancreatic islet function found in these animals.
The amino sulfonic acid is included, therefore, in the present review.
Taurine was reported to display insulinotropic action in fetal rat islets.76 In pancreatic
islets isolated from adult rats, however, taurine (1.0 to 10.0 mM) fails to affect insulin
release whether in the absence or presence of D-glucose (5.6 to 11.1 mM) or L-leucine
(10.0 mM).77 Likewise, the omission of taurine fails to affect significantly insulin release
evoked by 8.3 mM D-glucose in adult rat islets incubated in the presence of a mixture of
22 amino acids otherwise also containing 0.29 mM taurine.1
Incidentally, taurine supplementation for 11 days only caused minor and occasional
increases in plasma insulin concentration, insulinogenic index, FAD-linked glycerophos-
phate dehydrogenase activity in islet homogenates, and the in vitro secretory response of
intact islets to several nutrient secretagogues in either control or protein malnourished
rats.78
1382_C20.fm Page 333 Tuesday, October 7, 2003 6:50 PM

Chapter twenty: Amino acid-mediated insulin secretion 333

20.11 Concluding remarks


When compared to the knowledge of the effects of amino acids upon insulin release as
reviewed by Panten79 almost 30 years ago, the present review attests to both the progress
made on their mode of action in the insulin-producing B-cells of the pancreatic islets and
certain limitations still present in this respect.

Acknowledgments
I am most grateful to C. Demesmaeker for secretarial help.

References
1. Dura, E. et al., Insulinotropic action of amino acids at their physiological concentrations. I.
Experiments in incubated islets, Int. J. Mol. Med., 9, 527, 2002.
2. Yada, T., Action mechanisms of amino acid in pancreatic B-cells, in Frontiers of Insulin Secretion
and Pancreatic B-Cell Research, Flatt, P. and Lenzen, S., Eds., Smith-Gordon, London, 1994,
chap. 18.
3. Floyd, J.C. et al., Evidence that insulin release is the mechanism for experimentally induced
leucine hypoglycaemia in man, J. Clin. Invest., 42, 1714, 1963.
4. Malaisse, W.J. and Sener, A., Branched-chain amino and ketoacids: effects upon insulin
secretion, in Metabolism and Clinical Implications of Branched Chain Amino and Keto Acids,
Walser, M. and Williamson, J.R., Eds., Elsevier North-Holland, New York, 1981, p. 181.
5. Malaisse, W.J., Branched chain amino and keto acids as regulators of insulin and glucagon
release, in Branched Chain Amino and Keto Acids in Health and Disease, Adibi, S.A. et al. Eds.,
Karger, Basel, Switzerland, 1984, p. 119.
6. Malaisse, W.J., Branched-chain amino acid and keto acid metabolism in pancreatic islets,
Adv. Enzyme Regul., 25, 203, 1986.
7. Hutton, J.C., Sener, A., and Malaisse, W.J., The metabolism of 4-methyl-2-oxopentanoate in
rat pancreatic islets, Biochem. J., 184, 291, 1979.
8. Hutton, J.C., Sener, A., and Malaisse, W.J., The stimulus-secretion coupling of 4-methyl-2-
oxopentanoate-induced insulin release, Biochem. J., 184, 303, 1979.
9. Sener, A. and Malaisse, W.J., L-leucine and a nonmetabolized analogue activate pancreatic
islet glutamate dehydrogenase, Nature, 288, 187, 1980.
10. Sener, A., Malaisse-Lagae, F., and Malaisse, W.J., Stimulation of islet metabolism and insulin
release by a nonmetabolizable amino acid, Proc. Natl. Acad. Sci. U.S.A., 78, 5460, 1981.
11. Malaisse-Lagae, F. et al., The stimulus-secretion coupling of amino acid-induced insulin
release. IX. Influence of a nonmetabolized analog of leucine on the metabolism of glutamine
in pancreatic islets, J. Biol. Chem., 257, 3754, 1982.
12. Hutton, J.C., Sener, A., and Malaisse, W.J., Interaction of branched chain amino acids and
keto acids upon pancreatic islet metabolism and insulin release, J. Biol. Chem., 255, 7340, 1980.
13. Sener, A. et al., Mechanism of 3-phenylpyruvate-induced insulin release: secretory, ionic and
oxidative aspects, Biochem. J., 210, 913, 1983.
14. Malaisse, W.J. et al., Mechanism of 3-phenylpyruvate-induced insulin release: metabolic
aspects, Biochem. J., 210, 921, 1983.
15. Sener, A., Malaisse-Lagae, F., and Malaisse, W.J., Does leucine- and norleucine-induced
insulin release depend on amino acid aminotransferase activity? J. Biol. Chem., 258, 6693, 1983.
16. Sener, A. et al., The stimulus-secretion coupling of amino acid-induced insulin release. III.
Biosynthetic and secretory responses of rat pancreatic islet to L-leucine and L-glutamine,
Diabetologia, 21, 135, 1981.
17. Malaisse-Lagae, F. et al., The stimulus-secretion coupling of amino acid-induced insulin
release. XIII. Secretory and oxidative response of pancreatic islets to L-asparagine, Diabetes,
33, 464, 1984.
1382_C20.fm Page 334 Tuesday, October 7, 2003 6:50 PM

334 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

18. Maechler, P. and Wollheim, C.B., Mitochondrial glutamate acts as a messenger in glucose-
induced insulin exocytosis, Nature, 402, 685, 1999.
19. MacDonald, M.J. and Fahien, L.A., Glutamate is not a messenger in insulin secretion, J. Biol.
Chem., 275, 34025, 2000.
20. Brice, N.L. et al., Metabotropic glutamate and GABAB receptors contribute to the modulation
of glucose-stimulated insulin secretion in pancreatic beta cells, Diabetologia, 45, 242, 2002.
21. Malaisse, W.J. et al., The stimulus-secretion coupling of glucose-induced insulin release.
XLVI. Physiological role of L-glutamine as a fuel for pancreatic islets, Mol. Cell. Endocrinol.,
20, 171, 1980.
22. Sener, A., Malaisse-Lagae, F., and Malaisse, W.J., The stimulus-secretion coupling of glucose-
induced insulin release. LII. Environmental influences on L-glutamine oxidation in pancreatic
islets, Biochem. J., 202, 309, 1982.
23. Sener, A. and Malaisse, W.J., Stimulation of insulin release by L-glutamine, Mol. Cell. Biochem.,
33, 157, 1980.
24. Sener, A. et al., The stimulus-secretion coupling of amino acid-induced insulin release. XIV.
Metabolism of L-asparagine in pancreatic islets, Arch. Biochem. Biophys., 229, 155, 1984.
25. Malaisse, W.J., Malaisse-Lagae, F., and Sener, A., The stimulus-secretion coupling of amino
acid-induced insulin release. XV. Metabolic interaction of L-asparagine and L-leucine in
pancreatic islets, Biochim. Biophys. Acta, 797, 194, 1984.
26. Malaisse, W.J. et al., Stimulus-secretion coupling of arginine-induced insulin release: metab-
olism of L-arginine and L-ornithine in pancreatic islets, Biochim. Biophys. Acta, 1013, 133, 1989.
27. Herchuelz, A. et al., The mechanism of arginine-stimulated Ca2+ influx into the pancreatic
B-cell, Am. J. Physiol., 246, E38, 1984.
28. Carpinelli, A.R. and Malaisse, W.J., The stimulus-secretion coupling of glucose-induced
insulin release. XLIV. A possible link between glucose metabolism and phosphate flush,
Diabetologia, 19, 458, 1980.
29. Mathias, P.C.F., Best, L., and Malaisse, W.J., Stimulation by glucose and carbamylcholine of
phospholipase C in pancreatic islets, Cell Biochem. Funct., 3, 173, 1985.
30. Best, L. and Malaisse, W.J., Effects of nutrient secretagogues upon phospholipids metabolism
in rat pancreatic islets, Mol. Cell. Endocrinol., 32, 205, 1983.
31. Best, L. and Malaisse, W.J., Phosphatidylinositol and phosphatidic acid metabolism in rat
pancreatic islets in response to neurotransmitter and hormonal stimuli, Biochim. Biophys.
Acta, 750, 157, 1983.
32. Leclercq-Meyer, V. et al., Calcium deprivation enhances glucagon release in the presence of
2-ketoisocaproate, Endocrinology, 108, 2093, 1981.
33. Leclercq-Meyer, V., Marchand, J., and Malaisse, W.J., Role of glucose and insulin in the
dynamic regulation of glucagon release by the perfused rat pancreas, Diabetologia, 24, 191,
1983.
34. Sener, A., Blachier, F., and Malaisse, W.J., Production of urea but absence of urea cycle in
pancreatic islet cells, Med. Sci. Res., 16, 483, 1988.
35. Flodström, M., Morris, S.M., Jr., and Eizirik, D.L., Role of the citrulline-nitric oxide cycle in
the functional response of adult human and rodent pancreatic islets to cytokines, Cytokine,
8, 642, 1996.
36. Delcros, J.-G., Roch, A.-M., and Quash, G., The competitive inhibition of tissue transglutam-
inase by a-difluoromethylornithine, FEBS Lett., 171, 221, 1984.
37. Lenzen, S., Hickethier, R., and Panten, U., Interactions between spermine and Mg2+ on
mitochondrial Ca2+ transport, J. Biol. Chem., 261, 16478, 1986.
38. Thams, P., Capito, K., and Hedeskov, C.J., Polyamine-enhanced casein kinase II in mouse
pancreatic islets, Diabetologia, 29, 888, 1986.
39. Hougaard, D.M., Nielsen, J.H., and Larsson, L.I., Localization and biosynthesis of polyamines
in insulin-producing cells, Biochem. J., 238, 43, 1986.
40. Sener, A. et al., Stimulus-secretion coupling of arginine-induced insulin release: effects of
2-aminoisobutyric acid upon ornithine decarboxylase activity and insulin secretion, Res.
Commun. Chem. Pathol. Pharmacol., 65, 65, 1989.
1382_C20.fm Page 335 Tuesday, October 7, 2003 6:50 PM

Chapter twenty: Amino acid-mediated insulin secretion 335

41. Charles, S., Tamagawa, T., and Henquin, J.-C., A single mechanism for the stimulation of
insulin release and 86Rb+ efflux from rat islets by cationic amino acids, Biochem. J., 208, 301,
1982.
42. Henquin, J.-. and Meissner, H.P., Effects of amino acids on membrane potential and 86Rb+
fluxes in pancreatic b-cells, Am. J. Physiol., 240, E245, 1981.
43. Blachier, F. et al., Stimulus-secretion coupling of arginine-induced insulin release: uptake of
metabolized and nonmetabolized cationic amino acids by pancreatic islets, Endocrinology,
124, 134, 1989.
44. Schneid, C. et al., Effects of ornithine a-ketoglutarate on insulin secretion in rat pancreatic
islets: implication of nitric oxide synthase and glutamine synthetase pathways, Br. J. Nutr.,
89, 249, 2002.
45. Malaisse, W.J. and Malaisse-Lagae, F., Stimulation of insulin secretion by non-carbohydrate
metabolites, J. Lab. Clin. Med., 72, 438, 1968.
46. Floyd, J.C. et al., Stimulation of insulin secretion by amino acids, J. Clin. Invest., 45, 1487, 1966.
47. Sener, A. et al., Stimulus-secretion coupling of arginine-induced insulin release: comparison
with lysine-induced insulin secretion, Endocrinology, 124, 2558, 1989.
48. Lambert, A.E. et al., Organ culture of fetal rat pancreas. II. Insulin release induced by amino
and organic acids, by hormonal peptides, by cationic alterations of the medium and by other
agents, Biochim. Biophys. Acta, 174, 540, 1969.
49. Fajans, S.S. et al., Effect of amino acids and proteins on insulin secretion in man, Recent Prog.
Horm. Res., 23, 617, 1967.
50. Milner, R.D.G., The stimulation of insulin release by essential amino acids from rabbit
pancreas in vitro, J. Endocrinol., 47, 347, 1970.
51. Pontirolli, A.E.., Micossi, P., and Foa, P.P., Glucagon and insulin response to arginine in rat
pancreas in vitro: effect of histamine and serotonin, Horm. Metab. Res., 12, 703, 1980.
52. Ince, B.W., Amino acid stimulation of insulin secretion from the in situ perfused eel pancreas:
modification by somatostatin, adrenaline, and theophylline, Gen. Comp. Endocrinol., 40, 275,
1980.
53. Edgar, P., Rabinowitz, D., and Merimee, T.J., Effects of amino acids on insulin release from
excised rabbit pancreas, Endocrinology, 84, 835, 1969.
54. Sener, A. et al., Stimulus-secretion coupling of arginine-induced insulin release: comparison
with histidine-induced insulin release, Endocrinology, 127, 107, 1990.
55. Itatsu, T., Shibata, A., and Ukai, M., The histaminergic mechanism of neurotensin-induced
glucagon release from isolated rat pancreatic islets, Endocrinol. Jpn., 28, 631, 1981.
56. Sener, A. and Malaisse, W.J., The stimulus-secretion coupling of amino acid-induced insulin
release. II. Sensitivity of K+, NH4+ and H+ of leucine-stimulated islets, Diabetes Metab., 6, 97,
1980.
57. Bjaaland, T. and Howell, S.L., Stimulation of insulin secretion from electrically permeabilised
islets of Langerhans by L-arginine, Diabetologia, 32, 467A, 1989.
58. Pfeiffer, E.F. and Telib, M., Insulin secretion in vitro: studies in amphibians and mammalians,
Acta Diabetol. Lat., 5 (Suppl. 1), 30, 1968.
59. Hellman, B., Sehlin, J., and Täljedal, I.-B., Uptake of alanine, arginine and leucine by mam-
malian pancreatic b-cells, Endocrinology, 89, 1432, 1971.
60. Hellman, B. and Täljedal, I.-G., Histochemistry of the pancreatic islet cells, in Handbook of
Physiology, Steiner, D.F. and Freinkel, N., Eds., American Physiological Society, Washington,
D.C., 1972, p. 91.
61. Lernmark, Å., Specificity of leucine stimulation of insulin release, Hormones, 3, 14, 1972.
62. Lernmark, Å., Effects of neutral and dibasic amino acids on the in vitro release of insulin,
Hormones, 3, 22, 1972.
63. Lambert, A.E. et al., Stimulation of insulin release by natural amino acids and their non-
metabolized analogues, Diabetologia, 6, 635, 1970.
64. Philipps, A.F., Dubin, J.W., and Raye, J.R., Alanine-stimulated insulin secretion in the fetal
and neonatal lamb, Am. J. Obstet. Gynecol., 136, 597, 1980.
1382_C20.fm Page 336 Tuesday, October 7, 2003 6:50 PM

336 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

65. Sann, L. et al., Effect of intravenous L-alanine administration on plasma glucose, insulin and
glucagon, blood pyruvate, lactate and b-hydroxybutyrate concentrations in newborn infants:
study on term and preterm newborn infants, Acta Paediatr. Scand., 67, 297, 1978.
66. Dunne, M.J. et al., Effects of alanine on insulin-secreting cells: patch-clamp and single cell
intracellular Ca2+ measurements, Biochim. Biophys. Acta, 1055, 157, 1990.
67. McClenaghan, N.H. et al., Induction of a glucose-dependent insulin secretory response by
the nonmetabolizable amino acid alpha-aminoisobutyric acid, Pancreas, 14, 65, 1997.
68. McClenaghan, N.H., Barnett, C.R., and Flatt, P.R., Na+ cotransport by metabolizable and
nonmetabolizable amino acids stimulates a glucose-regulated insulin-secretory response,
Biochem. Biophys. Res. Commun., 249, 299, 1998.
69. Sener, A. and Malaisse, W.J., The stimulus-secretion coupling of amino acid-induced insulin
release: insulinotropic action of L-alanine, Biochim. Biophys. Acta, in press, 2002.
70. Malaisse, W.J. and Sener, A., Metabolic effects and fate of succinate esters in pancreatic islets,
Am. J. Physiol., 264, E434, 1993.
71. Malaisse-Lagae, F., Zähner, D., and Malaisse, W.J., NADP-malate dehydrogenase activity in
rat erythrocytes: comparison with pyruvate kinase in terms of coupling to lactate dehydro-
genase, Int. J. Biochem. Cell. Biol., 27, 905, 1995.
72. Bakkali Nadi, A., Malaisse-Lagae, F., and Malaisse, W.J., Is pyruvate channelled from pyru-
vate kinase to lactate dehydrogenase? Med. Sci. Res., 23, 329, 1995.
73. Alsever, R.N., Georg, R.H., and Sussman, K.E., Stimulation of insulin secretion by guanidino-
acetic acid and other guanidine derivatives, Endocrinology, 86, 332, 1970.
74. Sehlin, J., Transport and oxidation of glycine in mammalian pancreatic islets with reference
to the mechanism of amino acid-induced insulin release, Hormones, 3, 144, 1972.
75. Reusens, B. et al., Long-term consequences of diabetes and its complications may have a
fetal origin: experimental and epidemiological evidence, Nestlé Nutr. Workshop Ser., 35, 187,
1995.
76. Cherif, H. et al., Stimulatory effects of taurine on insulin secretion by fetal rat islets cultured
in vitro, J. Endocrinol., 151, 501, 1996.
77. Scruel, O., Sener, A., and Malaisse, W.J., Assay, plasma and tissue content and insulinotropic
action of taurine, Diabetes Res., 32, 257, 1997.
78. Scruel, O. et al., Taurine deficiency and supplementation in protein malnourished rats,
Diabetes Res., 32, 133, 1997.
79. Panten, U., Amino acids and insulin secretion, in Insulin, Part 2, Hasselblatt, A. and
Bruchhausen, F., Eds., Springer-Verlag, Berlin, 1975, p. 115.
1382_C21.fm Page 337 Tuesday, October 7, 2003 6:54 PM

Part III

Amino acid metabolism in disease


1382_C21.fm Page 338 Tuesday, October 7, 2003 6:54 PM
1382_C21.fm Page 339 Tuesday, October 7, 2003 6:54 PM

chapter twenty-one

Cancer-associated cachexia:
altered metabolism of protein
and amino acids
Michelle Mackenzie
University of Alberta
Vickie E. Baracos
University of Alberta

Contents
Summary.......................................................................................................................................340
21.1 Amino acid metabolism during progressive tumor growth .......................................340
21.1.1 Applications of tumor models ..........................................................................340
21.1.2 Amino acid metabolism in clinical studies .....................................................341
21.2 Alterations of amino acid metabolism in the tumor-bearing state...........................341
21.2.1 The total and relative amounts of essential amino acids required
are altered .............................................................................................................342
21.2.2 Amino acids normally considered nonessential for humans become
conditionally essential in the diet .....................................................................342
21.2.3 A deficit of available amino acid supplies from the diet, in the
presence of increased requirements for both essential and nonessential
amino acids, is a primary driver of the catabolism of endogenous
protein reserves in skeletal muscle...................................................................343
21.2.4 Amino acid mobilization may be driven by the secretion of different
tumor-specific catabolic mediators ...................................................................343
21.2.5 At substantial disease burden the tumor may become a quantitatively
important player in whole-body amino acid utilization ..............................343
21.2.6 Surgery, radiotherapy, and chemotherapy are associated with large
metabolic changes in substrate utilization ......................................................343
21.3 The metabolism of specific amino acids in cancer ......................................................343
21.3.1 Tumor metabolism of amino acids ...................................................................343
21.3.2 Amino acid metabolism in the tumor-bearing host ......................................344
21.3.2.1 Glutamine .............................................................................................345
21.3.2.2 Arginine.................................................................................................345

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 339
1382_C21.fm Page 340 Tuesday, October 7, 2003 6:54 PM

340 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

21.3.2.3 Sulfur amino acids and glutathione .................................................346


21.3.2.4 Alanine ..................................................................................................346
21.3.2.5 Branched-chain amino acids..............................................................346
21.3.2.6 Aromatic amino acids .........................................................................347
21.4 Manipulation of amino acid supplies in the tumor-bearing state ..............................347
21.4.1 Glutamine..............................................................................................................347
21.4.2 Arginine.................................................................................................................348
21.4.3 Cysteine .................................................................................................................348
21.5 Application of new methods in the study of amino acid metabolism in cancer ......348
21.5.1 Determination of amino acid requirements ....................................................348
21.5.1.1 Indicator amino acid oxidation .........................................................349
21.5.1.2 The plasma amino acid response to an infusion of an amino
acid mixture..........................................................................................349
References .....................................................................................................................................349

Summary
Changes in metabolism of amino acids are not fully described for the tumor-bearing state,
either for animal models or for cancer patients. Several amino acids appear to show
characteristic patterns of utilization in the tumor-bearing state, including aromatic, sulfur-
containing, and branched-chain amino acids, as well as the nonessential amino acids
alanine, glutamine, cysteine, and arginine. Trials of dietary supplementation have been
done more extensively for several individual amino acids in laboratory animal models,
and from these we can infer the presence of possible amino acid deficiencies characteristic
of the tumor-bearing state; few amino acid supplementation trials have been done on
cancer patients. Several minimally invasive approaches are available to determine amino
acid requirements in humans; however, these have yet to be used in cancer patient
populations.

21.1 Amino acid metabolism during progressive tumor growth


The current information set on protein and amino acid metabolism in cancer patients
consists of a relatively sparse group of data emerging from a spectrum of animal models;
human studies have employed a few highly defined patient subsets. This creates an
incomplete and somewhat sketchy picture of amino acid metabolism in the tumor-bearing
state.

21.1.1 Applications of tumor models


Cancer is an ensemble of diseases varying in biology, epidemiology, and prognosis. There
is no single tumor model to represent cancer, since cancer is not a single entity. Animal
models for the study of cancer-associated metabolism have been the subject of several
recent reviews from our laboratory.1–3 There is considerable diversity of tumor types, stages
of tumor growth, and background control diets used in studies of laboratory rodents.
Investigators working in this area have not yet worked together to develop a panel of
tumor models that is considered representative of cancer-associated nutrient metabolism
in man. The lack of a consensus set of animal models for investigations into metabolism
and nutrition in cancer is an impediment to current understanding because it is impossible
to reconcile results obtained in totally different models. Unfortunately, there is no single
1382_C21.fm Page 341 Tuesday, October 7, 2003 6:54 PM

Chapter twenty-one: Cancer-associated cachexia 341

animal model for which metabolic utilization of more than one or a few amino acids has
been studied. There exist a number of single reports, where the metabolism of one specific
amino acid has been determined in a tumor model, but very often that tumor has not also
been studied with regard to other amino acids or other aspects of metabolism.
Another important point is that the majority of work has focused on the tumor-bearing
state per se, and while this formally constitutes the cancer-associated metabolic change,
the clinical reality usually includes superimposed alterations in amino acid metabolism
attributable to radiotherapy, chemotherapy, and surgical intervention. Much less work has
focused on amino acid metabolism in the treated tumor-bearing state.
Amino acid metabolism is especially complex because the utilization of at least 20
amino compounds used for protein synthesis must be accounted for. Key alterations in
amino acid metabolism may additionally include compounds not used for protein syn-
thesis, such as ornithine and taurine. Because the metabolism of amino acids is interrelated,
a full description of amino acid metabolism in a tumor model would be of interest. Such
a global appreciation of amino acid metabolism in animal models remains elusive.
Animal studies can illuminate which amino acids may be preferentially utilized by
the host and tumor. A particular advantage of animal studies is that they permit assess-
ment of the ability of certain amino acids to promote or inhibit tumor growth. By contrast,
quantitative aspects of amino acid metabolism determined in animal studies are very
difficult to relate to cancer patients. It is especially important that consideration be given
to the magnitude of tumor burden in animal studies. We have estimated that a Morris
hepatoma 7777 in the rat, at 0.2% of body weight, captures 2.0% of the animal’s daily N
balance.4 Notably, this represents a 10-fold difference between the expected N capture by
the tumor based on its mass and its actual metabolic activity. This may be a characteristic
of hepatomas, which are derived from liver cells, the key site of amino acid catabolism.
As few detailed balance studies exist, the quantity of amino acid capture by other tumors
is unknown. At higher tumor burdens the tumor represents a significant N trap. For
example, at 8.8% of body weight the daily N balance of the tumor is equivalent to 150%
of the daily retention of N from the diet.4 Protein and amino acid metabolism in animals
bearing a tumor of up to 30% of body mass have been reported, but the clinical relevance
of these models is questionable, since human tumor burdens are usually in the range of
1% of body weight or less.

21.1.2 Amino acid metabolism in clinical studies


The generalization of many metabolic studies in cancer patients is affected by method-
ological issues such as recruitment of patients at different points in the course of their
disease and the difficulty in recruiting representative cohorts among these patients. Clin-
ical studies on amino acid metabolism in cancer patients are relatively few and, like the
animal models, usually concern a small sample from a particular patient population. For
example, elevated alanine metabolism has been described in early stage and advanced
gastrointestinal cancer patients5 and advanced non-small cell lung cancer patients,6 but it
is not clear whether this represents a generality for cancer at other sites or at what time
in the disease trajectory altered alanine metabolism may have become evident.

21.2 Alterations of amino acid metabolism in the tumor-bearing state


The various observations on amino acid metabolism in the tumor-bearing state remain to
be fully woven into a coherent mosaic. However, recognizing the limitations stated above,
1382_C21.fm Page 342 Tuesday, October 7, 2003 6:54 PM

342 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

it is possible to identify modifications of amino acid utilization in the tumor-bearing state,


in several global categories, based on available evidence.

21.2.1 The total and relative amounts of essential amino acids required are altered
The rate and type of protein synthesis would be expected to drive total amino acid
requirements. Protein synthesis, on a whole-body basis, is frequently elevated, and this is
one of several manifestations of a hypermetabolic state. Elevated whole-body protein
turnover has been reported7–9in tumor-bearing animals and in lung, colorectal, and other
cancer patients relative to healthy age-matched populations or patients with nonmalignant
disease.
There is a change in the relative amounts of different amino acids utilized, in associ-
ation with a shift from peripheral protein synthesis toward the viscera. Skeletal muscle
protein mass is progressively depleted, while hepatic protein mass is maintained or may
even increase. This redistribution has been known for about 30 years and was initially
described in animal models.7,10,11 Deficits of muscle protein synthesis have been verified
in human subjects using stable isotope approaches.12,13 More recently, numerous studies
have demonstrated increased synthesis of hepatic secretory proteins, including albumin,14
fibrinogen,15 and C-reactive protein.16–19 Pancreatic cancer patients experiencing weight
loss have also been reported to have elevated albumin synthetic rates as determined using
a flooding dose technique with 2H5-phenylalnine.14 Despite an increased synthetic rate,
the cancer patients had lower plasma albumin concentrations and intravascular albumin
mass compared to age- and height-matched healthy controls.14 As well, these same pan-
creatic cancer patients had elevated synthesis of the positive acute phase protein fibrinogen
and elevated concentration of C-reactive protein, suggesting an ongoing acute phase
response.15
Acute phase proteins contain a higher amount of aromatic and sulfur amino acids
than skeletal muscle protein.20 Albumin contains a high content of cysteine and methionine
(about 77 mg of sulfur amino acids per gram of albumin vs. 43 mg of sulfur amino acids
per gram of skeletal muscle protein).20,21 Where present, the acute phase response repre-
sents a factor promoting altered amino acid requirements.

21.2.2 Amino acids normally considered nonessential for humans become


conditionally essential in the diet
Amino acids such as glutamine, arginine, and cysteine may become conditionally essential
in the tumor-bearing state. This appears to be connected with elevated utilization of these
amino acids. For example, elevated metabolic energy expenditure in the presence of
anorexia creates an environment for high rates of gluconeogenesis, and the amino acids
alanine and glutamine are key precursors for this process. An early paper by Shaw and
Wolfe5 evaluated glucose and urea kinetics in healthy volunteers and in early stage and
advanced gastrointestinal cancer patients. These authors report a stage-dependent eleva-
tion of gluconeogenesis and ureagenesis that was not suppressed in response to glucose
infusion or total parenteral nutrition. Leij-Halfwerk et al.6 confirmed this result in
advanced non-small cell lung cancer patients using a primed constant infusion of glucose
and alanine and 31P magnetic resonance spectra in vivo to show elevated hepatic gluco-
neogenic intermediates. These illustrate a larger degree of enhancement of gluconeogen-
esis with more advanced disease and in patients with more intense rather than slower
weight loss. Elevated metabolic rate appears to be driven by pro-inflammatory cytokines
or by excess adrenergic stimulation.18,23,24
1382_C21.fm Page 343 Tuesday, October 7, 2003 6:54 PM

Chapter twenty-one: Cancer-associated cachexia 343

21.2.3 A deficit of available amino acid supplies from the diet, in the presence of
increased requirements for both essential and nonessential amino acids,
is a primary driver of the catabolism of endogenous protein reserves in
skeletal muscle
Skeletal muscle tissue comprises the body reserve of amino acids, which are mobilized
under conditions where the dietary supply does not match the demand for these com-
pounds. The major metabolic fates of mobilized muscle amino acids are protein synthesis
in extramuscular tissues and gluconeogenesis. The internal redistribution of amino acids
is not necessarily an efficient process, and there is some evidence that mobilized muscle
protein is not a perfect match for whole-body amino acid requirements in every physio-
logic or pathologic state.20 This has the same net effect of feeding an unbalanced amino
acid mixture, with amino acids present in excess being lost to oxidation.

21.2.4 Amino acid mobilization may be driven by the secretion of different


tumor-specific catabolic mediators
For example, a proteolysis-inducing glycoprotein of tumor origin provokes intense protein
catabolism in skeletal muscle.25,26 In animal models, tumor-derived cytokines and
eicosanoids have also been implicated in initiation of protein catabolism.27 The signals for
protein catabolism secreted by a tumor are not linked to a purposeful use of amino acids,
and since this protein mobilization is not necessarily coordinated with host protein syn-
thesis, oxidative losses will result.

21.2.5 At substantial disease burden the tumor may become a quantitatively


important player in whole-body amino acid utilization
As discussed above for animal models, there is a point in tumor progression where the
acquisition of amino acids by the tumor becomes quantitatively important. This may be
considered to occur only in advanced disease; however, its clinical relevance is unknown.

21.2.6 Surgery, radiotherapy, and chemotherapy are associated with large


metabolic changes in substrate utilization
It is important to appreciate the metabolic change due to treatment factors. Apart from a
very small number of cancers deemed untreatable or too advanced except for palliative
intervention, the vast majority of cancer patients receive aggressive therapy within the
limits of tolerance. Various specific amino acids may additionally be required for healing
after surgery, tissue injury in the gut, or bone marrow after systemic therapy;22,28 however,
the amino acid supply required to support such processes is not well defined.

21.3 The metabolism of specific amino acids in cancer


21.3.1 Tumor metabolism of amino acids
Tumors, as a class, appear to have unique patterns of amino acid metabolism. This is
related to their generally high proliferation rate and the use of amino compounds as
biosynthetic precursors of multiple classes of molecules (Table 21.1). Protein biosynthesis
is a primary determinant of tumor amino acid use and will implicate all 20 amino acids
used in this process. Several publications have shown that the fractional synthetic rate of
1382_C21.fm Page 344 Tuesday, October 7, 2003 6:54 PM

344 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 21.1 Tumor Utilization of Amino Acids


Process Amino Acids
Protein synthesis All
ATP production Glutamine
Nucleotide synthesis Glutamine
Polyamine synthesis Arginine, ornithine
Nitric oxide synthesis Arginine
Methyl group transfer Methionine
Serotonin synthesis Tryptophan

protein of tumors is high relative to other tissues in the tumor-bearing animal.7 The
fractional synthesis rate of colorectal tumors in humans was in the range of 17.2 to
33.9%/day, and in breast tumors the range of rates obtained was 5.3 to 15.9%/day.29 The
impact of this amino acid use will depend on tumor synthetic rate and mass.
Glutamine, arginine, and sulfur-containing amino acids are amino acids for which
elevated tumor utilization has been established for other processes in addition to protein
synthesis. Tumor cells obtain a relatively high proportion of fuel for energy metabolism
from complete and partial glutamine oxidation.4,30 However, tumor cells do not have a
high capacity to synthesize glutamine31 and rely on systemic glutamine from the host.32,33
The rapid proliferation rate of tumor cells also requires enhanced production of the
bioactive products of arginine metabolism, including nitric oxide and polyamines. Ele-
vated whole-body ornithine turnover in tumor-bearing rats34 supports the increase in
ornithine and polyamine concentrations during the tumor growth.35,36 Clinically,
polyamine excretion is associated with tumor burden.37,38 Unlike nitric oxide production,
the conversion of ornithine to polyamines results in arginine net loss. Similar to arginine,
methionine is required by tumor cells to support proliferation through its essential role
in methylation reactions, polyamine formation, and initiation of protein synthesis. Several
tumor cell lines are dependent on methionine uptake, attributed to low levels of methion-
ine synthase activity.39
Some tumors exhibit atypical amino acid use, for example, the carcinoid tumors, a
class of neuroendocrine tumors that produce amines or peptides depending on the site
of origin.40 Serotonin is among the major bioactive substances released by active carcinoid
tumors, and in carcinoid patients there is a massive diversion of tryptophan for serotonin
production.
Dependence upon various amino acids by some tumor lines has led to the investiga-
tion of amino acid deprivation to reduce tumor growth. An early nutritional approach
involved formulation of diets lacking amino acids essential for tumor growth. Diets
deficient in arginine or methionine slowed tumor growth in animal models; antimetabo-
lites blocking glutamine metabolism have been investigated as chemotherapeutic agents
and have been successful in reducing tumor growth in rats.33,41 These approaches are
associated with the problem of toxicity in the case of antimetabolites and of inducing
amino acid deficiency in the host as well as the tumor.42

21.3.2 Amino acid metabolism in the tumor-bearing host


The amino acid metabolism of the host and tumor interact. A tumor may capture amino
acids, reducing availability to host tissues, and selective capture may be especially prob-
lematic in a situation when total dietary intake of amino acids is reduced. The host animal
or person also responds to the presence of the tumor. Tumor-derived secretory products
may alter host metabolism of amino acids in various tissues. Host response to the tumor
may include endocrine and immunological changes that also impact metabolism.
1382_C21.fm Page 345 Tuesday, October 7, 2003 6:54 PM

Chapter twenty-one: Cancer-associated cachexia 345

The overall impact of a tumor on whole-body amino acid metabolism will inevitably
depend upon the size of the tumor. More often than not, the tumor burdens used in rodent
models are relatively large compared to the size of tumors in human cancer. Therefore,
caution needs to be taken in interpreting and applying the findings to the clinical setting.
These studies have, however, provided useful information on the pattern of amino acid
metabolism in the tumor-bearing state.

21.3.2.1 Glutamine
Uptake and utilization of glutamine by the tumor is a possible mechanism for low plasma
glutamine levels and compromised host glutamine availability in tumor-bearing ani-
mals.43–45 Clinically, plasma glutamine levels are reduced in patients with cancer of the
gastrointestinal tract46,47; however, inadequate dietary intake often accompanies these can-
cers, making it difficult to conclude that tumor glutamine metabolism is responsible for
alterations in plasma concentrations.
Intramuscular glutamine formation and release are increased in tumor-bearing
animals43 and are attributed to enhanced rates of muscle protein breakdown and subse-
quent nitrogen donation from elevated catabolism of intramuscular branched-chain amino
acids. Elevated turnover of glutamine in muscle from tumor-bearing rats48 is associated
with a reduction in intramuscular glutamine concentration.43 Intracellular glutamine is an
important regulator of muscle protein synthesis, and low concentrations may contribute
to muscle loss. The liver can either produce or consume glutamine depending on the
physiological state. However, the influence of tumors on hepatic glutamine metabolism
is unclear. Both hepatic glutamine uptake49–53 and release49 have been reported to be
increased in rats bearing large tumors, without an increase in hepatic glutamine oxida-
tion.50 Even more unclear is the effect that tumors have on other tissues that are high
consumers of glutamine, such as enterocytes and immune cells.

21.3.2.2 Arginine
Weight-losing cancer patients have lower plasma concentrations of arginine than well-
nourished and malnourished controls in the fasted state.46,48,54 In malnourished and well-
nourished patients with or without cancer, plasma arginine levels are not raised in
response to feeding.46 Elevated rates of protein catabolism12,13 and gluconeogenesis5,6
enhance urea production in cancer. Hepatic arginine uptake52 and arginase activity36 are
elevated in tumor-bearing rats, indicating an increase in urea cycle activity and arginine
demand. Unlike glutamine, the capacity for the host to increase arginine biosynthesis is
limited. Inadequate arginine intake is not associated with up-regulation of its synthesis,55
and its precursor, citrulline, is not formed at an elevated rate in tumor-bearing rats,56 which
further limits arginine production. As well, alterations in glutamine availability and uti-
lization in the tumor-bearing state (see Section 21.3.2.1) may be a factor compromising the
intestinal synthesis of citrulline.
A generalized stimulation of arginine use is associated with pro-inflammatory cyto-
kines, which mediate the expression of inducible nitric oxide synthase (iNOS) in macroph-
ages, other immune cells, Kupffer cells, and skeletal muscle.57,58 However, the quantitative
importance to arginine utilization is unknown. Muscle nitric oxide is associated with
impaired insulin-stimulated glucose uptake and oxidative stress,58,59 both of which are
observed in cancer. The role of nitric oxide in cancer-associated muscle wasting is not
known, yet the presence of a pro-inflammatory cytokine response in cancer suggests that
iNOS expression and nitric oxide production are stimulated. Nitric oxide synthase inhi-
bition prevents muscle wasting in a murine model of cachexia60; however, this effect has
not been shown clinically or in cachexia associated with cancer.
1382_C21.fm Page 346 Tuesday, October 7, 2003 6:54 PM

346 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

21.3.2.3 Sulfur amino acids and glutathione


Plasma cystine61 and glutathione62 levels are lowered in cancer patients, resulting in
alterations in redox status that have been associated with a loss in body cell mass in
subjects with various types of advanced cancer.61 These changes are reflective of a reduc-
tion in the availability of sulfur amino acids in the tumor-bearing state attributed to tumor
utilization of methionine and inadequate dietary intake. As well, the host response to
tumors increases the metabolic demand for sulfur-containing amino acids for the synthesis
of glutathione and acute phase proteins.63 Hepatic glutathione levels are increased in
tumor-bearing mice, while hepatic sulfate levels decrease, suggesting that an increase in
cysteine incorporation into glutathione reduces cysteine catabolism within the liver.64 By
contrast, intracellular glutathione is lowered in muscle from tumor-bearing mice,64 sug-
gesting that hepatic cysteine utilization receives priority in the tumor-bearing state when
cysteine availability is limited. Impaired intramuscular glutathione is associated with
reduced oxidative metabolism in tumor-bearing mice, which can be reversed by supple-
mentation with cysteine.65

21.3.2.4 Alanine
As with glutamine, alanine release from muscle is accelerated in the tumor-bearing state.66
Protein catabolism alone does not account for the relatively large release of alanine.
Increased glycolytic rate and subsequent alanine production from pyruvate enhances
alanine release from muscle. Hepatic conversion of alanine provides pyruvate for gluco-
neogenesis through the alanine–glucose cycle as well as nitrogen for urea synthesis.
Patients with advanced cancer have elevated rates of hepatic alanine conversion to
glucose6 and gluconeogenesis in general.5 Plasma alanine concentration has been reported
to be increased, decreased, or unchanged in cancer depending on the site of sampling, the
degree of malnutrition, and glycolytic rate. Alanine provision is not likely to reduce its
release from muscle; however, adequate nonprotein energy, particularly glucose, may be
beneficial in slowing alanine–glucose cycling.

21.3.2.5 Branched-chain amino acids


The plasma concentrations of the branched-chain amino acids (BCAA) — leucine, iso-
leucine, and valine — are lowered in patients with cancer of the gastrointestinal tract46,67
and in tumor-bearing animals.68 Leucine in particular has a key role in the regulation of
skeletal muscle protein metabolism.69 Leucine promotes muscle protein synthesis, and the
metabolites of leucine catabolism inhibit muscle proteolysis.70 Unlike other amino acids,
the catabolism of leucine, isoleucine, and valine takes place almost exclusively in extra-
hepatic tissue, including skeletal muscle. Transamination of the BCAA to their respective
a-keto acid derivatives by BCAA transferase (BCAAT) in muscle supplies amino N for
alanine, glutamate, or glutamine synthesis, providing a nontoxic transportation of ammo-
nia from the muscle to the liver for detoxification. Following transamination, the branched-
chain keto acids can be further catabolized for fuel within the muscle itself or transported
for utilization in other tissues.
Tumor-bearing rats have increased oxidation of leucine in vivo compared to pair-fed
controls.71,72 Increased activity of BCAAT and BCKD have been reported in tumor-bearing
rats.71,73 Enhanced leucine oxidation by skeletal muscle for use as fuel may be the result
of impaired glucose and fatty acid metabolism. Insulin resistance, decreased glucose
uptake by the muscle, and reduced mitochondrial transport of fatty acids in skeletal muscle
impede the use of glucose and fatty acids for fuel by muscle. BCAA supplementation vs.
feeding at requirement increased muscle protein synthesis and improved nitrogen balance
1382_C21.fm Page 347 Tuesday, October 7, 2003 6:54 PM

Chapter twenty-one: Cancer-associated cachexia 347

without changing tumor weight,74,75 suggesting that the requirements for one or more of
the BCAA is elevated in the tumor-bearing state.

21.3.2.6 Aromatic amino acids


It is generally accepted that tyrosine and phenylalanine plasma concentrations are elevated
in cancer76 and are a reflection of increased muscle proteolysis. On the other hand, con-
flicting reports regarding tryptophan metabolism reflect its more complex metabolism.
Studies have reported both elevated and lowered concentrations of tryptophan in a variety
of cancer types.77,78 Free tryptophan may be increased in cancer, but this is most likely
reflective of a decrease in the binding of tryptophan to albumin as total tryptophan is
normal or decreased.79,80 Cytokines enhance tryptophan catabolism to kynurenine81; thus,
immune stimulation in cancer may be partially responsible for the reduction in tryptophan
levels. Tryptophan is the direct precursor for serotonin formation. Lowered serotonin
levels have been associated with depression. In patients with colorectal cancer, a reduction
in serum tryptophan levels was correlated with lower quality of life.78 In contrast, elevated
levels of tryptophan in the brain and subsequent serotonin synthesis may have a role in
the loss of appetite common in cancer.73,80,82

21.4 Manipulation of amino acid supplies in the tumor-bearing state


Current nutritional approaches are based on the concept of manipulating nutrient mixtures
to alter the balance between the host and the tumor in a manner that favors the host
overall. Supplementation of amino acids that have become limiting for the function of the
host would be expected to improve nutritional status and tolerance to treatments, and to
limit morbidity and mortality. The identification and supplementation of limiting amino
acids in cancer patients has potential in alleviating muscle loss, antioxidant status, and
improving immunity. It has been suggested that supplementation may pose a risk of
enhancement of tumor growth. It has alternatively been suggested that appropriately
formulated amino acid mixtures may interfere with tumor metabolism or have immuno-
stimulatory properties, allowing more efficient antitumor immunity.
A factor in the use of supplementary amino acids is the administration by oral or
intravenous routes. Feeding amino acid supplements may be expected to have different
outcomes depending on the route of administration. Intravenous administration of amino
acids affects the requirements due to the bypass of first-pass metabolism in the gut and
liver. For example, the branched-chain amino acid requirement in parenterally fed piglets
is half of the requirement for piglets fed an elemental enteral diet.83 As well, bypassing
the gut with intravenous feeding may compromise the de novo synthesis of nonessential
amino acids, such as arginine.84

21.4.1 Glutamine
The size, protein synthetic rate, and DNA content of tumors are not affected by glutamine
administration compared to tumor-bearing animals given isonitrogenous, isocaloric
glutamine-free diets.85–87 This is not the outcome that would have been predicted based
on the observation that glutamine is required by tumor cells, suggesting that dietary
influences must be evaluated in vivo. Glutamine supplementation, however, did improve
whole-body nitrogen retention and increase protein synthetic rates and glutamine content
in muscle and small intestine of tumor-bearing rats.85–87
1382_C21.fm Page 348 Wednesday, October 8, 2003 1:33 PM

348 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

21.4.2 Arginine
Animal studies of arginine supplementation suggest that in at least some cases tumor
growth is stimulated. Increased tumor growth was observed with either arginine or
citrulline parenteral supplementation but not with ornithine in rats bearing the Ward colon
tumor.88,89 Arginine supplementation also increased the tumor protein synthetic rate in
human breast cancer90 but not head and neck cancer.91 Others have found that arginine
parenteral supplementation has no effect on tumor growth92–94 and that arginine supple-
mentation improves whole-body nitrogen retention and muscle protein synthesis in
tumor-bearing rats. Ornithine and a-ketoglutarate provide metabolic precursors for the
formation of arginine and glutamine, and a product for enteral nutrition containing this
mixture did not stimulate growth of either the Yoshida hepatoma or Morris hepatoma.4,95
An amino acid mixture containing glutamine and arginine promoted deposition of lean
body mass in non-small cell lung cancer patients without any reported side effects.96 There
is not a clear explanation why parenteral arginine would increase tumor growth in some
studies and not others, but it may be related to the type of tumor.

21.4.3 Cysteine
A concern with cysteine supplementation is that methionine will be spared and will in
turn promote tumor growth. This has not yet been investigated in animal models. Pro-
viding supplemental oral N-acetyl-cysteine for 4 weeks (400 mg three times a week) was
reported to improve quality of life, normalize redox state, and increase plasma albumin
levels and body cell mass in 50 patients with various forms of inoperable cancer,61 sug-
gesting that cysteine becomes conditionally essential in cancer. Importantly, the survival
curve from the start of treatment to 550 days was not different between the group that
received supplementation and cancer patients who did not, which indirectly suggests that
supplemental cysteine did not enhance tumor growth.

21.5 Application of new methods in the study of amino acid


metabolism in cancer
Several techniques have been applied to study the metabolism of protein and amino acids
in tumor-bearing animals and in clinical cancer populations, including plasma amino acid
profiles45–47 and protein turnover using both flooding dose and constant infusion of labeled
amino acids used in cancer patients9,13–15,29 to determine in vivo protein synthesis of liver,
muscle, tumors, and plasma proteins. However, there are a few recently developed meth-
odologies for identifying limiting amino acids and the quantification of amino acid require-
ments that have yet to be applied to cancer; they will be discussed further. An important
theme in the approaches used is to limit patient burden, as considerable symptoms and
functional loss are associated with advanced disease.

21.5.1 Determination of amino acid requirements


Despite the evidence for altered amino acid metabolism and suggestions of beneficial
effects of amino acid supplementation in cancer,96 amino acid requirements of cancer
patients have not been determined. Recent advances in methodology include minimally
invasive techniques for the determination of the requirement for specific amino acids, and
these merit utilization in cancer patients.
1382_C21.fm Page 349 Tuesday, October 7, 2003 6:54 PM

Chapter twenty-one: Cancer-associated cachexia 349

21.5.1.1 Indicator amino acid oxidation


This approach is based on the principle that the oxidation of an indicator amino acid
decreases once amino acid requirements are met.97,98 Using this technique, breath and urine
are the only samples required following the consumption of diet with a varying amount
of the study amino acid and administration of the indicator amino acid tracer.97,99 The
indicator amino acid oxidation (IAOO) technique has been used to determine amino acid
requirements in healthy adults, neonates, children with liver transplant, and individuals
with metabolic disorders.97,99

21.5.1.2 The plasma amino acid response to an infusion of an amino acid mixture
This method has been utilized to identify the amino acids that limit protein synthesis in
HIV/AIDS.100 This method is based on the principle that if an amino acid is limiting, its
plasma concentration will not rise during an amino acid infusion, because of its use for
protein synthesis. By contrast, infusion of an amino acid that is already present at or above
required amounts will result in a steep rise in its plasma concentration. A similar approach
has been developed to manipulate parenteral amino acid formulation to meet the specific
needs of hospitalized patients in an intensive care unit.101 The linear regression of plasma
plateau concentrations of amino acids in response to an amino acid infusion was used to
determine which amino acids were oversupplied or undersupplied in each individual
patient.101 The administration of a parenteral amino acid formulation that corrected these
imbalances was then given for 5 days and resulted in improved nitrogen balance.101 The
identification of limiting amino acids in cancer is an important step in determining appro-
priate dietary supplements that will promote lean tissue gain; however, to date this has
not been accomplished in cancer patients.

References
1. Baracos, V.E., Management of muscle wasting in cancer-associated cachexia: understanding
from experimental studies, Cancer, 92, 1669–1677, 2001.
2. Le Bricon, T., Robinson, L., and Baracos, V.E., Modéles animaux pour les études métaboliques
et nutritionelles lors de la croissance tumorale, Nutr. Clin. Métabol., 15, 273–285, 2001.
3. Baracos, V.E. and Le Bricon, T., Animal models for nutrition in cancer, in Cancer and Nutrition:
Prevention and Treatment, Mason, J.B. and Nitenberg, G., Eds., Nestle Nutrition Workshop
Series Clinical and Performance Program Vol. 4, Karger AG, Basel, Switzerland, 2000,
p. 167–182.
4. Le Bricon, T., Cynober, L., Field, C.J., and Baracos, V.E., Supplemental nutrition with ornithine
a-ketoglutarate in rats with cancer-associated cachexia: surgical treatment of the tumor
improves efficacy of nutritional support, J. Nutr., 125, 2999–3010, 1995.
5. Shaw, J.H.F. and Wolfe, R.R., Glucose and urea kinetics in patients with early and advanced
gastrointestinal cancer: the response to glucose infusion, parenteral feeding, and surgical
resection, Surgery, 101, 181–191, 1987.
6. Leif-Halfwerk, S., Dagnelie, P.C., van den Berg, J.W.O., Wilson, J.H.P., and Sijens, P.E., Hepatic
sugar phosphate levels reflect gluconeogenesis in lung cancer: simultaneous turnover mea-
surements and 31P magnetic resonance spectroscopy in vivo, Clin. Sci., 98, 167–174, 2000.
7. Norton, J.A., Shamberger, R., Stein, T.P, Milne, G.W.A., and Brennan, M.F., The influence of
tumor-bearing on protein metabolism in the rat, J. Surg. Res., 30, 456–462, 1981.
8. Jeevanandam, M., Lowry, S.F., Horowitz, G.D., and Brennan, M.F., Cancer cachexia and
protein metabolism, Lancet, 8392, 1423–1426, 1984.
9. Fearon, K.C.H., Hansell, D.T., Preston, T., Plumb, J.A., Davies, J., Shapiro, D., Shenkin, A.,
Calman, K.C., and Burns, H.J.G., Influence of whole body protein turnover rate on resting
energy expenditure in patients with cancer, Cancer Res., 48, 2590–2595, 1988.
1382_C21.fm Page 350 Tuesday, October 7, 2003 6:54 PM

350 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

10. Emery, P.W., Lovell, L., and Rennie, M.J., Protein synthesis measured in vivo in muscle and
liver of cachectic tumor-bearing mice, Cancer Res., 44, 2779–2784, 1984.
11. Kawamura, I., Moldawer, L.L., Keenan, R.A., Batist, G., Bothe, A., Bistrian, B.R., and
Blackburn, G.L., Altered amino acid kinetics in rats with progressive tumor growth, Cancer
Res., 42, 824–829, 1982.
12. Lundholm, K., Bylund, A.C., Holm, J., and Schersten, T., Skeletal muscle metabolism in
patients with malignant tumor, Eur. J. Cancer, 12, 465–473, 1976.
13. Emery, P.W., Edwards, R.H.T., Rennie, M.J., Souhami, R.L., and Halliday, D., Protein synthesis
in muscle measured in vivo in cachectic patients with cancer, Br. Med. J., 289, 584–586, 1984.
14. Fearon, K.C.H., Falconer, J.S., Slater, C., McMillan, D.C., Ross, J.A., and Preston, T., Albumin
synthesis rates are not decreased in hypoalbuminemic cachectic cancer patients with an
ongoing acute-phase protein response, Ann. Surg., 227, 249–254, 1998.
15. Preston, T., Slater, C., McMillan, D.C., Falconer, J.S., Shenkin, A., and Fearon, K.C.H., Fibrin-
ogen synthesis is elevated in fasting cancer patients with an acute phase response, J. Nutr.,
128, 1355–1360, 1998.
16. Wigmore, S.J., McMahon, A.J., Sturgeon, C.M., and Fearon, K.C.H., Acute-phase response,
survival and tumour recurrence in patients with colorectal cancer, Br. J. Surg., 88, 255–260,
2001.
17. Falconer, J.S., Fearon, K.C.H., Ross, J.A., Elton, R., Wigmore, S.J., Garden, O.J., and Carter,
D.C., Acute-phase protein response and survival duration of patients with pancreatic cancer,
Cancer, 75, 2077–2082, 1995.
18. Barber, M.D., Fearon, K.C.H., and Ross, J.A., Relationship of serum levels of interleukin-6,
soluble interleukin-6 receptor and tumour necrosis factor receptors to the acute-phase re-
sponse in advanced pancreatic cancer, Clin. Sci., 96, 83–87, 1999.
19. McMillan, D.C., Scott, H.R., Watson, W.S., Preston, T., Milroy, R., and McArdle, C.S., Longi-
tudinal study of body cell mass depletion and the inflammatory response in cancer patients,
Nutr. Cancer, 31, 101–105, 1998.
20. Reeds, P.J., Fjeld, C.R., and Jahoor, F., Do the differences between the amino acid compositions
of acute phase and muscle proteins have a bearing on nitrogen loss in traumatic states?
J. Nutr., 124, 906–910, 1997.
21. Barker, W.C. and Putnam, F.W., Amino acid sequences of plasma proteins, in The Plasma
Proteins: Structure, Function, and Genetic Control, Putnam, F.W., Ed., Academic Press, New
York, 1984, p. 361–397.
22. Le Bricon, T., Gugins, S., Cynober, L., and Baracos, V.E., Negative impact of cancer chemo-
therapy on protein metabolism in healthy and tumor-bearing rats, Metabolism, 44, 1340–1348,
1995.
23. Barber, M.D., McMillan, D.C., Preston, T., Ross, J.A., and Fearon, K.C.H., Metabolic response
to feeding in weight-losing pancreatic cancer patients and its modulation by a fish-oil-
enriched nutritional supplement, Clin. Sci., 98, 389–399, 2000.
24. Hyltander, A., Daneryd, P., Sandstrom, R., Korner, U., and Lundholm, K., B-adrenoceptor
activity and resting energy metabolism in weight losing cancer patients, Eur. J. Cancer, 36,
330–334, 2000.
25. Tisdale, M.J., Cachexia in cancer patients, Nat. Rev. Cancer, 2, 862–871, 2002.
26. Todorov, P., Cariuk, P., McDevitt, T., Coles, B., Fearon, K., and Tisdale, M., Characterization
of a cancer cachectic factor, Nature, 379, 739–742, 1996.
27. Ross, J.A. and Fearon, K.C., Eicosanoid-dependent cancer cachexia and wasting, Curr. Opin.
Clin. Nutr. Metab. Care, 5, 241–248, 2002.
28. Ziegler, T.R., Glutamine supplementation in bone marrow transplantation, Br. J. Nutr., 87,
S9–S15, 2002.
29. Heys, S.D., Park, K.G., McNurlan, M.A., Calder, A.G., Buchon, V., Blessing, K., Eremin, O.,
and Garlick, P.J., Measurement of tumor protein synthesis in vivo in human colorectal and
breast cancer and its variability in separate biopsies from the same tumor, Clin. Sci., 80,
587–593, 1991.
30. Lazo, P.A., Amino acids and glucose utilization by different metabolic pathways in ascites-
tumor cells, Eur. J. Biochem., 17, 19–25, 1981.
1382_C21.fm Page 351 Tuesday, October 7, 2003 6:54 PM

Chapter twenty-one: Cancer-associated cachexia 351

31. Matsuno, T. and Hirai, H., Glutamine synthetase and glutaminase activities in various
hepatoma cells, Biochem. Int., 19, 219–225, 1989.
32. Sauer, L.A., Stayman, J.W., and Dauchy, R.T., Amino acid, glucose, and lactic acid utilization
in vivo by rat tumors, Cancer Res., 42, 4090–4097, 1982.
33. Chance, W.T., Cao, L., Kim, M.W., Nelson, J.L., and Fischer, J.E., Reduction of tumor growth
following treatment with glutamine antimetabolite, Life Sci., 42, 87–94, 1988.
34. Buffkin, D.C., Webber, M.M., Davidson, W.D., Bassist, L.F., and Verma, R.C., Ornithine as a
possible marker of cancer, Cancer Res., 38, 3225–3229, 1978.
35. Seiler, N., Knodgen, B., and Bartholeyns, J., Polyamine metabolism and polyamine excretion
in normal and tumor bearing rodents, Anticancer Res., 5, 371–378, 1985.
36. Marquez, J., Mates, J.M., Quesada, A.R., Medina, M.A., Nunez de Castro, I., and Sanchez-
Jimenez, F., Altered ornithine metabolism in tumor-bearing mice, Life Sci., 45, 1877–1884,
1989.
37. Hyltander, A., Lind, A.K., Yoshikawa, T., Sandstrom, R., and Lundholm, K., Increased urinary
polyamine excretion in unselected cancer patients related to host factors, Acta Oncol., 37,
91–96, 1998.
38. Durie, B.G.M., Salmon, S.E., and Russell, D.H., Polyamines as markers of response and
disease activity in cancer chemotherapy, Cancer Res., 37, 214–221, 1977.
39. Kenyon, S.H., Waterfield, C.J., Timbrell, J.A., and Nicolaou, A., Methionine synthase activity
and sulfur amino acid levels in the rat liver tumor cells HTC and Phi-1, Biochem. Pharmacol.,
63, 381–391, 2002.
40. Jensen, R.T. and Norton, J.A., Carcinoid tumors and carcinoid syndrome, in Cancer: Principles
and Practice of Oncology, 5th ed., Vol. 2, DeVita, V.T., Hellman, S., and Rosenberg, S.A., Eds.,
Lippincott-Raven, Philadelphia, 1997, pp. 1704–1723.
41. Breillout, F., Antoine, E., and Poupon, M.F., Methionine dependency of malignant tumors:
a possible approach for therapy, J. Natl. Cancer Inst., 82, 1628–1632, 1990.
42. Harvie, M.N., Campbell, I.T., Howell, A., and Thatcher, N., Acceptability and tolerance of a
low tyrosine and phenylalanine diet in patients with advanced cancer: a pilot study, J. Hum.
Nutr. Diet., 15, 193–202, 2002.
43. Chen, M.K., Salloum, R.M., Austgen, T.R., Blamd, J.B., Bland, K.I., Copeland, E.M., and
Souba, W.W., Tumor regulation of hepatic glutamine metabolism, J. Parenter. Enteral Nutr.,
15, 159–164, 1991.
44. Chen, M.K., Espat, N.J., Bland, K.I., Copeland, E.M., and Souba, W.W., Influence of progres-
sive tumor growth on glutamine metabolism in skeletal muscle and kidney, Ann. Surg., 217,
655–667, 1993.
45. Souba, W.W., Strebel, F.R., Bull, J.M., Copeland, E.M., Teagtmeyer, H., and Cleary, K., Inter-
organ glutamine metabolism in the tumor-bearing rat, J. Surg. Res., 44, 720–726, 1988.
46. Bennegard, K., Lindmark, L., Eden, E., Svaninger, G., and Lundholm, K., Flux of amino acids
across the leg in weight-losing cancer patients, Cancer Res., 44, 386–393, 1984.
47. Kubota, A., Meguid, M.M., and Hitch, D.C., Amino acid profiles correlate diagnostically with
organ site in three kinds of malignant tumors, Cancer, 69, 2343–2348, 1992.
48. De Blaauw, I., Heeneman, S., Deutz, N.E.P., and Von Meyenfeldt, M.F., Increased whole-body
protein and glutamine turnover in advanced cancer is not matched by an increased muscle
protein and glutamine turnover, J. Surg. Res., 68, 44–55, 1997.
49. Inoue, Y., Bode, B.P., Copeland, E.M., and Souba, W.W., Enhanced hepatic amino acid trans-
port in tumor-bearing rats is partially blocked by antibody to tumor necrosis factor, Cancer
Res., 55, 3525–3530, 1995.
50. Fischer, C.P., Bode, B.P., Hurley, B.P., and Souba, W.W., Alterations in oxidative metabolism
and glutamine transport support glucose production in the tumor-influenced hepatocyte,
J. Surg. Res., 69, 379–384, 1997.
51. Easson, A.M., Bode, B.P., Fischer, C.P., and Souba, W.W., Effects of endotoxin challenge on
hepatic amino acid transport during cancer, J. Surg. Res., 77, 29–34, 1998.
52. Easson, A.M., Pawlik, T.M., Fischer, C.P., Conroy, J.L., Sgroi, D., Souba, W.W., and Bode, B.P.,
Tumor-influenced amino acid transport activities in zonal-enriched hepatocyte populations,
Am. J. Physiol., 279, G1209–G1218, 2000.
1382_C21.fm Page 352 Tuesday, October 7, 2003 6:54 PM

352 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

53. Inoue, Y., Bode, B.P., and Souba, W.W., Dietary regulation of the hepatic system n glutamine
transporter in tumor-bearing rats, Am. J. Surg., 169, 173–178, 1995.
54. Norton, J.A., Gorschboth, C.M., Wesley, R.A., Burt, M.E., and Brennan, M.F., Fasting plasma
amino acid levels in cancer patients, Cancer, 56, 1181–1186, 1985.
55. Castillo, L., Chapman, T.E., Sanchez, M., Yu, Y.M., Burke, J.F., Ajami, A.M., Vogt, J., and
Young, V.R., Plasma arginine and citrulline kinetics in adults given adequate and arginine-
free diets, Proc. Natl. Acad. Sci. U.S.A., 90, 7749–7753, 1993.
56. De Blaauw, I., Deutz, N.E.P., Van Der Hulst, R.R.W.W., and Von Meyenfeldt, M.F., Glutamine
depletion and increased permeability in nonanorectic, non-weight-losing tumor-bearing rats,
Gastroenterology, 112, 118–126. 1997.
57. Moncada, S. and Higgs, E.A., Endogenous nitric oxide: physiology, pathology and clinical
relevance, Eur. J. Clin. Invest., 21, 361–374, 1991.
58. Bedard, S., Marcotte, B., and Marette, A., Cytokines modulate glucose transport in skeletal
muscle by inducing expression of inducible nitric oxide synthase, Biochem. J., 325, 487–493,
1997.
59. Kapur, S., Bedard, S., Marcotte, B., Cote, C.H., and Marette, A., Expression of nitric oxide
synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action,
Diabetes, 46, 1691–1700, 1997.
60. Buck, M. and Chojkier, M., Muscle wasting and dedifferentiation induced by oxidative stress
in murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and anti-
oxidants, EMBO J., 15, 1753–1765, 1996.
61. Hack, V., Breitkreutz, R., Kinscherf, R., Rohrer, H., Bartsch, P., Taut, F., Benner, A., and Droge,
W., The redox state as a correlate of senescence and wasting and as a target for therapeutic
intervention, Blood, 92, 59–67, 1998.
62. Lang, C.A., Mills, B.J., Mastropaolo, W., and Liu, M.C., Blood glutathione decreases in chronic
diseases, J. Lab. Clin. Med., 135, 402–405, 2000.
63. Grimble, R.F., Nutrition and cytokine action, Nutr. Res. Rev., 3, 193–210, 1990.
64. Hack, V., Gross, A., Kinscherf, R., Bockstette, M., Fiers, W., Berke, G., and Droge, W., Abnor-
mal glutathione and sulfate levels after interleukin 6 treatment and in tumor-induced cachex-
ia, FASEB J., 10, 1219–1226, 1996.
65. Ushmorov, A.J., Hack, V., and Droge, W., Differential reconstitution of mitochondrial respi-
ratory chain activity and plasma redox state by cysteine and ornithine in a model of cancer
cachexia, Cancer Res., 59, 3527–3534, 1999.
66. Holm, E., Hack, V., Tokus, M., Breitkreutz, R., Babylon, A., and Droge, W., Linkage between
postabsorptive amino acid release and glutamate uptake in skeletal muscle tissue of healthy
young subjects, cancer patients, and the elderly, J. Mol. Med., 75, 454–461, 1997.
67. Clarke, E.F., Lewis, A.M., and Waterhouse, C., Peripheral amino acid levels in patients with
cancer, Cancer, 42, 2909–2913, 1978.
68. Kurzer, M., Janiszewski, J., and Meguid, M.M., Amino acid profiles in tumor-bearing and
pair-fed nontumor-bearing malnourished rats, Cancer, 62, 1492–1496, 1988.
69. Nair, K.S., Schwartz, R.G., and Welle, S., Leucine as a regulator of whole body and skeletal
muscle protein metabolism in humans, Am. J. Physiol., 263, E928–E934, 1992.
70. Mitch, W.E. and Clark, A.S., Specificity of the effects of leucine and its metabolites on protein
degradation in skeletal muscle, Biochem. J., 222, 579–586, 1984.
71. Paxton, K., Ward, L.C., and Wilce, P.A., Amino acid oxidation in the tumor-bearing rat, Cancer
Biochem. Biophys., 9, 343–351, 1988.
72. Goodlad, G.A.J., Tee, M.K., and Clark, C.M., Leucine oxidation and protein degradation in
the extensor digitorum longus and soleus of the tumor-bearing host, Biochem. Med., 26,
143–147, 1981.
73. Argiles, J.M. and Lopez-Soriano, F.J., Oxidation of branched-chain amino acids in tumour-
bearing rats, Biochem. Soc. Trans., 17, 1044–1045, 1989.
74. Kawamura, I., Sato, H., Ogoshi, S., and Blackburn, G.L., Use of an intravenous branched
chain amino acid enriched diet in the tumor-bearing rat, Jpn. J. Surg., 15, 471–476, 1985.
1382_C21.fm Page 353 Tuesday, October 7, 2003 6:54 PM

Chapter twenty-one: Cancer-associated cachexia 353

75. Schaur, R.J., Semmelrock, H.-J., Schreibmayer, W., Tillian, H.M., and Schauenstein, E., Nitro-
gen metabolism in yoshida sarcoma-bearing rats: reduction of growth rate and increase of
survival time by administration of physiological doses of branched-chain amino acids,
J. Cancer Res. Clin. Oncol., 97, 285–293, 1980.
76. Kokolis, N. and Ziegler, I., On the levels of phenylalanine, tyrosine and tetrahydrobiopterin
in the blood of tumor-bearing organisms, Cancer Biochem. Biophys., 2, 79–85, 1977.
77. Naini, A.B., Dickerson, J.W.T., and Brown, M.M., Preoperative and postoperative levels of
plasma protein and amino acid in esophageal and lung cancer patients, Cancer, 62, 355–360,
1988.
78. Huang, A., Fuchs, D., Widner, B., Glover, C., Henderson, D.C., and Allen-Mersh, T.G., Serum
tryptophan decrease correlates with immune activation and impaired quality of life in
colorectal cancer, Br. J. Cancer, 86, 1691–1696. 2002.
79. Cascino, A., Cangiano, C., Ceci, F., Franchi, F., Mineo, T., Mulieri, M., Muscaritoli, M., and
Rossi-Fanelli, F., Increased plasma free tryptophan levels in human cancer: a tumor related
effect? Anticancer Res., 11, 1313–1316, 1991.
80. Rossi-Fanelli, F., Cangiano, C., Ceci, F., Cellerino, R., Franchi, F., Menichetti, E.T., Muscaritoli,
M., and Cascino, A., Plasma tryptophan and anorexia in human cancer, Eur. J. Clin. Oncol.,
22, 89–95, 1986.
81. Murr, C., Widner, B., Sperner-Unterweger, B., Ledochowski, M., Schubert, C., and Fuchs, D.,
Immune reaction links disease progression in cancer patients with depression, Med. Hypoth.,
55, 137–140, 2000.
82. Meguid, M.M., Muscaritolo, M., Beverly, L., Yang, Z.J., Cangiano, C., and Rossi-Fanelli, F.,
The early cancer anorexia paradigm: changes in plasma free tryptophan and feeding indexes,
J. Parenter. Enteral Nutr., 16, 56S–59S, 1992.
83. Elango, R., Pencharz, P.B., and Ball, R.O., The branched-chain amino acid requirement of
parenterally fed neonatal piglets is less than the enteral requirement, J. Nutr., 132, 3123–3129,
2002.
84. Brunton, J.A., Bertolo, R.F.P., Pencharz, P.B., and Ball, R.O., Proline ameliorates arginine
deficiency during enteral but not parenteral feeding in neonatal piglets, Am. J. Physiol., 277,
E223–E231, 1999.
85. Kaibara, A., Yoshida, S., Yamasaki, K., Ishibashi, N., and Kakegawa, T., Effect of glutamine
and chemotherapy on protein metabolism in tumor-bearing rats, J. Surg. Res., 57, 143–149,
1994.
86. Austgen, T.R., Dudrick, P.S., Sitren, H., Bland, K.I., Copeland, E., and Souba, W.W., The effects
of glutamine-enriched total parenteral nutrition on tumor growth and host tissues, Ann.
Surg., 215, 107–113, 1992.
87. Yoshida, S., Kaibara, A., Yamasaki, K., Ishibashi, N., Noake, T., and Kakegawa, T., Effect of
glutamine supplementation on protein metabolism and glutathione in tumor-bearing rats,
J. Parenter. Enteral Nutr., 10, 492–497, 1995.
88. Grossie, V.B., Citrulline and arginine increase the growth of the ward colon tumor in parenter-
ally fed rats, Nutr. Cancer, 26, 91–97, 1996.
89. Grossie, V.B., Nishioka, K., Ajani, J.A., and Ota, D.M., Substituting ornithine for arginine in
total parenteral nutrition eliminates enhanced tumor growth, J. Surg. Oncol., 50, 161–167,
1992.
90. Parks, K.G., Heys, S.D., Blessing, K., Kelly, P., McNurlan, M.A., Eremin, O., and Garlick, P.J.,
Stimulation of human breast cancers by dietary L-arginine, Clin. Sci., 82, 413–417, 1992.
91. Caso, G., Matar, S., McNurlan, M., McMillan, D., Eremin, O., and Garlick, P., Metabolic effects
of arginine on malignant tissues, Clin. Nutr., 15, 89–90, 1996.
92. Oka, T., Ohwada, K., Nagao, M., Kitazato, K., and Kishino, Y., Arginine-enriched solution
induces a marked increase in muscle glutamine concentration and enhances muscle protein
synthesis in tumor-bearing rats, J. Parenter. Enteral Nutr., 18, 491–496, 1994.
93. Oka, T., Ohwada, K., Nagao, M., and Kitazato, K., Effect of arginine-enriched total parenteral
nutrition on the host-tumor interaction in cancer-bearing rats, J. Parenter. Enteral Nutr., 17,
375–383, 1993.
1382_C21.fm Page 354 Tuesday, October 7, 2003 6:54 PM

354 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

94. Ye, S.-L., Istfan, N.W., Driscoll, D.F., and Bistrian, B.R., Tumor and host response to arginine
and branched chain amino acid-enriched total parenteral nutrition, Cancer, 69, 261–270, 1992.
95. Robinson, L.E., Bussière, F.I., Le Boucher, J., Farges, M.C., Cynober, L.A., Field, C.J., and
Baracos, V.E., Amino acid nutrition and immune function in tumour-bearing rats: a compar-
ison of glutamine-, arginine- and ornithine 2-oxoglutarate-supplemented diets, Clin. Sci., 97,
657–669, 1999.
96. May, P.E., Barber, A., D’Olimpio, J.T., Hourihane, A., and Abumrad, N.N., Reversal of cancer-
related wasting using oral supplementation with combination of b-hydroxy-b-methyl-
butyrate, arginine and glutamine, Am. J. Surg., 183, 471–479, 2002.
97. Brunton, J.A., Ball, R.O., and Pencharz, P.B., Determination of amino acid requirements by
indicator amino acid oxidation: applications in health and disease, Curr. Opin. Clin. Nutr.
Metab. Care, 1, 449–453, 1998.
98. Zello, G.A., Wykes, L.J., Ball, R.O., and Pencharz, P.B., Recent advances in methods of
assessing dietary amino acid requirements for adult humans, J. Nutr., 125, 2907–2915, 1995.
99. Bross, R., Ball, R.O., and Pencharz, P.B., Development of a minimally invasive protocol for
the determination of pheylalanine and lysine kinetics in humans during the fed state, J. Nutr.,
128, 1913–1919, 1998.
100. Laurichesse, H., Tauveron, I., Gourdon, F., Cormerais, L., Champredon, C., Charrier, S.,
Rochon, C., Lamain, S., Bayle, G., Laveran, H., Thieblot, P., Beytout, J., and Grizard, J.,
Threonine and methionine are limiting amino acids for protein synthesis in patients with
AIDS, J. Nutr., 128, 1342–1348, 1998.
101. Berard, M.P., Pelletier, A., Ollivier, J.M., Gentil, B., and Cynober, L., Qualitative manipulation
of amino acid supply during total parenteral nutrition in surgical patients, J. Parenter. Enteral
Nutr., 26, 136–143, 2002.
1382_C22.fm Page 355 Tuesday, October 7, 2003 6:55 PM

chapter twenty-two

Diabetes mellitus
Ketan Dhatariya
Mayo Clinic and Foundation
K. Sreekumaran Nair
Mayo Clinic and Foundation

Contents
Introduction..................................................................................................................................355
22.1 Mechanisms of insulin action on protein metabolism ................................................356
22.1.1 Amino acid transport into skeletal muscle .....................................................356
22.1.2 Protein synthesis ..................................................................................................357
22.1.3 Protein breakdown ..............................................................................................358
22.2 Diabetes mellitus ...............................................................................................................358
22.2.1 In vivo studies: animal models ...........................................................................358
22.2.2 Type 1 diabetes......................................................................................................359
22.2.2.1 Amino acid metabolism during insulin deficiency .......................359
22.2.2.2 Amino acid metabolism during insulin therapy............................362
22.2.2.3 Regional differences in amino acid metabolism ............................364
22.2.3 Type 2 diabetes......................................................................................................364
22.2.4 The effects of other hormones on protein turnover in diabetes...................367
22.3 Newer technologies...........................................................................................................367
22.4 Conclusions and summary ..............................................................................................368
References .....................................................................................................................................368

Introduction
Diabetes mellitus is becoming an increasingly common metabolic disorder.1 The condition
is characterized by impaired glucose metabolism manifested by hyperglycemia. Diabetes
also affects fat and protein metabolism. Studies in humans have demonstrated that effects
on protein and amino acid metabolism differ in subjects with type 1 and type 2 diabetes.
In addition, the effects on protein metabolism in these conditions differ in humans from
the effects seen in vitro and in animal models of type 1 and type 2 diabetes.

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 355
1382_C22.fm Page 356 Tuesday, October 7, 2003 6:55 PM

356 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

In people with type 1 diabetes, cachexia and muscle wasting are completely prevented
by insulin replacement. No such dramatic changes in body composition are noted in
people with type 2 diabetes. Circulating amino acid levels, amino acid kinetics, protein
synthesis, and breakdown are different between people with type 1 diabetes and those
with type 2 diabetes. The current review will focus on the alterations in amino acid and
protein metabolism that occur in people with type 1 and type 2 diabetes.

22.1 Mechanisms of insulin action on protein metabolism


Insulin plays a pivotal role in the regulation of amino acid and protein metabolism in
health.2 In individuals without diabetes, plasma insulin concentration varies from 2 to
12 mU/l in the fasting state, to 30 to 100 mU/l in the postprandial state. In common with
many other hormones, insulin acts on the cells after binding to a specific receptor. The
receptor is a tetrameric glycoprotein, consisting of 2 a-subunits that anchor the receptor
to the cell membrane, and two intracellular b-subunits, to which tyrosine kinase domains
are attached. The a- and b-subunits are linked by sulfydryl bonds. Insulin binding causes
a conformational change to the receptor, leading to autophosphorylation of the tyrosine
residues. Other amino acid residues on the receptor, such as threonine and serine, may
also become phosphorylated.
Liver, fat, and skeletal muscle are the tissues in which insulin has its main effects. The
actions of insulin in the liver include changes in hepatic gluconeogenesis by its effect on
key enzymes such as phosphoenolpyruvate carboxykinase (PEPCK)3 and glucokinase.4
Within the nucleus, insulin acts on a number of genes via the insulin response element.
Insulin also acts on several pathways, possibly through its stimulatory effect on the insulin
receptor substrate 1 and 2 genes. These genes produce intracellular signaling proteins that
trigger the cascade that is responsible for many of the actions of insulin.5 In type 2 diabetes,
the chronic hyperinsulinemia leads to a downregulation of insulin receptor substrate 2
gene transcription, thus leading to abnormalities in many intracellular processes, including
a failure to suppress hepatic gluconeogenesis.6

22.1.1 Amino acid transport into skeletal muscle


In the postabsorptive state, intracellular transport of amino acids into skeletal muscle is
well below the maximum capacity of the transport systems. However, optimal amino acid
transport across the blood–brain barrier takes place at concentrations similar to those
found during fasting. This implies that when plasma amino acid concentrations rise after
a meal, transport of amino acids into skeletal muscle can increase relative to transport
across the blood–brain barrier. This allows for greater intracellular amino acid availability
for protein synthesis in skeletal muscle.7,8 There are several different amino acid transport
mechanisms, of which four — A, ASC, Nm, and Xc — are insulin responsive.7–9 These four
mechanisms are primarily responsible for the transport of nonessential amino acids. How-
ever, the mechanism responsible for the transport of some of the large neutral branched-
chain amino acids, L, is not sensitive to insulin. Insulin also effects amino acid transport
in several tissues, including cardiac and skeletal muscle, liver, and adipose tissue.10–12
Insulin has most of its effects on intracellular amino acid metabolism in a dose-
dependent manner. This occurs by altering cell surface receptor binding. This change
in binding inhibits the inactivation of amino acid uptake by the sodium- and pH-
dependent small neutral amino acid transport mechanism known as system A. Although
these transport systems are important in regulating the intracellular concentrations of
amino acids, no evidence is currently available to show that they limit protein synthesis
in any way.
1382_C22.fm Page 357 Tuesday, October 7, 2003 6:55 PM

Chapter twenty-two: Diabetes mellitus 357

Table 22.1 Mechanisms By Which Insulin Has Effects on Protein Synthesis

Total Cellular Protein Synthesis Can Be Enhanced By:


Stimulation of rRNA transcription
Increased translation of ribosomal protein mRNA with selective translation of previously
transcribed mRNA
Promotion of nuclear mRNA release
Increasing association of polysomes with cytoskeleton
Regulation of initiation and elongation of mRNA translation

Specific Proteins Can Be Affected By:


Specific gene transcription can be either up- or down-regulated by altering the activity of a variety
of transcription factors that are dependent on the hormone response element on the 5¢ region of
the gene
Increased association and enhanced efficiency of translation of specific mRNA with polysomes
Changes in the stability of specific mRNA molecules

Adapted from Kimball, S.R. et al., Annu. Rev. Physiol., 56, 348, 1994.

22.1.2 Protein synthesis


Insulin plays a major regulatory role in vivo protein synthesis in human and animals;
however, the precise role that insulin plays at various stages of regulation protein synthesis
and the interactions of insulin with other factors remain to be clearly defined. Stimulation
of protein synthesis in various in vitro cell lines and tissue models has been clearly
documented.3,13–17 These researchers reported divergent findings, largely related to meth-
odological limitations. For example, it is hard to demonstrate insulin effects separate from
the changes in concentrations of substrates such as amino acids, glucose, and free fatty
acids, or other hormones such as glucagon, cortisol, growth hormone, etc. Recent advances
in molecular techniques allow a better understanding of insulin’s specific effects at differ-
ent levels of protein synthesis.
Insulin has been shown to be a regulator of gene expression by altering mRNA
production, maintenance of mRNA stability, ribosomal biogenesis, imitation and elonga-
tion of mRNA translation, and regulation of preexisting enzymes.3,16 Table 22.1 summa-
rizes some of the steps where insulin has been shown to act.
The effect of insulin on gene transcription is thought to be based on intracellular trans-
acting factors bound to cis-acting DNA sites in the nucleus. The specific gene sites affected
by insulin are referred to as insulin response sequences or elements (IRSs/IREs) located
in the gene promoter region. Specific IREs such as glyceryldehydro-3-phosphate dehydro-
genase, pyruvate kinase, glucagon, PEPCK, and insulin-like growth factor protein-1 have
been identified.3 Insulin has been shown to selectively increase mRNA of eukaryotic elon-
gation factor-2 (eEF-2),18 phosphorylated heat-acid stable protein (PHAS-1),19 and myosin
heavy chain alpha.20 Recent studies using the gene array approach have demonstrated that
insulin down-regulates and up-regulates expression of several genes in human muscle.21
Insulin has been shown to reduce the degradation of transcripts (mRNA) of glyceryl-
3-glycogen phosphate dehydrogenase, glycogen phosphorylase, and GLUT-1.3,22 These are
examples of the role of insulin in stabilizing mRNAs. In contrast, there are examples of
insulin destabilizing mRNAs of PEPCK and GLUT-4.23,24 These data show that insulin can
stabilize some mRNA while destabilizing others within the same cell. These animal and
in vitro results are intriguing, as they show insulin’s divergent effects on the transcription
of many genes in skeletal muscle. Work has recently been done looking at these differences
in gene expression in humans.21
1382_C22.fm Page 358 Tuesday, October 7, 2003 6:55 PM

358 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

The mechanism of insulin’s effect on translation of messages has been extensively


reviewed.16,25,26 Insulin has been demonstrated to regulate translation initiation, in part,
by enhancing mRNA binding to the 40S ribosomal subunit. In this process, a key step is
the binding of the eIF-4E (cap binding protein) and eIF-4E binding protein (PHAS-I or
4E-BP1) and eIF-4G to the 7-methylguanosine 5'-triphosphate cap structure at the 5¢ end
of eukaryote mRNAs.15,27–29 Insulin enhances PHAS-1 action by dissociating it from eIF-4E,
so that eIF-4E forms a complex with eIF-4G that associates with the 40S ribosomal sub-
unit.27 Insulin deficiency has been shown to decrease eIF-4E binding to eIF-4G and increase
eIF-4E-4E-BP1 complex formation in skeletal muscle.30
Insulin selectively up-regulates translation initiation of mRNAs containing polypyri-
midine at the 5' transcriptional start site. These mRNAs encode important components of
translational elongation factors that would increase overall capacity for protein synthesis.
Insulin appears to promote elongation by inactivation of eukaryotic elongation factor-2
kinase through pathways sensitive to the antifungal agent rapamycin.31
Insulin thus promotes important steps in translation initiation and elongation. In
addition, insulin promotes the capacity of cells to synthesize proteins by increasing syn-
thesis of ribosomal proteins.32 The synthesis of mRNA species has also been shown to be
stimulated by insulin as well as the maintenance of ribosomal numbers.32–35 Insulin also
may diminish the rate of ribosomal degradation.16

22.1.3 Protein breakdown


The regulation of cellular protein breakdown is a highly complex process and is variable
in different tissues. The available information on insulin control of protein breakdown is
minimal. Insulin is known to influence cellular protein breakdown via several mecha-
nisms, including lysosomal pathways, ATP-dependent ubiquitin–proteosome pathway,
calcium-dependent proteosomes, and ATP-independent pathways. Approximately 10 to
20% of protein breakdown occurs via lysosomal pathways, which are primarily responsible
for degrading extracellular membrane and organellar proteins. The majority of intracel-
lular proteins are degraded by the ubiquitin–proteosome pathway. The ubiquitin–proteo-
some pathway is involved in accelerated protein breakdown in animal models of diabe-
tes.36 Inhibition of ATP synthesis reduces the increased muscle protein breakdown during
insulin deficiency. Selective inhibition of lysosomal functions or calcium-dependent pro-
teases does not result in reduction of protein breakdown during insulin deficiency. How-
ever, involvement of both ubiquitin–proteosome and calcium-dependent pathways is also
reported in streptozotocin-treated rats.37 Studies also have shown that adrenalectomy
prevented the increased muscle protein breakdown in streptozotocin-induced rats, with
muscle protein breakdown being restored with subsequent glucocorticoid administra-
tion.38 It remains to be established whether insulin has any direct control on the ubiq-
uitin–proteosome pathway and the underlying mechanism.

22.2 Diabetes mellitus


22.2.1 In vivo studies: animal models
In the rodent diabetic model it has been shown that protein synthesis is depressed in many
tissues.39,40 The effects of insulin deprivation on muscle protein breakdown in these animal
models were determined indirectly, as there is evidence for an increase in whole-body
leucine oxidation in streptozotocin-treated rats.41 The loss of muscle, disproportionate to
the decrease in synthesis, is assumed to be due to increased breakdown. Even within
1382_C22.fm Page 359 Tuesday, October 7, 2003 6:55 PM

Chapter twenty-two: Diabetes mellitus 359

muscle, the rates of protein degradation vary, with the relative preservation of muscle
with high oxidative capacity, such as the soleus.42
In rats, starvation has similar effects on muscle protein metabolism as insulin depri-
vation. There is loss of muscle protein and a depression in muscle protein synthesis. In
studies looking at the effects of refeeding after a period of prolonged starvation, muscle
protein synthesis is rapidly restored, with the rate of response proportional to the length
of starvation.43,44 As insulin can mimic this effect by stimulating protein synthesis in fasted
animals, it is thought that insulin may also be responsible for the increase in protein
synthesis seen in the fed state.45 Two studies support this hypothesis. The use of anti-
insulin antibodies has been shown to prevent the protein synthesis seen with insulin
infusions.46 In addition, it was demonstrated that the infusion of supraphysiological doses
of insulin in fed rats does not increase protein synthesis further, suggesting a plateau to
the dose–response relationship between insulin levels and protein synthesis rates.47,48
However, the addition of amino acids alters these variables, allowing maximal protein
synthesis rates to occur at lower insulin concentrations. This suggests that amino acids
increase the sensitivity of muscle protein synthesis to insulin.48 The infusion of amino
acids without insulin did not increase protein synthesis rates, showing that both insulin
and amino acids are necessary for synthesis to occur.46,48 This is the situation in nondia-
betics during feeding — both amino acid concentrations and insulin levels increase,
providing that there are optimal conditions for protein synthesis. Further work by Garlick
and Grant49 showed that it was only the branched-chain amino acids, with leucine in
particular, that stimulated protein synthesis in the presence of insulin.
Aging also has an effect on protein synthesis rates. There is a divergent effect of
diabetes on protein synthesis in growing rats vs. old rats. Later studies have shown that
in adult rats the response to insulin is altered, suggesting that age is a determinant in
insulin action on muscle protein synthesis.50 These results are more consistent with those
from humans. Studies performed in piglets demonstrated that there is an age-related
alteration of stimulation of muscle protein synthesis in response to insulin.51 These studies
clearly indicated that age, or stage of development, is important in determining the effect
of insulin on muscle protein synthesis.
Further work has suggested that the effects of insulin are tissue specific, as illustrated
in Figure 22.1A and B. Work in pigs has shown that insulin has a regulatory role in
mitochondrial protein synthesis.52 This effect was most pronounced in skeletal muscle and
was not found in other highly active tissues, such as liver or heart.52 This is shown in
Figure 22.1A. These studies also have shown that insulin has no effect on liver tissue
protein synthesis, whereas Figure 22.1B shows that insulin has an inhibitory effect on
fractional synthesis rates of fibrinogen and albumin.53 The same study indicated that
insulin administered at a high physiological level failed to alter the rate of liver tissue
protein synthesis.53 However, there are differences in protein synthesis rates between tissue
beds and also within specific tissues.54
There is some support for the hypotheses that tissue protein synthesis may be driven
by the high intracellular amino acid concentrations as a stimulus. There has been recent
work showing that the provision of leucine in postabsorbative rats had a stimulant effect
on skeletal muscle protein synthesis.55,56

22.2.2 Type 1 diabetes


22.2.2.1 Amino acid metabolism during insulin deficiency
Unlike in animal models, the stimulatory effect of insulin on protein synthesis has been
more difficult to demonstrate in humans. The effects of insulin withdrawal and restoration
on whole-body protein balance were originally studied in humans in the early 1930s by
1382_C22.fm Page 360 Tuesday, October 7, 2003 6:55 PM

360 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Skeletal muscle mitochondrial protein FSR


2.0 *
1.8
1.6 **
1.4
1.2

(%/h)
1.0
0.8
0.6
0.4
0.2
0.0
Control Insulin Insulin + amino acids
Liver mitochondrial protein FSR (%/h)

1.2

1
#
0.8

0.6

0.4

0.2

0
Control Insulin Insulin + amino acids
Heart mitochondrial protein FSR (%/h)

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0
(A) Control Insulin Insulin + amino acids

Figure 22.1 (A) Skeletal muscle, liver, and heart mitochondrial protein fractional synthesis rates
using [13C]leucine. The upper panel represents skeletal muscle. *, significant difference between the
control group and the insulin group (p < 0.04). **, significant difference between the control group
and the insulin with amino acid group (p = 0.05). The middle panel represents liver mitochondrial
protein fractional synthesis rates. #, significant difference between the control group and the insulin
group (p < 0.02). There was a nonsignificant difference between the control group and the insulin
with amino acid group (p < 0.08). The lower panel represents heart mitochondrial protein fraction
synthesis rates. There was no significant difference between the control group and the insulin group,
nor was there any significant difference between the control group and the insulin with amino acid
group (p > 0.1 for both). (Data from Boirie, Y. et al., Diabetes, 50, 2652, 2001.)

Atchley et al.57 This is illustrated in Figure 22.2. It has been known for some time that
stable insulin doses allow the maintenance of neutral nitrogen balance; however, on
withdrawal of insulin, nitrogen balance quickly becomes negative, indicating rapid net
protein breakdown.57 Restarting insulin therapy restored a neutral nitrogen balance within
1382_C22.fm Page 361 Tuesday, October 7, 2003 6:55 PM

Chapter twenty-two: Diabetes mellitus 361

25

Liver tissue FSR (%/h)


20

15

10

0
Control Insulin Insulin + amino acids

18
16
Albumin FSR (%/h)

14
12
10 *
8
6
4
2
0
Control Insulin Insulin + amino acids

90
80
Fibrinogen FSR (%/h )

70
*
60
*
50
40
30
20
10
0
(B) Control Insulin Insulin + amino acids

Figure 22.1 (continued) (B) Liver tissue, albumin, and fibrinogen fractional synthesis rate in a swine
model using leucyl-tRNA as the precursor. The upper panel shows that total liver protein synthesis
is not significantly different under control conditions or when insulin alone or with amino acids is
infused. The middle panel shows the albumin fractional synthesis rate. This shows that, unlike liver,
albumin synthesis is significantly decreased by insulin infusion but restored if amino acids are added
(p < 0.05). The lower panel shows fibrinogen fractional synthesis rate. This differs from the effects
on albumin. It can be seen that an insulin infusion significantly reduces the fibrinogen fractional
synthesis rate, but that amino acid infusion does not restore this (p < 0.05 for both). (Data from
Ahlman, B. et al., Diabetes, 50, 947, 2001.)

48 h. This is also illustrated in Figure 22.2. The changes associated with protein catabolism
occur within hours of changing the insulin status. In humans, as with animals, the post-
absorptive, insulin-deprived state is associated with a rise in plasma amino acid levels in
type 1 diabetic patients.58 These levels fall with the reintroduction of insulin.59–61 It is the
catabolism of these increased levels of circulating amino acids that accounts for higher
nitrogen excretion.
In contrast to animal models, where insulin stimulates protein synthesis as well as
preventing protein breakdown, human studies using isotopically labeled leucine, phenyl-
alanine, and tyrosine have shown that insulin deprivation in subjects with type 1 diabetes
leads predominantly to an increase in whole-body protein breakdown.60,62–65 Although
1382_C22.fm Page 362 Tuesday, October 7, 2003 6:55 PM

362 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Nitrogen balance (grams per 24 hours)


Insulin replete Insulin deprived Insulin restarted
2
1
0
-1
-2
-3
-4
-5
-6
-7
1 2 3 4 5
Time (days)

Figure 22.2 Effects of insulin deprivation on whole-body nitrogen balance. (Data from Atchley, D.W.
et al., J. Clin. Invest., 12, 297, 1933.)

there is an associated increase in protein synthesis during insulin deprivation, as measured


by a high nonoxidative leucine disposal rate, the rate of breakdown far exceeds that of
synthesis, resulting in net protein loss.60–68
With the addition of amino acids to insulin, studies of healthy volunteers showed that
there are more reductions in protein breakdown and increases in whole-body protein
synthesis rates than with insulin alone.69,70 These results have been reproduced in subjects
with type 1 diabetes.63,71 This suggests that the decline in protein synthesis seen with
insulin infusions in early studies was due to the lack of substrate availability.
These acute and subacute changes seen in protein turnover with underinsulinization
can lead to changes in body weight, such that children with poorly controlled type 1
diabetes may present with growth retardation, despite normal growth hormone levels.72

22.2.2.2 Amino acid metabolism during insulin therapy


Previous work by Luzi et al.63 showed that insulin had a differential effect on type 1
diabetes with respect to glucose and amino acid metabolism. The responses of leucine
flux, leucine oxidation, and nonoxidative disposal in response to insulin infusion were
similar in both diabetic and nondiabetic subjects. However, glucose uptake remained
initially suppressed by over 50%, suggesting differences in cellular response of glucose to
insulin.63 The baseline increase in leucine kinetics in poorly controlled type 1 diabetic
patients was normalized by 1 to 2 months of intensive insulin treatment.
Small changes in plasma insulin levels, e.g., from 20 to 70 pmol/l, can reduce the rate
of skeletal muscle protein breakdown by up to 40%.65,73
Although intensified insulin regimens may normalize glycemic control, the abnormal-
ities in leucine metabolism may take hours or days to resolve.64,73 This delay may be due
to the effects of insulin on inhibiting enzymes such as branched-chain keto acid dehydro-
genase, which is the rate-limiting enzyme for the catabolism for branched-chain amino
acids, which determine the rate of leucine oxidation in the insulin-deprived state. Alter-
natively, the delay may be due to the time taken to restore levels of counterregulatory
hormones once euglycemia is achieved. However, these effects may be additive, because
glucagon activates branched-chain keto acid dehydrogenase, and levels of this hormone
rise in absolute insulin deficiency.74
Studies in type 1 diabetic patients and in nondiabetic people have shown that insulin’s
main effect on leucine oxidation is at the level of leucine transamination.65 During insulin
1382_C22.fm Page 363 Tuesday, October 7, 2003 6:55 PM

Chapter twenty-two: Diabetes mellitus 363

Table 22.2 Percent Change in Amino Acid Balance Comparing Insulin Deprivation with
Insulin Treatment across the Leg and Splanchnic Beds
Across leg Across splanchnic bed
Aspartate 187.5 a –49.5d
Glutamate 37.2d –39.4d
Serine –192.9c 109.6c
Glutamine –70.7d 111.0d
Histidine –91.2a 129.8b
Glycine –64.7a 359.9a
Theronine –84.0c 145.4c
Alanine –58.8c 75.8a
Tyrosine –74.9a 30.2b
Valine –79.0d 108.3d
Methionine –63.1a 93.5a
Phenylalanine –83.4b 194.5b
Isoleucine –93.1a –174.2d
Leucine –79.0a 937.0b
Lysine –89.9c 369.9b
a-Ketoisocaproic acid –83.0b –2000.0b

Note: A negative number signifies a reduction in arteriovenous differences with insulin treatment,
consistent with a decrease in amino acid output, i.e., a net decrease in protein breakdown.
a p < 0.05.
b p < 0.01.
c p < 0.001.
d Not significant.
Source: Data from Nair, K.S. et al., J. Clin. Invest., 95, 2926, 1995.

deficiency leucine transamination increases severalfold and accounts for the accelerated
decarboxylation (oxidation) of leucine.
In the fed state, protein metabolism reflects the degree of insulinization and amino
acid availability. In subjects without diabetes, approximately 50% of ingested amino acids
are taken up and metabolized by the splanchnic bed after a meal. The branched-chain
amino acids, leucine, isoleucine, and valine, are not preferentially taken up by the liver,
leading to a greater rise in plasma levels with respect to other amino acids. These branched-
chain amino acids represent the major form of nitrogen transport between the gut and
the skeletal muscle after a meal. In underinsulinized subjects, this response is exaggerated,
with the liver failing to take up the majority of amino acids, leading to an approximately
50% rise in systemic plasma levels compared with either adequately insulinized subjects
or healthy controls.75 How insulin status alters plasma amino acid levels across the
splanchnic bed and across the leg is shown in Table 22.2.
There are other differences in the types of amino acid found in the plasma of subjects
with poorly controlled type 1 diabetes. Within the circulation, the ratio of gluconeogenic
amino acid to branched-chain amino acid changes with poorly controlled type 1 diabetes,
possibly due to increased hepatic or renal uptake to maintain the high rate of gluconeo-
genesis.75,76
In humans, the results suggesting that intracellular amino acid levels contribute to
muscle protein synthesis rates have been conflicting. They have shown that although
amino acid administration has an effect on decreasing protein breakdown and increasing
leucine oxidation,77–79 the effects on whole-body nonoxidative leucine disposal, i.e., protein
synthesis, have not been consistent.63,71,80–82 This issue remains to be fully clarified.
1382_C22.fm Page 364 Tuesday, October 7, 2003 6:55 PM

364 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

There have been several studies in healthy individuals to suggest that there is a decline
in whole-body protein breakdown with insulin.67,83–85 These findings have also been
reported in subjects with type 1 diabetes.65,86,87 Taken together, these studies suggest that
insulin inhibits skeletal muscle protein breakdown.

22.2.2.3 Regional differences in amino acid metabolism


Whole-body studies do not allow assessments to be made of the relative contribution
made to protein synthesis and breakdown by individual tissue beds. Regional differences
in protein flux have been demonstrated, based on isotopic and amino acid balance studies
performed in people with type 1 diabetes. There is a differential response of protein
dynamics in splanchnic and muscle beds.65,88 During the postabsorptive state, in both
nondiabetic people and patients with type 1 diabetes (insulin deprived), muscle protein
breakdown exceeds that of muscle protein synthesis. The difference between synthesis
and breakdown is greater in type 1 diabetic patients during insulin deprivation than in
nondiabetic people. Work looking at protein synthesis in mucosal tissue of the small
intestine showed that in the insulin-deprived state there was a 30% decline in protein
synthesis compared with the insulin-replete state.89 This is in contrast to an 82% increase
in protein synthesis across the whole of the splanchnic bed with insulin deprivation in
subjects with type 1 diabetes.65 Other work has shown that in vitro and in vivo the absence
of insulin led to a decline in albumin synthesis,16,90 with the restoration of albumin mRNA
expression being reliant in a dose-dependent fashion on the amount of insulin present.
At the same time, restoring insulin levels in insulin-deprived subjects with type 1 diabetes
can normalize fibrinogen levels.91
Muscle protein breakdown decreased in subjects with type 1 diabetes and in nondi-
abetic subjects with insulin treatment.65,83,88 In both groups there was minimal effect on
muscle protein synthesis. In contrast, splanchnic protein synthesis and breakdown
decreased with insulin in people with type 1 diabetes.65 This decrease in splanchnic protein
synthesis can explain the entire change that occurred in the whole-body protein synthesis
in people with type 1 diabetes. Although insulin did not stimulate muscle protein synthesis
in these experiments, the relative contribution of muscle protein synthesis rate to whole-
body protein synthesis increased significantly with insulin.92 These changes are illustrated
in Figure 22.3A and B. Another example of this differential effect includes myosin heavy
chain93 as well as mitochondrial and sarcoplasmic proteins found in skeletal muscle.52 In
type 1 diabetes, insulin deprivation in the short term had no effect on muscle myosin
heavy chain synthesis rates. Although the effects of insulin on the synthesis rates of
individual proteins have not been studied, studies have been conducted on the effects of
insulin in different tissues.92
Although the synthesis rates of individual proteins are measurable, there are no data
on the breakdown rates or of the effect insulin has on them, because there are no methods
currently available for such measurements. Although studies have been done to help
confirm the differential effects that insulin and amino acids have on the synthesis rates of
individual proteins, how this differential effect is regulated has yet to be elucidated. It is
possible that part of the differences seen with regional protein turnover may be due to
the differential effects of insulin on protein subfractions within a tissue bed. Examples of
this include the decrease in albumin synthesis with a simultaneous rise in fibrinogen
synthesis, as well as the effects on apolipoprotein B 100 and antithrombin III.94

22.2.3 Type 2 diabetes


Type 2 diabetes, formerly known as either maturity onset diabetes or non-insulin-depen-
dent diabetes, constitutes approximately 85% of all cases of diabetes. It is most commonly
1382_C22.fm Page 365 Tuesday, October 7, 2003 6:55 PM

Chapter twenty-two: Diabetes mellitus 365

14 P = NS P = < 0.05

12

10
micromol/kg/h 8

0
Off insulin On insulin Off insulin On insulin
skeletal muscle skeletal muscle
Protein synthesis Protein breakdown
(A)

P = < 0.01 P = < 0.05


45
40
35
micromol/kg/h

30
25
20
15
10
5
0
Off insulin On insulin Off insulin On insulin

splanchnic bed splanchnic bed

(B)
Protein synthesis Protein breakdown

Figure 22.3 (A) Effects of insulin on skeletal muscle protein dynamics. Insulin does not significantly
increase the rate of protein synthesis, but decreases the rate of protein breakdown. (Data from Nair,
K.S. et al., J. Clin. Invest., 95, 2926, 1995.) (B) Protein turnover in the splanchnic bed. This figure
demonstrates that in the splanchnic bed, insulin deprivation leads to higher protein synthesis rates
and higher protein breakdown rates. See text for explanation. (Data from Nair, K.S. et al., J. Clin.
Invest., 95, 2926, 1995.)
1382_C22.fm Page 366 Tuesday, October 7, 2003 6:55 PM

366 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

associated with obesity and may occur associated with a constellation of other conditions,
including hypertension and dyslipidemia, when it may be referred to as part of the
metabolic syndrome or syndrome X.95 Type 2 diabetes is characterized by the altered use
of fuel, with glucose being overproduced by the liver and underused by the peripheral
tissues. Although this condition is predominantly associated with abnormalities in carbo-
hydrate metabolism, individuals with type 2 diabetes may still have some changes in
protein metabolism that are associated with chronic complications seen with long-term,
poor glycemic control. Multiple hormones, and possibly substrates such as free fatty acids,
may affect protein metabolism in type 2 diabetes. Since type 2 diabetes increases with age,
aging per se may be a factor involved in protein metabolism in type 2 diabetes.
Although there is a rapid loss of lean body mass in type 1 diabetes when insulin is
withdrawn, there is no such lean tissue loss in type 2 diabetes. This may be due to the
protein-conserving effects of the insulin that is present. The quantity of insulin secreted
may be the same as that in subjects who do not have diabetes, but they have an inappro-
priately low level in the face of high blood glucose. Only very low concentrations of insulin
may be required to prevent proteolysis compared to those levels needed to stimulate
glucose uptake into cells in people with type 2 diabetes. A total of 10 mU/l of insulin is
also the plasma concentration needed to prevent ketosis from hepatic nonesterified fatty
acid metabolism by its effect on the mitochondrial carnitine shuttle, and this level may
be sufficient to normalize amino acid metabolism.
Protein metabolism in type 2 diabetes is relatively preserved. Henry et al.96 showed
that despite prolonged periods of hyperglycemia and dietary restriction, subjects with
type 2 diabetes preserve lean body mass. Furthermore, in age-matched controls, fasting
whole-body protein metabolism is no different in subjects with or without type 2 diabe-
tes.97–99 Leucine flux, leucine balance, and oxidation rates are normal in obese and nonobese
subjects with type 2 diabetes and are unaffected by an improvement in glycemic con-
trol.97–101 In addition, improving glycemic control by the use of sulphonylureas, biguanides,
or insulin has not been shown to improve either splanchnic or whole-body protein turn-
over.99,102 More evidence for this lack of difference came from work comparing amino acid
disposal in diabetic and nondiabetic subjects undergoing simultaneous amino acid and
insulin infusions. In both sets of subjects, whole-body protein turnover was reduced,
suggesting that the antiproteolytic effect of insulin in people with type 2 diabetes remains
intact.98 There is some evidence showing that protein turnover is in fact increased in type 2
diabetes.103,104 Several previous studies have demonstrated a nonsignificant trend toward
increased whole-body protein turnover. However, one study showed a significant 21%
rise in breakdown with a corresponding 16% increase in protein synthesis when comparing
protein kinetics during a period of poor glycemic control and then after 42 days of a very
low energy diet.105 The methodological differences among the studies may explain the
differences. Cross-sectional comparisons between type 2 diabetic subjects and controls are
not easy because of the difference in body composition and fat distribution. Comparison
of lean and obese people has demonstrated that obese people have higher whole-body
protein turnover than lean people.62 However, at the baseline state there are no reported
differences between obese (nondiabetic) controls and type 2 diabetic people.
Levels of branched-chain amino acids in subjects with type 2 diabetes vary according
to body habitus. Branched-chain amino acid levels in lean diabetic subjects are the same
as those in lean controls.97 In addition, although levels are different from those seen in
lean subjects, branched-chain amino acid levels were reported to be similar97 or higher106
in obese subjects with and without type 2 diabetes. When subjects were maintained on a
similar diet prior to the measurements, no differences between type 2 diabetic patients
(on and off treatment) and obese and lean controls could be detected.101 This study,
however, demonstrated that leucine transamination rates are slightly higher in type 2
1382_C22.fm Page 367 Tuesday, October 7, 2003 6:55 PM

Chapter twenty-two: Diabetes mellitus 367

diabetic people on poor glycemic control.101 Furthermore, the above study showed that
synthesis rates of various fractions of muscle proteins were unaffected by type 2 diabetes
or its treatment state.101 It may also be that although the antiproteolytic effects of insulin
at the whole-body level may be similar in subjects with type 2 diabetes and controls,98,101
the action of insulin in specific tissues to produce an antiproteolytic effect seems to be
significantly different. In lean control subjects, reductions in whole-body proteolysis by
high-dose insulin primarily result from suppression of proteolysis in skeletal muscle. This
is in contrast to type 2 diabetes, where despite the improvement in glycemic control to
almost normal levels, the hyperinsulinemia seems only to suppress nonmuscle proteo-
lysis.83 This could be because protein and glucose metabolism in skeletal muscle is resistant
to the effects of insulin in type 2 diabetes. An alternative explanation is that there is a
dose–response curve in different tissues for each of these effects, and that suppression of
proteolysis may be maximal with the hyperinsulinemic state seen in type 2 subjects and
in hyperinsulinemic clamp studies. This would explain the apparent low basal rates of
skeletal muscle proteolysis and the inability of higher doses of insulin to produce any
further suppression.107
Skeletal muscle protein turnover in healthy individuals is reduced with insulin infu-
sions,67,83 but in individuals with type 2 diabetes the effects of insulin on muscle protein
turnover are less clear.97,99,101,103 Gougeon et al.108 found that with high-protein isoenergetic
feeding of subjects with moderately well controlled type 2 diabetes, insulin therapy suf-
ficient to normalize plasma glucose levels was enough to induce nitrogen retention.
Recent work by Stump et al.123 has demonstrated that there is a defect in ATP produc-
tion capacity in the muscles of subjects with type 2 diabetes. In healthy volunteers, an
insulin infusion was associated with a 32 to 42% increase in skeletal muscle ATP produc-
tion. However, in these indivduals, this was reduced to a 16 to 26% increase. This is in
contrast to subjects with type 2 diabetes who showed no increase in skeletal muscle
mitochondrial ATP production with either low or high insulin levels.

22.2.4 The effects of other hormones on protein turnover in diabetes


Insulin deficiency is associated with increase in glucagon in all cases. Glucagon in the
postabsorptive state increases energy expenditure and leucine oxidation, especially when
insulin levels are low.109 When amino acids are administered, glucagon plays a pivotal
role in their disposal.110 Glucagon also plays a key role in amino acid catabolism in people
with type 1 diabetes.111 Growth hormone levels may increase in some cases. When given
systemically, growth hormone increases whole-body protein synthesis, but its effects on
muscle protein synthesis remain to be clarified.112,113 Insulin-like growth factor-1 (IGF-1)
has also been shown to increase muscle protein synthesis.114 IGFBP-1 levels increase
resulting in a decrease of free IGF-1 levels. In extreme situations, epinephrine, norepi-
nephrine, and cortisol levels also increase. Catecholamines are not thought to be protein
catabolic,115 and although cortisol levels do not rise in acute insulin deficiency, increased
circulating levels have been associated with increased protein breakdown.116 Glucocorti-
coids enhance proteolysis via the induction of the ATP–ubiquitin pathway in muscle,
leading to a decrease in intracellular muscle proteins with little or no effect being seen in
the liver.

22.3 Newer technologies


Over time, the ability of researchers to better understand protein metabolism has advanced.
Early work relied on crude estimations of whole-body nitrogen balance,57 followed by the
introduction of labeled tracers that allowed measurements at the whole-body level and
1382_C22.fm Page 368 Tuesday, October 7, 2003 6:55 PM

368 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

across tissue beds.117 Further refinement allowed the measurement of individual protein
subfractions within a tissue, such as myosin heavy chain, sarcoplasmic, and mitochondrial
proteins within skeletal muscle. Other advances allowed the measurements of synthesis
rates of circulating proteins.91,118–120 It is currently possible to better understand the specific
molecular level regulation of protein synthesis and breakdown. To understand the regula-
tion of amino acid and protein metabolism, it is important to study different tissues and
the regulation of transcriptional and translational levels by hormones, substrates, and other
factors, such as aging, exercise, etc. In addition, the study of amino acid and protein
metabolism is not complete until the factors involved in the posttranslational changes in
proteins are better understood. Furthermore, integration of studies of body functions with
changes in protein metabolism is also important. With the recent advances in genomics
and proteomics, the potential for studies in this area has never been greater. The new
microarrays and gene chips allow the assessment of several thousand gene products simul-
taneously. This can be done without having to specify which gene in particular needs to
be studied. These global approaches will help to further focus studies on patterns of changes
in gene transcript profiles121 and on specific genes involved in the regulation of protein
synthesis and breakdown in specific tissues. These changes can be followed over time in
the same subjects, leading to a better understanding of what changes occur at the level of
mRNA expression when insulin or other hormone or substrate levels alter. However,
mRNA expression often may not correspond to protein expression or function. It also
remains to be determined what constitutes normal and what is abnormal. The normal,
small differences in the genetic makeup of populations and individuals need to be distin-
guished from the small differences seen in disease states such as diabetes. Although there
are challenges in data interpretation, it is the progress made in the data management and
informatics areas of this technology that has enabled rapid improvement of our under-
standing. This issue is discussed in detail elsewhere.122

22.4 Conclusions and summary


Absolute insulin deficiency is associated with profound metabolic changes, including
increased circulating amino acid levels (especially branched-chain amino acids), net pro-
tein loss, and increases in whole-body protein catabolism. These effects can be reversed
by the administration of insulin. The causes of increased amino acid levels and nitrogen
loss associated with insulin deficiency have been investigated using isotopic techniques.
Most of the findings suggest that, in humans, the anabolic actions of insulin are mainly
in the prevention of protein breakdown, although not having an overall stimulatory effect
on whole-body protein synthesis unless in the presence of supplemental amino acids. It
is the plasma amino acid concentrations that determine the ultimate effects that insulin
has on protein synthesis rates. However, insulin is a key hormone involved in the regu-
lation of gene transcription and translation, and therefore plays a pivotal role in the
regulation of protein synthesis and breakdown. Insulin specifically stimulates synthesis
of proteins such as mitochondrial protein. In contrast to the dramatic changes of protein
turnover in type 1 diabetic patients, the changes in protein turnover are rather minimal
in type 2 diabetic people. In type 2 diabetes these effects on protein metabolism are less
impressive due to the probable high circulating levels of insulin already present.

References
1. Zimmet, P.Z., McCarty, D.J., and de Courten, M.P., The global epidemiology of non-insulin-
dependent diabetes mellitus and the metabolic syndrome, J. Diabetes Complications, 11, 60,
1997.
1382_C22.fm Page 369 Tuesday, October 7, 2003 6:55 PM

Chapter twenty-two: Diabetes mellitus 369

2. Cahill, G.F., The Banting Memorial Lecture 1971: physiology of insulin in man, Diabetes, 20,
785, 1971.
3. O’Brien, R.M. and Granner, D. K., Regulation of gene expression by insulin, Physiol. Rev., 76,
1109, 1996.
4. Magnuson, M.A., Glucokinase gene structure: functional implications of molecular genetic
studies, Diabetes, 39, 523, 1990.
5. White, M.F., The IRS-signalling system: a network of docking proteins that mediate insulin
action, Mol. Cell. Biochem., 182, 3, 1998.
6. Zhang, J. et al., Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin
response element (IRE) that resembles IREs of other insulin-repressed genes, Proc. Natl. Acad.
Sci. U.S.A., 98, 3756, 2001.
7. Christensen, H.N., Role of amino acid transport and countertransport in nutrition and
metabolism, Physiol. Rev., 70, 43, 1990.
8. Tovar, A.R. et al., Neutral amino acid transport into rat skeletal muscle: competition, adaptive
regulation, and effects of insulin, Metabolism, 40, 410, 1991.
9. Le Cam, A. and Freychet, P., Effect of insulin on amino acid transport in isolated rat hepat-
ocytes, Diabetologia, 15, 117, 1978.
10. Akedo, H. and Christensen, H.N., Nature of insulin action on amino acid uptake in isolated
rat diaphragm, J. Biol. Chem., 237, 118, 1962.
11. Manchester, K.L. and Wool, I.G., Insulin and incorporation of amino acids into protein of
muscle. 2. Accumulation and incorporation studies with the perfused rat heart, Biochem. J.,
89, 202, 1963.
12. Schwartz, A., Hormonal regulation of amino acid accumulation in human fetal liver explants:
effects of dibutyryl cyclic AMP, glucogon and insulin, Biochim. Biophys. Acta, 362, 276, 1974.
13. Jefferson, L.S., Koehler, J.O., and Morgan, H.E., Effect of insulin on protein synthesis in
skeletal muscle of an isolated perfused preparation of rat hemicorpus, Proc. Natl. Acad. Sci.
U.S.A., 69, 816, 1972.
14. Jefferson, L.S., Li, J.B., and Rannels, S.R., Regulation by insulin of amino acid release and
protein turnover in the perfused rat hemicorpus, J. Biol. Chem., 252, 1476, 1975.
15. Kimball, S.R., Horetsky, R.L., and Jefferson, L.S., Signal transduction pathways involved in
the regulation of protein synthesis by insulin in L6 myoblasts, Am. J. Physiol. Cell. Physiol.,
274, C221, 1998.
16. Kimball, S.R., Vary, T.C., and Jefferson, L.S., Regulation of protein synthesis by insulin, Annu.
Rev. Physiol., 56, 348, 1994.
17. Stirewalt, W.S. and Lo, R.B., Effects of insulin in vitro on protein turnover in rat epitrochlearis
muscle, Biochem. J., 210, 323, 1983.
18. Levenson, R.M., Nairn, A.C., and Blackshear, P.J., Insulin rapidly induces the biosynthesis
of elongation factor 2, J. Biol. Chem., 264, 11904, 1989.
19. Lin, T.A. et al., Control of PHAS-I by insulin in 3T3-L1 adipocytes: synthesis, degradation,
and phosphorylation by a rapamycin-sensitive and mitogen-activated protein kinase-inde-
pendent pathway, J. Biol. Chem., 270, 18531, 1995.
20. Dillmann, W.H., Barrieux, A., and Shanker, R., Influence of thyroid hormone on myosin
heavy chain mRNA and other messenger RNAs in the rat heart, Endocr. Res., 15, 565, 1989.
21. Sreekumar, R. et al., Gene expression profile in skeletal muscle of type 2 diabetes and the
effect of insulin therapy, Diabetes, 51, 1913, 2002.
22. Rao, P.V., Pugazhenthi, S., and Khandelwal, R.L., The effects of streptozotocin-induced dia-
betes and insulin supplementation on expression of the glycogen phosphorylase gene in rat
liver, J. Biol. Chem., 270, 24955, 1995.
23. Christ, B. and Nath, A., The glucagon-insulin antagonism in the regulation of cytosolic
protein binding to the 3' end of phosphoenolpyruvate carboxykinase mRNA in cultured rat
hepatocytes: possible involvement in the stabilization of the mRNA, Eur. J. Biochem., 215,
541, 1993.
24. Flores-Riveros, J.R. et al., Insulin down-regulates expression of the insulin-responsive glucose
transporter (GLUT4) gene: effects on transcription and mRNA turnover, Proc. Natl. Acad. Sci.
U.S.A., 90, 512, 1993.
1382_C22.fm Page 370 Tuesday, October 7, 2003 6:55 PM

370 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

25. Proud, C.G. and Denton, R.M., Molecular mechanisms for the control of translation by
insulin, Biochem. J., 328, 329, 1997.
26. Lin, T.A. et al., PHAS-I as a link between mitogen-activated protein kinase and translation
initiation, Science, 266, 653, 1994.
27. Azpiazu, I. et al., Regulation of both glycogen synthase and PHAS-I by insulin in rat skeletal
muscle involves mitogen-activated protein kinase-independent and rapamycin-sensitive
pathways, J. Biol. Chem., 271, 5033, 1996.
28. Kimball, S.R. et al., Insulin and diabetes cause reciprocal changes in the association of eIF-4E
and PHAS-I in rat skeletal muscle, Am. J. Physiol. Cell. Physiol., 270, C705, 1996.
29. Kimball, S.R. et al., Insulin stimulates protein synthesis in skeletal muscle by enhancing the
association of eIF-4E and eIF-4G, Am. J. Physiol. Cell. Physiol., 272, C754, 1997.
30. Sinaud, S. et al., Diazoxide-induced insulin deficiency greatly reduced muscle protein syn-
thesis in rats: involvement of eIF4E, Am. J. Physiol. Endocrinol. Metab., 276, E50, 1999.
31. Redpath, N.T., Foulstone, E.J., and Proud, C.G., Regulation of translation elongation factor-
2 by insulin via a rapamycin-sensitive signalling pathway, EMBO J., 15, 2291, 1996.
32. DePhilip, R.M. et al., Rapid stimulation by insulin of ribosome synthesis in cultured chick
embryo fibroblasts, Biochemistry, 18, 4812, 1979.
33. Hannan, K.M., Rothblum, L.I., and Jefferson, L.S., Regulation of ribosomal DNA transcription
by insulin, Am. J. Physiol. Cell. Physiol., 275, C130, 1998.
34. Hammond, M.L. and Bowman, L.H., Insulin stimulates the translation of ribosomal proteins
and the transcription of rDNA in mouse myoblasts, J. Biol. Chem., 263, 17785, 1988.
35. Antonetti, D.A. et al., Regulation of rDNA transcription by insulin in primary cultures of
rat hepatocytes, J. Biol. Chem., 268, 25277, 1993.
36. Price, S.R. et al., Muscle wasting in insulinopenic rats results from activation of the ATP-
dependent, ubiquitin-proteasome proteolytic pathway by a mechanism including gene tran-
scription, J. Clin. Invest., 98, 1703, 1996.
37. Pepato, M.T. et al., Role of different proteolytic pathways in degradation of muscle protein
from streptozotocin-diabetic rats, Am. J. Physiol. Endocrinol. Metab., 271, E340, 1996.
38. Mitch, W.E. et al., Evaluation of signals activating ubiquitin-proteasome proteolysis in a
model of muscle wasting, Am. J. Physiol. Cell. Physiol., 276, C1132, 1999.
39. Pain, V.M. and Garlick, P.J., Effect of streptozotocin diabetes and insulin treatment on the
rate of protein synthesis in tissues of the rat in vivo, J. Biol. Chem., 249, 4510, 1974.
40. McNurlan, M.A. and Garlick, P.J., Protein synthesis in liver and small intestine in protein
deprivation and diabetes, Am. J. Physiol. Endocrinol. Metab., 241, E238, 1981.
41. Garlick, P.J. et al., Protein turnover in tissues of diabetic rats, Acta Biol. Med. Ger., 40, 1301,
1981.
42. Flaim, K.E., Copenhaver, M.E., and Jefferson, L.S., Effects of diabetes on protein synthesis
in fast- and slow-twitch rat skeletal muscle, Am. J. Physiol. Endocrinol. Metab., 239, E88, 1980.
43. Garlick, P.J., Baillie, A.G., Grant, I., et al., In-vivo action of insulin on protein metabolism in
experimental animals, in Protein Metabolism in Diabetes Mellitus, Nair, K.S., Ed., Smith-Gordon
and Company Limited, London, 1992, p. 163.
44. Millward, D.J., Odedra, B., and Bates, P.C., The role of insulin, corticosterone and other
factors in the acute recovery of muscle protein synthesis on refeeding food-deprived rats,
Biochem. J., 216, 583, 1983.
45. Li, J.B., Higgins, J.E., and Jefferson, L.S., Changes in protein turnover in skeletal muscle in
response to fasting, Am. J. Physiol. Endocrinol. Metab., 236, E222, 1979.
46. Preedy, V.R. and Garlick, P.J., The response of muscle protein synthesis to nutrient intake in
postabsorptive rats: the role of insulin and amino acids, Biosci. Rep., 6, 177, 1986.
47. Garlick, P.J., Fern, M., and Preedy, V.R., The effect of insulin infusion and food intake on
muscle protein synthesis in postabsorptive rats, Biochem. J., 210, 669, 1983.
48. Garlick, P.J. and Grant, I., Amino acid infusion increases the sensitivity of muscle protein
synthesis in vivo to insulin: effect of branched-chain amino acids, Biochem. J., 254, 579, 1988.
49. Garlick, P.J. and Grant, I. Stimulation of muscle protein synthesis in vivo by leucine and
insulin, FASEB J., 3, A341, 1989 (abstract).
1382_C22.fm Page 371 Tuesday, October 7, 2003 6:55 PM

Chapter twenty-two: Diabetes mellitus 371

50. Baillie, A.G. and Garlick, P.J., Attenuated responses of muscle protein synthesis to fasting
and insulin in adult female rats, Am. J. Physiol. Endocrinol. Metab., 262, E1, 1992.
51. Wray-Cahen, D. et al., Response of skeletal muscle protein synthesis to insulin in suckling
pigs decreases with development, Am. J. Physiol. Endocrinol. Metab., 275, E602, 1998.
52. Boirie, Y. et al., Tissue-specific regulation of mitochondrial and cytoplasmic protein synthesis
rates by insulin, Diabetes, 50, 2652, 2001.
53. Ahlman, B. et al., Insulin’s effect on synthesis rates of liver proteins: a swine model comparing
various precursors of protein synthesis, Diabetes, 50, 947, 2001.
54. Baumann, P.Q. et al., Precursor pools of protein synthesis: a stable isotope study in a swine
model, Am. J. Physiol. Endocrinol. Metab., 267, E203, 1994.
55. Anthony, J.C. et al., Leucine stimulates translation initiation in skeletal muscle of postabsorp-
tive rats via a rapamycin-sensitive pathway, J. Nutr., 130, 2413, 2000.
56. Anthony, J.C. et al., Orally administered leucine stimulates protein synthesis in skeletal
muscle of postabsorptive rats in association with increased eIF4F formation, J. Nutr., 130,
139, 2000.
57. Atchley, D.W. et al., On diabetic acidosis: a detailed study of electrolyte balances following
the withdrawal and reestablishment of insulin therapy, J. Clin. Invest., 12, 297, 1933.
58. Felig, P. et al., Plasma amino acid levels in diabetic ketoacidosis, Diabetes, 19, 727, 1970.
59. Luck, J.M., Morrison, G., and Wilbur, L.F., The effect of insulin on the amino acid content of
blood, J. Biol. Chem., 77, 151, 1928.
60. Robert, J.J. et al., Whole body de novo amino acid synthesis in type I (insulin-dependent)
diabetes studied with stable isotope-labeled leucine, alanine, and glycine, Diabetes, 34, 67,
1985.
61. Felig, P., Amino acid metabolism in man, Annu. Rev. Biochem., 44, 955, 1975.
62. Nair, K.S. et al., Effect of poor diabetic control and obesity on whole body protein metabolism
in man, Diabetologia, 25, 400, 1983.
63. Luzi, L. et al., Leucine metabolism in IDDM: role of insulin and substrate availability, Diabetes,
39, 38, 1990.
64. Umpleby, A.M. et al., The effect of metabolic control on leucine metabolism in type 1 (insulin-
dependent) diabetic patients, Diabetologia, 29, 131, 1986.
65. Nair, K.S. et al., Protein dynamics in whole body and in splanchnic and leg tissues in type
I diabetic patients, J. Clin. Invest., 95, 2926, 1995.
66. Fukagawa, N.K. et al., Insulin-mediated reduction of whole body protein breakdown: dose-
response effects on leucine metabolism in postabsorptive men, J. Clin. Invest., 76, 2306, 1985.
67. Gelfand, R.A. and Barrett, E.J., Effect of physiologic hyperinsulinemia on skeletal muscle
protein synthesis and breakdown in man, J. Clin. Invest., 80, 1, 1987.
68. Tessari, P. et al., Effects of acute systemic hyperinsulinemia on forearm muscle proteolysis
in healthy man, J. Clin. Invest., 88, 27, 1991.
69. Castellino, P. et al., Effect of insulin and plasma amino acid concentrations on leucine
metabolism in man: role of substrate availability on estimates of whole body protein syn-
thesis, J. Clin. Invest., 80, 1784, 1987.
70. Tessari, P. et al., Differential effects of hyperinsulinemia and hyperaminoacidemia on leucine-
carbon metabolism in vivo: evidence for distinct mechanisms in regulation of net amino acid
deposition, J. Clin. Invest., 79, 1062, 1987.
71. Inchiostro, S. et al., Effects of insulin and amino acid infusion on leucine and phenylalanine
kinetics in type 1 diabetes, Am. J. Physiol. Endocrinol. Metab., 262, E203, 1992.
72. Tamborlane, W.V. et al., Insulin-infusion-pump treatment of diabetes: influence of improved
metabolic control on plasma somatomedin levels, N. Engl. J. Med., 305, 303, 1981.
73. Nair, K.S., Ford, G.C., and Halliday, D., Effect of intravenous insulin treatment on in vivo
whole body leucine kinetics and oxygen consumption in insulin-deprived type I diabetic
patients, Metabolism, 36, 491, 1987.
74. Block, K.P. et al., Activation of rat liver branched-chain 2-oxo acid dehydrogenase in vivo by
glucagon and adrenaline, Biochem. J., 232, 593, 1985.
75. Wahren, J., Felig, P., and Hagenfeldt, L., Effect of protein ingestion on splanchnic and leg
metabolism in normal man and in patients with diabetes mellitus, J. Clin. Invest., 57, 987, 1976.
1382_C22.fm Page 372 Tuesday, October 7, 2003 6:55 PM

372 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

76. Wahren, J. et al., Splanchnic and peripheral glucose and amino acid metabolism in diabetes
mellitus, J. Clin. Invest., 51, 1870, 1972.
77. Nair, K.S., Schwartz, R.G., and Welle, S., Leucine as a regulator of whole body and skeletal
muscle protein metabolism in humans, Am. J. Physiol. Endocrinol. Metab., 263, E928, 1992.
78. Nair, K.S. et al., Effect of leucine on amino acid and glucose metabolism in humans, Metab-
olism, 41, 643, 1992.
79. Louard, R.J., Barrett, E.J., and Gelfand, R.A., Effect of infused branched-chain amino acids
on muscle and whole-body amino acid metabolism in man, Clin. Sci., 79, 457, 1990.
80. Flakoll, P.J. et al., Amino acids augment insulin’s suppression of whole body proteolysis,
Am. J. Physiol. Endocrinol. Metab., 257, E839, 1989.
81. Bennet, W.M. et al., Inability to stimulate skeletal muscle or whole body protein synthesis
in type 1 (insulin-dependent) diabetic patients by insulin-plus-glucose during amino acid
infusion: studies of incorporation and turnover of tracer L-[1-13C]leucine, Diabetologia, 33,
43, 1990.
82. May, M.E. and Buse, M.G., Effects of branched-chain amino acids on protein turnover,
Diabetes Metab. Rev., 5, 227, 1989.
83. Denne, S.C. et al., Proteolysis in skeletal muscle and whole body in response to euglycemic
hyperinsulinemia in normal adults, Am. J. Physiol. Endocrinol. Metab., 261, E809, 1991.
84. McNurlan, M.A. et al., Measurement of protein synthesis in human skeletal muscle: further
investigation of the flooding technique, Clin. Sci., 81, 557, 1991.
85. Moller-Loswick, A.C. et al., Insulin selectively attenuates breakdown of nonmyofibrillar
proteins in peripheral tissues of normal men, Am. J. Physiol. Endocrinol. Metab., 266, E645,
1994.
86. Bennet, W.M. et al., Effects of insulin and amino acids on leg protein turnover in IDDM
patients, Diabetes, 40, 499, 1991.
87. Tessari, P. et al., Effects of insulin on whole body and forearm leucine and KIC metabolism
in type 1 diabetes, Am. J. Physiol. Endocrinol. Metab., 259, E96, 1990.
88. Meek, S.E. et al., Differential regulation of amino acid exchange and protein dynamics across
splanchnic and skeletal muscle beds by insulin in healthy human subjects, Diabetes, 47, 1824,
1998.
89. Charlton, M.R., Ahlman, B., and Nair, K.S., The effect of insulin on human small intestinal
mucosal protein synthesis, Gastroenterology, 118, 299, 2000.
90. Lloyd, C.E. et al., Stimulation of albumin gene transcription by insulin in primary cultures
of rat hepatocytes, Am. J. Physiol. Cell. Physiol., 252, C205, 1987.
91. De Feo, P., Gaisano, M.G., and Haymond, M.W., Differential effects of insulin deficiency on
albumin and fibrinogen synthesis in humans, J. Clin. Invest., 88, 833, 1991.
92. Nair, K.S. Regional protein dynamics in type 1 diabetic patients, in Amino Acid and Protein
Metabolism in Health and Disease, Tessari, P., Seters, P.B., Pittoni, G., et al., Eds., Smith-Gordon,
London, 1997, p. 133.
93. Charlton, M.R., Balagopal, P., and Nair, K.S., Skeletal muscle myosin heavy chain synthesis
in type 1 diabetes, Diabetes, 46, 1336, 1997.
94. De Feo, P. et al., Physiological increments in plasma insulin concentrations have selective
and different effects on synthesis of hepatic proteins in normal humans, Diabetes, 42, 995,
1993.
95. Reaven, G.M., The Banting Memorial Lecture 1988: role of insulin resistance in human
disease, Diabetes, 37, 1595, 1988.
96. Henry, R.R. et al., Metabolic consequences of very-low-calorie diet therapy in obese non-
insulin-dependent diabetic and nondiabetic subjects, Diabetes, 35, 155, 1986.
97. Staten, M.A., Matthews, D.E., and Bier, D.M., Leucine metabolism in type II diabetes mellitus,
Diabetes, 35, 1249, 1986.
98. Luzi, L., Petrides, A.S., and DeFronzo, R.A., Different sensitivity of glucose and amino acid
metabolism to insulin in NIDDM, Diabetes, 42, 1868, 1993.
99. Welle, S. and Nair, K.S., Failure of glyburide and insulin treatment to decrease leucine flux
in obese type II diabetic patients, Int. J. Obes., 14, 701, 1990.
1382_C22.fm Page 373 Tuesday, October 7, 2003 6:55 PM

Chapter twenty-two: Diabetes mellitus 373

100. Conway, J.M. et al., Whole-body lysine flux in young adult men: effects of reduced total
protein and of lysine intake, Am. J. Physiol. Endocrinol. Metab., 239, E192, 1980.
101. Halvatsiotis, P. et al., Synthesis rate of muscle proteins, muscle functions, and amino acid
kinetics in type 2 diabetes, Diabetes, 57, 2395, 2002.
102. Tessari, P. et al., Effects of metformin treatment on whole-body and splanchnic amino acid
turnover in mild type 2 diabetes, J. Clin. Endocrinol. Metab., 79, 1553, 1994.
103. Denne, S.C. et al., Skeletal muscle proteolysis is reduced in noninsulin-dependent diabetes
mellitus and is unaltered by euglycemic hyperinsulinemia or intensive insulin therapy,
J. Clin. Endocrinol. Metab., 80, 2371, 1995.
104. Gougeon, R., Pencharz, P.B., and Sigal, R.J., Effect of glycemic control on the kinetics of
whole-body protein metabolism in obese subjects with non-insulin-dependent diabetes mel-
litus during iso- and hypoenergetic feeding, Am. J. Clin. Nutr., 65, 861, 1997.
105. Gougeon, R., Pencharz, P.B., and Marliss, E.B., Effect of NIDDM on the kinetics of whole-
body protein metabolism, Diabetes, 43, 318, 1994.
106. Caballero, B. and Wurtman, R.J., Differential effects of insulin resistance on leucine and
glucose kinetics in obesity, Metabolism, 40, 51, 1991.
107. Louard, R.J. et al., Insulin sensitivity of protein and glucose metabolism in human forearm
skeletal muscle, J. Clin. Invest., 90, 2348, 1992.
108. Gougeon, R. et al., Effect of exogenous insulin on protein metabolism with differing non-
protein energy intakes in type 2 diabetes mellitus, Int. J. Obes., 22, 250, 1998.
109. Nair, K.S., Hyperglucagonemia increases resting metabolic rate in man during insulin defi-
ciency, J. Clin. Endocrinol. Metab., 64, 896, 1987.
110. Charlton, M.R., Adey, D.B., and Nair, K.S., Evidence for a catabolic role of glucagon during
an amino acid load, J. Clin. Invest., 98, 90, 1996.
111. Charlton, M.R. and Nair, K.S., Role of hyperglucagonemia in catabolism associated with type
1 diabetes: effects on leucine metabolism and the resting metabolic rate, Diabetes, 47, 1748,
1998.
112. Copeland, K.C. and Nair, K.S., Acute growth hormone effects on amino acid and lipid
metabolism, J. Clin. Endocrinol. Metab., 78, 1040, 1994.
113. Yarasheski, K.E. et al., Effect of growth hormone and resistance exercise on muscle growth
in young men, Am. J. Physiol. Endocrinol. Metab., 262, E261, 1992.
114. Fryburg, D.A., Insulin-like growth factor I exerts growth hormone- and insulin-like actions
on human muscle protein metabolism, Am. J. Physiol. Endocrinol. Metab., 267, E331, 1994.
115. Matthews, D.E., Pesola, G., and Campbell, R.G., Effect of epinephrine on amino acid and
energy metabolism in humans, Am. J. Physiol. Endocrinol. Metab., 258, E948, 1990.
116. Beaufrère, B. et al., Glucocorticosteroids increase leucine oxidation and impair leucine bal-
ance in humans, Am. J. Physiol. Endocrinol. Metab., 257, E712, 1989.
117. Halliday, D. et al., Rate of protein synthesis in skeletal muscle of normal man and patients
with muscular dystrophy: a reassessment, Clin. Sci., 74, 237, 1988.
118. Fu, A.Z. et al., Sequential purification of human apolipoprotein B-100, albumin, and fibrin-
ogen by immunoaffinity chromatography for measurement of protein synthesis, Anal.
Biochem., 247, 228, 1997.
119. Fu, A.Z. and Nair, K.S., Age effect on fibrinogen and albumin synthesis in humans, Am. J.
Physiol. Endocrinol. Metab., 275, E1023, 1998.
120. Hasten, D.L. et al., Isolation of human skeletal muscle myosin heavy chain and actin for
measurement of fractional synthesis rates, Am. J. Physiol. Endocrinol. Metab., 275, E1092, 1998.
121. Sreekumar, R., Halvatsiotis, P., Coenen Schimke, J.M., and Nair, K.S., Gene expression profile
in skeletal muscle of type 2 diabetes and the effect of insulin therapy, Diabetes, 51, 1913, 2002.
122. Tefferi, A. et al., Primer on medical genomics. III. Microarray experiments and data analysis,
Mayo Clin. Proc., 77, 927, 2002.
123. Stump, C.S. et al., Effect of insulin on human skeletal muscle mitochondrial ATP production,
protein synthesis, and mRNA transcripts, PNAS, 100, 7996, 2003.
1382_C22.fm Page 374 Tuesday, October 7, 2003 6:55 PM
1382_C23.fm Page 375 Tuesday, October 7, 2003 6:58 PM

chapter twenty-three

Acidosis and amino acid


metabolism
Tomas Welbourne
Louisiana State University/HSC
Itzhak Nissim
University of Pennsylvania School of Medicine

Contents
23.1 Physiological significance of acidosis and the importance
of glutamine/glutamate metabolism..............................................................................375
23.2 Renal glutamine/glutamate metabolism in defense of metabolic acidosis ..............376
23.3 Whole-body response to NH4Cl acid load .....................................................................378
23.4 Renal tubule cell response to an NH4Cl acid load ........................................................380
23.5 Glutamine/Glutamate metabolism in acidotic cells.....................................................382
23.6 Cellular acidosis and glutamine metabolism contribute to programmed
cell death .............................................................................................................................384
Acknowledgments ......................................................................................................................385
References .....................................................................................................................................385

23.1 Physiological significance of acidosis and the importance


of glutamine/glutamate metabolism
Amino acid metabolism in acidosis necessarily focuses upon glutamine, the predominant
extracellular amino acid, and glutamate, the most significant intracellular amino acid.
Metabolic acidosis, diagnosed as a reduction in plasma bicarbonate concentration, results
from enhanced nonvolatile acid production derived from the incomplete oxidation of
glucose yielding lactic acid and fatty acids yielding ketone bodies, and the complete
oxidation of sulfur-containing amino acids and phospholipids yielding sulfuric acid and
phosphoric acid, respectively. Thus a potential metabolic acidosis will be present in vir-
tually all catabolic states,1 e.g., injury, starvation, chronic illnesses such as AIDS, endocrine
disorders such as diabetes mellitus, and anaerobic episodes such as exercise and ischemia.
Metabolic acidosis, without an increase in nonvolatile acid production, is also ubiquitous

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 375
1382_C23.fm Page 376 Tuesday, October 7, 2003 6:58 PM

376 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

in the aged population,2 apparently reflecting diminished renal acid secretion and a
reduced plasma bicarbonate threshold.
The presence of cellular acidosis in association with the systemic metabolic acidosis
provides a direct stimulus to pathways of amino acid metabolism, acts as an allosteric
activator of acid extrusion,3 and signals adaptive responses through multiple-cell signaling
pathways.4,5 The critical importance of amino acid metabolism in response to metabolic
acidosis becomes apparent in the role played in preventing a precarious cellular acidosis.
When this response is not forthcoming, a catastrophic fall in cell pH ensues, activating
pathways leading to programmed cell death.6 Once triggered, these pathways lead to
impairment of oxidative metabolism and shift the cell to acid generating glycolysis and
lactic acidosis,7 a key indicator of multiple-organ failure.8
Thus, accelerating acid production without a concomitant increase in renal base pro-
duction would overwhelm the body fluid alkaline reserves, further escalating the threat
of cell death.

23.2 Renal glutamine/glutamate metabolism in defense of metabolic


acidosis
In response to metabolic acidosis, the kidneys must generate new bicarbonate at a rate
commensurate with acid production in order to stabilize the body fluid alkaline reserves.
To generate this new base, glutamine is extracted from the blood by the kidneys and in
turn metabolized to ammonium and bicarbonate, as shown in Figure 23.1. Note that there
are multiple metabolic pathways available to glutamine entering the kidney, emphasizing
the regulatory roles played by the glutamine/glutamate couplet.9 The basogenic pathway
(bold lines) begins with glutamine transport into the cytosol via the high-affinity SN1
transporter10 present on both the luminal and antiluminal tubule surfaces (1). Within the
cytosol, glutamine is converted to glutamate via the phosphate-dependent glutaminase
(PDG) (4), functionally active on the outer surface of the mitochondrial inner membrane;11
the formed glutamate, in turn, is coupled to the inner membrane glutamate/H+ transporter
(6), gaining entrance into the matrix space12 where glutamate undergoes deamination via
glutamate dehydrogenase (GDH) (7). The resulting alpha-ketoglutarate is subsequently
oxidized generating bicarbonate; secretion of NH4+ into the urine via the brush border
sodium hydrogen ion exchanger (NHE3) (11) and release of bicarbonate into the renal
vein via the basolateral sodium bicarbonate cotransporter (NBC) (12) ensure the net
formation of base. Thus, for every mole of glutamine metabolized by this pathway in the
functioning kidney, there are two moles of bicarbonate released into the renal vein13 for
every two moles of ammonium produced.13
Regulation of this pathway occurs at multiple levels, beginning first with the extra-
cellular glutamine hydrolysis via phosphate-independent glutaminase (PIG) (2), active on
both surfaces of the renal tubule.14 The glutamate formed is coupled to the high-affinity
glutamate transporters EAAC1 (3) and GLT1 (3) present on the luminal15 and basolateral16
borders of the renal tubules, respectively. The combined activity of these two transporters
functioning as a unit with the extracellular glutaminase contributes to maintaining a high
cytosolic glutamate concentration, which in turn effectively inhibits the cytosolic func-
tional PDG.11,17 Animals with the EAAC1 gene knocked out excrete far greater amounts
of glutamate18 than enters the kidney, consistent with luminal glutamine hydrolysis via
brush border PIG, while animals with the GLT1 knocked out have a nearly 50% reduction
in kidney glutamate content and a threefold increase in ammonium excretion.19 Since
extracellular glutamine conversion to glutamate is directly related to both the plasma
glutamine and bicarbonate concentrations,20 the metabolic acidosis-induced decrease in
1382_C23.fm Page 377 Tuesday, October 7, 2003 6:58 PM

Chapter twenty-three: Acidosis and amino acid metabolism 377

GLUÐ

GLN GLN GLUÐ GLUÐ 2 NH4+ Urine


+
2 HCO3 Renal
GLUÐ
+ Vein
NH4+ ALA

ALA

Transporters
1- SN1 Gin transporter,plasma membrane
3- XAG- GLU transporters, plasma membrane
6- GLUÐ/H+ mitochondrial membrane GLU transporter
10- GLN, GLN/GLUÐ mitochondrial membrane GLN transporter
11- NHE3 sodium/hydrogen ion exchanger, brush border
12- NBC sodium/bicarbonate cotransporter, basolateral membrane
Enzymes
2- phosphate independent glutaminase
4,9- phosphate dependent glutaminaes, 4 functional in cytosol, 9 latent in mitochondria
5,8-alanine aminotransferase,cytosolic,5, and intramitochondrial,8
7-glutamate dehdrogenase, mitochondrial matrix

Figure 23.1 Basogenic glutamine metabolism pathway (bold lines) and potential regulatory sites in
human kidney.

both bicarbonate and circulating glutamine meters the plasma membrane glutamate
uptake to the level of body fluid alkaline reserves. A second regulatory site at the plasma
membrane is the high-affinity glutamine uptake (1)10 that increases glutamine influx and
elevates cytosolic glutamine. As a consequence of the decreased glutamate uptake and
increased glutamine uptake into the cytosolic compartment, glutamine/glutamate content
favors glutamine flux through the functional PDG (4).
Cellular acidosis is coupled to metabolic acidosis as the result of base efflux out of
the cytosol via NBC (12) and subsequent rise in cytosolic H+ (decrease in pHi). The fall
in pHi has four important effects:

1. Activation of the inner mitochondrial membrane glutamate/H+ transporter


(6)-mediated glutamate uptake into the mitochondrial matrix space12
2. Inhibition of cytosolic ALT flux (5) (see below)
3. Activation of the brush border NHE3 (12)-mediated acid extrusion3
4. Activation of signaling pathways leading to adaptive mechanisms enhancing base
generation4,5

The cellular acidosis activation of glutamate uptake into the mitochondrial matrix has
two consequences: it (1) ensures that glutamate formed in the cytosol from glutamine via
number 4 is dedicated to base generation by metabolism through GDH (7), and (2) removes
a potential inhibitory effect of glutamate on functional PDG flux (4).
The cellular acidosis inhibition of cytosolic ALT flux (5) prevents glutamate runoff
into a nonbasogenic pathway and reinforces the coupling of the PDG flux (4) to the GDH
flux (7).
The metabolic acidosis-induced rise in cytosolic H+ allosterically activates NHE3 (11),
accelerating acid extrusion (H+ and NH4+) across the luminal membrane and thereby
countering the accelerated base extrusion across the basolateral membrane (12); this
1382_C23.fm Page 378 Tuesday, October 7, 2003 6:58 PM

378 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

moderates but, importantly, does not prevent the drop in pHi and ensures that cytosolic
NH4+ is preferentially secreted into the urine.
Finally, the decrease in pHi activates cell-signaling cascades, leading to up-regulation
of a number of transporters and enzymes involved in acid–base homeostasis.4,5

23.3 Whole-body response to NH4Cl acid load


Pitts et al.21 in his classical study of acid–base balance regulation used an NH4Cl acid
challenge to “frame” potential regulatory mechanisms. NH4Cl simulates an endogeous
nonvolatile acid load because of conversion to HCl. Accordingly, NH4+ enters the portal
blood and is transported into the hepatic perivenous cells as NH4+. Inside these cells NH3
is metabolically clear by conversion to glutamine, leaving the free H+ to lower the pHi
and provoke metabolic changes in the liver,22 with the net effect to shift nitrogen from the
ureagenic periportal to the basogenic perivenous glutamine synthesis pathway (see Chap-
ter 7). This rise in liver cell cytosolic H+ activates the basolateral NHE1, and acid is then
shifted from the intracellular into the extracellular compartment where H+ depletes the
alkaline reserves as reflected by the fall in plasma bicarbonate (Figure 23.2). This fall in
plasma bicarbonate becomes the signal that is transmitted to pHi-sensitive proximal tubule
cells’ glutamine/glutamate metabolism through two mechanisms: by movement of bicar-
bonate out of the cell via the NBC (12), resulting in a reduced cytosolic pH, and by reducing
activity of the bicarbonate-activated extracellular glutaminase (2). Reinforcing the reduced
glutamate formation via number 2 in Figure 23.2 is the decline in circulating plasma
glutamine concentration23; this resetting of glutamine homeostasis is mediated by the
lungs23 and directly related to arterial blood pCO2.23 Note that while glutamine flux
through the low-affinity glutaminase (2) is markedly reduced, glutamine uptake flux
through the high-affinity plasma membrane glutamine transporter (1) is enhanced. The
physiological role for resetting glutamine homeostasis downward by almost 50% becomes
apparent as an effect on nitrogen balance. Now, removal of circulating glutamine from
the blood by the small intestine falls in proportion to the blood glutamine concentration,23
while the portal blood nitrogen profile shifts23 from enriched alanine (ureagenic) to pre-
dominantly ammonium (glutaminogenic). As a consequence, whole-body glutamine
metabolism in metabolic acidosis is targeted to the kidneys and base generation, while
shifting nitrogen from hepatic ureagenesis, preventing a large negative nitrogen balance.23
Renal glutamine extraction and ammonium production for humans given an NH4Cl
load protocol similar to that shown in Figure 23.2 were performed in the classical studies
carried out by Tizianello et al.24 and are presented in Figure 23.3. They showed that even
in the absence of the exogenous acid load, the kidneys remove a significant amount of
glutamine from the blood, but utilizing only about half of its base-generating potential
(1.21 NH4+ produced for one glutamine extracted, consistent with a limiting GDH flux
(Figure 23.1 (7)); after 24 h of NH4Cl loading, glutamine extraction does not decrease
despite the 32% reduction in plasma glutamine concentration (Figure 23.2). Nevertheless,
ammonium production (reflecting base production) almost doubles because of the maxi-
mal utilization of glutamine (2.5 ammonium produced for 1 glutamine extracted), consis-
tent with cellular acidosis activation of the GDH flux (Figure 23.1 (7)); this also suggests
other potential ammoniagenic precursors, e.g., glutamate-generating peptides such as
GSH.25 Whole-kidney pHi measured in the rat after 24 h of NH4Cl loading falls from 7.39
± 0.04 to 7.16 ± 0.03,26 supporting the accelerated flux of glutamate into GDH driven by
reduced pHi via number 6 in Figure 23.1.12 After 3 days (Figure 23.3), glutamine extraction
is maintained despite the continued reduced plasma glutamine concentration (Figure 23.2),
with 2.5 ammonium produced for each glutamine extracted, again consistent with
glutamate flux via GDH and a persistent cellular acidosis, as well as catabolic (GSH?)
1382_C23.fm Page 379 Tuesday, October 7, 2003 6:58 PM

Chapter twenty-three: Acidosis and amino acid metabolism 379

Figure 23.2 Standard NH4Cl acid challenge for elucidating regulatory mechanisms on whole-body
and organ levels in humans. (Adapted from Sartorious, O.W. et al., J. Clin. Invest., 28, 423–439, 1949,
with plasma glutamine concentration and role of lung/pCO2 referenced in Welbourne and Joshi.23)

contributions to ammoniagenesis. After 4 to 6 days of the NH4Cl acid load, plasma


bicarbonate attains a steady state, at which point renal bicarbonate generation (as reflected
in the ammonium excreted, 190 mmol/day) matches the acid production (NH4Cl ingested,
190 mmol/day (Figure 23.2)). Driving the bicarbonate generation is the large glutamine
flux through the cytosolic PDG as the result of the adaptive increase in the high-affinity
glutamine transporter, SN1, appearing at both poles of the renal tubule cells, but now
significantly at the basolateral pole10,24 and increased PDG enzyme.27 Glutamine flux via
extracellular glutaminase is reduced due to the low bicarbonate and glutamine, so that
flux through the intracellular glutaminase is no longer checked by extracellular
glutamate.28 Consequently, the cytosolic glutamine/glutamate mix, as well as adaptive
increase in PDG, favors glutamine hydrolysis. On the other hand, glutamate flux through
the GDH pathway diminishes as indicated by only 1.3 ammonium produced per glutamine
extracted (Figure 23.3); that this reduced glutamate flux via the GDH pathway reflects a
rise in pHi is supported by a measured26 rise in pHi to 7.30 ± 0.02 vs. 7.16 ± 0.03 on day
1. However, this pHi, 7.30 ± 0.02, still represents a significant cellular acidosis (vs. 7.39 ±
0.04 for control). The partial restoration of pHi after 4 days of NH4Cl acid loading appar-
ently reflects the adaptive increase29,30 in NHE3-mediated acid/NH4+ secretion coupled to
sodium reabsorption as supported by the progressive decline in sodium excretion
(Figure 23.2). At this time the deficit in body fluid alkaline reserves, as reflected in the
1382_C23.fm Page 380 Tuesday, October 7, 2003 6:58 PM

380 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

175
Gln uptake
NH4Cl Acid Load
NH4+ produced NH4 + /GLN=1.30
150

NH4+ /GLN=2.51

125

100
um ole /m in

NH4+ /GLN=2.42

75
NH4 +/GLN=1.21

50

25

0
0 1 3 6
Da ys

Figure 23.3 Kidney glutamine extraction and ammonium production before and after standard
NH4Cl acid challenge in humans. (Adapted from Tizianello, A. et al., Contrib. Nephrol., 31, 40–46,
1982.)

plasma bicarbonate concentration (Figure 23.2), is maximal with the whole-body pHi,
indicating a significant intracellular acidosis (6.46 ± 0.036 vs. 6.70 ± 0.021 for 4-day standard
NH4Cl loading in humans vs. controls, respectively31). Thus, both the acute and chronic
renal metabolic responses to an exogenous NH4Cl acid load are associated with a reduction
in pHi, which, in the chronic phase, reflects the up-regulation of NHE3 (11), NBC (12),
PDG (4 and 9), and SN1 (1) protein levels.
Removing the exogenous NH4Cl acid load results in falling renal bicarbonate gener-
ation (ammonium excretion) with the rising of the alkaline reserves (Figure 23.2); declining
ammonium production reflects both the rise in plasma bicarbonate and glutamine con-
centration (the latter in parallel with the rise in pCO2 (Figure 23.2)) and the braking effect
of restored cytosolic glutamate and pHi on the rate-limiting functional glutaminase step
in the basogenic pathway (Figure 23.1 (4)).

23.4 Renal tubule cell response to an NH4Cl acid load


In order to study adaptive mechanisms on the molecular level, cellular models must
faithfully reflect in vivo acidosis in terms of both their acid–base balance and amino acid
metabolism. The acid–base balance response of cells derived from renal tubules to an
NH4Cl acid load can be studied32 in a manner analogous to that used for studying the
whole-body response to an oral NH4Cl load. As shown in Figure 23.4, proximal tubule-
like OK cells typically have a cytosolic pH ranging from approximately 7.21 to 7.1; expos-
ing them to an NH4Cl acid load leads immediately to a prompt rise in intracellular pHi
(pHi = 7.6) as a result of NH3 diffusion from the media into the cytosol where NH3 binds
free H+. Thereafter pHi declines in the presence of NH4Cl, reflecting the uptake of NH4+.
With the removal of the extracellular NH4Cl (recovery), intracellular NH3 rapidly
diffuses out of the cell, shifting accumulated cellular NH4+ to NH3 and free H+, causing a
plummeting of the pHi (pHi = 6.6). The free-falling pHi halts with H+ activation of the
NHE3 (Figure 23.1 (11)) at its pH-sensitive internal site3 and accelerated acid extrusion
(most rapid over the pHi range of 6.6 to 6.7) until the control pHi point is attained
(pHi = 7.18 after 12 min). Unlike the whole-kidney pHi and whole-body pHi responses
1382_C23.fm Page 381 Tuesday, October 7, 2003 6:58 PM

Chapter twenty-three: Acidosis and amino acid metabolism 381

Figure 23.4 pHi response to standard NH4Cl acid challenge in BCECF-loaded proximal tubule-like
OK cells. Cells-equilibrated HEPES-buffered Krebs Henseleit media (KHH) regulate pHi by NHE3-
mediated acid extrusion. Recovery (control) in KHH continuously monitored for 8 min, followed
by a 12-min measure. Second recovery in KHH plus 20 mM troglitazone. Rates pHi/min are super-
imposed over the lines drawn during recovery periods. Calibration performed with pH standards
in high potassium and 10 mM nigericin.

to the in vivo NH4Cl-induced extracellular acidosis (kidney pHi = 7.39 to 7.1926; whole-
body pHi = 6.76 to 6.4731), the response of pH-sensitive cultured cells to the NH4Cl-induced
cellular acidosis is a complete restoration of intracellular acid–base balance; that is, there
is no discernable acidosis error signal except for the very brief transient fall in pHi.
Nevertheless, this fleeting signal is sufficient to activate cell signaling pathways, e.g., c-Src,
a nonreceptor protein kinase.4,33 Even extracellular acidosis created by adding HCl to the
media and reducing pH to as low as 6.8 (far lower than blood pH observed with NH4Cl
loading in vivo (Figure 23.2)) fails to achieve a chronic cellular acidosis in either OKP,34
LLC-PK135 renal-derived cell lines, or osteoblasts36 because of their adaptive up-regulation
of the NHE-mediated acid extrusion. Consequently, attempts to study acidosis effects on
glutamine metabolism in pH-sensitive culture cell models must overcome the obstacle
that the expected decrease in pHi, as the result of either enhanced intracellular acid
production (NH4Cl loads, accelerated glycolysis) or exogenous acid loads (lowering media
pH to 6.8), activates NHE-mediated acid extrusion that returns the pHi to the normal
range. Because of the dominance of this acid extrusion response exhibited by pHi-respon-
sive cells in culture, it is difficult, at best, to assess the effects of cellular acidosis (clearly
shown to occur in vivo in both the kidney26 and whole-body intracellular pHi31 on amino
acid metabolism).
Fortunately, one can “clamp” this exaggerated NHE-mediated acid extrusion and
thereby reveal the direct effect of cellular acidosis on amino acid metabolism (and related
cellular processes), as shown in Figure 23.4. If one challenges these cells with a second
NH4Cl load (normally handled by NHE3 as effectively as the first — it would be of some
interest to perform this analogous experiment in vivo, applying a second NH4Cl load 24 h
after removing the first to test the acute response of the fully adapted kidney) but now
adds 20 mM troglitazone to the recovery media (troglitazone recovery), acid extrusion is
virtually eliminated. Note that after an initial attempted rally (first 15 sec), the pHi drops
to a level as low as, if not lower than, that observed after the first acid pulse. Clearly, this
abortive response is not for lack of H+ stimulus. Indeed, over the last 4 min of continuous
1382_C23.fm Page 382 Tuesday, October 7, 2003 6:58 PM

382 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 23.5 OK cells incubated for 18 h in DMEM containing 20 mM troglitazone and then loaded
with BCECF followed by equilibration in HEPES-buffered Krebs Henseleit media (KHH) plus
20 mM troglitazone. Subsequent acid challenge and recovery were in media containing 20 mM
troglitazone.

recording, the rate of acid secretion was 0.084 for the control recovery vs. 0.011 pHi/min
with troglitazone; after 12 min the pHi attained was only 6.67 compared to 7.18 for the
control recovery. These studies37 indicate that troglitazone-treated cells are unable to
recover their homeostatic pHi in response to an exogenous NH4Cl acid load. What happens
when these pHi-sensitive OK cells are chronically exposed to troglitazone? As shown in
Figure 23.5, surprisingly, pHi remains depressed, so that the pHi measured after 18 h,
6.68, is not different from that observed after 12 min. To determine whether this degree
of cellular acidosis resulted from inhibition of acid extrusion, the cells were pulsed with
NH4Cl and the NHE3-mediated acid extrusion monitored. As clearly seen, troglitazone
not only prevents the normal acid extrusion response to the NH4Cl load (Figure 23.4), but
also prevents expression of the adaptive response that normally occurs in response to
chronic acidosis.4 Acid extrusion in these cells, with NHE3 nonfunctional, presumably
relies upon proton-linked monocarboxylic acid transporters (MCT).

23.5 Glutamine/Glutamate metabolism in acidotic cells


The ability to “clamp” pHi at acidic levels provides a window through which to view the
molecular level effects of cellular acidosis on amino acid metabolism. Since cell lines such
as OK, LLC-PK1, and MDCK are renal tubule derived, the metabolic responses obtained
should measure up to those seen in situ for the acute metabolic acidosis phase (days 1
and 3 (Figure 23.2 and Figure 23.3)). The metabolic effects of troglitazone-induced cellular
acidosis on amino acid metabolism in several cell lines has previously been published.9,37
In the control state (pHi = 7.18), glutamine is metabolized predominantly to glutamate
and ammonium via number 4 and then to alanine via the transamination pathway (5), all
within the cytosol; at 20 mM troglitazone (pHi = 6.7) alanine formation is markedly reduced
1382_C23.fm Page 383 Tuesday, October 7, 2003 6:58 PM

Chapter twenty-three: Acidosis and amino acid metabolism 383

as ammonium formation increases, consistent with cytosolic acidosis shifting glutamate


into the mitochondrial GDH (7) ammonigenic pathway.9,37 Proof that cellular acidosis shifts
glutamine’s amino nitrogen into the mitochondrial GDH pathway (Figure 23.1, (7)) was
obtained using [2-15N] glutamine. At the normal pHi the predominant flux of glutamate
is via cytosolic ALT (Figure 23.1 (5)) and alanine formation9; cellular acidosis, pHi > 6.9,
induced by 20 mM troglitazone markedly inhibited alanine formed from [2-15N] glutamine
and proportionately increased ammonium formation.9,37 Note that other glitazones,
rosiglitazone, and ciglitazone had similar, but less potent, effects on both cell pHi and
ammoniagenesis/alanine formation.37 Since the acidosis-inhibitable ALT flux is predom-
inantly a cytosolic activity37,38 and GDH is exclusively a mitochondrial matrix activity,12
these findings are consistent with cytosolic acidosis shifting glutamate formed by the
functional glutaminase into the mitochondria, with both effects largely but not exclusively
(see effect of troglitazone on assayable ALT activity below) dependent upon the fall in
cytosolic pH. Troglitazone-induced cellular acidosis should provide a useful tool for the
effect of cellular acidosis on other pathways of amino acid metabolism in a variety of cell
lines.
In addition to peroxisome-proliferator-activated receptor (PPAR) gamma-independent
effects, troglitazone may alter gene expression via PPAR gamma transcriptional-related
regulation. For example, troglitazone decreases the assayable ALT activity in both proximal
tubule LLC-PK1-F+ cells9 and OK cells, but not in the MDCK37 cell line. Note that rates of
alanine formation in these cell lines are directly related to the level of assayable ALT,39
suggesting that troglitazone may act as a partial agonist40 via PPAR gamma signaling in
down-regulating the expression of cytosolic ALT; indeed, a PPRE promoter region is
present in the gene expressing cytosolic ALT.41 Troglitazone, a relatively weak PPAR
gamma ligand, is no longer prescribed as an antihyperglycemic therapy in patients with
type II diabetes mellitus because of hepatic toxicity of unknown origin.42 In this regard,
recent studies43,44 suggest that if alanine formed by the small intestine in vivo and released
into the portal vein becomes limiting in supporting urea synthesis (as a source of aspar-
tate), then the liver may undergo proteolysis in order to provide this nitrogen from
endogenous sources, a response not unlike that observed in metabolic acidosis.23
Whether hepatic toxicity due to troglitazone results from an imbalance between deliv-
eries of these ureagenic substrates, particularly in catabolic subjects, remains to be deter-
mined. Besides cytosolic ALT, another key enzyme, phosphoenolpyruvate carboxykinase
(PEPCK), which contributes to the adaptive response of glutamine metabolism during
metabolic acidosis, also contains a PPRE region in its promoter sequence.45 These responses
are reminiscent of the in situ renal tubule cell’s response to the chronic phase of acid
loading and suggest that endogenous prostanoids may play additional roles in acid–base
balance regulation.46 Extracellular acidosis, on the other hand, has far less of an effect on
intracellular glutamate metabolism, with only small increases in ammonium
formation20,39,47,48,49 and no change in alanine production20,49 or even an increase in alanine
production.39,47,48 The inability to observe the cellular acidosis-induced fall in cytosolic
alanine production may reflect adaptive increase in NHE-mediated acid extrusion and the
restoration of normal pHi in these cells as occurs in the parental line,34,35 or it may reflect
increased flux through the mitochondrial ALT (Figure 23.1 (8)) as the result of increased
mitochondrial glutamate uptake. Nevertheless, an adaptive increase in glutamine uptake
can be seen at both the apical and basolateral surfaces of the proximal tubule-like LLC-
PK1-F+ cell.20 In contrast, in vivo NH4Cl-induced metabolic acidosis produces a marked
decrease in pHi within the kidney26 and a large increase in ammonium production (Figure
23.3). Therefore, by suppressing the corrective NHE acid extrusion response in cultured
cells, troglitazone-induced cellular acidosis reveals potential direct effects of H+ on
1382_C23.fm Page 384 Tuesday, October 7, 2003 6:58 PM

384 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

glutamine/glutamate metabolism (as well as other pH-sensitive amino acid metabolic


pathways) and expression of various signaling pathways regulating gene expression.4,5,50

23.6 Cellular acidosis and glutamine metabolism contribute to


programmed cell death
Cells in culture are constantly under an acid challenge as a consequence of a high rate of
lactic acid production and a limited ability to generate net bicarbonate. Perhaps in order
to prevent a lethal fall in pHi in the face of this challenge, NHE-mediated acid extrusion
responds and maintains pHi within the normal range, as shown above. If this did not
occur, cellular acidosis would intervene, altering amino acid metabolism (as shown above)
and activating programmed cell death. Noteworthy is the fact that cellular acidosis by
itself can activate apoptosis at a pHi6,51,52 similar to the chronic pHi shown with troglitazone
in Figure 23.5. Cellular acidosis potentially plays important roles in activating apoptosis
via at least three sites by activating caspase 3,52,53 a proapoptotic protease that in turn
inactivates proteins vital for normal cellular function; activating endonucleases51 involved
in DNA laddering (for example, a number of nuclear localized endonucleases are activated
at pHi values of 6.9 to 7.0)51; and altering the mitochondrial inner membrane transmem-
brane potential (H+)6,52,54 directly or via promoting the binding of Bcl-2-like proteins that
lead to increased mitochondrial membrane permeability.55
An increase in mitochondrial permeability could have profound consequences for
amino acid metabolism and glutamine/glutamate specifically as depicted in Figure 23.6.
Because most of the mitochondrial PDG activity (Figure 23.1 (9)) is maintained latent
within the matrix space as a consequence of the high matrix space glutamate content,11,17
a permeability transition (PT) (Figure 23.6) that tends to equilibrate transmembrane H+
would be expected to activate this intramitochondrial glutaminase flux. Efflux of
glutamate from the matrix compartment as intramitochondrial pH falls would release
PDG from inhibition. At the accelerated glutaminase flux the additional glutamate efflux
could become coupled (10) to glutamine influx,12 resulting in an explosive metabolic
production of acid (NH4+) within the matrix space. Normally the glutaminase is functional
on the cytosolic surface (Figure 23.6, control) so that cytosolic ammonium formed is
released in the vicinity of NHE3. The ammonium formed within the alkaline matrix space,
pH = 8.0,52 is from the amino nitrogen of glutamate; this ammonium is converted to NH3
with the free H+ pumped out via the electron transport (ET) chain and the NH3 diffusing
into the acidic cytosol and secreted as NH4+. In contrast, in the proapoptotic condition
(Figure 23.6, apoptotic), the NH4+ formed from glutamine within the matrix presents an
endogenous acid and osmotic loads at the time when the electron transport chain capacity
is impaired and survival depends on the FoF1-ATPase/H+ pump operating in reverse.6
The metabolic cost of this stopgap maneuver is to accelerate nonvolatile acid production
(lactic acidosis) and accelerate influx of H+ into the matrix space via the PT pore. Conse-
quently, the stopgap pumping of protons out of the matrix would have to work against
the glutaminase-driven NH4+ production and accumulation contributing to matrix space
swelling, furthering the leakiness.56
Of note is the fact that the products of both glycolysis (acid) and glutaminase
(glutamate) accumulate in the media, reflecting the cellular acidosis and PT-mediated
effects.57 Whereas the lactate is symptomatic of multiple-organ failure in vivo,8 the buildup
of extracellular glutamate acid would activate excitotoxicity in the CNS 58,59 and might
even contribute to elevated systemic plasma glutamate concentrations.60 Thus, the activa-
tion of the mitochondrial glutaminase would deliver an acid load into the mitochondria
precisely at this critical juncture when mitochondrial integrity, and perhaps even cell death,
1382_C23.fm Page 385 Tuesday, October 7, 2003 6:58 PM

Chapter twenty-three: Acidosis and amino acid metabolism 385

Control Apoptotic
LAc- H+
MCT
LAcH
H+
ATP
Gluc
PT FoF1 ADP
GLN H+
NH4+ OH—
H+
GLN
4 GLU— NH NH4+ 9 10
3
H+ GLU—
H+ GLU—
GLU— 6 NH3
ET 6
+ +
NH4+ H
NH3+ H+
H+
11
11

NH4+
Na+ NH4+ Na+

Figure 23.6 Putative model of cellular acidosis-induced mitochondrial permeability increase and
intramitochondrial glutaminase flux activation. Left half (control) shows normal mitochondrial
function at cytosolic and matrix pH values of 7.20 and 8, respectively. Right half (apoptotic) shows
proapoptotic mitochondrial function as matrix pH approaches cytosolic pH lowered to approxi-
mately 6.6 with troglitazone. Numbers correspond to those listed in Figure 23.1.

hangs in the balance. If so, this perspective suggests that acid–base homeostasis and
glutamine metabolism may be interrelated on the whole-body, organ, cellular, and sub-
cellular levels in both physiological and pathophysiological conditions.

Acknowledgments
Studies were supported by the Southern Arizona Foundation (to T.W.) and NIH grants
DK-53761 and CA-79495 (to I.N.).

References
1. Kinney, J.M. and Elwyn, D.H., Protein metabolism and injury, Annu. Rev. Nutr., 3, 433–466,
1983.
2. Frassetto, L.A., Morris, C.R., and Sebastian, A., Effect of age on blood acid-base composition
in adult humans: role of age-related renal functional decline, Am. J. Physiol., 271, F1114–F1122,
1996.
3. Aronson, P.S., Nee, J., and Suhm, M.A., Modifier role of internal H+ in activating the Na+-
H+ exchanger in renal microvillus membrane vesicles, Nature (Lond.), 299, 161–163, 1992.
4. Alpern, R.J., Trade-offs in the adaptation to acidosis, Kidney Int., 47, 1205–1215, 1995.
5. Curthoys, N.P. and Gstraunthaler, G., Mechanism of increased renal gene expression during
metabolic acidosis, Am. J. Physiol., 281, F381–F390, 2001.
6. Matsuyama, S. and Reed, J.C., Mitochondria-dependent apoptosis and cellular pH regula-
tion, Cell Death Diff., 7, 1155–1165, 2000.
1382_C23.fm Page 386 Tuesday, October 7, 2003 6:58 PM

386 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

7. Tiefenthaler, M., Amberger, A., Bacher, N., Hartmann, B.L., Margeiter, R., Kofler, R., and
Konwalinka, G., Increased lactate production follows loss of mitochondrial membrane po-
tential during apoptosis of human leukemia cells, Br. J. Haemat., 114, 574–580, 2001.
8. Bakker, J., Gris, P., Coffernils, M., Kahn, R.J., and Vincent, J.-L., Serial blood lactate levels
can predict the development of multiple organ failure following septic shock, Am. J. Surg.,
171, 221–226, 1996.
9. Welbourne, T.C., Routh, R.E., Yudkoff, M., and Nissim, I., The glutamine/glutamate couplet
and cellular function, News Physiol. Sci., 16, 157–160, 2001.
10. Karinch A.M., Lin, C.-M., Wolfgang, C.L., Pan, M., and Souba, W.W., Regulation of expression
of the SN1 transporter during renal adaptation to chronic metabolic acidosis in rats, Am. J.
Physiol., 283, F1011–F1019, 2002.
11. Kvamme, E., Torgner, I.A., and Roberg, B., Evidence indicating that pig renal phosphate
dependent glutaminase has a functionally predominant external localization in the inner
mitochondrial membrane, J. Biol. Chem., 266, 12185–12192, 1991.
12. Schoolwerth, A.C. and LaNoue, K.F., Transport of metabolic substrates in renal mitochondria,
Annu. Rev. Physiol., 47, 143–171, 1985.
13. Welbourne, T.C. and Phromphetcharat, V.C., Renal glutamine metabolism and hydrogen ion
homeostasis, in Glutamine Metabolism in Mammalian Tissues, Haussinger, D. and Sies, H., Eds.,
Springer-Verlag, Berlin, 1984, pp. 161–177.
14. Heidiger, M.A. and Welbourne, T.C., Glutamate transport, metabolism and physiological
responses, Am. J. Physiol., 277, F477–F480, 1999.
15. Hediger, M.A., Glutamate transporters in kidney and brain, Am. J. Physiol., 277, F487–F492,
1999.
16. Welbourne, T. and Matthews, J., Glutamate transport and renal function, Am. J. Physiol., 277,
F501–F505, 1999.
17. Roberg, B., Torgner, I.A., Laake, J., Takumi, Y., Otterson, O.P., and Kvamme, E., Properties
and submitochondrial localization of pig and rat renal phosphate-activated glutaminase,
Am. J. Physiol., 279, C648–C657, 2000.
18. Peghini, P., Jaryen, J., and Stoffle, W., Glutamate transporter EAAC1 deficient mice develop
dicarboxylic aminoaciduria and behavioral abnormalities but no neural degeneration, EMBO
J., 16, 3822–3832, 1997.
19. Tanaka, K. and Welbourne, T., Enhanced ammonium excretion in mice lacking the glutamate
transporter GLT-1, J. Am. Soc. Nephrol., 12, AO287, 2001 (abstract).
20. Mu, X. and Welbourne, T., Response of LLC-PK1_F+ cells to metabolic acidosis, Am. J. Physiol.,
270, C920–C925, 1996.
21. Sartorious, O.W., Roemmmelt, J.C., and Pitts, R.F., The renal regulation of acid base balance
in man. IV. The nature of the renal compensations in ammonium chloride acidosis, J. Clin.
Invest., 28, 423–439, 1949.
22. Nissim, I., Yudkoff, M., and Brosnan, J.T., Regulation of [15] urea synthesis from [5-15N]
glutamine: role of pH, hormones, and pyruvate, J. Biol. Chem., 271, 31234–31242, 1996.
23. Welbourne, T.C. and Joshi, S., Interorgan glutamine metabolism during acidosis, J. Parenter.
Enteral Nutr., 14, 775–855, 1990.
24. Tizianello, A., DeFarrari, G., Garibotto, G., Robaudo, C., Bruzzone, M., and Passerone, G.C.,
Renal ammoniagenesis during the adaptation to metabolic acidosis in man, Contrib. Nephrol.,
31, 40–46, 1982.
25. Dass, P.D., Holmes, E.W., and Bermes, E.W., Hepatic and renal regulation of metabolite flow
in the remnant kidney model of chronic renal failure, Contrib. Nephrol., 92, 93–102, 1991.
26. Adam, W.R., Koretsky, A.P., and Weiner, M.W., 31P-NMR in vivo measurement of renal
intracellular pH: effects of acidosis and K+ depletion in rats, Am. J. Physiol., 251, F904–F910,
1986.
27. Curthoys, N.P. and Lowry, O.H., The distribution of glutaminase isoenzymes in the various
structures of the nephron in normal, acidotic, and alkalotic rat kidney, J. Biol. Chem., 248,
162–168, 1973.
28. Welbourne, T. and Nissim, I., Regulation of mitochondrial glutamine/glutamate metabolism
by glutamate transport: studies with 15N, Am. J. Physiol., 280, C1151–C1159, 2001.
1382_C23.fm Page 387 Tuesday, October 7, 2003 6:58 PM

Chapter twenty-three: Acidosis and amino acid metabolism 387

29. Kinsella, J., Cujdik, T., and Sacktor, B., Na+-H+ exchange activity in renal brush border
membrane vesicles in response to metabolic acidosis: the role of glucocorticoids, Proc. Natl.
Acad. Sci. U.S.A., 81, 630–634, 1984.
30. Amemiya, M., Yamaji, Y., Cano, A., Moe, O.W., and Alpern, R.J., Acid incubation increases
NHE-3 mRNA abundance in OKP cells, Am. J. Physiol., 269, C126–C133, 1995.
31. Tizianello, A., DeFerrari, G., Gurreri, G., and Acquarone, N., Effects of metabolic alkalosis,
metabolic acidosis and uraemia on whole-body intracellular pH in man, Clin. Sci. Mol. Med.,
52, 125, 1977.
32. Boron, W.F., Intracellular pH regulation in epithelial cells, Annu. Rev. Physiol., 48, 377–388,
1986.
33. Yamaji, Y., Tsuganewa, H., Moe, O.W., and Alpern, R.J., Intracellular acidosis activates c-Src,
Am. J. Physiol., 272, C886–C893, 1997.
34. Moe, O., Miller, T., Horie, S., Cano, A., Presig, P., and Alpern, R.J., Differential regulation of
Na/H antiporter by acid in renal epithelial cells and fibroblasts, J. Clin. Invest., 88, 1703–1708,
1991.
35. Igarashi, P., Freed, M.I., Ganz, M.B., and Reilly, R.F., Effects of chronic metabolism acidosis
on Na+-H+ exchangers in LLC-PK1 renal epithelial cells, Am. J. Physiol., 263, F83–F88, 1992.
36. Ori, Y., Lee, S.G., Krieger, N.S., and Bushinsky, D.A., Osteoblastic intracellular pH and
calcium in metabolic and respiratory acidosis, Kidney Intern., 47, 1790–1796, 1995.
37. Coates, G., Nissim, I., Battarbee, H., and Welbourne, T., Glitazones regulate glutamine me-
tabolism by inducing a cellular acidosis in MDCK cells, Am. J. Physiol., 283, E729–E737, 2002.
38. DeRosa, G. and Swick, R.W., Metabolic implications of the distribution of the alanine amino-
transferase isoenzymes, J. Biol. Chem., 250, 7961–7967, 1975.
39. Gstraunthaler, G.J.A., Ammoniagenesis in renal cell culture: a comparative study on ammo-
nia metabolism of renal epithelial cell lines, Contrib. Nephrol., 110, 88–97, 1993.
40. Camp, H.S., Ou, I., Wise, S.C., Hong, Y.H., Frankowski, C.I., Shen, X.Q., Vanbogelen, R., and
Leff, T., Differential activation of peroxisome proliferator activated receptor-gamma by tro-
glitazone and rosiglitazone, Diabetes, 49, 539–547, 2000.
41. Edgar, A.D., Tomkiewicz, C., Costet, P., Legendre, C., Aggerbeck, M., Bouguet, J., Staels, B.,
Guyomard, C., Pineau, T., and Barouki, R., Fenofibrate modifies transaminase gene expres-
sion via a proxisome proliferator activated receptor-dependent pathway, Toxicol. Lett., 98,
13–23, 1998.
42. Fujiwara, T. and Horikoshi, H., Troglitazone and related compounds: therapeutic potential
beyond diabetes, Life Sci., 67, 2405–2416, 2000.
43. Lopez, H.W., Moundras, C., Morand, C., Demigné, C., and Rémésy, C., Opposite fluxes of
glutamine and alanine in the splanchnic area are an efficient mechanism for nitrogen sparing
in rats, J. Nutr., 128, 1487–1494, 1998.
44. Brosnan, J.T., Brosnan, M.E., Yudkoff, M., Nissim, I., Daikhin, Y., Lazarow, A., Horyn, O.,
and Nissim, I., Alanine metabolism in the perfused rat liver: studies with 15N, J. Biol. Chem.,
276, 31876–31882, 2001.
45. Glorian, M., Duplus, E., Beale, E.G., Scott, D.K., Granner, D.K., and Forest, C., A single
element in the phosphoenolpyruvate carboxykinase gene mediates thiazolidinedione action
specifically in adipocytes, Biochimie, 83, 933–943, 2001.
46. Jones, E.R., Beck, T.R., Kapoor, S., Shay, R., and Narins, R.G., Prostaglandins inhibit renal
ammoniagenesis in the rat, J. Clin. Invest., 74, 992–1001, 1984.
47. Nissim, I., Sahai, A., Sandler, R., and Tannen, R.L., The intensity of acidosis differentially
alters the pathways of ammoniagenesis in LLC-PK1 cells, Kidney Int., 45, 1014–1019, 1994.
48. Nissim, I., States, B., Nissim, I., Lin, Z., and Yudkoff, M., Hormonal regulation of glutamine
metabolism by OK cells, Kidney Int., 47, 96–105, 1995.
49. Nissim, I. and States, B., Ammoniagenesis pathways in cultured human renal epithelial cells:
study with 15N, Am. J. Physiol., 286, F187–F196, 1989.
50. Gstraunthaler, G., Holcomb, T., Feifel, E., Liu, W., Spitaler, N., and Curthoys, N.P., Differential
expression and acid base regulation of glutaminase mRNAs in gluconeogenic LLC-PK1-
FBPase cells, Am. J. Physiol., 278, F227–F237, 2000.
1382_C23.fm Page 388 Tuesday, October 7, 2003 6:58 PM

388 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

51. Gottlieb, R.A., Nordberg, J., Skowronski, E., and Babior, B.M., Apoptosis induced in Jurkat
cells by several agents is preceded by intracellular acidification, Proc. Natl. Acad. Sci. U.S.A.,
93, 654–658, 1996.
52. Matsuyama, S., Llopis, J., Deveraux, Q.L., Tsien, R.Y., and Reed, J.C., Changes in intramito-
chondrial and cytosolic pH: early events that modulate caspase activation during apoptosis,
Nat. Cell Biol., 2, 318–325, 2000.
53. Segal, M. and Beem, E., Effect of pH, ionic charge, and osmolality on cytochrome c-mediated
caspase-3 activity, Am. J. Physiol., 281, C1196–C1204, 2001.
54. Adachi, S., Cross, A.R., Babior, B.M., and Gottlieb, R.A., Bcl-2 and the outer mitochondrial
membrane in the activation of cytochrome c during Fas-mediated apoptosis, J. Biol. Chem.,
272, 21878–21882, 1997.
55. Kubasiak, L.A., Hernandez, O.M., Bishopric, N.H., and Webster, K.A., Hypoxia and acidosis
activate cardiac myocyte death through the Bcl-2 family protein BNIP3, Proc. Natl. Acad. Sci.
U.S.A., 99, 1285, 2002.
56. Zieminska, E., Dolinska, M., Lazarewicz, J.W., and Albrect, J., Induction of permeability
transition and swelling of rat brain mitochondria by glutamine, Neurotoxicology, 21, 295–300,
2000.
57. Langford, M.P., Chen, D., Welbourne, T.C., Redens, T.B., and Ganley, J.P., Stereoisomer
specific induction of renal cell apoptosis by synthetic muramyl dipeptide (N-acetylmuramyl-
L-alanyl-D-isoglutamine), Mol. Cell Biochem., 236, 63–73, 2002.
58. Newcomb, R., Sun, X., Taylor, L., Curthoys, N., and Giffard, R.G., Increased production of
extracellular glutamate by the mitochondrial glutaminase following neuronal death, J. Biol.
Chem., 272, 11276–11282, 1997.
59. Mena, F.V., Baab, P.J., Zielke, C.L., and Zielke, H.R., In vivo glutamine hydrolysis in the
formation of extracellular glutamate in the injured rat brain, J. Neurosci. Res., 60, 632–641,
2000.
60. Droge, W., Eck, H.P., Betzler, M., and Naher, H., Elevated plasma glutamate levels in col-
orectal carcinoma patients and in patients with acquired immunodeficiency syndrome
(AIDS), Immunobiology, 174, 473–479, 1987.
1382_C24.fm Page 389 Tuesday, October 7, 2003 7:00 PM

chapter twenty-four

Muscle protein and amino acid


metabolism with respect to
age-related sarcopenia
Stéphane Walrand
Centre de Recherche en Nutrition Humaine
Yves Boirie
Centre de Recherche en Nutrition Humaine

Contents
Introduction: definition and consequences of sarcopenia during aging.............................389
24.1 Whole-body protein turnover in elderly ........................................................................390
24.2 Muscle protein turnover in aged individuals................................................................392
24.2.1 Total mixed muscle proteins..............................................................................392
24.2.2 Specific muscle proteins .....................................................................................393
24.2.2.1 Myofibrillar proteins ...........................................................................393
24.2.2.2 Mitochondrial proteins .......................................................................395
24.2.2.3 Sarcoplasmic proteins .........................................................................396
24.3 Amino acid metabolism and therapy during physiological and pathological
muscle loss in aged individuals.....................................................................................396
24.3.1 Effect of amino acid intervention on sarcopenia............................................396
24.3.2 Amino acid intervention during stress in aging individuals: impact
of glutamine..........................................................................................................398
24.4 Concluding remarks.........................................................................................................399
References .....................................................................................................................................399

Introduction: definition and consequences of sarcopenia during aging


The Greek word sarco refers to flesh and penia indicates a deficiency. Sarcopenia is a generic
term for the loss of skeletal muscle tissue that comes with age. Sarcopenia is distinct from
wasting, which is largely due to inadequate nutritional intake and malabsorption associ-
ated with starvation, cancer, or acquired immunodeficiency syndrome, and cachexia,

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 389
1382_C24.fm Page 390 Tuesday, October 7, 2003 7:00 PM

390 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

which primarily represents an inflammatory cytokine-driven process resulting in acceler-


ated muscle protein degradation.1,2
Sarcopenia is not a disease but rather refers specifically to the involuntary decline in
lean body mass. The cumulative decline in muscle mass reaches 40% from 20 to 80 years.3,4
The depletion of muscle mass does not result in weight loss, suggesting that a correspond-
ing accumulation of body fat occurs; hence, fat mass increases from 18 to 36% in elderly
men.5 These comparative data involving various classes of age have been recently con-
firmed in longitudinal studies, suggesting that like bone mass decline, muscle mass reduc-
tion might be modelized.6–8 Qualitatively, this muscle atrophy corresponds to a gradual
and selective loss of muscle fibers leading to a modification of muscle constitution,
although still debated. The number of fibers IIb (glycolytic) and IIa (oxido-glycolytic) is
decreased, whereas the number of fibers I (oxidative) remains unchanged.9,10 This results
in a relative elevation in fiber I density related with a preservation of muscle endurance
and a reduction in muscle strength in elderly persons. In addition to the muscle mass
decline, there is also an increase in fat and collagen contents between fibers. These changes
in contractile tissue decrease locomotive abilities but may also alter metabolic functions
of muscles in aged individuals.
Sarcopenia has important consequences in terms of muscle strength. Healthy people
in the seventh and eighth decades score on average 20 to 40% less during isometric and
concentric strength tests than young adults, and the very old show even greater (50% or
more) reduction. As a result, and because most of the loss of muscle mass with age occurs
in the lower body, many older individuals have impaired mobility and an increased risk
of falls and hip fractures, and a considerable number require assistance with everyday
activities.11,12 The functional consequences of the loss in muscle mass in the elderly place
those with more than 2 standard deviations below young controls at a three to four times
greater risk of disability and a two to three times greater risk of falls. In all developed
countries, the number of elderly is increasing, and the effects on the health care system
may be substantial in the years to come.13 Therefore, sarcopenia may now be considered
as one of the major public health problems in the aging population.
Unlike fat, which is truly stored as a reserve for energy disposal and eventually times
of starvation, body proteins are involved in many functional activities such as contractile
proteins in muscle, enzymes, hormones, antibodies, etc.1 Thus, loss of proteins also means
loss of function. Sarcopenia may therefore contribute to the reduced ability to withstand
physical, environmental, or immunological stresses in old age. In addition, the reduction
in muscle mass accelerates muscle fatness, leading to the well-known sarcopenic obesity.
This phenomenon results from a number of factors, but chief among these causes are a
declining metabolic rate and activity level coupled with an energy intake that does not
match the reduced need for energy.14 Finally, skeletal muscle and its age-related decreased
metabolism may contribute to the changes in insulin sensitivity. It is well established that
aging is associated with decreased glucose tolerance that is related to changes in body
composition and physical activity, but the impact of insulinoresistance on amino acid
metabolism, together with the sensitivity to substrates, still needs to be defined.
For these reasons, strategies for prevention of muscle mass and strength losses with
advancing age may be an important way to decrease the prevalence of many age-associ-
ated chronic diseases and increase functional independence and quality of life.

24.1 Whole-body protein turnover in elderly


Protein mass results from a continuous equilibrium between protein synthesis and
breakdown, which together constitute protein turnover. Many studies (for reviews see
1382_C24.fm Page 391 Tuesday, October 7, 2003 7:00 PM

Chapter twenty-four: Muscle protein and amino acid metabolism 391

Beaufrère and Boirie15 and Millward et al.16) have concluded that there is a decline in
whole-body protein turnover per kilogram body weight with advancing age in both
sexes. For example, Young and co-workers17 first reported in humans a decrease in whole-
body protein synthesis from birth to old age. Others18–20 described a significant reduction
of whole-body protein synthesis in healthy, well-nourished elderly compared to young
adults. Nevertheless, the studies vary considerably in terms of methodology and design
so that comparisons are difficult. These include different tracer methods (15N-glycine end
products, 13C-leucine precursor) and different nutritional states (postabsorptive vs. fed
states) or dietary intake on previous days. In addition, because of differences in body
composition between young and elderly adults, whole-body protein synthesis and break-
down have to be examined in relation to indices of lean body mass. When expressed
per kilogram of body weight, whole-body turnover declines with age, but remains
constant or slightly higher when normalized per unit of lean body mass.16,21–23 Thus,
because older people lose fat-free mass but not weight and because adipose tissue
contributes little, if any, to protein turnover, it is possible that the difference observed
in whole-body protein synthesis in the elderly is due to their increased fat mass. In
addition, the apparent discrepancy in the above outcome can also be explained by the
fact that whole-body protein turnover represents the average of many different protein
pools that turn over at different rates. For example, heart, liver, kidney, intestine, and
stomach tissues have 2 to 10 times higher protein synthesis rates than skeletal muscle.24,25
While the muscle accounts for 45 to 50% of total body mass and 80 to 85% of fat-free
mass in young, lean healthy subjects, it generally contributes less than 30% to whole-
body protein turnover.25 Therefore, small changes in muscle protein synthesis and break-
down are difficult to detect with measurement of whole-body protein turnover.26 This
effect is magnified in older individuals since muscle mass accounts for less than 35% of
body mass and only 40% of fat-free mass in those over 65 years of age.26 The smaller
total amount of proteins turning over in elderly people results in a reduced contribution
of muscle to total body protein metabolism from 30 to 20%, suggesting a redistribution
of proteins from muscle to splanchnic organs.27,28 Other data confirmed the decrease in
muscle participation to whole-body protein turnover and also demonstrated the relative
preservation of the activity of protein metabolism within the gut, liver,21,27,29 and white
blood cells (Walrand and Boirie, unpublished data). These studies21,29,30 reported or
suggested firmly that the nitrogen splanchnic extraction, i.e., the fraction of dietary amino
acids that is taken up by the gut and liver on its first pass, was markedly increased in
elderly people, probably due to the amino acid needs for protein and energy metabolism
in these compartments. This might also result in a lower availability of dietary amino
acids to peripheral tissues, i.e., muscle, although it has not been confirmed from phenyl-
alanine metabolism study.29
A study by Balagopal et al.31 found an age-related decline in whole-body protein
turnover even correcting the results for fat-free mass. From young to old, whole-body
protein synthesis decreased by 19%. These authors highlighted the importance of care in
the selection of volunteer populations and body composition methods in aging population
studies.32 Whole-body measurements of protein turnover are susceptible to be influenced
by protein intake on the previous days. Without careful control, variability related to
dietary habits of the subjects may mask the changes in protein metabolism with aging. In
the report by Balagopal et al.,31 diet and physical activity were closely controlled during
the week prior to the measurements. In addition, the decline in muscle mass with aging
may be underestimated by the use of dual-energy X-ray absorptiometry (DEXA) when
compared with estimates based on urinary creatinine output.33
Finally, concerning the relationship between whole-body protein turnover and energy
expenditure, it is noticeable that energetic cost of protein turnover during aging is not
1382_C24.fm Page 392 Tuesday, October 7, 2003 7:00 PM

392 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

changed compared to young adults.34 This observation implies that reduction of energy
expenditure with age may involve other aspects of substrate metabolism such as a decrease
in fatty acid oxidation.35

24.2 Muscle protein turnover in aged individuals


24.2.1 Total mixed muscle proteins
In aged animals, data have indicated that muscle protein synthesis is reduced in the
postabsorptive state either in relative (fractional protein synthesis rate, percent per hour36)
or in absolute values (milligrams per hour37,38). In humans, sophisticated techniques using
a combination of microbiopsy samples or arteriovenous catheterization across the limb in
conjunction with labeled amino acid infusions allow examination of the regulation of
protein turnover within skeletal muscle. These studies reported that elderly people have
reduced rates of mixed muscle protein synthesis (Table 24.1).31,39–46 Yarasheski et al.40 found
that the total mixed protein synthesis rate was 38% lower in the elderly. However, other
authors42 using arteriovenous balance and measurement of fractional synthesis rate did
not find any modification with age. These results are still debated, especially with regards
to the population selected.47
The molecular alterations of muscle protein synthesis are still poorly understood.
Welle et al.48 have demonstrated that the total mRNA content in aged muscle was identical
to that observed in young controls. This would indicate that transcription is not affected
in human muscle as it was already described in animals.49 From in vitro data,49 it seems
that aging results in a decline in the efficiency and accuracy of ribosomes, and in alterations
of tRNAs and elongation factors. Much work remains to be done to describe better the
mechanism of mixed muscle protein synthesis reduction in aged people. In addition,
because the mixed muscle protein synthesis rate represents the average measurement of
many different proteins within the muscle pool, i.e., myofibrillar, mitochondrial, and
sarcoplasmic protein fractions, this approach represented only the first step in understand-
ing muscle protein regulation. Finally, it is likely that posttranslational modifications of
proteins occur with aging. The impact of theses changes on protein function and metab-
olism is a growing field of challenging studies (for a review see Tavernarakis and
Driscoll50).

Table 24.1 Muscle Protein Synthesis Changes in Elderly Subjects


Aged vs. Young Adults References
Total mixed muscle proteins Ø 39–41
Æ 42
Mixed myofibrillar proteins Ø 43
MHC Ø 31, 44
Actin Æ 44
Mixed mitochondrial proteins Ø 45
Mixed sarcoplasmic proteins Æ 31
Ryanodine receptora Ø 46
Ca2+-ATPasea Ø 46
53-kDa glycoproteina Æ 46
Calsequestrina ≠ 46
a Measured in rats.
1382_C24.fm Page 393 Tuesday, October 7, 2003 7:00 PM

Chapter twenty-four: Muscle protein and amino acid metabolism 393

Basal
AA administration

0.12
* *

0.1 *
*
Protein fractional synthesis rate (%/h)

0.08

0.06

0.04

0.02

0
Elderly Young Elderly Young Elderly

IV AA PO AA PO AA + glucose

Figure 24.1 Response of mixed muscle protein anabolism to amino acid infusion, oral amino acid,
or oral amino acid–glucose mixture administrations in young and elderly healthy subjects.29,52,53,
*p < .05 vs. basal situation. IV, intravenous; PO, per os; AA, amino acid.

24.2.2 Specific muscle proteins


24.2.2.1 Myofibrillar proteins
Halliday and McKeran51 were among the first to measure the synthesis rate of myofibrillar
proteins, a subcellular pool of several proteins, comprised mainly in the muscle contractile
apparatus but possibly also containing some mitochondrial elements.32 Welle et al.,43 using
the same approach, have shown that the fractional synthesis rate of myofibrillar proteins
is reduced by about 28% in 62- to 81-year-old adults (Table 24.1). These findings are similar
to those reported by Yarasheski et al.40 for mixed muscle proteins. Nair’s group (for a
review see Short and Nair32) determined the fractional synthesis rate of individual con-
tractile muscle proteins (Figure 24.1).29,52,53 These authors observed that the synthesis rate
of myosin heavy chain (MHC) is significantly reduced in middle-aged (31%) and older
(44%) men and women.31 MHC is the principal molecular motor of contractile activity
based on its ability to hydrolyze ATP to generate force. The synthesis rate of MHC was
correlated to muscle strength,31 demonstrating the importance of this mechanism for the
reduction of contractile capacity in elderly. Another key observation from these studies is
that aging effects on MHC fractional synthesis rate were detected by middle age (45 to 55
years), which is earlier than previously reported for myofibrillar synthesis rate studies
comparing young and old groups.40,41,43,54 A recent interesting work by Hasten and co-
workers44 compared the fractional synthesis rate of mixed muscle proteins with the rates
of MHC and actin in 23- to 32-year-olds and 78- to 84-year-olds, by using electrophoretic
procedures to isolate individual contractile proteins (Figure 24.1). The younger subjects
had greater mixed protein and MHC synthesis rates than the older participants. In
1382_C24.fm Page 394 Tuesday, October 7, 2003 7:00 PM

394 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

addition, the basal MHC synthesis rate was equivalent to 80% of the corresponding mixed
muscle protein synthesis rate in the young adults, whereas MHC synthesis was equivalent
to only 65% of the mixed muscle protein synthesis rate in the older group. The rate of
actin synthesis was similar in the younger and older groups and faster than mixed or
MHC protein synthesis rates.44 However, this finding was limited to a small number of
subjects, i.e., four older individuals, leading to a considerably greater measurement vari-
ability for actin.44 As a result of their lower whole-body skeletal muscle mass, elderly
people had lower absolute rates (grams of protein synthesized per day) of mixed, MHC,
and actin protein synthesis than young adults.44
The decrease in myofibrillar protein synthesis rate is not caused by reduced mRNA
availability encoding MHC or actin,48 revealing again the potential role of posttranslational
events in this alteration. The slowing rate of protein synthesis associated with aging in
some animal models has been attributed to reduced expression of elongation factor-1a
(which catalyzes the binding of aminoacyl-tRNAs to ribosomes), but in human muscle
the expression of this enzyme does not decline with age.55
Another important aspect of protein turnover is the rate of protein degradation
through specific proteolytic pathways. A recent work56 evaluated the relationship between
age-related muscle atrophy and increased rate of protein degradation in extensor digitorum
longus muscles from young (2 to 4 months), middle-aged (12 to 17 months), and old (22
to 24 months) mice. In this study, the rate of protein degradation, assessed by tyrosine
release during in vitro incubations, was higher in muscles from old and middle-aged
animals than it was in those from the younger group. In addition, an inverse relationship
between muscle mass and proteolysis rate was observed.56 Recently, various pathways of
protein degradation have also been studied at the molecular level in relation to aging. For
example, studies on the activities of calpains57 and lysosomes58 have shown the accumu-
lation of calpains II and inactive cathepsin D in muscles of old rats, respectively. Dardevet
et al.59 did not find any changes in the ubiquitin–proteasome pathway of old rat muscles
compared to those of adult animals, but another study60 reported that 20S proteasome
proteolytic activity in the muscle of very old rats declined along with changes in the level
of 20S subunits, whereas the quantity of ubiquitin-linked protein remained constant.58
This new area of research is promising, especially in relation to the changes in posttrans-
lational modifications of individual muscle proteins.
In humans, the output of 3-methylhistidine (3-MH) serves as a noninvasive index of
the rate of myofibrillar protein breakdown in vivo. The daily excretion of 3-MH in urine
is quantitatively related to its rate of release from the myofibrillar proteins in the skeletal
musculature since it is not used in any metabolic pathway. Results on 3-MH excretion in
elderly are conflicting. The urinary excretion of 3-MH in groups of healthy young adults
and elderly people was determined by Uauy et al.28 in the 1970s. These authors reported
that urinary output of this amino acid was lower for elderly subjects. However, this
appeared to be due to the reduced muscle mass, because 3-MH excretion per unit of
creatinine output (3-MH/creatinine), which estimated the size of muscle mass, did not
differ significantly between the two age groups.28,61 A more recent work44 based on
3-MH/creatinine determinations showed that myofibrillar proteolysis was significantly
increased in elderly in comparison with young adults. These authors44 postulated that in
combination with the lower rates of contractile protein synthesis observed in old subjects,
the elevated myofibrillar protein breakdown contributed to the muscle wasting that
accompanies advanced age. The discrepancies between these studies may be explained
by the fact that sources other than muscles, such as flesh-containing diet or smooth
musculature, can also contribute to the total daily output of this amino acid. Sjölin et al.62
determined the release of 3-MH from the splanchnic region and from the leg, and the
contributions to the increase in urinary 3-MH excretion during infection by using
1382_C24.fm Page 395 Tuesday, October 7, 2003 7:00 PM

Chapter twenty-four: Muscle protein and amino acid metabolism 395

arteriovenous catheterizations. These authors concluded that skeletal muscle is the source
of 3-MH and concluded that this amino acid can be used as a marker of myofibrillar
protein catabolism.

24.2.2.2 Mitochondrial proteins


Taking advantage of recent technical progress in mass spectrometry, Rooyackers et al.45
published the first in vivo determination of muscle mitochondrial protein synthesis rate
in vastus lateralis muscle from healthy elderly (Table 24.1). These authors reported a
pronounced decline (40%) in mitochondrial protein synthesis rates of middle-aged
(54 years old) subjects in comparison with adult (24 years old) individuals, but no further
decrease in seven elderly (73 years old) people. Mitochondrial proteins consist, in large
part, of enzymes involved in energy production (b-oxidation, tricarboxylic acid cycle,
electron transfer chain, and oxidative phosphorylation). It is therefore likely that the
reduction in the mitochondrial protein turnover rate is associated with a decline in skeletal
muscle oxidative capacity63 and mitochondrial function.64 For example, in most of the
biochemical studies devoted to muscle mitochondrial respiratory chain, an age-related
decline in bioenergetic activity has been observed.65,66 Cytochemical-immunocytochemical
studies of the respiratory chain enzymes from old monkeys reported defects of ubiquinone
cytochrome-c-oxidoreductase (complex III), of cytochrome C oxidase (COX) (complex IV),
and of ATP-synthase (complex V) in the limb muscles, diaphragm, heart, and extraocular
muscles.67 In addition, an almost 10-fold increase in the incidence of COX defective fibers
was noted in limb muscle and diaphragm from humans in their eighth and ninth decades
compared with those between their third and sixth decades.67–69 The affected muscle fibers
showed normal succinate dehydrogenase (complex II) activity, a protein encoded by the
nuclear genome.68 Since COX is encoded by mitochondrial genome, these authors con-
cluded that reduced mitochondrial DNA (mtDNA) copy number, which is proportional
to muscle oxidative capacities,70 and increased mtDNA mutation during aging could have
major effects on mitochondrial encoded transcript levels and enzyme activities in muscle.
Several studies have reported increased damage of mtDNA in different tissues with aging
(for reviews see Lenaz et al.63 and Brand71), including human skeletal muscle mitochon-
dria.72 Increased oxidative damage has been suggested as a potential candidate for mtDNA
mutations during aging.73 Mitochondrial DNA damage could limit mitochondrial gene
expression at the level of transcription. Welle et al.74 used a large-scale screening approach
to identify several mitochondrial and nuclear encoded mRNAs for mitochondrial proteins
that were present at reduced levels in aging human skeletal muscle. The 20% lower
abundance of many of these gene transcripts in old vs. young subjects was also confirmed
by quantitative polymerase chain reaction assays.74 Therefore, it is concevable that the
elevated mtDNA damage is the proximate cause of the reduced protein synthesis rate in
mitochodrion and the consequent decline in muscle mitochondrial content and function
in elderly.45 These conclusions are in agreement with the free radical theory of aging75 if
mtDNA mutations are related to increased reactive oxygen species (ROS) production or
decreased capacity to protect the organite from its toxicity by an altered ROS trapping
system.76
It is intriguing that the mitochondrial enzyme activities continue to decline after
middle age, whereas mitochondrial protein synthesis rates did not decrease any further.45
This may be due to an elevated mitochondrial protein breakdown or an increased damage
to mitochondrial protein, leading to reduced enzyme activities at an older age. Anyway,
the alteration of the synthesis steps of the muscle remodeling process is likely to reduce
the quality and quantity of mitochondrial proteins. Such a decline may impair oxidative
and endurance capacities and then may alter muscle performance. Moreover, a great part
of the mitochondrial proteins need to be imported into the organite and associated with
1382_C24.fm Page 396 Tuesday, October 7, 2003 7:00 PM

396 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

the mitochondria encoded proteins to be functional. However, studies dealing with protein
import into mitochondria during aging are missing.

24.2.2.3 Sarcoplasmic proteins


Balagopal et al.31 reported that the sarcoplasmic protein synthesis rate in quadriceps
muscle did not vary in young, middle-aged, and old subjects (Table 24.1). Nevertheless,
in this study, the ratio of fractional synthesis rate of sarcoplasmic proteins to total mixed
muscle protein increased from young to old-age volunteers. These data indicated that
contribution of the synthesis rate of sarcoplasmic protein increased with age, whereas
contribution of synthesis rate of MHC and mitochondrial proteins decreased.31
Sarcoplasmic protein fraction is involved in many functions such as anaerobic ATP
production, intracellular transport, and many other enzyme activities. More effort directed
at purification of individual sarcoplasmic proteins is then warranted. For example, a
report46 has shown that the major calcium regulatory proteins of the sarcoplasmic reticu-
lum (Ca2+-ATPase and ryanodine) from skeletal muscle of aged rats exhibited 25% slower
protein turnover in vivo relative to that from young muscle. This observation may explain
the slower relaxation rates and times, decreased sarcoplasmic reticulum Ca2+ uptake, and
Ca2+-ATPase activity after percutaneous electrical stimulation of the quadiceps femoris in
elderly subjects.77 More interestingly, Ferrington et al.46 observed that the effect of aging
on the protein synthesis rate in skeletal muscle sarcoplasmic reticulum was specific to
individual proteins; the ryanodine receptor and Ca2+-ATPase experienced slower relative
rates of turnover, whereas other sarcoplasmic reticulum proteins, such as 53-kDa glyco-
protein or calsequestrin, exhibited higher or no changed protein synthesis rate, respec-
tively. These results emphasize the importance of examining individual proteins as
opposed to mixtures of proteins because the significant changes in turnover of specific
proteins that may occur with age could be masked by other proteins that are found in
greater abundance or have significantly different half-lives. Conversely, the consequences
of alterations in individual protein synthesis should be analyzed at the tissue level, i.e.,
in an integrative manner, to consider the complexity and the regulation of the systems.

24.3 Amino acid metabolism and therapy during physiological


and pathological muscle loss in aged individuals
24.3.1 Effect of amino acid intervention on sarcopenia
As it has been mentioned above, basal protein metabolism is lower or unchanged in muscle
of aging individuals. A defect in postprandial anabolism with age has been also reported
in rodents78 and humans.79 It has been proposed to be one of the mechanisms responsible
for the loss of muscle mass during aging. Postprandial stimulation of muscle protein
synthesis in rats originated mainly from absorbed amino acids because this stimulation
was observed after feeding a high-protein meal but not after an isoenergetic protein-free
meal.80 Similar observations have been made in humans81,82 and suggest that amino acids
are essential in the regulation of postprandial muscle protein metabolism. From these
studies, some authors have advocated the use of amino acid supplements to limit sar-
copenia.
By using femoral arteriovenous catheterization and quadricep muscle biopsies, Volpi
and colleagues52 measured muscle protein synthesis and breakdown, and amino acid
transport during intravenous infusion of an amino acid mixture in young and elderly
subjects (Figure 24.1). Peripheral amino acid infusion significantly increased amino acid
delivery to the leg, amino acid transport, and muscle protein synthesis whatever the age
1382_C24.fm Page 397 Tuesday, October 7, 2003 7:00 PM

Chapter twenty-four: Muscle protein and amino acid metabolism 397

of the volunteers. Despite no change in protein breakdown during amino acid infusion,
a positive net balance of amino acids across the muscle was achieved. The authors con-
cluded that although muscle mass is decreased in the elderly, muscle protein anabolism
can nonetheless be stimulated by increased amino acid availability.52 The same group29
determined the muscle protein turnover and amino acid transport in healthy young and
elderly people during an oral administration of an amino acid mixture (Figure 24.1). As
already showed by Boirie et al.,21 amino acid first-pass splanchnic extraction was signifi-
cantly higher in the elderly during ingestion of amino acids, but the delivery to the leg
increased to the same extent in both groups in Volpi et al.’s study. In addition, amino acid
transport into muscle, muscle protein synthesis, and net balance increased similarly in
both the young and the elderly.29 Thus, despite an increased splanchnic extraction, muscle
protein anabolism can be stimulated by oral amino acids in the elderly as well as in young
subjects. Interestingly, muscle protein synthesis increased to the same extent after an oral
intake of either balanced amino acids or essential amino acids in healthy elderly.83 There-
fore, nonessential amino acids appear not to be required to stimulate muscle protein
anabolism in older adults. In a third experimentation, Volpi et al.53 evaluated the same
parameters during the oral administration of an amino acid–glucose mixture (Figure 24.1).
The nutritional intervention increased amino acid delivery and transport into the muscle
and decreased muscle protein breakdown in both groups. However, the stimulation of
muscle protein synthesis in the young was not depicted in the elderly despite the amino
acid–glucose mixture ingestion.53 The response of muscle protein anabolism to hyperam-
inoacidemia with endogenous hyperinsulinemia seems to be impaired in healthy elderly
as a result of a blunted response of protein synthesis. Studies by Volpi et al.29,53 help to
understand the effects of the route and the nonprotein substrates added to amino acids
on net muscle protein anabolism in young and elderly subjects.Whereas in young adults
the addition of glucose to an amino acid mixture increased the effect of amino acids alone
in stimulating muscle protein anabolism, such a combination in the elderly did not add
a real benefit compared with that of amino acids alone. These studies lead us to open the
question of muscle sensitivity to hormones like insulin or substrates like amino acids
during aging. These observations may explain the lack of anabolic response to complete
meal in aged muscle,78 which is likely to contribute to the development, over the long
term, of sarcopenia in the elderly.
The alteration of muscle protein response to anabolic signals may be counteracted by
nutritional strategies aimed at increasing amino acid availability to the muscle tissue or
improving amino acid efficiency via specific signaling of branched-chain amino acids.38,84
Dardevet et al.85 reported that physiological concentration of leucine reproduced the
stimulating effect of total amino acids on adult rat muscle protein synthesis in vitro.
Because leucine reproduced the anabolic effect of total amino acids, these authors hypo-
thetized that this effect was not dependent on the amino acid concentration itself but on
a specific signal initiated by leucine. Using specific inhibitors, Dardevet et al.85 showed
that the intracellular targets of this amino acid were phosphatidylinositol 3¢ kinase and
the rapamycin-sensitive pathway involving mammalian target of rapamycin (mTOR) and
p70 S6 kinase. These observations confirmed that leucine stimulated muscle protein syn-
thesis by enhancing the efficiency of protein translation.86 Dardevet et al.85 also reported
that in vitro muscle protein synthesis still responded to the leucine signal in old rats but
required a two to three times greater leucine concentration than in young adult rodents.
More recently, the same authors38 observed that in vivo leucine supplementation had no
additional effect on postprandial muscle protein synthesis in adult rats, but totally restored
its stimulation in old rats. In this experiment, leucine concentrations in plasma reached
supraphysiologic levels in both age groups (two times the control postprandial values).
This study confirmed in vivo the hypothesis that old rat muscles are less sensitive to leucine
1382_C24.fm Page 398 Tuesday, October 7, 2003 7:00 PM

398 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

but are still able to respond when the concentration of this amino acid is greatly
increased.38,85 In a recent report, Guillet et al.87 also revealed that leucine-supplemented
diet improved the age-related alteration of myofibrillar and sarcoplasmic protein synthesis
response after meal intake in rat. Taken as a whole, these data demonstrate that a nutri-
tional manipulation increasing the availability of leucine to muscle could be beneficial in
maintaining the postprandial stimulation of protein synthesis during aging.
Taurine is also important for normal development and proper function of the excitable
tissues of mammals, such as skeletal muscle.88 Pierno et al.89 postulated that decline in
plasma and tissue taurine content during aging90 may contribute to the abnormalities in
the morphology and function of skeletal muscle. They demonstrated that in vivo taurine
supplementation restored taurine content in old rat muscles toward that found in adult
rodents and ameliorated the electrical and contractile properties of this tissue.89 Therefore,
taurine may also have a potential application in ensuring normal muscle function in the
elderly.
Other nutritional strategies have been used to improve muscle protein anabolism
during aging. For instance, to overcome the decreased sensitivity of muscle protein syn-
thesis to feeding, Arnal et al.91 modulated the daily protein feeding pattern in old rats by
regrouping 66% of dietary proteins in one meal for 21 days. This pulse pattern restored
a significant response to feeding of gastrocnemius muscle protein synthesis without effect
on muscle breakdown.91 Thus, using a pulse pattern could be useful in preventing the
age-related loss of muscle in humans by increasing feeding-induced stimulation of muscle
protein synthesis as it is at the whole-body level.79 Likewise, the slow and fast protein
concepts92 may be applied to the aged population to optimize muscle protein anabolism
in a period of potential muscle wasting.84 Other studies are now required to confirm the
anabolic effects of such nutritional strategies on the muscle protein turnover in healthy
elderly humans.

24.3.2 Amino acid intervention during stress in aging individuals: impact


of glutamine
Muscle amino acid metabolism is altered during stress conditions. For example, glutamine
release from muscle is increased in catabolic states, such as burn, trauma, or sepsis, due
to the activation of muscle protein breakdown and of glutamine synthetase activity (for
a review see Griffiths93). Although glutamine synthetase activity increases during aggres-
sion, muscle glutamine concentrations are often depleted because of accelerated efflux of
this amino acid.94 In these conditions, glutamine taken up from blood has been suggested
to serve as an energetic substrate for a number of tissues, including intestine, immune
cells, and kidney. Glutamine is also the main carrier of a-amino nitrogen between tissues
of the body and is therefore important in interorgan traffic and acid–base homeostasis.
Finally, glutamine may promote protein synthesis95 and inhibit protein catabolism in
muscle.96 The decrease in muscle mass in aged individuals may limit the amount of
glutamine available during stress conditions. Meynial-Denis et al.97 reported that the
glutamine synthetase responsiveness (i.e., mRNA expression and enzyme activity) to an
excess of glucocorticoids was not modified in old rats in either tibialis anterior or soleus
muscles. In addition, glucocorticoid treatment resulted in a significant increase in the
circulating concentrations of glutamine and in a simultaneous drop in intramuscular
glutamine concentration whatever the age of animals.97 Interestingly, glutamine synthetase
was also not affected by sex steroid hormone changes that may occur with age.98 With the
assumption that the rate of removal of this amino acid by other tissues is constant in such
a catabolic situation, the efflux of glutamine from muscle should be similar in adult and
aged rats. Minet-Quinard et al.99 reported converse observations showing that muscle
1382_C24.fm Page 399 Tuesday, October 7, 2003 7:00 PM

Chapter twenty-four: Muscle protein and amino acid metabolism 399

glutamine metabolism was not altered in the same way in adult and old rats during
glucocorticoid treatment. The glutamine plasma pool was not depleted in adult rats after
glucocorticoid infusion, whereas it was lower in old animals, despite the same activation
of muscle glutamine synthetase activity. Consequently, these data suggest that either aged
rats have a greater central utilization of glutamine than adult animals or endogenous
production and peripheral release are not sufficient to supply the organism. Irrespective
of the mechanism involved, hypoglutaminemia in aged stressed rats was strongly related
to their mortality.99
These observations emphasized the importance of evaluating the beneficial effect of
glutamine supplementation in aged injured individuals. Oral supplementation with
glutamine in old rats did not prevent muscle glutamine depletion and atrophy induced
by lipopolisaccharide challenge.100 Conversely, a mixture of b-hydroxy-b-methylbutyrate,
arginine, and glutamine was effective in increasing fat-free mass of advanced cancer
65-year-old patients.101 The authors postulated that the reason for this improvement could
be attributed to the observed properties of b-hydroxy-b-methylbutyrate on slowing the
rate of protein breakdown102 and to the anabolic effects observed with arginine and
glutamine.103
These findings are promising and highlight the need to test the impact of other
compounds, such as ornithine alpha-ketoglutarate, a precursor of both glutamine and
arginine,104 or leucine, on muscle metabolism in elderly subjects under stress conditions.

24.4 Concluding remarks


Sarcopenia is an important feature of aging, which is associated with loss of strength,
decreased protein reserves, and increased disability. There are many potential mechanisms
leading to sarcopenia, but one of the most important is probably the alteration of muscle
protein turnover. Extending our reductionist view to the determination of subcellular or
individual muscle protein synthesis rates by using new methodological approaches could
be useful in aging studies. In addition, decreased protein turnover rate may lead to the
accumulation of altered and abnormal proteins within the muscle cell with many conse-
quences. Further studies are required to unravel the steps of transcription and translation,
as well as the impact of potential age-accumulated DNA and protein damages in the
pathogenesis of sarcopenia.
Concerning interventional strategies, the main objective is to give back mobility to
elderly subjects in order to offer a better quality of life. As is often the case in biomedicine,
we know more about the treatment of sarcopenia than about its etiology. The treatment
of muscle loss in the aged population seems to be feasible by using nutritional, hormonal,
or resistance training approaches. In Western countries, sarcopenia and its consequences
on mobility are one of the first health problems of this fastest-growing segment of the
population. Our efforts are to be focused in the basic direction of understanding the
biology of sarcopenia, but also in a more applied direction. There is an urgent need to
translate the current research findings into public health programs that may prevent an
epidemic of sarcopenia-related disability, together with its health and societal costs, for
the 21st century.

References
1. Roubenoff, R. and Castaneda, C., Sarcopenia—understanding the dynamics of aging muscle,
JAMA, 286, 1230, 2001.
2. Waters, D.L., Baumgartner, R.N., and Garry, P.J., Sarcopenia: current perspectives, J. Nutr.
Health Aging, 4, 133, 2000.
1382_C24.fm Page 400 Tuesday, October 7, 2003 7:00 PM

400 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

3. Cohn, S.H., Vartsky, D., Yasumura, S., Sawitsky, A., Zanzi, I., Vaswani, A., and Ellis, K.J.,
Compartmental body composition based on total-body nitrogen, potassium, and calcium,
Am. J. Physiol., 239, E524, 1980.
4. Forbes, G.B. and Reina, J.C., Adult lean body mass declines with age: some longitudinal
observations, Metabolism, 19, 653, 1970.
5. Novak, L.P., Aging, total body potassium, fat-free mass, and cell mass in males and females
between ages 18 and 85 years, J. Gerontol., 27, 438, 1972.
6. Gallagher, D., Ruts, E., Visser, M., Heshka, S., Baumgartner, R.N., Wang, J., Pierson, R.N.,
Pi-Sunyer, F.X., and Heymsfield, S.B., Weight stability masks sarcopenia in elderly men and
women, Am. J. Physiol., 279, E366, 2000.
7. Kyle, U.G., Genton, L., Hans, D., Karsegard, V.L., Michel, J.P., Slosman, D.O., and Pichard,
C., Total body mass, fat mass, fat-free mass, and skeletal muscle in older people: cross-
sectional differences in 60-year-old persons, J. Am. Geriatr. Soc., 49, 1633, 2001.
8. Kyle, U.G., Unger, P., Dupertuis, Y.M., Karsegard, V.L., Genton, L., and Pichard, C., Body
composition in 995 acutely ill or chronically ill patients at hospital admission: a controlled
population study, J. Am. Diet. Assoc., 102, 944, 2002.
9. Larsson, L., Sjodin, B., and Karlsson, J., Histochemical and biochemical changes in human
skeletal muscle with age in sedentary males, age 22–65 years, Acta Physiol. Scand., 103, 31,
1978.
10. Lexell, J., Henriksson-Larsen, K., Winblad, B., and Sjostrom, M., Distribution of different
fiber types in human skeletal muscles: effects of aging studied in whole muscle cross sections,
Muscle Nerve, 6, 588, 1983.
11. Giampaoli, S., Ferrucci, L., Cecchi, F., Lo Noce, C., Poce, A., Dima, F., Santaquilani, A., Vescio,
M.F., and Menotti, A., Hand-grip strength predicts incident disability in non-disabled older
men, Age Ageing, 28, 283, 1999.
12. Launer, L.J., Harris, T., Rumpel, C., and Madans, J., Body mass index, weight change, and
risk of mobility disability in middle-aged and older women: the epidemiologic follow-up
study of NHANES I, JAMA, 271, 1093, 1994.
13. Porter, E.J., Non-equilibrium systems theory: some applications for gerontological nursing
practice, J. Gerontol. Nurs., 21, 24, 1995.
14. Evans, W., Functional and metabolic consequences of sarcopenia, J. Nutr., 127, 998S, 1997.
15. Beaufrère, B. and Boirie, Y., Aging and protein metabolism, Curr. Opin. Clin. Nutr. Metab.
Care, 1, 85, 1998.
16. Millward, D.J., Fereday, A., Gibson, N., and Pacy, P.J., Aging, protein requirements, and
protein turnover, Am. J. Clin. Nutr., 66, 774, 1997.
17. Young, V.R., Steffee, W.P., Pencharz, P.B., Winterer, J.C., and Scrimshaw, N.S., Total human
body protein synthesis in relation to protein requirements at various ages, Nature, 253, 192,
1975.
18. Golden, M.H. and Waterlow, J.C., Total protein synthesis in elderly people: a comparison of
results with [15N]glycine and [14C]leucine, Clin. Sci. Mol. Med., 53, 277, 1977.
19. Lehmann, A.B., Johnston, D., and James, O.F., The effects of old age and immobility on
protein turnover in human subjects with some observations on the possible role of hormones,
Age Ageing, 18, 148, 1989.
20. Winterer, J.C., Steffee, W.P., Davy, W., Perera, A., Uauy, R., Scrimshaw, N.S., and Young, V.R.,
Whole body protein turnover in aging man, Exp. Gerontol., 11, 79, 1976.
21. Boirie, Y., Gachon, P., and Beaufrère, B., Splanchnic and whole-body leucine kinetics in young
and elderly men, Am. J. Clin. Nutr., 65, 489, 1997.
22. Morais, J.A., Gougeon, R., Pencharz, P.B., Jones, P.J., Ross, R., and Marliss, E.B., Whole-body
protein turnover in the healthy elderly, Am. J. Clin. Nutr., 66, 880, 1997.
23. Robert, J.J., Bier, D., Schoeller, D., Wolfe, R., Matthews, D.E., Munro, H.N., and Young, V.R.,
Effects of intravenous glucose on whole body leucine dynamics, studied with 1-13C-leucine,
in healthy young and elderly adults, J. Gerontol., 39, 673, 1984.
24. Boirie, Y., Short, K.R., Ahlman, B., Charlton, M., and Nair, K.S., Tissue-specific regulation of
mitochondrial and cytoplasmic protein synthesis rates by insulin, Diabetes, 50, 2652, 2001.
1382_C24.fm Page 401 Tuesday, October 7, 2003 7:00 PM

Chapter twenty-four: Muscle protein and amino acid metabolism 401

25. Short, K.R. and Nair, K.S., Mechanisms of sarcopenia of aging, J. Endocrinol. Invest., 22,95,
1999.
26. Bross, R., Storer, T., and Bhasin, S., Aging and muscle loss, Trends Endocrinol. Metab., 10, 194,
1999.
27. Morais, J.A., Ross, R., Gougeon, R., Pencharz, P.B., Jones, P.J., and Marliss, E.B., Distribution
of protein turnover changes with age in humans as assessed by whole-body magnetic
resonance image analysis to quantify tissue volumes, J. Nutr., 130, 784, 2000.
28. Uauy, R., Winterer, J.C., Bilmazes, C., Haverberg, L.N., Scrimshaw, N.S., Munro, H.N., and
Young, V.R., The changing pattern of whole body protein metabolism in aging humans,
J. Gerontol., 33, 663, 1978.
29. Volpi, E., Mittendorfer, B., Wolf, S.E., and Wolfe, R.R., Oral amino acids stimulate muscle
protein anabolism in the elderly despite higher first-pass splanchnic extraction, Am. J. Phys-
iol., 277, E513, 1999.
30. Walrand, S., Chambon-Savanovitch, C., Felgines, C., Chassagne, J., Raul, F., Normand, B.,
Farges, M.C., Beaufrère, B., Vasson, M.P., and Cynober, L., Aging: a barrier to renutrition?
Nutritional and immunologic evidence in rats, Am. J. Clin. Nutr., 72, 816, 2000.
31. Balagopal, P., Rooyackers, O.E., Adey, D.B., Ades, P.A., and Nair, K.S., Effects of aging on in
vivo synthesis of skeletal muscle myosin heavy-chain and sarcoplasmic protein in humans,
Am. J. Physiol., 273, E790, 1997.
32. Short, K.R. and Nair, K.S., The effect of age on protein metabolism, Curr. Opin. Clin. Nutr.
Metab. Care, 3, 39, 2000.
33. Proctor, D.N., O’Brien, P.C., Atkinson, E.J., and Nair, K.S., Comparison of techniques to
estimate total body skeletal muscle mass in people of different age groups, Am. J. Physiol.,
277, E489, 1999.
34. Boirie, Y., Beaufrère, B., and Ritz, P., Energetic cost of protein turnover in healthy elderly
humans, Int. J. Obes. Relat. Metab. Disord., 25, 601, 2001.
35. Morio, B., Hocquette, J.F., Montaurier, C., Boirie, Y., Bouteloup-Demange, C., McCormack,
C., Fellmann, N., Beaufrère, B., and Ritz, P., Muscle fatty acid oxidative capacity is a deter-
minant of whole body fat oxidation in elderly people, Am. J. Physiol., 280, E143, 2001.
36. Baillie, A.G. and Garlick, P.J., Responses of protein synthesis in different skeletal muscles to
fasting and insulin in rats, Am. J. Physiol., 260, E891, 1991.
37. Mosoni, L., Patureau Mirand, P., Houlier, M.L., and Arnal, M., Age-related changes in protein
synthesis measured in vivo in rat liver and gastrocnemius muscle, Mech. Ageing Dev., 68, 209,
1993.
38. Dardevet, D., Sornet, C., Bayle, G., Prugnaud, J., Pouyet, C., and Grizard, J., Postprandial
stimulation of muscle protein synthesis in old rats can be restored by a leucine-supplemented
meal, J. Nutr., 132,95, 2002.
39. Balagopal, P., Ljungqvist, O., and Nair, K.S., Skeletal muscle myosin heavy-chain synthesis
rate in healthy humans, Am. J. Physiol., 272, E45, 1997.
40. Yarasheski, K.E., Zachwieja, J.J., and Bier, D.M., Acute effects of resistance exercise on muscle
protein synthesis rate in young and elderly men and women, Am. J. Physiol., 265, E210, 1993.
41. Yarasheski, K.E., Zachwieja, J.J., Campbell, J.A., and Bier, D.M., Effect of growth hormone
and resistance exercise on muscle growth and strength in older men, Am. J. Physiol., 268,
E268, 1995.
42. Volpi, E., Sheffield-Moore, M., Rasmussen, B.B., and Wolfe, R.R., Basal muscle amino acid
kinetics and protein synthesis in healthy young and older men, JAMA, 286, 1206, 2001.
43. Welle, S., Thornton, C., Jozefowicz, R., and Statt, M., Myofibrillar protein synthesis in young
and old men, Am. J. Physiol., 264, E693, 1993.
44. Hasten, D.L., Pak-Loduca, J., Obert, K.A., and Yarasheski, K.E., Resistance exercise acutely
increases MHC and mixed muscle protein synthesis rates in 78–84 and 23–32 yr olds, Am.
J. Physiol., 278, E620, 2000.
45. Rooyackers, O.E., Adey, D.B., Ades, P.A., and Nair, K.S., Effect of age on in vivo rates of
mitochondrial protein synthesis in human skeletal muscle, Proc. Natl. Acad. Sci. U.S.A., 93,
15364, 1996.
1382_C24.fm Page 402 Tuesday, October 7, 2003 7:00 PM

402 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

46. Ferrington, D.A., Krainev, A.G., and Bigelow, D.J., Altered turnover of calcium regulatory
proteins of the sarcoplasmic reticulum in aged skeletal muscle, J. Biol. Chem., 273, 5885, 1998.
47. Yarasheski, K.E., Welle, S., and Sreekumaran Nair, K., Muscle protein synthesis in younger
and older men, JAMA, 287, 317, 2002.
48. Welle, S., Bhatt, K., and Thornton, C., Polyadenylated RNA, actin mRNA, and myosin heavy
chain mRNA in young and old human skeletal muscle, Am. J. Physiol., 270, E224, 1996.
49. Rattan, S.I., Synthesis, modifications, and turnover of proteins during aging, Exp. Gerontol.,
31, 33, 1996.
50. Tavernarakis, N. and Driscoll, M., Caloric restriction and lifespan: a role for protein turnover?
Mech. Ageing Dev., 123, 215, 2002.
51. Halliday, D. and McKeran, R.O., Measurement of muscle protein synthetic rate from serial
muscle biopsies and total body protein turnover in man by continuous intravenous infusion
of L-(alpha-15N)lysine, Clin. Sci. Mol. Med., 49, 581, 1975.
52. Volpi, E., Ferrando, A.A., Yeckel, C.W., Tipton, K.D., and Wolfe, R.R., Exogenous amino acids
stimulate net muscle protein synthesis in the elderly, J. Clin. Invest., 101, 2000, 1998.
53. Volpi, E., Mittendorfer, B., Rasmussen, B.B., and Wolfe, R.R., The response of muscle protein
anabolism to combined hyperaminoacidemia and glucose-induced hyperinsulinemia is im-
paired in the elderly, J. Clin. Endocrinol. Metab., 85, 4481, 2000.
54. Welle, S., Thornton, C., and Statt, M., Myofibrillar protein synthesis in young and old human
subjects after three months of resistance training, Am. J. Physiol., 268, E422, 1995.
55. Welle, S., Cellular and molecular basis of age-related sarcopenia, Can. J. Appl. Physiol., 27,
19, 2002.
56. Reynolds, T.H., IV, Krajewski, K.M., Larkin, L.M., Reid, P., Halter, J.B., Supiano, M.A., and
Dengel, D.R., Effect of age on skeletal muscle proteolysis in extensor digitorum longus
muscles of B6C3F1 mice, J. Gerontol. A Biol. Sci. Med. Sci., 57, B198, 2002.
57. Johnson, P. and Hammer, J.L., Cardiac and skeletal muscle enzyme levels in hypertensive
and aging rats, Comp. Biochem. Physiol. B, 104, 63, 1993.
58. Carmeli, E., Coleman, R., and Reznick, A.Z., The biochemistry of aging muscle, Exp. Gerontol.,
37, 477, 2002.
59. Dardevet, D., Sornet, C., Taillandier, D., Savary, I., Attaix, D., and Grizard, J., Sensitivity and
protein turnover response to glucocorticoids are different in skeletal muscle from adult and
old rats: lack of regulation of the ubiquitin-proteasome proteolytic pathway in aging, J. Clin.
Invest., 96, 2113, 1995.
60. Bardag-Gorce, F., Farout, L., Veyrat-Durebex, C., Briand, Y., and Briand, M., Changes in 20S
proteasome activity during ageing of the LOU rat, Mol. Biol. Rep., 26, 89, 1999.
61. Young, V.R., Protein and amino acid metabolism with reference to aging and the elderly,
Prog. Clin. Biol. Res., 326, 279, 1990.
62. Sjölin, J., Stjernstrom, H., Henneberg, S., Andersson, E., Martensson, J., Friman, G., and
Larsson, J., Splanchnic and peripheral release of 3-methylhistidine in relation to its urinary
excretion in human infection, Metabolism, 38, 23, 1989.
63. Lenaz, G., D’Aurelio, M., Merlo Pich, M., Genova, M.L., Ventura, B., Bovina, C., Formiggini,
G., and Parenti Castelli, G., Mitochondrial bioenergetics in aging, Biochim. Biophys. Acta, 1459,
397, 2000.
64. Ames, B.N., Shigenaga, M.K., and Hagen, T.M., Mitochondrial decay in aging, Biochim.
Biophys. Acta, 1271, 165, 1995.
65. Cottrell, D.A. and Turnbull, D.M., Mitochondria and ageing, Curr. Opin. Clin. Nutr. Metab.
Care, 3, 473, 2000.
66. Short, K.R. and Nair, K.S., Does aging adversely affect muscle mitochondrial function? Exerc.
Sport Sci. Rev., 29, 118, 2001.
67. Muller-Hocker, J., Schafer, S., Link, T.A., Possekel, S., and Hammer, C., Defects of the respi-
ratory chain in various tissues of old monkeys: a cytochemical-immunocytochemical study,
Mech. Ageing Dev., 86, 197, 1996.
68. Muller-Hocker, J., Cytochrome c oxidase deficient fibres in the limb muscle and diaphragm
of man without muscular disease: an age-related alteration, J. Neurol. Sci., 100, 14, 1990.
1382_C24.fm Page 403 Tuesday, October 7, 2003 7:00 PM

Chapter twenty-four: Muscle protein and amino acid metabolism 403

69. Skorjanc, D., Dunstl, G., and Pette, D., Mitochondrial enzyme defects in normal and low-
frequency-stimulated muscles of young and aging rats, J. Gerontol. A Biol. Sci. Med. Sci., 56,
B503, 2001.
70. Barazzoni, R., Short, K.R., and Nair, K.S., Effects of aging on mitochondrial DNA copy
number and cytochrome c oxidase gene expression in rat skeletal muscle, liver, and heart,
J. Biol. Chem., 275, 3343, 2000.
71. Brand, M.D., Uncoupling to survive? The role of mitochondrial inefficiency in ageing, Exp.
Gerontol., 35, 811, 2000.
72. DiMauro, S., Tanji, K., Bonilla, E., Pallotti, F., and Schon, E.A., Mitochondrial abnormalities
in muscle and other aging cells: classification, causes, and effects, Muscle Nerve, 26, 597, 2002.
73. Van Remmen, H. and Richardson, A., Oxidative damage to mitochondria and aging, Exp.
Gerontol., 36, 957, 2001.
74. Welle, S., Bhatt, K., and Thornton, C.A., High-abundance mRNAs in human muscle: com-
parison between young and old, J. Appl. Physiol., 89, 297, 2000.
75. Beckman, K.B. and Ames, B.N., The free radical theory of aging matures, Physiol. Rev., 78,
547, 1998.
76. Ozawa, T., Mechanism of somatic mitochondrial DNA mutations associated with age and
diseases, Biochim. Biophys. Acta, 1271, 177, 1995.
77. Hunter, S.K., Thompson, M.W., Ruell, P.A., Harmer, A.R., Thom, J.M., Gwinn, T.H., and
Adams, R.D., Human skeletal sarcoplasmic reticulum Ca2+ uptake and muscle function with
aging and strength training, J. Appl. Physiol., 86, 1858, 1999.
78. Mosoni, L., Valluy, M.C., Serrurier, B., Prugnaud, J., Obled, C., Guezennec, C.Y., and Patureau
Mirand, P., Altered response of protein synthesis to nutritional state and endurance training
in old rats, Am. J. Physiol., 268, E328, 1995.
79. Arnal, M.A., Mosoni, L., Boirie, Y., Houlier, M.L., Morin, L., Verdier, E., Ritz, P., Antoine,
J.M., Prugnaud, J., Beaufrère, B., and Patureau-Mirand, P., Protein pulse feeding improves
protein retention in elderly women, Am. J. Clin. Nutr., 69, 1202, 1999.
80. Yoshizawa, F., Kimball, S.R., Vary, T.C., and Jefferson, L.S., Effect of dietary protein on
translation initiation in rat skeletal muscle and liver, Am. J. Physiol., 275, E814, 1998.
81. Bennet, W.M., Connacher, A.A., Scrimgeour, C.M., Smith, K., and Rennie, M.J., Increase in
anterior tibialis muscle protein synthesis in healthy man during mixed amino acid infusion:
studies of incorporation of [1- 13C]leucine, Clin. Sci. (Lond.), 76, 447, 1989.
82. Fryburg, D.A., Jahn, L.A., Hill, S.A., Oliveras, D.M., and Barrett, E.J., Insulin and insulin-
like growth factor-I enhance human skeletal muscle protein anabolism during hyperami-
noacidemia by different mechanisms, J. Clin. Invest., 96, 1722, 1995.
83. Volpi, E., Kobayashi, H., Sheffield-Moore, M., Mittendorfer, B., and Wolfe, R.R., Essential
amino acids are primarily responsible for the amino acid stimulation of muscle protein
anabolism in healthy elderly adults, Am. J. Clin. Nutr., 78(2), 250, 2003.
84. Dangin, M., Boirie, Y., Guillet, C., and Beaufrère, B., Influence of the protein digestion rate
on protein turnover in young and elderly subjects, J. Nutr., 132, 3228S, 2002.
85. Dardevet, D., Sornet, C., Balage, M., and Grizard, J., Stimulation of in vitro rat muscle protein
synthesis by leucine decreases with age, J. Nutr., 130, 2630, 2000.
86. Anthony, J.C., Yoshizawa, F., Anthony, T.G., Vary, T.C., Jefferson, L.S., and Kimball, S.R.,
Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a
rapamycin-sensitive pathway, J. Nutr., 130, 2413, 2000.
87. Guillet, C., Rousset, P., Giraudet, C., Sornet, C., Dardevet, D., and Boirie, Y., Age-related
alteration of myofibrillar and sarcoplasmic proteins synthesis after meal intake is prevented
by a leucine supplemented diet, Clin. Nutr., 21, 2, 2002.
88. Huxtable, R.J., Physiological actions of taurine, Physiol. Rev., 72, 101, 1992.
89. Pierno, S., De Luca, A., Camerino, C., Huxtable, R.J., and Camerino, D.C., Chronic admin-
istration of taurine to aged rats improves the electrical and contractile properties of skeletal
muscle fibers, J. Pharmacol. Exp. Ther., 286, 1183, 1998.
90. Wallace, D.R. and Dawson, R., Jr., Decreased plasma taurine in aged rats, Gerontology, 36, 19,
1990.
1382_C24.fm Page 404 Tuesday, October 7, 2003 7:00 PM

404 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

91. Arnal, M.A., Mosoni, L., Dardevet, D., Ribeyre, M.C., Bayle, G., Prugnaud, J., and Patureau
Mirand, P., Pulse protein feeding pattern restores stimulation of muscle protein synthesis
during the feeding period in old rats, J. Nutr., 132, 1002, 2002.
92. Boirie, Y., Dangin, M., Gachon, P., Vasson, M.P., Maubois, J.L., and Beaufrère, B., Slow and
fast dietary proteins differently modulate postprandial protein accretion, Proc. Natl. Acad.
Sci. U.S.A., 94, 14930, 1997.
93. Griffiths, R.D., The evidence for glutamine use in the critically-ill, Proc. Nutr. Soc., 60, 403,
2001.
94. Ardawi, M.S. and Jamal, Y.S., Glutamine metabolism in skeletal muscle of glucocorticoid-
treated rats, Clin. Sci. (Lond.), 79, 139, 1990.
95. Jepson, M.M., Bates, P.C., Broadbent, P., Pell, J.M., and Millward, D.J., Relationship between
glutamine concentration and protein synthesis in rat skeletal muscle, Am. J. Physiol., 255,
E166, 1988.
96. Smith, R.J., Glutamine metabolism and its physiologic importance, J. Parenter. Enteral Nutr.,
14, 40S, 1990.
97. Meynial-Denis, D., Mignon, M., Miri, A., Imbert, J., Aurousseau, E., Taillandier, D., Attaix,
D., Arnal, M., and Grizard, J., Glutamine synthetase induction by glucocorticoids is preserved
in skeletal muscle of aged rats, Am. J. Physiol., 271, E1061, 1996.
98. Verdier, L., Boirie, Y., Van Drieesche, S., Mignon, M., Bègue, R.J., and Meynial-Denis, D., Do
sex steroids regulate glutamine synthesis with age? Am. J. Physiol., 282, E215, 2002.
99. Minet-Quinard, R., Moinard, C., Villié, F., Walrand, S., Vasson, M.P., Chopineau, J., and
Cynober, L., Kinetic impairment of nitrogen and muscle glutamine metabolisms in old
glucocorticoid-treated rats, Am. J. Physiol., 276, E558, 1999.
100. Farges, M.C., Bérard, M.P., Raul, F., Cézard, J.P., Joly, B., Davot, P., Vasson, M.P., and Cynober,
L., Oral administration of a glutamine-enriched diet before or after endotoxin challenge in
aged rats has limited effects, J. Nutr., 129, 1799, 1999.
101. May, P.E., Barber, A., D’Olimpio, J.T., Hourihane, A., and Abumrad, N.N., Reversal of cancer-
related wasting using oral supplementation with a combination of beta-hydroxy-beta-meth-
ylbutyrate, arginine, and glutamine, Am. J. Surg., 183, 471, 2002.
102. Knitter, A.E., Panton, L., Rathmacher, J.A., Petersen, A., and Sharp, R., Effects of beta-
hydroxy-beta-methylbutyrate on muscle damage after a prolonged run, J. Appl. Physiol., 89,
1340, 2000.
103. De Bandt, J.P. and Cynober, L.A., Amino acids with anabolic properties, Curr. Opin. Clin.
Nutr. Metab. Care, 1, 263, 1998.
104. Cynober, L.A., The use of alpha-ketoglutarate salts in clinical nutrition and metabolic care,
Curr. Opin. Clin. Nutr. Metab. Care, 2, 33, 1999.
1382_C25.fm Page 405 Tuesday, October 7, 2003 7:02 PM

chapter twenty-five

Gastrointestinal disease
Peter B. Soeters
Academic Hospital, Maastricht
Karel W. Hulsewe
Academic Hospital, Maastricht
Nicolaas E.P. Deutz
Academic Hospital, Maastricht
Yvette Luiking
Academic Hospital, Maastricht
Cornelis H.C. Dejong
Academic Hospital, Maastricht

Contents
Introduction..................................................................................................................................406
25.1 Intermediary amino acid metabolism in the intestine..................................................406
25.1.1 Glutamine metabolism in the intestine and intestinal integrity..................406
25.1.1.1 Glutamine metabolism in the intestine............................................406
25.1.1.2 Glutamine metabolism in the intestine after trauma and
in models of sepsis ..............................................................................407
25.1.1.3 Glutamine uptake in solid tumors of the colon.............................408
25.1.2 The role of the intestine in the routing of nitrogen and carbon..................408
25.1.2.1 The role of the intestine in the routing of nitrogen.......................408
25.1.2.2 The role of the intestine in the routing of amino acid-derived
carbon skeletons...................................................................................409
25.1.3 The role of the intestine in the production of specific amino acids ...........409
25.1.3.1 Intestinal production of citrulline and arginine .............................409
25.1.3.2 Intestinal production of taurine and glycine ..................................410
25.2 Intermediary protein metabolism in the intestine ........................................................410
25.2.1 The biological value of protein .........................................................................410
25.2.1.1 The appearance of amino acids in the portal vein after a
protein-containing bolus meal...........................................................411
25.2.1.2 The regulation of protein accretion in the gut ...............................412
25.2.2 What do the protein kinetic data signify in biological terms? ....................413
25.2.2.1 The nature of the labile protein pool in the intestine ...................413

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 405
1382_C25.fm Page 406 Tuesday, October 7, 2003 7:02 PM

406 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

25.2.2.2 The role of the labile protein pool in old age and disease...........414
25.3 Summary and conclusions ...............................................................................................415
References .....................................................................................................................................415

Introduction
In this chapter we will try to define the role of the intestine in the intermediary metabolism
of amino acids.
For a long time it has been recognized that the intestine is not a passive receptacle
that indiscriminately absorbs food, regardless of its composition and quantity. In the 1970s,
several authors demonstrated that apart from its absorptive capacity, the intestine played
an important role in the intermediary metabolism of macronutrients. In this chapter, we
focus on the intestinal metabolism of protein and amino acids. It was found that entero-
cytes metabolized in vitro significant quantities of glutamine and stoichiometrically pro-
duced alanine and ammonia, regardless of its site of entry, be it the intestinal lumen or
the arterial inflow. Windmueller and Spaeth performed landmark investigations regarding
the contribution of amino acids and carbohydrates to the intermediary metabolism of the
intestine. They and others established that ammonia, alanine, and glutamic acid were the
main products of degradation of glutamine in enterocytes. Ammonia generation as an end
point of protein metabolism was in the 1960s considered to arise from bacterial degradation
of food protein and urea, because antibiotic therapy lowered plasma ammonia levels and
improved hepatic encephalopathy. Due to the work of several authors, it became clear
that the intestine of germ-free animals also produced significant quantities of ammonia,
which must therefore have arisen from intermediary metabolism of protein by the gut
wall itself without the interference of bacteria. This subject will be dealt with elsewhere
in this book (Chapter 26).
In recent years it has been postulated that short bowel or diseased bowel not only
failed to absorb sufficient quantities of protein or would ineffectively absorb bolus feeding,
but would also fail to produce crucial amino acids derived from the degradation of other
amino acids presented to the enterocytes from the gut lumen or arterial supply.
In more recent years, it has been recognized that quantitatively important recycling
of the products of protein degradation occurs in the splanchnic tissues, which may have
important implications for the efficiency with which protein can be utilized for growth
and the preservation of body protein mass. This may have important consequences for
the way in which we estimate protein quality and for the way in which we must supple-
ment protein in the food in different diseases and in different stages of life. In this chapter,
we will focus on the role of the intestine in efficient protein absorption and on the role of
the intestine in the degradation and production of potentially harmful or useful amino
acids.

25.1 Intermediary amino acid metabolism in the intestine


25.1.1 Glutamine metabolism in the intestine and intestinal integrity
25.1.1.1 Glutamine metabolism in the intestine
Glutamine is an important substrate for the intestine and plays a central role in interme-
diary amino acid metabolism in the gut. It serves as a fuel, as a precursor of protein,
glutathione, polyamines, and nucleotide synthesis, and as a nitrogen carrier. The degra-
dation of glutamine in the gut wall yields as main nitrogenous substances ammonia,
1382_C25.fm Page 407 Tuesday, October 7, 2003 7:02 PM

Chapter twenty-five: Gastrointestinal disease 407

proline, alanine, glutamine, and citrulline. Its role as a carrier of nitrogen and deliverer
of ammonia to the portal circulation will be discussed in Chapter 26. Japanese
investigators1 reported that enterocytes incubated in vitro in a glutamine-containing
medium produced glutamic acid, alanine, and ammonia in a time- and concentration-
dependent manner. Watford et al.2 reported similar results. Windmueller and Spaeth3–5
assessed in a large series of semi-in vivo experiments the fate of glutamine-derived nitrogen
and the contribution of glutamine in the provision of energy to the intestine. They found
that glutamine was taken up from the intestine in a concentration-dependent manner and
was metabolized to other amino acids and ammonia. The nitrogen of glutamine was found
to end up for 36% in alanine, 7% in proline, 10% in citrulline, 11% in ornithine, and 36%
in ammonia. Energy coverage was found to be derived as 35% from glutamine carbon,
26% from 3-hydroxybutyrate, 24% from acetoacetate, 7% from glucose, and the remainder
from lactate and unesterified fatty acids. Despite these important findings, it took another
decade before the clinical relevance of these findings was appreciated by clinicians and
before research was initiated to explore the potential benefits of glutamine supplementa-
tion in the experimental in vivo6 or clinical setting.7,8

25.1.1.2 Glutamine metabolism in the intestine after trauma and in models


of sepsis
Souba and Wilmore and others9–17 have given great impetus to this area of research and
found that in experimental animals, endotoxin greatly stimulated the uptake of glutamine
by the intestine and by the liver. We found in similar experiments in pigs that both after
surgical trauma and after endotoxin challenge, net release of glutamine by the hindquarter
increased, whereas the net uptake by the intestine decreased.18,19 Concomitantly, uptake
by the liver and the spleen increased. In fact, in these two organs the metabolism of
glutamine changed from a modest net release in the control nonstressed situation to net
uptake after trauma or endotoxin challenge.18 This increased net flux of glutamine is
supported by the finding that after trauma and sepsis the A-V difference across the hind-
quarter or forearm increases.20–24 This must imply that the glutamine released by the
hindquarter is taken up by central organs like liver and spleen, as found in our experi-
ments.18 Rather unexpectedly, whole-body tracer data do not indicate a substantial increase
in the turnover of glutamine after trauma or during sepsis.25–27 This should probably be
taken to imply that the increased net flux of glutamine from peripheral tissues to central
tissues is not generated by increased production in muscle and increased uptake in central
tissues but rather by decreased uptake in muscle and decreased production in central
organs.
The suggestion, put forward by Souba and Wilmore and others,9–11,13 that peripheral,
predominantly muscle tissue produces glutamine that is subsequently taken up by the
gut deserves further explanation. The intestine itself is in this process a rather passive
organ that takes up less glutamine in the fasted or traumatized state, whereas the liver
and the spleen take up more glutamine even in the presence of lower plasma levels.18,19
Plasma glutamine level has been suggested to be an important factor, determining uptake
of glutamine by the intestine in the semi-in vivo experimental setting.3–5 We found in pigs
that the first few days after trauma, coinciding with relative starvation, glutamine levels
drop and uptake across the intestine decreases substantially.18 A-V differences across the
jejunum and ileum in humans during abdominal operations were found to correlate with
plasma glutamine levels.28 The fractional extraction of glutamine was by far the highest
(30%) in the jejunum. This was paralleled by the A-V differences of the main products of
glutamine degradation: ammonia, citrulline, proline, ornithine, and alanine.28 The release
of these degradation products of glutamine was much larger in the jejunum than in the
1382_C25.fm Page 408 Tuesday, October 7, 2003 7:02 PM

408 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

ileum. In the colon, there was some uptake of glutamine but little nonbacterial interme-
diary metabolism. Only glutamic acid and ammonia were released in the venous effluent
of the colon, but not in a concentration-dependent manner. It is likely that ammonia release
in the colon is effectuated by a modest degradation of glutamine in the colonocyte and
by bacterial degradation of urea and probably amino acids.
It is of interest that uptake of glutamine by the intestine appears to be concentration
dependent,28 and that the negative effects of starvation on intestinal integrity can be
counteracted by infusion of glutamine.29 This raises the question of which factors deter-
mine glutamine levels. In the past, we have claimed that the depleted state causes plasma
glutamine levels to drop. Most clinical patients that have lost weight and are considered
to be depleted also exhibit symptoms of chronic inflammation. It is therefore often difficult
to separate the influences of chronic inflammation and depletion. To try to elucidate the
contribution of each of these factors, we reviewed our data and found that patients
exhibiting signs of inflammatory activity had low plasma glutamine levels and increased
permeability of the bowel. It is especially in this subgroup of patients that glutamine
supplementation appears to be effective in reducing intestinal permeability (unpublished
results).

25.1.1.3 Glutamine uptake in solid tumors of the colon


In vitro studies on cancer cell lines have provided evidence that some cell types degrade
large quantities of glutamine.30 It has been suggested that some cancer types may therefore
act as a “sink” for glutamine. In the same experiment in surgical patients in which we
studied A-V concentration differences in different parts of the intestine, we also assessed
A-V differences across parts of the colon containing malignant tumors. In accordance with
observations by Holm’s group,31 we found that there was no preferential uptake of
glutamine by these tumors.28 For coverage of their energy requirements, colonic cancers
appeared to rely on glycolysis, because there was a substantially increased uptake of
glucose and stoichiometric release of lactate compared to healthy parts of the colon.

25.1.2 The role of the intestine in the routing of nitrogen and carbon
25.1.2.1 The role of the intestine in the routing of nitrogen
Trauma and disease put a metabolic burden on the organism, which through its mere
existence implicitly has proven that it can adapt to this changing environment. One of the
metabolic challenges to the organism is that in disease and after trauma, the body needs
to generate a host response for which it needs substrate. The substrate arises largely from
peripheral tissues but needs to be released in the circulation and taken up by the central
and crucial organs like liver, immune system, and wound in which this host response
occurs. The transport needs to be nontoxic, and the waste products arising from these
substrates need to be presented to the organs involved in their clearance. The two main
categories of substrates necessary to sustain a host response consist of appropriate fuel
and amino acids necessary for synthesis of proteins that play important roles in host
response.
It is generally acknowledged that glutamine carbon is an important fuel for white
cells in liver, spleen, and the remainder of the immune system.32,33 The glutamine released
by peripheral tissues, largely muscle tissue, is derived from three sources: from the free
intracellular pool, from protein breakdown, and from new formation. The quantities of
glutamine released from the periphery can only be derived from the free pool to a very
limited degree, as this pool would suffice for only 1 day to deliver the amount of glutamine
taken up in the splanchnic area. In addition, the amount of glutamine derived from protein
degradation can only be modest. On average, glutamine constitutes only 5% of human
1382_C25.fm Page 409 Tuesday, October 7, 2003 7:02 PM

Chapter twenty-five: Gastrointestinal disease 409

muscle protein, and the amount of amino acid-contained nitrogen exported from muscle
consists of 30% glutamine. The new formation of glutamine in the periphery arises from
ammonia taken up in the periphery by glutamic acid, which in turn arises from transam-
ination of branched-chain amino acids to a-ketoglutarate, largely resulting from the deg-
radation of glucose. The branched-chain amino acids serve as fuel for muscle tissue after
their transamination. The beauty of this arrangement is that the catabolic process in muscle
furnishes amino acids that function as building blocks for protein synthesis in the liver,
wound, and immune system, and glutamine that serves as fuel for white cells in liver,
spleen, wound, and immune system. Glutamine is ideally suited for this purpose, because
its concentration can differ without toxic side effects. The carbon skeleton of glutamine
can easily be oxidized to generate energy, and the sites where it is degraded are ideally
located, so that the ammonia resulting from the first step in its breakdown is presented
to the liver or the kidney. These organs can adequately deal with this ammonia by the
formation of urea or by the excretion in the urine, respectively (see Chapter 26).

25.1.2.2 The role of the intestine in the routing of amino acid-derived carbon
skeletons
The role of the intestine in the routing of amino acid-derived carbon is not completely
elucidated. Part of the glutamine skeleton is oxidized; part is degraded to yield alanine
and citrulline. Ubiquitously 14C-labeled glutamine carbon was shown to be metabolized
in a semi-in vivo intestinal preparation for 55% to CO2, 8 to 15% lactate, 2% citrate, 1%
other organic acids, 5% citrulline, 4% proline, 4% alanine, and 4% glucose.34 The formation
of alanine can be considered part of the Cori cycle in that it serves as precursor of
gluconeogenesis. Peripheral tissues take up the glucose thus produced in the liver where
it can yield anaplerotic substrate for the Krebs cycle or where it serves glycolysis. Pyruvate
resulting from glycolysis in turn can be transaminated with branched-chain amino acids
to yield alanine, which is released in the circulation and in turn can participate in renewed
Cori cycling. Cori cycling is an expensive but useful way to provide energy to badly
perfused tissues, but it is also operative and apparently effective in severely stressed or
septic states. Sepsis or severe illness induces and apparently requires an increased glyco-
lytic flux, which provides energy in compromised tissues but needs to be fueled in organs
that still have preserved Krebs cycle activity. The main organ performing this function is
the liver.

25.1.3 The role of the intestine in the production of specific amino acids
25.1.3.1 Intestinal production of citrulline and arginine
The intestine is the only site where substantial amounts of citrulline are produced by way
of the enzymes glutaminase, ornithine–oxoacid aminotransferase, and ornithine transcar-
bamylase, which degrade glutamine to produce citrulline via glutamate and ornithine (see
Chapter 9 for more details on this pathway). The production of citrulline is crucial because
it is released in the circulation and can be taken up by the kidney and produce arginine
via argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). These pathways
are probably very important because the length of the remaining small bowel in short
bowel syndrome has been demonstrated to correlate with citrulline levels.35 In case reports,
hyperammonemic encephalopathy had been described in the patient presenting healthy
livers but short bowel.36 Similarly, focal tubulo-interstitial nephritis has been suggested to
result from short bowel and low citrulline and, consequently, arginine levels.37 Supple-
mentation with arginine reversed the renal pathology. In contradistinction with what is
generally claimed, in our patients we found consistently negative arterio-portal
1382_C25.fm Page 410 Tuesday, October 7, 2003 7:02 PM

410 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

concentration differences for arginine, implying that there is substantial production of


arginine in the gut in the fasted state.28 The arginine is produced in the gut because the
gut has modest ASS and ASL activity. This arginine did not arise from net negative protein
balance or from the fact that the patients may not have been completely fasting, because
net production by the gut was not found for other (conditionally) essential amino acids.
Arginine may serve an important role in the urea cycle, and is therefore unlikely to be
released into the general circulation because in this process it is avidly taken up and
metabolized in the liver via arginase. It may therefore be crucial that at times of dietary
shortage the kidney serves as an extrasplanchnic producer of arginine, which can be made
available to the systemic circulation and to peripheral organs for protein synthesis and
NO production. In this context, it is worthwhile to mention that citrulline passes through
the liver without significant uptake. In addition, Cynober38 has pointed out repeatedly
that in times when arginine intake is high, it is taken up as such from the gut and released
into the portal vein, where it is then taken up by the liver and degraded in the urea cycle.34
On the other hand, when omnivorous mammals are kept on a low arginine diet, the
intestinal machinery adapts and converts most of the enterally administered arginine to
citrulline. This then passes through the liver without significant uptake and reaches the
kidney, where conversion to arginine takes place. Obviously, this elegant biological
arrangement has an arginine-sparing effect in times of arginine scarcity. It should be
stipulated that theoretically this role could be taken over by glutamine, since this is
converted to a certain degree to citrulline.

25.1.3.2 Intestinal production of taurine and glycine


The entero-hepatic cycling of bile acids has received much interest in the past decades for
its role in several digestive and metabolic processes, which are not the subject of this
chapter. In a study in which we assessed in surgical patients the concentration differences
of amino acids in different parts of the intestine, we found unexpectedly high concentration
differences across the jejunum and ileum of glycine and taurine.28 The release of taurine
into the venous effluent of the ileum reached levels of an order of magnitude similar to
those of the uptake of glutamine and the release of alanine. The release of taurine and
glycine cannot be derived from protein degradation or from the diet, because this did not
occur with most other amino acids, essential or nonessential. An obvious explanation is
that in the process of reabsorption of conjugated bile acids in the small bowel, these
conjugates are de-conjugated and released into the portal vein as bile acids and free glycine
and taurine. It demonstrates that entero-hepatic cycling of bile salts is not restricted to the
bile acid part but also applies to these amino acids. The consequences of bile acid malab-
sorption and the coinciding loss of glycine and especially taurine have not received much
attention in the literature.

25.2 Intermediary protein metabolism in the intestine


25.2.1 The biological value of protein
For more than three decades, and especially since the advent of tracer techniques allowing
for the assessment of protein synthesis and turnover, it has been known that after a meal
in a healthy subject there is net protein synthesis, implying that at the whole-body level
more protein is synthesized than is degraded. The negativity and the positivity neutralize
each other after 24 h in healthy organisms. In the last decade many investigators have
suggested39 and provided evidence that the splanchnic area and specifically the intestine
itself40–44 play an important role in accumulating protein after a meal and releasing amino
acids from this protein in the postabsorptive period. The assessment of protein kinetics
1382_C25.fm Page 411 Tuesday, October 7, 2003 7:02 PM

Chapter twenty-five: Gastrointestinal disease 411

after a meal in the splanchnic area is complicated by several factors. However, the dis-
tinction between intestinal and hepatic utilization of meal-derived protein is difficult for
methodological reasons. In addition, the assessment of protein kinetics by the gut is
hampered by the fact that amino acids can be metabolized that are derived from the
intestinal lumen and from the arterial inflow of amino acids. Finally, the intestine stores,
synthesizes, and releases protein in the gut lumen and the gut wall. All these factors
complicate the precise assessment of what happens with meal-derived protein. Neverthe-
less, we will, in the following part of this chapter, try to provide evidence for the presence
of a major part of the labile protein pool in the intestine, as well as propose a role for this
pool and its relevance to the biological value of protein, and its role in several disease
states.

25.2.1.1 The appearance of amino acids in the portal vein after a protein-containing
bolus meal
The quality of a protein has been claimed to be dependent on two factors: the digestibil-
ity/absorption and the composition of the protein. Applying these criteria to different
proteins attributed much weight to the amino acid composition of the protein because
most proteins for human use are easily digested. In fact, in the past decades some protein
formula diets have been claimed to have superior quality because after ingestion, amino
acids derived from digestion of the protein rapidly appeared in the portal vein. In the
following paragraphs we will try to explain why this is a misconception.
Waterlow39 proposed that a substantial part of the labile protein pool, accumulating
during and immediately after a meal, resided in the splanchnic area, but due to lack of
sophisticated techniques was not yet able to prove this and to specify its exact location.
Consequently, it was also impossible to define its relevance for protein quality and for the
efficiency of the utilization of specific food proteins. In the last decade, several investigators
have contributed to a new approach to this field. Reeds and others41,45,46 performed animal
research and assessed the handling of specific amino acids by different organs, including
the intestine. Beaufrère and others44,47,48 developed techniques to measure the first-pass
metabolism of protein-derived amino acids in the splanchnic area since 1996. He devel-
oped the concept of slow and fast proteins and applied his techniques to different proteins
and in different age groups.
We have since developed, in more than a decade, a view on protein quality, originally
inspired by the effect of different proteins on ammonia generation in the gut in liver
patients.49 In an attempt to explain the dismal effect of bleeding on neurological state in
liver patients, we found that hemoglobin, infused as packed erythrocytes, yielded high
levels of ammonia and free amino acids in the portal vein with one exception: isoleucine,
which dropped to almost undetectable levels. This was suggested to be caused by the fact
that adult hemoglobin does not contain isoleucine. Supplementation with isoleucine led
to much lower rates of appearance of amino acids after packed erythrocytes and to lower
portal ammonia levels. The amino acid imbalance of the protein ingested apparently led
to an increased release of amino acids, whereas balancing the protein led to lower appear-
ance rates.
We hypothesized that a balanced amino acid profile was more suitable for protein
synthesis from absorbed amino acids inside the intestinal wall or lumen, which then would
be temporarily retained. To test this hypothesis, we assessed the influence of other methods
to optimize protein synthesis on the appearance of amino acids in the portal vein. Adding
calories to protein in the meal is known to optimize protein synthesis, and this intervention
decreased the appearance of amino acids in the portal vein after bolus feeding. Again, we
interpreted these data to imply that the amino acids derived from protein digestion are
1382_C25.fm Page 412 Tuesday, October 7, 2003 7:02 PM

412 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

partly reutilized for protein synthesis in the gut wall and in the gut lumen if circumstances
for protein synthesis are optimal. The addition of the lacking essential amino acid
isoleucine and the addition of calories to a protein meal lead not only to lower appearance
rates of free amino acids in the portal vein but also to decreased ureagenesis. High portal
concentrations of amino acids and, consequently, high fluxes of amino acids are known
to increase urea formation. The delayed appearance of amino acids and the lower initial
urea formation after the bolus meal may spare protein, provided the remaining amino
acids derived from digestion and apparently reutilized for the synthesis of proteins in the
gut wall or lumen are in turn digested and absorbed to appear in the portal vein and to
be made available to the organism.

25.2.1.2 The regulation of protein accretion in the gut


Many authors have investigated postprandial protein turnover and confirmed a positive
protein balance at the whole-body level.50–53 Although the location of the protein accumu-
lated could not be specified, in most reports whole-body level protein degradation was
diminished, whereas protein synthesis decreased or did not exhibit changes.50,51,53,54 The
kinetics of the labile protein pool in the gut have only been investigated in a few labora-
tories in experimental settings. In our own laboratory, we tried to assess via tracer tech-
niques across different organs the changes of protein synthesis and degradation.55 After
a meal containing either casein or soy as a protein we found that feeding self-evidently
increased the net appearance of amino acids in the portal vein. If it is assumed that
phenylalanine is hardly oxidized in the intestine, its appearance reflects the rate at which
protein from intestinal wall sources or from luminal, meal-derived origin is degraded.
The appearance of phenylalanine across the intestine as a marker of protein degradation
greatly increased to a similar degree in casein and in soy protein-containing meals. Protein
synthesis increased, however, to a greater degree after casein than after soy. This difference
was not significant, but the difference between appearance (protein degradation) and
disappearance (protein synthesis) of phenylalanine was lower in the casein group than in
the soy group. This implies that less of the meal-derived protein appears in the portal
vein and, therefore, that more of the casein protein is temporarily retained in the intestine.
Simultaneously, urea production was greatly enhanced in the first few hours after initiating
tube feeding in these pigs in the soy group, whereas this was not the case in the casein
group. This may be partly because the protein-containing meals that were administered
via tube feeding to the experimental animals were given at low rates. When more protein
is administered, more of the protein-derived amino acids will be oxidized.45 Other groups
reported similar findings.41,42,56–58
Boirie et al.47 and Arnal et al.48 employed a dual-tracer technique with two leucine
tracers given with the meal either intravenously or intragastrically. This is an established
technique to estimate splanchnic extraction of the protein in the meal. They compared
whey and casein protein in an otherwise balanced diet and found that more of the whey
protein appeared in the first few hours after the meal in the portal vein, that amino acid
concentrations were higher in the whey group, and that the protein synthesis rate was
higher. After 2 h, however, protein synthesis dropped in the whey group, whereas in the
casein group synthesis remained higher than in the whey group and protein degradation
remained suppressed longer in the casein group. All these findings are consistent with
the interpretation that despite the fact that the meals are identical, isocaloric, and isonitro-
genous, different proteins are utilized differently. The rapid appearance and high oxidation
of whey proteins have led to the designation fast dietary protein, but implies that it has
a short-lasting anabolic effect, whereas a slow dietary protein like casein is oxidized to a
lesser extent and has a longer-lasting anabolic effect, which makes it a protein with a
higher biological value.
1382_C25.fm Page 413 Tuesday, October 7, 2003 7:02 PM

Chapter twenty-five: Gastrointestinal disease 413

25.2.2 What do the protein kinetic data signify in biological terms?


25.2.2.1 The nature of the labile protein pool in the intestine
The fact that meal protein-derived amino acids are temporarily retained in the intestine
or in the splanchnic area during and immediately after a meal raises the question of what
the nature of these proteins is.
The first possibility includes differences in the rate of digestion and absorption.44 This
may apply for casein, which is known to coagulate in the stomach and, consequently, to
be digested at a much slower rate than most other proteins. In the study that we performed
looking at differences of protein kinetics in pigs fed either casein or soy protein, we found
that the rate of absorption did not significantly differ between these two proteins but that
the appearance in the portal vein was different. In addition, the differences in appearance
that occur when circumstances for protein synthesis are not optimal cannot be explained
by bad digestibility of casein.
We suggest that the explanation for the findings reviewed in this chapter is more
complex. After a bolus meal, a concerted action of amino acids, glucose, insulin, and
cholecystokinin stimulates the secretion and synthesis of pancreatic and intestinal diges-
tive enzymes.59,60 Part of these enzymes is already present in the pancreas as zymogen
stores and is waiting to be released during the meal. Control of this process occurs at the
posttranslational level.60 This process adds enzyme protein to the total protein and amino
acid pool in the intestine, which complicates interpretation of tracer studies during enteral
feeding but does not explain the retention of meal-derived amino acids, because the
enzymes released were already present and were not synthesized from amino acids from
the meal. Also, newly synthesized pancreatic enzymes can only be derived from amino
acids taken up from the systemic circulation, because of the anatomic location of the
pancreas. These considerations do not fully apply to the synthesis and secretion of
enzymes by the intestinal mucosa, because at this location amino acids utilized for enzyme
protein synthesis may be derived both from the intestinal lumen and from the systemic
circulation.61,62 For this to be the case, it must be possible that goblet cells and enterocytes
take up amino acids from the intestinal lumen and from the baso-lateral membrane. Only
part of the labile protein pool in the intestine may therefore be located in the enzymes,
newly synthesized in response to a meal. Similar considerations apply to the synthesis of
mucin in the intestine, pancreas, and bile. The question is whether mucin is directly
synthesized from gut-derived substrate or from substrate derived from the systemic cir-
culation.
A second potential store of amino-nitrogen consists of di- or tripeptides absorbed from
the intestinal lumen and subsequently released as such into the portal vein. Claims have
been made that part of protein-derived amino-nitrogen is released in this manner, but
very little reliable data are available. Another possibility is that the intestine produces
proteins that are released into the portal vein in a manner similar to that of the production
of acute phase proteins synthesized by the liver. At present, an estimate of the quantity
of protein secreted into the portal vein by the gut cannot be made.
A third and very likely factor contributing to retention of meal-derived protein inside
the intestine is proliferation of bacteria and bacterial protein during and after the meal,
which subsequently is digested and absorbed by the enterocytes.
These three potential stores of acutely produced protein may form a substantial part
of the labile protein pool that accumulates during and after the meal, and in the post-
absorptive phase is degraded, furnishing amino acids to the organism. The result is that
after 24 h a zero protein balance is reached.
1382_C25.fm Page 414 Tuesday, October 7, 2003 7:02 PM

414 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

The presence of a labile protein pool in the intestine has beneficial effects to the body.
It prevents rapid degradation of protein in bolus meals and improves the efficiency of
protein utilization. This is beneficial at times when food is scarce and when the organism
has to rely on the unforeseen moment that food is available. The ability of the human
organism to retain protein in this labile protein pool is very modest, however, compared
with reptiles like the python, which can live on one meal per 2 months. This achievement
is also effectuated by a labile protein pool, consisting of the meal itself, which is very
slowly digested and absorbed from the gut, and also the intestine, which greatly hyper-
trophies after the meal and, consequently, greatly increases its protein content.63
The data reviewed also indicate that for the labile protein pool to expand maximally,
a protein with a high biological value should be consumed, combined with calories and
other essential nutrients to promote maximal protein synthesis. We reported similar find-
ings with regard to casein and soy protein. Soy appeared much faster in the portal vein
and stimulated urea production to a much greater extent than casein. These findings have
defined casein as a slow protein and whey and soy protein as fast proteins and support
the claim that casein is a better protein in healthy organisms consuming bolus meals.
There are few data shedding light on these aspects of protein quality in disease states.
However, we will try to formulate suggestions as to how the principles outlined above
may apply to disease states.

25.2.2.2 The role of the labile protein pool in old age and disease
The scarce data that exist with regard to the retention or utilization of meal-derived protein
in the splanchnic area are obtained in elderly people. The difference in appearance rates
of leucine, obtained via the dual-tracer approach, is a measure for the splanchnic extraction
of the meal-derived amino acids. The splanchnic extraction is the sum of amino acids
degraded and used for protein synthesis during their first pass from food into the portal
circulation and to the liver. These techniques were applied in senescence and compared
with a younger age group.44,64,65 It was found that splanchnic extraction decreased in
elderly people when assessed with labeled phenylalanine or leucine. Muscle protein
synthesis increased less in elderly people than in younger people, and protein degradation
was less suppressed. Beaufrère65 suggested that increased splanchnic extraction of meal-
derived protein contributes to the sarcopenia of old age (see Chapter 24 for more details).
The unanswered question remains: what happens to the protein-derived amino acids
extracted during their first pass in the splanchnic area? One possibility is that in old age
there is a state of continuous inflammation leading to the synthesis of acute phase proteins
in the splanchnic area from meal-derived amino acids. Although this is a possibility, at
present there are no data supporting this hypothesis. Albumin synthesis has been shown
not to differ between young and old people.64
Very little data exist regarding protein kinetics in critically ill patients and in patients
with compromised intestinal function, including short bowel syndrome. It has been
reported, however, that digestion and absorption are compromised in severely ill
patients,66,67 and that they often do not tolerate their full daily requirements enterally.68
Bolus feeding leads even more to nausea, vomiting, and diarrhea. The disturbances in
digestion and absorption reduce the ability of the bowel to adequately take up nutrients
in a given period. This type of clinical experience therefore dictates spreading of nutrient
intake to continuous infusions of liquid formulas via fine-bore tubes. Another reason why
this may be indicated is the likelihood that bolus feeding will fail to raise an anabolic
response in the intestine and to expand the labile protein pool in periods of sepsis and
critical illness. This will lead to inefficient utilization of protein and to increased ureagen-
esis and oxidation of amino acid-derived carbon skeletons.
1382_C25.fm Page 415 Tuesday, October 7, 2003 7:02 PM

Chapter twenty-five: Gastrointestinal disease 415

Another disease state in which enteral nutrition should be optimized to fulfill nutri-
tional requirements is the short bowel syndrome. It can be assumed that for the bowel to
act as a reservoir of protein, successful recycling of protein and amino acids is necessary.
For that purpose, a critical intestinal mass for retaining labile protein and sufficient intes-
tinal length are necessary to allow complete digestion and reabsorption of recycled protein.
Some short bowel patients only manage to maintain adequate body cell mass if they
receive, in addition to their normal daily bolus meals, nocturnal continuous enteral feeding
through fine-bore feeding tubes. They also have been shown to benefit from more frequent
but smaller meals.

25.3 Summary and conclusions


In the past three decades it has become clear from flux studies across the gut that the
intestine plays a role not only in the absorption of food but also in the intermediary and
interorgan metabolism of amino acids. There are still gaps in our current understanding
of the exact mass flux of amino-N, because the studies hitherto performed do not take
into account the flux of oligopeptides. In addition, the role of bacteria and the cycling of
nitrogen through bile salts or intestinal enzymes remain to be quantitated.
These factors have an important bearing on the assessment of protein quality, because
they are very likely to be an important component of the labile protein pool, which
accumulates after food ingestion.
Improving our understanding of the complex role of the intestine in amino acid and
protein handling is crucial in designing optimal nutrition in health and disease.

References
1. Matsutaka, H., Aikawa, T., Yamamoto, H., and Ishikawa, E., Gluconeogenesis and amino
acid metabolism. 3. Uptake of glutamine and output of alanine and ammonia by non-hepatic
splanchnic organs of fasted rats and their metabolic significance, J. Biochem. (Tokyo), 74,
1019–1029, 1973.
2. Watford, M., Lund, P., and Krebs, H.A., Isolation and metabolic characteristics of rat and
chicken enterocytes, Biochem. J., 178, 589–596, 1979.
3. Windmueller, H.G. and Spaeth, A.E., Uptake and metabolism of plasma glutamine by the
small intestine, J. Biol. Chem., 249, 5070–5079, 1974.
4. Windmueller, H.G. and Spaeth, A.E., Respiratory fuels and nitrogen metabolism in vivo in
small intestine of fed rats: quantitative importance of glutamine, glutamate, and aspartate,
J. Biol. Chem., 255, 107–112, 1980.
5. Windmueller, H.G. and Spaeth, A.E., Intestinal metabolism of glutamine and glutamate from
the lumen as compared to glutamine from blood, Arch. Biochem. Biophys., 171, 662–672, 1975.
6. Yoshida, S., Leskiw, M.J., Schluter, M.D., Bush, K.T., Nagele, R.G., Lanza-Jacoby, S., and Stein,
T.P., Effect of total parenteral nutrition, systemic sepsis, and glutamine on gut mucosa in
rats, Am. J. Physiol., 263, E368–E373, 1992.
7. Ziegler, T.R., Young, L.S., Benfell, K., Scheltinga, M., Hortos, K., Bye, R., Morrow, F.D., Jacobs,
D.O., Smith, R. J., Antin, J.H., et al., Clinical and metabolic efficacy of glutamine-supple-
mented parenteral nutrition after bone marrow transplantation: a randomized, double-blind,
controlled study, Ann. Intern. Med., 116, 821–828, 1992.
8. Scheltinga, M.R., Young, L.S., Benfell, K., Bye, R.L., Ziegler, T.R., Santos, A.A., Antin, J.H.,
Schloerb, P.R., and Wilmore, D.W., Glutamine-enriched intravenous feedings attenuate extra-
cellular fluid expansion after a standard stress, Ann. Surg., 214, 385–393, 1991.
9. Souba, W.W. and Wilmore, D.W., Postoperative alteration of arteriovenous exchange of
amino acids across the gastrointestinal tract, Surgery, 94, 342–350, 1983.
10. Souba, W.W., Smith, R.J., and Wilmore, D.W., Effects of glucocorticoids on glutamine me-
tabolism in visceral organs, Metabolism, 34, 450–456, 1985.
1382_C25.fm Page 416 Tuesday, October 7, 2003 7:02 PM

416 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

11. Souba, W.W., Smith, R.J., and Wilmore, D.W., Glutamine metabolism by the intestinal tract,
J. Parenter. Enteral Nutr., 9, 608–617, 1985.
12. Souba, W.W., Scott, T.E., and Wilmore, D.W., Intestinal consumption of intravenously ad-
ministered fuels, J. Parenter. Enteral Nutr., 9, 18–22, 1985.
13. Souba, W.W., Roughneen, P.T., Goldwater, D.L., Williams, J.C., and Rowlands, B.J., Post-
operative alterations in interorgan glutamine exchange in enterectomized dogs, J. Surg. Res.,
42, 117–125, 1987.
14. Souba, W.W., Klimberg, V.S., Plumley, D.A., Salloum, R.M., Flynn, T.C., Bland, K.I., and
Copeland, E.M., III, The role of glutamine in maintaining a healthy gut and supporting the
metabolic response to injury and infection, J. Surg. Res., 48, 383–391, 1990.
15. Souba, W.W., Total parenteral nutrition with glutamine in bone marrow transplantation and
other clinical applications, J. Parenter. Enteral Nutr., 17, 403, 1993.
16. Souba, W.W., Glucocorticoids alter amino acid metabolism in visceral organs, Surg. Forum,
79, 1983.
17. Sarac, T.P., Souba, W.W., Miller, J.H., Ryan, C.K., Koch, M., Bessey, P.Q., and Sax, H.C.,
Starvation induces differential small bowel luminal amino acid transport, Surgery, 116,
679–685, 1994.
18. Deutz, N.E., Reijven, P.L., Athanasas, G., and Soeters, P.B., Post-operative changes in hepatic,
intestinal, splenic and muscle fluxes of amino acids and ammonia in pigs, Clin. Sci., 83,
607–614, 1992.
19. Bruins, M.J., Soeters, P.B., and Deutz, N.E., Endotoxemia affects organ protein metabolism
differently during prolonged feeding in pigs, J. Nutr., 130, 3003–3013, 2000.
20. Fong, Y.M., Tracey, K.J., Hesse, D.G., Albert, J.D., Barie, P.S., and Lowry, S.F., Influence of
enterectomy on peripheral tissue glutamine efflux in critically ill patients, Surgery, 107,
321–326, 1990.
21. Clowes, G.H., Jr., Randall, H.T., and Cha, C.J., Amino acid and energy metabolism in septic
and traumatized patients, J. Parenter. Enteral Nutr., 4, 195–205, 1980.
22. Carli, F., Webster, J., Ramachandra, V., Pearson, M., Read, M., Ford, G.C., McArthur, S.,
Preedy, V.R., and Halliday, D., Aspects of protein metabolism after elective surgery in patients
receiving constant nutritional support, Clin. Sci. (Lond.), 78, 621–628, 1990.
23. Mjaaland, M., Unneberg, K., Larsson, J., Nilsson, L., and Revhaug, A., Growth hormone after
abdominal surgery attenuated forearm glutamine, alanine, 3-methylhistidine, and total ami-
no acid efflux in patients receiving total parenteral nutrition, Ann. Surg., 217, 413–422, 1993.
24. Brown, J.A., Gore, D.C., and Jahoor, F., Catabolic hormones alone fail to reproduce the stress-
induced efflux of amino acids, Arch. Surg., 129, 819–824, 1994.
25. van Acker, B.A., Hulsewe, K.W., Wagenmakers, A.J., Soeters, P.B., and von Meyenfeldt, M.F.,
Glutamine appearance rate in plasma is not increased after gastrointestinal surgery in hu-
mans, J. Nutr., 130, 1566–1571, 2000.
26. Gore, D.C. and Jahoor, F., Glutamine kinetics in burn patients: comparison with hormonally
induced stress in volunteers, Arch. Surg., 129, 1318–1323, 1994.
27. Jackson, N.C., Carroll, P.V., Russell-Jones, D.L., Sonksen, P.H., Treacher, D.F., and Umpleby,
A.M., The metabolic consequences of critical illness: acute effects on glutamine and protein
metabolism, Am. J. Physiol., 276, E163–E170, 1999.
28. van der Hulst, R.R., von Meyenfeldt, M.F., Deutz, N.E., and Soeters, P.B., Glutamine extrac-
tion by the gut is reduced in depleted [corrected] patients with gastrointestinal cancer, Ann.
Surg., 225, 112–121, 1997.
29. van der Hulst, R.R., van Kreel, B.K., von Meyenfeldt, M.F., Brummer, R.J., Arends, J.W.,
Deutz, N.E., and Soeters, P.B., Glutamine and the preservation of gut integrity, Lancet, 341,
1363–1365, 1993.
30. Bode, B.P., Fuchs, B.C., Hurley, B.P., Conroy, J.L., Suetterlin, J.E., Tanabe, K.K., Rhoads, D.B.,
Abcouwer, S.F., and Souba, W.W., Molecular and functional analysis of glutamine uptake in
human hepatoma and liver-derived cells, Am. J. Physiol., 283, G1062–G1073, 2002.
31. Holm, E., Hagmuller, E., Staedt, U., Schlickeiser, G., Gunther, H.J., Leweling, H., Tokus, M.,
and Kollmar, H.B., Substrate balances across colonic carcinomas in humans, Cancer Res., 55,
1373–1378, 1995.
1382_C25.fm Page 417 Tuesday, October 7, 2003 7:02 PM

Chapter twenty-five: Gastrointestinal disease 417

32. Newsholme, E.A., A role for muscle in the immune system and its importance in surgery,
trauma, sepsis and burns, Nutrition, 4, 261–268, 1988.
33. Newsholme, E.A., Glutamine and immune cells, Front. Clin. Nutr., 4, 2–5, 1992.
34. Windmueller, H.G., Glutamine utilization by the small intestine, Adv. Enzymol. Relat. Areas
Mol. Biol., 53, 201–237, 1982.
35. Crenn, P., Coudray-Lucas, C., Thuillier, F., Cynober, L., and Messing, B., Postabsorptive
plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal
failure in humans, Gastroenterology 119, 1496–1505, 2000.
36. Yokoyama, K., Ogura, Y., Kawabata, M., Hinoshita, F., Suzuki, Y., Hara, S., Yamada, A.,
Mimura, N., Nakayama, M., Kawaguchi, Y., and Sakai, O., Hyperammonemia in a patient
with short bowel syndrome and chronic renal failure, Nephron, 72, 693–695, 1996.
37. Hebiguchi, T., Kato, T., Yoshino, H., Mizuno, M., Wakui, H., Komatsuda, A., and Imai, H.,
Renal focal tubulointerstitial fibrosis with short bowel syndrome: report of a case, Surg. Today,
32, 646–650, 2002.
38. Cynober, L., Can arginine and ornithine support gut functions? Gut, 35 (Suppl.), S42–S45,
1994.
39. Waterlow, J.C., Whole-body protein turnover in humans: past, present, and future, Annu.
Rev. Nutr., 15, 57–92, 1995.
40. Soeters, P.B., de Jong, C.H., and Deutz, N.E., The protein sparing function of the gut and the
quality of food protein, Clin. Nutr., 20, 97–99, 2001.
41. Van Der Schoor, S.R., Reeds, P.J., Stoll, B., Henry, J.F., Rosenberger, J.R., Burrin, D.G., and
Van Goudoever, J.B., The high metabolic cost of a functional gut, Gastroenterology, 123,
1931–1940, 2002.
42. Volpi, E., Lucidi, P., Cruciani, G., Monacchia, F., Reboldi, G., Brunetti, P., Bolli, G.B., and De
Feo, P., Contribution of amino acids and insulin to protein anabolism during meal absorption,
Diabetes, 45, 1245–1252, 1996.
43. Mariotti, F., Huneau, J.F., Mahe, S., and Tome, D., Protein metabolism and the gut, Curr.
Opin. Clin. Nutr. Metab. Care, 3, 45–50, 2000.
44. Dangin, M., Boirie, Y., Garcia-Rodenas, C., Gachon, P., Fauquant, J., Callier, P., Ballevre, O.,
and Beaufrère, B., The digestion rate of protein is an independent regulating factor of
postprandial protein retention, Am. J. Physiol., 280, E340–E348, 2001.
45. van der Schoor, S.R., van Goudoever, J.B., Stoll, B., Henry, J.F., Rosenberger, J.R., Burrin, D.G.,
and Reeds, P.J., The pattern of intestinal substrate oxidation is altered by protein restriction
in pigs, Gastroenterology, 121, 1167–1175, 2001.
46. Stoll, B., Henry, J., Reeds, P.J., Yu, H., Jahoor, F., and Burrin, D.G., Catabolism dominates the
first-pass intestinal metabolism of dietary essential amino acids in milk protein-fed piglets,
J. Nutr., 128, 606–614, 1998.
47. Boirie, Y., Dangin, M., Gachon, P., Vasson, M.P., Maubois, J.L., and Beaufrère, B., Slow and
fast dietary proteins differently modulate postprandial protein accretion, Proc. Natl. Acad.
Sci. U.S.A., 94, 14930–14935, 1997.
48. Arnal, M.A., Mosoni, L., Boirie, Y., Gachon, P., Genest, M., Bayle, G., Grizard, J., Arnal, M.,
Antoine, J.M., Beaufrère, B., and Patureau Mirand, P., Protein turnover modifications induced
by the protein feeding pattern still persist after the end of the diets, Am. J. Physiol., 278,
E902–E909, 2000.
49. Olde Damink, S.W., Dejong, C.H., Deutz, N.E., van Berlo, C.L., and Soeters, P.B., Upper
gastrointestinal bleeding: an ammoniagenic and catabolic event due to the total absence of
isoleucine in the haemoglobin molecule, Med. Hypotheses, 52, 515–519, 1999.
50. Quevedo, M.R., Price, G.M., Halliday, D., Pacy, P.J., and Millward, D.J., Nitrogen homo-
eostasis in man: diurnal changes in nitrogen excretion, leucine oxidation and whole body
leucine kinetics during a reduction from a high to a moderate protein intake, Clin. Sci., 86,
185–193, 1994.
51. Pacy, P.J., Price, G.M., Halliday, D., Quevedo, M.R., and Millward, D.J., Nitrogen homeostasis
in man: the diurnal responses of protein synthesis and degradation and amino acid oxidation
to diets with increasing protein intakes, Clin. Sci., 86, 103–116, 1994.
1382_C25.fm Page 418 Tuesday, October 7, 2003 7:02 PM

418 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

52. Millward, D.J., Price, G.M., Pacy, P.J., and Halliday, D., Whole-body protein and amino acid
turnover in man: what can we measure with confidence? Proc. Nutr. Soc., 50, 197–216, 1991.
53. Melville, S., McNurlan, M.A., McHardy, K.C., Broom, J., Milne, E., Calder, A.G., and Garlick,
P.J., The role of degradation in the acute control of protein balance in adult man: failure of
feeding to stimulate protein synthesis as assessed by L-[1-13C]leucin infusion, Metabolism
38, 248–255, 1989.
54. Cayol, M., Tauveron, I., Rambourdin, F., Prugnaud, J., Gachon, P., Thieblot, P., Grizard, J.,
and Obled, C., Whole-body protein turnover and hepatic protein synthesis are increased by
vaccination in man, Clin. Sci., 89, 389–396, 1995.
55. Deutz, N.E., Bruins, M.J., and Soeters, P.B., Infusion of soy and casein protein meals affects
interorgan amino acid metabolism and urea kinetics differently in pigs, J. Nutr., 128,
2435–2345, 1998.
56. van Goudoever, J.B., Stoll, B., Henry, J.F., Burrin, D.G., and Reeds, P.J., Adaptive regulation
of intestinal lysine metabolism, Proc. Natl. Acad. Sci. U.S.A., 97, 11620–11625, 2000.
57. Mahe, S., Roos, N., Benamouzig, R., Davin, L., Luengo, C., Gagnon, L., Gausserges, N.,
Rautureau, J., and Tome, D., Gastrojejunal kinetics and the digestion of [15N]beta-lactoglo-
bulin and casein in humans: the influence of the nature and quantity of the protein, Am. J.
Clin. Nutr., 63, 546–552, 1996.
58. Gaudichon, C., Mahe, S., Benamouzig, R., Luengo, C., Fouillet, H., Dare, S., Van Oycke, M.,
Ferriere, F., Rautureau, J., and Tome, D., Net postprandial utilization of [15N]-labeled milk
protein nitrogen is influenced by diet composition in humans, J. Nutr., 129, 890–895, 1999.
59. O’Keefe, S.J., Bennet, W.M., Zinsmeister, A.R., and Haymond, M.W., Pancreatic enzyme
synthesis and turnover in human subjects, Am. J. Physiol., 266 (Pt. 1), G816–G821, 1994.
60. Bragado, M.J., Tashiro, M., and Williams, J.A., Regulation of the initiation of pancreatic
digestive enzyme protein synthesis by cholecystokinin in rat pancreas in vivo, Gastroenter-
ology, 119, 1731–1739, 2000.
61. Nakshabendi, I.M., Obeidat, W., Russell, R.I., Downie, S., Smith, K., and Rennie, M.J., Gut
mucosal protein synthesis measured using intravenous and intragastric delivery of stable
tracer amino acids, Am. J. Physiol., 269, E996–E999, 1995.
62. Bouteloup-Demange, C., Boirie, Y., Dechelotte, P., Gachon, P., and Beaufrère, B., Gut mucosal
protein synthesis in fed and fasted humans, Am. J. Physiol., 274, E541–E546, 1998.
63. Holmberg, A., Kaim, J., Persson, A., Jensen, J., Wang, T., and Holmgren, S., Effects of digestive
status on the reptilian gut, Comp. Biochem. Physiol. A Mol. Integr. Physiol., 133, 499–518, 2002.
64. Boirie, Y., Gachon, P., Cordat, N., Ritz, P., and Beaufrère, B., Differential insulin sensitivities
of glucose, amino acid, and albumin metabolism in elderly men and women, J. Clin. Endo-
crinol. Metab., 86, 638–644, 2001.
65. Boirie, Y., Gachon, P., and Beaufrère, B., Splanchnic and whole-body leucine kinetics in young
and elderly men, Am. J .Clin. Nutr., 65, 489–495, 1997.
66. Thompson, J.S., The intestinal response to critical illness, Am. J. Gastroenterol, 90, 190–200,
1995.
67. Sodeyama, M., Gardiner, K.R., Regan, M.C., Kirk, S.J., Efron, G., and Barbul, A., Sepsis
impairs gut amino acid absorption, Am. J. Surg., 165, 150–154, 1993.
68. Montejo, J.C., Enteral nutrition-related gastrointestinal complications in critically ill patients:
a multicenter study. The Nutritional and Metabolic Working Group of the Spanish Society
of Intensive Care Medicine and Coronary Units, Crit. Care Med., 27, 1447–1453, 1999.
1382_C26.fm Page 419 Tuesday, October 7, 2003 7:04 PM

chapter twenty-six

Amino acids and ammonia


in liver disease
Cornelis H.C. Dejong
Academic Hospital, Maastricht
S.W.M. Olde Damink
Academic Hospital, Maastricht
R. Jalan
University College Medical School and UCLH Hospitals
Nicolaas E.P. Deutz
Academic Hospital, Maastricht
Peter B. Soeters
Academic Hospital, Maastricht

Contents
General introduction...................................................................................................................419
26.1 Ammonia and glutamine..................................................................................................420
26.1.1 Glutamine and ammonia exchange across the gut........................................421
26.1.2 Glutamine and ammonia exchange across the liver .....................................422
26.1.3 Glutamine and ammonia exchange across muscle........................................423
26.1.4 Glutamine and ammonia exchange across the brain ....................................424
26.1.5 Glutamine and ammonia exchange across the kidney .................................425
26.1.6 Other organs .........................................................................................................428
26.2 Effects of gastrointestinal bleeding .................................................................................428
26.3 Does the prevailing ammonia hypothesis of hepatic encephalopathy require
modification? ......................................................................................................................429
Acknowledgments ......................................................................................................................429
References .....................................................................................................................................429

General introduction
The liver has a key function in nitrogen metabolism. Liver disease and the resulting
hepatocellular failure adversely affect these processes and induce profound disturbances

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 419
1382_C26.fm Page 420 Tuesday, October 7, 2003 7:04 PM

420 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

among others in nitrogen homeostasis. A crucial feature in this context is the diminished
hepatic urea synthesis capacity, leading to an impaired capacity to detoxify ammonia. This
diminished ammonia detoxification capacity combined with the usually existing intra- or
extrahepatic shunting contributes to the development of systemic hyperammonemia,1,2
which may induce the much feared complication of hepatic encephalopathy.3,4 Hyperam-
monemia may be further aggravated by esophageal variceal bleeds in patients with cir-
rhosis and portal hypertension, leading to an even more pronounced disturbance of
nitrogen homeostasis.
Glutamine synthesis has been suggested to be the most important alternative pathway
for ammonia detoxification during diminished urea synthesis. Therefore, systemic hyper-
ammonemia has a considerable impact on the metabolism of the nonessential amino acid
glutamine. These disturbances in nitrogen homeostasis have been and still are subject to
extensive research. Specifically, the relation between ammonia and glutamine metabolism
in several organs has received considerable attention. However, relatively little attention
has been paid to the interaction between these organs during liver failure. Especially, few
attempts have been made to quantify the exchange of nitrogen and specifically ammonia
and glutamine between organs. In the past 15 years, we have focused on interorgan
exchange of amino acids and ammonia during acute and chronic liver failure and other
hyperammonemic states with particular emphasis on the effects of gastrointestinal bleed-
ing and the role of the gut, liver, muscle, brain, and kidney (reviewed in Olde Damink et
al.5,7 and Dejong et al.6).
Therefore, our first aim is to address ammonia and amino acid metabolism during
acute and chronic liver failure with special emphasis on interorgan exchange of ammonia
and glutamine. Second, the effects of gastrointestinal bleeding will be addressed. Finally,
the prevailing hypothesis on the pathogenesis of hepatic encephalopathy will be discussed
against the background of data from our own group, suggesting that this hypothesis might
require modification.

26.1 Ammonia and glutamine


Ammonia in this chapter refers to total ammonia concentrations, i.e., NH3 + NH4+. Ammo-
nium is formed from ammonia and vice versa in the equilibrium reaction NH3 + H+ =
NH4+.8,9 At physiological pH, 99% is in the form of ammonium (NH4+).10 Ammonia (NH3)
is gaseous and lipophilic and easily diffuses across cell membranes, whereas ammonium
(NH4+) is nondiffusible and can only be transported across biological membranes by
carrier-mediated processes.9–11
Although ammonia is important in several biochemical pathways, it is toxic at elevated
levels.10,12 Therefore, it must be converted to a nontoxic compound, and several pathways
serve this purpose. In most mammals, this takes place mainly in the liver by synthesis of
urea in the urea cycle.2,10 During liver failure, diminished urea synthesis capacity and
portasystemic shunting1,2,13 impair or bypass the main route of ammonia detoxification
(urea synthesis), leading to hyperammonemia.2 In this situation the synthesis of glutamine
(see below) from equimolar amounts of glutamate and ammonia becomes the most impor-
tant, though temporary, pathway for ammonia detoxification.1,10,14 Glutamine is a non-
essential amino acid commonly available in many dietary sources.15 It serves as an oblig-
atory fuel for the gut16–18 and the immune system,10,15 and plays an important role in the
regulation of acid–base balance by providing the most important substrate for renal
ammoniagenesis in many mammals9 (for more details on the role of glutamine in nucleic
acid biosynthesis and protein synthesis, the interested reader can look at Chapter 11 in
this book).
1382_C26.fm Page 421 Tuesday, October 7, 2003 7:04 PM

Chapter twenty-six: Amino acids and ammonia in liver disease 421

During acute and chronic liver failure, ammonia detoxification via urea synthesis fails
and portasystemic shunting is a common feature. As a consequence, ammonia accumulates
in the body as evidenced by hyperammonemia. As stated, the most important alternative
route of ammonia detoxification in this situation is the formation of excess quantities of
glutamine leading to high plasma glutamine levels accompanying the hyperammonemia.
In the following, we will address the role of various organs in this new nitrogen equilib-
rium characterized by hyperammonemia 19and hyperglutaminemia.20,21

26.1.1 Glutamine and ammonia exchange across the gut


In the physiological state, glutamine is a crucial source of energy for the large and espe-
cially small intestine. Besides glucose, short-chain fatty acids, and ketone bodies, the
intestines take up glutamine in large quantities from either the bloodstream or the intes-
tinal lumen.16,17,22,23 This is a concentration-dependent process, with a Vmax at plasma
concentrations of approximately 600 mM.17 Most of the glutamine consumed by the intes-
tine is utilized in the small intestinal mucosa (predominantly jejunum), constituting 75%
of small intestinal weight.24
The initial step in intestinal glutamine breakdown is by conversion of glutamine to
glutamate and ammonia, a reaction catalyzed by the enzyme glutaminase. The intestines
contain high glutaminase activity and only minimal glutamine synthetase activity,16,17,25
which makes this organ particularly suitable to use glutamine as an energy source. On
the other hand, it should be stressed that ammonia is a toxic product of this reaction.
Therefore, from a teleological point of view, it is extremely interesting and important that
the liver is situated immediately downstream of the vascular bed of the intestine. It is
only because of this particular biological arrangement that the intestine can utilize without
adverse effects for the organism a fuel that produces a potentially harmful end product.
The large bowel utilizes less glutamine, but instead uses other substrates, e.g., glucose,
short-chain fatty acids, and ketone bodies.26 Apart from this small contribution to
glutamine metabolism-derived ammonia, the colon contributes significantly to portal
venous ammonia generation by bacterial splitting of urea and possibly amino acids.26–28
The ratio between small intestinal glutamine-derived ammonia and colonic urea-derived
ammonia released into the portal vein is probably approximately 3:2.26
During liver failure, ammonia derived from intestinal glutamine metabolism and
bacterial urea and amino acid breakdown is only partly taken up from the portal vein
and cleared by hepatic urea synthesis. As a consequence of hepatocellular failure and
intra- and extrahepatic portasystemic shunting, gut-derived ammonia escapes hepatic
clearance, resulting in systemic hyperammonemia. The elevated glutamine levels encoun-
tered during liver failure29 have been suggested to enhance intestinal glutamine consump-
tion and thus ammonia production. However, in experimental animal studies, we were
unable to demonstrate this.30 Intestinal glutamine uptake and ammonia production did
not parallel arterial glutamine levels if the latter exceeded 600 mM.30 This is in agreement
with the notion that intestinal glutamine uptake is concentration dependent up to arterial
concentrations of 600 mM.17 Overall, the data suggested that systemic ammonia levels
were merely determined by portasystemic shunting and not by increased intestinal ammo-
nia generation due to increased gut glutamine consumption during high systemic
glutamine levels.7 Recent data from human studies by our group support these experi-
mental animal findings.31
High ammonia levels have been repeatedly implicated in the pathogenesis of hepatic
encephalopathy,1,8,32 a neuropsychiatric syndrome often accompanying hepatic failure.33
Hepatic encephalopathy is an important cause of morbidity and mortality in patients with
acute and chronic liver failure.33 Hyperammonemia and hepatic encephalopathy are often
1382_C26.fm Page 422 Tuesday, October 7, 2003 7:04 PM

422 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

precipitated by gastrointestinal hemorrhage (or other accidental high protein loads)32 or


protein overload of endogenous origin during catabolic illness.33
The association between high protein loads, hyperammonemia, and hepatic enceph-
alopathy in patients with hepatic failure has led to the generally accepted idea that
enhanced intestinal ammonia liberation is an important factor in the pathogenesis of
hepatic encephalopathy.1,32 Consequently, standard therapies focus on removing intestinal
contents, accelerating intestinal transit time by cathartic agents, reducing protein loads,
lowering systemic ammonia levels by acidification of intestinal contents with lactulose,
and reducing bacterial flora with poorly resorbable antibiotics (for review see Record32).
Insight in the role of the intestine in interorgan nitrogen exchange during several hyper-
ammonemic states could therefore be valuable in developing new therapeutic strategies
in the treatment of the complications of liver failure.

26.1.2 Glutamine and ammonia exchange across the liver


In the physiological situation, ammonia and amino acids produced by the gut are released
into the portal blood and transported to the liver.16 Here, ammonia is taken up34,35 and
enters the urea cycle, leading to its detoxification. In addition, several amino acids are
taken up from the portal bloodstream, of which glutamine deserves specific mention in
the context of this chapter.
Ammonia detoxification in the liver occurs mainly in periportal hepatocytes in the
urea cycle, leading to urea formation. Urea is subsequently released into the hepatic veins36
and then excreted in the urine. In addition, part of the urea synthesized participates in an
enterohepatic cycle, since colonic bacteria generate ammonia by splitting luminal urea.26–28
Hepatic urea synthesis is a low-affinity, high-capacity detoxification system,36 and nor-
mally very little ammonia escapes this process. Any ammonia escaping detoxification in
the periportal hepatocytes is usually trapped in the perivenous hepatocytes, where it is
incorporated in glutamine in the glutamine synthetase reaction (a high-affinity, low-capac-
ity system)36 (for more details on the zonation and regulation of ammonia, glutamine, and
urea metabolism in the liver, refer to Chapter 7).
The liver contains both glutaminase and glutamine synthetase activity.25,34,35,37 Thus,
the liver is capable of both synthesizing and degrading glutamine. Whether net production
or consumption of glutamine by the liver occurs is dependent on tissue ammonia and
glutamine concentrations and pH.37 Ammonia activates hepatic glutaminase, providing a
feed-forward mechanism for urea synthesis, whereas a fall in pH is associated with
decreased glutaminase flux and increased glutamine production.37 Also, protons consume
bicarbonate, reducing the bicarbonate available for urea synthesis. Therefore, during acute
and chronic acidosis, the liver becomes an organ of enhanced net glutamine release.34,35,37
In the physiological state, periportal glutamine breakdown and perivenous glutamine
synthesis are probably of equal magnitude, and therefore the liver glutamine balance is
close to zero.36 The role of Kupffer cells in this context is still unclear. As a consequence
of its high glutaminase and glutamine synthetase content25,34 and the way these enzymes
are regulated,36,38 however, the liver can rapidly respond to changes in systemic ammonia
levels and acid–base status by changing from glutamine uptake to release.34–37
Liver disease, whether acute or chronic, leads to impaired hepatic metabolism of
various metabolites, among others ammonia.1,2 Several studies have demonstrated dimin-
ished urea synthesis and glutamine synthesis capacity during liver failure.13,36,38–41 This
reduced ammonia detoxification combined with the usually existing portasystemic shunts
have been proposed to contribute to systemic hyperammonemia.1,2 Very little literature
has appeared on hepatic ammonia and glutamine metabolism during acute or chronic
liver failure. Interestingly, flux through the glutaminase reaction was shown to be
1382_C26.fm Page 423 Tuesday, October 7, 2003 7:04 PM

Chapter twenty-six: Amino acids and ammonia in liver disease 423

increased four- to sixfold in patients with chronic liver failure.36,38 Kaiser et al.38 suggested
that this would lead to enhanced local ammonia formation, acting as a compensatory
mechanism for maintenance of a life-compatible urea cycle flux in a situation of reduced
urea synthetic capacity. However, despite its pivotal role in nitrogen metabolism and
ammonia detoxification, virtually no research has been done on the role of the diseased
liver in interorgan nitrogen transport and ammonia and glutamine exchange.
Recently, we studied hepatic ammonia, glutamine, and urea exchange in 24 patients
with stable hepatic cirrhosis and previous treatment with a transjugular intrahepatic
portasystemic stent shunt (TIPSS) undergoing TIPSS portography to check shunt patency.31
The cirrhotic liver still removed ammonia from the circulation in significant quantities. In
addition, glutamine and alanine uptake from the portal bloodstream accounted for 50%
of total amino acid uptake by the liver.31 Hepatic urea synthesis was significantly correlated
with glutamine and alanine uptake.

26.1.3 Glutamine and ammonia exchange across muscle


In a situation where the liver fails to clear the ammonia generated within the splanchnic
bed, other organs will be forced to adapt to a situation of high systemic ammonia levels.
Glutamine synthesis is the most important alternative detoxification pathway in this
situation, and muscle and brain have traditionally been proposed to play a key role in this.
Glutamine synthetase activity in skeletal muscle is low,25 but by virtue of its mass,
muscle is one of the principal glutamine synthesizing organs.14 Skeletal muscle glutami-
nase activity42 is negligible compared to glutamine synthetase activity, and therefore,
muscle should be viewed as a glutamine-synthesizing organ.
Ammonia can be taken up or released by skeletal muscle. Ammonia release has been
demonstrated during exercise, probably related to purine nucleotide cycle activation.18
Ammonia uptake by skeletal muscle was shown in various hyperammonemic states in
rats, dogs, and man,43–47 but also in healthy human volunteers44,45 and normal control rats.43
This ammonia uptake was suggested to lead to glutamine synthesis in the glutamine
synthetase reaction resulting in an augmentation of the skeletal muscle free glutamine
pool and glutamine release from skeletal muscle.1,14,29
The discussion of whether muscle ammonia uptake always leads to glutamine release
and whether ammonia uptake is of equal magnitude as glutamine release (the stoichiom-
etry issue) is confounded by contrasting reports. Thus, enhanced muscle ammonia uptake
was found during ammonia infusion in rats,43,48 monkeys,46 and dogs.46,47 This ammonia
uptake enhanced skeletal muscle glutamine release only in some of these studies,46,48
whereas glutamine release remained unchanged in others.43,47 Ammonia uptake either
exceeded or balanced the enhanced glutamine release.46
These literature data demonstrate that stoichiometry cannot consistently be shown.
An explanation could be that most of the above mentioned were acute hyperammonemia
experiments. In such acute experiments, steady state is lacking and part of the ammonia
could be trapped in augmented tissue ammonia and glutamine pools. This does not
necessarily lead to glutamine release. During chronic hyperammonemia experiments the
situation is different because hyperammonemia is continuously present and ammonia
trapping in tissues might not be an important phenomenon.
In the context of the stoichiometry issue, it is important to briefly discuss the role of
skeletal muscle in glutamine metabolism in nonhyperammonemic states. Skeletal muscle
is an organ of net glutamine release in the physiological situation18,49 as well as in various
disease states.18,49 Skeletal muscle contains 70 to 80% of the total body free amino acid
pool, and glutamine constitutes 60% of this muscle pool (excluding taurine, which is not
incorporated in protein).50 Thus, apart from being potentially explainable by enhanced
1382_C26.fm Page 424 Tuesday, October 7, 2003 7:04 PM

424 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

glutamine synthesis, any glutamine release from skeletal muscle can also be caused by
changes in muscle protein turnover and release from the free glutamine pool.51 It has been
shown that the composition of amino acids released from muscle due to proteolysis52 is
not representative of skeletal muscle protein composition.53 Glutamine constitutes approxi-
mately 30% of the amino acids released from skeletal muscle, although it constitutes only
5% of skeletal muscle protein.53 For this reason, glutamine is commonly viewed as a carbon
and nitrogen carrier from skeletal muscle to splanchnic organs.50,53 Any attempt to study
muscle glutamine synthesis from blood-borne ammonia during hyperammonemia related
to liver failure must take into account this commonly observed muscle glutamine release
during catabolic states.50,51
During hyperammonemia due to liver failure, unidirectional ammonia uptake by
human skeletal muscle has been shown by tracer techniques.14 As stated before, net ammo-
nia uptake by skeletal muscle has been demonstrated by measurements of arteriovenous
(AV) differences in fasted healthy human volunteers across the leg,44,45,54 but not across
the forearm (reviewed in Olde Damink et al.7). In fasted humans with liver disease,
ammonia was taken up by the leg54,55 and forearm. In experimental studies in rats with
acute or chronic liver failure, we were unable to demonstrate net ammonia uptake across
the hindquarter.56,57 Glutamine release did not increase during acute liver failure and only
minimally increased in rats with chronic liver failure.56,57 However, in more recent studies
by our group in patients with stable cirrhosis undergoing TIPSS check, significant ammo-
nia uptake by the leg was observed and glutamine release in this study exceeded ammonia
uptake.31
The considerable difference between unidirectional uptake and net AV differences for
ammonia across muscle could mean that muscle takes up and releases ammonia.14 The
fact that these AV differences are often small may indicate that muscle ammonia uptake
is only important because skeletal muscle constitutes over 40% of body mass. This also
means that muscle wasting during liver disease could reduce the potential role of muscle
in ammonia detoxification14 and provides an argument for adequate nutritional support
in patients with liver failure.58

26.1.4 Glutamine and ammonia exchange across the brain


The brain has traditionally been thought to play a crucial role in glutamine synthesis
during hyperammonemia due to liver failure. The brain contains appreciable amounts of
both glutamine synthetase8,25 and glutaminase.8 These two enzymes are compartmental-
ized in brain: astrocytes contain most of total brain glutamine synthetase while neurons
contain virtually all brain glutaminase.8 Neurons contain only maximally 20% of total
brain glutamine synthetase.
The normal brain is generally viewed as an organ of ammonia uptake and glutamine
release.8,14 Ammonia uptake has been suggested to occur in healthy human volunteers44,45
and in some animal species.8 However, net ammonia uptake by the brain has never been
proven in normal rats8 despite extensive research and reports confirming that net cerebral
glutamine release occurs in normal rats are scarce.29
Unidirectional ammonia uptake by the normal brain has been observed in various
mammals using tracer techniques,14,59 and similar observations were made in various
hyperammonemic states.14,60 Net cerebral ammonia uptake during chronic hyperammone-
mia has been demonstrated in humans.44,45,61 Also, net ammonia uptake by the brain was
observed in rats made acutely hyperammonemic62 and in portacaval shunted rats.29,63
Ammonia uptake in these studies was accompanied by increased glutamine release.62,63 It
has been proposed that the differences between unidirectional and net ammonia exchange
can be explained, in analogy to skeletal muscle, by assuming that brain continuously uses
1382_C26.fm Page 425 Tuesday, October 7, 2003 7:04 PM

Chapter twenty-six: Amino acids and ammonia in liver disease 425

and produces ammonia. The ultimate result of this process is probably a fractional ammo-
nia extraction of between 11 and 20%.7,14,59
Tracer studies using 13N-ammonia have demonstrated that in the normal brain ammo-
nia is rapidly incorporated in glutamine (t1/2 < 3 sec),59 suggesting that cerebral ammonia
detoxification is by the glutamine synthetase reaction.8,59,60 This takes place in a small (20%
volume) compartment of the brain, probably the astrocytes.8,59,60 During hyperammone-
mia, ammonia is less rapidly incorporated into glutamine (t1/2 = 9 sec).60 The hyperam-
monemic brain has a higher glutamine content than the normal rat brain,12 as well as a
lower content of glutamate. Such observations could be explained by an augmented
enzymatic machinery incorporating ammonia at a lesser velocity.8
Ammonia probably plays a key role in the pathogenesis of hepatic encephalopathy.1,8,32
It is not the purpose of this chapter to review the pathogenesis of hepatic encephalopathy
in great detail. However, the interorgan exchange of ammonia and glutamine may have
two important consequences for brain metabolism that may play a role in the pathogenesis
of hepatic encephalopathy. First, the net effect of increased cerebral conversion of ammonia
to glutamine may be a loss of tissue glutamate. As glutamate is an important excitatory
neurotransmitter,8 an imbalance in transmitters could ensue, which could contribute to
the pathogenesis of hepatic encephalopathy.32 Second, accumulation of glutamine in the
brain may act as an osmolyte and could lead to cerebral edema due to cell swelling, a
hypothesis that recently has received considerable attention.64–66
In conclusion, concerning their relative roles in interorgan ammonia metabolism and
alternative detoxification pathways, brain glutamine synthetic capacity should be negli-
gible compared with that of skeletal muscle, in view of their respective relative masses.
However, the contribution of brain glutamine efflux to blood glutamine concentrations in
man remains to be elucidated.

26.1.5 Glutamine and ammonia exchange across the kidney


The kidneys play a pivotal role in waste nitrogen excretion. Also, renal glutamine and
ammonia metabolism plays an important role in acid–base regulation.9 With respect to
both nitrogen excretion and acid–base equilibrium, the kidneys and liver interact in a very
sophisticated manner to maintain whole-body homeostasis (Figure 26.1). It is therefore
surprising that renal ammonia and glutamine metabolism have received relatively little
attention in patients with liver disease.
The kidneys contain both glutaminase and glutamine synthetase,34 and therefore, the
kidneys are capable of both synthesizing and degrading glutamine. In the postabsorptive
state, glutamine is taken up by the mammalian kidney.34,35,37,45,49,67–70 Glutamine constitutes
the main substrate for renal ammoniagenesis.9 Following uptake from the bloodstream,
it is metabolized by intramitochondrial phosphate-dependent glutaminase,9,68 and the
ammonia produced is either excreted in the urine or released back into the renal vein.9,69,71
For the residual glutamate, three possible fates remain.9 First, the glutamate can be
released into the renal vein.72 Second, it can be used in transamination reactions, yielding
predominantly alanine.9 After being released into the bloodstream,67 alanine can be taken
up by the liver, where it can be used for gluconeogenesis and urea synthesis.35 Finally, the
amine moiety can be split in the glutamate dehydrogenase reaction, yielding ammonia
and a-ketoglutarate, which can be oxidized in the tricarboxylic acid (TCA) cycle.9,68 As
we and others have pointed out previously, this metabolic route results in bicarbonate
synthesis, which can be released into the bloodstream (reviewed in Dejong et al.6 and Olde
Damink et al.7).
In the physiological situation, the mammalian kidneys excrete only minor amounts
of ammonia in the urine:71 30% of total renal ammonia production is released into the
1382_C26.fm Page 426 Tuesday, October 7, 2003 7:04 PM

426 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

UREA CYCLE

AMM
AMM 70%

G-ase

G-ase GLN

AMM 30% UREA

Figure 26.1 The role of the kidney in interorgan glutamine (GLN) exchange under physiological
circumstances. GLN is taken up by the gut and kidney and metabolized by the glutaminase (G-ase)
pathway to yield ammonia (AMM). AMM generated by the kidney is excreted in the urine or
released into the renal vein. Renal AMM released into the circulation as well as AMM generated by
the gut is metabolized in the liver in periportal hepatocytes to form urea, the latter being excreted
in the urine. (The role of muscle and brain in GLN synthesis has been omitted deliberately from
the graph.)

urine in rats; the remainder is released into the renal vein.9,71 These figures illustrate that
the normal kidney is an organ of net ammonia addition to the body. During acidosis, this
situation reverses because total renal ammoniagenesis is enhanced and 70% of this
enhanced amount is excreted in the urine to dispose the acid load.9,35,67,68 This is accom-
panied by an increase in renal glutamine extraction.35,67 Ammonia excretion in the urine
increases at the expense of urea,34,35 but total urinary nitrogen (ammonia plus urea) remains
constant.34,35
Glutamine breakdown and subsequent ammonia excretion in the urine are a means
of excreting protons and generating bicarbonate.9 Bicarbonate is used in hepatic urea
synthesis, which therefore also constitutes a pH-regulating modality.2 This illustrates the
sophisticated way in which liver and kidney act in concert to maintain nitrogen and
acid–base homeostasis.
To our knowledge no data are available concerning the effects of chronic alkalosis on
renal glutamine and ammonia metabolism. Hepatic coma is often accompanied by chronic
respiratory alkalosis.33 This has led some investigators to assess the effect of hyperventi-
lation on ammonia exchange across the kidney.55 In this study it was demonstrated that
ammonia release into the renal vein increased after hyperventilation in cirrhotic patients,
suggesting that hyperventilation impairs renal urinary ammonia excretion. However, the
latter was not measured in that study.55
In the past, high glutamine and ammonia concentrations were suggested to favor
urinary ammonia excretion,54,73 and it was shown that the kidney releases ammonia into
the circulation in cirrhotic patients.54,55 This renal ammonia release into the circulation
decreased at elevated ammonia concentrations.54 Similarly, artificially elevated arterial
1382_C26.fm Page 427 Tuesday, October 7, 2003 7:04 PM

Chapter twenty-six: Amino acids and ammonia in liver disease 427

UREA CYCLE

GS
AMM 30%

AMM

G-ase

G-ase GLN

AMM 70% UREA

Figure 26.2 The proposed role of the kidney in interorgan glutamine exchange during hyperam-
monemia due to liver failure (cf. also Figure 26.1). Urea synthesis is diminished, and ammonia
(AMM) coming from the gut or kidneys escapes periportal urea synthesis. This AMM is subsequently
scavenged in the perivenous hepatocytes in the glutamine synthetase pathway (GS) to form
glutamine (GLN). GLN in turn is released back into the circulation and subsequently undergoes
degradation by G-ase in gut and kidney. During hyperammonemia, a greater fraction of the AMM
generated in the kidney is excreted in the urine, which turns the kidney into an organ of net ammonia
removal from the body. (The role of muscle and brain in GLN synthesis has been omitted deliberately
from the graph.)

ammonia levels in human volunteers induced the kidney to take up ammonia from the
circulation and to increase urinary ammonia excretion.73 A problem of these studies is that
most of them do not provide data on renal glutamine metabolism.
In the early 1990s of the past century, we have conducted two rat experiments during
acute74 and chronic75 hyperammonemia. In these experiments, 70% of total renal ammo-
niagenesis was excreted in the urine and only 30% was released back into the renal vein.
This reversal of the urinary excretion/renal venous release ratio from 30/70 to 70/30 made
the kidneys change from ammonia-producing organs in the body to ammonia-eliminating
organs. Similar observations were made in a methionine sulfoximine model of subacute
hyperammonemia.76 This beneficial adaptation helps to lower systemic ammonia levels6,7
(Figure 26.2). Some incidental support for these observations comes from studies on the
effects of chronic metabolic acidosis,35,67 where enhanced renal ammoniagenesis and uri-
nary ammonia excretion were observed at elevated arterial ammonia levels due to admin-
istration of NH4Cl and NH4HCO3 (reviewed in Dejong et al.6). Such observations could
indicate that arterial ammonia itself regulates urinary ammonia excretion, rather than pH.
Subsequent experiments during feeding in pigs have shown that renal ammoniagen-
esis increases following a protein meal and that the kidneys contribute more to systemic
ammonia levels than the entire hepatosplanchnic area.77 These results have recently been
confirmed in patients with hepatic cirrhosis studied under control conditions31 and during
an actual as well as a simulated gastrointestinal bleed.78
1382_C26.fm Page 428 Tuesday, October 7, 2003 7:04 PM

428 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

26.1.6 Other organs


In the past, some attention has also been paid to the role of the lung in glutamine and
ammonia homeostasis.79,80 Some authors have suggested that the normal79 as well as
injured80 lung is an organ of glutamine release, although we were unable to confirm this.81
Incidentally, ammonia uptake by the lung in septic patients has been observed.80 To the
best of our knowledge, no reports have appeared in literature concerning pulmonary
ammonia and glutamine metabolism during acute or chronic liver failure. Considering
the enzymatic characteristics of lung tissue (lungs contain glutamine synthetase), a role
for the lungs in ammonia uptake and glutamine release during liver failure would hypo-
thetically be possible.25,79
Literature concerning the role of the heart in ammonia and glutamine exchange is
scarce. Considering the enzymatic properties of myocardial cells,25,42 the heart could con-
tribute to glutamine production, as has been demonstrated in the cayman heart,82 as well
as breakdown. To our knowledge, no data are available concerning ammonia and
glutamine metabolism by the heart during liver failure.
Evidence is accumulating from in vitro and in vivo studies that cells from the immune
system consume glutamine and produce ammonia.10,83,84 The importance of the immune
system for glutamine and ammonia metabolism in interorgan nitrogen exchange, however,
remains to be established.

26.2 Effects of gastrointestinal bleeding


It is well known that a protein meal may precipitate hepatic encephalopathy in patients
with chronic liver failure. This has traditionally been attributed to the aggravation of
hyperammonemia that usually accompanies meal-induced encephalopathy. In this con-
text, the ammoniagenic potential of different meals is known to vary considerably, blood
having the highest capacity to precipitate hyperammonemia. The cause of this ammoni-
agenicity has long been unknown, but in the late 1980s, we found by serendipity that
blood protein does not contain the essential amino acid isoleucine.85 This makes blood a
protein with low biological value that cannot completely be utilized for protein synthetic
purposes. The consequence would theoretically be that most of the proteins contained in
blood will have to be broken down and further degraded through the urea cycle if blood
enters the digestive tract, unless isoleucine is provided from a different source.5 This would
explain protein breakdown elsewhere in the body during gastrointestinal bleeding and
would be compatible with the notion that an upper GI bleed in a patient with liver cirrhosis
is a catabolic event.
In agreement with this, ingestion of blood protein by pigs was followed by hyperam-
monemia, uremia, and hypo-isoleucinemia.5,85 In keeping with the above hypothesis, this
could be reversed by intravenous isoleucine administration simultaneously with the blood
protein meal.86 Similar observations were later made in rats following gavage feeding of
blood.87,88 In patients with normal liver function and in a pilot study in patients with liver
cirrhosis, the occurrences of hypo-isoleucinemia, increased plasma urea levels, and hyper-
ammonemia (in cirrhotics) were subsequently confirmed during actual gastrointestinal
bleeding.89
The next step was to prove or disprove this hypothesis in patients with cirrhosis of
the liver during an actual or simulated gastrointestinal bleed. To this purpose, studies
were conducted using stable isotopes in order to measure protein synthesis and break-
down on a whole-body level as well as in several organs in patients with hepatic cirrhosis
during GI bleeds. Furthermore, similar studies were then conducted supplementing
1382_C26.fm Page 429 Tuesday, October 7, 2003 7:04 PM

Chapter twenty-six: Amino acids and ammonia in liver disease 429

isoleucine or placebo. The results of these studies are currently being analyzed and will
hopefully be published in the next 2 years.

26.3 Does the prevailing ammonia hypothesis of hepatic


encephalopathy require modification?
In the pathogenesis of hepatic encephalopathy, ammonia is believed to play a crucial role.33
The currently held concept is that in the fasted state ammonia is generated in the gut from
bacterial degradation of urea and intestinal glutamine breakdown. Food intake further
increases the portal ammonia load. Ammonia then escapes detoxification by the liver as
a consequence of hepatocellular failure and intra- and extrahepatic shunting. This then
leads to systemic hyperammonemia and cerebral ammonia uptake and metabolism. The
latter gives rise to cerebral neurotransmitter changes and cerebral edema, contributing to
the neurological state known as hepatic encephalopathy.66
As detailed above, we have generated data in the past 10 years suggesting that the
kidney may play a crucial role in ammonia metabolism during acute and chronic liver
failure by changing from ammonia production in the body to ammonia excretion from
the body.74,75 These results have recently been confirmed in patients with hepatic cirrhosis
studied under control conditions31 and during an actual as well as a simulated gastrointes-
tinal bleed.78 Taken together, these animal and human studies constitute a considerable
and consistent body of evidence that provide us with a basis for suggesting two important
modifications of the prevailing ammonia hypothesis of hepatic encephalopathy:

• Fasted systemic ammonia levels during liver disease are probably determined by
intestinal and renal ammonia metabolism.
• Hyperammonemia following an intestinal protein load (meal, gastrointestinal
bleed) not only is the consequence of intestinal ammonia generation but also is
caused by renal ammonia generation.

Clearly, this modified hypothesis provides us with a unique opportunity to start a


search for new ammonia-lowering therapeutic modalities focusing on renal ammonia
metabolism.

Acknowledgments
C.H.C. Dejong expresses his gratitude to the Dutch Organization for Scientific Research
(NWO) for financial support as an NWO clinical fellow.

References
1. Zieve, L., Pathogenesis of hepatic encephalopathy, Metab. Brain Dis., 2, 147–165, 1987.
2. Meijer, A.J., Lamers, W.H., and Chamuleau, R.A.F.M., Nitrogen metabolism and ornithine
cycle function, Physiol. Rev., 70, 701–748, 1990.
3. Butterworth, R.F., Pathogenesis of acute hepatic encephalopathy, Digestion, 59 (Suppl. 2),
16–21, 1998.
4. Riordan, S.M. and Williams, R., Treatment of hepatic encephalopathy, N. Engl. J. Med., 337,
473–479, 1997.
5. Olde Damink, S.W.M., Dejong, C.H.C., Deutz, N.E.P., van Berlo, C.L.H., and Soeters, P.B.,
Upper gastrointestinal bleeding: an ammoniagenic and catabolic event due to the total
absence of isoleucine in the haemoglobin molecule, Med. Hypotheses, 52, 515–519, 1999.
1382_C26.fm Page 430 Tuesday, October 7, 2003 7:04 PM

430 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

6. Dejong, C.H.C., Deutz, N.E.P., and Soeters, P.B., Ammonia and glutamine metabolism during
liver insufficiency: the role of kidney and brain in interorgan nitrogen exchange, Scand. J.
Gastroenterol., 218, 61–77, 1996.
7. Olde Damink, S.W., Deutz, N.E., Dejong, C.H., Soeters, P.B., and Jalan, R., Interorgan am-
monia metabolism in liver failure, Neurochem. Int., 41, 177–188, 2002.
8. Cooper, A.J.L. and Plum, F., Biochemistry and physiology of brain ammonia, Physiol. Rev.,
67, 440–519, 1987.
9. Halperin, M.L., Kamel, K.S., Ethier, J.H., Stinebaugh, B.J., and Jungas, R.L., Biochemistry and
Physiology of Ammonium Excretion, 2nd ed., Raven Press, New York, 1992.
10. Newsholme, E.A. and Leech, A.R., Biochemistry for the Medical Sciences, John Wiley & Sons,
New York, 1983.
11. Kikeri, D., Sun, D., Zeidel, M.L., and Hebert, S.C., Cellular NH4/K transport pathways in
mouse medullary thick limb of Henle: regulation by intracellular pH, J. Gen. Physiol., 99,
435–461, 1992.
12. Lin, S. and Raabe, W., Ammonia intoxication: effects on cerebral cortex and spinal cord,
J. Neurochem., 44, 1252–1258, 1985.
13. Rudman, D., DiFulco, T.J., Galambos, J.T., Smith, R.B., Salam, A.A., and Warren, W.D.,
Maximal rates of excretion and synthesis of urea in normal and cirrhotic subjects, J. Clin.
Invest., 52, 2241–2249, 1973.
14. Lockwood, A.H. et al., The dynamics of ammonia metabolism in man: effects of liver disease
and hyperammonemia, J. Clin. Invest., 63, 449–460, 1979.
15. Lacey, J.M. and Wilmore, D.W., Is glutamine a conditionally essential amino acid? Nutr. Rev.,
48, 297–309, 1990.
16. Windmueller, H.G., Glutamine utilization by the small intestine, Adv. Enzym., 53, 201–237,
1982.
17. Windmueller, H.G. and Spaeth, A.E., Uptake and metabolism of plasma glutamine by the
small intestine, J. Biol. Chem., 249, 5070–5079, 1974.
18. Souba, W.W., Interorgan ammonia metabolism in health and disease: a surgeon’s view,
J. Parenter. Enteral Nutr., 11, 569–579, 1987.
19. Plauth, M., Roske, A.E., Romaniuk, P., Roth, E., Ziebig, R., and Lochs, H., Post-feeding
hyperammonaemia in patients with transjugular intrahepatic portosystemic shunt and liver
cirrhosis: role of small intestinal ammonia release and route of nutrient administration, Gut,
46, 849–855, 2000.
20. Clemmesen, J.O., Kondrup, J., and Ott, P., Splanchnic and leg exchange of amino acids and
ammonia in acute liver failure, Gastroenterology, 118, 1131–1139, 2000.
21. Clemmesen, J.O., Larsen, F.S., Kondrup, J., Hansen, B.A., and Ott, P., Cerebral herniation in
patients with acute liver failure is correlated with arterial ammonia concentration, Hepatology,
29, 648–653, 1999.
22. Windmueller, H.G. and Spaeth, A.E., Intestinal metabolism of glutamine and glutamate from
the lumen as compared to glutamine from the blood, Arch. Biochem. Biophys., 171, 662–672,
1975.
23. Windmueller, H.G. and Spaeth, A.E., Identification of ketone bodies and glutamine as the
major respiratory fuels in vivo for postabsorptive rat small intestine, J. Biol. Chem., 253, 69–76,
1978.
24. Steiner, M., Bourges, H.R., Freedman, L.S., and Gray, S.J., Effect of starvation on the tissue
composition of the small intestine in the rat, Am. J. Physiol., 215, 75–77, 1968.
25. Lund, P., A radiochemical assay for glutamine synthetase, and activity of the enzyme in rat
tissue, Biochem. J., 118, 35–39, 1970.
26. Weber, F.L. and Veach, G.L., The importance of the small intestine in gut ammonium pro-
duction in the fasting dog, Gastroenterology, 77, 235–240, 1979.
27. Weber, F.L., Friedman, D.W., and Fresard, K.M., Ammonium production from intraluminal
amino acids in canine jejunum, Am. J. Physiol., 254, G264–268, 1988.
28. Imler, M. and Schlienger, J.L., The effect of chronic uremia on portal and systemic ammonia
in normal and portal-strictured rats, J. Lab. Clin. Med., 94, 872–878, 1979.
1382_C26.fm Page 431 Tuesday, October 7, 2003 7:04 PM

Chapter twenty-six: Amino acids and ammonia in liver disease 431

29. Gjedde, A., Lockwood, A.H., Duffy, T.E., and Plum, F., Cerebral blood flow and metabolism
in chronically hyperammonemic rats: effect of an acute ammonia challenge, Ann. Neurol., 3,
325–330, 1978.
30. Dejong, C.H.C., Deutz, N.E.P., and Soeters, P.B., Intestinal glutamine and ammonia metab-
olism during chronic hyperammonaemia induced by liver insufficiency, Gut, 34, 1112–1119,
1993.
31. Olde Damink, S.W., Jalan, R., Redhead, D.N., Hayes, P.C., Deutz, N.E., and Soeters, P.B.,
Interorgan ammonia and amino acid metabolism in metabolically stable patients with cir-
rhosis and a TIPSS, Hepatology, 36, 1163–71, 2002.
32. Record, C.O., Neurochemistry of hepatic encephalopathy, Gut, 32, 1261–1263, 1991.
33. Sherlock, S., Diseases of the Liver and Biliary System, 8th ed., Blackwell Scientific Publications,
Oxford, 1989, pp. 116–128.
34. Welbourne, T.C., Phromphetcharat, V., Givens, G., and Joshi, S., Regulation of interorgan
glutamine flow in metabolic acidosis, Am. J. Physiol., 250, E457–E463, 1986.
35. Welbourne, T.C., Childress, D., and Givens, G., Renal regulation of interorganal glutamine
flow in metabolic acidosis, Am. J. Physiol., 251, R859–R866, 1986.
36. Haussinger, D., Kaiser, S., Stehle, T., and Gerok, W., Structural and functional organization
of hepatic ammonia metabolism: pathophysiological consequences, in Advances in Ammonia
Metabolism and Hepatic Encephalopathy, Soeters, P.B., Wilson, J.H.P., Meijer, A.J., and Holm, E.,
Eds., Excerpta Medica, Amsterdam, 1988, pp. 26–36.
37. Welbourne, T.C., Hepatic glutaminase flux regulation of glutamine homeostasis, Biol. Chem.
Hoppe Seyler, 367, 301–305, 1986.
38. Kaiser, S., Gerok, W., and Haussinger, D., Ammonia and glutamine metabolism in liver slices:
new aspects on the pathogenesis of hyperammonemia in chronic liver disease, Eur. J. Clin.
Invest., 18, 535–542, 1988.
39. Steele, R.D., Hyperammonemia and orotic aciduria in portacaval shunted rats, J. Nutr., 114,
210–216, 1984.
40. Bianchi, G. et al., Hepatic amino-nitrogen clearance to urea-nitrogen in control subjects and
in patients with cirrhosis: a simplified method, Hepatology, 13, 460–466, 1991.
41. Hamberg, O., Nielsen, K., and Vilstrup, H., Effects of an increase in protein intake on hepatic
efficacy for urea synthesis in healthy subjects and in patients with cirrhosis, J. Hepatol., 14,
237–243, 1992.
42. Nelson, D., Rumsey, W.L., and Erecinska, M., Glutamine catabolism by heart muscle, Biochem.
J., 282, 559–564, 1992.
43. Chabrier, G., Schlienger, J.L., and Imler, M., Etude du metabolisme musculaire de l’ammo-
niaque sur le train posterieur de rat intact, C. R. Soc. Biol., 176, 716–722, 1982.
44. Warter, J.M. et al., Sodium valproate associated with phenobarbital: effects on ammonia
metabolism in humans, Epilepsia, 24, 628–633, 1983.
45. Warter, J.M. et al., The renal origin of sodium valproate-induced hyperammonemia in fasting
humans, Neurology, 33, 1136–1140, 1983.
46. Hills, A.G., Reid, E.L., and Kerr, W.D., Circulatory transport of L-glutamine in fasted mam-
mals: cellular sources of urine ammonia, Am. J. Physiol., 223, 1470–1475, 1972.
47. Fine, A., The effects of ammonia infusion on ammonia and glutamine metabolism by liver
and muscle in the normal dog, Contrib. Nephrol., 47, 1–8, 1985.
48. Ruderman, N.B. and Lund, P., Amino acid metabolism in skeletal muscle, Isr. J. Med. Sci., 8,
295–302, 1972.
49. Schrock, H., Cha, C.-J.M., and Goldstein, L., Glutamine release from hindlimb and uptake
by the kidney in the acutely acidotic rat, Biochem. J., 188, 557–560, 1980.
50. Souba, W.W., Smith, R.J., and Wilmore, D.W., Glutamine metabolism by the intestinal tract,
J. Parenter. Enteral Nutr., 9, 608–617, 1985.
51. Rennie, M.J., Babij, P., Taylor, P.M., Hundal, H.S., MacLennan, P.A., and Watt, P.W., Charac-
teristics of a glutamine carrier in skeletal muscle have important consequences for nitrogen
loss in injury, infection and chronic disease, Lancet, ii, 1008–1011, 1986.
1382_C26.fm Page 432 Tuesday, October 7, 2003 7:04 PM

432 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

52. Lowell, B.B., Ruderman, N.B., and Goodman, M.N., Regulation of myofibrillar protein deg-
radation in rat skeletal muscle during brief and prolonged starvation, Metabolism, 35,
1121–1127, 1986.
53. Ruderman, N.D. and Berger, M., The formation of glutamine and alanine in skeletal muscle,
J. Biol. Chem., 249, 5500–5506, 1974.
54. Tyor, M.P., Owen, E.E., Berry, J.N., and Flanagan, J.F., The relative role of extremity, liver and
kidney as ammonia receivers and donors in patients with liver disease, Gastroenterology, 39,
420–424, 1960.
55. Berry, J.N., Flanagan, J.F., Owen, E.E., and Tyor, M.P., The kidney as a source of blood
ammonia in resting and hyperventilated cirrhotics, Clin. Res., 7, 154–155, 1959.
56. Dejong, C.H.C., Kampman, M.T., Deutz, N.E.P., and Soeters, P.B., Altered glutamine metab-
olism in rat portal drained viscera during hyperammonemia, Gastroenterology, 102, 936–948,
1992.
57. Dejong, C.H.C., Deutz, N.E.P., and Soeters, P.B., Muscle ammonia and glutamine exchange
during chronic liver insufficiency in the rat, J. Hepatol., 21, 299–307, 1994.
58. Nompleggi, D.J. and Bonkovsky, H.L., Nutritional supplementation in chronic liver disease:
an analytical review, Hepatology, 19, 518–533, 1994.
59. Cooper, A.J.L., McDonald, J.M., Gelbard, A.S., Gledhill, R.F., and Duffy, T.E., The metabolic
fate of 13N-labeled ammonia in rat brain, J. Biol. Chem., 254, 4982–4992, 1979.
60. Cooper, A.J.L., Mora, S.N., Cruz, N.F., and Gelbard, A.S., Cerebral ammonia metabolism in
hyperammonemic rats, J. Neurochem., 44, 1716–1723, 1985.
61. Bessman, S.P. and Bradley, J.E., Uptake of ammonia by muscle: its implications in ammoni-
agenic coma, N. Engl. J. Med., 253, 1143–1147, 1955.
62. Dejong, C.H.C., Kampman, M.T., Deutz, N.E.P., and Soeters, P.B., Cerebral cortex ammonia
and glutamine metabolism during liver insufficiency-induced hyperammonemia in the rat,
J. Neurochem., 59, 1071–1079, 1992.
63. Dejong, C.H.C., Deutz, N.E.P., and Soeters, P.B., Cerebral cortex ammonia and glutamine
metabolism in two rat models of chronic liver insufficiency-induced hyperammonemia:
influence of pair feeding, J. Neurochem., 60, 1047–1057, 1993.
64. Ganz, R., Swain, M., Traber, P., DalCanto, M., Butterworth, R.F., and Blei, A.T., Ammonia-
induced swelling of rat cerebral cortical slices: implications for the pathogenesis of brain
edema in acute hepatic failure, Metab. Brain Dis., 4, 213–223, 1989.
65. Swain, M., Butterworth, R.F., and Blei, A.T., Ammonia and related amino acids in the patho-
genesis of brain edema in acute ischemic liver failure in rats, Hepatology, 15, 449–453, 1992.
66. Blei, A.T., Pathophysiology of brain edema in fulminant hepatic failure, revisited, Metab.
Brain Dis., 16, 85–94, 2001.
67. Welbourne, T.C., Effect of metabolic acidosis on hindquarter glutamine and alanine release,
Metabolism, 35, 614–618, 1986.
68. Welbourne, T.C. and Dass, P.D., Gamma glutamyl transferase contribution to renal ammo-
niagenesis in vivo, Pflugers Arch., 411, 573–578, 1988.
69. Hallemeesch, M.M., Cobben, D.C., Dejong, C.H.C., Soeters, P.B., and Deutz, N.E.P., Renal
amino acid metabolism during endotoxemia in the rat, J. Surg. Res., 92, 193–200, 2000.
70. Dejong, C.H.C., Welters, C.F.M., Deutz, N.E.P., Heineman, E., and Soeters, P.B., Renal arginine
metabolism in fasted rats with subacute short bowel syndrome, Clin. Sci., 95, 409–418, 1998.
71. Vinay, P., Allignet, E., Pichette, C., Watford, M., Lemieux, G., and Gougoux, A., Changes in
renal metabolite profile and ammoniagenesis during acute and chronic metabolic acidosis
in dog and rat, Kidney Int., 17, 312–325, 1980.
72. Cooper, A.J.L., Filc-DeRicco, S., and Gelbard, A.S., L-[13N]-glutamate metabolism in normal
rat kidney, in Progress in Hepatic Encephalopathy and Metabolic Nitrogen Exchange, Bengtsson,
F., Almdal, T., Jeppsson, B., and Vilstrup, H., CRC Press, Boca Raton, FL, 1991, pp. 341–351.
73. Owen, E.E., Johnson, J.H., and Tyor, M.P., The effect of induced hyperammonemia on renal
ammonia metabolism, J. Clin. Invest., 40, 215–221, 1961.
74. Dejong, C.H.C., Deutz, N.E.P., and Soeters, P.B., Renal ammonia and glutamine metabolism
during liver insufficiency-induced hyperammonemia in the rat, J. Clin. Invest., 92, 2834–2840,
1993.
1382_C26.fm Page 433 Tuesday, October 7, 2003 7:04 PM

Chapter twenty-six: Amino acids and ammonia in liver disease 433

75. Dejong, C.H.C., Deutz, N.E.P., and Soeters, P.B., Metabolic adaptation of the kidney to
hyperammonemia during chronic liver insufficiency in the rat, Hepatology, 18, 890–902, 1993.
76. Heeneman, S., Dejong, C.H.C., Deutz, N.E.P., and Soeters, P.B., Effects of methionine sulfox-
imine treatment on renal amino acid and ammonia metabolism in the rat, Pflugers Arch., 427,
524–532, 1994.
77. Welters, C.F.M., Deutz, N.E.P., Dejong, C.H.C., and Soeters, P.B., Enhanced renal vein am-
monia efflux after a protein meal in the pig, J. Hepatol., 31, 489–496, 1999.
78. Olde Damink, S.W. et al., The kidney plays a major role in the hyperammonemia seen after
simulated or actual GI bleeding in patients with cirrhosis, Hepatology, 37, 1277–1285, 2003.
79. Welbourne, T.C., Role of the lung in glutamine homeostasis, Contrib. Nephrol., 63, 178–182,
1988.
80. Plumley, D.A. et al., Accelerated lung amino acid release in hyperdynamic septic surgical
patients, Arch. Surg., 125, 57–61, 1990.
81. van Berlo, C.L.H. et al., Lung glutamine metabolism: effects of starvation, parenteral and
enteral nutrition: a study in man, Clin. Nutr., 15, 86–88, 1996.
82. Coulson, R.A. and Hernandez, T., Site of synthesis of amino acids in the intact cayman, Am.
J. Physiol., 213, 411–417, 1967.
83. Kelso, T.B., Shear, C.R., and Max, S.R., Enzymes of glutamine metabolism in inflammation
associated with skeletal muscle hypertrophy, Am. J. Physiol., 257, E883–E894, 1989.
84. Deutz, N.E.P., Reijven, P.L.M., Athanasas, G., and Soeters, P.B., Post-operative changes in
hepatic, intestinal, splenic and muscle fluxes of amino acids and ammonia in pigs, Clin. Sci.,
83, 607–614, 1992.
85. van Berlo, C.L.H. et al., Is increased ammonia liberation after bleeding in the digestive tract
the consequence of complete absence of isoleucine in hemoglobin? A study in pigs, Hepatol-
ogy, 10, 315–323, 1989.
86. Deutz, N.E.P., Reijven, P.L.M., Bost, M.C.F., van Berlo, C.L.H., and Soeters, P.B., Modification
of the effects of blood on amino acid metabolism by intravenous isoleucine, Gastroenterology,
101, 1613–1620, 1991.
87. Olde Damink, S.W.M., Dejong, C.H.C., Deutz, N.E.P., and Soeters, P.B., Decreased plasma
and tissue isoleucine levels after simulated gastrointestinal bleeding by blood gavages in
chronic portacaval shunted rats, Gut, 40, 418–424, 1997.
88. Olde Damink, S.W.M., Dejong, C.H.C., Deutz, N.E.P., and Soeters, P.B., Effects of simulated
upper gastrointestinal hemorrhage on ammonia and related amino acids in blood and brain
of chronic portacaval shunted rats, Metab. Brain Dis., 12, 121–132, 1997.
89. Dejong, C.H.C., Meijerink, W.J.H.J., van Berlo, C.L.H., Deutz, N.E.P., and Soeters, P.B.,
Decreased plasma isoleucine concentrations after upper gastrointestinal haemorrhage in
humans, Gut, 39, 13–17, 1996.
1382_C26.fm Page 434 Tuesday, October 7, 2003 7:04 PM
1382_C27.fm Page 435 Tuesday, October 7, 2003 7:08 PM

Requirements and Supply


1382_C27.fm Page 436 Tuesday, October 7, 2003 7:08 PM
1382_C27.fm Page 437 Tuesday, October 7, 2003 7:08 PM

Part IV

Amino acid requirements


1382_C27.fm Page 438 Tuesday, October 7, 2003 7:08 PM
1382_C27.fm Page 439 Tuesday, October 7, 2003 7:08 PM

chapter twenty-seven

Nutritional essentiality of amino


acids and amino acid requirements
in healthy adults
Vernon R. Young
Massachusetts Institute of Technology
John F. Tharakan
Massachusetts Institute of Technology

Contents
Introduction..................................................................................................................................440
27.1 Some historical milestones ...............................................................................................440
27.2 Essential and nonessential, indispensable and dispensable........................................441
27.3 Indispensable amino acid requirements in adults ........................................................443
27.3.1 Methods and approaches for estimating amino acid requirements
in adults.................................................................................................................445
27.3.1.1 Nitrogen balance..................................................................................445
27.3.1.2 Plasma amino acid concentration–intake response .......................447
27.3.1.3 Tracer techniques .................................................................................448
27.3.1.3.1 DAAO and DAAB............................................................448
27.3.1.3.2 IAAO ..................................................................................449
27.3.1.3.3 24-h IAAO and 24-h IAAB .............................................451
27.3.1.3.4 Postprandial protein utilization .....................................451
27.3.1.3.5 Summary of tracer protocols ..........................................452
27.3.1.4 Factorial prediction of amino acid requirements ...........................452
27.3.1.5 Summary of approaches for estimation of amino acid
requirements in adults........................................................................453
27.3.2 Estimates of the requirements for specific indispensable amino acids
in healthy adults ..................................................................................................454
27.3.2.1 Lysine.....................................................................................................454
27.3.2.2 Phenylalanine and tyrosine ...............................................................455
27.3.2.2.1 Tyrosine sparing ...............................................................456
27.3.2.3 Threonine ..............................................................................................458
27.3.2.4 Branched-chain amino acids..............................................................459
27.3.2.4.1 Leucine ...............................................................................459

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 439
1382_C27.fm Page 440 Tuesday, October 7, 2003 7:08 PM

440 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

27.3.2.4.2 Valine and isoleucine .......................................................460


27.3.2.5 Tryptophan ...........................................................................................460
27.3.2.6 Sulfur amino acids...............................................................................460
27.3.2.7 Histidine................................................................................................462
27.3.3 Summary and tentative recommendations for indispensable amino
acid requirements in healthy adults.................................................................462
27.3.4 Indispensable amino acid requirements in other adult age and
physiological groups ...........................................................................................462
27.4 The nonspecific nitrogen requirement............................................................................463
References .....................................................................................................................................464

Introduction
During the course of biological evolution, animal cells lost their enzymatic capacity to
synthesize certain organic molecules that were required for their growth and maintenance
of tissue function.1 These are the essential dietary nutrients that we now recognize and
classify as vitamins, certain fatty acids, and specific amino acids. In this chapter we will
focus our attention on the amino acids, especially those indispensable amino acids that
are an obligatory component of an adequate diet in adults. Whereas all plants can syn-
thesize the 18 amino acids and 2 amides commonly found in proteins, the animal kingdom,
from protozoa up to mammals, is dependent on at least 9 of the amino acids being supplied
from exogenous sources.2 Depending on the pathophysiological condition of the individ-
ual, not only these 9 but a number of the other 11 common, protein-bound amino acids
may be required to maintain body protein homeostasis. These are the “conditionally
indispensable” amino acids, which we will not discuss in any depth, since they are covered
extensively in other contributions to this treatise. This chapter can be supplemented by
consultation of a number of recent reviews on the amino acid requirements in adults.3–5

27.1 Some historical milestones


It is worthwhile first to give a brief, historical account of the discovery of the amino acids
and some of the major developments that led to the earlier assessments of their nutritional
significance. Thus, cystine was the first amino acid to be discovered, apparently by Woo-
laston in 1810.6 It was later given its name by Swedish chemist Jac Berzelius in about 1833.
Berzelius also proposed the name “protein” in a letter dated July 10, 1838, and sent to the
Dutch chemist Mulder in Rotterdam; he formally accepted this term in his communication
in the Bulletin des Sciences physiques et naturelles en Néerlande, July 30, 1838.7
In their review on the discovery of the amino acids, Vickery and Schmidt6 in 1931 list
methionine as being, at that time, the most recently discovered amino acid, which had
been announced in 1922 by Mueller. Further, with the increasing number of amino acids
being identified as products of the hydrolysis of proteins, there was a growing recognition
of the marked differences in the amino acid content of proteins. In 1907, Osborne8 con-
cluded his monograph The Proteins of the Wheat Kernel with the following statement:

The proportion of lysine (in wheat gluten) is likewise small, especially com-
pared with that obtained from leguminous seeds. The amount of histidine,
however, does not differ very greatly from that of the other seed proteins. What
significance these differences have in respect to the nutritive value of these
different proteins must be determined by future investigation, for it has only
recently been discovered that such differences exist.
1382_C27.fm Page 441 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 441

Indeed, the classical studies by Osborne and Mendel on the nutritive value of purified
proteins and protein-containing foods (see Block and Mitchell9 for a concise review) were
of fundamental importance for improving our understanding of the nutritional signifi-
cance of the different amino acids.
In the first half of the 19th century the earlier views emphasizing the importance of
the quantity of nitrogen in foods as the primary basis of their protein nutritional value
gave way to a focus on the nutritional properties of the different amino acids. According
to McCay,10 it was the gelatin-feeding, tyrosine supplementation experiments in dogs and
pigs carried out between 1869 and 1874 by T. Escher that marked the advent of the idea
of “essential” amino acids. Then in 1906, Hopkins and Cole, who had isolated tryptophan
in 1901, became familiar with the work of Escher, and together with Edith Wilcock,
Hopkins devised diets for mice based on zein, which were improved by supplementation
with tryptophan. The era of the nutritional study of essential amino acids then began in
full, although by 1932 only tryptophan, lysine, and histidine had been shown unequivo-
cally to be indispensable dietary components for the growing rat (see Rose11 for a review).
Specifically, of interest here is that Cox and Rose,12 in 1926, had defined “an indispensable
dietary component as one which cannot be synthesized by the species in question from
materials ordinarily available to the cells at a rate commensurate with the needs for optimal
growth.”
The discovery of threonine in 1935 by Rose and his coworkers13,14 opened the way for
major advances in the study of protein and amino acid nutrition, since this permitted
preparation of defined mixtures of all of the nutritionally important amino acids. In turn,
this made it possible and relatively simple, in Rose’s11 opinion, to establish the dietary
significance of each amino acid. Through investigations with the weanling rat, Rose et
al.15 showed that only 10 of the amino acids ordinarily found in proteins were necessary
for maximum gain of weight. These were (see Table 27.1) valine, leucine, isoleucine,
methionine, threonine, lysine, phenylalanine, tryptophan, histidine, and arginine. How-
ever, it might be noted that in these earlier studies the average growth rate of their
weanling rats was about 4 g daily when consuming adequate, purified L-amino acid-based
diets,15 a rate considerably below that which can be achieved with modern, well-formu-
lated L-amino acid diets.18,19 This fact might well have had an influence on the assessment
of the nutritional/dietary significance of the different amino acids. For example, it can
now be anticipated that proline might have been shown by Rose and coworkers15 to be
required, if the growth rates of the rats had approached their genetic potential. In fact, it
has since been concluded that proline is a necessary component of the diet for the rapidly
growing pig,20, rat,21 and chick.22 We will return to this matter of effects of metabolic state
and age of the organism, when the requirements for the specific amino acids are discussed
in greater detail.

27.2 Essential and nonessential, indispensable and dispensable


The qualitative, dietary significance of the individual amino acids in human nutrition was
first explored in depth by Rose and coworkers over about a 10-year period beginning in
1942. In these studies, often involving only two or three healthy, male graduate students,
the experimental diet supplied 95% of the total nitrogen intake (about 7 g daily) via a
purified amino acid mixture that contained only the 10 amino acids that had been found
to be essential dietary constituents for the rat. The status of body N balance served as the
criteria of the adequacy or inadequacy of the diet in these human studies. After apparent
nitrogen balance had been established, single amino acids were then removed from the
diet, one at a time, while total N intake was maintained at a constant level.16 From these
studies, Rose11 concluded, because it was possible to establish nitrogen equilibrium with
1382_C27.fm Page 442 Tuesday, October 7, 2003 7:08 PM

442 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 27.1 Classification of Amino Acids with Respect to Their


Growth Effects in the Rat and to Their Dietary Role in
Maintenance of Nitrogen Equilibrium in Normal Young Men
Essential Nonessential
Lysine Glycine
Tryptophan Alanine
Histidinee Serine
Phenylalanine Cystinea
Leucine Tyrosineb
Isoleucine Aspartic acid
Threonine Glutamic acidc
Methionine Prolinec
Valine Hydroxyproline
Arginined Citrulline
Histidinef
Argininef
a Cystine can replace about one sixth of the methionine requirement,
but has no growth effect in the absence of methionine.
b Tyrosine can replace about one half of the phenylalanine requirement,
but has no growth effect in the absence of phenylalanine.
c Glutamic acid and proline can serve individually as rather ineffective
substitutes for arginine in the diet. This property is not shared by
hydroxyproline.
d Arginine can be synthesized by the rat, but not at a sufficiently rapid
rate to meet the demands of maximum growth. Its classification, there-
fore, as essential or nonessential is purely a matter of definition.
e Only in rat.
f In man.
Source: Growth effects in rat data from Rose, W.C. et al., J. Biol. Chem.,
182, 541, 1950; dietary role in normal young men from Rose, W.C. et al.,
J. Biol. Chem., 206, 421, 1954.

the diet of indispensable amino acids, “the ten amino acids which are indispensable for
the rat and the dog are also indispensable for adult man.” These investigators also noted
in their paper16 that they found it “quite remarkable” that their experimental subject came
into a slight positive balance with this diet, since the latter only furnished 10 amino acids.
We also find their results rather surprising and difficult to interpret for their nutritional
relevance. However, as summarized in Table 27.1, in 1954 Rose et al.17 presented their
“final” classification of the dietary significance of the individual amino acids, as judged
by their capacity to maintain nitrogen equilibrium in young men. The amino acids were,
as noted here, distributed into one of two categories, essential or nonessential. Further, it
can be seen that this classification (Table 27.1) differed from that for the growing rat only
in respect to arginine and histidine.
The report of the first international committee (Food and Agriculture Organization
(FAO)) on protein requirements, published in 1957,23 presented estimates of the average
minimal requirements for these “essential” amino acids in adult men, based on the data
of Rose et al.24 Subsequent United Nations, FAO/WHO,25,26 and FAO/WHO/UNU27 expert
committee reports all have continued to focus attention on these same essential amino
acids (Table 27.1), with histidine being added to the list in the 1985 report.27 Little attention
was given by the 1985 UN committee27 to the other common dietary amino acids or their
possible nutritional significance. Although a primary focus on the so-called essential amino
acids, as proposed for adults by Rose11 and then extended via the studies in infants by
Holt and Snyderman28 and their colleagues, is appropriate from a general public health,
1382_C27.fm Page 443 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 443

human nutrition standpoint, the other amino acids also deserve attention in a number of
contexts, as discussed elsewhere in this treatise.
Several developments over the past two decades as reviewed, in part, by Laidlaw and
Koppe,29 have been responsible for a further and more complete examination of the role
of indispensable and dispensable amino acids in human nutrition and metabolism. These
include (1) development and successful clinical application of parenteral and enteral
feeding techniques requiring specific, chemically defined formulations, (2) the possibility
of maintaining patients on highly regulated and well-defined feeding regiments for pro-
longed periods, and (3) an increased understanding of the metabolism and function of
the amino acids. These have diverted major attention away from the earlier and narrower
focus on nitrogen balance, as a dominant technique and criterion for establishing nutri-
tional indispensability, toward a more comprehensive evaluation of the consequences of
altered amino acid levels and balance of intake on the metabolic and functional status of
individual subjects.
The strict nutritional classification of the common amino acids made in Table 27.1 is
no longer acceptable or of much value for a better understanding of how the protein
component of the diet serves to meet nutritional needs and supports the health of the host
under various pathophysiological states. However, to give due credit to Rose and his
colleagues,15 they did state that “No longer is one warranted in referring to amino acids
as dispensable or indispensable without designating the species in which the tests were
made, and indicating the criterion used as the basis of the classification. The conclusions
presented in Table V [see the present Table 27.1] apply to the growth of the rat ‘only’.”
For summary purposes, in Table 27.2 we present the basis of and resulting classification
of the different amino acids for four historically important schemes, and we also include
the more recent 1995 classification by Young and El-Khoury.32

27.3 Indispensable amino acid requirements in adults


A reasonable starting point for a review of the indispensable amino acid requirements in
adults is in reference to the 1985 FAO/WHO/UNU report.27 In this report the amino acid
requirement values for adults were based on the combined data available for both sexes
that were presented in the 1973 FAO/WHO report.26 The values for men were taken from
the N balance studies of Rose.11 These values represented double the estimate of the
requirement found to be the highest for an individual, in a study group, to achieve positive,
apparent nitrogen balance. The values for women were taken from a series of investiga-
tions, which have been reviewed by Irwin and Hegsted.33 These are stated by the 1973
FAO/WHO Expert Committee26 to have been estimated by the authors of the original
investigations to be the highest estimate of individual requirement to achieve the zone of
nitrogen equilibrium (balance of 0 ± 5% of intake). Some of the values for women also
were based on a reanalysis by Hegsted34 of the published data, in which regression analysis
was used to estimate the average requirement to achieve nitrogen equilibrium (without
accounting for skin and unmeasured losses). It must be emphasized that the amino acid
values given in column 5 of the original Table 4 in the 1985 FAO/WHO/UNU report27
for adults, except for the addition of a requirement for histidine, were exactly the same
as those given in the 1973 FAO/WHO report. 26 The values proposed by the
FAO/WHO/UNU27 for adults are given in Table 27.3.
It was noted in the 1985 UN report27 that there was further scope for research on
amino acid requirements. Hence, beginning in the early 1980s a number of tracer tech-
niques and approaches were initiated at the Massachusetts Institute of Technology (MIT),
with the purpose of estimating the efficiency of amino acid utilization and the require-
ments for specific indispensable amino acids in healthy adults. Tracer-based experimental
1382_C27.fm Page 444 Tuesday, October 7, 2003 7:08 PM

444 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 27.2 Some Earlier and More Recent Schemes for Classification of Amino Acids
in Human Nutrition
1. Rose et al. (1954)17
Basis: Dietary presence critical for supporting maintenance of nitrogen balance in healthy
adults.
Classification: Essential amino acids (valine, leucine, isoleucine, threonine, methionine,
phenylalanine, lysine, and tryptophan) and nonessential amino acids (glycine, alanine, serine,
tyrosine, aspartic acid, glutamic acid, proline, hydroxyproline, histidine, citrulline, and
arginine).
2. Chipponi et al. (1982)30
Basis: Observations from chronically ill patients supported via total parenteral nutrition.
Classification: These investigators promote the concept of conditionally essential nutrients;
included are cystine, tyrosine, and taurine.
3. Jackson (1983)31
Basis: Ability to synthesize the carbon skeleton together with ability to aminate carbon
skeleton.
Classification: (1) Nonessential, e.g., alanine, glutamic, and aspartic acid (carbon skeletons
readily available and metabolically active); (2) essential carbon skeletons required (e.g.,
branched-chain amino acids); (3) essential amino acids — neither synthesis of carbon skeleton
nor amination (lysine, threonine); (4) carbon skeleton readily synthesized but inadequate
amination (serine, glycine).
4. Laidlaw and Kopple (1987)29
Basis: Data from subjects who fall outside the range of the normal healthy adult volunteers.
Classification: (1) Totally indispensable (no metabolic precursor — lysine, threonine); (2) carbon
skeleton indispensable (keto acid or hydroxyacid can be substituted; e.g., histidine, isoleucine,
tryptophan); (3) conditionally indispensable — reduce need for indispensable amino acids or
may become indispensable in absence of precursor (tyrosine, cysteine); (4) acquired
indispensable — become indispensable in state of metabolic disorder, stress, or immaturity
(e.g., tyrosine, cysteine, arginine, taurine, perhaps citrulline); (5) dispensable (alanine,
glutamate, aspartate).
5. Young and El-Khoury (1995)32
Indispensable:
(a) Carbon skeleton cannot be synthesized.
(b) Rate-limiting enzymes of catabolism regulated in relation to adequacy of intake and
tissue supply.
Conditionally indispensable:
(a) Indispensable amino acid is a precursor (i.e., methionine cysteine) and/or
(b) Synthesis, and in particular, degradation is modulated by dietary supply (i.e., arginine,
glycine).
(c) Stressful states cause tissue depletion where rates of synthesis are insufficient to meet
increased metabolic demand rate (proline, glutamine, arginine, glycine, taurine) or
match the catabolic rate.
Dispensable: Rate of synthesis is not down-regulated by intake of amino acid; metabolism
largely a function of metabolic status of major energy-yielding substrates and overall
nutritional status (alanine, glutamate, aspartate).

approaches have accounted for much of the new research on human amino acid require-
ments carried out since that time. Therefore, before considering the absolute amino acid
requirement values for adults, particularly those that have been suggested since the 1985
FAO/WHO/UNU27 recommendations, it is worthwhile to begin with a brief critique of
the various experimental approaches that have been or may be used to arrive at amino
acid requirement values in adults. For purposes of discussion in this review, the minimum
physiological requirement for an indispensable amino acid might be defined as:
1382_C27.fm Page 445 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 445

Table 27.3 1985 FAO/WHO/UNU Recommendations for the Indispensable


Amino Acid Requirements in Adults
Requirement
Amino acid mg·kg–1·day–1 mg·g of protein–1
Histidine 8–12 16
Isoleucine 10 13
Leucine 14 19
Lysine 12 16
Methionine + cystine 13 17
Phenylalanine + tyrosine 14 19
Threonine 7 9
Tryptophan 3.5 5
Valine 10 13
Total (minus histidine) 83.5 111

Source: From FAO/WHO/UNU, Energy and Protein Requirements, Report of a Joint


FAO/WHO/UNU Expert Consultation, Technical Report Series 724, World Health Organi-
zation, Geneva, 1985, 206 pp.

the continuing dietary intake of an indispensable amino acid that is just suffi-
cient to balance whole-body irreversible losses in an initially healthy individual
at energy balance under conditions of moderate physical activity and as deter-
mined after a brief period of adjustment to a new quasi-steady state with a
change in test amino acid intake. For pregnant and lactating women the amino
acid requirement is taken to also include the extra dietary need associated with
the deposition of protein in tissues or secretion of milk at rates consistent with
good health.

27.3.1 Methods and approaches for estimating amino acid requirements


in adults
Determinations of adult human amino acid requirements have been based principally on
application of (1) the nitrogen balance method, (2) plasma amino acid responses, and (3) a
number of stable isotope tracer approaches. The latter largely include (1) direct amino
acid oxidation (DAAO), (2) indicator amino acid oxidation (IAAO), (3) indicator amino
acid balance (IAAB), and (4) direct amino acid balance (DAAB) approach. In addition, a
factorial prediction of amino acid requirements has been used, and this is based on
estimates of obligatory amino acid oxidative losses (OAALs).32 The principles underlying
each method, as well as their possible advantages and limitations, will be reviewed here.

27.3.1.1 Nitrogen balance


Nitrogen balance is the difference between the nitrogen intake and total nitrogen excretion
and, as such, is a deceptively simple concept. The balance technique has been used for
many years in all aspects of protein and amino acid nutrition research, and it remains the
principal method used to estimate adult human protein (nitrogen) needs, as noted above.
Manatt and Garcia35 have reviewed, in some detail, the concepts underlying the N balance
method and the experimental techniques used to investigate the concepts. Therefore, we
will address here, more selectively, a number of issues in specific reference to the use of
N balance in studies of adult human amino acid requirements.
The technical problems associated with measuring intake and all of the routes of N
loss accurately have been discussed by Manatt and Garcia.35 However, of particular impor-
tance in amino acid requirements studies is the difficulty of determining the integumental
1382_C27.fm Page 446 Tuesday, October 7, 2003 7:08 PM

446 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

and other minor or unmeasured routes of N loss. Although these losses are usually
relatively small, in relation to the total N intake and total N output, small differences can
affect profoundly the interpretation of N balance data in any individual study, for example,
when Hegsted34 reanalyzed some published N balance data from amino acid requirement
studies in adults. For this purpose, he assumed an additional and unmeasured N loss of
0.5 g of N daily, in effect, allowing for an apparent +0.5 g of N daily balance. The revised
requirement values were significantly increased above those proposed by the original
authors of the various studies; the range of increase was from by about 150% for tryp-
tophan to nearly 500% for threonine and methionine. Millward3 has reassessed the calcu-
lations made by Hegsted34 by using an assumed +0.3 g of N daily miscellaneous loss
(approximately 5 mg of N·kg–1·day–1) rather than the +0.5 g of N value used by Hegsted.34
Millward’s3 recalculated values are generally much lower than those of Hegsted,34 illus-
trating the fact that the magnitude of these unmeasured or assumed additional N losses
can have a profound influence on the estimated amino acid requirement. Rand and Young36
made a point of this after their analysis of the N balance data published by Jones et al.37
Further, if the regression of N balance on intake has a relatively low slope, the effect on
the amino acid requirement estimate of adjusting for a given and different value for
unmeasured N losses, or for a specified positive N balance, is even more dramatic.
The precise magnitude of these losses cannot be stated with any confidence. The 1981
FAO/WHO/UNU Expert Consultation27 proposed a value of 8 mg of N·kg–1·day–1 for
adults and 10 mg of N·kg–1·day–1 for children up to the age of 12 years as estimates of
miscellaneous losses. In a comprehensive and detailed study, Calloway et al.38 concluded
that dermal and miscellaneous losses amounted to about 0.5 g of N daily (~7 mg·kg–1·day–1)
in sedentary, healthy young men. In a single study, older men losses of N via sweat, nail,
and hair were determined to amount to 2.7 mg·kg–1·day–1.39 The magnitude of these losses
is not known with sufficient confidence, but the UN value of 8 mg was chosen in a
relatively recent reanalysis36 of “apparent” lysine intake–N balance data of Jones et al.37
Millward and Roberts40 consider the value of 8 mg to be too high, and Millward3 chose
5 mg·kg–1·day–1 for his reevaluation of the Hegsted34 N balance data. A recent meta-analysis
of N balance studies by Rand et al.41 suggests that dermal and miscellaneous loss approx-
imate 5 and 11 mg of N·kg–1·day–1 for subjects studied in geographically temperate (mainly
U.S.) and tropical areas of the world, respectively.
In addition to N balance estimations being compounded by errors involved in over-
estimating intake and underestimating loss via urine and feces,35 they are also affected by
various dietary/metabolic factors, for example, the effect of dietary energy, since N balance
is highly sensitive to changes in energy intake above and below that required to just
achieve an equilibrium with energy expenditure. There are problems associated with the
high-energy intakes in the N balance–amino acid requirement studies by Rose and his
coworkers, and these have been commented upon by Rose11 and by the MIT group.42–44
Another example is the length of the dietary adjustment period and impact of the prior
dietary intake on the estimation of the steady-state N balance status. In N balance studies
where total protein intake levels have been changed, the MIT group has observed that a
new and relatively steady state of N excretion is achieved usually within about 4 to 6
days.45 Where the N balance period is more prolonged, there has seemed to be little further
change in N excretion.46,47 Quevedo et al.48 suggest that a period of about 2 weeks or longer
is necessary to achieve a new steady-state N output with a change in total N intake.
However, these specific N balance results may be confounded by the low and possibly
inadequate level of energy intake that was given to their experimental subjects, as dis-
cussed elsewhere.4
In view of these various problems with the N balance technique, the amino acid
requirement values that were derived from earlier N balance studies must be viewed with
1382_C27.fm Page 447 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 447

considerable circumspection. It is now reasonably clear that the earlier requirement values
for adults as proposed and used by FAO/WHO/UNU27 were significantly underesti-
mated. This problem and the limitations of the earlier N balance–amino acid requirement
studies apply also to the recalculated N balance–amino acid requirement estimates as
conducted by Hegsted,34 Millward,3 and Rand and Young.36 Based on these considerations,
we do not recommend using N balance-derived estimates of amino acid requirements in
adults as a primary basis for establishing an updated amino acid requirement pattern for
adults. Nevertheless, we should reemphasize that N balance, when obtained via appro-
priately designed experiments, measured carefully and the results analyzed appropriately,
can be a useful marker of the relative adequacy of dietary nitrogen or even of a specific,
indispensable amino acid intake in comparison with an acceptable reference point.

27.3.1.2 Plasma amino acid concentration–intake response


Plasma amino acid concentrations change in response to various dietary factors, including
the levels and sources of energy yielding substrates and the amount and profile of the
amino acid intake.49,50 The latter response can be somewhat paradoxical51,52 in that high-
protein intakes lower the plasma concentrations of a number of indispensable and con-
ditionally indispensable amino acids, including threonine, glutamine, and taurine. Further,
the concentrations of free amino acids in the circulating plasma vary according to the time
of day,53 the extent of which depends on the protein or amino acid intake level54,55 and is
modulated according to the pattern of meal consumption.56 In addition, plasma levels of
amino acids are affected by disease, physical trauma, genetic factors, and developmental
age.49 Excessive or inadequate intakes of single essential amino acids are reflected in
increases or decreases, respectively, in the plasma concentration of the amino acid.57
Studies in experimental animals suggest that for some essential amino acids these changes
may be associated with an even greater alteration in their concentration in the free amino
acid pool of body tissues, mainly in skeletal muscle.58
Although the qualitative relationship between the adequacy of dietary amino acid
intakes and plasma amino acid concentrations has been reasonably well established,50 the
more important issue here concerns the quantitative relationship between the intake of a
specific amino acid and the level of that amino acid in blood plasma. Therefore, a series
of studies, in young and elderly human adults, was conducted at MIT to explore the
relationships between specific amino acid intake, plasma amino acid concentration, and
the possible requirement for the amino acid. Initial studies in healthy young adult men
were carried out with tryptophan as the test amino acid,59 and these revealed a complex
relationship between dietary tryptophan intake and the plasma tryptophan concentration.
Nevertheless, it was concluded that the plasma tryptophan response curve could provide
a basis for assessing the minimum tryptophan requirement in healthy adults. A subsequent
study in healthy elderly men and women was carried out, with generally comparable
results.60 While the plasma tryptophan response curve could be analyzed in terms of a
“breakpoint” response, the plasma threonine response curve was not as easy to interpret
in a similar way.61 Further, studies by Young et al.62 with valine and lysine as test amino
acids generated new data on the relationships between their intakes and plasma concen-
trations. It now appears that the plasma response curve estimates of the requirements
tend to be lower than those derived from tracer studies, as discussed below. It is possible
that this may, in part at least, be due to the fact that total nitrogen intake in the plasma
response series of studies carried out at MIT was relatively low or equivalent to 0.5 g of
protein·kg–1·day–1. This intake level was thought to be close to the average protein require-
ment at the time of those studies, but from the more recent N balance analyses described
above,41 it is likely that a limiting N intake may have been a confounding factor for
precise interpretation of those studies. In any event, the sensitivity of the approach and
1382_C27.fm Page 448 Tuesday, October 7, 2003 7:08 PM

448 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

interpretation of minimum requirement significance of the response curve appeared to be


open to a great deal of subjective judgment. Hence, while the plasma amino acid response
approach appeared to have utility and precision when applied to the rapidly growing
organism,49,50 a focus on exploring further, alternative approaches for estimation of the
amino acid requirements of healthy adults appeared to be more worthwhile.

27.3.1.3 Tracer techniques


With advances in the mass spectrometric measurement of stable isotope enrichment in
biological matrices and the expanded use of tracers enriched with these isotopes in human
metabolic research, a series of tracer studies was begun at MIT in the early 1980s to revisit
the determination and estimation of the amino acid requirements in adults. Since that
time a number of different paradigms have been used in tracer-based studies of human
amino acid requirements. These can be distinguished according to the choice of tracer and
the protocol design applied as follows:

1. Studies involving use of a tracer of the test dietary amino acid, with a measure of
its rate of oxidation at various test intake levels (the DAAO approach) or of the
body 13C-amino acid balance (the DAAB technique). These techniques have been
used to assess the requirements for leucine, valine, lysine, threonine,43 and pheny-
lalanine,63 as summarized later.
2. Studies involving use of an indicator tracer to assess the status of whole-body
amino acid oxidation (IAAO) or of IAAB at varying levels of a test dietary amino
acid. An example of the IAAO approach is given in the study by Zello et al.,64
which included a determination of the rate of 13C-phenylalanine oxidation (the
indicator) at varying levels of lysine intake.
3. Kinetic studies designed to assess the retention of protein during the postprandial
phase of amino acid metabolism, using 13C-leucine as a tracer.65,66 This is the
postprandial protein utilization (PPU) approach.

The general limitations and problems associated with the use of stable isotope tracers for
estimating amino acid requirements have been considered by Millward.67 Each of these
main methods will be critiqued briefly below.

27.3.1.3.1 DAAO and DAAB. The potential advantage of the DAAO/DAAB tech-
nique is that the rate of oxidation of the dietary amino acid of interest is directly estimated;
it is then possible to evaluate both the pattern of change in the oxidation rate of the test
amino acid and the body balance of the dietary amino acid under study. The MIT group
began to explore and apply this approach in studies of adult human amino acid require-
ments in the early 1980s, using stable isotopically labeled tracers. However, there are a
number of major limitations and disadvantages to this specific tracer approach. First,
precise determination of the rate of tracer oxidation is difficult to achieve since for most
amino acids the isotopic enrichment of the pool directly supplying substrate for oxidation
is not actually known. For practical reasons, the isotopic enrichment of the venous plasma
free amino acid pool is routinely sampled and analyzed for purposes of determining the
oxidation rate. Except for leucine68 and possibly methionine, where the plasma enrich-
ments of a-ketoisocaproate and homocysteine,69 respectively, can be used as an index of
labeling in the intracellular pool of the parent amino acid, it is likely that the DAAO and
DAAB approaches, to date, have underestimated oxidation and overestimated balance.
This may not necessarily be a major problem where the pattern of response in oxidation
rate to altered intake levels is of primary interest, but it definitely is a problem for the
accurate estimation of amino acid balance.
1382_C27.fm Page 449 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 449

A second limitation of the DAAO and DAAB approaches is that the labeled amino
acid is given in finite or tracerless amounts that, therefore, contribute to the effective test
amino acid intake with possible modification of the status of endogenous amino acid
metabolism, especially where the tracer is given by vein. Again, such effects may require
relatively high tracer intakes, possibly equivalent to more than 10% of the plasma amino
acid flux. In recent DAAO and DAAB 13C-tracer studies, the tracer input has been included
in the estimation of total test amino acid intake for purposes of estimating amino acid
balance. This appears to be fully justified, at least for leucine,70 but this problem deserves
further investigation.
With reference to the DAAB and DAAO approaches, the initial MIT tracer protocols
were of relatively short duration (3 h) and were conducted in subjects who were in the
fed state.71–74 To estimate the daily amino acid balance, assumptions had to be made about
the rates of amino acid oxidation during the remaining 9 h of the fed period and then also
during the 12-h postabsorptive period. These assumptions have been described,71 but a
precise estimate of daily balance ideally would require use of a 24-h tracer protocol. A
further reason is that the rate of amino acid oxidation during the fed period does not
necessarily remain constant but can vary throughout this 12-h phase of the fed–fast cycle,
with the rhythm changing according to the adequacy of intake of the test amino acid.75,76
Therefore, the original, short-term DAAB technique has been substantially modified to
include 24-h tracer balance studies.68,76,77 This 24-h approach has been validated using
leucine as the tracer/test amino acid.68 These studies are complex and difficult to carry
out, often limiting the number of repeat 24-h studies that can be conducted in any one
single subject or the total number of 24-h studies that are feasible in any one investigation.
Despite this limitation. the 24-h tracer balance approach might be regarded currently as
the most rigorous, tracer-based paradigm for determining amino acid balance and, there-
fore, requirements in adults. We refer to this paradigm as the 24-h DAAB (24-h DAAO)
technique.

27.3.1.3.2 IAAO. The indicator amino acid oxidation method was applied initially
in studies of amino acid requirements in young growing pigs78 and validated against
traditional approaches based on criteria of growth, N balance, and body composition. The
underlying concept of this technique has been discussed in detail by Zello et al.64 Thus,
the requirement for an indispensable amino acid (e.g., lysine) is determined from the
pattern or rate of change in oxidation of another (indicator) amino acid (e.g., 13C-phenyl-
alanine). The approach was first applied in adult humans by the Toronto group in a study
designed to determine the dietary requirement for lysine.79 Pencharz, Ball, and their
colleagues in Toronto have since extended this approach to estimate the tryptophan,80
threonine,81and methionine82,83 requirements of healthy adults, as well as in a follow-up
studies on the lysine requirements of adults84,85 and a study on tyrosine–phenylalanine
relationships.86
The experimental approach followed by the Toronto group has involved giving sub-
jects an adequate, constant diet for a few days followed by 13C-phenylalanine (or 13C-lysine)
tracer study at a test intake level of the amino acid whose requirement is being estimated.
The tracer protocol involves giving subjects small hourly meals for 7 h, beginning 3 h
before the infusion of labeled indicator tracer. Isotopic data for the last 2 h of the 4-h tracer
period are used to estimate the indicator amino acid oxidation rate. Individual subjects
are studied at multiple test amino acid intake levels, with as many as six or more levels
in some of their investigations.
There are a number of advantages to this short-term IAAO approach, including (1) the
possibility of carrying out a relatively large number of these short-term tracer studies
within the same subject; (2) problems arising from changes in pool sizes and kinetics that
1382_C27.fm Page 450 Tuesday, October 7, 2003 7:08 PM

450 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

might affect the behavior of a direct tracer and interpretation of the isotopic data obtained
are, presumably, obviated or largely avoided when an indicator amino acid tracer is used;
and (3) there is no a priori reason to determine the absolute rate of indicator amino acid
oxidation since the pattern of release of the 13C-label in expired air can provide the basis
for requirement estimate derived from the so-called breakpoint analysis on the intake–oxi-
dation response curve. This pattern of 13C-appearance should parallel that for the absolute
oxidation rate of the indicator, according to the principle on which this approach is based.
However, in the study by Duncan et al.,84 concerned with the lysine requirement of adult
males, this was not found to be the case; while the absolute rate of phenylalanine oxidation
showed a pattern generally similar to that of 13CO2 release, the variation precluded use
of the former parameter to estimate the requirement for lysine.
There are disadvantages of the IAAO method, as originally and usually conducted.
First, it is a short-term, fed-state tracer model. Therefore, there is uncertainty as to whether
the same pattern of change or at least breakpoint in IAAO response would apply similarly
to a later (or even earlier) period within the 12-h fed phase, compared with the specific
2-h period that has been used to elaborate the relationship between amino acid intake,
oxidation, and requirements. From 24-h tracer studies it is now clear that the rate of amino
acid oxidation changes throughout a 12-h fed period in a complex way, depending upon
the adequacy of amino acid intake.75,76 In summary, it is not entirely certain whether the
time frame chosen for detailed study in the Toronto investigations is necessarily optimal,
although they have given results generally consistent with those obtained using the 24-h
DAAO/24-h DAAB and the 24-h IAAO/IAAB approaches, described below. Second, Zello
et al.64 state that the IAAO technique has the advantage of permitting oxidation measure-
ments to be taken with no prior adaptation to the level of the test amino acid, in contrast
to the DAAO and DAAB studies, where adaptation periods of about 6 to 7 days have
been included in the study design. This may not be a particular advantage of the IAAO
technique for two reasons: (1) the DAAO procedure could be similarly applied without a
period of dietary adaptation, just as is the case of the Toronto studies; and (2) perhaps
more importantly, the lack of a period of adaptation to a test amino acid intake level is
potentially a serious design limitation, at least in terms of how the Toronto group applied
the IAAO approach.79,85,86 Millward87 has argued that without a suitable adaptation period
to a specific and lower test lysine intake, the IAAO approach effectively would give a
higher value than the minimum physiological requirements for lysine. On the other hand,
Young4 has argued the opposite, namely, that the minimum requirement might, in theory,
be underestimated when applying the IAAO approach under conditions where there is
no adaptation to a lower-than-usual intake. Recent studies by Millward et al.65,66 on the
postprandial utilization of milk and wheat proteins in nonadapted subjects would support
this view, since their estimate of the nutritional quality of wheat protein was higher than
the authors had predicted, presumably due to the buffering effect of a significant and
replete free tissue (possibly muscle) lysine pool over the course of their short-term tracer
study. Nevertheless, there is a need to establish whether and for how long an adaptation
period should be included in studies involving a short-term, fed-state tracer and IAAO
technique. A third limitation of the short-term, fed-state IAAO method is that the approach
has not been validated directly in healthy adult humans. Direct support for the concept
is based essentially on studies in piglets whose growth rate and intensity of protein
metabolism are profoundly different from those in human adults.88 The question to be
raised, therefore, is whether the breakpoint in the indicator amino acid oxidation–test
amino acid intake response relationship as applied in adult humans is actually that intake
that just meets the requirement for maintenance of nutritional status.
1382_C27.fm Page 451 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 451

27.3.1.3.3 24-h IAAO and 24-h IAAB. To circumvent some of the limitations of the
short-term IAAO technique, as specifically applied by the Toronto group, a 24-h indicator
amino acid oxidation and balance approach has been developed in a collaboration between
the MIT group and Anura Kurpad, St. John’s Medical College, Bangalore, India.89 It has
now been applied in 13C-leucine, indicator tracer studies of the lysine requirement of adult
Indian subjects,89–91 the threonine requirement in U.S.92 and Indian93 subjects, and the
methionine requirement in Indian subjects.94 The approach is identical in concept to that
of the short-term IAAO technique, but is based on a 24-h indicator amino acid oxida-
tion–daily balance protocol. It has the advantage of providing a direct estimate of both
24-h indicator amino acid oxidation and balance. We regard the 24-h IAAB as a functional
criterion of dietary amino acid adequacy, in contrast to a measure of the short-term, fed-
state indicator amino acid oxidation rate that is essentially a kinetic marker of adequacy.
The disadvantage of the 24-h IAAO and 24-h IAAB approaches relates to the complexity
of the 24-h tracer study and the stringent demands and restraints that it places on the
experimental subject. Furthermore, the 24-h paradigm has been most often conducted
using a 12-h fed-state period that involves giving small, frequent, isocaloric, isonitrogenous
meals. We have determined95,96 that leucine oxidation is lower when three discrete meals
vs. a multiple frequent meal schedule is used. This has been observed for generous and
limited intakes of leucine. Hence, this raises the question as to what is the most appropriate
meal pattern for estimating amino acid requirements in adults when using the IAAO and
IAAB techniques.
Another unsolved issue is the molecular form of the amino acid intake. The earlier
amino acid requirement studies of Rose11 and of many of the other investigators33 used
L-amino acid mixtures as a principal source of amino acids. The more recent 13C-tracer
studies have generally used mixtures of L-amino acids. However, it is not clear whether
there are differences in the efficiency of amino acid utilization over a 24-h period between
amino acids supplied as mixtures or in protein-bound form, or whether the requirement
for the indispensable amino acid is different when consumed via three discrete meals or
multiple frequent meals.
On the basis of these various considerations, we conclude that the amino acid require-
ment estimates generated from the 13C-tracer 24-h DAAB, 24-h IAAB, and 24-h IAAO
collectively provide the best primary estimates of the minimum physiological require-
ments for the indispensable amino acids. At this time the 24-h IAAB technique (with
13C-leucine as indicator and when leucine is given at an approximate requirement intake)

represents the state of the art and can be regarded as the gold standard. It might therefore
be used as a basis for validating other and possibly less complex tracer paradigms.

27.3.1.3.4 Postprandial protein utilization. Millward et al.65,66 have conducted two,


short-term fast/fed tracer [1-13C]leucine balance experiments to evaluate the utilization
of wheat compared with milk protein, with calculation of the average requirement for
lysine. In theory, this approach could be adapted to estimate the requirements for other
indispensable amino acids, especially if L-amino acid mixtures, or combinations of proteins
and L-amino acids, were used in place of intact proteins. As carried out by these investi-
gators, the tracer protocol lasts for 9 h with three consecutive 3-h phases, a postabsorptive
phase, and then a low-protein meal phase followed and terminated by a higher-protein
meal phase. The lysine requirement is derived from an estimate of the relative efficiency
of wheat nitrogen retention compared with milk, assuming that lysine limits wheat uti-
lization. The indicator tracer amino acid in this case is [13C]leucine. Thus, it has the
potential for providing a reliable means of estimating the change in protein balance with
protein intake and of comparing the efficiency of protein utilization (PPU) from different
protein sources. However, the specific experimental design that has been used to estimate
1382_C27.fm Page 452 Tuesday, October 7, 2003 7:08 PM

452 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

PPU has not been critically optimized for purposes of estimating the minimum daily
requirement for an amino acid. For example, Young et al.97 found by limiting intakes of
an indispensable amino acid that the estimate of PPU can differ with the passage of time
within the meal-feeding phase. Further, it seems likely that a low-protein meal phase
would influence the leucine balance that occurs during the succeeding high-protein meal
phase, so affecting the value of the PPU obtained. Also, in the study by Millward et al.65
there were changes in the size of the free leucine pool that were not, apparently, taken
into account in estimating the change in protein-bound leucine balance. In this case, the
relative PPU may have been overestimated, and so the lysine requirement would have
been underestimated. A more recent study by these investigators66 suffers from the same
limitations and from a number of other assumptions that need validation and that, as
pointed out by Kurpad and Young,98 may lead to an underestimation of the lysine require-
ment. Finally, when conducted in nutritionally replete individuals, the PPU of wheat
protein, as assessed during an acute experiment, would be expected to be higher than that
for individuals whose body, especially muscle, free lysine pool is lower as a consequence
of a habitually lower intake of lysine. Indeed, there was a much higher relative efficiency
of utilization (0.68) of wheat as determined in the study by Millward et al.,65 compared
with a far lower predicted value (0.22). This observation supports the view that the lysine
requirement may have been underestimated by Millward et al.65 In any event, this point
underscores the fact that it is necessary to evaluate more rigorously the need for and the
length of an adaptation period, as mentioned above in reviewing the IAAO technique.
Millward et al.65 dismissed this as a problem because they state that, in their model, the
amplitude of the metabolic demand (24 times hourly postabsorptive loss determined from
the rate of leucine oxidation) would decrease with a lower previous lysine intake. The
argument put forth was that this would effectively reduce the requirement for lysine to
achieve a given balance since it would mean that there is a smaller net protein deposition
in the fed phase required to compensate for the lower loss during the fast phase. There
is little direct evidence to support this contention, as far as amino acid requirement studies
are concerned. Indeed, in contrast, in a relatively recent study, leucine oxidation data92
obtained with subjects receiving inadequate, sufficient, and generous intakes of threonine,
as test amino acid, do not show any changes in the rate of postabsorptive leucine losses
between 6 and 13 days of the test dietary periods. Similarly, postabsorptive leucine losses
did not change between 7 and 20 days in subjects given varying test intakes of lysine.91
However, postabsorptive leucine losses were higher at the lower vs. high lysine intakes,
presumably reflecting higher lysine losses during the postabsorptive state.91 This response
would complicate the estimate of lysine intake required to satisfy the so-called metabolic
demand.

27.3.1.3.5 Summary of tracer protocols. In summary, a number of different tracer


techniques and protocols have been applied with the purpose of determining the require-
ment for specific indispensable amino acids in healthy adults. None are without important
limitations. The 24-h IAAO and IAAB techniques would appear to be the best current
tracer-based approaches to date for estimation of adult amino acid requirements, although
the short-term IAAO studies have provided generally similar amino acid requirement
estimates.

27.3.1.4 Factorial prediction of amino acid requirements


For those amino acids that have not yet been examined in tracer studies, an estimate of
the obligatory amino acid losses and the minimum dietary intake required to balance them
can be made.43,99,100 Here, it is assumed that the amounts of the different IAAs contributing
to these N losses occur in proportion to their concentrations in body mixed proteins,
1382_C27.fm Page 453 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 453

Table 27.4 Comparison of the Pattern of Indispensable Amino Acids in


Body Mixed Proteins and Proposed Pattern of Indispensable Amino Acid
Requirements in Adults
Body proteinsa Requirementb
Amino acid (% of total) (% of total)
Lysine 22 20
Phenylalanine 12 12c
Methionine 6 6d
Valine 14 13
Isoleucine 10 13
Leucine 23 26
Threonine 12 10
a Based on Reeds, P.J., J. Nutr., 130, 1835S, 2000.
b Underlined values have been based on interpretation of data from tracer studies.
c Assuming 50% of the total aromatic amino acid requirement as phenylalanine.
d Assuming methionine is required at 75% of the total sulfur amino acid needed.

providing that those proteins contributing to the major proportion of the total N loss do
not have amino acid patterns (concentrations) that differ markedly from the average of
the body mixed proteins.
Although the major route of obligatory loss of these amino acids is via oxidative
catabolism, they are also lost in small quantities via urine or the intestine (particularly for
threonine) in the ileal digesta.101 However, this is not a crucial issue for estimating OAALs
since they are predicted from total ONL. Thus, after estimation of the OAALs it is then
necessary to assume, from whole-body N balance studies in humans,27 that at about a
requirement level of intake individual IAAs would be retained with a specified efficiency.
Research is needed to determine whether the 70% value that has been assumed earlier100
for this purpose is valid for all indispensable amino acids. The overall efficiency of
utilization of good-quality dietary protein, according to the analyses of N balance data
summarized earlier in this paper, approximates a 50% value. There is no assurance,
however, that this would be a more appropriate figure for use in this factorial procedure
since the relative efficiencies, at different intakes relative to physiological requirements,
of total, indispensable, and a-amino nitrogen for maintenance are not known.
The factorial prediction assumes that the amino acid requirement pattern for mainte-
nance of protein nutritional status in adults is similar to that for mixed proteins in the
whole body. This assumption has been criticized.87,102–104 It might be of interest, however,
that we compare in Table 27.4 the relative concentrations of specific amino acids in mixed
body proteins with requirement estimations derived from tracer studies (to be discussed
below). There is a reasonable agreement between the body mixed protein amino acid
pattern and our estimations from interpretation of the amino acid requirements in adults.
Nevertheless, given these various uncertainties, this predictive approach cannot be
used confidently for primary purposes of deriving amino acid requirement values.

27.3.1.5 Summary of approaches for estimation of amino acid requirements


in adults
The foregoing are the major methods that have been used to estimate the amino acid
requirements in adults. All of the approaches are problematical in various contexts. How-
ever, the 24-h IAAO and 24-h IAAB approaches appear to be the best current basis for
establishing indispensable amino acid requirements.
1382_C27.fm Page 454 Tuesday, October 7, 2003 7:08 PM

454 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 27.5 Estimates of Mean Requirement for Lysine: N Balance and Tracer Studies
Mean requirement
Author(s) (mg·kg–1 ·day–1) Comments

Nitrogen Balance
Rose et al.105 8.8 N balance: multilevels
Jones et al.37 6.5–8 N balance: multilevels
Fisher et al.106 <1 mg N balance: variable intakes
Hegsted34 29.2 Reevaluation by regression (+0.5 g of N)
Millward87 18.6 Reevaluation by curvilinear regression and
+0.3 g of N
Rand and Young36 30.6 Reevaluation by nonlinear regression and
+~0.5 g of N

Tracer Approaches
Meredith et al.73 ~30 Fed state: multilevels, DAAO
Meredith et al.73 20 < 30 Estimated DAAB (from fed state)
El-Khoury et al.107,108 30 Three levels: 24-h DAAB; confirmation
Kurpad et al.90 29 24-h IAAB; four levels
Kurpad et al.91 31 24-h IAAB; four levels; 21 days
Zello et al.79 37 IAAO: multilevels (fed state)
Duncan et al.84 45 IAAO: multilevels (fed state)
Kriengsinyos et al.85 37 IAAO: multilevels (fed state), oral tracer and
intravenous tracer

We will now summarize and critique the requirement values that have been proposed
based on application of these various approaches.

27.3.2 Estimates of the requirements for specific indispensable amino acids


in healthy adults
Because there are insufficient data to permit a determination of whether differences in the
quantitative requirements for the individual, indispensable amino acids exist between
adult men and women, we have considered requirements for adults without reference to
gender.

27.3.2.1 Lysine
In Table 27.5 we summarize the results from the major studies concerned with estimation
of the mean requirement for lysine in adults. The data from the earlier balance studies in
men105 and women37 indicated a mean requirement of about 8 mg·kg–1·day–1. The short-
term N balance studies by Fisher et al.106 suggested a lower mean requirement. The latter
estimate is probably complicated by an inadequate experimental design that involved
consecutive short N balance periods when during some both lysine and total N intake
levels were changed simultaneously.
Three mathematical analyses of original N balance data have led to much higher
requirements, after taking into account unmeasured miscellaneous N losses. The analyses
by Hegsted34 and Rand and Young36 of the N balance data of Jones et al.37 gave a mean
lysine requirement of about 30 mg·kg–1·day–1; the analysis by Millward87 yielded a mean
requirement of 18.6 mg·kg–1·day–1, due to the fact that the allowance for unmeasured N
losses made was assumed to be somewhat lower than that included in the analyses by
Hegsted34 and Rand and Young.36
1382_C27.fm Page 455 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 455

Table 27.6 Estimates of Mean Requirement for Phenylalanine: N Balance, Tracer Studies, and Other
Mean requirement
Author(s) (mg·kg–1 ·day–1) Comments

Nitrogen Balance
Rose et al.109 13.2 N balance: no tyrosine
Tolbert and Watts110 ~10.7 N balance: no tyrosine
Leverton et al.111 ~13 N balance: no tyrosine
Leverton et al.111 ~3.7 N balance: with tyrosine
Hegsted34 9.1 Reassess of Leverton et al.111
Millward87 ~19.7 Assumed value from Tolbert and Watts110

Tracer Studies
Zello et al.63 9.1 DAAO: with high tyrosine
Basile-Filho et al.112 >18.5, £36 24-h IAAB (13C-tyrosine): no tyrosine
Sánchez et al.76 >22 24-h DAAB: single Phe intake of 21.9
mg·kg–1·day–1
Basil-Filho et al.113 £39 24-h DAAB: single test intake predicted to be
requirement
Roberts et al.86 15 IAAO: mean aromatic amino acid

Other
Young and El-Khoury100 38 Obligatory amino acid losses

Results from series of tracer studies, despite their individual limitations and different
designs, have yielded mean lysine requirement values ranging from greater than
20 mg·kg–1·day–1 up to 45 mg·kg–1·day–1, with a number of these studies indicating a mean
value of approximately 30 mg·kg–1·day–1. Probably, the most satisfactory estimates are
those by Kurpad et al.,90,91 who used the 24-h IAAO and 24-h IAAB technique and four
test lysine intake levels. The mean requirement, obtained by regression analysis of both
the lysine intake–leucine oxidation and lysine intake–leucine balance responses, was
approximately 30 mg·kg–1·day–1. The results of the short-term IAAO studies79,84,85 and the
short-term and 24-h DAAO and DAAB studies73,107,108 are all consistent with this mean
estimate (30 mg) of the lysine requirement in healthy adults.

27.3.2.2 Phenylalanine and tyrosine


In the absence of a dietary supply of tyrosine, the minimum phenylalanine requirement
would be that intake be just sufficient to meet the metabolic needs for these two aromatic
amino acids. When tyrosine is present in the diet, it might effectively spare part of the
requirement for total phenylalanine. From the earlier N balance studies, with no dietary
tyrosine, the mean requirement for phenylalanine appears to be in the range of 13 to
19 mg·kg–1·day–1 (Table 27.6).
A limited number of tracer studies have been carried out at different intakes of
phenylalanine in the absence of dietary tyrosine. Using an oral tracer of 13C-phenylalanine
given over a 24-h period (DAAB),76,112–114 daily phenylalanine balance was determined to
be negative in the entire group of seven subjects when phenylalanine intake was
22 mg·kg–1·day–1.76 A subsequent study113 with an oral tracer of 13C-phenylalanine and
[2H3]tyrosine indicated that subjects were in approximate neutral body phenylalanine
balance when given a daily intake of 39 mg·kg–1·day–1, supporting the prediction made
from OAAL that the total aromatic amino acid approaches 39 mg·kg–1·day–1. The findings
from these 24-h DAAB studies, despite model limitations, were consistent with those of
the short-term, fed-state DAAO study by Zello et al.63 These latter investigators concluded
1382_C27.fm Page 456 Tuesday, October 7, 2003 7:08 PM

456 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

that the total aromatic amino acid requirement should be 30 mg·kg–1·day–1 and that the
FAO/WHO/UNU27 recommendation of 14 mg·kg–1·day–1 is an underestimate.
A series of 24-h IAAB 13C-tyrosine experiments by Basile-Filho et al.112 revealed that
tyrosine and total aromatic amino acid balance were negative at a phenylalanine intake of
18.5 kg–1·day–1, with an equilibrium being achieved at a phenylalanine intake of 35.6
kg–1·day–1 and tyrosine intake of 6.8 mg·kg–1·day–1. It appears to us that most tracer studies
would indicate a total mean aromatic amino acid requirement in excess of 20 mg·kg–1·day–1
and that it probably falls in the range of about 30 to 40 mg·kg–1·day–1. A possible exception
is the study by Roberts et al.,86 which we will discuss below.

27.3.2.2.1 Tyrosine sparing. The question of the quantitative extent of sparing effect
of tyrosine on the requirement for phenylalanine deserves comment and assessment.
Leverton et al.111 observed that the amount of phenylalanine needed for nitrogen equilib-
rium in the presence of a generous intake of tyrosine (900 mg or about 16 mg·kg–1·day–1)
was about 220 mg daily (~3.7 mg·kg–1·day–1). These investigators were not able to establish
a quantitative relationship between the requirements for the two amino acids but stated
that a sparing effect of tyrosine was apparent. Tolbert and Watts110 concluded from studies
in three subjects that tyrosine exerted a sparing effect of at least 70% on the phenylalanine
requirement. However, only one of their subjects was in true N balance equilibirum at the
low phenylalanine intakes when tested in the presence of tyrosine. This study is not
definitive. Similarly, Rose and Wixom115 concluded, from N balance experiments in two
subjects, that tyrosine is capable of a sparing effect of 70 to 75% upon the phenylalanine
needs. Again, it may be questioned as to whether their subjects were in true N balance at
the low phenylalanine intake level of about 3.1 to 4.5 mg·kg–1·day–1. This latter intake had
been judged by the authors to be the requirement in the presence of abundant quantities
of tyrosine. We have noted above that this requirement estimate was undoubtedly too low.
The 13C-phenylalanine short-term, fed-state DAAO tracer study by Zello et al.63 was
carried out at different phenylalanine intakes in the presence of generous tyrosine
(40 mg·kg–1·day–1). From the breakpoint analysis of the phenylalanine oxidation–intake
response relationship, a requirement of about 9 mg·kg–1·day–1 was proposed. Assuming a
daily OAAL of phenylalanine of 14 mg, or >7 mg·kg–1·day–1, for the fast 12-h period, then
an intake of about or slightly greater than 9 mg of phenylalanine·kg–1·day–1might be
sufficient to sustain whole-body phenylalanine homeostasis in the presence of an adequate
tyrosine intake, if the fed-state rate of phenylalanine oxidation is actually as low as their63
estimate suggests, or approximately 1.2 mm kg–1h–1 (2.4 mg of phenylalanine per kg when
extrapolated to the 12-h fed period of the day). However, a different conclusion would be
obtained if the plasma 13C-tyrosine enrichment values reported by Zello et al.63 had been
used to estimate rate of phenylalanine oxidation rate, since the 13C-label from phenylala-
nine initially passes through the tyrosine pool before being liberated as 13CO2. In that case,
the oxidation rate would have been considerably higher and there would have been a
lower, apparent fed-state retention of phenylalanine for the 9-mg intake level. Since the
tyrosine enrichment data were not reported by Zello et al.,63 we cannot estimate exactly
what the differential in the phenylalanine oxidation rates would have been.
Finally, the concentration of tyrosine in whole-body mixed proteins is somewhat less
than that for phenylalanine.116 Hence, we estimate an OAAL of 12 mg·kg–1·day–1 for
tyrosine, giving a total aromatic OAAL of 26 mg·kg–1·day–1. On this basis, a total dietary
aromatic amino acid requirement of 30 mg·kg–1·day–1 as proposed by Zello et al.63 would
appear to be reasonable, although perhaps somewhat low.
A direct assessment of the tyrosine sparing of the phenylalanine requirement has
been reported by Roberts et al.86 These investigators gave a constant phenylalanine
intake of 9 mg·kg–1 over a 12-h period, and they studied the effects of increasing tyrosine
1382_C27.fm Page 457 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 457

intake on the oxidation of L-[1-13C]lysine as an indicator. The 9 mg·kg–1 intake used in


this experiment was based on the results of an earlier study in which L-[1-13C]phenyl-
alanine oxidation was measured at varying phenylalanine intakes with constant excess
tyrosine (40 mg·kg–1). As discussed above, it had been determined that a breakpoint
in the phenylalanine intake–oxidation curve occurred at a phenylalanine intake of
9.1 mg·kg–1·day–1. In the study by Roberts et al.,86 a mean breakpoint in the lysine oxida-
tion–tyrosine intake response curve occurred at 6 mg tyrosine·kg–1·day–1. Also, at this
intake it can be calculated from the balance between lysine intake–oxidation that lysine
retention approximated 11.3 mmol·kg–1h–1. Assuming a molar ratio of 0.58 for the phenyl-
alanine/lysine concentration ratio in mixed body proteins, this would predict a phenyl-
alanine retention of 6.6 mmol·kg–1h–1 or approximately 13 mg of phenylalanine·kg–112 h–1.
Because the absolute rate of lysine oxidation was probably underestimated in the study
by Roberts et al.86 and also because some phenylalanine oxidation occurs at the 9-mg
intake level, it is likely that this retention figure of 13 mg is too high; in reality, it would
be lower and perhaps much below the test intake of 9 mg of phenylalanine·kg–1·day–1,
given over a 12-h period. Therefore, it seems reasonable for us to conclude that an intake
of 9 mg of phenylalanine·kg–1·day–1 was in all likelihood limiting. Thus, no further
improvement in overall amino acid utilization, as reflected by lysine oxidation, could be
expected with a further increase in tyrosine intake above 6 mg·kg–1·day–1, since the molar
tyrosine:lysine concentration ratio in mixed body proteins is less than the phenylala-
nine:lysine ratio. It follows that the total aromatic amino acid intake of 15 mg (9 mg of
phenylalanine and 6 mg of tyrosine) proposed by Roberts et al.86 is limiting or insufficient
to meet the physiological, mean total aromatic amino acid requirement.
There are data in support as well as in contradiction of the foregoing interpretation.
In reference to the latter possibility, the earlier N balance data in women110 were interpreted
to indicate a tyrosine-sparing effect of at least 70% on the phenylalanine requirement.
However, this conclusion was based on a study in three college students, two of whom
received relatively high-energy intakes that would have confounded the interpretation of
the N balance findings. Leverton and coworkers111 interpreted their N balance data to
suggest a phenylalanine requirement of 220 mg daily (or 3.4 mg of phenyl-
alanine·kg–1·day–1, assuming a body weight of 65 kg) in the presence of a generous tyrosine
intake of about 14 mg·kg–1·day–1. In a later experiment115 with two young men, it was
stated that “tyrosine is capable of exerting a sparing effect of 70–75% upon the phenyl-
alanine needs of the organism.” Thus, N balance studies clearly reveal a sparing effect of
tyrosine, but there are a number of uncertainties. First, what is the quantitative relationship
between the total aromatic amino acid requirement and those for phenylalanine and
tyrosine at intake ratios that are more likely to reflect those in common food protein
sources, including milk proteins?116 Intakes of tyrosine that are about two to three times
that of phenylalanine, as in the case of the studies by Tolbert and Watts110 and Rose and
Wixom,115 would not be likely in practice. The results of their experiments would seem to
have limited practical significance.
Second, there is the question of the quantitative requirement for total aromatic amino
acids, when they are met either via phenylalanine alone or via a combination of phenyl-
alanine and tyrosine. The study by Roberts et al.86 is interpreted by these investigators to
indicate that the mean requirement for total aromatic amino acids is 15 mg·kg–1·day–1. On
the other hand, Young et al.43,100 predicted, from obligatory amino acid losses, that the
requirement for total aromatic amino acids would approximate about 39 mg·kg–1·day–1.
Using L-[1-13C]tyrosine as a tracer, Basile-Filho et al.112 observed that at a phenylalanine
intake of 18.5 mg·kg–1·day–1 (or with the phenylalanine and tyrosine tracers the total
aromatic amino acid intake was 25.3 mg·kg–1·day–1) subjects were in distinctly negative,
whole-body aromatic amino acid balance; the subjects were at equilibrium at a total
1382_C27.fm Page 458 Tuesday, October 7, 2003 7:08 PM

458 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 27.7 Estimates of Mean Requirement for Threonine: N Balance and Tracer
Studies
Mean requirement
Author(s) (mg·kg–1·day–1) Comments

Nitrogen Balance
Rose et al.118 6.5 N balance
Leverton et al.119 ~3.5 N balance
Fisher et al.120 ~1.7–3.3 N balance
Millward3 15.7 Reevaluation of Hegsted36
Hegsted34 29.1 Reevaluation of Leverton et al.119

Tracer Approaches
Zhao et al.74 10–20 (19.7) IAAB; short term
Wilson et al.81 19.0 IAAB; short term
Borgonha et al.92 15 24-h IAAB; three levels tested
Kurpad et al.93 15 24-h IAAB

aromatic amino acid intake of 42.4 mg·kg–1·day–1. This investigation supports a mean
aromatic amino acid requirement in the region of 39 mg·kg–1·day–1, but it perhaps falls
between 25 and 39 mg, since an intermediate level of intake between these has not been
studied.
Additional support, however, for a mean aromatic amino acid requirement exceeding
that suggested by Roberts et al.86 can be obtained from the studies of Kindt and
Halvorsen117 in two children, about 6 years old, with phenylketonuria (PKU) and one
3 year old with a tyrosine aminotransferase defect (TATD). Intake of phenylalanine that
kept values for plasma phenylalanine and tyrosine within a clinically acceptable range
while supporting normal growth in the PKU children was approximately 20 mg·kg–1·day–1,
and the combined phenylalanine and tyrosine intake in the child with TATD was about
60 mg·kg–1·day–1. It is accepted that the major utilization of metabolic need for amino acids
in young children is for maintenance. By age 3 years maintenance would account for about
85% and at 6 years for about 90% of the daily requirement.27 Hence, these genetic nutrition
studies offer further support of our view that the mean requirement for aromatic amino
acids for maintenance in the healthy adult exceeds, probably by a considerable margin,
the mean value of 15 mg·kg–1·day–1 proposed by Roberts et al.86

27.3.2.3 Threonine
The mean requirement for threonine was estimated to be in the range of 2.0 to
6.5 mg·kg–1·day–1 by three groups of investigators118–120 who carried out the early N balance
studies (Table 27.7). The reanalysis of the data from Leverton et al.119 by Hegsted34 gave
a mean requirement estimate of 29.1 mg·kg–1·day–1, while Millward’s87 modification of
Hegsted’s regression equation gave a mean requirement estimate of 15.7 mg·kg–1·day–1.
Both reassessments lead to profoundly different requirement estimates compared with
those proposed earlier.
A number of tracer studies have been carried out to assess the threonine requirement.
The earlier short-term DAAB studies by Zhao et al.74 put the mean requirement in the
range of 10 to 20 mg·kg–1·day–1. Wilson et al.,81 using the short-term IAAO technique,
conclude that the requirement is 19.0 mg·kg–1·day–1. Results from recent 24-h IAAB
studies92,93 indicate that the FAO/WHO/UNU27 proposed upper requirement level of
7 mg·kg–1·day–1 is insufficient. On the other hand, the tracer studies indicate that a mean
intake of 15 mg·kg–1·day–1 would be sufficient to maintain amino acid homeostasis.92,93
1382_C27.fm Page 459 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 459

Table 27.8 Estimates of Mean Requirement for Leucine, Valine, and Isoleucine: N Balance,
Tracer Studies, and Others
Mean requirement
Author(s) (mg·kg–1 ·day–1) Comments

Leucine
Nitrogen Balance
Rose et al.121 9.9 N balances
Leverton et al.122 <10 N balances
Hegsted34 43 Reevaluation of Leverton et al.111
Millward87 26.3 Reevaluation of Hegsted34
Tracer Approaches
Meguid et al.71 >20 < 40 DAAB; short term
Kurpad et al.77 37.3 24-h DAAB at four intakes
El-Khoury et al.68,75 40 24-h DAAB; three levels, confirmatory

Valine
Nitrogen Balance
Rose et al.123 8.8 N balance
Leverton et al.124 ~11 N balance
Hegsted34 17 Reanalysis of Leverton et al.124
Millward87 13.5 Reanalysis of Hegsted34
Tracer Studies
Meguid et al.72 ≥16 DAAB; short term
Others
Young et al.43 24 Obligatory amino acid losses
Meguid et al.72 ~20 Plasma amino acid response
This review 25 Proportionate to leucine requirement

Isoleucine
Nitrogen Balance
Rose et al.121 10.5 N balance
Swendseid and Dunn125 7.5 N balance
Hegsted34 28 Reanalysis of Swendseid and Dunn125
Millward87 18.1 Reanalysis of Hegsted34
Other
Young et al.43 23 Obligatory amino acid losses
This review 19 Proportionate to leucine requirement

27.3.2.4 Branched-chain amino acids


27.3.2.4.1 Leucine. From the earlier N balance studies, in men121 and women122 it
was concluded that the mean requirement for leucine approximated 10 mg·kg–1·day–1 or
less (Table 27.8). Reassessment of these data by Hegsted34 gave a requirement value of
43 mg·kg–1·day–1 and by Millward87 a lower value of 26.3 mg·kg–1·day–1.
Earlier tracer studies involving the short-term DAAB technique suggested that the
mean requirement fell in the range of 20 to 40 mg·kg–1·day–1.71 The 24-h DAAB tracer
studies by El-Khoury et al.68,75 confirmed that the FAO/WHO/UNU27 upper requirement
of 14 mg of leucine·kg–1·day–1 was too low. Their results supported the prediction of a
leucine requirement of about 40 mg·kg–1·day–1. More recently Kurpad et al.,77 using four
test intake levels of leucine and the 24-h DAAB approach, have estimated the mean leucine
requirement to be 37.3 mg·kg–1·day–1.
From these data it would appear that the mean requirement for leucine is close to
40 mg·kg–1·day–1.
1382_C27.fm Page 460 Tuesday, October 7, 2003 7:08 PM

460 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

27.3.2.4.2 Valine and isoleucine. The earlier N balance studies on the valine and
isoleucine needs in healthy adults suggested requirements in the region of 10 mg·kg–1·day–1
for each of these amino acids (Table 27.8). Reanalysis of the N balance data by Hegsted34
gave mean requirement estimates of 17 and 28 mg·kg–1·day–1 for valine and isoleucine,
respectively, with Millward’s87 reanalysis giving values of 13.5 and 18.1 mg·kg–1·day–1.
Short-term DAAB studies72 suggest a valine requirement of about 16 mg·kg–1·day–1 or
greater. A value of 24 mg·kg–1·day–1 is predicted from OAAL.43
No tracer studies have been conducted to assess the isoleucine requirements. The
prediction from OAAL gives a value of 23 mg·kg–1·day–1.43
Because the data are limited, considerably more judgment is needed to propose a
mean requirement for these two branched-chain amino acids. For both valine and isole-
ucine the mean requirement may be in the region of 20 to 30 mg·kg–1·day–1, when the
reassessed N balance and more recent metabolic data are considered. To arrive at a
requirement for each amino acid, it is reasonable to assume that the requirement for these
two branched-chain amino acids is in proportion to their relative concentrations in body
proteins compared to that for leucine. The leucine, valine, and isoleucine concentrations
(mg/g of protein) of mixed body proteins are about 75, 47, and 35, respectively.116 Hence,
using a leucine requirement of 40 mg·kg–1·day–1, the valine and isoleucine requirements
would be 25 and 19 mg·kg–1·day–1, respectively.

27.3.2.5 Tryptophan
The requirements for tryptophan obtained from the reassessment of the earlier N balance
studies126,127 provide estimates of mean requirements of 3.787 and 4.5 mg·kg–1·day–1.34 These
values are supported by the short-term IAAO tracer studies of Lazaris-Brunner et al.,80
and they are complemented by an earlier interpretation of plasma amino acid data59 and
considerations of estimated intakes to balance OAAL of tryptophan.43
Thus, in spite of the limited database available, it would seem reasonable to suggest
that the mean tryptophan requirement be set, tentatively, at 5 mg·kg–1·day–1.

27.3.2.6 Sulfur amino acids


The requirements for the sulfur amino acids (methionine and cystine) might be met from
methionine alone or more usually from a combination of methionine and cystine.
The nitrogen balance data provide highly variable estimates (Table 27.9). The studies
by Rose et al.118 suggested a mean methionine requirement of 13.2 mg·kg–1·day–1, when
no dietary cystine was present. Requirement estimates for women, again without dietary
cystine, were somewhat less than those for men.33,128,129 Hegsted’s reassessment of the N
balance data34 gave a very high mean requirement value when methionine was given
alone. When cystine was present, the mean methionine requirement was estimated via
this reanalysis to be 6.3 mg·kg–1·day–1.34
The limited number of tracer studies that have been carried out suggest that the
requirement for methionine and cystine is not met when the methionine intake is
~6 mg·kg–1·day–1 in the presence of a cystine intake.131,132 It appears that there is no signif-
icant cystine sparing at this lower methionine intake131,133,134 when cystine intakes are
sufficient to achieve a total SAA intake of about 13 mg·kg–1·day–1. Hence, methionine and
presumably cystine balance is achieved only when methionine intake is in the region of
13 mg·kg–1·day–1 in the absence of cystine. Recently, using the IAAO paradigm, the Toronto
group82 has confirmed and extended these observations, and it has been concluded that
the requirement for methionine, in the absence of dietary cyst(e)ine, is 13 mg·kg–1·day–1.
This is the requirement intake Young et al.43 predicted 12 years ago from OAAL. Whether
S amino acid status would be improved with an additional intake of cystine above the
1382_C27.fm Page 461 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 461

Table 27.9 Estimates of Mean Requirement for Methionine (with or without Cystine): N Balance,
Tracer Studies, and Others
Mean requirement
Author(s) (mg·kg–1 ·day–1) Comments

Nitrogen Balance
Rose et al.118 13.2 N balance; no cystine
Swendseid et al.128 ~9 N balance; small level of cystine
Reynolds et al.129 ~9.2 N balance; small level of cystine
Hegsted34 ~6.3 Reanalysis of Reynolds129 with cystine
Hegsted34 ~57.5 Reanalysis of Reynolds129 without cystine

Tracer Studies
Young et al.130 ~13 DAAB; short-term protocol; single test intake;
no cystine
Hiramatsu131 >6, £13 DAAB: short-term protocol cystine, no sparing
Fukagawa et al.132 >6, £13 DAAB: short-term protocol cystine, no sparing
Di Buono et al.82 13 IAAO: short-term, no dietary cystine
Kurpad et al.94 15 24-h IAAO/IAAB

Other
Millward87 16 Obligatory amino acid losses
Young et al.43 13 Obligatory amino acid losses

13 mg·kg–1·day–1 intake level of methionine (cystine) is not fully known. We comment on


a new study by the Toronto group below.
A sparing effect of dietary cystine on the methionine requirement has long been
accepted. However, the quantitative relationships between dietary methionine and cystine
in reference to the maintenance of S amino acid balance and homeostasis are not well
established. From the earlier human N balance studies, sparing of the methionine require-
ment by dietary cystine ranged widely from about 16 to 90%.11,33,135 Tracer studies have
not revealed measurable sparing of methionine oxidation at intakes of methionine in the
6 to 13 mg·kg–1·day–1 range, as mentioned above. The quantitative extent to which a dietary
level of cystine spares methionine loss remains undetermined. In the N balance studies
by Rose and Wixom,135 for example, the high sparing by cystine was noted at a methion-
ine/cystine intake ratio that would be far lower than ever would be the case in practice.
Hence, these earlier N balance studies must be interpreted with considerable caution and
circumspection.
A recent short-term IAAO study by Di Buono et al.83 is also instructive. These inves-
tigators gave six healthy individuals intakes of methionine ranging from 0 to
13.0 mg·kg–1·day–1 in the presence of a constant cysteine intake of 21 mg·kg–1·day–1. Each
diet was given for 2 days prior to an IAAO tracer study with 13C-phenylalanine, carried
out in the fed state. The results showed that the breakpoint in the 13CO2 excretion–methion-
ine intake response relationship occurred at a methionine intake of 4.5 mg·kg–1·day–1. It
was concluded that dietary cysteine is capable of reducing the exogenous requirement for
methionine in adult males. However, there are a number of limitations to this study:
(1) The rate of 13CO2 release from the L-[1-13C]phenylalanine tracer at the breakpoint and
at higher levels of methionine was approximately 0.5 mmol·kg–1h–1 in this study and
0.4 mmol·kg–1h–1 in the earlier study, in which methionine was given in absence of cys-
teine.83 Hence, it is difficult to compare closely these two Toronto studies, especially since
the dietary conditions and tracer infusion rates were otherwise identical. However, it is
possible that the difference in 13CO2 release rates between these studies is not statistically
1382_C27.fm Page 462 Tuesday, October 7, 2003 7:08 PM

462 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

significant. (2) There was apparently no sparing by cysteine of methionine at the zero and
very low intakes of methionine, when the 13CO2 release rates in the two related studies82,83
are compared. This in contrast to the earlier observations of Storch et al.136 (3) Even
assuming that a sparing of methionine has been definitively shown, the practical impli-
cations of these observations remain unclear because, as already noted above for the N
balance studies, the level of cysteine intake (21 mg·kg–1·day–1) relative to that of methionine
at the breakpoint level, or 4.5 mg·kg–1·day–1, would not be characteristic of natural diets
where the relative intakes of each of these sulfur amino acids are much more similar.
Hence, this study83 does not provide definitive evidence that the methionine requirement,
as determined in the absence of cyst(e)ine, can be reduced to a substantial degree when
the ratio of its intake approximates that of usual diets.
In summary, the present but incomplete, data set suggests a mean requirement for
the S amino acids of about 13 to 15 mg·kg–1·day–1 for meeting body amino acid balance.
Further, it appears that the greater proportion of this intake should be derived from
methionine when the methionine/cystine ratio is similar to that in usual diets. The opti-
mum ratio of methionine to cystine and the desirable intake level of cystine per se remain
to be determined, especially in reference to the nonproteinogenic functions of methionine
and cyst(e)ine, such as the support and maintenance of glutathione homeostasis and
synthesis.

27.3.2.7 Histidine
There have not been recent investigations of the minimum physiological requirements for
histidine in adults since the investigations by Kopple and Swendseid137 and Kriengsinyos
et al.138 On the basis of this earlier study, the 1981 UN consultation proposed a requirement
of between 8 and 12 mg·kg–1·day–1 for healthy adults.

27.3.3 Summary and tentative recommendations for indispensable amino acid


requirements in healthy adults
There is now substantial evidence to indicate that the amino acid requirements in healthy
adults were underestimated by FAO/WHO/UNU.27 Further, it is again emphasized that
the consultation had simply followed the recommendations given in the 1973 FAO/WHO
report.
The 24-h indicator amino acid oxidation and balance techniques may be regarded as
the gold standard, at the present time, for estimating amino acid requirements in healthy
adults.
Our recommendations for these amino acids (mg·kg–1·day–1), based on the foregoing,
are, therefore, as follows: lysine, 30; leucine, 40; threonine, 15; aromatic amino acids
(phenylalanine and tyrosine), 39; valine, 25; isoleucine, 19; tryptophan, 5; sulfur amino
acids (methionine + cystine), 15. These estimates can be compared in Table 27.10 with
recommendations made recently by the U.S. Food and Nutrition Board5 and by Young
and Borgonha.139

27.3.4 Indispensable amino acid requirements in other adult age and


physiological groups
There have not yet been any substantive tracer studies, of the kind discussed above for
young adults, in elderly subjects or in pregnant or lactating women. Therefore, specific
recommendations for these groups cannot be made in the same context as is now possible
1382_C27.fm Page 463 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 463

Table 27.10 Comparison of Indispensable Amino Acid Requirement Pattern (mg·kg–1·day–1) in


Healthy Adults as Proposed by IOM (FNB), Young and Borgonha, and in this Review
Young and
Amino acid IOM/FNB (2002)5 Borgonha (2000)139 This review
Histidine 11 — 8–12
Isoleucine 15 23 19
Leucine 34 40 40
Lysine 31 30 30
Methionione + cystine 15 13 15
Phenylalanine + tyrosine 27 39 39
Threonine 16 15 15
Tryptophan 4 6 5
Valine 19 20 25

for young adults. In the interim it is assumed, relative to the requirements established for
healthy young adults, that the amino acid requirements in these other groups would
change in proportion to any change in the requirement for total protein.

27.4 The nonspecific nitrogen requirement


The two components of the total protein requirement are (1) for the indispensable amino
acids and (2) for a utilizable source of so-called nonspecific nitrogen (NSN)25 needed for
the synthesis of the dispensable and conditionally indispensable amino acids and other
physiologically important nitrogen-containing compounds, such as purines and pyrim-
idines, glutathione, creatine. In an early FAO/WHO25 report it was stated that “mixtures
containing the essential amino acids at minimal levels supplemented with one or more
simple nitrogen-containing compounds, such as glycine and ammonium citrate, can effec-
tively support growth and maintain N balance.… It seems, therefore, that within wide
limits the body is indifferent to the make-up of the non-essential nitrogen component.”
Thus, simple sources of nitrogen and a mixture of diammonium citrate and urea or
diammonium citrate and glycine were assumed to be sufficient to meet the NSN require-
ment. There was some but not necessarily definitive evidence at that time that this assump-
tion was justified. However, there has been increasing research interest in the importance
of the qualitative nature of the NSN requirement. A more contemporary view would be
that there is a specificity to this component of the total protein requirement. This introduces
an expanded perspective on the understanding of the underlying metabolic basis of the
total nitrogen requirement.
Two significant developments are relevant here. First, there is the proposal by
Jackson140 that the endogenous synthesis rate of glycine could be rate limiting, especially
in rapidly growing babies 141 or where the metabolic demand for glycine was
increased.142–144 Further, the hypothesis was that a preformed source of dietary glycine was
needed in these circumstances, although it was not directly tested to a sufficient extent
by these investigators. Nevertheless, glycine synthesis has been shown to be reduced when
N and dispensable amino acid intakes are low.145 Even at an adequate dietary N level, a
lower glycine intake results in an increased excretion of urinary L-oxoproline taken to be
an index of glycine adequacy.146 Also, low dietary glycine results in a lower rate of whole-
blood glutathione synthesis (Castillo and Young, unpublished MIT data), which is also
reduced when the sulfur amino acid intake is similarly restricted.147 This supports the
view that metabolic need for glycine might be most effectively met via a still-to-be-defined
minimum intake level of this amino acid.
1382_C27.fm Page 464 Tuesday, October 7, 2003 7:08 PM

464 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

A second development concerns the glutamate dehydrogenase reaction that, in theory,


can be a net route of fixation of ammonium ions. However, it is now considered unlikely
to play a quantitatively important role in this context because of the high Km for (NH4
(>1 mM).148 Thus, under normal circumstances ammonia levels would not be high enough
so that the direction of the reaction would favor release of the amino N from glutamate.
The other potential route of net ammonium ion entry into the amino acid pool is via the
glycine cleavage system with the formation of glycine and its subsequent conversion to
serine. This latter amino acid releases its nitrogen as ammonium via the serine dehydratase
reaction. The formation of serine occurs via the transamination (from glutamate) of 3-phos-
phoglycerate, and the predominant direction of the glycine cleavage reaction is in favor
of the degradation of glycine.149
Therefore, it can be hypothesized that the human subject requires a preformed source
of a-amino nitrogen in addition to that supplied by the indispensable amino acids.150
Under usual dietary conditions this would be met by the dispensable or conditionally
indispensable amino acids from food proteins. However, in experimental circumstances
it seems possible that glutamate alone or possibly glutamate plus glycine would serve as
an efficient source of a-amino nitrogen. The relative efficacy of these two sources in
comparison with other simple N-containing mixtures151 deserves careful study, possibly
using a 24-h IAAO approach.
We conclude that the total nitrogen requirement is effectively met through the provi-
sion of an appropriate intake level and balance of indispensable amino acids together with
an additional source of a-amino nitrogen, with glutamate possibly being the simplest,
effective form of this component. Based on our tentative mean requirement estimates for
the indispensable amino acids and assuming a mean total nitrogen requirement of 106 mg
of N·kg–1·day–1,41 intakes of about 20 and 86 mg of N·kg–1·day–1 of indispensable amino N
and of preformed a-amino N, respectively, would be sufficient to maintain body N homeo-
stasis in healthy adults.

References
1. Scrimshaw, N.S. and Young, V.R., The requirements of human nutrition, Sci. Am., 235, 50,
1976.
2. Munro, H.N., Evolution of protein metabolism in mammals, in Mammalian Protein Metabolism,
Vol. III, Munro, H.N., Ed., Academic Press, New York, 1959, chap. 25.
3. Millward, D.J., The nutritional value of plant-based diets in relation to human amino acid
and protein requirements, Proc. Nutr. Soc., 58, 249, 1999.
4. Young, V.R., Amino acid flux and requirements: counterpoint: tentative estimates are feasible
and necessary, in The Role of Protein and Amino Acids in Sustaining and Enhancing Performance,
Committee on Military Nutrition/Food and Nutrition Board/Institute of Medicine, Eds.,
National Academy Press, Washington, D.C., 1999, p. 217.
5. IOM/FNB (Institute of Medicine/Food and Nutrition Board), Dietary Reference Intakes: En-
ergy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein and Amino Acids, The National
Academy Press, Washington, D.C., 2002, chap. 10 (prepublication copy, unedited proofs).
6. Vickery, H.B. and Schmidt, C.L.A., The history of the discovery of the amino acids, Chem.
Rev., 9, 169, 1931.
7. Jorpes, J.E. and Berzelius, J., His Life and Work, Almquist and Wiksells Boktryckeri, A.B.,
Uppsala, Sweden, 1970, 256 pp.
8. Osborne, T.B., The Proteins of the Wheat Kernel, Carnegie Institute Publication 84, Washington,
D.C., 1970.
9. Block, R.R. and Mitchell, H.H., The correlation of the amino acid composition of proteins
with their nutritive value, Nutr. Abstr. Rev., 16, 249, 1946.
10. McCay, C.M., Notes on the History of Nutrition Research, Verzar, F., Ed., Hans Hubner Publish-
ers, Bern, Switzerland, 1973, 234 pp.
1382_C27.fm Page 465 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 465

11. Rose, W.C., The amino acid requirements of adult man, Nutr. Abstr. Rev., 27, 631, 1957.
12. Cox, G.J. and Rose, W.C., The availability of synthetic imidazoles in supplementing diets
deficient in histidine, J. Biol. Chem., 68, 781, 1926.
13. McCoy, R.H., Meyer, C.E., and Rose, W.C., Feeding experiments with mixtures of highly
purified amino acids. VIII. Isolation and identification of a new essential amino acid, J. Biol.
Chem., 112, 283, 1935–1936.
14. Meyer, C.E. and Rose, W.C., The spatial configuration of a-amino-b-hydroxy-n-butyric acid,
J. Biol. Chem., 115, 721, 1936.
15. Rose, W.C., Oesterling, M.J., and Womack, M., Comparative growth on diets containing ten
and nineteen amino acids, with further observations upon the role of glutamic and aspartic
acids, J. Biol. Chem., 176, 753, 1948.
16. Rose, W.C., Johnson, J.E., and Haines, W.J., The amino acid requirements of man. I. The role
of valine and methionine, J. Biol. Chem., 182, 541, 1950.
17. Rose, W.C., Haines, W.J., and Warner, D.T., The amino acid requirements of man. V. The role
of lysine, arginine and tryptophan, J. Biol. Chem., 206, 421, 1954.
18. Rogers, Q.R. and Harper, A.E., Amino acid diets and maximal growth in the rat, J. Nutr., 87,
267, 1965.
19. Young, V.R. and Zamora, J., Effects of altering the proportions of essential to nonessential
amino acids on growth and plasma amino acid levels in the rat, J. Nutr., 96, 21, 1968.
20. Ball, R.O., Atkinson, J.L., and Bailey, H.S., Proline as an essential amino acid for the young
pig, Br. J. Nutr., 55, 659, 1986.
21. Heger, J., Non-essential nitrogen and protein utilization in the growing rat, Br. J. Nutr., 64,
653, 1990.
22. Austic, R.E., Nutritional and metabolic interrelationships of arginine, glutamic acid and
proline in the chicken, Fed. Proc., 35, 1914, 1976.
23. FAO, Protein Requirements, Report of the FAO Committee, FAO Nutritional Studies 16, Food
and Agriculture Organization of the United Nations, Rome, October 24–31, 1955; 1957, 52 pp.
24. Rose, W.C. et al., The amino acid requirements of man. XV. The valine requirement: summary
and final observations, J. Biol. Chem., 217, 987, 1955.
25. FAO/WHO, Protein Requirements, Report of a Joint FAO/WHO Expert Group, FAO Nutrition
Meetings Report Series 37, Food and Agriculture Organization of the United Nations, Rome,
1965, 69 pp.
26. FAO/WHO, Energy and Protein Requirements, Report of a Joint FAO/WHO Ad Hoc Commit-
tee, Report Series 522, World Health Organization, Geneva, 1973, 118 pp.
27. FAO/WHO/UNU, Energy and Protein Requirements, Report of a Joint FAO/WHO/UNU
Expert Consultation, Technical Report Series 724, World Health Organization, Geneva, 1985,
206 pp.
28. Holt, L.E., Jr. and Snyderman, S.E., Protein and amino acid requirements of infants and
children, Nutr. Abstr. Rev., 35, 1, 1965.
29. Laidlaw, S.A. and Kopple, J.D., New concepts of the indispensable amino acids, Am. J. Clin.
Nutr., 46, 593, 1987.
30. Chipponi, J.X. et al., Deficiencies of essential and conditionally essential nutrients, Am. J.
Clin. Nutr., 35, 1112, 1982.
31. Jackson, A.A., Amino acids: essential and non-essential? Lancet, i, 1034, 1983.
32. Young, V.R. and El-Khoury, A.E., The notion of the nutritional essentiality of amino acids,
revisited, with a note on the indispensable amino acid requirements in adults, in Amino Acid
Metabolism and Therapy in Health and Nutritional Disease, Cynober, L.A., Ed., CRC Press, Boca
Raton, FL, 1995, p. 191.
33. Irwin, M.I. and Hegsted, D.M., A conspectus of research on amino acid requirements of man,
J. Nutr., 101, 539, 1971.
34. Hegsted, D.M., Variation in requirements of nutrients: amino acids, Fed. Proc., 22, 1424, 1963.
35. Manatt, M.W. and Garcia, P.A., Nitrogen balance: concepts and techniques, in Modern Methods
in Protein Nutrition Metabolism, Nissen, S., Ed., Academic Press, San Diego, 1992, p. 9.
36. Rand, W.M. and Young, V.R., Statistical analysis of N balance data with reference to the
lysine requirement in adults, J. Nutr., 129, 1920, 1999.
1382_C27.fm Page 466 Tuesday, October 7, 2003 7:08 PM

466 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

37. Jones, E.M. Bauman, C.A., and Reynolds, M.S., Nitrogen balances of women maintained on
various levels of lysine, J. Nutr., 60, 549, 1956.
38. Calloway, D.H., Odell, A.C.F., and Margen, S., Sweat and miscellaneous nitrogen losses in
human balance studies, J. Nutr., 101, 775, 1971.
39. Zanni, E., Calloway, D.H., and Zezulka, A.Y., Protein requirements of elderly men, J. Nutr.,
109, 513, 1979.
40. Millward, D.J. and Roberts, S.B., Protein requirements for older individuals, Nutr. Res. Rev.,
9, 67, 1996.
41. Rand, W.R., Pellett, P.L., and Young, V.R., A meta-analysis of nitrogen balance studies for
estimating protein requirements in healthy adults, Am. J. Clin. Nutr., 77, 109, 2003.
42. Young, V.R. and Marchini, J.S., Mechanisms and nutritional significance of metabolic re-
sponses to altered adaptation in humans, Am. J. Clin. Nutr., 51, 270, 1990.
43. Young, V.R., Bier, D.M., and Pellett, P.L., A theoretical basis for increasing current estimates
of the amino acid requirements in adult man, with experimental support, Am. J. Clin. Nutr.,
50, 80, 1989.
44. Young, V.R., McCollum Award Lecture: kinetics of human amino acid metabolism: nutri-
tional implications and some lessons, Am. J. Clin. Nutr., 46, 709, 1987.
45. Rand, W.M., Young, V.R., and Scrimshaw, N.S., Change of urinary nitrogen excretion in
response to low-protein diets in adults, Am. J. Clin. Nutr., 29, 639, 1976.
46. Rand, W.M., Scrimshaw, N.S., and Young, V.R., An analysis of temporal patterns in urinary
nitrogen excretion of young adults receiving constant diets at two nitrogen intakes for 8 to
11 weeks, Am. J. Clin. Nutr., 32, 1408, 1979.
47. Rand, W.M., Scrimshaw, N.S., and Young, V.R., A retrospective analysis of long term meta-
bolic balance studies: implications for understanding dietary nitrogen and energy utilization,
Am. J. Clin. Nutr., 42, 1329, 1985.
48. Quevedo, M.R. et al., Nitrogen homeostasis in man: diurnal changes in nitrogen excretion,
leucine oxidation and whole body leucine kinetics during a reduction from a high to a
moderate protein intake, Clin. Sci., 86, 185, 1994.
49. Munro, H.H., Free amino acid pools and their role in regulation, in Mammalian Protein
Metabolism, Vol. IV, Munro, N.H., Ed., Academic Press, New York, 1970, p. 299.
50. Young, V.R. and Scrimshaw, N.S., The nutritional significance of plasma and urinary amino
acids, in Protein and Amino Acid Functions, Bigwood, E.J., Ed., Pergamon Press, New York,
1972, chap. 11.
51. Moundras, C., Remesy, C., and Demigne, C., Dietary protein paradox: decrease of amino
acid availability induced by high protein diets, Am. J. Physiol., 264, G1057, 1993.
52. Forslund, A.H. et al., Inverse relationship between protein intake and plasma free amino
acids in healthy men at physical exercise, Am. J. Physiol., 278, E857, 2000.
53. Wurtman, R.J., Diurnal rhythms in mammalian protein metabolism, in Mammalian Protein
Metabolism, Munro, H.N., Ed., Academic Press, New York, 1970, chap. 36.
54. Fernstrom, J.D. et al., Diurnal variations in plasma concentrations of tryptophan, tyrosine
and other neutral amino acids: effect of dietary protein intake, Am. J. Clin. Nutr., 32, 1912,
1979.
55. Hussein, M.A. et al., Daily fluctuations of plasma amino acids in adult men: effect of tryp-
tophan intake and distribution of meals, J. Nutr., 101, 61, 1971.
56. Young, V.R. et al., Tryptophan intake, spacing of meals and diurnal fluctuations of plasma
tryptophan in men, Am. J. Clin. Nutr., 22, 1563, 1969.
57. Harper, A.E., Benevenga, N.J., and Wohlhueter, R.M., Effects of injection of disproportionate
amounts of amino acids, Physiol. Rev., 50, 428, 1970.
58. Pion, R., The relationship between the levels of free amino acids in blood and muscle and
the nutritive value of proteins, in Protein in Human Nutrition, Porter, J.W.G. and Rolls, B.A.,
Eds., Academic Press, New York, 1973, p. 392.
59. Young, V.R. et al., Plasma tryptophan response curve and its relation to tryptophan require-
ments in young men, J. Nutr., 101, 45, 1971.
60. Tontisirin, K. et al., Plasma tryptophan response curve and tryptophan requirements of
elderly people, J. Nutr., 103, 1220, 1973.
1382_C27.fm Page 467 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 467

61. Tontisirin, K. et al., Plasma threonine response curve and threonine requirements of young
men and elderly women, J. Nutr., 104, 495, 1974.
62. Young, V.R. et al., Plasma amino acid response curve and amino acid requirements in young
men: valine and lysine, J. Nutr., 102, 1159, 1972.
63. Zello, G.A., Pencharz, P.B., and Ball, R.O., Phenylalanine flux, oxidation and conversion to
tyrosine in human studies with L-[1-13C]phenylalanine, Am. J. Physiol., 259, E835, 1990.
64. Zello, G.A. et al., Recent advances in method of assessing dietary amino acid requirements
for adult humans, J. Nutr., 125, 2907, 1995.
65. Millward, D.J. et al., Human adult amino acid requirements: [1-13C]leucine balance evaluation
of the efficiency of utilization and apparent requirements for wheat protein and lysine
compared with those for milk protein in healthy adults, Am. J. Clin. Nutr., 72, 112, 2000.
66. Millward, D.J., Fereday, A., Gibson, N.R., Cox, M.C., and Pacy, P.J., Efficiency of utilization
of wheat and milk protein in healthy adults and apparent lysine requirements determined
by a single-meal [1-13]leucine balance protocol, Am. J. Clin. Nutr., 76, 1326, 2002.
67. Millward, D.J., Methodological issues, Proc. Nutr. Soc., 60, 3, 2001.
68. El-Khoury, A.E. et al., Validation of the tracer-balance concept with reference to leucine: 24
hour intravenous tracer studies with L-(1-13C)leucine and (15N-15N)urea, Am. J. Clin. Nutr.,
59, 1000, 1994.
69. MacCross, M.J., Fukagawa, N.K., and Matthews, D.E., Measurement of homocysteine con-
centrations and stable isotope tracer enrichments in human plasma, Anal. Chem., 71, 4527,
1999.
70. Kurpad, A.V. et al., Intravenously infused 13C-leucine retained in fasting healthy adult men,
J. Nutr., 132, 1906, 2002.
71. Meguid, M.M. et al., Leucine kinetics at graded leucine intakes in young men, Am. J. Clin.
Nutr., 43, 770, 1986.
72. Meguid, M.M. et al., Valine kinetics at graded valine intakes in young men, Am. J. Clin. Nutr.,
43, 781, 1986.
73. Meredith, C.N. et al., Lysine kinetics at graded lysine intakes in young men, Am. J. Clin.
Nutr., 43 787, 1986.
74. Zhao, X.-H. et al., Threonine kinetics at graded threonine intakes in young men, Am. J. Clin.
Nutr., 43, 795, 1986.
75. El-Khoury, A.E. et al., The 24 hour pattern and rate of leucine oxidation, with particular
reference to tracer estimates of leucine requirements in healthy adults, Am. J. Clin. Nutr., 59,
1012, 1994.
76. Sánchez, M. et al., Phenylalanine and tyrosine kinetics in young men throughout a contin-
uous 24-h period, at a low phenylalanine intake, Am. J. Clin. Nutr., 61, 555, 1995.
77. Kurpad, A.V. et al., Daily requirement for and splanchnic uptake of leucine in adult healthy
Indians, Am. J. Clin. Nutr., 74, 747, 2001.
78. Kim, K.-I., McMillan, I., and Bayley, H.S., Determination of amino acid requirements of young
pigs using an indicator amino acid, Br. J. Nutr., 50, 369, 1983.
79. Zello, G.A., Pencharz, P.B., and Ball, R.O., Dietary lysine requirement of young adult males
determined by oxidation of L-[1-13C]phenylalanine, Am. J. Physiol., 264, E677, 1993.
80. Lazanis-Brunner, G. et al., Tryptophan requirement in young adult women as determined
by indicator amino acid oxidation with L-[13C]phenylalanine, Am. J. Clin. Nutr., 68, 303, 1998.
81. Wilson, D.C. et al., Threonine requirement of young men determined by indicator amino
acid oxidation with use of L-[1-13C]phenylalanine, Am. J. Clin. Nutr., 71, 757, 2000.
82. Di Buono, M. et al., Total sulfur amino acid requirement in young men as determined by
indicator amino acid oxidation with L-[1-13C]phenylalanine, Am. J. Clin. Nutr., 74, 756, 2001.
83. Di Buono, M. et al., Dietary cysteine reduces the methionine requirement in men, Am. J. Clin.
Nutr., 74, 761, 2001.
84. Duncan, A.M., Ball, R.O., and Pencharz, P.B., Lysine requirement of adult males is not affected
by decreasing dietary protein, Am. J. Clin. Nutr., 64, 718, 1996.
85. Kriengsinyos, W. et al., Oral and intravenous tracer protocols of the indicator amino acid
oxidation provide the same estimate of the lysine requirements in healthy men, J. Nutr., 132,
2251, 2002.
1382_C27.fm Page 468 Tuesday, October 7, 2003 7:08 PM

468 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

86. Roberts, S.B. et al., Tyrosine requirement of healthy men receiving a fixed phenylalanine
intake determined by using indicator amino acid oxidation, Am. J. Clin. Nutr., 73, 276, 2001.
87. Millward, D.J., Metabolic demands for amino acids and the human dietary requirement:
Millward and Rivers (1988) revisited, J. Nutr., 128, 2563S, 1998.
88. Young, V.R., Nutrient interactions with reference to amino acid and protein metabolism in
non-ruminants: particular emphasis on protein-energy relationships in man, Z. Ernahrungs-
weiss, 30, 239, 1991.
89. Kurpad, A.V. et al., An initial assessment, using 24 hour 13C-leucine kinetics, of the lysine
requirement of healthy adult Indian subjects, Am. J. Clin. Nutr., 67, 58, 1998.
90. Kurpad, A.V. et al., Lysine requirements of healthy adult Indian subjects, measured by an
indicator amino acid balance technique, Am. J. Clin. Nutr., 73, 900, 2001.
91. Kurpad, A.V. et al., Lysine requirements of healthy adult Indian subjects receiving longer
term feeding, measured with a 24h indicator amino acid oxidation and balance technique,
Am. J. Clin. Nutr., 76, 404, 2002.
92. Borgonha, S. et al., Threonine requirement of healthy adults, derived with a 24h indicator
amino acid balance technique, Am. J. Clin. Nutr., 75, 698, 2002.
93. Kurpad, A.V. et al., Threonine requirements of healthy Indian men, measured by a 24h
indicator amino acid oxidation and balance technique, Am. J. Clin. Nutr., 76, 789, 2002.
94. Kurpad, A.V. et al., Daily methionine requirements of healthy adult Indian men measured
by a 24h indicator amino acid oxidation and balance approach, Am. J. Clin. Nutr., 77, 1198,
2003.
95. El-Khoury, A.E. et al., The 24 hour kinetics of leucine oxidation in healthy adults receiving
a generous leucine intake via three discrete meals, Am. J. Clin. Nutr., 62, 579, 1995.
96. Raguso, C., El-Khoury, A.E., and Young, V.R., Leucine kinetics in reference to the effect of
the feeding mode as three discrete meals, Metabolism, 48, 1378, 1999.
97. Young, V.R., Yu, Y.-M., and Borgonha, S., Proteins, peptides and amino acids in enteral
nutrition: overview and some research challenges, in Proteins, Peptides and Amino Acids in
Enteral Nutrition, Fürst, P. and Young, V.R., Eds., S. Karger AG, Basel, Switzerland, 2000, p. 1.
98. Kurpad, A.V. and Young, V.R., What is apparent is not always real: lessons from lysine
requirement studies in adult humans, J. Nutr., 133, 1227, 2003.
99. Young, V.R. and El-Khoury, A.E., Human amino acid requirements: a reevaluation, Food Nutr.
Bull., 17, 191, 1996.
100. Young, V.R. and El-Khoury, A.E., Can amino acid requirements for nutritional maintenance
in adult humans be approximated from the amino acid composition of body mixed proteins?
Proc. Natl. Acad. Sci. U.S.A., 92, 300, 1995.
101. Fuller, M.F. et al., Amino acid losses in ileostomy fluid on a protein-free diet, Am. J. Clin.
Nutr., 59, 70, 1994.
102. Waterlow, J.C., The requirements of adult man for indispensable amino acids, Eur. J. Clin.
Nutr., 50, S151, 1996.
103. Millward, D.J. et al., Maintenance protein requirements: the need for conceptual re-evalua-
tion, Proc. Nutr. Soc., 49, 473, 1990.
104. Reeds, P.J., Dispensable and indispensable amino acids for humans, J. Nutr., 130, 1835S, 2000.
105. Rose, W.C. et al., The amino acid requirements of man. X. The lysine requirement, J. Biol.
Chem., 214, 579, 1955.
106. Fisher, H., Brush, M.K., and Griminger, P., Reassessment of amino acid requirements of young
women on low nitrogen diets. 1. Lysine and tryptophan, Am. J. Clin. Nutr., 22, 1190, 1969.
107. El-Khoury, A.E. et al., Twenty-four hour intravenous and oral tracer studies with L-[1-13C]-
2-aminoadipic acid and L-(1-13C)lysine as tracers at generous nitrogen and lysine intakes in
healthy adults, Am. J. Clin. Nutr., 68, 827, 1998.
108. El-Khoury, A.E. et al., Twenty-four hour oral studies with L-[1-13C][lysine at a low (15
mg·kg–1·d–1) and intermediate (29 mg·kg–1·d–1)lysine intake in healthy adults, Am. J. Clin.
Nutr., 72, 122, 2000.
109. Rose, W.C. et al., The amino acid requirements of man. IX. The phenylalanine requirement,
J. Biol. Chem., 213, 913, 1955.
1382_C27.fm Page 469 Tuesday, October 7, 2003 7:08 PM

Chapter twenty-seven: Amino acid requirements in adults 469

110. Tolbert, B. and Watts, J.H., Phenylalanine requirement of women consuming a minimal
tyrosine diet and the sparing effect of tyrosine on the phenylalanine requirement, J. Nutr.,
80, 111, 1980.
111. Leverton, R.M. et al., The quantitative amino acid requirements of young women. IV.
Phenylalanine, with and without tyrosine, J. Nutr., 58, 341, 1956.
112. Basile-Filho, A. et al., Twenty-four hour L-[1-13C]tyrosine and L-[3,3-2H3]phenylalanine oral
tracer studies at generous, intermediate and low phenylalanine intakes to estimate aromatic
amino acid requirements in adults, Am. J. Clin. Nutr., 67, 640, 1998.
113. Basile-Filho, A. et al., Continuous twenty-four hour L-1-13C]phenylalanine and L-[3,3-2H2]ty-
rosine oral tracer studies at an “intermediate” phenylalanine intake, to estimate requirements
in adults, Am. J. Clin. Nutr., 65, 473, 1997.
114. Sánchez, M. et al., Twenty-four hour intravenous and oral studies with L-[1-13C]phenylalanine
and L-[3-3-2H2]tyrosine at a tyrosine-free generous phenylalanine intake in adults, Am. J. Clin.
Nutr., 63, 532, 1996.
115. Rose, W.C. and Wixom, R.L., The amino acid requirements of man. XIV. The sparing effect
of tyrosine on the phenylalanine requirement, J. Biol. Chem., 217, 95, 1955.
116. Davis, T.A., Fiorotto, M.L., and Reeds, P.J., Amino acid compositions of body and milk protein
change during the suckling period in rats, J. Nutr., 123, 947, 1993.
117. Kindt, E. and Halvorsen, A., The need for essential amino acids in children: an evaluation
based on the intake of phenylalanine, tyrosine, leucine, isoleucine and valine in children
with phenylketonurea, tyrosine amino transferase defect and maple syrup urine disease,
Am. J. Clin. Nutr., 33, 279, 1980.
118. Rose, W.C. et al., The amino acid requirements of man. XI. The threonine and methionine
requirements, J. Biol. Chem., 215, 101, 1955.
119. Leverton, R.M. et al., The quantitative amino acid requirements of young women. I. Threo-
nine, J. Nutr., 58, 59, 1956.
120. Fisher, H., Brush, M.K., and Griminger, P., Reassessment of amino acid requirements of young
women on low nitrogen diets. III. Isoleucine, threonine, phenylalanine and summation, Am.
J. Clin. Nutr., 27, 130, 1974.
121. Rose, W.C. et al., The amino acid requirements of man. XII. The leucine and isoleucine
requirements, J. Biol. Chem., 216, 225, 1955.
122. Leverton, R.M. et al., The quantitative amino acid requirements of young women. V. Leucine,
J. Nutr., 58, 355, 1956.
123. Rose, W.C. et al., The amino acid requirements of man. XV. The valine requirement: summary
and final observations, J. Biol. Chem., 217, 987, 1955.
124. Leverton, R.M. et al., The quantitative amino acid requirements of young women. II. Valine,
J. Nutr., 58, 83, 1956.
125. Swendseid, M.E. and Dunn, M.S., Amino acid requirements of young women based on
nitrogen balance data. II. Studies on isoleucine and on minimum amounts of eight essential
amino acids fed simultaneously, J. Nutr., 58, 507, 1956.
126. Rose, W.C., Lambert, G.F., and Coon, M.J., The amino acid requirements of man. VII. General
procedures: the tryptophan requirement, J. Biol. Chem., 211, 815, 1954.
127. Leverton, R.M. et al., The quantitative amino acid requirements of young women. III. Tryp-
tophan, J. Nutr., 58, 219, 1956.
128. Swendseid, M.E., Williams, I., and Dunn, M.S., Amino acid requirements in young women
based on nitrogen balance data. I. The sulfur-containing amino acid, J. Nutr., 58, 495, 1956.
129. Reynolds, M.S. et al., Nitrogen balances of women maintained on various levels of methion-
ine and cystine, J. Nutr., 64, 99, 1958.
130. Young, V.R. et al., Methionine kinetics and balance at the 1985 FAO/WHO/UNU.I. Intake
requirement in adult men studied with L-[2H3-methyl-1-13C]methionine as tracer, Am. J. Clin.
Nutr., 54, 377, 1991.
131. Hiramatsu, T. et al., Methionine and cysteine kinetics at different intakes of cystine in healthy
adult men, Am. J. Clin. Nutr., 60, 525, 1994.
132. Fukagawa, N.K., Yu, Y.-M., and Young, V.R., Methionine and cysteine kinetics at different
intakes of methionine and cystine in elderly men and women, Am. J. Clin. Nutr., 68, 380, 1998.
1382_C27.fm Page 470 Tuesday, October 7, 2003 7:08 PM

470 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

133. Raguso, C. et al., Effect of cystine intake on methionine kinetics and oxidation, using oral
tracers of methionine and cystine in healthy adults, Am. J. Clin. Nutr., 66, 283, 1997.
134. Raguso, C., Regan, M.M., and Young, V.R., Cysteine kinetics and oxidation at different intakes
of methionine and cystine in young adults, Am. J. Clin. Nutr., 71, 491, 2000.
135. Rose, W.C. and Wixom, R.L., The amino acid requirements of man. XII. The sparing effect
of cystine on the methionine requirement, J. Biol. Chem., 216, 763, 1955.
136. Storch, K.J. et al., [1-13C; methyl-2H3]methionine kinetics in humans: methionine conservation
and cystine sparing, Am. J. Physiol., 258, E790, 1990.
137. Kopple, J.D. and Swendseid, M.E., Evidence that histidine is an essential amino acid in
normal and chronically uremic man, J. Clin. Invest., 55, 881, 1975.
138. Kriengsinyos, W. et al., Long-term effects of histidine depletion on whole-body protein
metabolism in healthy adults, J. Nutr., 132, 3340, 2002.
139. Young, V.R. and Borgonha, S., Nitrogen and amino acid requirements: the Massachusetts
Institute of Technology amino acid requirement pattern, J. Nutr., 130, 1841S, 2000.
140. Jackson, A.A., The glycine story, Eur. J. Clin. Nutr., 45, 59, 1991.
141. Jackson, A.A., Show, J.C., and Barber, A., Nitrogen metabolism in pre-term infants fed human
donor breast milk: the possible essentiality of glycine, Pediatr. Res., 15, 1454, 1981.
142. Jackson, A.A. et al., Urinary excretion of 5-oxoproline (pyroglutamic aciduria) as an index
of glycine insufficiency in normal man, Br. J. Nutr., 58, 207, 1987.
143. Meakins, T.S., Persaud, C., and Jackson, A.A., Dietary supplementation with L-methionine
impairs the utilization of urea-nitrogen and increases 5-L-oxoprolinurea in normal women
consuming a low protein diet, J. Nutr., 128, 720, 1998.
144. Persaud, C., Forrester, T., and Jackson, A.A., Urinary excretion of 5-L-oxoproline (pyroglutam-
ic acid) is increased during recovery from severe childhood malnutrition and responds to
supplemental glycine, J. Nutr., 126, 2823, 1996.
145. Yu, Y.-M. et al., Quantitative aspects of glycine and alanine metabolism in post absorptive
young men: effects of level of nitrogen and dispensable amino acid intake, J. Nutr., 115, 399,
1985.
146. Metges, C.C. et al., Oxoproline kinetics and oxoproline excretion during glycine- or sulfur
amino acid-free diets in adults, Am. J. Physiol., 278, E868, 2000.
147. Lyons, J. et al., Blood glutathione synthesis rates in healthy adults receiving a sulfur amino
acid-free diet, Proc. Natl. Acad. Sci. U.S.A., 97, 5071, 2000.
148. Katagiri, M. and Nakamura, M., Is there really any evidence indicating that animals synthe-
size glutamate? Biochem. Educ., 27, 83, 1999.
149. Kikuchi, G., The glycine cleavage system: composition, reaction mechanism and physiolog-
ical significance. Mol. Cell. Biochem., 1, 169, 1973.
150. Katagiri, M. and Naramura, M., Animals are dependent on preformed alpha-amino nitrogen
as an essential nutrient, IUBMB Life, 53, 125, 2002.
151. Jackson, A.A., Nitrogen trafficking and recycling throughout the human bowel, in Proteins,
Peptides and Amino Acids in Enteral Nutrition, Fürst, P. and Young, V.R., Eds., S. Karger A.G.,
Basel, Switzerland, 2000, p. 89.
1382_C28.fm Page 471 Tuesday, October 7, 2003 7:12 PM

chapter twenty-eight

Neonatal requirements
for amino acids
David K. Rassin
The University of Texas Medical Branch at Galveston

Contents
Introduction..................................................................................................................................471
28.1 Amino acids and development........................................................................................473
28.2 Amino acids in human milk, formulas, and parenteral solutions..............................474
28.3 Enteral vs. parenteral nutrition: tyrosine and phenylalanine .....................................478
28.4 Conclusion: amino acids and behavior...........................................................................479
Acknowledgment ........................................................................................................................479
References .....................................................................................................................................480

Introduction
Concepts of neonatal requirements for amino acids have changed as a greater understand-
ing of biochemical and physiologic development has occurred over the last 30 years. In
particular, recently more attention has been given to the role of nutrients in cognitive
development. This emphasis has been stimulated by the numerous studies that have
demonstrated improved cognitive performance in breastfed infants; such performance has
been noted to persist into adulthood.1 It has been suggested that the mechanism by which
such improved function may occur may reflect the differential socioeconomic status of
breastfed babies, the bonding between mother and infant, and the effect of long-chain
polyunsaturated fatty acids, especially docosahexaenoic and arachidonic acid, found in
human milk. In this review the basis for amino acids playing a role in this function has
been laid. It is clear that the amino acid biochemical milieu of the breastfed infant is
different than that of the formula-fed infant, and the mechanism for such a difference to
be reflected in cognitive development exists.2
The background for the recommended protein intakes of infants has been extensively
reviewed.3,4 For the term infant, human milk remains the standard,3 while for the preterm
infant there is still no clear standard.4 Protein, the macromolecular carrier of amino acids,
is still the nutrient that receives primary attention rather than the individual amino acids
with respect to neonatal needs. The consensus of various reports that have addressed

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 471
1382_C28.fm Page 472 Tuesday, October 7, 2003 7:12 PM

472 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

neonatal protein requirements (Recommended Dietary Allowances,5 The Food and Agricul-
ture Organization of the World Health Organization,6,7 The European Society for Pediatric
Gastroenterology and Nutrition,8–10 and the Committee on Nutrition of the American
Academy of Pediatrics11) appears to be that term infants need 2.0 to 2.2 g of protein per
kilogram of body weight per day for the first 4 months of life, declining by about
0.2 g/kg/day after 4 months, and that preterm infants need about 1 g/kg/day more
protein than term neonates. All these recommendations assume that a complete protein
preparation is being used, one that contains sufficient amounts of the amino acids that
have classically been defined as essential.
Further, although all these groups recommend breastfeeding as the first choice for the
healthy term neonate, they do not address the paradox that human milk probably does
not fall within their recommended guidelines for protein intake (based upon the fact that
human milk generally contains about 1 g of protein per decaliter of milk and would have
to be fed at a rate of approximately 200 ml/kg/day to supply 2 g/kg/day of protein).
This potential deficiency becomes even more apparently acute if one subtracts all the
proposed biologically active proteins from the nutritionally available pool. However, it is
clear that term infants efficiently use the proteins of their own species to support optimal
growth and development.
The original classification of amino acids as essential or nonessential was based upon
the requirement for maintenance of growth and nitrogen balance. This classification then
came to be modified as new understanding developed of the functions of amino acids as
well as their biochemistry. Thus, a variety of categories of essentiality have emerged and
can be compiled as follows:12–14 nonessential amino acids, such as glutamate and aspartate,
that can be completely synthesized; essential carbon skeleton amino acids, such as valine
and leucine, that have a carbon skeleton that can be aminated but not synthesized; semi-
essential amino acids, such as glycine and serine, that can undergo carbon skeleton syn-
thesis but cannot be aminated; and essential amino acids, such as lysine, that have a carbon
skeleton that cannot be synthesized or aminated.
Additional categories include genetically required amino acids, such as tyrosine in
phenylketonuria or cysteine in homocystinuria, that cannot be synthesized due to an
inherited metabolic defect; disease-induced essential amino acids, such as the branched-
chain amino acids in hepatic dysfunction, that cannot be synthesized due to a disease not
specifically related to amino acid metabolism; nutritionally induced requirements, such
as arginine during total parenteral nutrition administration, that appear to be required
due to special nutritional circumstances; and developmentally required amino acids, such
as cysteine in the premature infant, that cannot be sufficiently synthesized due to the
biochemical immaturity of the neonate.
The net result of this increased understanding of neonatal requirements for amino
acids is that almost all, if not all, the amino acids probably ought to be provided in the
diet to support optimal growth and development. Even those amino acids that are not
considered essential or semiessential ought to be included in the diet to make sure that a
properly balanced intake occurs. Those amino acids that are still classified as nonessential
may only be so categorized because all their functions are not yet completely understood.
In those situations in which some nonessential amino acids have been omitted from the
diet of neonates, it has usually been due to concerns regarding toxicity; for example,
glutamate is usually not included in parenteral nutrition solutions due to potential neu-
rotoxicity.15 This consideration raises the issue that when discussions of requirements take
place the complete spectrum of intake ought to be considered, from deficiency to suffi-
ciency to excess. Potential excess of amino acids causing toxicity may be as problematic
as a deficiency, particularly in the vulnerable developing human being.
1382_C28.fm Page 473 Tuesday, October 7, 2003 7:12 PM

Chapter twenty-eight: Neonatal requirements for amino acids 473

In the following discussion, emphasis will be on the role of amino acids in early
biochemical development or, as in the category described previously, as developmentally
required.

28.1 Amino acids and development


One of the first suggestions that acceptance of the original list of essential amino acids
was not sufficient for neonates emerged as a result of studies by Snyderman and col-
leagues.16,17 These investigators evaluated growth, nitrogen retention, and plasma proteins
in infants receiving formulas prepared with individual amino acids deleted. By these
parameters, cysteine and histidine appeared to be essential for the neonate, because growth
and nitrogen retention decreased when they were each removed from the diet.16,17 Histi-
dine depletion was also associated with a rash that appeared to reflect malnutrition.16
Similar studies with arginine did not result in responses that supported a requirement for
this amino acid.18 Thus, by the classical criteria of growth and nitrogen balance, histidine
and cysteine appeared to be developmentally essential.
The next stage in refining neonatal amino acid requirements emerged as an improved
understanding of biochemical development was achieved. Three metabolic pathways in
particular appear to undergo developmental changes that impact neonatal requirements
— those that involve the aromatic amino acids, the sulfur-containing amino acids, and
the urea cycle.
Phenylalanine is the precursor for tyrosine in a reaction catalyzed by phenylalanine
hydroxylase. Tyrosine is catabolized to its a-keto acid analog in a reaction dependent upon
tyrosine aminotransferase. The p-hydroxyphenylpyruvic acid formed is then broken down
by a series of oxidation steps. In evaluating the capacity of this pathway to function in
the neonate, it was found that phenylalanine hydroxylase functions in fetal liver at about
59% of the activity found in adult liver, while tyrosine aminotransferase functions at about
7% and the oxidizing system at about 8% of adult activity.19–21 Thus, the infant has less
capacity to synthesize tyrosine than the adult, but perhaps more important, the infant has
an extremely limited capacity to catabolize tyrosine.
The lack of adequate capacity to further metabolize tyrosine may be directly respon-
sible for the cases of transient tyrosinemia that have been documented in neonates.22–24 At
one time, this biochemical response was believed to reflect an increased neonatal require-
ment for ascorbic acid, but this explanation no longer appears to be correct.23–25 Rather,
neonatal tyrosinemia is a direct reflection of the type of protein fed to neonates, particularly
proteins with a high content of aromatic amino acids such as the cow milk caseins.26,27 The
fact that tyrosinemia is observed rather than hyperphenylalaninemia (or that plasma
tyrosine is more dramatic in its response than plasma phenylalanine) implies that a
relatively adequate capacity exists to synthesize tyrosine, but that the capacity to break-
down tyrosine is severely limited.26 As will be discussed later, this situation may vary
depending upon the route of administration of amino acids.
The urea cycle represents a critical metabolic process for the removal of nitrogen waste
products that result from protein catabolism. While all the enzymes responsible for cata-
lyzing the steps in this pathway are present in the liver of the fetus,28,29 the fact that preterm
infants fed arginine-free total parenteral nutrition develop hyperammonemia implies that
the cycle is not completely active.30 While ornithine transcarbamylase, argininosuccinase,
and arginase in fetal liver have 31, 17, and 46% of adult liver activity, respectively, argin-
inosuccinate lyase only has 2% of adult activity.28,29 Supplementation with arginine appears
to successfully compensate for the effects of this metabolic block; however, citrulline only
has a limited effect, and ornithine has no protective effect against the hyperammonemia
induced by limited dietary arginine.31 The metabolic dependency on arginine manifested
1382_C28.fm Page 474 Tuesday, October 7, 2003 7:12 PM

474 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

in the hyperammonemia described previously was not reflected in changes in nitrogen


balance or growth in the studies performed by Snyderman et al.18 Thus, dependency on
arginine may be better measured by protection from the biochemical insult of excess
ammonia than by the classical measures of amino acid requirements, growth, and nitrogen
balance. Also, just as there may be adverse effects from excess tyrosine, excess arginine
may be associated with pancreatic toxicity.32 Parenteral solutions need to have arginine in
them, but as is usually true in nutrition, more is not necessarily better.
The sulfur amino acids have been of particular interest to those investigating the
nutrient needs of newborns since the early work of Snyderman et al.,16 which showed
growth slow down when cysteine was omitted from the diet. This work fit nicely with
the finding that no activity (indeed no protein) could be identified in fetal liver for
cystathionase, the enzyme responsible for catalyzing cysteine synthesis from cystathion-
ine.33,34 These findings have prompted suggestions that cysteine is essential for the neo-
nate.35 Cystathionase can be identified in other fetal tissues,36 but the liver appears to be
the major site of cysteine synthesis.
Other enzymes in the methionine metabolic pathway are also reduced in the fetal liver
compared to that of the adult. Cystathionine b-synthase, the enzyme that catalyzes the
synthesis of cystathionine from homocysteine, has about 1.5% of adult activity, and
methionine adenosyltransferase, the enzyme that catalyzes S-adenosylmethionine forma-
tion from methionine, has about 30% of adult activity.33 These data may be interpreted to
mean that the fetus primarily remethylates methionine (methionine to S-adenosylmethion-
ine to S-adenosylhomocysteine to homocysteine to methionine), while early in neonatal
life the synthesis of cysteine and taurine (from homocysteine via cystathionine) begins to
take place. Thus, at the least, the premature infant may require cysteine for protein
synthesis.
Taurine, on the other hand, is not well synthesized by the human at any stage during
development. Although cysteine sulfinic acid decarboxylase (the enzyme that catalyzes
taurine synthesis via hypotaurine) activity is present in fetal liver at 81% of the activity
of adult liver, this activity is astonishingly low compared to all other species, including
the cat, which is known to have a requirement for dietary taurine.37 Taurine is ubiquitous
in most animal tissues and therefore is present in the diet of most humans. However, until
the mid 1980s, infant formulas did not contain this amino acid, and infants fed such
formulas became taurine deficient, as measured by their plasma concentrations of this
amino acid compared to infants fed human milk, which contains ample taurine.38 Although
taurine is not a constituent of proteins, it appears to have important functions in the
neonate and must be supplied in the diet.
Each of the biochemical pathways mentioned previously demonstrates steps that are
biochemically immature in the fetus, preterm infant, and term infant. These developmental
aspects of neonatal biochemistry result in the responses to various feeding regimens
discussed in the following section.

28.2 Amino acids in human milk, formulas, and parenteral solutions


The findings described previously regarding the potential for certain amino acids to be
essential during development due to the immature biochemical status of the neonate led
to a number of investigations of the effect of various feeding regimens on the newborn
infant. The first studies26,37,39,40 were designed to explore the role of cysteine nutrition in
the neonate because of the relatively low amount of cysteine contained in casein protein-
predominant formulas compared to human milk proteins (Figure 28.1).
An examination of the amino acid composition of the two commercial protein sources
most frequently used to feed neonates (casein protein and whey protein from cow milk)
1382_C28.fm Page 475 Tuesday, October 7, 2003 7:12 PM

Chapter twenty-eight: Neonatal requirements for amino acids 475

Figure 28.1 The amino acids supplied by the protein in either a casein protein (open symbols)- or
a whey protein (closed symbols)-predominant formula expressed either as a percent of the amount
supplied by an equal amount of human milk protein (circles) or as a percent of the amount of human
milk protein corrected to actual daily protein intakes (triangles), which were 2.7 g/kg/day for the
formulas and 1.54 g/kg/day for human milk. (Data from Rassin, D.K. et al., Pediatrics, 90, 356, 1977;
Järvenpää, A.-L. et al., Pediatrics, 70, 221, 1982; Gaull, G.E. et al., J. Pediatr., 90, 348, 1977; Rassin, D.K.
et al., Pediatrics, 59, 407, 1977.)

shows that on a gram-for-gram basis, most amino acids are fairly similar in content to
human milk, but with several notable exceptions. Threonine and methionine are particu-
larly high, and tryptophan, cysteine, and taurine are particularly low in the whey protein-
predominant preparations (unless taurine is supplemented) (Figure 28.1). Cysteine, tau-
rine, and tryptophan are particularly low in the casein protein-predominant formulas.
When the actual amino acid intake, as opposed to a gram-for-gram comparison, is calcu-
lated (Figure 28.1) for the formulas compared to human milk, it is clear that almost all
amino acids are supplied in considerable excess, with the notable exception of cysteine in
the casein protein-predominant formula. This difference reflects the differing protein con-
tent of formulas (about 1.5 g/dl) vs. human milk (about 1.0 g/dl), a difference that is
apparently not compensated for by increased volume intake in breastfed infants.
The high amino acid intakes of the formulas are reflected in increased plasma amino
acid concentrations in both preterm and term infants (Figure 28.2 and Figure 28.3)26,37,40,41
The preterm infant appears less able than the term infant to metabolize the excess of amino
acids being administered, as reflected by the generally more increased plasma concentra-
tions in these infants (compare Figure 28.2 and Figure 28.3).
Of interest is the fact that the whey protein-predominant formulas result in particularly
increased plasma threonine, while the casein protein-predominant formulas result in
particularly increased plasma phenylalanine and tyrosine, and tyrosine appears to accu-
mulate particularly in the preterm infant. Even manipulating the relative amounts of whey
and casein in the formula does not correct these differences.42 Also, these differences
emerge rapidly (in the first 24 h of life) and are marked by 72 h of life.43 Thus, plasma
threonine may serve as a biochemical marker for infants fed whey protein-predominant
formulas, and tyrosine may fill the same role for casein protein-predominant formulas.
1382_C28.fm Page 476 Tuesday, October 7, 2003 7:12 PM

476 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 28.2 The plasma amino acid concentrations of preterm infants fed either a casein protein- or
a whey protein-predominant formula (1.5 g of protein per decaliter) for 4 weeks; expressed as a
percent of the plasma amino acid concentrations of similar infants fed pooled human milk. (Data
from Rassin, D.K. et al., Pediatrics, 90, 356, 1977; Järvenpää, A.-L. et al., Pediatrics, 70, 221, 1982;
Rassin, D.K. et al., Pediatrics, 59, 407, 1977.)

Figure 28.3 The plasma amino acid concentrations of term infants fed either a casein protein- or a
whey protein-predominant formula (1.5 g of protein per decaliter) for 4 weeks; expressed as a percent
of the plasma amino acid concentrations of similar infants who were breastfed. (Adapted from
Järvenpää, A.-L. et al., Pediatrics, 70, 221, 1982.)

The notable findings of these investigations were that formulas appear to subject the
neonate to increased loads of amino acids compared to human milk. If taurine is not
supplied in the formula, decreased plasma concentrations are observed, potentially reflect-
ing a deficiency state. In fact, these studies partially resulted in taurine being added to
formulas. Despite the low amount of cysteine supplied by the casein protein-predominant
formulas, the neonate appears capable of maintaining plasma concentrations of this amino
1382_C28.fm Page 477 Tuesday, October 7, 2003 7:12 PM

Chapter twenty-eight: Neonatal requirements for amino acids 477

Figure 28.4 The amino acid concentrations of two different pediatric and one adult total parenteral
nutrition formulations currently in use; expressed as a percent of an equal amount of protein
supplied by human milk. (Adapted from Rassin, D.K., Total Parenteral Nutrition: Indications, Utiliza-
tion Complications, and Pathophysiological Considerations, Lebenthal, H., Ed., Raven Press, New York,
1986, p. 5; Rassin, D.K., Absorption and Utilization of Amino Acids, Vol. 2, Friedman, M., Ed., CRC
Press, Boca Raton, FL, 1939, p. 71.)

acid. Last, there are some studies indicating that the formula-fed infant may be at some
risk of receiving a reduced amount of tryptophan as reflected by plasma concentrations,
even though the formula composition appears to be sufficient.42,44
The lack of clear-cut findings with respect to cysteine led to further studies of
parenteral nutrition, because the unique composition of the preparations administered to
infants results in an even greater risk for cysteine deficiency. The development of
parenteral solutions and their composition has been reviewed;45,46,67 suffice it to say that
currently most neonates are maintained on preparations designated for pediatric use.
These amino acid solutions reflect problems with the physical characteristics of amino
acids (for example, the poor solubility of tyrosine and poor stability of cysteine)45 and
concerns regarding the neurotoxicity of some amino acids (glutamate and aspartate).15,47
Both adult and pediatric amino acid solutions have minimal or no tyrosine or cysteine
and tend to have little glutamate or aspartate (Figure 28.4). Methionine is often included
in excess to try to compensate for the lack of cysteine. Glycine and alanine are often fed
in excess to increase overall nitrogen intake, and arginine is usually fed in excess because
of concerns that deficient arginine in parenteral nutrition will result in hyperammoni-
emia.48 However, excessive arginine may also have some toxic effects.32 Particular care with
respect to arginine administration ought to be taken due to its role as a precursor of nitric
oxide, a potent regulator of blood vessel contractility.
The pattern of amino acids in parenteral solutions is reflected in the plasma concen-
trations of amino acids in neonates maintained on these solutions. Methionine, serine,
arginine, and glycine are often relatively high, while cystine and particularly tyrosine are
relatively low (Figure 28.5). The low cystine observed in parenterally maintained neonates
has led one investigator to suggest that this finding is sufficient to declare cysteine essential
to the neonate.35,68 The very unusual amino acid patterns observed in these neonates are
also accompanied by tissue changes that are not obvious from the plasma findings. For
example, in animal studies, cystathionine, a metabolite of methionine, is dramatically
1382_C28.fm Page 478 Tuesday, October 7, 2003 7:12 PM

478 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Figure 28.5 The plasma amino acid concentrations of preterm infants fed 6 days with an adult total
parenteral nutrition solution (second generation) at two different protein intakes; expressed as a
percent of the plasma amino acid concentrations of similar preterm infants fed pooled human milk.
(Adapted from Malloy, M.H. et al., J. Pediatr. Gastroenterol. Nutr., 3, 239, 1984.)

increased in the brain,49,50 and glutathione, an endogenous antioxidant tripeptide depen-


dent upon cysteine availability, is reduced in the liver.50 Thus, the infant may be at risk
for these changes also. The reduced availability of glutathione may increase the risk of
oxidant damage in the sick newborn that may also be treated with oxygen therapy, further
stressing this vulnerable metabolic antioxidant system. Animal studies have suggested
that the combination of low glutathione and high oxygen in the neonate may result in
increased rates of programmed cell death in the central nervous system.51

28.3 Enteral vs. parenteral nutrition: tyrosine and phenylalanine


One of the paradoxes observed in investigating neonatal responses to various types of
feeding has been the manner in which tyrosine reflects the route of amino acid nutrition.
Preterm infants fed enterally receive approximately equimolar amounts of phenylalanine
and tyrosine from human milk (614 and 652 mmol/2 g of protein, respectively), whey
protein-predominant formulas (605 and 559), and casein protein-predominant formulas
(669 and 677), even though the total aromatics differ. When corrected for actual intakes,
the casein protein-predominant formulas supply almost twice as much in the way of
aromatic amino acids as does human milk (1817 vs. 975 mmol/kg/day, respectively).
When the concentrations of phenylalanine and tyrosine are measured in the infant, it
is apparent that tyrosine responds far more dramatically than phenylalanine. For example,
after 4 weeks of feeding a casein protein-predominant formula, plasma tyrosine is 235%
of that in infants fed human milk, while phenylalanine is only 157%. The implication of
these findings is that phenylalanine is readily converted to tyrosine in enterally fed preterm
neonates.
In contrast, parenterally fed preterm neonates characteristically have low plasma
tyrosine, sometimes approaching the barely detectable, despite the fact that ample phenyl-
alanine is usually administered and plasma phenylalanine is usually greater in parenter-
ally fed than human milk-fed infants (see Figure 28.5). Indeed, even in the adult rat, which
has ample capacity to synthesize tyrosine from phenylalanine,52 parenteral nutrition
1382_C28.fm Page 479 Tuesday, October 7, 2003 7:12 PM

Chapter twenty-eight: Neonatal requirements for amino acids 479

induces a reduced plasma tyrosine.53 Thus, the route of administration of amino acids
appears to have a regulating effect on the capacity of the infant to metabolize phenyl-
alanine. The difference persists despite stable isotope evidence for the conversion of
parentally fed phenylalanine to tyrosine; however, a major portion of such phenylalanine
may go to alternative metabolites.54,55 This finding merits further exploration for other
amino acids.

28.4 Conclusion: amino acids and behavior


Finally, it is necessary to evaluate the implications of these various amino acid responses
to diet in the neonate outlined above. Increased plasma tyrosine concentrations in the
neonate, a phenomenon described as transient neonatal hypertyrosinemia, have been
associated with adverse neurologic outcome.56,57 These biochemical changes appear to
directly reflect the use of casein protein-predominant formulas prepared from cow milk.
Reduced dietary taurine intake has been associated with retinal and cerebellar dys-
function in cats and Rhesus monkeys,58–60 raising concerns that like phenomena may occur
in human infants. A few cases of retinal dysfunction associated with administration of
taurine-deficient parenteral nutrition have been reported.61
Preterm infants behave differently when fed relatively low protein-containing diets.62,63
These behavioral changes are associated with the plasma concentrations of the large
neutral amino acids.63 These large neutral amino acids are particularly important due to
their roles as neurotransmitter precursors and their common transport mechanisms into
the central nervous system. Thus, plasma amino acid modifications may influence brain
development. These modifications may provide one potential mechanism for the
improved intellectual outcome described for human milk-fed preterm infants compared
to similar infants fed formulas.64
Evidence for this association has been developed in acute experiments in which
manipulations of the amino acid tryptophan in the diet have been associated with mod-
ifications in sleep behavior.65,66 Tryptophan induced sleep more rapidly than an unmodified
formula, while administration of a blood–brain barrier transport competitor for tryp-
tophan, valine, lengthened the time of induction of sleep.65 Thus, infant behavior may well
reflect modulations in dietary amino acid intake.
In conclusion, human milk is the standard for determining amino acid intake in the
healthy term infant. Utilization of formulas and parenteral solutions in both term and
preterm infants results in a variety of biochemical modifications that reflect the type of
nutrition, the developmental status of the infant, and the route of administration. These
biochemical changes have the potential to influence the immediate behavior as well as
the neurologic development of the neonate. Administration of amino acids to neonates
ought to conform to amounts supplied by human milk until the consequences of deviating
from this standard are fully understood. Long-term evaluation of neonatal amino acid
requirements must consider growth, nitrogen balance, biochemical response, and cognitive
development in order to ensure optimal outcome. As recently as the 1950s, formula feeding
was associated with increased mortality in the U.S.;69 such is no longer the case, but there
is a persistent association of formula feeding with increased morbidity. Of particular
interest, however, are the repeated findings of greater cognitive development in human
milk-fed infants.

Acknowledgment
The author is grateful for the expert secretarial assistance of Mrs. Deborah LaVictoire.
1382_C28.fm Page 480 Tuesday, October 7, 2003 7:12 PM

480 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

References
1. Mortensen, E.L., Michaelsen, K.F., Sanders, S.A., and Reinisch, J.M., The association between
duration of breastfeeding and adult intelligence, JAMA, 287, 2365, 2002.
2. Rassin, D.K., Essential and non-essential amino acids in neonatal nutrition, in Protein
Metabolism during Infancy, Räihä, N.C.R., Ed., Raven Press, New York, 1994, p. 183.
3. Raiten, D.J., Talbot, J.M., and Waters, J.H., Assessment of nutrient requirements for infant
formulas, J. Nutr., 128, 2110S, 1998.
4. Klein, C.J., Nutrient requirements for preterm infant formulas, J. Nutr., 132, 1415S, 2002.
5. Food and Nutrition Board, Recommended Dietary Allowances, 9th ed., National Academy of
Sciences–National Research Council, Washington, D.C., 1980.
6. WHO/FAO, Energy and Protein Requirements, WHO Technical Report Series 522, World Health
Organization, Geneva, 1973.
7. WHO/FAO, Energy and Protein Requirements, Technical Report Series 724, World Health
Organization, Geneva, 1985.
8. ESPGAN Committee on Nutrition, Guidelines on infant nutrition. I. Recommendations for
the composition of an adapted formula, Acta Paediatr. Scand. Suppl., 1, 20, 1977.
9. ESPGAN Committee on Nutrition, Guidelines on infant nutrition. II. Recommendations for
the composition of a follow-up formula and beikost, Acta Paediatr. Scand. Suppl., 287, 1, 1981.
10. ESPGAN Committee on Nutrition, Nutrition and feeding of preterm infants, Acta Paediatr.
Scand. Suppl., 336, 1, 1987.
11. Committee on Nutrition, American Academy of Pediatrics, commentary on breastfeeding
and infant formulas including proposed standards for formulas, Pediatrics, 57, 278, 1976.
12. Irwin, M.I. and Hegsted, D.M., A conspectus of research on amino acid requirements of man,
J. Nutr., 101, 539, 1971.
13. Jackson, A.A., Shaw, J.C.L., Barker, A., and Golden, M.H.N., Nitrogen metabolism in preterm
infants fed donor breast milk: the possible essentiality of glycine, Pediatr. Res., 15, 1454, 1981.
14. Laidlaw, S.A. and Kopple, J.D., Newer concepts of the indispensable amino acids. Am. J.
Clin. Nutr., 46, 593, 1987.
15. Olney, J.W., Sharpe, L.G., and Feigin, R.D., Glutamate-induced brain damage in infant pri-
mates, J. Neuropathol. Exp. Neurol., 31, 464, 1972.
16. Snyderman, S.E., Boyer, A., Roitman, E., and Holt, L.E., Jr., The histidine requirement of the
infant, Pediatrics, 31, 786, 1963.
17. Snyderman, S.E., The protein and amino acid requirements of the premature infant, in
Metabolic Processes in the Foetus and Newborn Infant, Jonxis, J.H.P., Visser, H.K.A., and Troelstra,
J.A., Eds., Leiden, Stenfert Kroese, 1971, p. 128.
18. Snyderman, S.E., Boyer, A., and Holt, L.E., The arginine requirement of an infant, J. Dis.
Child., 97, 192, 1959.
19. Del Valle, J.A. and Greengard, O., Phenylalanine hydroxylase and tyrosine aminotransferase
in human fetal and adult liver, Pediatr. Res., 11, 2, 1976.
20. Kretchmer, N., Levine, S.Z., McNamara, H., and Barnett, H.L., Certain aspects of tyrosine
metabolism in the young. 1. The development of the tyrosine oxidizing system in human
liver, J. Clin. Invest., 35, 236, 1956.
21. Kretchmer, N., Levine, S.Z., and McNamara, H., The in vitro metabolism of tyrosine and its
intermediates in the liver of the premature infant, J. Dis. Child, 93, 19, 1957.
22. Levine, S., Marples, E., and Gordon, H., A defect in the metabolism of tyrosine and phenyl-
alanine in premature infants. I. Identification and assay of intermediary products, J. Clin.
Invest., 20, 199, 1957.
23. Mathews, J. and Partington, M.W., The plasma tyrosine levels of premature babies, Arch.
Dis. Child., 39, 371, 1964.
24. Avery, M.E., Clow, C.L., Menkes, J.H., Ramos, A., Scriver, C.R., Stern, L., and Wasserman,
B.P., Transient tyrosinemia of the newborn dietary and clinical aspects, Pediatrics, 39, 378,
1967.
1382_C28.fm Page 481 Tuesday, October 7, 2003 7:12 PM

Chapter twenty-eight: Neonatal requirements for amino acids 481

25. Bakker, H.D., Wadman, S.K., van Sprang, F.S., van Der Heiden, C., Ketting, D., and DeBree,
P.K., Tyrosinemia and tyrosinuria in healthy prematures: time courses not vitamin C-depen-
dent, Clin. Chim. Acta, 61, 73, 1975.
26. Rassin, D.K., Gaull, G.E., Räihä, N.C.R., and Heinonen, K., Milk protein quantity and quality
in low birth-weight infants. IV. Effects on tyrosine and phenylalanine in plasma and urine,
Pediatrics, 90, 356, 1977.
27. Järvenpää, A.-L., Rassin, D.K., Räihä, N.C.R., and Gaull, G.E., Milk protein quantity and
quality in the term infant. II. Effects on acidic and neutral amino acids, Pediatrics, 70, 221, 1982.
28. Räihä, N.C.R. and Suihkonen, J., Development of urea-synthesizing enzymes in human liver,
Acta Paediatr. Scand., 57, 121, 1968.
29. Räihä, N.C.R. and Suihkonen, J., Factors influencing the development of urea-synthesizing
enzymes in rat liver, Biochem. J., 107, 793, 1968.
30. Heird, W.C., Total parenteral nutrition, in Textbook of Gastroenterology and Nutrition in Infancy,
Lebenthal, E., Ed., Raven Press, New York, 1981, p. 659.
31. Czarncki, G.L. and Baker, D.J., Urea cycle function in the dog with emphasis on the role of
arginine, J. Nutr., 114, 581, 1984.
32. Mizunuma, T., Kawamura, S., and Kishino, V., Effects of ingesting excess arginine on rat
pancreas, J. Nutr., 114, 467, 1984.
33. Sturman J.A., Gaull, G.E., and Räihä, N.C.R., Absence of cystathionase in human fetal liver:
is cysteine essential? Science, 169, 74, 1970.
34. Pascal, T.A., Gillam, B.M., and Gaull, G.E., Cystathionase: immunochemical evidence for
absence from human fetal liver, Pediatr. Res., 6, 773, 1972.
35. Pohlandt, F., Cysteine: a semi-essential amino acid in the newborn infant, Acta Paediatr. Scand.,
63, 801, 1974.
36. Zlotkin, S.H. and Anderson, C.H., The development of cystathionase activity during the first
year of life, Pediatr. Res., 16, 65, 1982.
37. Gaull, G.E., Rassin, D.K., Räihä, N.C.R., and Heinonen, K., Milk protein quantity and quality
in low-birth-weight infants. III. Effects on sulfur-containing amino acids in plasma and urine,
J. Pediatr., 90, 348, 1977.
38. Rassin, D.K., Sturman, J.A., and Gaull, G.E., Taurine and other free amino acids in milk of
man and other mammals, Early Hum. Dev., 2, 1, 1978.
39. Räihä, N.C.R., Heinonen, K., Rassin, D.K., and Gaull, G.E., Milk protein quantity and quality
in low birth-weight infants. I. Metabolic responses and effects on growth, Pediatrics, 57, 659,
1976.
40. Rassin, D.K., Gaull, G.E., Heinonen, K., and Räihä, N.C.R., Milk protein quantity and quality
in low-birth-weight infants. II. Effects on selected essential and non-essential amino acids in
plasma and urine, Pediatrics, 59, 407, 1977.
41. Järvenpää, A.-L., Rassin, D.K., Räihä, N.C.R., and Gaull, G.E., Milk protein quantity and
quality in the term infant. II. Effects on acidic and neutral amino acids, Pediatrics, 70, 221, 1982.
42. Picone, T.A., Benson, J.D., Moro, G., Minoli, I., Fulconis, F., Rassin, D.K., and Räihä, N.C.R.,
Growth and serum biochemistries and amino acids of term infants fed formulas with amino
acid and protein concentrations similar to human milk, J. Pediatr. Gastroenterol. Nutr., 9, 351,
1989.
43. Cho, F., Bhatia, J., and Rassin, D.K., Amino acid responses to dietary intake in the first 72
hours of life, Nutrition, 6, 449, 1990.
44. Janas, L.M., Picciano, M.F., and Hatch, T.F., Indices of protein metabolism in term infants
fed human milk, whey-predominant formula, or cow’s milk formula, Pediatrics, 75, 775, 1985.
45. Stegink, L.O., Amino acids in pediatric parenteral nutrition, J. Dis. Child., 137, 1008, 1983.
46. Rassin, D.K., Amino acid requirements and profiles in total parenteral nutrition, in Total
Parenteral Nutrition: Indications, Utilization Complications, and Pathophysiological Considerations,
Lebenthal, E., Ed, Raven Press, New York, 1986, p. 5.
47. Olney, J.D., Ho, O.L., and Rhee, V., Cytotoxic effects of acidic and sulfur containing amino
acids on the infant mouse central nervous system, Exp. Brain Res., 14, 61, 1971.
1382_C28.fm Page 482 Tuesday, October 7, 2003 7:12 PM

482 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

48. Anderson, T.L., Heird, W.C., and Winters, R.W., Clinical and physiological consequences of
total parenteral nutrition in the pediatric patient, in Current Concepts in Parenteral Nutrition,
Greef, J.M., Soeterz, B., Wesdorp, R.I.C., PhAF, C.W.C., and Fischer, J.E., Eds., Martinus
Nijshoff, The Hague, 1977, p. 111.
49. Malloy, M.H., Rassin, D.K., Heird, W.C., and Gaull, G.E., Transsulfuration in parenterally
nourished beagle pups, Am. J. Clin. Nutr., 34, 1520, 1981.
50. Malloy, M.H. and Rassin, D.K., Cysteine supplementation of total parenteral nutrition: the
effect in beagle pups, Pediatr. Res., 18, 741, 1984.
51. Taglialatela, G., Perez-Polo, J.R., and Rassin, D.K. Induction of apoptosis in the CNS during
development by the combination of hyperoxia and inhibition of glutathione synthesis, Free
Rad. Biol. Med., 25, 936, 1998.
52. Wurtman, R.J., Aspartame effects on brain serotonin, Am. J. Clin. Nutr., 45, 799, 1987.
53. Rivera, A., Bhatia, J., Rassin, D.K., Gourley, W.K., and Catarau, E., In vivo biliary function
in the adult rat: the effect of parenteral glucose and amino acids, J. Parenter. Enteral Nutr.,
13, 240, 1989.
54. Roberts, S.A., Ball, R.O., Filler, R.M., Moore, A.M., and Penchaz, P.B., Phenylalanine and
tyrosine metabolism in neonates receiving parenteral nutrition differing in pattern of amino
acids, Pediatr. Res., 44, 907, 1998.
55. Roberts, S.A., Ball, R.O., Miller, A.M., Filler, R.M., and Penchaz, P.B., The effect of graded
intake of glycyl-L-tyrosine on phenylalanine and tyrosine metabolism in parenterally fed
neonates with an estimation of tyrosine requirement, Pediatr. Res., 49, 111, 2001.
56. Mamunes, P., Prince, P.E., Thornton, H.H., Hunt, P.A., and Hitchcock, F.S., Intellectual deficits
after transient tyrosinemia in the term neonate, Pediatrics, 57, 675, 1976.
57. Menkes, J.H., Welcher, D.W., Levi, H.S., Dallas, J., and Gretsky, N.E., Relationship of elevated
blood tyrosine to the ultimate intellectual performances of premature infants, Pediatrics, 49,
218, 1972.
58. Hayes, K.C., Carey, R.E., and Schmidt, S.Y., Retinal degeneration associated with taurine
deficiency in the cat, Science, 188, 949, 1975.
59. Sturman, S.A., Wen, G.Y., Wisniewski, H.M., and Neuringer, M.D., Retinal degeneration in
primates raised on a synthetic human infant formula, Int. J. Dev. Neurosci., 2, 121, 1984.
60. Sturman, S.A., Moretz, R.C., French, J.H., and Wisniewski, H.M., Postnatal taurine deficiency
in the kitten results in a persistence of the cerebellar external granule cell layer: correction
by taurine feeding, J. Neurosci. Res., 13, 521, 1985.
61. Geggel, H.S., Ament, M.F., Heckenlively, J.R., Martin, D.A., and Kopple, J.D., Nutritional
requirement for taurine in patients receiving long-term parenteral nutrition, N. Engl. J. Med.,
312, 142, 1985.
62. Tyson, J.E., Lasky, R.E., Mize, C.E., Richards, C.J., Blair-Smith, N., Whyte, R., and Beer, A.E.,
Growth, metabolic response, and development in very-low-birth-weight infants fed banked
human milk or enriched formula. 1. Neonatal findings, J. Pediatr., 103, 95, 1983.
63. Bhatia, J., Rassin, D.K., Cerreto, M.C., and Bee, D.E., Effect of protein/energy ratio on growth
and behavior of premature infants: preliminary findings, J. Pediatr., 119, 103, 1991.
64. Lucas, A., Morley, R., Cole, T.J., Lister, G., and Leeson-Payne, C., Breast milk and subsequent
intelligence quotient in children born preterm, Lancet, 339, 261, 1992.
65. Yogman, M.W. and Zeisel, S.H., Diet and sleep patterns in newborn infants, N. Engl. J. Med.,
309, 1147, 1983.
66. Steinberg, L.A., O’Connell, N.C., Hatch, T.F., Picciano, M.F., and Birch, L.L., Tryptophan
intake influences infants’ sleep latency, J. Nutr., 122, 1781, 1992.
67. Rassin, D.K., Amino acid metabolism in total parenteral nutrition during development, in
Absorption and Utilization of Amino Acids, Vol. 2, Friedman, M., Ed., CRC Press, Boca Raton,
FL, 1989, p. 11.
68. Malloy, M.H., Rassin, D.K., and Richardson, C.J., Total parenteral nutrition in sick preterm
infants: effects of cysteine supplementation with nitrogen intakes of 240 and 400 mg/kg/day,
J. Pediatr. Gastroenterol. Nutr., 3, 239, 1984.
69. Robinson, M., Infant morbidity and mortality: A study of 3266 infants, Lancet, 1, 788–794,
1951.
1382_C29.fm Page 483 Tuesday, October 7, 2003 7:15 PM

chapter twenty-nine

Amino acid requirements


in the elderly
P. Patureau Mirand
Unité de Nutrition et Métabolisme Protéique
L. Mosoni
Unité de Nutrition et Métabolisme Protéique
D. Rémond
Unité de Nutrition et Métabolisme Protéique

Contents
Introduction..................................................................................................................................484
29.1 Main consequences of aging on protein metabolism ...................................................484
29.1.1 Whole-body protein metabolism ......................................................................484
29.1.2 Degradation and nonprotein roles of amino acids ........................................485
29.1.3 Specific impact of amino acids in the regulation of protein metabolism
during aging .........................................................................................................485
29.1.4 Consequences for amino acid requirements ...................................................486
29.2 Amino acid requirements .................................................................................................486
29.2.1 Methods used to determine amino acid requirements in the elderly ........486
29.2.1.1 Indirect methods..................................................................................486
29.2.1.2 Nitrogen balance method...................................................................487
29.2.1.3 The plasma amino acid response curve method ...........................487
29.2.1.4 The tracer balance method.................................................................488
29.2.2 Discrepancies in the estimations of amino acid requirements
in the elderly ........................................................................................................488
29.2.2.1 Estimations derived from protein requirements and minimal
nitrogen losses......................................................................................488
29.2.2.2 Sulfur amino acid requirements........................................................490
29.2.2.3 Leucine requirements..........................................................................491
29.2.2.4 Other amino acid requirements ........................................................491
29.3 Conclusion ..........................................................................................................................492
Acknowledgment ........................................................................................................................492
References .....................................................................................................................................492

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 483
1382_C29.fm Page 484 Tuesday, October 7, 2003 7:15 PM

484 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Introduction
Protein requirement is an integrated expression of a complex demand for amino acids.
These latter molecules are precursors of tissue proteins and also of a number of essential
metabolites (endocrine, neuro- or immunomediators, cofactors, etc.). Therefore, protein
requirement consists in the simultaneous presence in balanced amounts of the 20 amino
acids that are constitutive of proteins, at the site where tissue proteins are synthesized.
The need induced by their role as precursors for various metabolites has to be added to
this demand.1 Nine amino acids (indispensable amino acids) cannot be synthesized in
sufficient amounts in human tissues to meet the requirements. Each of them needs to be
supplied by food. The supply of each of the others (dispensable amino acids) does not
depend strictly on feeding insofar as the diet includes dietary proteins. They can be
synthesized from other amino acids and intermediary metabolites (see Chapter 27 for
more details). This irreducible complexity is worsened by the fact that some dispensable
amino acids can be indispensable in specific physiopathological conditions (conditionally
indispensable amino acids). Furthermore, the conditions of the amino acid supply may
interfere to modify the need for amino acids (e.g., imbalances, physiological properties of
some proteins or peptides, timing of protein feeding).2
Research on protein nutrition in the elderly has not taken into account all these aspects,
which are still controversial in young adults, whose protein and amino acid requirements
are already better known. The aim of this review is to present and discuss reasons to
believe why amino acid requirements of elderly people may be different from those of
young adults. This relies on evidence from comparisons of amino acid and protein metab-
olism in young adults and elderly people to detect the modifications that result from aging.

29.1 Main consequences of aging on protein metabolism


29.1.1 Whole-body protein metabolism
Most cross-sectional studies have shown that fat mass (mainly internal adipose tissue)
increases during aging (from 15 to 36% of whole-body mass between 25 and 75 years)
and that fat-free mass decreases. The rate of fat-free mass loss could reach 320 g per year
after 30 years of age, and could be higher later.3 However, longitudinal studies that report
similar tendencies indicate that this rate could be less extreme.4 This age-related decrease
of fat-free mass corresponds to muscle (see Chapter 24) and bone losses, whereas most
visceral organs are less affected.5 The variations of organ size during aging reflect modi-
fications in the main pathways of protein metabolism.
Whole-body protein turnover expressed per kilogram of body weight is reduced6–16
or maintained17 during aging. This is a consequence of the decrease in fat-free mass, which
is the main component of whole-body protein turnover. Indeed, it is maintained if
expressed per lean body mass unit except in two studies,12,18 where it was decreased. This
lack of effect of aging on whole-body protein turnover suggests that amino acid require-
ments are unchanged with aging.
Protein turnover variations may also be a consequence of age-related protein alter-
ations. During aging, like in many pathological conditions, proteins undergo alterations
resulting from oxidative stress caused by free radicals. Most of these modifications, which
result in protein carbonylation or glycation,19,20 have complex consequences. They can
stimulate protein degradation because modified proteins are more prone to degradation.
But the increase in modified proteins during aging indicates that some of them can
accumulate either because they are more resistant to degradation or because of alterations
in the efficiency of proteolytic systems. The consequences of oxidative stress on protein
1382_C29.fm Page 485 Tuesday, October 7, 2003 7:15 PM

Chapter twenty-nine: Amino acid requirements in the elderly 485

may increase amino acid requirements by inducing specific losses: (1) the accumulation
of nondegradable modified proteins, which reduces recycling of their constitutive amino
acids, corresponds to an irreversible loss; (2) the increased turnover of the oxidized pro-
teins that are prone to degradation can also be responsible for specific amino acid losses,
since amino acids released by proteolysis are not fully reutilized by protein synthesis; and
(3) irreversibly oxidized or glycated amino acids cannot be reutilized and they are lost.
The last two reasons for an increase in amino acid losses, caused by oxidative stress, result
in an increase in nitrogen losses. In summary, higher oxidative damages like those
described during aging or inflammatory diseases are expected to increase amino acid
losses. However, measurements of minimal nitrogen losses, which take into account the
last two types of losses, failed to detect clear differences between young and old adults
(see Kurpad and Vaz21 for a review).

29.1.2 Degradation and nonprotein roles of amino acids


The nonprotein utilization of some amino acids could be different in young adults and
elderly people. A reduction in methionine intake, which decreases methionine catabolism
and cysteine synthesis through the transsulfuration pathway in young adults, is less
effective in sparing methionine in elderly people.22 This suggests an increased cysteine
requirement in the elderly, probably to maintain glutathione pool size despite oxidative
damage,23,24 resulting from a low-grade inflammatory state frequently observed in elderly
people. Similarly, the decrease in taurine levels in serum and in several tissues suggests
that there is an increased requirement for cysteine resulting from the stimulation of
glutathione synthesis.25 Other amino acids like histidine and two related peptides (anserine
and carnosine), aromatic amino acids, and several of their derivatives also directly take
part in the control of oxidative stress. An increasing need for such compounds can be
expected during aging.
In opposition, leucine oxidation in the postabsorptive state was lower in old people
than in young adults,13,26,27 which is in keeping with a lower transamination rate in old
individuals than in young adults.28 However, leucine oxidation may have been more
extensively underestimated in older than in younger adults. Leucine oxidation was cal-
culated from the oxidation of peripherally infused 1-13C leucine, which underestimates
dietary leucine oxidation.13,29 This may have a higher impact in the elderly than in young
adults since it was shown that leucine first-pass extraction in the splanchnic area was
higher in old than in young men.13

29.1.3 Specific impact of amino acids in the regulation of protein metabolism


during aging
The whole-body protein mass rises during the postprandial period and decreases during
the postabsorptive period, so the variations of protein mass result from the difference
between the postprandial gains and the postabsorptive losses. The postprandial gains
result mainly from the inhibition of protein degradation. This inhibition is blunted in
elderly compared to young adults.13,27 Furthermore, experiments in rats have also shown
that the stimulation of protein synthesis in muscle by food intake was less intensive in
old than in mature rats.30–32 This defect in protein synthesis stimulation and of protein
degradation inhibition during the postprandial period contributes to the explanation of
muscle protein loss because of an incomplete recovery of protein lost during the post-
absorptive period.
A number of experiments in the elderly have shown that nitrogen balance increased
when protein intake is increased.21,33 Even in the frail elderly, a protein supplement
1382_C29.fm Page 486 Tuesday, October 7, 2003 7:15 PM

486 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

increased whole-body protein content by stimulating postprandial protein anabolism.14


However, the efficiency of dietary nitrogen to improve nitrogen balance appears to be
lower in elderly (31%) than in young adults (48%).33 This is consistent with the decreased
capability of skeletal muscle protein synthesis to be stimulated in response to the post-
prandial increase in extracellular free amino acid levels. The variations of plasma free
leucine levels appear to be the main determinants of this stimulation.34 Experiments in
rats have shown that the responsiveness of muscle protein synthesis to the increase in
postprandial levels of plasma free leucine is lower in old animals than in younger ones.35
Consequently, old subjects require a higher plasma leucine level to obtain the same stim-
ulatory effect than in young adults, as it was shown recently in rats.32 This mechanism
may explain why two nutritional practices that result in a sharp increase in plasma leucine
levels promoted protein anabolism in elderly subjects. The consumption of quickly
digested proteins, high in leucine, was more efficient in improving leucine balance in the
elderly than slowly digested proteins.36 The pulse feeding pattern, in which 80% of daily
protein intake was concentrated in one meal (at noon), was more efficient in increasing
lean body mass and whole-body protein mass than a spread pattern, in which daily protein
intake was spread over four meals, in old women.37
In contrast, no difference in dietary protein utilization was reported in two studies:
one using the nitrogen balance method38 and the other based on leucine kinetics.18,26 The
reasons for such discrepancies are not clear. They could result from differences in health
status. Age-related inflammatory diseases like rheumatoid arthritis are known to induce
alterations in the control of protein degradation,39 and dietary protein efficiency is mainly
determined by whole-body protein degradation rate.40

29.1.4 Consequences for amino acid requirements


This comparison between protein metabolism in young adults and elderly unveils three
kinds of observations that may have an impact on the nature and the level of protein
requirements in the elderly.

1. In a basal state, there are only small differences in the activities of the main
pathways of protein metabolism between young and old adults. This suggests that
amino acid requirements would not be affected by aging.
2. The consequences of oxidative damage (oxidation, glycation, etc.) on protein me-
tabolism increase during aging. This may induce specific amino acid requirements
in the elderly to promote antioxidant defenses.
3. The decrease in protein anabolism response to protein intake suggests higher
protein or amino acid requirement in elderly people.

The consequences of these observations on amino acid requirements appear to be


rather conflicting, and this may be a reason for a great deal of uncertainty about the protein
and amino acid requirements of elderly people.

29.2 Amino acid requirements


29.2.1 Methods used to determine amino acid requirements in the elderly
29.2.1.1 Indirect methods
Several approaches have been used to estimate amino acid requirements in the elderly.
The less specific ones are indirect methods. They are based on protein requirement deter-
mination and on an estimation of an adequate indispensable amino acid pattern like the
1382_C29.fm Page 487 Tuesday, October 7, 2003 7:15 PM

Chapter twenty-nine: Amino acid requirements in the elderly 487

Food and Agriculture Organization (FAO) pattern.41 This approach assumes that the con-
ventional pattern defined for young adults is not affected by aging and that protein
requirement is known; however, both points are questionable. Another method is based
on the determination of the minimal obligatory nitrogen losses. It is assumed that these
losses correspond to the oxidative degradation of amino acids released from body proteins
and that the oxidation pattern of these amino acids is in proportion to the amino acid
pattern of mixed body proteins.42,43 Both methods, which derive from the determinations
of protein requirement or losses, were used to estimate the requirements of most indis-
pensable amino acids.

29.2.1.2 Nitrogen balance method


Attempts to directly measure amino acid requirements in the elderly have mostly been
performed by the nitrogen balance method, which remains, until now, the reference
method to determine protein requirements in adults.33 It has been used to determine
requirements in the elderly for sulfur amino acids,44 lysine,44,45 tryptophan,45,46 and threo-
nine.47 This method implies that maintenance of whole-body protein mass (i.e., nitrogen
balance equilibrium) indicates that amino acid intake is adequate. It is flawed by several
shortcomings.48 Some are related to the principle of the method. Amino acid requirements
correspond to complex needs, and maintenance of whole-body protein mass is assumed
to integrate all these needs. However, it is not clear whether the nonprotein roles of amino
acids are correctly taken into account; indeed, there are situations in which amino acid
requirements were not met despite nitrogen balance equilibrium, for example, in the case
of histidine deficiency. A second point is related to the nonlinearity of the nitrogen balance
response to the variations of indispensable amino acid intake.49 Other difficulties are
related to practical aspects of the method. It requires very careful measurements of dietary
intake and nitrogen output; otherwise, nitrogen balances are generally overestimated
(because of overestimation of intake and underestimation of losses). This results in under-
estimation of amino acid requirements. Measurements have to be performed on at least
four consecutive days (in order to limit the consequences of daily variations). It is necessary
to know individual requirement variability to determine the safe amino acid intake from
mean amino acid requirement. Therefore, nitrogen balance must be determined in the
same individual at several amino acid intake levels, but only few individual multipoint
experiments have been published.44–47 The time required to obtain a steady state of nitrogen
excretion is variable after changes in amino acid intake levels. A quasi-steady state is
observed within a week.41 However long-term multipoint studies are hardly feasible.
Nitrogen balance measurements for amino acid requirement determination have to be
performed in energy balance conditions since nitrogen excretion is increased when energy
intake does not meet energy requirements and is reduced when energy intake is increased.
Furthermore, Millward and Roberts50 reported that specific problems are encountered
in determining protein requirement in the elderly by the nitrogen balance method; these
problems may also affect amino acid requirement determinations. Briefly, the energy
requirements are often underestimated, leading to an overestimation of protein require-
ment. Miscellaneous nitrogen losses could be overestimated. Finally, the time required to
obtain a steady state in nitrogen losses with low protein intake is longer than that in young
adults.51,52

29.2.1.3 The plasma amino acid response curve method


Direct estimations of tryptophan46 and threonine47 requirements in the elderly have been
performed by this method. It is based on the fact that when the intake of the test amino
acid is below requirement, its plasma level is low and rather constant, despite increasing
1382_C29.fm Page 488 Tuesday, October 7, 2003 7:15 PM

488 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

intake, because this limiting amino acid is used in increasing amounts for protein synthesis.
By contrast, when intake is increased above requirement, this amino acid is used at a
maximal but constant level for protein anabolism since it is not limiting, and its plasma
level rises sharply because of excessive intake. The amino acid intake corresponding to
the breakpoint in the response curve is supposed to indicate the requirement. This assump-
tion has not been clearly demonstrated for most amino acids in humans. Furthermore, it
may be difficult for some amino acids to detect a clear breakpoint in the plasma response
curve.

29.2.1.4 The tracer balance method


This method has been used to determine sulfur amino acid requirements in elderly men
and women.22 This method is based on the determination of the intake that is necessary
to balance the daily amino acid losses (see Chapter 27 for further details). This method
relies on assumptions that have been discussed previously (see Waterlow48 for a review).
A potential specific problem is expected in the elderly. The first-pass utilization of dietary
amino acids in the splanchnic area is not fully taken into account when the tracer is
intravenously infused. In this condition, the amino acid balance is overestimated, and the
amino acid requirement is underestimated. This could be the case in elderly people because
it has been reported that splanchnic extraction is high. For leucine, it was twice as high
in old men than it was in young men.13 However, this may have only limited impact in
methionine balance determinations since it was found that methionine splanchnic extrac-
tion in young adults fed a low methionine diet was very low in the fed state.53 A specific
problem of sulfur amino acids is to be certain that sulfur amino acid requirement is met
when methionine balance is achieved. A cysteine balance study in young adults seems to
validate this assumption.54

29.2.2 Discrepancies in the estimations of amino acid requirements in the elderly


29.2.2.1 Estimations derived from protein requirements and minimal nitrogen
losses
Estimates of amino acid requirements based on protein requirement are rather uncertain
because there is no agreement on protein requirement in elderly people.
In 1985, the FAO/WHO/UNU Expert Consultation41 set the mean daily protein
requirement of healthy elderly at 0.6 g of protein·kg of body weight–1·day–1, and the safe
protein intake at 0.75 g of protein·kg of body weight–1·day–1. These values were the same
in young adults. Although it recognized that various changes occur during aging, the
consultation found that insufficient data were available to establish a specific protein
requirement with confidence in the elderly.
A new analysis of the same data but including two further studies55,56 led to a reas-
sessment of protein requirement for the elderly (0.8 g of protein·kg–1·day–1), which
indicates a safe level of 1.0 g of protein·kg–1·day–1.57 This was questioned by Millward
and Roberts,50 who looked at the methodology of protein requirement assessments and
concluded that they “cannot identify any studies which unequivocally demonstrate either
a change with age of the requirement or a mean requirement value which is higher than
the values defined by FAO/WHO/UNU (1985).” Another review21 of all the previous
data, with one further balance study added,58 suggests that the mean protein requirement
in healthy elderly is greater than 0.8 g of protein·kg–1·day–1. More recently, the follow-up
of nitrogen balance measurements indicates a shift toward positive values when experi-
ments lasted more than 2 weeks.51,52 Measurements at week 2 indicate that mean protein
requirement is close to 0.75 g of protein·kg–1·day–1, and measurements at week 3 indicated
1382_C29.fm Page 489 Tuesday, October 7, 2003 7:15 PM

Chapter twenty-nine: Amino acid requirements in the elderly 489

Table 29.1 Estimates of Amino Acid Requirements Derived from the Amounts Fed in Different Diets
and from Obligatory Nitrogen Losses
Western dieta 22% IAAd 44% IAAd
Amino acids 0.6 g·kg–1 0.74 g·kg–l ONLb RDAc (0.65 g·kg–1) (0.65 g·kg–1)
Histidine 16 20 13 14 6.5 13
Isoleucine 29 36 17 19 19 38
Leucine 45 56 36 42 26 51
Lysine 44 54 35 38 21 41
Methionine + 21 26 17 19 15 30
cystine
Phenylalanine + 48 59 35 33 23 46
tyrosine
Threonine 25 31 20 20 11 23
Tryptophan 7 8.5 6 5 3-6 6
Valine 33 41 23 24 19 38
a Usual western diet: animal protein, 67%; plant protein, 33%.
b Obligatory nitrogen losses.
c Recommended Dietary Allowances for adults 19 years and older.60
d Protein intake consisted of amino acid mixture containing 22 or 44% indispensable amino acids (IAA) patterned
as egg proteins.61

that it was 0.56 g of protein·kg–1·day–1.51 A later experiment indicated that a protein intake
of 0.8 g of protein·kg–1·day–1, which equilibrated nitrogen balances at week 2, resulted in
a positive nitrogen balance in week 6 or 14.52 This restates the problem of adaptation or
accommodation and the need for additional criteria. Finally, a very extensive review on
adult protein requirements was based on the simultaneous analysis of the nitrogen balance
data in 235 adults.33 The median dietary nitrogen requirements were established at 103.9
mg of nitrogen·kg–1·day–1 for young adults (<55 years) and 130.5 mg of nitrogen·kg–1·day–1
for old adults (>55 years), in keeping with the difference in dietary protein efficiency.
However, the authors concluded that since the difference was not significant, the median
protein requirement for adults is the same whatever the age: 105 mg of nitrogen·kg–1·day–1,
i.e., 0.65 g of protein·kg–1·day–1. The variability of protein requirement assessments for
elderly based on nitrogen balance studies shows that it is not possible to establish a unique
protein requirement level. It is likely that there are elderly populations who have the same
protein requirement as young adults and others who have higher requirements. To take
into account this variability, the amounts of indispensable amino acid supplied by either
0.6 or 0.75 g·kg–1·day–1 of mixed proteins (two thirds animal protein, one third plant
protein) were calculated and are shown in Table 29.1. Nitrogen balance equilibrium was
observed in elderly healthy women with the higher level.37 Such estimates, which are
consistent with nitrogen equilibrium, do not provide an index of the minimum required
for most amino acids.
The estimates of amino acid requirements derived from minimal nitrogen losses
(54 mg of N·kg–1·day–1, retention efficiency (70%)) and the amino acid composition of body
proteins59 are shown in Table 29.1. They are slightly different from results published in a
recent review21 because these estimates included data obtained in tracer studies for lysine,
threonine, sulfur amino acids, and valine43 and because whole-body protein composition
was derived from beef composition.42 It is not clear whether minimal nitrogen losses in
the elderly are similar to or lower than those in young adults. In any case, the retained
value can be considered a maximum. The calculation of retention efficiency is much more
questionable. First, it is likely that the efficiency is different according to the amino acids,
like it is in young adults. Second, the lower efficiency of dietary protein for retention33
1382_C29.fm Page 490 Tuesday, October 7, 2003 7:15 PM

490 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 29.2 Comparison of Direct Estimates of Some Amino Acid Requirements in the Elderly
Obtained by Different Methods with Mean Requirements Determined in Young Adults
Mean
Elderly Young Methodsa References requirementb
Sulfur amino >32 NB 44
acids 5–7 or 17 NB 45 15
13 13 TB 22, 62
Lysine 45 30 NB 44, 49 31
Threonine 8 7 PAA 47 16
>14 NB 47
Tryptophan 5 NB 45
>4.4 NB 46 4
2 3 PAA 46

Note: Estimates of requirements are expressed in mg·kg–1·day–1.


a NB = nitrogen balance reanalyzed; TB = tracer balance; PAA = plasma free amino acid response curve.
b For adults 19 years and older.60

suggests that these efficiencies can be lower in elderly than in young adults for at least
one amino acid. On the other hand, Fereday et al.26 reported that the postprandial
efficiency of leucine was not different in young adults and elderly, but in this case leucine
oxidation may have been more extensively underestimated in elderly than in young
adults (as previously explained). However, these estimates compare with recommended
dietary allowances for adults 19 years old and older.60 They can be considered minimal
requirements.
The comparison of these values with the amounts of indispensable amino acid fed
during different nitrogen balance studies61 can be used to try to detect which amino acid
could be limiting in a low-protein western diet. Indeed, these estimates were compared
with data obtained in five men (52 to 68 years old) who were fed 0.65 g of protein·kg–1·day–1
in which there was either 22 or 44% indispensable amino acids patterned as in egg proteins.
All the subjects went into negative nitrogen balance when the proportion of indispensable
amino acids was 22%; they equilibrated when this proportion was doubled. The amounts
of branched-chain and sulfur amino acids fed by this diet were higher than the amounts
supplied by the low occidental diet. It is unlikely that isoleucine and valine are limiting
since their content in the occidental diet is markedly higher than the recommended dietary
allowances for young adults,60 but it is not unlikely for sulfur amino acids and leucine. A
few experiments were designed to study specific amino acid requirements in the elderly.
The main results are shown in Table 29.2, and we compared them with the recently
published mean requirement estimates for adults.59

29.2.2.2 Sulfur amino acid requirements


Nitrogen and tracer balance studies have been performed to determine sulfur amino acid
requirements in the elderly. The daily intake of sulfur amino acid required to achieve
nitrogen balance was above 32 mg·kg–1·day–1 in six men (58 to 73 years old) fed diets of
purified amino acid mixtures containing variable amounts of methionine as the sole sulfur
amino acid source.44 The authors suggested that such a high need could be due to a
decreased efficiency in the conversion of methionine to cysteine. This may be the conse-
quence of inadequate vitamin B6 intake. Deficiencies in methyl donors like choline or in
folates and vitamin B12 may also lead to enhanced methionine requirements. In another
nitrogen balance study with semipurified diets with no histidine in the indispensable
amino acid mixture, the results were rather inconsistent. Corrected nitrogen balance for
miscellaneous nitrogen losses was achieved when sulfur amino acid intake was
1382_C29.fm Page 491 Tuesday, October 7, 2003 7:15 PM

Chapter twenty-nine: Amino acid requirements in the elderly 491

17 mg·kg–1·day–1 for four subjects, but two required less than 7 or 5 mg·kg–1·day–1.45 More
recently, estimates of sulfur amino acid requirement were derived from a study on
methionine and cysteine kinetics using the tracer balance method.22 In healthy elderly
subjects a mean intake of 13 mg·kg–1·day–1 for total sulfur amino acids appeared to be
sufficient to achieve body methionine balance. This is similar to what was obtained in
young adults.62 However, there is some concern about the methionine balance estimation
since the increase in methionine balance between the low and the adequate methionine
diets was higher than the increase in methionine intake. This may result from differences
in the estimation of methionine oxidation rate depending on the amount fed. In young
adults, it was shown that the splanchnic extraction of methionine during the fast period
was higher for the adequate methionine diet than for the low methionine diet.53 Further-
more, methionine balance was not achieved when sulfur amino acid intake consisted of
6.5 mg·kg–1·day–1 methionine with either 5.2 or 21 mg·kg–1·day–1 cystine. The authors
concluded that “a total sulfur amino acid requirement of 13 mg·kg–1·day–1 may not be met
if dietary cystine accounts for as much as half of the total sulfur amino acid intake.”22

29.2.2.3 Leucine requirements


The beneficial effect of a sharp increase in postprandial plasma free leucine levels on
protein anabolism could suggest that elderly people have a higher leucine requirement
than young people. Unfortunately, no experiments were designed to evaluate the leucine
requirement in the elderly, and it is difficult to estimate the consequences of leucine intake
variations on leucine balance despite numerous studies of leucine kinetics. In the post-
absorptive state, leucine oxidation on body weight basis appears to be lower in elderly
than in young adults.13,26,27 Estimates of postprandial efficiency for leucine retention were
similar at both ages.26 This would indicate a lower leucine requirement in elderly people
than in young adults. However, the age-related differences in the first-pass utilization of
leucine through the splanchnic area13 and the incomplete measurement of splanchnic
leucine oxidation with peripherally infused tracer may have biased this comparison.
Furthermore, if higher postprandial plasma leucine levels are required to stimulate muscle
protein synthesis, this does not necessarily imply that higher daily leucine intakes are
required. It seems that the postprandial increase in leucine concentrations acts as a tem-
porary signal to stimulate muscle protein synthesis. This explains why in elderly people
fed usual diets (1.05 g of protein·kg–1·day–1) with the same leucine content (72 mg/g of
protein), the pulse pattern for protein feeding was more efficient to improve protein
retention and lean body mass than a spread pattern.37

29.2.2.4 Other amino acid requirements


Lysine requirements were determined by the nitrogen balance method in four men, 53 to
64 years old, fed two levels of lysine. A reanalysis of the data, including miscellaneous
losses, indicates that a mean daily intake of 45 mg·kg–1·day–1 lysine achieved nitrogen
balance equilibrium.44
Tryptophan requirement was determined in two nitrogen balance method studies. In
one experiment with six men, 65 to 84 years old, the estimate of tryptophan requirement
was close to 5 mg·kg–1·day–1 when miscellaneous nitrogen losses were taken into account.45
This is consistent with the value found in another experiment performed with 10 women
and 4 men (55 to 82 years old). It was above 4.4 mg·kg–1·day–1, correcting for miscellaneous
losses and excluding the initial period data because it was the adaptation period.46 The
plasma response curve method was also used in this experiment. A breakpoint was found
by statistical analysis of the response curve for a daily intake of 2 mg·kg–1. This was lower
than the estimates obtained by the same method in young adults (3 mg·kg–1·day–1).46
1382_C29.fm Page 492 Tuesday, October 7, 2003 7:15 PM

492 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Threonine requirement was also determined in elderly women and in young men by
the nitrogen balance and the plasma response curve methods.47 The results of the nitrogen
balance study were rather inconsistent in the elderly, and there was no clear breakpoint
in the plasma response curve. However, the regression analysis suggested that threonine
requirement was 8 mg·kg–1·day–1, not different from what was found in young men
(7 mg·kg–1·day–1) during this experiment by the same approach. These values are much
lower than the requirement inferred from reanalyzed nitrogen balance data in young men
(>14 mg·kg –1 ·day –1 ) and the mean requirement recently proposed for adults
(16 mg·kg–1·day–1).60

29.3 Conclusion
Some metabolic studies indicate that there are several reasons to think that some amino
acid requirements could be higher in elderly people than in young adults, but it is not
clear whether it is a general tendency because other studies do not find any differences.
Overall, it appears that the data on amino acid requirements in the elderly are scarce and
conflicting. Many factors are involved (methods, health status, lean body mass), which
may explain the discrepancies in the mean requirement estimates.
However, the amino acid requirements in the elderly appear to be at least as much
as in young adults and presumably higher. The variability of amino acid requirement
estimates for elderly suggests that it is not possible to establish a unique amino acid
requirement level. It is likely that there are elderly populations who have amino acid
requirements similar to those of young adults, whereas others may have higher require-
ments. Furthermore, in elderly people more than in young adults, the effectiveness of
dietary proteins depends on their form and their feeding pattern.

Acknowledgment
We thank Susan Samuels for helpful advice.

References
1. Reeds, P.J. and Biolo, G., Non-protein roles of amino acids: an emerging aspect of nutrient
requirements, Curr. Opin. Clin. Nutr. Metab. Care, 5, 43, 2002.
2. Mosoni, L. and Patureau Mirand, P., Type and timing of protein feeding to optimize anab-
olism, Curr. Opin. Clin. Nutr. Metab. Care, 6, 301, 2003.
3. Cohn, S.H. et al., Compartmental body composition based on total-body nitrogen, potassium,
and calcium, Am. J. Physiol., 239, E524, 1980.
4. Guo, S.S. et al., Aging, body composition, and lifestyle: the Fels longitudinal study, Am. J.
Clin. Nutr., 70, 405, 1999.
5. Korenchevsky, V., Chemical changes with ageing, in Physiological and Pathological Ageing,
Bourne, G.H., Ed., Karger, Basel, Switzerland, chap. 7, p. 87.
6. Sharp, G.S. et al., Studies of protein retention and turnover using nitrogen-15 as a tag, J.
Nutr., 63, 155, 1957.
7. Uauy, R., Scrimshaw, N.S., and Young, V.R., Human protein requirements: nitrogen balance
response to graded levels of egg protein in elderly men and women, Am. J. Clin. Nutr., 31,
779, 1978.
8. Welle, S. et al., Myofibrillar protein synthesis in young and old men, Am. J. Physiol., 264,
E693, 1993.
9. Welle, S. et al., Postprandial myofibrillar and whole body protein synthesis in young and
old human subjects, Am. J. Physiol., 30, E599, 1994.
1382_C29.fm Page 493 Tuesday, October 7, 2003 7:15 PM

Chapter twenty-nine: Amino acid requirements in the elderly 493

10. Welle, S., Thornton, C., and Statt, M., Myofibrillar protein synthesis in young and old human
subjects after three months of resistance training, Am. J. Physiol., 268, E422, 1995.
11. Pannemans, D.L.E., Halliday, D., and Westerterp, K.R., Whole-body protein turnover in
elderly men and women: responses to two protein intakes, Am. J. Clin. Nutr., 61, 33, 1995.
12. Balagopal, P. et al., Effects of aging on in vivo synthesis of skeletal muscle myosin heavy-
chain and sarcoplasmic protein in humans, Am. J. Physiol., 273, E790, 1997.
13. Boirie, Y., Gachon, P., and Beaufrère, B., Splanchnic and whole-body leucine kinetics in young
and elderly men, Am. J. Clin. Nutr., 65, 489, 1997.
14. Bos, C. et al., Short-term protein and energy supplementation activates nitrogen kinetics and
accretion in poorly nourished elderly subjects, Am. J. Clin. Nutr., 71, 1129, 2000.
15. Morais, J.A. et al., Whole-body protein turnover in the healthy elderly, Am. J. Clin. Nutr., 66,
880, 1997.
16. Morais, J.A. et al., Distribution of protein turnover changes with age in humans as assessed
by whole-body magnetic resonance image analysis to quantify tissue volumes, J. Nutr., 130,
784, 2000.
17 Benedek, C. et al., Resting metabolic rate and protein turnover in apparently healthy elderly
Gambian men, Am. J. Physiol., 268, E1083, 1995.
18. Millward, D.J. et al., Aging, protein requirements, and protein turnover, Am. J. Clin. Nutr.,
66, 774, 1997.
19. Grune, T. et al., Age-related changes in protein oxidation and proteolysis in mammalian
cells, J. Gerontol. A Biol. Sci. Med. Sci., 56, B459, 2001.
20. Stadtman, E.R., Importance of individuality in oxidative stress and aging, Free Radic. Biol.
Med., 33, 597, 2002.
21. Kurpad, A.V. and Vaz, M., Protein and amino acid requirements in the elderly, Eur. J. Clin.
Nutr., 54, S131, 2000.
22. Fukagawa, N.K., Yu, Y.M., and Young, V.R., Methionine and cysteine kinetics at different
intakes of methionine and cystine in elderly men and women, Am. J. Clin. Nutr., 68, 380, 1998.
23. Breuillé, D. and Obled, C., Cysteine and glutathione in catabolic states, in Protein, Peptides
and Amino-Acids in Enteral Nutrition, 3rd Nestlé Nutrition Workshop, Stockholm, Fürst, P. and
Young, V.R., Eds., Karger, Basel, Switzerland, 2000, p. 173.
24. Sastre, J. et al., Mitochondria, oxidative stress and aging, Free Radic. Res., 32, 189, 2000.
25. Eppler, B. and Dawson, R., Dietary taurine manipulations in aged male Fischer 344 rat tissue:
taurine concentration, taurine biosynthesis, and oxidative markers, Biochem. Pharmacol., 62,
29, 2001.
26. Fereday, A. et al., Protein requirements and ageing: metabolic demand and efficiency of
utilization, Br. J. Nutr., 77, 685, 1997.
27. Arnal, M.A. et al., Protein turnover modifications induced by the protein feeding pattern
still persist after the end of the diets, Am. J. Physiol., 278, E902, 2000.
28. Tessari, P., Changes in protein, carbohydrate, and fat metabolism with aging: possible role
of insulin, Nutr. Rev., 58, 11, 2000.
29. Kurpad, A.V. et al., Daily requirement for and splanchnic uptake of leucine in healthy adult
Indians, Am. J. Clin. Nutr., 74, 747, 2001.
30. Mosoni, L. et al., Altered response of protein synthesis to nutritional state and endurance
training in old rats, Am. J. Physiol., 268, E328, 1995.
31. Arnal, M.A. et al., Pulse protein feeding pattern restores stimulation of muscle protein
synthesis during the feeding period in old rats, J. Nutr., 132, 1002, 2002.
32. Dardevet, D. et al., Postprandial stimulation of muscle protein synthesis in old rats can be
restored by a leucine-supplemented meal, J. Nutr., 132, 95, 2002.
33. Rand, W.M., Pellett, P.L., and Young, V.R., Meta-analysis of nitrogen balance studies for
estimating protein requirements in healthy adults, Am. J. Clin. Nutr., 77, 109, 2003.
34. Anthony, J.C. et al., Orally administered leucine stimulates protein synthesis in skeletal
muscle of postabsorptive rats in association with increased eIF4F formation, J. Nutr., 130,
139, 2000.
35. Dardevet, D. et al., Stimulation of in vitro rat muscle protein synthesis by leucine decreases
with age, J. Nutr., 130, 2630, 2000.
1382_C29.fm Page 494 Tuesday, October 7, 2003 7:15 PM

494 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

36. Dangin, M. et al., Influence of the protein digestion rate on protein turnover in young and
elderly subjects, J. Nutr., 132, 3228S, 2002.
37. Arnal, M.A. et al., Protein pulse feeding improves protein retention in elderly women, Am.
J. Clin. Nutr., 69, 1202, 1999.
38. Cheng, A.H.R. et al., Comparative nitrogen balance study between young and aged adults
using three levels of protein intake from a combination wheat-soy-milk mixture, Am. J. Clin.
Nutr., 31, 12, 1978.
39. Rall, L.C. et al., Protein metabolism in rheumatoid arthritis and aging: effects of muscle
strength training and tumor necrosis factor alpha, Arthritis Rheum., 39, 1115, 1996.
40. Fereday, A. et al., Variation in the apparent sensitivity of the insulin-mediated inhibition of
proteolysis to amino acid supply determines the efficiency of protein utilization, Clin. Sci.,
95, 725, 1998.
41. FAO/WHO/UNU, Energy and Protein Requirements, WHO, Geneva, 1985.
42. Young, V.R., Bier, D.M., and Pellett, P.L., A theoretical basis for increasing current estimates
of the amino acid requirements in adult man with experimental support, Am. J. Clin. Nutr.,
50, 80, 1989.
43. Young, V.R. and El-Khoury, A.E., Can amino acid requirements for nutritional maintenance
in adult humans be approximated from the amino acid composition of body mixed proteins?
Proc. Natl. Acad. Sci. U.S.A., 92, 300, 1995.
44. Tuttle, S.G. et al., Further observation on amino acid requirements of older men. II. Methion-
ine and lysine, Am. J. Clin. Nutr., 16, 229, 1965.
45. Watts, J.H. et al., Nitrogen balances of men over 65 fed the FAO and milk patterns of essential
amino acids, J. Gerontol. A Biol. Sci. Med. Sci., 19, 370, 1964.
46. Tontsirin, K. et al., Plasma tryptophan response curve and tryptophan requirements of
elderly people, J. Nutr., 103, 1220, 1973.
47. Tontsirin, K. et al., Plasma threonine response curve and threonine requirements of young
men and elderly women, J. Nutr., 104, 495, 1974.
48. Waterlow, J.C., The requirements of adult man for indispensable amino acids, Eur. J. Clin.
Nutr., 50, S151, 1996.
49. Rand, W.M. and Young, V.R., Statistical analysis of nitrogen balance data with reference to
the lysine requirement in adults, J. Nutr., 129, 1920, 1999.
50. Millward, D.J. and Roberts, S.B., Protein requirements of older individuals, Nutr. Res. Rev.,
9, 67, 1996.
51. Morse, M.H. et al., Protein requirement of elderly women: nitrogen balance responses to
three levels of protein intake, J. Gerontol. A Biol. Sci. Med. Sci., 56, M724, 2001.
52. Campbell, W.W. et al., Dietary protein adequacy and lower body versus whole body resistive
training in older humans, J. Physiol. (Lond.), 542, 631, 2002.
53. Raguso, C.A. et al., Effect of cystine intake on methionine kinetics and oxidation determined
with oral tracers of methionine and cysteine in healthy adults, Am. J. Clin. Nutr., 66, 283, 1997.
54. Raguso, C.A., Regan, M.M., and Young, V.R., Cysteine kinetics and oxidation at different
intakes of methionine and cystine in young adults, Am. J. Clin. Nutr., 71, 491, 2000.
55. Castaneda, C. et al., Protein turnover and energy metabolism of elderly women fed a low-
protein diet, Am. J. Clin. Nutr., 62, 40, 1995.
56. Campbell, W.W. et al., Increased protein requirements in elderly people: new data and
retrospective reassessments, Am. J. Clin. Nutr., 60, 501, 1994.
57. Campbell, W.W. and Evans, W.J., Protein requirements of elderly people, Eur. J. Clin. Nutr.,
50, S180, 1996.
58. Pannemans, D.L.E. et al., The effect of an increase of protein intake on whole body protein
turnover in elderly women is tracer dependent, J. Nutr., 127, 1788, 1997.
59. Davis, T.A. et al., Amino acid composition of human milk is not unique, J. Nutr., 124, 1126,
1994.
60. FNB/IOM, Protein and amino acids, in Dietary Reference Intakes for Energy, Carbohydrates,
Fiber, Fat, Protein and Amino Acids (Macronutrients), National Academies Press, Washington,
D.C., 2002, chap. 10, p. 465.
1382_C29.fm Page 495 Tuesday, October 7, 2003 7:15 PM

Chapter twenty-nine: Amino acid requirements in the elderly 495

61. Tuttle, S.G. et al., Study of the essential amino acid requirements of men over fifty, Metabolism,
6, 564, 1957.
62. Hiramatsu, T. et al., Methionine and cysteine kinetics at different intakes of cystine in healthy
adult men, Am. J. Clin. Nutr., 60, 525, 1994.
1382_C29.fm Page 496 Tuesday, October 7, 2003 7:15 PM
1382_C30.fm Page 497 Tuesday, October 7, 2003 7:16 PM

chapter thirty

Amino acid requirements in sport


Michael Gleeson
Loughborough University
Asker E. Jeukendrup
University of Birmingham

Contents
Introduction..................................................................................................................................498
30.1 Exercise and protein metabolism ....................................................................................498
30.1.1 Effects of acute exercise on amino acid and protein metabolism ...............498
30.1.2 Effects of exercise training on protein metabolism........................................499
30.2 Diet–exercise interactions and protein metabolism......................................................500
30.3 Amino acid supplements..................................................................................................501
30.3.1 Amino acids as ergogenic aids..........................................................................502
30.3.1.1 Arginine.................................................................................................502
30.3.1.2 Aspartate ...............................................................................................503
30.3.1.3 Glutamine .............................................................................................503
30.3.1.3.1 Glutamine supplements and fluid balance..................504
30.3.1.3.2 Glutamine supplements and exercise performance ...504
30.3.1.3.3 Glutamine supplements and muscle anabolic
processes ............................................................................504
30.3.1.3.4 Glutamine supplements and muscle soreness ............504
30.3.1.3.5 Glutamine supplements and immune function ..........504
30.3.1.3.6 Is glutamine safe?.............................................................505
30.3.1.4 Isoleucine, leucine, and valine: the branched-chain
amino acids (BCAAs)..........................................................................505
30.3.1.4.1 BCAAs as a fuel for exercise ..........................................505
30.3.1.4.2 BCAAs to reduce net protein breakdown....................505
30.3.1.4.3 BCAAs and central fatigue .............................................506
30.3.1.4.4 BCAAs and endurance performance ............................506
30.3.1.4.5 BCAAs and immune responses to exercise .................508
30.3.1.5 Ornithine ...............................................................................................508
30.3.1.6 Taurine...................................................................................................508
30.3.1.7 Tyrosine .................................................................................................509
30.3.1.8 Tryptophan ...........................................................................................509

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 497
1382_C30.fm Page 498 Tuesday, October 7, 2003 7:16 PM

498 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

30.4 Do excessive intakes of protein or individual amino acids pose health risks
for athletes?.........................................................................................................................510
References .....................................................................................................................................510

Introduction
Many athletes believe that a high-protein diet will improve their performance. But what
is the evidence that athletes need to consume additional protein for optimal performance
or to maximize adaptation to training? Are supplements of individual amino acids effec-
tive as ergogenic aids or as boosters of immune function? Are excessive intakes of protein
and amino acids a risk to health? These are questions that are often asked, and the answers
are still subject to considerable debate.
Muscle contains a large proportion of the total protein in a human body (about 40%).
Muscle also accounts for 30 to 50% of all protein turnover in the body. Both the structural
proteins that make up the myofibrillar proteins and the proteins that act as enzymes within
a muscle cell change during adaptation to exercise training. Indeed, muscle mass, muscle
protein composition, and muscle protein content all change in response to training. There-
fore, it is not surprising that meat as a protein source for athletes (especially strength
athletes) has been very popular. There is an enormous interest among athletes in protein.
There are strong beliefs, which mainly originate from wishful thinking, that a large protein
intake or certain protein or amino acid supplements will increase muscle mass and
strength. Despite this long history of protein use in sports, there is still continuous debate
even about simple questions such as whether protein requirements are increased in ath-
letes compared with sedentary people. There are several reasons for this. First, protein
and amino acid metabolism is very complex, and many organs and tissues are involved.
Second, there are also several associated methodological difficulties. For example, the use
of different techniques to estimate protein turnover may give different results. This chapter
examines protein metabolism during acute bouts of exercise and the dietary protein
requirements of endurance and strength-training athletes. The known effects of supple-
menting the diet with various individual amino acids will be described, and the possible
impact of amino acid supplements on immune responses to exercise will be discussed.

30.1 Exercise and protein metabolism


Exercise has a number of specific effects on protein metabolism. Strength training results
in an increase in muscle mass,1 indicating increased formation of actin and myosin, and
it is tempting to assume that this process is dependent on the availability of dietary protein.
Endurance training is not associated with muscle hypertrophy, but there is an increase in
the muscle content of mitochondrial protein resulting in an increased capacity for oxidative
metabolism.2 Hard exercise and unaccustomed or eccentric muscle actions result in tem-
porary muscle damage, and there is clearly a role for protein in the repair and recovery
process. Adaptations to training are specific to the nature of the exercise stimulus and also
depend on an adequate supply of protein in the diet. However, the adaptive process cannot
be accelerated by increasing the protein intake above normal levels.

30.1.1 Effects of acute exercise on amino acid and protein metabolism


During exercise the muscle uptake of glutamate and branched-chain amino acids (BCAAs)
from the blood is increased; at the same time, the production and release of alanine and
glutamine by the muscle increases almost linearly with increasing exercise intensity.3 At
1382_C30.fm Page 499 Tuesday, October 7, 2003 7:16 PM

Chapter thirty: Amino acid requirements in sport 499

exercise intensities below 70% of the maximum oxygen uptake (VO2max), there is little
or no change in the concentration of amino acids in the muscle. At intensities above 70%
VO2max, a sharp decrease in the intramuscular glutamate and glutamine concentration
occurs.4 In prolonged exercise or very high intensity exercise, the net negative protein
balance that is normally observed in the hours after eating is increased.5 There is an
increased accumulation of amino acids that are not metabolized in the muscle (e.g., lysine
and threonine). Whether this is the result of increased protein breakdown, decreased
protein synthesis, or both is still unclear.
Rates of protein turnover appear to be increased after high-intensity exercise. There
are reports of increased protein breakdown after leg resistance exercise.6,7 Muscle protein
breakdown was increased after a resistance exercise session but to a smaller degree than
muscle protein synthesis.6–9 The elevations in protein degradation and synthesis are tran-
sient. Protein breakdown and synthesis after exercise were still elevated at 3 and 24 h after
exercise but returned to baseline levels after 48 h.8 These results seem to apply to resistance
exercise or dynamic exercise at a relatively high intensity. Low- to moderate-intensity
dynamic endurance exercise does not seem to have the same effects on muscle protein
breakdown, although studies have shown that endurance exercise may result in increased
protein oxidation, especially during the later stages of very prolonged exercise and in
conditions of glycogen depletion (i.e., reduced carbohydrate availability).5,10
During endurance exercise there is an increased rate of oxidation of leucine.11 Because
leucine is an essential amino acid that cannot be synthesized within the body, some authors
have interpreted this to imply that the dietary protein requirements are increased. It has
been estimated that protein may contribute about 5 to 15% to energy expenditure in resting
conditions. During exercise this relative contribution is likely to decrease to about 3 to 6%
because of the increasing importance of carbohydrate and fat as fuels. During very pro-
longed exercise, when carbohydrate availability becomes limited, the contribution of pro-
tein to energy expenditure may amount to about 10% of total energy expenditure.10 Thus,
although protein oxidation is increased during endurance exercise, the relative contribu-
tion of protein to energy expenditure remains small. Furthermore, the oxidized amino
acids do not appear to be derived from degradation of myofibrillar proteins.12 In fact, only
6 of the 20 available amino acids available are oxidized by muscle.

30.1.2 Effects of exercise training on protein metabolism


Adaptation to exercise training results from the cumulative effects of a series of exercise
bouts. During the first few weeks of a resistance-training program, there is little change
in muscle mass and initial strength increase seems to be mostly attributable to neural
mechanisms.1 Over the next few months there is a further increase in muscle strength,
which is associated with an increase in muscle size. However, after a year or so of such
a program, there is little further change in muscle mass. Endurance exercise training
stimulates increases in mitochondrial proteins but with little change in total muscle protein
content or muscle mass.2
Because the oxidation of some essential amino acids is increased during aerobic
exercise,11 it has been suggested that the dietary protein requirements are increased in
endurance athletes. Studies in which a nitrogen balance technique was used confirmed
that the dietary protein requirements for athletes involved in prolonged endurance train-
ing are higher than those for sedentary individuals.13,14 However, these results have been
questioned by other authors who did not find such differences, or even reported improved
nitrogen and leucine balance in more active individuals.15 A recent study found that men
could maintain a nitrogen and leucine balance when exercising a total of 3 h per day at
50% VO2max when ingesting a diet containing either a moderate protein intake (1 g/kg
1382_C30.fm Page 500 Tuesday, October 7, 2003 7:16 PM

500 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

of body mass) or a high protein intake (2.5 g/kg of body mass).16 Even though most
researchers agree that exercise will increase protein oxidation to some extent and that this
is accompanied by increased nitrogen losses, there is still controversy about whether
athletes have to eat more protein than less active individuals. Several research groups
claim that there is evidence that this is the case.5,8,17 Others believe that there is not enough
evidence to make such a statement.15 The research groups that advocate an increased
protein intake for endurance athletes usually recommend an intake of 1.2 to 1.8 g/kg of
body mass (as opposed to the recommended intake of 0.8 g/kg of body mass for the
average population).
Even if protein requirements are increased by exercise, in practice it should be no
problem for athletes to meet these needs. As an extreme example, we could look at the
Tour de France. Cyclists in this 3-week event compete for 3 to 7 h per day and maintaining
energy balance is often problematic.18,19 Nevertheless, in this situation they seem to have
no problems in maintaining nitrogen balance.20 With increasing food intake, the intake of
protein automatically increases because many food products contain at least some protein.
A study by van Erp-Baart and colleagues21 showed a linear relationship between energy
intake and protein intake. Tour de France cyclists consumed 12% of their daily energy
intake (26 MJ, 6500 kcal) in the form of protein, and they easily met the suggested increased
requirements (about 2.5 g/kg of body mass/day).20 These results suggest that provided
that energy intake matches energy expenditure on a daily basis, there is no need for
endurance athletes to supplement their diets with protein. In spite of this, however, many
athletes ingest large quantities of protein-containing foods and expensive protein supple-
ments. Indeed, daily protein intakes of up to 400 g are known in some sports.21,22
Hypertrophy resulting from strength training must be caused by increased protein
synthesis. This protein synthesis must occur in the recovery phase in between training
sessions. Studies have clearly shown that the body seems to adapt to training by being
more efficient with protein.16 Protein turnover seems to be decreased after training, and
there is less net protein degradation.9 In other words, after training athletes become more
efficient and will “waste” less protein. To further support this, it has been shown that
BCAA oxidation at the same relative workload is the same in untrained and trained
individuals.23 So, although initially there may be an increased protein requirement, after
adaptation to the training this seems to disappear. The nitrogen balance studies on resis-
tance-training individuals that have been done have been criticized because they generally
have been of short duration and a steady-state situation may not have been established.15
Gontzea et al.13 showed that the negative nitrogen balance that is used by many authors
to indicate an increased protein requirement disappears after approximately 12 days of
training. The protein requirements may therefore only be temporarily elevated. It is likely,
however, that with increasing training stimuli, the protein requirements may increase
again. The recommendation for strength athletes is therefore generally to consume 1.6 to
1.7 g of protein/kg of body mass/day.5 Again, this can be easily met with a normal diet
(see Table 30.1), and no extra attention to protein intake is needed.

30.2 Diet–exercise interactions and protein metabolism


Nutrition always plays a very important role in the establishment of training adaptations.
In the hours after exercise, protein synthesis may exceed protein degradation, but this will
only occur after feeding. If feeding is delayed by 24 to 48 h, net protein balance will remain
negative and no muscle hypertrophy will occur.15 Feeding a mixed diet not only will
provide substrates but also will result in a favorable hormonal milieu for protein synthesis.
In resting conditions, higher amino acid concentrations in plasma have a stimulatory effect
on protein synthesis.24,25 Rises in the circulating concentrations of glucose and amino acids
1382_C30.fm Page 501 Tuesday, October 7, 2003 7:16 PM

Chapter thirty: Amino acid requirements in sport 501

Table 30.1 Daily Protein Intake of Athletes with Different Levels of Energy Intake
Energy intake Protein intake
60-kg athlete (typical female) 8 MJ (2000 kcal) = 60–75 g of protein = 1.0–1.3 g/kg
12 MJ (3000 kcal) = 90–112 g of protein = 1.5–1.9g/kg
68-kg athlete (typical male) 12 MJ (3000 kcal) = 90–112 g of protein = 1.3–1.6 g/kg
20 MJ (5000 kcal) = 150–188 g protein = 2.1–2.7 g/kg
26 MJ (6500 kcal) = 195–244 g of protein = 2.9–3.6 g/kg
90-kg athlete (e.g., heavy male 12 MJ (3000 kcal) = 90–112 g of protein = 1.0–1.2 g/kg
resistance exercise trainer) 16 MJ (4000 kcal) = 120–148 g of protein = 1.3–1.7 g/kg
20 MJ (5000 kcal) = 150–188 g of protein = 1.7–2.1 g/kg

Note: These calculations assume that the diet contains 12–15% of energy as protein.

will also result in an increased plasma insulin concentration, which in turn will cause a
significant reduction of protein breakdown and a small increase in protein synthesis.6,25
Increased availability of amino acids immediately after exercise has a larger effect on
protein synthesis than resting conditions.6 Amino acids and exercise thus seem to have an
additive effect on net protein synthesis. It must be noted, however, that in these studies
amino acids were infused and plasma amino acid concentrations were elevated to very
high levels. Intravenous infusion bypasses the liver, and the liver normally extracts 20 to
90% of all amino acids after absorption (first-pass splanchnic extraction). It was therefore
not clear if similar effects were to be expected after oral ingestion of amino acids. A follow-
up study investigated this question.26 In this study a relatively large amount of amino
acids was ingested after resistance exercise. Postexercise muscle protein balance was
negative after placebo ingestion, but when amino acids were ingested, the net balance
was positive. This was mainly caused by an increased muscle protein synthesis. From this
study and a limited number of other studies we can conclude that ingestion of amino
acids after exercise will enhance net protein synthesis.
Carbohydrate ingestion per se may not have an effect on protein synthesis. However,
carbohydrate ingestion may elevate the plasma insulin concentration and thereby reduce
the breakdown of protein.25 It seems that the combined ingestion of amino acids (protein)
and carbohydrate is the preferred meal after exercise. The protein will deliver the substrate
(amino acids), and carbohydrate will further increase the anabolic hormonal milieu
required for net protein synthesis. Direct evidence in support of this hypothesis was
recently provided by a study by Rasmussen et al.27 After a strenuous bout of resistance
exercise subjects were fed 6 g of essential amino acids plus 35 g of glucose. Plasma amino
acids levels increased 3-fold and the plasma insulin concentration increased 10-fold. Mus-
cle protein synthesis was increased 3.5-fold, while there was no increase in protein break-
down. In the control condition a net protein breakdown was observed in the first 3 h after
exercise. These results confirm that the ingestion of a relatively small amount of amino
acids with a larger amount of carbohydrate can increase net muscle protein synthesis.

30.3 Amino acid supplements


In the past the amino acid needs of the body were primarily met by ingestion of whole
proteins in the diet. However, over the last few years the supplementation of individual
amino acids has become increasingly popular. Technological advances have made it pos-
sible to manufacture food-grade pure amino acids. The individual amino acids, called
free-form amino acids, are mostly produced by bacterial fermentation.
1382_C30.fm Page 502 Tuesday, October 7, 2003 7:16 PM

502 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Scientific studies are focusing more and more on the pharmacologic and metabolic
interactions of free-form amino acids. Considerable progress has been made in the area
of clinical nutrition, where individual amino acids, among others, are used to reduce
nitrogen losses and improve organ functions in traumatized and critically ill patients.
Individual amino acids also are marketed these days as supplements for athletes and
healthy individuals. Intake of certain individual amino acids is often claimed to improve
exercise performance, stimulate hormone release, and improve immune function among
a variety of other positive effects. This section will review the facts and fallacies of these
claims.

30.3.1 Amino acids as ergogenic aids


In recent years amino acid supplements have become increasingly available and popular
in certain athletic circles. Weight lifters and body builders consume various amino acids
in attempts to stimulate the release of growth hormone from the pituitary gland, hoping
that the growth hormone will then stimulate muscle development. Amino acids have also
been used to stimulate the release of insulin from the pancreas. Insulin is considered an
anabolic hormone because it facilitates the uptake of amino acids as well as glucose by
muscle cells.25 In addition to these effects on protein synthesis, certain amino acids are
claimed to provide an extra fuel to the muscle and to prevent fatigue by changing the
concentrations of brain neurotransmitters. Other amino acids have been used in attempts
to reduce immunosuppression during strenuous training, to increase adenosine triphos-
phate (ATP) and phosphocreatine (PCr) levels in the muscle, and to help athletes lose
body weight.
Scientific studies have shown that ingestion of amino acids can have profound phys-
iological effects. However, amino acid metabolism is very complex. One amino acid can
be converted into another, and amino acids may influence nerve impulse transmission as
well as hormone secretion. Large intakes of individual amino acids or even high-protein
diets may lead to nutritional imbalances because overload of one amino acid may reduce
the absorption of other amino acids.
The following section will discuss the latest scientific findings with regard to the intake
of individual amino acids or combinations of amino acids.

30.3.1.1 Arginine
Research has shown that infusion of arginine (ARG) into the blood can stimulate the
release of growth hormone from the pituitary gland.28,29 ARG is not the only amino acid
that can have such an effect; lysine and ornithine may also stimulate the release of
hormones from endocrine glands. The intravenous administration of ARG to adult humans
in doses of 30 g in 30 min caused a marked increase in the secretion of human pituitary
growth hormone.28,29 Intravenous and oral ARG administration also resulted in a marked
insulin release from the b-cells of the pancreas in humans.30,31 The contention that ARG
increases the secretion of anabolic hormones such as human growth hormone and insulin
has made ARG a very popular supplement among body builders and strength athletes.
The doses of ARG present in sports nutritional supplements are usually small in compar-
ison with the intravenous doses that have been shown to have potent secretagogue actions.
Well-controlled, double-blind, crossover studies32,33 failed to show an effect of oral ARG
supplementation taken in these low quantities on the daily variation (24-h profile) and
concentrations of growth hormone and insulin in male competitive weight lifters and
body builders. The ingestion of larger doses of ARG can cause severe gastrointestinal
discomfort.34
1382_C30.fm Page 503 Tuesday, October 7, 2003 7:16 PM

Chapter thirty: Amino acid requirements in sport 503

In conclusion, although ARG infused in large quantities can have anabolic properties,
oral ingestion of tolerable amounts (amounts that do not cause gastrointestinal problems)
will not result in secretion of human growth hormone and insulin. Certainly large increases
in insulin secretion can be obtained by ingestion of carbohydrate, and much larger
increases in plasma growth hormone are observed after exercise than with even large
doses of ARG and other individual amino acids.34

30.3.1.2 Aspartate
It is often claimed that aspartate (ASP) improves aerobic exercise performance. ASP is a
precursor of the tricarboxylic acid (TCA) cycle intermediates and reduces plasma ammonia
accumulation during exercise. Since ammonia formation is associated with the develop-
ment of fatigue, ASP supplementation could theoretically be ergogenic. In a study by
Maughan and Sadler,35 eight subjects rode to exhaustion at 75 to 80% VO2max after
ingestion of 6 g of ASP (as magnesium and potassium salts) or placebo over 24 h. No
effect was observed on the plasma ammonia concentration or exercise time to exhaustion.
One study36 reported a 15% improvement in performance when subjects rode to exhaus-
tion at 75% VO2max after ingestion of 10 g of ASP in the 24 h prior to the test. This was
associated with decreased plasma ammonia levels and increased plasma fatty acid levels
with ASP ingestion. However, Tuttle et al.37 reported no effect of ASP supplementation on
plasma ammonia responses to a bout of resistance exercise.

30.3.1.3 Glutamine
Glutamine (GLN) is important as a constituent of proteins and as a means of nitrogen
transport between tissues. It is also important in acid–base regulation and as a precursor
of the antioxidant glutathione. GLN is the most abundant free amino acid in human muscle
and plasma. Its alleged effects can be classified as anabolic and immunostimulatory.
Loosely based on an uncritical evaluation of the scientific literature, GLN supplements
are claimed by various manufacturers and suppliers to have the following effects that may
benefit the athlete:

• More rapid water absorption from the gut


• Improved intracellular fluid retention (i.e., a volumizing effect)
• Improved gut barrier function and reduced risk of endotoxemia
• Nutritional support for immune system and prevention of infection
• Stimulation of muscle protein synthesis and muscle tissue growth
• Stimulation of muscle glycogen resynthesis
• Reduction in muscle soreness and improved muscle tissue repair
• Enhanced buffering capacity and improved high-intensity exercise performance

The normal daily intake of GLN from dietary protein is about 3 to 6 g per day
(assuming a daily intake of 0.8 to 1.6 g of protein/kg of body mass for a 70-kg individual).
Researchers examining the effects of GLN on the postexercise decline in plasma GLN
concentration report that a dose of about 0.1 g of GLN per kg of body mass has to be
given every 30 min over a 2- to 3-h period to prevent the fall in the plasma GLN concen-
tration.38
GLN is utilized at high rates by white blood cells (particularly lymphocytes) to provide
energy and optimal conditions for nucleotide biosynthesis and hence cell proliferation.39
Indeed, GLN is considered important, if not essential, to lymphocytes and other rapidly
dividing cells, including the gut mucosa and bone marrow stem cells. Prolonged exercise
is associated with a fall in the intramuscular and plasma concentration of GLN, and it has
1382_C30.fm Page 504 Tuesday, October 7, 2003 7:16 PM

504 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

been hypothesized that this decrease in GLN availability could impair immune function.40
Periods of very heavy training are associated with a chronic reduction in plasma GLN
levels, and it has been suggested that this may be partly responsible for the immunosup-
pression apparent in many endurance athletes.40 The intramuscular concentration of GLN
is known to be related to the rate of net protein synthesis,41 and there is also some evidence
for a role for GLN in promoting glycogen synthesis.42 However, the mechanisms under-
lying these alleged anabolic effects of GLN remain to be elucidated.

30.3.1.3.1 Glutamine supplements and fluid balance. GLN is not included in commer-
cial sports drinks mainly because of its relative instability in solution. Water transport
from the gut into the circulation is known to be promoted by the presence of glucose and
sodium in drinks consumed. This is because water movement is determined by osmotic
gradients, and the cotransport of sodium and glucose into the gut epithelial cells is
accompanied by the osmotic movement of water molecules in the same direction. GLN
is transported into gut epithelial cells by both sodium-dependent and sodium-indepen-
dent mechanisms, and the addition of GLN to oral rehydration solutions has been shown
to increase the rate of fluid absorption above that of ingested water alone.43 However, the
potential benefits of adding GLN to commercially available sports drinks have not been
adequately tested, and any additional benefit in terms of increased rate of fluid absorption
and retention is likely to be very small indeed.

30.3.1.3.2 Glutamine supplements and exercise performance. Placebo-controlled stud-


ies that have investigated the effects of GLN supplementation on extracellular buffering
capacity and high-intensity exercise performance have not found any beneficial effect.44

30.3.1.3.3 Glutamine supplements and muscle anabolic processes. Muscle protein break-
down occurs in the fasted state. Recent research indicates that resistance exercise reduces
the extent of this protein catabolism, but an anabolic (muscle growth) response requires
an intake of essential amino acids (dietary protein) in the recovery period after exer-
cise.15,26,27 This promotes amino acid uptake into muscle and increases tissue protein
synthesis rate without affecting the rate of protein breakdown. Provided that ingested
protein contains the eight essential amino acids, taking supplements of individual non-
essential amino acids at this time is unlikely to be of any additional benefit. Protein
synthesis in the tissues of the body requires the simultaneous presence of all 20 amino
acids. There is some evidence for an effect of GLN supplements in promoting glycogen
synthesis in the first few hours of recovery after exercise,42 but further work using optimal
amounts of carbohydrate feeding after exercise needs to be done to substantiate this
finding and give it practical relevance. Thus, a postexercise meal consisting of predomi-
nantly carbohydrate with some protein would seem to be the best strategy to promote
both glycogen and protein synthesis in muscle after exercise.

30.3.1.3.4 Glutamine supplements and muscle soreness. Eccentric exercise-induced


muscle damage does not affect the plasma GLN concentration.45 There is no scientific
evidence for a beneficial effect of oral GLN supplementation on muscle repair after exer-
cise-induced damage and no evidence of reduced muscle soreness when consuming GLN
compared with placebo.

30.3.1.3.5 Glutamine supplements and immune function. Several scientists have sug-
gested that exogenous provision of GLN supplements may be beneficial by preventing
the impairment of immune function following endurance exercise. Prolonged exercise at
50 to 70% VO2max has been shown to cause a 10 to 30% fall in plasma GLN concentration
1382_C30.fm Page 505 Tuesday, October 7, 2003 7:16 PM

Chapter thirty: Amino acid requirements in sport 505

that may last for several hours during recovery.38,40,46 This fall in plasma GLN coincides
with the window of opportunity for infection following prolonged exercise when an
athlete is more susceptible to opportunist infections.38 One study showed that an oral GLN
supplement (5 g in 330 ml of water) consumed immediately after and 2 h after a marathon
reduced the incidence of upper respiratory tract infection in the 7 days following the race.47
However, it is unlikely that the dose given in that study was sufficient to prevent the
postexercise fall in the plasma GLN concentration. Furthermore, several recent studies
that have investigated the effect of GLN supplementation during exercise on various
indices of immune function have failed to find any beneficial effect. A larger dose of GLN
(0.1 g per kg of body mass) than that given by Castell and Newsholme47 ingested at 0, 30,
60, and 90 min following a marathon race prevented the fall in the plasma GLN concen-
tration but did not prevent the fall in mitogen-induced lymphocyte proliferation and
lymphokine-activated killer cell activity.46 Similarly, maintaining the plasma GLN concen-
tration by consuming glutamine in drinks taken both during and after a prolonged bout
of cycling did not affect leukocyte subset trafficking or prevent the exercise-induced fall
in neutrophil function, lymphocyte proliferative response, natural killer cell activity, and
salivary immunoglobulin (Ig) A secretion rate.48–50 Unlike the feeding of carbohydrate51
during exercise, it seems that GLN supplements do not affect the immune function per-
turbations that have been examined to date. A recent review by Hiscock and Pedersen52
concluded that the small (~20%) and temporary fall in the plasma glutamine concentration
after exercise is unlikely to play a mechanistic role in exercise-induced immunosuppres-
sion.

30.3.1.3.6 Is glutamine safe? GLN is thought to be relatively safe and well tolerated
by most people. Administration of GLN to people with kidney disorders is not recom-
mended. No adverse reactions to short-term GLN supplementation have been reported.
Excessive doses may cause gastrointestinal problems. No information is available on long-
term use of supplements exceeding 1 g per day.

30.3.1.4 Isoleucine, leucine, and valine: the branched-chain amino acids (BCAAs)
The three BCAAs — leucine, isoleucine, and valine — are not synthesized in the body.
Yet, they are oxidized during exercise and must therefore be replenished by the diet. In
the late 1970s it was suggested that BCAAs were the third fuel for skeletal muscle after
carbohydrate and fat.53 BCAAs are nowadays often sold to athletes as part of energy drinks
to provide extra fuel. Claims have also been made that BCAA supplementation can reduce
net protein breakdown in muscle during exercise and may reduce fatigue and enhance
performance via effects on the brain.

30.3.1.4.1 BCAAs as a fuel for exercise. As mentioned above, it has been suggested
that BCAAs could act as a fuel during exercise in addition to carbohydrate and fat.53 More
recently, however, it was shown that the activities of the enzymes involved in the oxidation
of BCAAs are too low to allow a major contribution of BCAAs to energy expenditure.54
Detailed studies with 13C-labeled BCAAs (e.g., 13C-leucine) have shown that the oxidation
of BCAAs only increases 2- to 3-fold during exercise, whereas the oxidation of carbohy-
drate and fat increases 10- to 20-fold.11,55 It has also been shown that carbohydrate ingestion
during exercise can prevent the increase in BCAA oxidation.10 The BCAAs, therefore, do
not seem to play an important role as a fuel during exercise, and from this point of view,
there is no reason for the supplementation of BCAAs during exercise.34

30.3.1.4.2 BCAAs to reduce net protein breakdown. The claims that BCAAs reduce
protein breakdown are mainly based on early in vitro studies that showed that adding
1382_C30.fm Page 506 Tuesday, October 7, 2003 7:16 PM

506 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

BCAAs to an incubation or perfusion medium stimulated tissue protein synthesis and


inhibited protein degradation. Several in vivo studies in healthy individuals56–58 failed to
confirm this positive effect on protein balance that had been observed in vitro. No studies
to date have demonstrated an improved nitrogen balance during or after exercise with
BCAA supplements. Therefore, it must be concluded that no valid scientific evidence exists
to support the commercial claims that orally ingested BCAAs have an anticatabolic effect
during and after exercise or that BCAA supplements may accelerate the repair of muscle
damage after exercise.34

30.3.1.4.3 BCAAs and central fatigue. In 1987, the central fatigue hypothesis was
put forward as an important mechanism contributing to the development of fatigue during
prolonged exercise.59 An illustration of this hypothesis is given in Figure 30.1a. During
exercise free fatty acids (FFAs) are mobilized from adipose tissue and are transported via
the blood to the muscles to serve as fuel. Because the rate of mobilization is greater than
the rate of uptake by the muscle, the blood FFA concentration increases. Both FFAs and
the free amino acid tryptophan (fTRP) bind to albumin and compete for the same binding
sites. TRP is displaced from binding to albumin by the increasing FFA concentration, and
therefore, the fTRP concentration and the fTRP:BCAA ratio in the blood rise. Experimental
studies in humans have confirmed that these events occur.60 The central fatigue hypothesis
predicts that the increase in this ratio results in an increased fTRP transport across the
blood–brain barrier, because BCAA and fTRP compete for carrier-mediated entry into the
central nervous system by the large neutral amino acid (LNAA) transporter.61,62 Once taken
up, the conversion of fTRP to serotonin (also known as 5-hydroxy tryptamine (5-HT))
occurs and leads to a local increase of this neurotransmitter.61 It has been well established
that serotonin plays a role in the onset of sleep and that it is a determinant of mood and
aggression. It was therefore hypothesized that the increase in serotoninergic activity sub-
sequently leads to central fatigue, forcing athletes to stop exercise or reduce the exercise
intensity.59 Of course, it is a rather large “leap of faith” to assume that increased fTRP
uptake will lead to increased serotonin synthesis and activity of serotoninergic pathways
(i.e., increased synaptic serotonin release).
The involvement of plasma fTRP and BCAAs in the central fatigue hypothesis also
predicts that ingestion of BCAAs will raise the plasma BCAA concentration and hence
reduce transport of fTRP into the brain (Figure 30.1b). Subsequent reduced formation of
serotonin may alleviate sensations of fatigue and hence improve endurance exercise per-
formance.63 If the central fatigue hypothesis is correct and the ingestion of BCAAs reduces
the exercise-induced increase of brain fTRP uptake and thus delays fatigue, the opposite
must also be true. That is, ingestion of TRP before exercise should reduce the time to
exhaustion. A few studies have supplemented human subjects with TRP before or during
exercise,64,65 and from these it must be concluded that there are no effects on exercise
performance (see Section 30.3.1.8).

30.3.1.4.4 BCAAs and endurance performance. The effect of BCAA ingestion on


physical performance was investigated for the first time in a field test by Blomstrand
et al.60 One hundred and ninety-three male subjects were studied during a marathon in
Stockholm. Subjects were randomly divided into an experimental group receiving BCAAs
in plain water and a placebo group receiving flavored water. The subjects also had free
access to carbohydrate-containing drinks. No difference was observed in the marathon
time of the two groups. However, when the original subject group was divided into groups
of fast and slower runners, then a small significant reduction in marathon time was
observed in subjects supplemented with BCAAs in the slower runners only. This study
has since been criticized for its design and statistical analysis. Later studies with various
1382_C30.fm Page 507 Tuesday, October 7, 2003 7:16 PM

Chapter thirty: Amino acid requirements in sport 507

Blood Brain

fTrp
LNAA
fTrp fTrp fTrp
BCAA
fTrp
Serotonin
FFA FFA
Alb FFA
FFA

(a) FATIGUE

Blood Brain

fTrp
LNAA
BCAA BCAA fTrp
BCAA
BCAA
Serotonin
FFA
Trp
Alb
FFA

(b) FATIGUE
Figure 30.1 Central fatigue hypothesis. (a) The central fatigue hypothesis59 proposes that during
exercise, rises in the plasma concentration of FFAs displace TRP bound to albumin (Alb) so that the
fTRP concentration and the fTRP:BCAA ratio in the blood rises. The increase in this ratio results in
an increased fTRP transport across the blood–brain barrier, because BCAAs and fTRP compete for
carrier-mediated entry into the central nervous system by the LNAA transporter. Once taken up,
the conversion of fTRP to serotonin (5-HT) occurs and leads to a local increase of this neurotrans-
mitter. An increase in serotoninergic activity subsequently leads to central fatigue, forcing athletes
to stop exercise or reduce the exercise intensity. (b) The involvement of plasma fTRP and BCAAs
in the central fatigue hypothesis also predicts that ingestion of BCAAs will raise the plasma BCAA
concentration and hence reduce transport of fTRP into the brain. Subsequent reduced formation of
serotonin may alleviate sensations of fatigue and hence improve endurance exercise performance.

exercise and treatment designs and several forms of administration of BCAAs (infusion,
oral, with and without carbohydrate) all failed to find a performance effect of BCAAs.65–68
Blomstrand et al.66 compared a placebo (water) with a solution of BCAAs in seven trained
1382_C30.fm Page 508 Tuesday, October 7, 2003 7:16 PM

508 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

endurance cyclists and did not find an effect on total work performed during a 20-min
time trial after 1 h of exercise. Van Hall et al.65 studied time trial performance in trained
cyclists consuming carbohydrate during exercise with and without BCAAs. In this study
a high and a low dose of BCAAs was given, but there were no significant differences in
time trial performance.

30.3.1.4.5 BCAAs and immune responses to exercise. Prolonged strenuous exercise is


associated with a temporary immunosuppression, which affects macrophages, neutro-
phils, and lymphocytes.51,52 The mechanisms involved are not fully established and appear
to be multifactorial, including hormonal actions (e.g., of catecholamines and cortisol),
inhibition of macrophage and T-cell cytokine production, altered heat shock protein
expression, and a fall in the plasma concentration of glutamine (see Section 30.3.1.3 on
glutamine).51 The amino group from BCAAs can be donated to glutamate to form
glutamine, and some studies have evaluated the effectiveness of BCAA supplements
during exercise to maintain the plasma glutamine concentration and modify immune
responses to exercise. One recent study69 showed that BCAA supplementation (6 g/day)
for 2 to 4 weeks and a 3-g dose 30 min before a long-distance run or triathlon race prevented
the 24% fall in the plasma glutamine concentration observed in the placebo group and
also modified the immune response to exercise. These authors reported that BCAA sup-
plementation did not affect the lymphocyte proliferative response to mitogens before
exercise but did prevent the 40% fall in lymphocyte proliferation observed after exercise
in the placebo group. Furthermore, blood mononuclear cells obtained from athletes in the
placebo group after exercise presented a reduction in the production of several cytokines,
including tumour necrosis factor-a (TNF-a), interferon-g (IFN-g), interleukin-1 (IL-1), and
IL-4 compared with before exercise. BCAA supplementation restored the production of
TNF-a and IL-1 and increased that of IFN-g. However, athletes given BCAA supplements
presented an even greater reduction in IL-4 production after exercise. There were, however,
flaws in the experimental design and statistical analysis of the data in this study, and the
results need to be confirmed in more controlled studies. Since several previous studies
have indicated that glutamine supplementation during exercise does not prevent the
exercise-induced fall in lymphocyte proliferation,46,50 these findings must be viewed with
some caution.

30.3.1.5 Ornithine
It has been suggested that ornithine (ORN) stimulates growth hormone release from the
pituitary gland70 and insulin from the pancreas. Growth hormone release after infusion
of ORN was even higher than that observed after ARG infusion. However, most com-
mercially available ORN supplements contain only 1 to 2 g of ORN, and this does not
affect the 24-h hormone profile.32 Therefore, ORN does not seem to increase growth
hormone release, muscle mass, or strength. Although it is often claimed that ORN
increases the secretion of insulin from the pancreas, such an effect has not been demon-
strated in controlled experimental studies.71 (See Chapter 37 for more details and addi-
tional discussion.)

30.3.1.6 Taurine
Taurine (TAU) is a nonprotein amino acid and a derivative of cysteine. TAU has recently
become a popular ingredient of many sports and disco drinks. The concentrations of TAU
in the brain, heart, and muscle are high, but its role is not clearly understood (see Chapters
39 and 44 for more details). It has been suggested to act as a membrane stabilizer, an
1382_C30.fm Page 509 Tuesday, October 7, 2003 7:16 PM

Chapter thirty: Amino acid requirements in sport 509

antioxidant, and a neuromodulator.72 TAU plays an undefined role in calcium currents in


cells, influences ionic conductance in excitable membranes, and plays a role in the regu-
lation of cell volume (see Chapter 44 for more details). Its value as a nutrition supplement
is unresolved.

30.3.1.7 Tyrosine
Several studies have shown that oral doses (5 to 10 g) of tyrosine (TYR) result in increases
in circulating levels of adrenaline, noradrenaline, and dopamine, hormones that are impor-
tant in the regulation of body function during physical stress and exercise.73,74 TYR sup-
plements are especially used by strength athletes because of the supposed effect of
activating metabolic pathways. One study by Struder et al.75 found no effect of ingestion
of 20 g of TYR on exercise performance, although the exercise-induced increase in plasma
prolactin concentration was higher with TYR supplementation, suggesting that changes
in the monoamiergic system were produced. Most other studies that have investigated
the effects of TYR supplements have used very low dosages (milligrams), whereas prob-
ably large doses are required to alter hormone levels. It is important to note that regular
supplementation of large amounts (more than 5 g) may have adverse health effects in the
long term, since TYR affects sympathetic nervous system activity.

30.3.1.8 Tryptophan
Tryptophan (TRP) is one of the amino acids that was suggested to stimulate the release
of growth hormone. The most common proposed ergogenic effect, however, is based on
another function: TRP is the precursor of serotonin, a neurotransmitter in the brain that
may induce sleepiness, decrease aggression, and elicit a mellow mood. It has also been
suggested that serotonin decreases the perception of pain. Segura and Ventura76 hypoth-
esized that TRP supplementation may increase serotonin levels and the tolerance of pain,
and thereby improve exercise performance. They studied 12 subjects during running to
exhaustion at 80% VO2max with ingestion of TRP or placebo. TRP was supplemented in
four doses of 300 mg in the 24 h prior to the endurance test, with the last dose being
ingested 1 h prior to the test (total TRP ingestion, 1200 mg). They observed a 49% improve-
ment in endurance capacity and decreased ratings of perceived exertion following TRP
ingestion. Since a 49% performance improvement seemed somewhat unrealistic, several
other investigators have challenged the results of this study.63,64 In a study by Stensrud
et al.,64 49 well-trained male runners were exercised to exhaustion at 100% VO2max and
no significant effect of TRP supplementation on time to fatigue was found. A very well
controlled study by Van Hall et al.65 supplemented eight cyclists with TRP and found no
effect on endurance time at 70% VO2max.
It is interesting that both TRP and BCAAs (see Section 30.3.1.4.3) have been suggested
as supplements to reduce central fatigue. Yet the BCAAs and TRP have opposite effects.
Whereas some have claimed that TRP reduces central fatigue,76 others have associated it
with the development of central fatigue63 (see Section 30.3.1.4 on BCAAs). It must also be
noted that TRP could exert some negative effects, including a blocking of gluconeogenesis
and decreased alertness. Based on these studies, TRP does not seem to be ergogenic and
may even be ergolytic in prolonged exercise. It is important to note that in 1989 in the
U.S. an epidemic growth of the eosinophilia-myalgia syndrome (EMS), a neuromuscular
disorder characterized by weakness, fever, edema, rashes, bone pain, and various other
symptoms, was attributed to the excessive intake of TRP. TRP has been classified as
neurotoxic and is now banned in the U.S.
1382_C30.fm Page 510 Tuesday, October 7, 2003 7:16 PM

510 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

30.4 Do excessive intakes of protein or individual amino acids pose


health risks for athletes?
Excessive protein intake (more than 3 g/kg of body mass/day) may have various negative
effects on health, including kidney damage, increased blood lipoprotein levels (which has
been associated with the development of artheriosclerosis), and dehydration.77 The latter
may occur as a result of increased nitrogen (ammonia and urea) excretion in urine. This
will result in increased urinary volume and dehydration if not compensated by an
increased fluid intake. Athletes consuming a high-protein diet therefore have to increase
their fluid intake in order to prevent dehydration. The recommended protein intakes for
athletes (1.2 to 1.8 g/kg of body mass/day) and up to approximately 2 g/kg of body
mass/day do not seem to be harmful.
It is only recently that individual amino acids have become commercially available.
Intake of individual amino acids has no added nutritional value compared with the intake
of proteins containing these amino acids. A possible advantage of the intake of individual
amino acids is that larger amounts can be ingested. Purified amino acids were developed
for clinical use in intravenous infusion of patients for adequate protein nutrition (partic-
ularly when oral consumption is compromised). Individual amino acids were also used
as food additives to enhance the protein balance in case the diet was deficient in certain
amino acids. Because amino acids are often ingested in pharmacological doses and the
effects of the ingestion of large doses of individual amino acids are largely unknown,
amino acids supplements should be treated as drugs.

References
1. Jones, D.A. and Rutherford, O.M., Human muscle strength training: the effects of three
different regimens and the nature of the resultant changes, J. Physiol. (Lond.), 391, 1, 1987.
2. Holloszy, J.O. and Coyle, E.F., Adaptations of skeletal muscle to endurance exercise and their
metabolic consequences, J. Appl. Physiol., 56, 831, 1984.
3. Wagenmakers, A.J., Protein and amino acid metabolism in human muscle, Adv. Exp. Med.
Biol., 441, 307, 1998.
4. Blomstrand, E., Andersson, S., Hassmen, P., Ekblom, B., and Newsholme, E.A., Effect of
branched-chain amino acid and carbohydrate supplementation on the exercise-induced
change in plasma and muscle concentration of amino acids in human subjects, Acta Physiol.
Scand., 153, 87, 1995.
5. Lemon, P.W., Effects of exercise on dietary protein requirements, Int. J. Sport Nutr., 8, 426,
1998.
6. Biolo, G., Maggi, S.P., Williams, B.D., Tipton, K.D., and Wolfe, R.R., Increased rates of muscle
protein turnover and amino acid transport after resistance exercise in humans, Am. J. Physiol.,
268, E514, 1995.
7. Biolo, G., Williams, B.D., Fleming, R.Y., and Wolfe, R.R., Insulin action on muscle protein
kinetics and amino acid transport during recovery after resistance exercise, Diabetes, 48, 949,
1999.
8. Phillips, S.M., Tipton, K.D., Aarsland, A., Wolf, S.E., and Wolfe, R.R., Mixed muscle protein
synthesis and breakdown after resistance exercise in humans, Am. J. Physiol., 273, E99, 1997.
9. Phillips, S.M., Tipton, K.D., Ferrando, A.A., and Wolfe, R.R., Resistance training reduces the
acute exercise-induced increase in muscle protein turnover, Am. J. Physiol., 276, E118, 1999.
10. Wagenmakers, A.J.M., Beckers, E.J., Brouns, F., Kuipers, H., Soeters, P.B., van der Vusse, G.J.,
and Saris, W.H.M., Carbohydrate supplementation, glycogen depletion, and amino acid
metabolism during exercise, Am. J. Physiol., 260, E883, 1991.
11. Knapik, J., Meredith, C., Jones, B., Fielding, R., Young, V., and Evans, W., Leucine metabolism
during fasting and exercise, J. Appl. Physiol., 70, 43, 1991.
1382_C30.fm Page 511 Tuesday, October 7, 2003 7:16 PM

Chapter thirty: Amino acid requirements in sport 511

12. Kasperek, G.J. and Snider, R.D., Total and myofibrillar protein degradation in isolated soleus
muscles after exercise, Am. J. Physiol., 257, E1, 1989.
13. Gontzea, I., Sutzeescu, R., and Dumitrache, S., The influence of adaptation to physical effort
on nitrogen balance in man, Nutr. Rep. Int., 11, 231, 1975.
14. Phillips, S.M., Atkinson, S.A., Tarnopolsky, M.A., and MacDougall, J.D., Gender differences
in leucine kinetics and nitrogen balance in endurance athletes, J. Appl. Physiol., 75, 2134, 1993.
15. Rennie, M.J. and Tipton, K.D., Protein and amino acid metabolism during and after exercise
and the effects of nutrition, Annu. Rev. Nutr., 20, 457, 2000.
16. Butterfield, G.E. and Calloway, D.H., Physical activity improves protein utilization in young
men, Br. J. Nutr., 51, 171, 1984.
17. Tarnopolsky, M., Atkinson, S., MacDougall, J., Chesley, A., Philips, S., and Schwarz, H.,
Evaluation of protein requirements for trained strength athletes, J. Appl. Physiol., 73, 1986,
1992.
18. Jeukendrup, A.E., Craig, N., and Hawley, J.A.H., Bioenergetics of world class cycling, J. Sci.
Med. Sport, 3, 414, 2000.
19. Saris, W.H.M., van Erp-Baart, M.A., Brouns, F., Westerterp, K.R., and ten Hoor, F., Study on
food intake and energy expenditure during extreme sustained exercise: the Tour de France,
Int. J. Sports Med., 10, S26, 1989.
20. Brouns, F., Saris, W.H.M., Stroecken, J., Beckers, E., Thijssen, R., Rehrer, N.J., and ten Hoor,
F., Eating, drinking, and cycling: a controlled Tour de France simulation study, part I, Int. J.
Sports Med., 10, S32, 1989.
21. van Erp-Baart, A.M.J., Saris, W.H.M., Binkhorst, R.A., Vos, J.A., and Elvers, J.W.H., Nation-
wide survey on nutritional habits in elite athletes. Part I. Energy, carbohydrate, protein, and
fat intake, Int. J. Sports Med., 10, S3, 1989.
22. Daly, J.M., Reynolds, J., Sigal, R.K., Shou, J., and Liberman, M.D., Effect of dietary protein
and amino acids on immune function, Crit. Care Med., 18 (Suppl.), S86, 1990.
23. Lamont, L.S., McCullough, A.J., and Kalhan, S.C., Comparison of leucine kinetics in endur-
ance-trained and sedentary humans, J. Appl. Physiol., 86, 320, 1999.
24. Bennet, W.M., Connacher, A.A., Scrimgeour, C.M., and Rennie, M.J., The effect of amino acid
infusion on leg protein turnover assessed by L-[15N]phenylalanine and L-[1-13C]leucine
exchange, Eur. J. Clin. Invest., 20, 41, 1990.
25. Bennet, W.M. and Rennie, M.J., Protein anabolic actions of insulin in the human body, Diabetic
Med., 8, 199, 1991.
26. Tipton, K.D., Ferrando, A.A., Phillips, S.M., Doyle, D., Jr., and Wolfe, R.R., Postexercise net
protein synthesis in human muscle from orally administered amino acids, Am. J. Physiol.,
276, E628, 1999.
27. Rasmussen, B.B., Tipton, K.D., Miller, S.L., Wolf, S.E., and Wolfe, R.R., An oral essential amino
acid-carbohydrate supplement enhances muscle protein anabolism after resistance exercise,
J. Appl. Physiol., 88, 386, 2000.
28. Knopf, R.F., Conn, J.W., Floyd, J.C., Jr., Fajans, S.S., Rull, J.A., Guntsche, E.M., and Thiffault,
C.A., The normal endocrine response to ingestion of protein and infusions of amino acids:
sequential secretion of insulin and growth hormone, Trans. Assoc. Am. Physicians, 79, 312,
1966.
29. Merimee, T.J., Lillicrap, D.A., and Rabinowitz, D., Effect of arginine on serum-levels of human
growth-hormone, Lancet, 2, 668, 1965.
30. Dupre, J., Curtis, J.D., Waddell, R.W., and Beck, J.C., Alimentary factors in the endocrine
response to administration of arginine in man, Lancet, 2, 28, 1968.
31. Floyd, J.C., Jr., Fajans, S.S., Conn, J.W., Knopf, R.F., and Rull, J., Stimulation of insulin
secretion by amino acids, J. Clin. Invest., 45, 1487, 1966.
32. Fogelholm, G.M., Naveri, H.K., Kiilavuori, K.T., and Harkonen, M.H., Low dose amino acid
supplementation: no effects on serum growth hormone and insulin in male weightlifters,
Int. J. Sports Nutr., 3, 290, 1993.
33. Lambert, M.I., Hefer, J.A., Millar, R.P., and Macfarlane, P.W., Failure of commercial oral amino
acid supplements to increase serum growth hormone concentrations in male body-builders,
Int. J. Sport Nutr., 3, 298, 1993.
1382_C30.fm Page 512 Tuesday, October 7, 2003 7:16 PM

512 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

34. Wagenmakers, A.J., Amino acid supplements to improve athletic performance, Curr. Opin.
Clin. Nutr. Metab. Care, 2, 539, 1999.
35. Maughan, R.J. and Sadler, D.J., The effects of oral administration of salts of aspartic acid on
the metabolic response to prolonged exhausting exercise in man, Int. J. Sports Med., 4, 119,
1983.
36. Wesson, M., Effects of oral administration of aspartic acid salts on the endurance capacity
of trained athletes, Res. Quart. Exerc. Sport, 59, 234, 1988.
37. Tuttle, J.L., Potteiger, J.A., Evans, B.W., and Ozmun, J.C., Effect of acute potassium-magne-
sium aspartate supplementation on ammonia concentrations during and after resistance
training, Int. J. Sport Nutr., 5, 102, 1995.
38. Walsh, N.P., Blannin, A.K., Robson, P.J., and Gleeson, M., Glutamine, exercise and immune
function: links and possible mechanisms, Sports Med., 26, 177, 1998.
39. Ardawi, M.S.M. and Newsholme, E.A., Glutamine metabolism in lymphoid tissues, in
Glutamine Metabolism in Mammalian Tissues, Haussinger, D. and Sies, H., Eds., Springer-
Verlag, Berlin, 1994, p. 235.
40. Parry-Billings, M., Budgett, R., Koutedakis, Y., Blomstrand, E., Brooks, S., Williams, C.,
Calder, P.C., Pilling, S., Baigrie, R., and Newsholme, E.A., Plasma amino acid concentrations
in the overtraining syndrome: possible effects on the immune system, Med. Sci. Sports Exerc.,
24, 1353, 1992.
41. Rennie, M.J., MacLellan, P.A., Hundal, H.S., Weryl, B., Smith, K., Taylor, P.M., Egan, C., and
Watt, P.W., Skeletal muscle glutamine concentration and muscle protein turnover, Clin. Exp.,
38, 47, 1989.
42. Bowtell, J.L., Gelly, G., Jackman, M.L., Patel, A., Simeoni, M., and Rennie, M.J., Effect of oral
glutamine on whole body carbohydrate storage during recovery from exhaustive exercise,
J. Appl. Physiol., 86, 1770, 1999.
43. Silva, A.C., Santos-Neto, M.S., Soares, A.M., Fonteles, M.C., Guerrant, R.L., and Lima, A.A.,
Efficacy of a glutamine-based oral rehydration solution on the electrolyte and water absorp-
tion in a rabbit model of secretory diarrhea induced by cholera toxin, J. Pediatr. Gastroenterol.
Nutr., 26, 513, 1998.
44. Haub, M.D., Potteiger, J.A., Nau, K.L., Webster, M.J., and Zebas, C.J., Acute L-glutamine
ingestion does not improve maximal effort exercise, J. Sports Med. Phys. Fitness, 38, 240, 1998.
45. Gleeson, M., Walsh, N.P., Blannin, A.K., Robson, P.J., Cook, L., Donnelly, A.E., and Day S.H.,
The effect of severe eccentric exercise-induced muscle damage on plasma elastase, glutamine
and zinc concentrations, Eur. J. Appl. Physiol., 77, 543, 1998.
46. Rhode, T., Asp, S., MacLean, D.A., and Pedersen, B.K., Competitive sustained exercise in
humans, lymphokine activated killer cell activity, and glutamine: an intervention study, Eur.
J. Appl. Physiol., 78, 448, 1998.
47. Castell, L.M. and Newsholme, E.A., Does glutamine have a role in reducing infections in
athletes? Eur. J. Appl. Physiol., 73, 488, 1996.
48. Krzywkowski, K., Petersen, E.W., Ostrowsi, K., Link-Amster, H., Boza, J., Halkjaer-Kristens-
en, J., and Pedersen, B.K., Effect of glutamine and protein supplementation on exercise-
induced decreases in salivary IgA, J. Appl. Physiol., 91, 832, 2001.
49. Walsh, N.P., Blannin, A.K., Bishop, N.C., Robson, P.J., and Gleeson, M., Effect of oral
glutamine supplementation on human neutrophil lipopolysaccharide-stimulated degranu-
lation following prolonged exercise, Int. J. Sport Nutr., 10, 39, 2000.
50. Krzywkowski, K., Petersen, E.W., Ostrowsi, K., Halkjaer-Kristensen, J., Boza, J., and Pedersen,
B.K., Effect of glutamine supplementation on exercise-induced changes in lymphocyte func-
tion, Am. J. Physiol., 281, C1259, 2001.
51. Gleeson, M. and Bishop, N.C., Elite athlete immunology: importance of nutrition, Int. J. Sports
Med., 21 (Suppl. 1), S44, 2000.
52. Hiscock, N. and Pedersen, B.K., Exercise-induced immunodepression: plasma glutamine is
not the link, J. Appl. Physiol., 93, 813, 2002.
53. Goldberg, A.L. and Chang, T.W., Regulation and significance of amino acid metabolism in
skeletal muscle, Fed. Proc., 37, 2301, 1978.
1382_C30.fm Page 513 Tuesday, October 7, 2003 7:16 PM

Chapter thirty: Amino acid requirements in sport 513

54. Wagenmakers, A.J.M., Brookes, J.H., Coakley, J.H., Reilly, T., and Edwards, R.H.T., Exercise-
induced activation of branched-chain 2-oxo acid dehydrogenase in human muscle, Eur. J.
Appl. Physiol., 59, 159, 1989.
55. Wolfe, R.R., Goodenough, R.D., Wolfe, M.H., Royle, G.T., and Nadel, E.R., Isotopic analysis
of leucine and urea metabolism in exercising humans, J. Appl. Physiol., 52, 458, 1982.
56. Frexes-Steed, M., Lacy, D.B., Collins, J., and Abumrad, N.N., Role of leucine and other amino
acids in regulating protein metabolism in vivo, Am. J. Physiol., 262, E925, 1992.
57. Louard, R.J., Barrett, E.J., and Gelfand, R.A., Effect of infused branched-chain amino acids
on muscle and whole-body amino acid metabolism in man, Clin. Sci., 79, 457, 1990.
58. Nair, K.S., Matthews, D.E., Welle, S.L., and Braiman, T., Effect of leucine on amino acid and
glucose metabolism in humans, Metabolism, 41, 643, 1992.
59. Newsholme, E.A., Acworth, I.N., and Blomstrand, E., Amino acids, brain neurotransmitters
and a functional link between muscle and brain that is important in sustained exercise, in
Advances in Myochemistry, Benzi, G., Ed., John Libby Eurotext, London, 1987, p. 127.
60. Blomstrand, E., Hassmen, P., Ekblom, B., and Newsholme, E.A., Administration of branched-
chain amino acids during sustained exercise: effects on performance and on plasma concen-
tration of some amino acids, Eur. J. Appl. Physiol., 63, 83, 1991.
61. Chaouloff, F., Kennett, G.A., Serrurrier, B., Merino, D., and Curzon, G., Amino acid analysis
demonstrates that increased plasma free tryptophan causes the increase of brain tryptophan
during exercise in the rat, J. Neurochem., 46, 1647, 1986.
62. Hargreaves, K.M. and Pardridge, W.M., Neutral amino acid transport at the human blood-
brain barrier, J. Biol. Chem., 263, 19392, 1988.
63. Newsholme, E.A., Blomstrand, E., and Ekblom, B., Physical and mental fatigue: metabolic
mechanisms and importance of plasma amino acids, Br. Med. Bull., 48, 477, 1992.
64. Stensrud, T., Ingjer, F., Holm, H., and Strømme, S.B., L-tryptophan supplementation does not
improve running performance, Int. J. Sports Med., 13, 481, 1992.
65. Van Hall, G., Raaymakers, J.S.H., Saris, W.H.M., and Wagenmakers, A.J.M., Ingestion of
branched-chain amino acids and tryptophan during sustained exercise in man: failure to
affect performance, J. Physiol., 486, 789, 1995.
66. Blomstrand, E., Hassmen, P., Ek, S., Ekblom, B., and Newsholme, E.A., Influence of ingesting
a solution of branched-chain amino acids on perceived exertion during exercise, Acta Physiol.
Scand., 159, 41, 1997.
67. Madsen, K., MacLean, D.A., Kiens, B., and Christensen, D., Effects of glucose, glucose plus
branched-chain amino acids, or placebo on bike performance over 100 km, J. Appl. Physiol.,
81, 2644, 1996.
68. Varnier, M., Sarto, P., Martines, D., Lora, L., Carmignoto, F., Leese, G.P., and Naccarato, R.,
Effect of infusing branched-chain amino acid during incremental exercise with reduced
muscle glycogen content, Eur. J. Appl. Physiol., 69, 26, 1994.
69. Bassit, R.A., Sawada, L.A., Bacurau, R.F.P., Navarro, F., Martins, E., Santos, R.V.T., Caperuto,
E.C., Rogeri, P., and Costa Rosa, L.F.B.P., Branched-chain amino acid supplementation and
the immune response of long-distance athletes, Nutrition, 18, 376, 2002.
70. Evain-Brion, D., Donnadieu, M., Roger, M., and Job, J.C., Simultaneous study of soma-
totrophic and corticotrophic pituitary secretions during ornithine infusion test, Clin. Endo-
crinol., 17, 119, 1982.
71. Bucci, L.R., Hickson J.F., Jr., Wolinsky, I., and Pivarnik, J.M., Ornithine supplementation and
insulin release in bodybuilders, Int. J. Sport Nutr., 2, 287, 1992.
72. Matsuzaki, Y., Miyazaki, T., Miyakawa, S., Bouscarel, B., Ikegami, T., and Tanaka, N., De-
creased taurine concentration in skeletal muscles after exercise for various durations, Med.
Sci. Sports Exerc., 34, 793, 2002.
73. Lehnert, H., Reinstein, D.K., Strowbridge, B.W., and Wurtman, R.J., Neurochemical and
behavioural consequences of acute uncontrollable stress: effects of dietary tyrosine, Brain
Res., 303, 215, 1984.
74. Owasoyo, J.O., Neri, D., and Lamberth, J.G., Tyrosine and its potential use as a countermea-
sure to performance decrement in military sustained operations, Aviat. Space Environ. Med.,
63, 364, 1992.
1382_C30.fm Page 514 Tuesday, October 7, 2003 7:16 PM

514 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

75. Struder, H.K., Hollman, W., Platen, P., Donike, M., Gotzmann, A., and Weber, K., Influence
of paroxetine, branched-chain amino acids and tyrosine on neuroendocrine system responses
and fatigue in humans, Horm. Metab. Res., 30, 188, 1998.
76. Segura, R. and Ventura, J.L., Effect of L-tryptophan supplementation on exercise performance,
Int. J. Sports Med., 9, 301, 1988.
77. Matthews, D.E., Proteins and amino acids, in Modern Nutrition in Health and Disease, Shils,
M.E., Olson, J.A., Shike, M., and Ross, A.C., Eds., Williams & Wilkins, Baltimore, 1999, p. 11.
1382_C31.fm Page 515 Tuesday, October 7, 2003 7:18 PM

Part V

Amino acid supply in diseases


1382_C31.fm Page 516 Tuesday, October 7, 2003 7:18 PM
1382_C31.fm Page 517 Tuesday, October 7, 2003 7:18 PM

section A

Quantitative and qualitative aspects


1382_C31.fm Page 518 Tuesday, October 7, 2003 7:18 PM
1382_C31.fm Page 519 Wednesday, October 8, 2003 1:35 PM

chapter thirty-one

Quantitative and qualitative


amino acid intake by the parenteral
route
Gaetano Iapichino
Universitá degli studi di Milano
Danilo Radrizzani
Universitá degli studi di Milano
Luc Cynober
Paris 5 University

Contents
Introduction..................................................................................................................................519
31.1 Analysis of protein wasting in patients..........................................................................520
31.2 Effects of nitrogen administration...................................................................................521
31.3 Available solutions ............................................................................................................524
31.4 Conclusions.........................................................................................................................525
References .....................................................................................................................................525

Introduction
Increased nitrogen losses are a common finding after trauma, surgery, or sepsis. They
result from a gap between protein synthesis and breakdown, which is dependent on the
severity of injuries, the age, and the previous nutritional status of the patient.1 Body protein
mass bears the effects of these increased losses as evidenced by a clinically appreciable
wasting of muscle mass and by a reduction of plasma protein content. Metabolic support
to these patients is devoted to reduce this protein wasting.
Patients generally defined as malnourished have already experienced an important
reduction of body protein stores, either because of a reduced nutrient intake or of a
previous metabolic stress. In these patients, metabolic support is mainly thought to replace
body protein mass. Whatever the goal of metabolic support, nitrogen supply is funda-
mental for an adequate therapy. A number of studies have investigated the amount and
amino acid (AA) composition of nitrogen ideal to obtain the objectives of metabolic

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 519
1382_C31.fm Page 520 Tuesday, October 7, 2003 7:18 PM

520 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

support. However, because historically the technical aspects were mastered before knowl-
edge of the physiopathological alterations became fully known, the specific AA needs of
ICU patients unfortunately long went unrecognized, and recommended requirements
were merely adapted from those set for healthy subjects. Most solutions for parenteral
nutrition (PN) provide a mixture of free AAs that reproduce the composition of high-
quality reference proteins (egg and cow milk proteins), namely, ª45% essential AAs (EAAs)
and a ratio of EAAs to total N of ª3.1 mg/g.

31.1 Analysis of protein wasting in patients


Nitrogen balance is the accepted method for investigating the bulk action of nitrogen
supply. From the mathematical point of view, it represents merely the difference between
input and output, while from the biological point of view, it is the expression of the
difference between protein synthesis and catabolism.
Without any intake, nitrogen balance is equal to nitrogen losses. In these conditions,
it is affected by two main sources of variation. The severity of the illness tends to increase
nitrogen losses (roughly 30 g in burn, 20 g in severe trauma, 10 g after elective surgery)
with respect to healthy persons. On the other hand, malnutrition per se tends to decrease
protein wasting, at least in absolute amounts. These two factors combine to depict the
wide variety of clinical situations and the specific nitrogen needs.
Plasma amino acid content is determined by the difference between amino acids
entering the plasma pool (exogenous supply, synthesis of not indispensable amino acids,
and protein catabolism) and those leaving it (protein synthesis and amino acid catabolism).
Plasma amino acid concentration responds very quickly to the variation of its determi-
nants, because of its relative smallness with respect to the total amino acid content of the
body, thus allowing some inference about the needs of single amino acids. During fasting,
stressed nonmalnourished patients show a significantly reduced plasma concentration of
total and nonessential amino acids compared to healthy subjects. The branched-chain
amino acids are slightly decreased. Phenylalanine, methionine, and cysteine are normal
or increased, particularly in septic patients. The variation of branched-chain amino acids,
phenylalanine, and methionine masks the reduction of other essential amino acids, so that
the sum of essential amino acids is unaffected. The greatest reduction is seen for glutamine
and alanine.3–5 Malnourished patients show a pattern characterized with a reduction of
almost all amino acids and without the increases due to stress derangement.6
Arteriovenous difference of amino acid concentrations is a qualitative method that
tells us whether an amino acid is taken up or released by a specific organ or system; when
coupled with blood flow measurement, it becomes a quantitative method (see Chapter 3
for more details). Because of easy access, these methods have been more frequently applied
to the limbs and have been of importance in understanding the needs of muscle tissue.
Stressed patients release amino acids from muscle tissue, taking up only glutamic acid.7,8
About 70% of the nitrogen released from muscle tissue is made of nonessential amino
acids, which are mainly represented by glutamine and alanine; essential amino acids
account only for about 30%, and half of this is made of lysine. Branched-chain amino acids
represent less than 10% of total nitrogen released from muscle tissue.9 In stressed patients,
the overall amino acid release is considerably higher than after elective surgery10 or in
normal subjects.11
Interorgan amino acid fluxes have been investigated in order to measure the clearance
of amino acid by the splanchnic compartment or to quantitize the fluxes for determining
amino acid requirements of each compartment and their interrelationship. This was made
by assuming that total body is divided into two compartments — the first made of muscle
tissue and the second representing the other tissues, of which the splanchnic region,
1382_C31.fm Page 521 Tuesday, October 7, 2003 7:18 PM

Chapter thirty-one: Quantitative and qualitative amino acid intake by the parenteral route 521

Figure 31.1 Effect of nitrogen intake on nitrogen balance in malnourished and stressed critically ill
patients. (Calculated from Radrizzani, D. et al., Intensive Care Med., 12, 308, 1986; Iapichino, G. et al.,
Intensive Care Med., 10, 251, 1984.)

especially the liver, is the more active metabolically. By doing this, it was possible to
demonstrate that central tissues (liver, etc.) retain about 50% of the nitrogen released from
muscle tissue during fasting.13

31.2 Effects of nitrogen administration


Even if nitrogen excretion and retention are affected by caloric amount in either stressed,
well-nourished or malnourished, nonstressed patients, the main determinant of nitrogen
balance is nitrogen intake.14,15 In both types of patients, the effect of caloric intake is
comparable and accounts for the retention of about 1 mg of nitrogen per kilogram per
kilocalorie per day. Also, the effect of nitrogen intake is comparable and gives rise to the
retention of about 60% of nitrogen supply. As malnourished patients show nitrogen losses
that are consistently less than those of stressed patients, it is easier to obtain nitrogen
balance in this clinical condition (Figure 31.1). Nitrogen retention cleared of the calorie
effect is the net protein utilization, and a figure of 50 to 80% is reported by many authors
in different clinical settings and with different nitrogen intakes.16,17 Then the simple
replacement of fasting nitrogen losses would lead to a negative nitrogen balance due to
imperfect utilization. It may be necessary to add a further amount to fasting losses,
depending on patient metabolic environment (stress or malnutrition), nitrogen clearance
ability (renal and hepatic function), and the planned nitrogen balance (replacement or
maintainance). Even in the most favorable conditions (nonstressed, depleted patient),
however, it is impossible to obtain a nitrogen balance greater than 2 to 3 g/day. A limit
(0.24 g of nitrogen/kg/day) beyond which there is no further improvement of nitrogen
balance has been advocated for stressed patients.18–21 Other authors indicate the optimal
nitrogen intake between 0.28 and 0.35 g of nitrogen/kg/day.13,15,22–25 This discrepancy could
be explained by different nitrogen needs. National and international consensus provide
the same range of values.26
1382_C31.fm Page 522 Tuesday, October 7, 2003 7:18 PM

522 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 31.1 Range of Dose (mg/kg/day) of Essential and Semiessential


Amino Acids Able to Maintain Plasma Concentration between 1 and 1.5
Times Normal in Injured and Malnourished Patients
Malnourished Injured
Arginine 200–310 125–200
Histidine 180–320 95–145
Isoleucine 85–195 85–150
Leucine 180–360 200–330
Lysine 220–410 255–485
Methionine 60–100 60–90
Phenylalanine 15–95 50–95
Threonine 95–180 70–95
Valine 145–250 195–250

Source: Adapted from Colombo, A. et al., Minerva Chir., 47, 1489, 1992; Colombo,
A. et al., RINPE, 10, 258, 1992.

Nevertheless, the studies supporting a lower nitrogen figure were all conducted with
a mixed system, and the others used a glucose system, thus suggesting a different nitrogen
utilization. 3-Methylhistidine (3MH) excretion is an indirect marker of protein catabolism,
and it is reduced during nitrogen and calorie administration. A positive relationship
between nitrogen balance and 3MH excretion has been demonstrated. The relationship is
still present if nitrogen balance is split into its two constituents: nitrogen input and output.
In particular, 3MH excretion increases when nitrogen output increases and decreases as
input increases, meaning that nitrogen supply per se affects proteolysis.27
In injured and depleted patients, amino acid intake increases plasma amino acid
concentration, and the increase is generally proportional to the intake: the greater the
input, the higher the plasma value.6,28 Two of us29,30 obtained 83 plasma amino acid profiles
during steady-state infusion of different amino acid solutions, either in stressed or in
malnourished patients, and were able to calculate the correlation between infusion and
plasma level for each essential amino acid. The relationships are exponential, and the
correlation coefficients range from 0.56 to 0.89. From these data,29,30 the mean input nec-
essary to obtain a plasma level between 1 and 1.5 times the normal values was also
calculated (Table 31.1).
We28,31 and others32 have found that during continuous parenteral administration of
AAs there is at first a sharp increase in AA levels and then a plateau lasting several hours.
The level of the plateau, which reflects the balance between rate of appearance (Ra) and
rate of disappearance (Rd), appears to be related to the rate of perfusion of each AA for
any given subject.33
This finding prompted one of us to construct a one-compartment model with first-
order elimination kinetics34 to study the relationship between the increase in plasma AA
level (pAAl) and the rate of perfusion for a given AA (AAx):

(AAxt3 – AAxt0) = f (rate of perfusion)

where AAxt3 is the pAAl of a given AA after 3 h of perfusion and AAxt0 is the pAAl of
the same AA at the postabsorptive state. There is a consensus34,35 that nutrition must be
interrupted 3 h before the start of the test infusion, this washout period representing a
compromise between too short and too long fasting.
This model was tested in healthy subjects receiving a new amino acid solution (10%
AFD, B. Braun) for parenteral nutrition.31 The plasma concentrations of the infused AAs
were closely (r2 = 0.92) correlated to their infusion rate (see Cynober2 for more details). In
1382_C31.fm Page 523 Tuesday, October 7, 2003 7:18 PM

Chapter thirty-one: Quantitative and qualitative amino acid intake by the parenteral route 523

addition, as expected, the steady state was reached within 3 h on AA infusion (except for
glycine and lysine, 6 h). Renal reabsorption was over 99% for most of the AAs.
This hypothesis and its related model were also tested in an interventional study in
ICU patients36 to determine whether a qualitative manipulation of AA intake could
improve the nutritional status of patients. Surgical patients received total PN for two
consecutive 5-day periods. The patients were randomized into two groups. The control
group received the same standard AA solution for the full 10 days. The experimental
group received the standard solution during the first 5 days, but was switched to a more
individualized solution during the last 5 days. The composition of the second solution
was determined from the dynamic test described above, i.e., choosing a solution available
on the market that provided less of the AAs found to be oversupplied and more of those
found to be undersupplied. AAs were defined as oversupplied or undersupplied when
outside the 95% confidence interval (above or below the curve, respectively). Thus, the
selected solution provided (per gram of N) less of the AAs given in excess and more of
those that were short.
During the second 5-day period (the test was performed again on day 8), imbalances
persisted in the control group but were almost abolished in the experimental group. In
addition, the mean of 5-day N balance was significantly higher during the second period
in the experimental group than in the control group. These findings suggest that the
relationship between rate of infusion and plasma AA variations may offer a rational basis
for choosing the most appropriate AA mixture for catabolic patients.
In the study on ICU patients,36 the relationship between [Aaxt3 – AAxt0] and rate of
perfusion was not as close (r2 = 0.45 to 0.88, with no relationship in one patient) as in the
study on healthy subjects,31 and varied from one patient to another. This emphasizes the
fact that the behavior of perfused AAs is patient specific and argues for setting intake
rates on a patient-by-patient basis, or at least according to pathology.
Notably, in 5 of the 12 patients, alanine (ALA) increase was appreciably lower than
predicted from its rate of perfusion, which was very high. This evidently reflects a very
high utilization of this AA in gluconeogenesis. The search for a relationship between this
observation and overproduction of hormones (e.g., glucagon) and mediators (e.g., tumor
necrosis factor-a, interleukin-6) in the concerned patients would be useful and deserves
future study. Also of major interest was the observation on day 3 that all but one of the
analyzed patients displayed abnormally high variations in levels of lysine (LYS), which
was apparently infused in too high amounts in this population of catabolic patients. This
was accompanied by a surprisingly high enrichment of plasma arginine (ARG), although
data from the literature37 would have on the contrary led to an expectation that standard
solutions would undersupply this AA. This may be because LYS and ARG share a common
transport system, called CAT.38 Therefore, oversupplying LYS may be responsible for a
decrease in ARG uptake and further metabolism with an accumulation of both AAs in
plasma. Decreasing LYS intake in the experimental group also normalized ARG levels.36
A study of particular interest is that of Lerebours et al.39 Gastroenterological patients
received the same AA solution in two crossover periods over 16 or 24 h. Plotting the
plasma variations of each AA against the infusion rate showed a clear-cut relationship
between the two parameters. (Note: In this study AAt0 was obtained after 7.5 h withdrawal
of the perfusion and sample at steady state after 15.5 h of perfusion). Increasing the
perfusion rate 1.5-fold (i.e., the same amount over 16 h instead of over 24 h) resulted in
2.5-, 4.5-, 2.0-, and 2.2-fold increases in proline (PRO), ALA, valine (VAL), and leucine
(LEU), respectively. Increases for most of the other AAs were proportional to the increases
in infusion rate. This suggests that PRO, ALA, VAL, and LEU intakes are excessive
compared with intake of other AAs in these patients. However, urinary elimination of
AAs did not differ between the two rates of perfusion, indicating that the imbalance
1382_C31.fm Page 524 Tuesday, October 7, 2003 7:18 PM

524 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

remained in the homeostatic range. Evidently then, a standard AA solution undersupplies


ALA in ICU patients36 but oversupplies it in gastrointestinal patients.39
Thus a number of factors may affect the relationship between rate of perfusion and
pAAl enrichment. These factors may include the type of pathology (depleted vs. hyper-
metabolic)6 and the level of concomitant energy administration.6,33 Notably, the rate of
plasma clearance after TPN cessation also depends on the underlying pathology.35,36
Synthesis of nonessential amino acids from their forerunners seems to be variably
impaired, as inferred by a generally low level of these amino acids.3,6 A low cysteine level
coupled with a high value of cystathionine, as well as low citrulline and high ornithine
levels,40 suggests a block along these pathways. However, a dependence of the plasma
level of the amino acid upon the concentration of the direct forerunner is demonstrated
for serine, ornithine, tyrosine, cysteine, and taurine.3 Metabolic blocks are easily overcome
by the infusion of the end product. This would be particularly useful for cysteine and
tyrosine, allowing a lower supply of methionine and phenylanine, which are poorly
managed and whose increased concentration is associated with untoward neurologic
effects.
Arteriovenous differences across the leg are reduced during amino acid infusion.7,8
As a consequence, total amino acid release is reduced during parenteral infusion of
nitrogen and calories to injured or septic patients.9,41,42 This is an overall nonselective
reduction that does not modify the amino acid composition. Its quantitative effect is calorie
dependent, being 60 and 40% of fasting release with 15 or 30 kcal/kg/day, respectively.9
However, this kind of therapy is not able to induce an amino acid retention in immobilized,
stressed patients, except for branched-chain amino acids when they are infused in consis-
tent amounts (0.9 g/kg/day). On the contrary, in healthy individuals11,41 and in malnour-
ished patients,43,44 it is possible to replenish muscle tissue. In stressed patients, the reduc-
tion of amino acid release induced by the therapy does not reduce the amino acid flux to
the central tissue when 0.30 g/kg/day of nitrogen is provided. However, the composition
of the mixture reaching the central tissue is appreciably different with respect to fasting
conditions, influencing a substantially lower utilization.12

31.3 Available solutions


Studies comparing different amino acid solutions must provide the same amount of
nitrogen, and this simple rule makes it difficult to investigate single amino acid needs. In
fact, it implies that in order to change the concentration of an amino acid, it is necessary
to modify the concentration of at least another amino acid. Despite sophisticated multi-
variate analysis, it is very difficult to discriminate the effect of each amino acid on the
variable under investigation. Moreover, self-controlled studies are almost impossible in
patients whose metabolic condition varies with time. For these reasons, net protein utili-
zation of commercially available solutions cannot be improved, at the moment, over the
reported figures. From this point of view, amino acid solutions are all unbalanced some-
how and show comparable effects on nitrogen balance despite wide differences in amino
acid content. One noticeable exception is the recent study by Bérard et al.45 comparing a
standard solution (E/T  3) with a solution (Hyperamine‚, B. Braun) richer in nonessential
AAs (E/T  2) in ICU patients. Nitrogen balance was better with the latter solution
supporting the idea (see above) that nonessential AA intake is a key point.
Finally, studying two different solutions requires to render the solutions isonitroge-
nous. Since neutral nitrogen does not exist, there is no ideal issue. This problem has not
been often addressed in the literature.46
1382_C31.fm Page 525 Tuesday, October 7, 2003 7:18 PM

Chapter thirty-one: Quantitative and qualitative amino acid intake by the parenteral route 525

Special solutions for particular pathologies (e.g., renal or hepatic failure solutions) and
amino acids (e.g., GLN, ARG) given in pharmacological amounts are beyond the scope
of this paper and are discussed in other chapters of this book.

31.4 Conclusions
From these data, it is possible to understand that muscle tissue releases a considerable
amount of amino acids during fasting. Central tissue utilizes a consistent part (about 50%)
of these amino acids for visceral protein synthesis.47 The remaining amino acid nitrogen
is wasted with urine as urea. The two processes are responsible for the resultant plasma
amino acid concentration. In these conditions, the plasma amino acid free pool is reduced
by about 15 to 20%, reflecting the imbalance between input and output either in the pool
or in the total body. Alterations in plasma amino acid concentration are not due to
hemodilution because different amino acids show different changes.
Plasma amino acid content during infusion of amino acids is modified, and generally
the greater the input, the higher the concentration, despite the differences in clearance of
single amino acids from the plasma pool and the evidence of metabolic blocks along the
synthetic pathway of some dispensable amino acids. Moreover, amino acid infusion seems
to induce a reduction of muscle protein catabolism. During parenteral nutrition, the
composition of the amino acid mixture presented to the central tissue varies signifícantly,
being affected either by the amino acid released by muscle tissue or by the formula of the
infused solution. Central nitrogen utilization depends on this final composition. It would
be very interesting to know the exact composition of amino acid nitrogen necessary for
each patient. The dynamic model presented above should allow us to do it.
Bearing in mind the amounts necessary to obtain nitrogen balance in stressed and in
malnourished patients, the amino acid composition should be mixed as indicated in Table
31.1 for essential and semiessential amino acids, integrated for the remaining quote by
nonessential amino acids, of which cysteine and tyrosine seem of importance in overcom-
ing metabolic blocks. Glutamine, arginine, glutamic acid, and aspartic acid (the major
participants in transamination reactions), as well as serine, proline, and glycine, might
form the other constituents up to the desired nitrogen infusion.45,48

References
1. Obled, C., Papet, I., and Breuille, D., Metabolic bases of amino acid requirements in acute
diseases, Curr. Opin. Clin. Nutr. Metab. Care, 5, 189, 2002.
2. Cynober, L., Lessons from pharmacokinetics in the design of new nutrition formulas for
critically ill patients, in Nutrition and Critical Care, Cynober, L. and Moore, F.A., Eds., Nestlé
Nutrition Workshop Series Vol. 8, Karger, Basel, Switzerland, 2003, p. 265.
3. Iapichino, G., Radrizzani, D., Scherini, A., Malacrida, R., Bonetti, G., Della Torre, P., et al.,
Essential and non-essential amino acid requirement in injured patients receiving total
parenteral nutrition, Intensive Care Med., 14, 399, 1988.
4. Jeevanandam, M., Ramias, L., and Schiller, W.R., Altered plasma free amino acid levels in
obese traumatized man, Metabolism, 40, 385, 1991.
5. Vente, J.P., Von Meyenfeldt, M.F., Van Ejik, H.M.H., Van Berlo, C.L.H., Gourma, D.J., Van Der
Linden, C.J., et al., Plasma amino acid profiles in sepsis and stress, Ann. Surg., 209, 57, 1989.
6. Iapichino, G., Radrizzani, D., Bonetti, G., Bressani Doldi, S., Della Torre, P., Ferrero, P., et al.,
Dispensable and indispensable amino acid requirements in depleted patients receiving total
parenteral nutrition, Clin. Nutr., 6, 5, 1987.
1382_C31.fm Page 526 Tuesday, October 7, 2003 7:18 PM

526 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

7. Iapichino, G., Radrizzani, D., Bonetti, G., Colombo, A., Damia, G., Della Torre, P., et al.,
Parenteral nutrition of injured patients: effect of manipulation of amino acid infusion (in-
creasing branched chain while decreasing aromatic and sulfurated amino acids), Clin. Nutr.,
4, 121, 1985.
8. Cynober, L., Blonde, F., Lioret, N., Coudray-Lucas, C., Saizy, R., and Giboudeau, J., Arterio-
venous differences in amino acids, glucose, lactate and fatty acids in burn patients: effect of
ornithine alpha-ketoglutarate, Clin. Nutr., 5, 221, 1986.
9. Iapichino, G., Radrízzani, D., Bonetti, G., Colombo, A., Leoni, L., Ronzoni, G., and Damia,
G., Influence of parenteral nutrition on leg nitrogen exchange in injured patients, Crit. Care
Med., 18, 1367, 1990.
10. Stehle, P., Zander, J., Mertens, N., Albers, S., Puchstein, C.H., Lawin, P., and Fürst, P., Effect
of parenteral glutamine peptide supplements on muscle glutamine loss and nitrogen balance
after major surgery, Lancet, i, 231, 1989.
11. Elia, M., Folmer, P., Schlattmann, A., Goren, A., and Austin, S., Amino acid metabolism in
muscle and in the whole body of man before and after ingestion of a single mixed meal,
Am. J. Clin. Nutr., 49, 1203, 1989.
12. Radrizzani, D., Iapichino, G., Cambisano, M., Bonetti, G., Ronzoni, G., and Colombo, A.,
Peripheral, visceral and body nitrogen balance of catabolic patients, without and with
parenteral nutrition, Intensive Care Med., 14, 212, 1988.
13. Clowes, G.H.A., Heideman, M., Lindberg, B., Randall, H.T., Hirsch, E.T., Cha, C.J., et al.,
Effects of parenteral alimentation on amino acid metabolism in septic patients, Surgery, 88,
531, 1980.
14. Radrizzani, D., Iapichino, G., Scherini, A., Ferrero, P., Bressani Doldi, S., Solca, M., et al.,
Main nitrogen balance determinants in malnourished patients, Intensive Care Med., 12, 308,
1986.
15. Iapichino, G., Radrizzani, D., Solca, M., Pesenti, A., Gattinoni, A., Ferro, A., et al., The main
determinants of nitrogen balance during total parenteral nutrition in critically ill patients,
Intensive Care Med., 10, 251, 1984.
16. Rutten, P., Blackburn, G.L., Flatt, J.P., Hallowell, E., and Cochran, D., Determination of
optimal hyperalimentation infusion rate, J. Surg. Res., 18, 477, 1975.
17. Iapichino, G., Solca, M., Radrizzani, D., Zucchetti, M., and Damia, G., Net protein utilization
during total parenteral nutrition of critically injured patients: an original approach, J. Parenter.
Enteral Nutr., 5, 317, 1981.
18. Larsson, J., Lemmarken, C., Martensson, J., Sandstcdt, S., and Vinnars, E., Nitrogen require-
ments in severely injured patients, Br. J. Surg., 77, 413, 1990.
19. Greig, P.D., Elwyn, D.H., Askanazi, J., and Kinney, J.M., Parenteral nutrition in septic patients:
effect of increasing nitrogen intake, Am. J. Clin. Nutr., 46, 1040, 1987.
20. Shaw, J.H.F., Widbore, M., and Wolfe, R.R., Whole body protein kinetics in severely septic
patients, Ann. Surg., 205, 288, 1987.
21. Wolfe, R.R., Goodenough, R.D., Burke, J.F., and Wolfe, M.H., Response of protein and urea
kinetics in burn patients to different levels of protein intake, Ann. Surg., 197, 163, 1983.
22. Iapichino, G., Gattinoni, L., Solca, M., Radrizzani D., Zucchetti, M., Langer, M., Vesconi, S.,
et al., Protein sparing and protein replacement in acutely injured patients during TPN with
and without amino acid supply, Intensive Care Med., 8, 25, 1982.
23. Humberstone, D.A., Koea, J., and Shaw, J.H.F., Relative importance of amino acid infusions
as a means of sparing protein in surgical patients, J. Parenter. Enteral Nutr., 13, 223, 1989.
24. Cerra, F., Hirsh, J., Mullen, K., Blackburn, G., and Luther, W., The effect of stress level, amino
acid formula, and nitrogen dose on nitrogen retention in traumatic and septic patients, Ann.
Surg., 205, 282, 1987.
25. Cerra, F.B., How nutrition intervention changes what getting sick means, J. Parenter. Enteral
Nutr., 14, 164s, 1990.
26. Bozzetti, F. and Allaria, B., Nutritional support in ICU patients: position of scientific societies,
in Nutrition and Critical Care, Cynober, L. and Moore, F.A., Eds., Nestlé Nutrition Workshop
Series Vol. 8, Karger, Basel, 2003, p. 279.
1382_C31.fm Page 527 Tuesday, October 7, 2003 7:18 PM

Chapter thirty-one: Quantitative and qualitative amino acid intake by the parenteral route 527

27. Iapichino, G., Radrizzani, D., Solca, M., Bonetti, G., Leoni, L., and Ferro, A., Influence of total
parenteral nutrition on protein metabolism following acute injury: assessment by urinary
3-methylhistidine excretion and nitrogen balance, J. Parenter. Enteral Nutr., 9, 42, 1985.
28. Iapichino, G., Radrizzani, D., Colombo, A., Ronzoni, G., Pasetti, G., Bonetti, G., et al., Plasma
amino acid concentration changes during total parenteral nutrition in critically ill patients,
Clin. Nutr., 11, 358, 1992.
29. Colombo, A., Radrizzani, D., Bonetti, C., Pasetti, G., Rigoli, A., and Ronzoni, G., Dos ottimale
di aminoacidi somministrati durante nutrizione parenterale totale (NPT) di pazienti malnu-
triti, Minerva Chir., 1489, 47, 1992.
30. Colombo, A., Radrizzani, D., Bonetti, C., Ciceri, R., Guarnerio, C., Rigoli, A., et al., Dose
ottimale di aminoacidi somministrati durante nutrizione parenterale totale (NPT) di pazienti
catabolici, RINPE, 10, 258, 1992.
31. Bérard M.P., Hankard, R., and Cynober, L., Amino acid metabolism during total parenteral
nutrition in healthy volunteers: evaluation of a new amino acid solution, Clin. Nutr., 20, 407,
2001.
32. Carpentier, Y.A., Richelle, M., Rubin, M., Rossle, C., Dahlan, W., Bosson, D., et al., Stabilisation
of plasma substrate concentrations: a model for conducting metabolic studies, Clin. Nutr., 9,
313, 1990.
33. Waterhouse, C., Clarke, E.F., Heinig, R.E., Lewis, A.M., and Jeanpretre, N., Free amino acid
levels in the blood of patients undergoing parenteral alimentation, Am. J. Clin. Nutr., 32,
2423, 1979.
34. Mosebach, K.O., Stoeckel, H., Caspari, R., Muller, R., Schulte, J., Lippoldt, R., et al., Pharma-
cokinetic evaluation of a new maintenance solution for severely injured patients, J. Parenter.
Enteral Nutr., 4, 346, 1980.
35. Radrizzani, D., Iapichino, G., Bonetti, G., Bozzetti, F., Ammatuna, M., and Colombo, A.,
Plasma amino acid concentration changes after total parenteral nutrition (TPN) interruption
in critically ill and surgical neoplasic patients, Clin. Nutr., 6, 201, 1987.
36. Bérard, M.P., Pelletier, A., Ollivier, J.M., Gentil, B., and Cynober, L., Qualitative manipulation
of amino acid supply during total parenteral nutrition in surgical patients, J. Parenter. Enteral
Nutr., 26, 136, 2002.
37. Yu, Y.M., Ryan, C.M., Castillo, L., Lu, X.M., Beaumier, L., Tompkins, R.G., et al., Arginine
and ornithine kinetics in severely burned patients: increased rate of arginine disposal, Am.
J. Physiol., 280, E509, 2001.
38. Morris, S.M., Regulation of arginine availability and its impact on NO synthesis, in Nitric
Oxide: Biology and Pathobiology, Ignarro L.J., Ed., Academic Press, San Diego, 2000, p.187.
39. Lerebours, E., Colin, R., Hecketsweiler, B., Matray, F., Plasma amino acids in total parenteral
nutrition comparison of continuous and cyclic parenteral nutrition, Clin. Nutr., 6, 143, 1987.
40. Bonetti, G., Iapichino, G., Radrizzani, D., Malacrida, R., Ronzoni, G., and Damia, G.,
Methionine, cystathionine and cystine increased urinary losses during total parenteral nu-
trition of adult patients, Clin. Nutr., 1, 43, 1988.
41. Clowes, G.H.A., Randall, H.T., and Cha, C.J., Amino acid and energy metabolism in septic
and traumatized patients, J. Parenter. Enteral Nutr., 4, 195, 1980.
42. Finley, R.J., Inculet, R.L., Pace, R., Holliday, R., Rose, C., Duff, J.H., et al., Major operative
trauma increases peripheral amino acids release during steady state infusion of total
parenteral nutrition in man, Surgery, 99, 491, 1986.
43. Legaspi, A., Roberts, J.P., Albert, J.D., Tracey, K.J., Shires, G.T., and Lowry, S.F., The effect of
starvation and total parenteral nutrition on skeletal muscle amino acid content and mem-
brane potential difference in normal man, Surg. Gynecol. Obstet., 166, 233, 1988.
44. Loder, P.B., Smith, R.C., Kee, A.J., Kohlhardt, S.R., Fisher, M., Jones, M., et al., What rate of
infusion of intravenous nutrition solution is required to stimulate uptake of amino acids by
peripheral tissues in depleted patients, Ann. Surg., 211, 360, 1990.
45. Bérard, M.P., Zazzo, J.F., Condat, P., Vasson, M.P., and Cynober, L., Total parenteral nutrition
enriched with arginine and glutamate generates glutamine and limits protein catabolism in
surgical patients hospitalized in intensive care units, Crit. Care Med., 28, 3637, 2000.
1382_C31.fm Page 528 Tuesday, October 7, 2003 7:18 PM

528 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

46. Cynober, L., Les pharmaconutriments azotés: du tube à essai à la pratique clinique, Nutr.
Clin. Métabol., 15, 131, 2001.
47. Hasselgreen, P.O., Pedersen, P., Sax, H.C., Warner, B.W., and Fisher, J.E., Current concepts
of protein turnover and amino acid transport in liver and skeletal muscle during sepsis,
Arch. Surg., 123, 992, 1988.
48. Fürst, P., Old and new substrates in clinical nutrition, J. Nutr., 128, 789, 1998.
1382_C32.fm Page 529 Tuesday, October 7, 2003 7:20 PM

chapter thirty-two

Quantitative and qualitative


aspects of nitrogen supply in
enteral nutrition in relation to free
amino acids and peptides
George K. Grimble
University of Surrey Roehampton

Contents
Introduction..................................................................................................................................530
32.1 Quantitative aspects of intestinal amino acid metabolism ..........................................531
32.2 Zonation of the human intestine .....................................................................................533
32.3 Mechanisms of protein assimilation ...............................................................................534
32.3.1 Gastric and pancreatic phases of digestion.....................................................534
32.3.2 Small intestinal nitrogen assimilation ..............................................................535
32.3.3 Colonic protein digestion ...................................................................................535
32.3.4 Urea recycling.......................................................................................................536
32.4 Absorption of the products of luminal digestion..........................................................536
32.4.1 Free amino acid transport ..................................................................................536
32.4.2 Peptide transport .................................................................................................536
32.5 Quantitative and qualitative comparisons of amino acid and peptide transport ....537
32.5.1 Relative rates of assimilation of proteins, peptides, and amino acids .......538
32.5.2 Effects of malnutrition on absorptive function ..............................................540
32.5.3 Effects of critical illness on absorptive function.............................................540
32.5.4 Effect of short bowel syndrome on absorptive function ..............................540
32.6 Comparative feeding studies of proteins, peptides, and amino acids.......................540
32.6.1 Feeding trials in healthy humans and animals ..............................................540
32.6.2 Feeding trials during recovery from starvation .............................................544
32.6.3 Acute feeding studies in humans and animals ..............................................544
32.7 Comparative feeding trials of proteins, peptides, and amino acids in clinical
situations .............................................................................................................................545
32.7.1 Patients with impaired gastrointestinal absorptive area ..............................545
32.7.2 Patients with impaired pancreatic function ....................................................545

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 529
1382_C32.fm Page 530 Tuesday, October 7, 2003 7:20 PM

530 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

32.7.3 Critically ill patients ............................................................................................545


32.7.4 Neonates................................................................................................................546
32.8 Perspectives ........................................................................................................................546
References .....................................................................................................................................547

Introduction
A patient with “severely impaired gastrointestinal function” who had only 15 cm of
remaining jejunum anastomosed to 30 cm of midtransverse colon was described by
Althausen and colleagues in 1949.1 Balance studies showed that despite a limited absorp-
tive surface, the patient was able to maintain stable body weight on an oral intake of
1600 kcal/day of hospital diet or a synthetic diet containing glucose, cream, hydrolyzed
protein, and vitamins and minerals. This was a clinical tour de force, but the authors
noted that flatulence, a symptom of malabsorption, disappeared when the patient con-
sumed the synthetic diet. The patient assimilated more than 70% of carbohydrate and
50% of nitrogen from the synthetic diet and was maintained in slight positive nitrogen
balance. This study, the first of many on synthetic or elemental diets,2 provided directions
for the ways diet can be chemically tailored for patients with digestive and absorptive
malfunction.
Present ideas on dietary protein assimilation owe much to Otto Loewi’s finding that
dietary amino acids given orally or parenterally in the form of “peptones” (now known
as protein hydrolysates) could stimulate protein synthesis.3,4 This new concept was applied
experimentally to intravenous nutrition of a goat by Henriques and Andersen5 and of
human subjects by van Slyke and Meyer6 and had reached clinical practice by the early
1940s, mainly through the efforts of Robert Elman.7 The widely accepted theory of protein
nutrition has three central tenets concerning the role of amino acids as metabolic currency
(Figure 32.1). First, they are the predominant form in which dietary protein is assimilated.
Second, they are the form in which interorgan amino acid flux occurs (because protein
meals increase blood amino acids). Lastly, they are the precursors for protein synthesis
itself (because the triplet code specifies only amino acids). This theory was a reaction
against the unpalatable concept that the peptone products of protein digestion and absorp-
tion were incorporated intact into nascent peptides during protein synthesis (Figure 32.1).
Of course, the proponents of the earlier view very sensibly based it on the grounds of
chemical and thermodynamic efficiency.

It may be a priori doubted on teleological grounds, whether under normal


circumstances the amount of amido-acids [sic] formed in the intestine is a large
one. It would be a waste of chemical potential energy, which would serve no
purpose when converted into kinetic energy by their decomposition, and a
reunion of the products of such a profound decomposition is highly improbable.8

The discoverer of pepsin’s mechanism of action thought that the present view triumphed
because new data made

it clear that proteins are nearly completely digested in the intestine to amino
acids, that these amino acids can replace intact dietary proteins, and that at-
tention had to be paid to the “quality” of a dietary protein and not merely its
nitrogen content.9
1382_C32.fm Page 531 Wednesday, October 8, 2003 1:37 PM

Chapter thirty-two: Quantitative and qualitative aspects of nitrogen supply 531

Intestine lumen Extraintestinal tissue


A
Newly
Dietary protein Peptones synthesized
protein

B
Newly
Dietary protein Amino acids synthesized
protein

Peptides Peptides Peptides


C

Dietary protein Newly


Amino acids Amino acids synthesized
protein

Figure 32.1 Development of ideas on protein nutrition and metabolism. (A) Nineteenth century
scientists believed that dietary protein was digested by the intestine to peptones that were incor-
porated intact into blood protein. This view was commended because of its thermodynamic effi-
ciency.8 Twentieth century scientists thought that amino acids were the currency of intestinal
absorption and protein synthesis. (B) This view was commended because the genetic code is equiv-
alent to single amino acids, not peptides. (C) Twenty-first century scientists may come to appreciate
the dual hypothesis of amino acid/peptide absorption and interorgan trafficking.

This prescient statement is clearly relevant to this chapter, but in fact, only the last tenet
of the dogma is completely secure. First, the intestine absorbs lots of peptides, so in
rejecting any peptone theories, the protein turnover pioneers threw the baby out with the
bathwater. Second, substrate-level interorgan flux of amino acids as small peptides
occurs,10 and to add insult to injury, the end products of proteasome-mediated intracellular
protein breakdown, like the products of intestinal protein degradation, not only are small
peptides that can be degraded to amino acids by cytosolic peptidases,11 but also are
absorbed by lysosomes via a membrane-bound peptide transporter.12 Although intact
peptides are not direct precursors for protein synthesis, the constituent amino acids of
circulating small peptides can be preferential substrates for intracellular protein synthe-
sis.13 An integrated view of this is given in Figure 32.1.
As the pendulum has swung away from peptones to amino acids and may yet swing
back to peptides, should this be considered in relation to the formulation of enteral diets?
Is it possible to modulate amino acid absorption for clinical benefit? These questions
should be put in the context of new ideas about the intestine, particularly metabolism of
dietary amino acids and the zonation of the intestine.

32.1 Quantitative aspects of intestinal amino acid metabolism


The intestine avidly metabolizes dietary amino acids that are a valuable and often scarce
food component. While this is not economical, according to Bunge’s views on metabolic
efficiency, nevertheless the consensus is that the intestine not only is appropriately sized
to the metabolic mass of each species14 but also has a digestive safety margin that is
“enough, but not too much.”15
Glutamine is extensively metabolized by intestinal tissues after uptake from luminal
and arterial sources as described in more detail in Chapter 11. It is worth noting that the
1382_C32.fm Page 532 Tuesday, October 7, 2003 7:20 PM

532 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

intestine, like the liver, may switch between glucose and glutamine as fuels and that this
determines whether it consumes glutamine or allows its portal efflux. Under normal
circumstances, CO2 production by the gut comes mainly from enteral glutamate and
enteral and arterial glucose, with a minor contribution from glutamine,16 which may be
suppressed in clinical situations such as endotoxemia.17 Glutamate is avidly metabolized
by the intestine18 and may therefore seem to be a more rational choice for a gut-specific
fuel, together with its deaminated product, a-ketoglutarate, which is equally voraciously
metabolized.19 A minor concern with this approach is that either acidic compound added
to enteral diets requires neutralization with a countercation (e.g., 10 g/l of glutamic acid
requires 78 mmol/l Na+). Additionally, neither compound is completely stable to heat
sterilization, and glutamate will cyclize to pyroglutamic acid, which will be reconverted
to glutamate by pyroglutamate hydrolase, which is abundant in the intestine.20 Serious
concerns about glutamate toxicity have produced a large body of literature. High plasma
concentrations of glutamic acid are neurotoxic (e.g., see Stegink et al.21), but it is not clear
whether this might ever be achieved during artificial nutrition support. Bolus adminis-
tration of glutamic acid by gavage or intravenous injection will produce focal necrosis in
the arcuate nucleus of the hypothalamus in sensitive species (e.g., mice) but rarely in
primates. In man, the blood concentrations required to produce lesions in mice (neonates,
1 mmol/l; adults, >6.3 mmol/l) are never observed, even after oral bolus administration.
Furthermore, when glutamate was administered orally to humans as part of a protein
hydrolysate, it failed to raise plasma glutamate concentration, whereas an equivalent oral
dose of monosodium glutamate did.22 Two recent reviews suggest that glutamate toxicity
is unlikely to be of serious concern.23,24
Dietary arginine is converted to ornithine and citrulline,25,26 which may be either
partially metabolized by the liver or reconverted to arginine by the kidneys.27 In addition,
intestinal metabolism of dietary lysine accounts for 14% of total body lysine oxidation,28
while leucine oxidation accounted for 18% of splanchnic CO2 production.29 Finally, there
is a chronological zonation of appearance of dietary amino acids in blood. An early portion
appears in portal blood while the remainder is either metabolized in the mucosa or
incorporated into newly synthesized and secreted mucoproteins, which are salvaged in
the distal small intestine.30 In summary, approximately half of amino acids in dietary
protein are available to peripheral tissues and show how the intestine is both a digestive
organ and one that is metabolically very active and engaged in high rates of protein
synthesis31 that are modulated by gastrointestinal disease.32
Common sense suggests that oral feeding must maintain protein synthesis in the
intestine because an exclusive parenteral intake leads to some villous atrophy in humans,
which is reversed by oral feeding.33 Indeed, mucosal atrophy in pigs occurs when enteral
intake falls below 40% of the total, the remainder being given parenterally.34 The exact
effect of luminal nutrition on gut protein metabolism is still unknown because zoned
fluxes of amino acids in the splanchnic region prevent accurate determination of labeling
of the true precursor for protein synthesis.31 In addition, these studies do not take into
account those amino acids absorbed into portal blood as intact peptides, which therefore
escape splanchnic metabolism (Figure 32.2). Estimates of the proportion of portal blood
amino acids in this form after casein feeding range from 10% in dogs35 and guinea pigs36
up to 66% in the portal vein of calves.37 In peripheral circulation, the proportion has been
estimated at 10% in humans38 and 51% in rats.37 Gardner39 carefully reviews the evidence
for this because there are formidable problems of artefacts, which are described in detail
by Backwell40 and Seal and Parker41 in a symposium on the subject.10 As Christensen et al.38
wrote, “peptidemia is a recurring concept of pathological chemistry which has never been
either completely established or disproved,” or more recently:
1382_C32.fm Page 533 Tuesday, October 7, 2003 7:20 PM

Chapter thirty-two: Quantitative and qualitative aspects of nitrogen supply 533

Lumen
1 4

5
Portal blood
Figure 32.2 Peptide and amino acid assimilation in the small intestine. An idealized view of the
intestinal wall is shown. Luminal digestion by pancreatic proteases produces di- and tripeptides
that are absorbed by PEPT1 (1) and hydrolyzed by intracellular peptidases (2). Tetra- and higher
peptides are hydrolyzed by brush border peptidases (3). Free amino acids are absorbed by one of
the specific active L-amino acid transporters (4). Some dietary peptides will be released intact into
the portal circulation via PEPT1 (e.g., carnosine, collagen peptides) (5).

The confident assumption of the last forty years that only free amino acids were
present in the plasma in significant amounts and that peptides, if present, were
of no importance, led to reliance on analytical methods, such as routine ion-
exchange chromatography, which in plasma reveal and measure only free ami-
no acids. If we continue to look only for free amino acids, we shall find only
free amino acids: peptides cannot be expected to declare their presence. Further
progress in this area will require improved and more convenient methods of
analysis. (Matthews,42 p. 356)

32.2 Zonation of the human intestine


The human intestine is zoned in terms of anatomy, digestive and absorptive function, and
metabolism. Anatomically, it is a good textbook model for describing transport physiology
in a linear fashion — an intuitive approach in which specialized compartments with
different digestive and absorptive functions succeed each other. This approach, however,
gives us few clues about improving the quality of protein in enteral diets. Stevens and
Hume43 have brilliantly enlisted comparative anatomy to show how dietary carbohydrate,
as the most significant dietary component, is associated with dramatic variations in gut
anatomy and morphology. For example, of two apparently similar lemur species from
Madagascar, Lepilemur (cactus-like plant feeder from Madagascar) has an enlarged hind-
gut, and Microcebus (mouse lemur — primarily insectivorous) has a generally small gut,
all round.14 In other words, Lepilemur is a hindgut fermenter, while Microcebus is a foregut
digester, and humans are mixed digesters. In contrast, the anatomical requirement for
efficient protein digestion in a carnivore is defined in negative terms as the absence of a
hindgut in which dietary protein may be fermented.
Digestive and absorptive zonation is a more helpful concept. Clinical situations that
impair gut function (with the exception of pancreatic insufficiency) result in fat and energy
1382_C32.fm Page 534 Tuesday, October 7, 2003 7:20 PM

534 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

malabsorption rather than poor protein digestion (e.g., short bowel syndrome44,45 or
HIV/AIDS46). This is because the duodenum/proximal jejunum is the main site of starch
assimilation, as suggested by the rapidity of appearance of glucose in blood after a starch
meal and by the longitudinal distribution or zonation of brush border glucosidases and
the sodium glucose-linked transporter (SGLT1). Ileal resection will leave the patient at
risk of fat malabsorption,47 whereas very extensive resection may finally lead to carbohy-
drate malabsorption.48 There is no hard and fast rule about this because adaptation of the
remaining intestine after resection is highly variable,48 and there is one report in which,
following resection of all but the duodenum and proximal 20 cm of jejunum, significant
fat absorption still occurred.49
Although molecular biology techniques have identified most apical and basolateral
mucosal transporters (see Chapter 5), it should be emphasized that dietary protein assim-
ilation is not only a luminal/mucosal event because membrane transporters and hydro-
lases that are normally associated with the intestinal brush border membrane are found
in nonintestinal tissues. This suggests that dietary protein assimilation may be completed
outside the gut, a concept extensively developed by Ugolev et al.50 as one of “distributed
digestion.” If true, it helps explain the phenomenon of peptiduria. For example, significant
amounts of orally ingested “resistant” dipeptide carnosine (L-beta-alanyl-L-histidine)
appear in urine. If an intestinal peptide transport system did not exist, none would be
excreted; if no plasma/tissue peptidases were active, then all would be excreted.51 Peptides
present in portal blood after a meal will require hydrolysis at the liver plasma membrane,
by plasma peptidases or by the renal tubular epithelium. The importance of this zoned
approach to enteral amino acid and protein metabolism has been demonstrated by the
fact that intravenously infused synthetic dipeptides are rapidly hydrolyzed at a rate that
seems unaltered by renal failure,52 implying that tissue and blood peptidases are important
sites of hydrolysis.53 Thus, one could consider enteral and parenteral metabolism as two
parts of a spectrum of peptide disposal.
An attempt to define the optimum formulation for enteral nitrogen could therefore
consider the mechanisms of intestinal protein assimilation and how this is altered by the
form of dietary nitrogen. Second, we could consider the way different proteins are assim-
ilated in healthy people and how this is affected by critical illness before finally considering
evidence for efficacy of special enteral diets.

32.3 Mechanisms of protein assimilation


Assimilation of dietary protein proceeds by two complementary phases. Luminal digestion
produces small peptides and free L-amino acids that are then subject to further hydrolysis
or absorption at the enterocyte brush border.

32.3.1 Gastric and pancreatic phases of digestion


Acid secretion in the stomach denatures dietary protein by protonating dicarboxylic amino
acid side chains. The gastric pepsins, which have a functional pH optimum at about 2.0,
will therefore convert this denatured protein to large soluble oligopeptides.54 These oli-
gopeptides are substrates for pancreatic endo- and exopeptidases, whose pH optimum is
much higher, and which comprise three endopeptidases (toward basic, hydrophobic, and
uncharged adjacent residues55) and two exopeptidases (toward C-terminal hydrophobic
or basic amino acids56). Their products are significant amounts of free amino acid and
small peptides that are completely soluble. This is not only of academic interest because
an undigested protein like casein clots quite readily at acid pH and has been shown to
1382_C32.fm Page 535 Tuesday, October 7, 2003 7:20 PM

Chapter thirty-two: Quantitative and qualitative aspects of nitrogen supply 535

block nasogastric tubes following retrograde reflux of gastric contents along the feeding
tube,57 to form gastric lactobezoars, or even to lead to esophageal obstruction.58 This can
be obviated by substituting whey protein (which has greater resistance to coagulation
during heat processing and in the stomach) or by using partial hydrolysates of protein
that can reduce gastric residuals and vomiting.59

32.3.2 Small intestinal nitrogen assimilation


Whereas glycemia following a starch meal consists of a rapid peak and exponential decline,
aminoacidemia following a protein meal is sustained; this suggests both rapid jejunal
absorption and more distal absorption.60,61 Adibi and Mercer61 observed that after a meal
of bovine serum albumin (BSA), undigested protein was observed in the ileum at a time
when the peak of blood amino acids had passed (60 min) and was declining slowly.
Furthermore, the majority of the BSA digestion products were peptides, and in the absence
of protein in the meal, the contribution of digestion products of secreted pancreatic pro-
teins to aminoacidemia was minimal.61 This suggested that peptide uptake is important
and that much dietary protein is assimilated in the distal small intestine. Certainly, the
positive longitudinal gradient of brush border peptidases in the human intestine would
support this interpretation.62,63 Nixon and Mawer64 could, like Adibi and Mercer,61 find no
contribution of pancreatic proteins to intestinal protein uptake; indeed, postprandial
plasma amino acid kinetics generally reflected the amino acid composition of the ingested
protein.65 The fate of endogenous proteins in the gut lumen was thus mysterious and has
remained so. On the one hand, 30% of the intestinal luminal nitrogen flow after a meal
comprises endogenous proteins66 and the stomal output of ileostomists contains significant
amounts of protein in the form of peptides.67 It has been inferred that this endogenous
protein will be recaptured after hydrolysis, by the ileum or by the colon after colonic
fermentation, along with other endogenously derived protein (intestinal secretions,
secreted plasma proteins, and desquamated cells). A more radical explanation by Rothman
and colleagues68 is that pancreatic proteins are absorbed intact and recycled to the pancreas
(i.e., analogous to the enterohepatic circulation of bile salts). This writer finds that their
most compelling arguments are that intravenously injected labeled pancreatic enzymes
can be recovered in the intestinal lumen and that the pancreas neither consumes enough
energy nor has sufficient ribosomes to produce pancreatic enzymes at the rate secreted
each day if they were to be destroyed in the small intestine.68 Their premise is that intestinal
digestion of pancreatic enzymes is wasteful, and in this respect, Rothman and colleagues
clearly support Bunge’s thermodynamic parsimony.8

32.3.3 Colonic protein digestion


Malabsorbed protein and carbohydrate have synergistic effects on growth of colonic
bacteria that ferment them to short-chain fatty acids (SCFAs), isomeric SCFAs, and copious
amounts of NH4+. The presence of excess carbohydrate (e.g., lactulose or starch) inhibits
the complete fermentation of protein to NH4+ in vitro and in vivo.69,70 This provides an
explanation of the effectiveness of lactulose/lactitol in preventing hyperammonemia in
alcoholic patients who have frequent variceal bleeds. However, the fate of malabsorbed
protein is still unclear. The presence of colonic mucosal peptidases71 led this writer to
predict that some peptides produced by colonic luminal hydrolysis would be salvaged
by the mucosa,72 as proved to be the case when peptide transporters were identified in
human colonic mucosa.73,74 These data highlight the considerable potential for salvage of
malabsorbed protein through competing human and microbial systems.
1382_C32.fm Page 536 Tuesday, October 7, 2003 7:20 PM

536 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

32.3.4 Urea recycling


Plasma urea may permeate into the colonic lumen to be hydrolyzed to NH3 and enter
either bacterial or host pathways of amino acid synthesis. Some groups75,76 suggest that
this is quantitatively important in neonates and children, although not all laboratories
obtain similar results.77 However, if endogenous urea can be reutilized, then it would
follow that antibiotic therapy during enteral nutrition will reduce the efficiency of colonic
luminal NH4+ generation (cf. antibiotic therapy in hepatic encephalopathy). It is tempting
to suggest that this is why noncritically ill but antibiotic-treated enterally fed patients
require high protein intakes (>16 g/day) to maintain nitrogen balance.78

32.4 Absorption of the products of luminal digestion


32.4.1 Free amino acid transport
Christensen developed classical methods to analyze amino acid transport functions by
treating the transporter as an enzyme that could not be isolated (since that would destroy
its activity) but that could be identified by its kinetic characteristics. For example, ionic
specificity and mutual competition for uptake between three amino acids would suggest
a common transport pathway. On this basis, several Na+-dependent or Na+-independent
systems were identified that were shown to be very substrate stereospecific, of high
specificity but often overlapping in function with another system. A fuller account of the
molecular biology of these transporters is given in Chapter 5, and it helps explain why
amino acid mixtures, protein hydrolysates, or their starter protein are all equally effective
in maintaining peptide transport, even though they may not be substrates for peptide
transport.79 It has been discovered that the heteromeric amino acid transporters (HATs),
of which the dibasic amino acid–cysteine transporter is an example, contain the heavy
subunit (HSHAT) which is a regulatory component linked by disulfide bond to the light
transport pore subunit (LSHAT). HSHAT has important chaperone functions in transporter
trafficking at the cell membrane and regulates transport characteristics of LSHAT (e.g.,
HSHAT is mutation site in type 1 cystinuria80). Thus, we and others have found that
ornithine, lysine, and arginine uptake in the perfused human intestine comprises saturable
and passive nonsaturable processes.81,82 In addition, dibasic amino acid transporters can,
when they are in heteromeric form, countertransport unrelated amino acids such as leu-
cine.83 Thus, when applying this information to therapeutic supplemental enteral appli-
cations of glutamine or the dibasic amino acids arginine or ornithine (as in ornithine
a-ketoglutarate), it is possible that benefit may arise from active uptake of one and
transstimulation of efflux of another. One example of this is polyamine efflux during
arginine or ornithine uptake,84 which may elicit local effects on epithelial lymphocytes.85
A second example is transport system x–c, which exchanges glutamate efflux for cysteine
influx80 and may thus increase enterocyte intracellular glutathione synthesis following
trauma or surgery. These examples of transport are directly relevant to amino acid sup-
plementation of enteral diets.

32.4.2 Peptide transport


In 1959, Newey and Smyth86 demonstrated that dipeptides could be absorbed intact across
everted sacs of rat intestine, but it was considered merely to be a physiological curiosity
as reviews of the time made clear: “these experiments (i.e., those of Prockop showing
proline peptiduria following gelatin ingestion87) conclusively demonstrated that some
1382_C32.fm Page 537 Tuesday, October 7, 2003 7:20 PM

Chapter thirty-two: Quantitative and qualitative aspects of nitrogen supply 537

peptide absorption may occur normally in man.”88 The evidence that changed this view
was that patients with cystinuria, in whom intestinal dibasic amino acid uptake was
absent, suffered neither from deficiency of these amino acids nor from the metabolic
disturbances of lysinuric protein intolerance. Thus, dietary dibasic amino acids must have
been absorbed by an unknown mechanism. When the small intestines of patients with
cystinuria were perfused with dipeptides containing arginine, normal arginine uptake
was observed.89 The dual hypothesis of dietary nitrogen assimilation that developed
(Figure 32.2) entailed uptake of intact di- or tripeptides by a system distinct from any
amino acid transporter and subsequent intracellular peptide hydrolysis. The second route
was that luminal amino acids or amino acids released by brush border hydrolysis of
luminal peptides would be absorbed by the amino acid transport systems.
The di- and tripeptide transport has proven to be most unusual. Uptake was shown
to be electrogenic and related to H+ cotransport.90 Since it occurs maximally if external
pH is less than intravesicular pH, the peptide transporter “acid-loads” the mucosal cell
from the slightly acidic submucosal microclimate, and the H+ is then exported back via
the Na+/H+ antiporter.91 The transporter also has interesting substrate specificity:92

• Di- or tripeptide, not tetrapeptide (unless it is a fatty acid amide)


• Free amino and carboxyl terminus (unless it is a cyclic peptide)
• Alpha orientation of peptide bond and a-amino group (valacyclovir is not a
peptide)
• Trans, not cis peptide (but not always)
• Preference for L- over D-amino acids (but not always)
• If above are satisfied, hydrophobicity governs rate of uptake

The transporter is therefore promiscuous, but molecular virtuosity such as this is not
surprising when one considers the diverse chemical structures that arise from luminal
and brush border protein digestion. From 20 protein amino acids one could, in theory,
generate 400 dipeptides and 8000 tripeptides.
Since the first report of expression cloning of the peptide transporter (PEPT1) in 1994,93
there have been over 200 papers on its function and tissue distribution, especially in
relation to peptidomimetic drugs, which can target different tissues.94 The transporter
should be considered with the peptidases associated with the membrane. Wells et al.95
first showed that enterocyte cystosolic and brush border peptidases were specific toward
di- and tripeptides and larger peptides, respectively. This suggested that one driving force
for peptide uptake was a “downhill” transmembrane peptide concentration gradient, to
which was later added the submucosal H+ gradient. PEPT1 is a symmetrical transporter
since peptides present on both sides of a membrane can be exchanged, but the direction
of net flow depends on membrane potential.96 The wide distribution of PEPT1 and
PEPT273,97 may explain how small peptides, which clearly originate from intracellular
protein breakdown, can exit cells and appear in urine98–100 (Figure 32.1).

32.5 Quantitative and qualitative comparisons of amino acid


and peptide transport
Before considering clinical studies that define the optimum form of enteral nitrogen to
use in different situations, the quantitative importance of peptide and amino acid absorp-
tion should be considered.
1382_C32.fm Page 538 Tuesday, October 7, 2003 7:20 PM

538 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

32.5.1 Relative rates of assimilation of proteins, peptides, and amino acids


Many human and animal small intestine perfusion studies have shown that jejunal uptake
of nitrogen is faster when presented in the form of a partially hydrolyzed protein than
that of the equivalent amino acid mixture. Furthermore, in most cases, the rate of absorp-
tion of individual amino acid residues was shown to be faster or more even.101–107 These
data suggested that peptide uptake was quantitatively significant and could be used to
provide a kinetic advantage to amino acid uptake. We investigated the effects of the
starter protein, the method of enzymic hydrolysis, and the chain length of constituent
peptides and showed that they all had profound effects on uptake of amino acid resi-
dues.104 Since there were several variables that could be investigated, we used Occam’s
Razor to select the simplest, that is, peptide chain length and its distribution. It proved
very difficult to measure this because the number of peptides possible at each chain
length is 20n (where n is the chain length) and each peptide has a unique pKa and
hydrophobicity. Furthermore, molecular weight methods do not separate on the basis of
chain length since triglycine (a tripeptide) has a lower molecular weight than methionyl-
methionine. Hence, the analytical problem was quite difficult. We therefore turned to
class separation methods such as Cu(II)-chelation chromatography and, subsequently,
the use of sequencing of the entire hydrolysate. Both methods gave similar results, and
this topic is reviewed in detail elsewhere.108,109 This is an important issue because a simple
measurement like degree of hydrolysis (DH) can encompass different chain-length pro-
files (e.g., DH = 20 is equivalent to 20% free amino acid, the remainder being undigested
protein or representing a mixture of pentapeptides). It was found that the peptide-based
enteral diets available at the time had remarkably heterogenous chain-length profiles,
which was not indicated by the DH value (Table 32.1). Since at the time it could be
deduced that there was a di- and tripeptide transporter, we investigated the effect of
changes in chain-length profile on nitrogen absorption in the perfused human jejunum.
A partial hydrolysate of lactalbumin (DH = 28.5, 32% di- and tripeptides) was signifi-
cantly better absorbed than one that was hydrolyzed to a slight extent (DH = 14.3, 98%
> pentapeptides). Two studies with ovalbumin and casein hydrolysates showed that
when the chain length was reduced from mainly tetra- and pentapeptides (DH = 30 to
33, 24 to 35% di- and tripeptides) to mainly di- and tripeptides (DH = 42 to 44, 70 to 75%
di- and tripeptides) there was a significant increase in nitrogen assimilation.106,109–111
Others have found the same relationship between DH and nitrogen absorption.103,112 These
studies were conducted under conditions in which pancreatic secretions were excluded
from the perfused segment and indicate that nitrogen uptake could be increased by 30
to 46% if chain length were reduced to di- and tripeptides.
Investigation of the effect of other hydrolysis variables could be a profitable means
of altering the sequence and hydrophobicity of the constituent peptides of a protein
hydrolysate. In general, the triple-enzyme method (neutrase, alcalase, and pancreatic
enzyme mixture) produces hydrolysates comprising mainly di- and tripeptides, many of
which are relatively resistant to brush border hydrolysis113 and which could even be used
in parenteral nutrition.109 These preparations may be absorbed predominantly via PEPT1,
thus promoting peptide efflux into portal blood, while reducing the amount available for
mucosal amino acid oxidation. One report suggests that the method of hydrolysis of casein
(neutrase or triple-enzyme method) alters its metabolic disposal in endotoxin-treated rats.
Protein synthesis in jejunum, liver, and spleen was higher in the triple-enzyme protein
hydrolysate-fed group.114 To my knowledge, this approach has only been incorporated
into one diet, Tipeptid®, which was briefly tested in France with encouraging results.115
Table 32.1 Peptide Chain-Length Profile of Several Protein Hydrolysates Used in Enteral Diets
Chapter thirty-two:

Peptide chain length (%)


L-Amino Degree of hydrolysis
Diet acids 2 3 4 5 6 7 8 9 10 (%)
Survimeda,e 18.0 5.0 5.0 1.0 1.0 24.0 10.0 2.0 17.0 17.0 31.9
Amirigeb,e 15.0 5.0 21.0 12.0 12.0 1.0 2.0 2.0 16.5 16.5 34.1
1382_C32.fm Page 539 Tuesday, October 7, 2003 7:20 PM

Reabilanc,e 2.0 11.0 21.0 1.0 17.0 17.0 15.0 16.0 — — 25.2
Peptisorbb,e 28.0 11.0 17.0 11.0 12.0 1.0 10.0 10.0 — — 47.2
Travasorb HNc,e 20.0 15.0 5.0 1.0 1.0 29.0 29.0 — — — 38.6
Pepti 2000b,f 17.0 11.0 16.0 14.0 14.0 13.0 15.0 — — — 38.4
Steraldietd,f 25.0 1.0 28.0 18.0 17.0 11.0 — — — — 44.6
Tipeptidf,g 8.0 34.0 35.0 16.0 7.0 — — — — — 42.1
Elemental 028e,h 100.0 — — — — — — — — — 100.0

Note: Peptide profile measured by Cu(II)-sephadex chromatography.179


a Fresenius-Kabi, Bad Homburg, Germany.
b Nutricia, Zoetermeer, Holland.
c Nestlé Clinical Nutrition, Deerfield, IL.
d Dubernard, Paris, France.
e Currently available.
f No longer available.
g Laboratoires Roger Bellon, Neuilly-sur-Seine, France.
Quantitative and qualitative aspects of nitrogen supply

h SHS International Ltd., Liverpool, U.K.


539
1382_C32.fm Page 540 Tuesday, October 7, 2003 7:20 PM

540 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

32.5.2 Effects of malnutrition on absorptive function


During early growth of animals, dipeptide transport is of greater quantitative significance
than free amino acid transport.116,117 Protein restriction or semistarvation reduces intestinal
mass, but transport of amino acids118 and peptides117,119 is maintained when expressed per
unit of body mass. Starvation leads to the ileum becoming a more significant site for
dipeptide and amino acid uptake.117 PEPT1 expression also moves “down” the villus and
is expressed more intensely in cells that have matured earlier.119,120 Overall, the effect of
malnutrition on peptide transport and intestinal peptidase activity seems to be stronger
than that for amino acid transport.121,122 These data confirm that the intestinal response to
starvation is physiological and that there is always a basal requirement for efficient capture
of amino acid and peptide released from endogenous sources.

32.5.3 Effects of critical illness on absorptive function


Few studies in man have investigated absorption of amino acids in response to trauma.
It is established that switching from enteral to parenteral nutrition causes a physiological
decline in small intestinal amino acid transport capacity.123 Sepsis also markedly inhibits
uptake of amino acids (including glutamine) and glucose by BBMV,124,125 and this would
be expected since intestinal energy consumption also decreases during sepsis.126,127 The
data on peptide transport is sparse, and one perfusion study of Zambian men with acute
bacterial pneumonia showed up-regulation of histidine uptake, but glycylglycine uptake
was unaltered.128 In rats, endotoxemia reduced mucosal PEPT1 expression,129 whereas
chemotherapeutic agents that cause intestinal damage led to profound down-regulation
of sugar and amino acid transporters, but not of PEPT1, which was up-regulated.130,131

32.5.4 Effect of short bowel syndrome on absorptive function


Following radical small bowel resection, there is considerable functional adaptation of the
remaining intestine over a period of a few months,132 but there are little data on how
peptide and amino acid transport adapts. One study in rabbits suggests that following
mid-intestinal resection, increases in amino acid and dipeptide transport never completely
compensate for the loss of absorptive area. However, intravenous epidermal growth factor
and growth hormone treatment up-regulated PEPT1 alone.133

32.6 Comparative feeding studies of proteins, peptides,


and amino acids
The literature abounds with trials comparing clinical benefits from enteral diets based on
protein hydrolysates, free L-amino acids, or whole proteins, but because these are essen-
tially formula comparisons, controls and experimental groups often differ in amino acid
pattern and energy or nitrogen intake. Changes in amino acid composition have important
and often overlooked effects on intermediary metabolism, which may invalidate such
studies. For example, in one comparative study of the effect of a high-fat diet on insulin
resistance, variation in the protein intake between cod, soy, or casein protein diets led to
very marked differences in muscle insulin insensitivity.134 Therefore, I shall try to compare
only trials that have appropriate control groups; these are summarized in Table 32.2.

32.6.1 Feeding trials in healthy humans and animals


Diets based on lactalbumin, partially hydrolyzed lactalbumin, or an equivalent amino acid
mixture yielded similar nitrogen balance during a chronic feeding study.135 Similarly, a
Table 32.2 Experimental Feeding Trials of Amino Acids, Peptides, or Proteins in Healthy Humans, Patients, or Other Animal Species
Subjects and Duration Comparison Results Reference
Humans (three 10-day periods, n = 2) Lactalbumin, P or H No difference in nitrogen balance 135
Humans Casein, P or H Hydrolysate evoked higher aminoacidemia, 145
protein synthesis, and leucine oxidation
Chapter thirty-two:

Humans Casein or whey protein More rapid aminoacidemia after whey protein 147
but leucine balance better with casein
Humans Lactalbumin, pea protein, and Hydrolysates evoked insulinemia, 146
whole milk, P or H glucagonemia, and aminoacidemia
Pigs (8-h enteral infusion) Milk protein, H or AA Higher portal amino acidemia during early 180
infusion period; integrated response the same
1382_C32.fm Page 541 Tuesday, October 7, 2003 7:20 PM

during entire period


Mice Casein, P, H, or AA No relative effects on intestinal amino acid and 79
dipeptide absorption and brush border
peptidases
Rats (growing) Casein, gluten, soybean, egg No significant differences in nitrogen balance or 138
albumin, P or AA growth rates
Rats (space-pair feeding to ensure no Casein, P or AA Equal growth and additional ammonia to render 136
bias due to food aversion/preference) AA isonitrogenous with P had no effect
Rats (pair-fed) Lactalbumin, H or AA Same growth but cecal enlargement in 181
hydrolysate-fed group
Rats Casein, P or AA Poorer nitrogen utilization and weight gain in 139
amino acid group
Rats (starved and refed) Casein, soy, P or H Protein synthesis, casein H > P; protein 142
breakdown, casein H and P > soy H
Rats (starved and refed) Lactalbumin, P, H, or AA Hydrolysate evoked faster weight gain and 122, 140
higher nitrogen retention; protein restored
Quantitative and qualitative aspects of nitrogen supply

villus height better


Rats (weaning, starved, and refed) Whey protein, casein, P or H Higher nitrogen retention in hydrolysate group 141
Rats fed by gavage (twice daily) for Milk protein, H or AA Net protein utilization better in hydrolysate-fed 143
10 days animals (0.144 ± 0.011 vs. 0.277 ± 0.027); higher
aminoacidemia and insulinemia (48.4 ± 11.2 vs.
20.0 ± 11.1 mIU/ml)
541
542

Table 32.2 Experimental Feeding Trials of Amino Acids, Peptides, or Proteins in Healthy Humans, Patients, or Other Animal Species (Continued)
Subjects and Duration Comparison Results Reference

Short Bowel Syndrome


Short bowel patients (jejunostomy) Lactalbumin, P + H or H Improved nitrogen absorption and increased 182
blood urea nitrogen with H alone
Short bowel patients (50–80cm, Lactalbumin, H or AA No difference in nitrogen absorption, nitrogen 152
1382_C32.fm Page 542 Tuesday, October 7, 2003 7:20 PM

crossover design) balance, or whole-body protein turnover


Short bowel patients (n = 10, young Lactalbumin, P or H No difference in total diet intake, nitrogen 183
children) balance, or intestinal permeability to sugar
probes
Rats (80% intestinal resection, 12-day Casein, H (two different chain No differences in food intake, weight gain, 153
feeding) lengths) or AA nitrogen balance
Rats (60% jejunoileal resection) Casein, P or H No differences in intestinal mucosal weight, 154
DNA, or glucose uptake

Pancreatic Disease
Total pancreatectomy for pancreatic Enteral diet, lactalbumin, P or H Higher nitrogen absorption from hydrolysate (91 158
cancer (n = 6) ± 2% vs. 61 ± 6%), but nitrogen balance similar

Cystic Fibrosis
Infants (n = 23, < 6 months of age) Whole-milk formula (P) vs. casein Similar growth rates and nutrient absorption 157
(H + AA)
Infants (n = 21, crossover study, no Whole milk or lactalbumin Protein absorption improved in patients 156
pancreatic enzyme supplements) hydrolysate absorbing <75% dietary fat

Critical Illness
Abdominal surgery (crossover design) Lactalbumin, P or H Hydrolysate evoked higher aminoacidemia and 162
insulinemia and reduced 3-methylhistidine
excretion; plasma proteins increased
Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition
Burned patients (crossover design) Lactalbumin, H or AA Nitrogen balance not different; some plasma 164
proteins higher in amino acid group
Abdominal surgery (crossover design) Lactalbumin, P or H Hydrolysate provoked increased aminoacidemia 163
and insulinemia and reduced 3-methylhistidine
excretion; plasma proteins increased

Neonates
Chapter thirty-two:

Premature infants (n = 100) Breast milk vs. casein, P or H Highest rates of weight gain with hydrolysate 184
Healthy infants (n = 60, 8-week Whole-milk protein, P and H Intake, weight gain, and length gain were 185
crossover design) comparable but more regurgitation in
hydrolysate group
Healthy infants (n = 45, 13 weeks) Whey-dominant formula and Similar weight and length gain 186
lactalbumin, H
1382_C32.fm Page 543 Tuesday, October 7, 2003 7:20 PM

Newborn infants (n = 59, 4 weeks) Human milk vs. lactalbumin, H Growth rate; two thirds H same as human milk, 187,188
(three types) one worse; H generally less effective than whey
formulae in preterm and term infants
Preterm infants (n = 61, 12 weeks) Fortified breast milk, standard Hydrolysates and standard formula evoked 170
formula, and partial or extensive higher weight gain, blood urea nitrogen, and
hydrolysates aminoacidemia
Very low birth weight infants (n = 87, 4 Preterm infant formula and Time to establish full milk feeding reduced 171
weeks) hydrolysate
Neonatal piglets (n = 24) Soy protein, P or H Similar portal substrate flows 189

Note: The protein used in the study is indicated and the form used is shown: P = whole protein; H = partially hydrolyzed protein; AA = equivalent amino acid mixture.
Quantitative and qualitative aspects of nitrogen supply
543
1382_C32.fm Page 544 Tuesday, October 7, 2003 7:20 PM

544 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

very careful study in young rats showed that casein or the identical amino acid mixture
produced equal growth rates.136 We also compared the effect of a whey hydrolysate-based
diet and an equivalent amino acid diet (without the nitrogen correction) in pair-fed rats
and found the hydrolysate as effective in maintaining growth rates, food intake, or the
composition of peripheral tissues,137 but it caused marked cecal hypertrophy, which could
have been due to the extra glutamine content of the hydrolysate or to induced malabsorp-
tion of some dietary component that was fermented in the large bowel. Forsum and
Hambraeus138 were also unable to observe any marked differences in nitrogen balance or
growth rates of rats fed one of several proteins or their equivalent free amino acid mixtures.
In contrast, Daenzer and colleagues139 found that protein balance was better in rats fed a
casein-based diet rather than the equivalent amino acid mixture. Intestinal amino acid
and dipeptide absorption and brush border peptidases seem to be unaffected by the form
of dietary nitrogen79 (Table 32.2).

32.6.2 Feeding trials during recovery from starvation


If animals were stressed by prior starvation, before refeeding with diets containing whole
protein, partial enzymic protein hydrolysate, or the equivalent amino acid mixture, then
differences were observed. Intact protein led to faster restoration of intestinal atrophy
(i.e., villus height), while weight gain and nitrogen balance were highest in the hydrolysate-
fed animals.122,140,141 Nielsen and colleagues142 also showed that refeeding starved rats with
casein hydrolysate or casein-based diets yielded the same nitrogen balance, but there were
differential effects on protein synthesis and degradation measured by 15N-glycine infusion.

32.6.3 Acute feeding studies in humans and animals


Acute feeding studies have highlighted metabolic differences between whole protein and
protein hydrolysates that seem to reside as much in the rapidity with which they are
emptied from the stomach as in their rates of small bowel assimilation. Rérat and
colleagues143,144 demonstrated that twice-daily gavage of a milk protein hydrolysate led to
a doubling of carcass protein accretion compared to the equivalent amino acid control
diet. More rapid growth was accompanied by hyperinsulinemia in the hydrolysate-fed
group. A comparable study in humans showed that the casein hydrolysate diet was
associated with higher rates of protein synthesis and leucine oxidation, but lower efficiency
of protein deposition.145 The relationship between the gastric emptying rate and secreta-
gogue effects has been demonstrated by Calbet and MacLean.146 Although the amino acid
composition of this comparative study was not controlled, it established that rapid gastric
emptying of dietary nitrogen leads to insulinemia and glucagonemia that is proportional
to the ability of the diet to raise plasma concentrations of the secretagogue amino acids,
leucine, isoleucine, valine, phenylalanine, and arginine.146 Finally, Beaufrère’s group
defined the metabolic characteristics of whole protein according to its rate of gastric
emptying: the fast–slow protein concept.147 More efficient protein balance was obtained
by slower gastric emptying and lower aminoacidemia, which was associated with reduced
amino acid oxidation.148 In elderly subjects, however, the opposite was the case.149
One interpretation of this conflicting data is that in the rat, which never stops growing,
hyperinsulinemia will stimulate growth and amino acid deposition in skeletal muscle,
whereas in adult humans it will not. Thus, in healthy adults, “fast” protein feeding leads
to inefficiency of protein deposition, whereas in elderly subjects who are more insulin
resistant and have lost relative muscle mass, stimulation of insulin secretion may promote
muscle protein deposition.150 The next section will therefore analyze clinical trials in this
context.
1382_C32.fm Page 545 Tuesday, October 7, 2003 7:20 PM

Chapter thirty-two: Quantitative and qualitative aspects of nitrogen supply 545

32.7 Comparative feeding trials of proteins, peptides, and amino acids


in clinical situations
The historical development of modern “elemental” diets is detailed in an excellent review.2
Efficacy of each form of enteral nitrogen will be reviewed according to the same criteria
as above.

32.7.1 Patients with impaired gastrointestinal absorptive area


We have shown that in patients with moderately impaired gastrointestinal function, pro-
tein hydrolysates have no nutritional advantages over whole protein,151 as is the case in
patients with 60 to 150 cm of the jejunum remaining.45 When absorptive area was more
severely reduced (50 to 80 cm of jejunum), no difference was observed in N balance, N
absorption, or 13C-leucine kinetics when patients received a whey–protein–hydrolysate-
based or equivalent amino acid-based enteral diet.152 Animal studies also suggest that
following very significant small bowel resection, whole protein, protein hydrolysate, or
free amino acids were equally well utilized and had no differential effects on gut morph-
ology.153,154 This does not mean that small bowel absorption differences did not exist,
because rats survive massive resection/reanastomosis quite well because they can effi-
ciently ferment and salvage nutrient that overspills into the colon.15

32.7.2 Patients with impaired pancreatic function


The earliest studies showed that protein malabsorption only occurred if pancreatic enzyme
secretion was impaired by more than 90%,155 and that in cystic fibrosis patients, nitrogen
absorption could be improved by using whey–hydrolysate-based diets.156 Improvements
in pancreatic enzyme supplements have negated this advantage and allow patients to eat
more normal diets.157 The intestinal brush border peptidases have a remarkable capacity
to digest significant amounts of whole protein in the absence of measurable luminal
pancreatic enzymes, as was shown by Steinhardt and colleagues158 in patients who had
undergone pancreatectomy for malignancy. Over half the protein was assimilated and the
malabsorbed portion was partially reutilized after colonic fermentation.

32.7.3 Critically ill patients


Insulin resistance, hyperglycemia, and muscle wasting, which commonly occur during
the early phase of critical illness, are associated with poor outcome,159 but during longer-
term critical illness, such as burns injury, there appears to be a failure of anabolic drive,
which leads to muscle wasting.160,161 If this analysis is correct, then it would be logical to
consider enteral nutritional therapy, which maximizes insulin and hypothalamic drive.
The ability to modulate gastric emptying of dietary nitrogen and insulinemia is an exciting
concept. Three studies have investigated this by comparing diets based on lactalbumin,
lactalbumin hydrolysate, or the equivalent amino acid mixture (Table 32.2). In patients
who underwent gastrointestinal surgery, lactalbumin hydrolysate was associated with
better muscle protein dynamics (arteriovenous differences, 3-methylhistidine excretion),
higher nitrogen balance, and higher insulinemia, which correlated most closely with
plasma leucine concentration,162,163 as it does in healthy individuals following protein
meals that have different gastric emptying rates.146 A third trial of burned patients com-
pared a lactalbumin hydrolysate with the equivalent amino acid mixture, but no consistent
pattern emerged to suggest that either formula was more beneficial.164 It would be of great
interest to consider the use of formulae that provoke the most rapid aminoacidemia and
1382_C32.fm Page 546 Tuesday, October 7, 2003 7:20 PM

546 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

insulinemia in burned patients because this seems to lead to the highest rates of muscle
protein repletion.160

32.7.4 Neonates
Protein hydrolysates have been used for nutrition of neonates for many years. The key
issue is whether in children with hypersensitivity to cow’s milk it is possible to reduce
protein antigenicity by partial hydrolysis, in order to prevent life-threatening immuno-
logical reactions. Considerable progress has been made in defining the broad characteris-
tics of protein hydrolysates for this application and ways in which antigenicity can be
tested in vitro.165,166 These protein hydrolysate formulae are also considered to be prophy-
lactic against development of cow’s milk intolerance (milk hypersensitivity), and the
reader is directed to some recent reviews on this topic.165,167,168 However, in nonhypersen-
sitive neonates, the metabolic and nutritional effects of different forms of enteral nitrogen
are relevant to this chapter, and some trials are shown in Table 32.2. The earliest prepa-
ration, Aminosol‚, made by the Vitrum Institute in Sweden, was a high-temperature
dialysate of casein digested with fresh pancreas. In the U.S., the Mead Johnson Company
also succeeded in producing a similar hydrolysate. According to several Pharmacopoeia
standards, they had low antigenicity and were successful amino acid sources for intrave-
nous nutrition, comprising mainly free amino acids. Activated charcoal processing could
deplete the tyrosine content, and most constituent glutamine/glutamate was cyclized to
pyroglutamic acid.20 The earliest trial that Arvid Wretlind, the inventor, conducted clearly
suggested that the hydrolysate was superior to whole-protein formulae or breast milk for
premature infants. Although comparable trials have been hard to find (i.e., formulae of
similar energy content and amino acid composition), the most recent suggest that peptide
diets may give advantage in terms of more rapid gastric emptying, aminoacidemia, weight
gain, and time to establish full milk feeding.169–171 This is a complex subject area because
growth rate is altered by the mode of infant feeding. Bolus and continuous nasogastric
feeding have different effects in term and preterm infants,172 and it seems that bolus feeding
per se promotes higher growth rates.173,174 It would clearly be profitable to determine
whether peptide-based formulae can improve rates of growth above those obtained with
bolus feeding.

32.8 Perspectives
Since the last edition of this book, there have been further advances in knowledge of
amino acid and peptide nutrition. In particular, the widespread tissue distribution of the
peptide transporter suggests that the human body operates a dual metabolic economy of
amino acids and peptides, just as the intestine has dual modes of nitrogen uptake. It was
originally suggested that peptide-based diets might have beneficial effects on protein
metabolism because their assimilation in the perfused small intestine led to faster and
more even amino acid uptake. One early study had shown that synchronous delivery of
all dietary amino acids yielded the highest weight regain after protein depletion,175 and
the authors had noted that “for effective tissue synthesis all essential amino acids must
be available in the tissues practically simultaneously; otherwise the first group absorbed
is not stored long enough to enable its essential amino acids to combine with those of the
second group for the synthesis of complete tissue-proteins.” This has proved almost
prophetic because the discovery of slow and fast proteins means that the efficiency of
whole-body protein metabolism may be altered by adjusting the form of dietary protein.
The clinical studies described here suggest very strongly that peptide diets contain “fast-
est” protein, which appears to stimulate aminoacidemia, growth, and better nutritional
1382_C32.fm Page 547 Tuesday, October 7, 2003 7:20 PM

Chapter thirty-two: Quantitative and qualitative aspects of nitrogen supply 547

parameters in critically ill patients. In addition, uptake of peptide diets can be modulated
by adjusting the chain length of constituent peptides.176 To my knowledge, the only enteral
diet whose nitrogen source comprised mainly di- and tripeptides was developed from
these studies. The uptake of intact peptides into portal blood may provide a “black
economy” of amino acids whose intestinal metabolism is different from the bulk of dietary
amino acids. Carnosine is an extreme example because of its resistance to enterocyte brush
border and cytosolic peptidases. Similarly, oral ingestion of collagen hydrolysates increases
urinary excretion of proline- and hydroxyproline-containing peptides.87 One intriguing
study showed that oral 14C-proline-labeled collagen hydrolysate preferentially labeled
cartilage compared to oral 14C-proline.177 One might ask whether this represents targeting
of oral proline peptides to a tissue adapted to removal of proline peptides after collagen
breakdown. Could a similar approach be used to deliver amino acids by combining them
into resistant dipeptides?
Finally, there have been considerable developments in manufacture of peptide prep-
arations. The term protein hydrolysate merely describes what was done to what, rather than
the nature of the product, which might be a component of food stock cubes or shampoo
or a surfactant to stabilize oil-in-water emulsions. The nutritional hydrolysates are rather
complex entities that tend to taste bitter (although this can be addressed by taste masking
or enzyme treatment165) and have variable chain length and antigenicity. There is tremen-
dous scope for “tailoring” through biotechnology113,178 to produce di- and tripeptide mix-
tures for maximal amino acid uptake in clinical conditions.

References
1. Althausen, T.L., Uyeyama, K., and Simpson, R.G., Digestion and absorption after massive
resection of the small intestine. I. Utilization of food from a “natural” versus a “synthetic”
diet and a comparison of intestinal absorption tests with nutritional balance studies in a
patient with only 45 cm of small intestine, Gastroenterology, 12, 795, 1949.
2. Silk, D.B.A., Enteral diets: clinical uses and formulation, in Artificial Nutrition Support in
Clinical Practice, 2nd ed., Payne-James, J.J., Grimble, G.K., and Silk, D.B.A., Eds., Greenwich
Medical Media, London, 2001, p. 303.
3. Loewi, O., Über Eiweisssynthese im Tierkörper [On protein synthesis in the animal body],
Arch. Exp. Path. Pharmakol., 48, 303, 1901.
4. Loewi, O., Zur frage nach der bildung von zucker aus fett, Arch. Exp. Path. Pharmakol., 47,
68, 1901.
5. Henriques, V. and Andersen, A.C., Über parenterale Ernährung durch intravenöse Injektion
[On parenteral nutrition through intravenous injection], Hoppe Seylers Z. Physiol. Chem., 88,
357, 1913.
6. van Slyke, D.D. and Meyer, G.M., The fate of protein digestion products in the body. III. The
absorption of amino acids from the blood by the tissues, J. Biol. Chem., 16, 197, 1913.
7. Elman, R., Parenteral Alimentation in Surgery, with Special Reference to Proteins and Amino Acids,
Paul B. Hoeber Inc., New York, 1947.
8. Bunge, C., Lehrbuch Der Physiologischen Und Pathologischen Chemie, in Zwanzig Vorlesun-
gen Für Ärzte Und Studierende, 4th ed., Verlag von F.C.W.Vogel, Leipzig, Germany, 1887, p. 168.
9. Fruton, J.S., Molecules and Life: Historical Essays on the Interplay of Chemistry and Biology, Wiley
Interscience, New York, 1972.
10. Grimble, G.K. and Backwell, F.R.C., Peptides in Mammalian Protein Metabolism: Tissue Utilisa-
tion and Clinical Targeting, Portland Press, London, 1997.
11. Botbol, V. and Scornik, O.A., Measurement of instant rates of protein degradation in the
livers of intact mice by the accumulation of bestatin-induced peptides, J. Biol. Chem., 266,
2151, 1991.
12. Zhou, X., Thamotharan, M., Gangopadhyay, A., Serdikoff, C., and Adibi, S.A., Characteriza-
tion of an oligopeptide transporter in renal lysosomes, Biochim. Biophys. Acta, 1466, 372, 2000.
1382_C32.fm Page 548 Tuesday, October 7, 2003 7:20 PM

548 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

13. Shennan, D.B., Calvert, D.T., Backwell, F.R., and Boyd, C.A.R., Peptide aminonitrogen trans-
port by the lactating rat mammary gland, Biochim. Biophys. Acta, 1373, 252, 1998.
14. MacLarnon, A.M., Martin, R.D., Chivers, D.J., and Hladnik, C.M., Some aspects of gastro-
intestinal allometry in primates and other mammals, in Definition et Origines de l’Homme, 1st
ed., Sakka, M., Ed., CNRS, Paris, 1983, p. 293.
15. O’Connor, T.P., Lam, M.M., and Diamond, J., Magnitude of functional adaptation after
intestinal resection, Am. J. Physiol., 276, R1265, 1999.
16. Stoll, B., Burrin, D.G., Henry, J., Yu, H., Jahoor, F., and Reeds, P.J., Substrate oxidation by the
portal drained viscera of fed piglets, Am. J. Physiol., 277, E168, 1999.
17. Austgen, T.R., Chen, M.K., Flynn, T.C., and Souba, W.W., The effects of endotoxin on the
splanchnic metabolism of glutamine and related substrates, J. Trauma, 31, 742, 1991.
18. Reeds, P.J., Burrin, D.G., Jahoor, F., Wykes, L., Henry, J., and Frazer, E.M., Enteral glutamate
is almost completely metabolized in first pass by the gastrointestinal tract of infant pigs, Am.
J. Physiol., 270, E413, 1996.
19. Lambert, B.D., Stoll, B., Niinikoski, H., Pierzynowski, S., and Burrin, D.G., Net portal ab-
sorption of enterally fed alpha-ketoglutarate is limited in young pigs, J. Nutr., 132, 3383, 2002.
20. Grimble, G., Glutamine, glutamate and pyroglutamate: facts and fantasies, Clin. Nutr., 12,
66, 1993.
21. Stegink, L.D., Shepherd, J.A., Brummel, M.C., and Murray, L.M., Toxicity of protein hydro-
lysate solutions: correlation of glutamate dose and neuronal necrosis to plasma amino acid
levels in young mice, Toxicology, 2, 285, 1974.
22. Marrs, T.C., Salmona, M., Garattini, S., Burston, D., and Matthews, D.M., The absorption by
human volunteers of glutamic acid from monosodium glutamate and from a partial enzymic
hydrolysate of casein, Toxicology, 11, 101, 1978.
23. Geha, R.S., Beiser, A., Ren, C., Patterson, R., Greenberger, P.A., Grammer, L.C., Ditto, A.M.,
Harris, K.E., Shaughnessy, M.A., Yarnold, P.R., Corren, J., and Saxon, A., Review of alleged
reaction to monosodium glutamate and outcome of a multicenter double-blind placebo-
controlled study, J. Nutr., 130, 1058S, 2000.
24. Walker, R. and Lupien, J.R., The safety evaluation of monosodium glutamate, J. Nutr., 130,
1049S, 2000.
25. Rérat, A., Nunes, C.S., Mendy, F., and Roger, L., Amino acid absorption and production of
pancreatic hormones in non-anaesthetized pigs after duodenal infusions of a milk enzymic
hydrolysate or of free amino acids, Br. J. Nutr., 60, 121, 1988.
26. Wu, G., Borbolla, A.G., and Knabe, D.A., The uptake of glutamine and release of arginine,
citrulline and proline by the small intestine of developing pigs, J. Nutr., 124, 2437, 1994.
27. O’Sullivan, D., Brosnan, J.T., and Brosnan, M.E., Hepatic zonation of the catabolism of
arginine and ornithine in the perfused rat liver, Biochem. J., 330, 627, 1998.
28. Van Goudoever, J.B., Stoll, B., Henry, J.F., Burrin, D.G., and Reeds, P.J., Adaptive regulation
of intestinal lysine metabolism, Proc. Natl. Acad. Sci. U.S.A., 97, 11620, 2000.
29. van der Schoor, S.R., Van Goudoever, J.B., Stoll, B., Henry, J.F., Rosenberger, J.R., Burrin,
D.G., and Reeds, P.J., The pattern of intestinal substrate oxidation is altered by protein
restriction in pigs, Gastroenterology, 121, 1167, 2001.
30. van der Schoor, S.R., Reeds, P.J., Stoll, B., Henry, J.F., Rosenberger, J.R., Burrin, D.G., and
Van Goudoever, J.B., The high metabolic cost of a functional gut, Gastroenterology, 123, 1931,
2002.
31. Bouteloup-Demange, C., Boirie, Y., Déchelotte, P., Gachon, P., and Beaufrère, B., Gut mucosal
protein synthesis in fed and fasted humans, Am. J. Physiol., 274, E541, 1998.
32. Heys, S.D., Park, K.G., McNurlan, M.A., Keenan, R.A., Miller, J.D., Eremin, O., and Garlick,
P.J., Protein synthesis rates in colon and liver: stimulation by gastrointestinal pathologies,
Gut, 33, 976, 1992.
33. Buchman, A.L., Moukarzel, A.A., Bhuta, S., Belle, M., Ament, M.E., Eckhert, C.D., Hollander,
D., Gornbein, J., Kopple, J.D., and Vijayaroghavan, S.R., Parenteral nutrition is associated
with intestinal morphologic and functional changes in humans, J. Parenter. Enteral. Nutr., 19,
453, 1995.
1382_C32.fm Page 549 Tuesday, October 7, 2003 7:20 PM

Chapter thirty-two: Quantitative and qualitative aspects of nitrogen supply 549

34. Burrin, D.G., Stoll, B., Jiang, R., Chang, X., Hartmann, B., Holst, J.J., Greeley, G.H., Jr., and
Reeds, P.J., Minimal enteral nutrient requirements for intestinal growth in neonatal piglets:
how much is enough? Am. J. Clin. Nutr., 71, 1603, 2000.
35. Christensen, H.N., Conjugated amino acids in portal plasma of dogs after protein feeding,
Biochem. J., 44, 333, 1949.
36. Gardner, M.L.G., Lindblad, B.S., Burston, D., Matthews, D.M., and Gardner, M.L., Trans-
mucosal passage of intact peptides in the guinea-pig small intestine in vivo: a reappraisal,
Clin. Sci., 64, 433, 1983.
37. Seal, C.J. and Parker, D.S., Isolation and characterization of circulating low molecular weight
peptides in steer, sheep and rat portal and peripheral blood, Comp. Biochem. Physiol. B Biochem.
Mol. Biol., 99, 679, 1991.
38. Christensen, H.N., Decker, D.G., Lynch, E.L., Mackenzie, T.M., and Powers, J.H., The conju-
gated, non-protein, amino acids of plasma. V. A study of the clinical significance of pep-
tidaemia, J. Clin. Invest., 26, 853, 1947.
39. Gardner, M.L.G., Transmucosal passage of intact peptides, in Peptides in Mammalian Protein
Metabolism: Tissue Utilisation and Clinical Targeting, 1st ed., Grimble, G.K. and Backwell, F.R.C.,
Eds., Portland Press, London, 1997, p. 11.
40. Backwell, F.R.C., Circulating peptides and their role in milk protein synthesis, in Peptides in
Mammalian Protein Metabolism: Tissue Utilisation and Clinical Targeting, 1st ed., Grimble, G.K.
and Backwell, F.R.C., Eds., Portland Press, London, 1997, p. 69.
41. Seal, C.J. and Parker, D.S., Methodological approaches for the quantitation of peptide ap-
pearance across the gastrointestinal tract, in Peptides in Mammalian Protein Metabolism: Tissue
Utilisation and Clinical Targeting, 1st ed., Grimble, G.K. and Backwell, F.R.C., Eds., Portland
Press, London, 1997, p. 43.
42. Matthews, D.M., Protein Absorption: Development and Present State of the Subject, 1st ed., Wiley-
Liss, New York, 1991.
43. Stevens, C.E. and Hume, I.D., Contributions of microbes in vertebrate gastrointestinal tract
to production and conservation of nutrients, Physiol. Rev., 78, 393, 1998.
44. Woolf, G.M., Miller, C., Kurian, R., and Jeejeebhoy, K.N., Nutritional absorption in short
bowel syndrome: evaluation of fluid, calorie, and divalent cation requirements, Dig. Dis. Sci.,
32, 8, 1987.
45. McIntyre, P.B., Fitchew, M., and Lennard-Jones, J.E., Patients with a high jejunostomy do not
need a special diet, Gastroenterology, 91, 25, 1986.
46. Kapembwa, M.S., Fleming, S.C., Griffin, G.E., Caun, K., Pinching, A.J., and Harris, J.R., Fat
absorption and exocrine pancreatic function in human immunodeficiency virus infection, Q.
J. Med., 74, 49, 1990.
47. Jeejeebhoy, K.N., Short bowel syndrome: a nutritional and medical approach, CMAJ, 166,
1297, 2002.
48. Nightingale, J.M.D. and Lennard-Jones, J., Nutrition support in short-bowel syndrome, in
Artificial Nutrition Support in Clinical Practice, 2nd ed., Payne-James, J.J., Grimble, G.K., and
Silk, D.B.A., Eds., Greenwich Medical Media, London, 2001, p. 701.
49. Booth, C.C., Alldis, D., and Read, A.E., Studies on the site of fat malabsorption. 2. Fat balances
after resection of varying amounts of the small intestine in man, Gut, 2, 168, 1961.
50. Ugolev, A.M., Timofeeva, N.M., Smirnova, L.F., Delaey, P., Gruzdkov, A.A., Iezuitova, N.N.,
Mityushova, N.M., Roshchina, G.M., Gurman, E.G., Gusev, V.M., Tsvetkova, V.A., and
Scherbakov, G.G., Membrane and intracellular hydrolysis of peptides: differentiation, role
and interrelations with transport, Ciba Found. Symp., 221, 1977.
51. Gardner, M.L., Illingworth, K.M., Kelleher, J., and Wood, D., Intestinal absorption of the
intact peptide carnosine in man, and comparison with intestinal permeability to lactulose,
J. Physiol. (Lond.), 439, 411, 1991.
52. Druml, W., Lochs, H., Roth, E., Hubl, W., Balcke, P., and Lenz, K., Utilization of tyrosine
dipeptides and acetyltyrosine in normal and uremic humans, Am. J. Physiol., 260, E280, 1991.
53. Stehle, P. and Fürst, P., In vitro hydrolysis of glutamine-, tyrosine- and cystine-containing
short-chain peptides, Clin. Nutr., 9, 37, 1990.
54. Fruton, J.S., A history of pepsin and related enzymes, Q. Rev. Biol., 77, 127, 2002.
1382_C32.fm Page 550 Tuesday, October 7, 2003 7:20 PM

550 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

55. Desnuelle, P., Chemistry and enzymology of pancreatic endopeptidases, in Molecular and
Cellular Basis of Digestion, Desnuelle, P., Sjöström, H., and Norén, O., Eds., Elsevier Science
Publishers B.V., Amsterdam, 1986, p. 195.
56. Puigserver, A., Chapus, C., and Kerfelec, B., Pancreatic exopeptidases, in Molecular and
Cellular Basis of Digestion, Desnuelle, P., Sjöström, H., and Norén, O., Eds., Elsevier Science
Publishers B.V., Amsterdam, 1986, p. 235.
57. Marcuard, S.P. and Perkins, A.M., Clogging of feeding tubes, J. Parenter. Enteral Nutr., 12,
403, 1988.
58. Turner, J.S., Fyfe, A.R., Kaplan, D.K., and Wardlaw, A.J., Oesophageal obstruction during
nasogastric feeding, Intensive Care Med., 17, 302, 1991.
59. Viall, C., Porcelli, K., Teran, J.C., Varma, R.N., and Steffee, W.P., A double-blind clinical trial
comparing the gastrointestinal side effects of two enteral feeding formulas, J. Parenter. Enteral
Nutr., 14, 265, 1990.
60. Chung, Y.C., Kim, Y.S., Shadchehr, A., Garrido, A., MacGregor, I.L., and Sleisenger, M.H.,
Protein digestion and absorption in human small intestine, Gastroenterology, 76, 1415, 1979.
61. Adibi, S.A. and Mercer, D.W., Protein digestion in human intestine as reflected in luminal,
mucosal, and plasma amino acid concentrations after meals, J. Clin. Invest., 52, 1586, 1973.
62. Triadou, N., Bataille, J., and Schmitz, J., Longitudinal study of the human intestinal brush
border membrane proteins: distribution of the main disaccharidases and peptidases, Gastro-
enterology, 85, 1326, 1983.
63. Darmoul, D., Voisin, T., Couvineau, A., Rouyer-Fessard, C., Salomon, R., Wang, Y., Swallow,
D.M., and Laburthe, M., Regional expression of epithelial dipeptidyl peptidase IV in the
human intestines, Biochem. Biophys. Res. Commun., 203, 1224, 1994.
64. Nixon, S.E. and Mawer, G.E., The digestion and absorption of protein in man. 1. The site of
absorption, Br. J. Nutr., 24, 227, 1970.
65. Marrs, T.C., Addison, J.M., Burston, D., and Matthews, D.M., Changes in plasma amino acid
concentrations in man after ingestion of an amino acid mixture simulating casein, and a
tryptic hydrolysate of casein, Br. J. Nutr., 34, 259, 1975.
66. Baglieri, A., Mahé, S., Zidi, S., Huneau, J.F., Thuillier, F., Marteau, P., and Tomé, D., Gastro-
jejunal digestion of soya-bean-milk protein in humans, Br. J. Nutr., 72, 519, 1994.
67. Chacko, A. and Cummings, J.H., Nitrogen losses from the human small bowel: obligatory
losses and the effect of physical form of food, Gut, 29, 809, 1988.
68. Rothman, S., Liebow, C., and Isenman, L., Conservation of digestive enzymes, Physiol. Rev.,
82, 1, 2002.
69. Mortensen, P.B., The effect of oral-administered lactulose on colonic nitrogen metabolism
and excretion, Hepatology, 16, 1350, 1992.
70. MacFarlane, G.T. and Macfarlane, S., Human colonic microbiota: ecology, physiology and
metabolic potential of intestinal bacteria, Scand. J. Gastroenterol. Suppl., 222, 3, 1997.
71. Hutter, H.J., Egorova, V.V., Nikitina, A.A., Zvetkova, V.A., and Ugolev, A.M., Proteolytic
activities of colonic mucosa, Z. Med. Lab. Diagn., 31, 27, 1990.
72. Grimble, G.K., Colonic fermentation of starch and protein: an end to all resistance? Nutrition,
11, 173, 1995.
73. Ziegler, T.R., Fernandez-Estivariz, C., Gu, L.H., Bazargan, N., Umeakunne, K., Wallace, T.M.,
Diaz, E.E., Rosado, K.E., Pascal, R.R., Galloway, J.R., Wilcox, J.N., and Leader, L.M., Distri-
bution of the H+/peptide transporter PepT1 in human intestine: up-regulated expression in
the colonic mucosa of patients with short-bowel syndrome, Am. J. Clin. Nutr., 75, 922, 2002.
74. Herrera-Ruiz, D., Wang, Q., Gudmundsson, O.S., Cook, T.J., Smith, R.L., Faria, T.N., and
Knipp, G.T., Spatial expression patterns of peptide transporters in the human and rat gastro-
intestinal tracts, Caco-2 in vitro cell culture model, and multiple human tissues, AAPS Pharm.
Sci., 3, E9, 2001.
75. Wheeler, R.A., Griffiths, D.M., and Jackson, A.A., Urea kinetics in neonates receiving total
parenteral nutrition, Arch. Dis. Child, 69, 24, 1993.
76. Badaloo, A., Boyne, M., Reid, M., Persaud, C., Forrester, T., Millward, D.J., and Jackson, A.A.,
Dietary protein, growth and urea kinetics in severely malnourished children and during
recovery, J. Nutr., 129, 969, 1999.
1382_C32.fm Page 551 Tuesday, October 7, 2003 7:20 PM

Chapter thirty-two: Quantitative and qualitative aspects of nitrogen supply 551

77. Fomon, S.J., Matthews, D.E., Bier, D.M., Rogers, R.R., Rebouche, C.J., Edwards, B.B., and
Nelson, S.E., Bioavailability of dietary urea nitrogen in the infant, J. Pediatr., 111, 221, 1987.
78. Rees, R.G., Cooper, T.M., Beetham, R., Frost, P.G., and Silk, D.B., Influence of energy and
nitrogen contents of enteral diets on nitrogen balance: a double blind prospective controlled
clinical trial, Gut, 30, 123, 1989.
79. Ferraris, R.P., Kwan, W.W., and Diamond, J., Regulatory signals for intestinal amino acid
transporters and peptidases, Am. J. Physiol., 255, G151, 1988.
80. Chillaron, J., Roca, R., Valencia, A., Zorzano, A., and Palacin, M., Heteromeric amino acid
transporters: biochemistry, genetics, and physiology, Am. J. Physiol., 281, F995, 2001.
81. Payne-James, J., Grimble, G., Cahill, E., and Silk, D.B.A., Jejunal absorption of ornithine-
oxoglutarate (OKGA) in man, J. Parenter. Enteral Nutr., 13 (Suppl.), 22S, 1989.
82. Hellier, M.D., Holdsworth, C.D., and Perrett, D., Dibasic amino acid absorption in man,
Gastroenterology, 65, 613, 1973.
83. Chillaron, J., Estevez, R., Mora, C., Wagner, C.A., Suessbrich, H., Lang, F., Gelpi, J.L., Testar,
X., Busch, A.E., Zorzano, A., and Palacin, M., Obligatory amino acid exchange via systems
bo,+-like and y+L-like: a tertiary active transport mechanism for renal reabsorption of cystine
and dibasic amino acids, J. Biol. Chem., 271, 17761, 1996.
84. Medina, M.A., Urdiales, J.L., Nunez de Castro, I., and Sánchez Jimenez, F., Diamines interfere
with the transport of L-ornithine in Ehrlich-cell plasma-membrane vesicles, Biochem. J., 280,
825, 1991.
85. ter Steege, J.C.A., Buurman, W.A., and Forget, P.P., Spermine induces maturation of the
immature intestinal immune system in neonatal mice, J. Pediatr. Gastroenterol. Nutr., 25, 332,
1997.
86. Newey, H. and Smyth, D.H., The intestinal absorption of some dipeptides, J. Physiol. (Lond.),
145, 48, 1959.
87. Prockop, D.J., Keiser, H.R., and Sjoerdsma, A., Gastrointestinal absorption and renal excretion
of hydroxyproline peptides, Lancet, ii, 527, 1962.
88. Saunders, S.J. and Isselbacher, K.J., Intestinal absorption of amino acids, Gastroenterology, 50,
586, 1966.
89. Silk, D.B.A., Perrett, D., and Clark, M.L., Jejeunal and ileal absorption of dibasic amino acids
and an arginine containing dipeptide in cystinuria, Gastroenterology, 68, 1426, 1975.
90. Ganapathy, V. and Leibach, F.H., Role of pH gradient and membrane potential in dipeptide
transport in intestinal and renal brush-border membrane vesicles from the rabbit: studies
with L-carnosine and glycyl-L-proline, J. Biol. Chem., 258, 14189, 1983.
91. Daniel, H. and Herget, M., Intestinal and renal transport of peptides at the cellular and
molecular level, in Peptides in Mammalian Protein Metabolism: Tissue Utilisation and Clinical
Targeting, 1st ed., Grimble, G.K. and Backwell, F.R.C., Eds., Portland Press, London, 1997,
p. 91.
92. Daniel, H., Morse, E.L., and Adibi, S.A., Determinants of substrate affinity for the oligopep-
tide/H+ symporter in the renal brush border membrane, J. Biol. Chem., 267, 9565, 1992.
93. Fei, Y.J., Kanai, Y., Nussberger, S., Ganapathy, V., Leibach, F.H., Romero, M.F., Singh, S.K.,
Boron, W.F., and Hediger, M.A., Expression cloning of a mammalian proton-coupled oli-
gopeptide transporter, Nature, 368, 563, 1994.
94. Brodin, B., Nielsen, C.U., Steffansen, B., and Frokjaer, S., Transport of peptidomimetic drugs
by the intestinal di/tri-peptide transporter, PepT1, Pharmacol. Toxicol., 90, 285, 2002.
95. Wells, G.P., Nicholson, J.A., and Peters, T.J., Subcellular localisation of di- and tripeptidases
in guinea pig and rat enterocytes, Biochim. Biophys. Acta, 569, 82, 1979.
96. Kottra, G. and Daniel, H., Bidirectional electrogenic transport of peptides by the proton-
coupled carrier PEPT1 in Xenopus laevis oocytes: its asymmetry and symmetry, J. Physiol.
(Lond.), 536, 495, 2001.
97. Chen, H., Wong, E.A., and Webb, K.E., Jr., Tissue distribution of a peptide transporter mRNA
in sheep, dairy cows, pigs, and chickens, J. Anim. Sci., 77, 1277, 1999.
98. Asatoor, A.M., Milne, M.D., and Walshe, J.M., Peptiduria in the Fanconi syndrome, Ciba
Found. Symp., 287, 1977.
1382_C32.fm Page 552 Tuesday, October 7, 2003 7:20 PM

552 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

99. Nam, T.J., Noguchi, T., and Naito, H., Changes in the urinary excretion of acid-soluble
peptides in rats injected with streptozotocin or dexamethasone: a trial to estimate the changes
in the rate of whole-body protein degradation in those rats, Br. J. Nutr., 65, 37, 1991.
100. Solaas, K.M., Skjeldal, O., Gardner, M.L., Kase, F.B., and Reichelt, K.L., Urinary peptides in
Rett syndrome, Autism, 6, 315, 2002.
101. Silk, D.B.A., Clark, M.L., Marrs, T.C., Addison, J.M., Burston, D., Matthews, D.M., and Clegg,
K.M., Jejunal absorption of an amino acid mixture simulating casein and an enzymic hydro-
lysate of casein prepared for oral administration to normal adults, Br. J. Nutr., 33, 95, 1975.
102. Silk, D.B.A., Fairclough, P.D., Clark, M.L., Hegarty, J.E., Marrs, T.C., Addison, J.M., Burston,
D., Clegg, K.M., and Matthews, D.M., Use of a peptide rather than free amino acid nitrogen
source in chemically defined “elemental” diets, J. Parenter. Enteral Nutr., 4, 548, 1980.
103. Friedrich, M., Noack, J., Proll, J., and Noack, R., Absorption of enzymatic protein hydrolysates
and equimolar amino acid mixtures in the perfused small intestine of the rat [Untersuchun-
gen zur Absorption enzymatischer Proteinhydrolysate sowie aquimolarer Aminosauremis-
chungen am perfundierten Dunndarm der Ratte], Biomed. Biochim. Acta, 43, 117, 1984.
104. Keohane, P.P., Grimble, G.K., Brown, B., Spiller, R.C., and Silk, D.B.A., Influence of protein
composition and hydrolysis method on intestinal absorption of protein in man, Gut, 26, 907,
1985.
105. Rérat, A., Simoes-Nuñes, C., Lacroix, M., Vaugelade, P., and Vaissade, P., Cinétique comparée
d’apparition dans la veine porte de l’azote a-aminé provenant de mélanges de petits peptides
ou d’acides aminés libres de même composition introduits dans le duodenum chez le porc
éveillé [Comparative kinetics of appearance in the portal vein of a-amino nitrogen from
mixtures of small peptides of free amino acids of the same composition introduced in the
duodenum of conscious pigs], C. R. Acad. Sci. III, 300, 293, 1985.
106. Grimble, G.K., Rees, R.G., Keohane, P.P., Cartwright, T., Desreumaux, M., and Silk, D.B.A.,
The effect of peptide chain-length on absorption of egg-protein hydrolysates in the normal
human jejunum, Gastroenterology, 92, 136, 1987.
107. Ehrlein, H. and Haas-Deppe, B., Comparison of absorption of nutrients and secretion of
water between oligomeric and polymeric enteral diets in pigs, Br. J. Nutr., 80, 545, 1998.
108. Beaumont, C., Grimble, G.K., and Welham, K.J., A novel method for the identification of
peptides in complex mixtures by tandem mass-spectrometry with atmospheric pressure
chemical ionization, in Peptides in Mammalian Protein Metabolism: Tissue Utilisation and Clinical
Targeting, 1st ed., Grimble, G.K. and Backwell, F.R.C., Eds., Portland Press, London, 1997,
p. 167.
109. Grimble, G.K., Protein hydrolysates as vehicles for the application of di- and tripeptides in
clinical nutrition, in Peptides in Mammalian Protein Metabolism: Tissue Utilisation and Clinical
Targeting, 1st ed., Grimble, G.K. and Backwell, F.R.C., Eds., Portland Press, London, 1997,
p. 119.
110. Grimble, G.K., Keohane, P., Higgins, B.E., Kaminski, M.V., and Silk, D.B.A., Effect of peptide
chain-length on amino acid and nitrogen absorption from two lactalbumin hydrolysates in
the normal human jejunum, Clin. Sci., 71, 65, 1986.
111. Rees, R.G., Raimundo, A.H., Grimble, G.K., Hunjan, M.K., and Silk, D.B.A., Peptide based
nitrogen source of enteral diets: studies with casein hydrolysates in man, J. Parenter. Enteral
Nutr., 12 (Suppl.), 21S, 1988.
112. Friedrich, M., Noack, J., Proll, J., and Noack, R., Comparative study of the absorption of
enzymatic protein hydrolysates in the small intestine of the rat. 2. The absorption of tryptic,
thermitatic and tryptic-thermitatic casein hydrolysates compared with the equimolar com-
position of free amino acids [Vergleichende Untersuchungen zur Absorption enzymatischer
Proteinhydrolysate am Dunndarm der Ratte. Mitt. Die Absorption von tryptischen, thermi-
tatischen und tryptisch-thermitatischen Caseinhydrolysaten im Vergleich zum aquimolaren
Gemisch freier Aminosauren], Nahrung, 29, 167, 1985.
113. Chataud, J., Desreumaux, S., and Cartwright, T., French Patent Application, 86 17516, De-
cember 15, 1986.
114. Kondrup, J. and Frokjaer, S., Effect of endotoxin on tissue protein synthesis in rats is modified
by method of casein hydrolysis, Clin. Nutr., 18 (Suppl.), 41, 1999.
1382_C32.fm Page 553 Tuesday, October 7, 2003 7:20 PM

Chapter thirty-two: Quantitative and qualitative aspects of nitrogen supply 553

115. Dufrene, C., Berta, J.L., and Poutignat, N., Results of a multicenter study on tolerance of a
semi-elemental diet administered enterally, Ann. Gastroenterol. Hepatol., 25, 163, 1989.
116. Guandalini, S. and Rubino, A., Development of dipeptide transport in the intestinal mucosa
of rabbits, Pediatr. Res., 16, 99, 1982.
117. Miller, P.M., Burston, D., Brueton, M.J., and Matthews, D.M., Kinetics of uptake of L-leucine
and glycylsarcosine into normal and protein malnourished young rat jejunum, Pediatr. Res.,
18, 504, 1984.
118. Lis, M.T., Matthews, D.M., and Crampton, R.F., Effects of dietary restriction and protein
deprivation on intestinal absorption of protein digestion products in the rat, Br. J. Nutr., 28,
443, 1972.
119. Ogihara, H., Suzuki, T., Nagamachi, Y., Inui, K., and Takata, K., Peptide transporter in the
rat small intestine: ultrastructural localization and the effect of starvation and administration
of amino acids, Histochem. J., 31, 169, 1999.
120. Cheeseman, C.I., Expression of amino acid and peptide transport systems in rat small
intestine, Am. J. Physiol., 251, G636, 1986.
121. Ihara, T., Tsujikawa, T., Fujiyama, Y., Ueyama, H., Ohkubo, I., and Bamba, T., Enhancement
of brush border membrane peptidase activity in rat jejunum induced by starvation, Pflugers
Arch., 440, 75, 2000.
122. Poullain, M.G., Cézard, J.P., Marche, C., Roger, L., Mendy, F., and Broyart, J.P., Dietary whey
proteins and their peptides or amino acids: effects on the jejunal mucosa of starved rats, Am.
J. Clin. Nutr., 49, 71, 1989.
123. Inoue, Y., Espat, N.J., Frohnapple, D.J., Epstein, H., Copeland, E.M., and Souba, W.W., Effect
of total parenteral nutrition on amino acid and glucose transport by the human small
intestine, Ann. Surg., 217, 604, 1993.
124. Salloum, R.M., Copeland, E.M., and Souba, W.W., Brush border transport of glutamine and
other substrates during sepsis and endotoxemia, Ann. Surg., 213, 401, 1991.
125. Souba, W.W., Herskowitz, K., Klimberg, V.S., Salloum, R.M., Plumley, D.A., Flynn, T.C., and
Copeland, E.M.I., The effects of sepsis and endotoxemia on gut glutamine metabolism, Ann.
Surg., 211, 543, 1990.
126. Pastores, S.M., Katz, D.P., and Kvetan, V., Splanchnic ischemia and gut mucosal injury in
sepsis and the multiple organ dysfunction syndrome, Am. J. Gastroenterol., 91, 1697, 1996.
127. Vallet, B., Lund, N., Curtis, S.E., Kelly, D., and Cain, S.M., Gut and muscle tissue PO2 in
endotoxemic dogs during shock and resuscitation, J. Appl. Physiol., 76, 793, 1994.
128. Cook, G.C., Effect of systemic bacterial infection on absorption rates of L-histidine and
glycylglycine from the human jejunum in vivo, Am. J. Clin. Nutr., 30, 1994, 1977.
129. Shu, H.J., Takeda, H., Shinzawa, H., Takahashi, T., and Kawata, S., Effect of lipopolysaccha-
ride on peptide transporter 1 expression in rat small intestine and its attenuation by dexa-
methasone, Digestion, 65, 21, 2002.
130. Hosoda, T., Bamba, T., and Hosoda, S., Ileal absorption of various amino acids and dipeptides
in rats administered cyclophosphamide: using the short-circuit current method, Jpn. J. Gas-
troenterol., 88, 2837, 1991.
131. Tanaka, H., Miyamoto, K.I., Morita, K., Haga, H., Segawa, H., Shiraga, T., Fujioka, A., Kouda,
T., Taketani, Y., Hisano, S., Fukui, Y., Kitagawa, K., and Takeda, E., Regulation of the PepT1
peptide transporter in the rat small intestine in response to 5-fluorouracil-induced injury,
Gastroenterology, 114, 714, 1998.
132. Cosnes, J., Carbonnel, F., Beaugerie, L., Ollivier, J.M., Parc, R., Gendre, J.P., and Le Quintrec,
Y., Functional adaptation after extensive small bowel resection in humans, Eur. J. Gastroen-
terol. Hepatol., 6, 197, 1994.
133. Avissar, N.E., Ziegler, T.R., Wang, H.T., Gu, L.H., Miller, J.H., Iannoli, P., Leibach, F.H.,
Ganapathy, V., and Sax, H.C., Growth factors regulation of rabbit sodium-dependent neutral
amino acid transporter ATB0 and oligopeptide transporter 1 mRNAs expression after enter-
ectomy, J. Parenter. Enteral Nutr., 25, 65, 2001.
134. Lavigne, C., Tremblay, F., Asselin, G., Jacques, H., and Marette, A., Prevention of skeletal
muscle insulin resistance by dietary cod protein in high fat-fed rats, Am. J. Physiol., 281, E62,
2001.
1382_C32.fm Page 554 Tuesday, October 7, 2003 7:20 PM

554 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

135. Moriarty, K.J., Hegarty, J.E., Fairclough, P.D., Kelly, M.J., Clark, M.L., and Dawson, A.M.,
Relative nutritional value of whole protein, hydrolysed protein and free amino acids in man,
Gut, 26, 694, 1985.
136. Itoh, H., Kishi, T., and Chibata, I., Comparative effects of casein and amino acid mixture
simulating casein on growth and food intake in rats, J. Nutr., 103, 1709, 1973.
137. Grimble, G.K., Preedy, V., Garlick, P., and Silk, D.B.A., Trophic effects of dietary peptides on
the rat intestinal tract, J. Parenter. Enteral Nutr., 13 (Suppl.), 6S, 1989.
138. Forsum, E. and Hambraeus, L., Effects of proteins and their corresponding amino acid
mixtures on nitrogen balance and body composition in the growing rat, J. Nutr., 108, 1518,
1978.
139. Daenzer, M., Petzke, K.J., Bequette, B.J., and Metges, C.C., Whole-body nitrogen and splanch-
nic amino acid metabolism differ in rats fed mixed diets containing casein or its correspond-
ing amino acid mixture, J. Nutr., 131, 1965, 2001.
140. Poullain, M.G., Cézard, J.P., Roger, L., and Mendy, F., Effect of whey proteins, their oligopep-
tide hydrolysates and free amino acid mixtures on growth and nitrogen retention in fed and
starved rats, J. Parenter. Enteral Nutr., 13, 382, 1989.
141. Boza, J.J., Martinez Augustin, O., Baro, L., Suarez, M.D., Gil, A., and Martinez-Augustin, O.,
Protein v. enzymic protein hydrolysates: nitrogen utilization in starved rats, Br. J. Nutr., 73,
65, 1995.
142. Nielsen, K., Kondrup, J., Elsner, P., Juul, A., and Jensen, E.S., Casein and soya-bean protein
have different effects on whole body protein turnover at the same nitrogen balance, Br. J.
Nutr., 72, 69, 1994.
143. Monchi, M. and Rérat, A.A., Comparison of net protein utilization of milk protein mild
enzymatic hydrolysates and free amino acid mixtures with a close pattern in the rat,
J. Parenter. Enteral Nutr., 17, 355, 1993.
144. Monchi, M., Vaugelade, P., Vaissade, P., and Rérat, A., Net protein utilisation after duodenal
infusion of small peptides or free amino acids in growing rats, Clin. Nutr., 10 (Suppl.), 31,
1991.
145. Collin-Vidal, C., Cayol, M., Obled, C., Ziegler, F., Bommelaer, G., and Beaufrère, B., Leucine
kinetics are different during feeding with whole protein or oligopeptides, Am. J. Physiol.,
267, E907, 1994.
146. Calbet, J.A.L. and MacLean, D.A., Plasma glucagon and insulin responses depend on the
rate of appearance of amino acids after ingestion of different protein solutions in humans,
J. Nutr., 132, 2174, 2002.
147. Boirie, Y., Dangin, M., Gachon, P., Vasson, M.P., Maubois, J.L., and Beaufrère, B., Slow and
fast dietary proteins differently modulate postprandial protein accretion, Proc. Natl. Acad.
Sci. U.S.A., 94, 14930, 1997.
148. Arnal, M.A., Mosoni, L., Boirie, Y., Houlier, M.L., Morin, L., Verdier, E., Ritz, P., Antoine,
J.M., Prugnaud, J., Beaufrère, B., and Patureau-Mirand, P., Protein feeding pattern does not
affect protein retention in young women, J. Nutr., 130, 1700, 2000.
149. Arnal, M.A., Mosoni, L., Boirie, Y., Houlier, M.L., Morin, L., Verdier, E., Ritz, P., Antoine,
J.M., Prugnaud, J., Beaufrère, B., and Patureau-Mirand, P., Protein pulse feeding improves
protein retention in elderly women, Am. J. Clin. Nutr., 69, 1202, 1999.
150. Boirie, Y., Gachon, P., Cordat, N., Ritz, P., and Beaufrère, B., Differential insulin sensitivities
of glucose, amino acid, and albumin metabolism in elderly men and women, J. Clin. Endo-
crinol. Metab., 86, 638, 2001.
151. Rees, R.G.P., Hare, W.R., Grimble, G.K., Frost, P.G., and Silk, D.B.A., Do patients with
moderately impaired gastrointestinal function requiring enteral nutrition need a predigested
nitrogen source? A prospective crossover controlled clinical trial, Gut, 33, 877, 1992.
152. Rees, R.G., Grimble, G., Halliday, D., Ford, C., and Silk, D.B.A., Influence of orally admin-
istered amino acids and peptides on protein turnover kinetics in the short-bowel syndrome,
Gut, 28, A1397, 1987.
1382_C32.fm Page 555 Tuesday, October 7, 2003 7:20 PM

Chapter thirty-two: Quantitative and qualitative aspects of nitrogen supply 555

153. Sales, M.G., de Freitas, O., Zucoloto, S., Okano, N., Padovan, G.J., Dos Santos, J.E., and
Greene, L.J., Casein, hydrolyzed casein, and amino acids that simulate casein produce the
same extent of mucosal adaptation to massive bowel resection in adult rats, Am. J. Clin. Nutr.,
62, 87, 1995.
154. Vanderhoof, J.A., Grandjean, C.J., Burkley, K.T., and Antonson, D.L., Effect of casein versus
casein hydrolysate on mucosal adaptation following massive bowel resection in infant rats,
J. Pediatr. Gastroenterol. Nutr., 3, 262, 1984.
155. Crane, C.W., Studies on the absorption of 15N labelled yeast in normal subjects and patients
with malabsorption, in The Role of the Gastrointestinal Tract in Protein Metabolism, Munro, H.N.,
Ed., F.A. Davis, Philadelphia, 1964, p. 333.
156. Canciani, M. and Mastella, G., Absorption of a new semielemental diet in infants with cystic
fibrosis, J. Pediatr. Gastroenterol. Nutr., 4, 735, 1985.
157. Ellis, L., Kalnins, D., Corey, M., Brennan, J., Pencharz, P., and Durie, P., Do infants with cystic
fibrosis need a protein hydrolysate formula? A prospective, randomized, comparative study,
J. Pediatr., 132, 270, 1998.
158. Steinhardt, H.J., Wolf, A., Jakober, B., Schmuelling, R.M., Langer, K., Brandl, M., Fekl, W.E.,
and Adibi, S.A., Nitrogen absorption in pancreatectomised patients: protein versus protein
hydrolysate as substrate, J. Lab. Clin. Med., 113, 162, 1989.
159. Van den Berghe, G., Wouters, P., Weekers, F., Verwaest, C., Bruyninckx, F., Schetz, M.,
Vlasselaers, D., Ferdinande, P., Lauwers, P., and Bouillon, R., Intensive insulin therapy in
the critically ill patients, N. Engl. J. Med., 345, 1359, 2001.
160. Hart, D.W., Wolf, S.E., Zhang, X.J., Chinkes, D.L., Buffalo, M.C., Matin, S.I., DebRoy, M.A.,
Wolfe, R.R., and Herndon, D.N., Efficacy of a high-carbohydrate diet in catabolic illness, Crit.
Care Med., 29, 1318, 2001.
161. Van den Berghe, G., Novel insights into the neuroendocrinology of critical illness, Eur. J.
Endocrinol., 143, 1, 2000.
162. Ziegler, F., Ollivier, J.M., Cynober, L., Masini, J.P., Coudray-Lucas, C., Levy, E., and
Giboudeau, J., Efficiency of enteral nitrogen support in surgical patients: small peptides v.
non-degraded proteins, Gut, 31, 1277, 1990.
163. Ziegler, F., Nitenberg, G., Coudray-Lucas, C., Lasser, P., Giboudeau, J., and Cynober, L.,
Pharmacokinetic assessment of an oligopeptide-based enteral formula in abdominal surgery
patients, Am. J. Clin. Nutr., 67, 124, 1998.
164. Badetti, C., Cynober, L., Bernini, V., Garabedian, M., and Manelli, J.C., Nutrition proteins
and muscular catabolism in severely burnt patients: comparative effects of small peptides
or free amino acids, Ann. Fr. Anesth. Reanim., 13, 654, 1994.
165. Lee, Y.H., Food-processing approaches to altering allergenic potential of milk-based formula,
J. Pediatr., 121, S47, 1992.
166. Docena, G., Rozenfeld, P., Fernandez, R., and Fossati, C.A., Evaluation of the residual anti-
genicity and allergenicity of cow’s milk substitutes by in vitro tests, Allergy, 57, 83, 2002.
167. Moro, G.E., Warm, A., Arslanoglu, S., and Miniello, V., Management of bovine protein allergy:
new perspectives and nutritional aspects, Ann. Allergy Asthma Immunol., 89, 91, 2002.
168. Terracciano, L., Isoardi, P., Arrigoni, S., Zoja, A., and Martelli, A., Use of hydrolysates in the
treatment of cow’s milk allergy, Ann. Allergy Asthma Immunol., 89, 86, 2002.
169. Tolia, V., Lin, C.H., and Kuhns, L.R., Gastric emptying using three different formulas in
infants with gastroesophageal reflux, J. Pediatr. Gastroenterol. Nutr., 15, 297, 1992.
170. Szajewska, H., Albrecht, P., Stoitiska, B., Prochowska, A., Gawecka, A., and Laskowska-Klita,
T., Extensive and partial protein hydrolysate preterm formulas: the effect on growth rate,
protein metabolism indices, and plasma amino acid concentrations, J. Pediatr. Gastroenterol.
Nutr., 32, 303, 2001.
171. Mihatsch, W.A., Franz, A.R., Hogel, J., and Pohlandt, F., Hydrolyzed protein accelerates
feeding advancement in very low birth weight infants, Pediatrics, 110, 1199, 2002.
172. Lucas, A., Bloom, S.R., and Aynsley-Green, A., Metabolic and endocrine events at the time
of the first feed of human milk in preterm and term infants, Arch. Dis. Child, 53, 731, 1978.
173. Dollberg, S., Kuint, J., Mazkereth, R., and Mimouni, F.B., Feeding tolerance in preterm infants:
randomized trial of bolus and continuous feeding, J. Am. Coll. Nutr., 19, 797, 2000.
1382_C32.fm Page 556 Tuesday, October 7, 2003 7:20 PM

556 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

174. Schanler, R.J., Shulman, R.J., Lau, C., Smith, E.O., and Heitkemper, M.M., Feeding strategies
for premature infants: randomized trial of gastrointestinal priming and tube-feeding method,
Pediatrics, 103, 434, 1999.
175. Cannon, P.R., Steffee, C.H., Frazier, L.J., Rowley, D.A., and Steptoe, R.C., The influence of
time on ingestion of essential amino acids upon utilization in tissue synthesis, Fed. Proc., 6,
390, 1947.
176. Grimble, G.K. and Silk, D.B.A., Peptides in human nutrition, Nutr. Res. Rev., 2, 87, 1989.
177. Oesser, S., Adam, M., Babel, W., and Seifert, J., Oral administration of 14C labeled gelatin
hydrolysate leads to an accumulation of radioactivity in cartilage of mice (C57/BL), J. Nutr.,
129, 1891, 1999.
178. Cowan, D.A., Andersen, J.A., Grimble, G.K., and Jaenicke, T.A., International Application,
WO97/13139.5, 1997.
179. Rothenbuhler, E., Waibel, R., and Solms, J., An improved method for the separation of
peptides and a-amino acids on copper sephadex, Anal. Biochem., 97, 367, 1979.
180. Rérat, A., Simoes-Nuñes, C., Mendy, F., Vaissade, P., and Vaugelade, P., Splanchnic fluxes of
amino acids after duodenal infusion of carbohydrate solutions containing free amino acids
or oligopeptides in the non-anaesthetised pig, Br. J. Nutr., 68, 111, 1992.
181. Grimble, G.K., Preedy, V., Garlick, P., and Silk, D.B.A., Trophic effects of dietary peptides on
the rat intestinal tract, Gut, 30, A1454, 1989.
182. Cosnes, J., Evard, D., Beaugerie, L., Gendre, J.P., and Le Quintrec, Y., Improvement in protein
absorption with a small-peptide-based diet in patients with high jejunostomy, Nutrition, 8,
406, 1992.
183. Ksiazyk, J., Piena, M., Kierkus, J., and Lyszkowska, M., Hydrolyzed versus nonhydrolyzed
protein diet in short bowel syndrome in children, J. Pediatr. Gastroenterol. Nutr., 35, 615, 2002.
184. Jorpes, J.E., Magnusson, J.H., and Wretlind, A., Casein hydrolysate; a supplementary food
for premature infants, Lancet, ii, 228, 1946.
185. Medjad-Guillou, N., Henocq, A., and Arnaud-Battandier, F., Does the hydrolysis of proteins
change the acceptability and the digestive tolerance of milk for infants? The results of a
comparative and randomized prospective study, Ann. Pediatr. (Paris), 39, 202, 1992.
186. Vandenplas, Y., Hauser, B., Blecker, U., Suys, B., Peeters, S., Keymolen, K., and Loeb, H., The
nutritional value of a whey hydrolysate formula compared with a whey-predominant for-
mula in healthy infants, J. Pediatr. Gastroenterol. Nutr., 17, 92, 1993.
187. Rigo, J., Salle, B.L., Putet, G., and Senterre, J., Nutritional evaluation of various protein
hydrolysate formulae in term infants during the first month of life, Acta Paediatr. Suppl., 402,
100, 1994.
188. Rigo, J., Salle, B.L., Picaud, J.C., Putet, G., and Senterre, J., Nutritional evaluation of protein
hydrolysate formulas, Eur. J. Clin. Nutr., 49 (Suppl. 1), S26, 1995.
189. Zijlstra, R.T., Mies, A.M., McCracken, B.A., Odle, J., Gaskins, H.R., Lien, E.L., and Donovan,
S.M., Short-term metabolic responses do not differ between neonatal piglets fed formulas
containing hydrolyzed or intact soy proteins, J. Nutr., 126, 913, 1996.
1382_C33.fm Page 557 Tuesday, October 7, 2003 7:23 PM

chapter thirty-three

Branched-chain amino
and keto acids in renal failure
Noël Cano
School of Pharmacy and Clinique Résidence du Parc, Marseille

Contents
Introduction..................................................................................................................................558
33.1 The kidney and branched-chain amino metabolism ....................................................558
33.1.1 Role of normal kidney in branched-chain amino acid metabolism............558
33.1.2 Amino and keto acids during renal failure.....................................................558
33.1.2.1 Circulating and cellular BCAAs during uremia ............................558
33.1.2.2 Muscle branched-chain amino acid and keto acid
metabolism during renal failure .......................................................560
33.1.2.2.1 Muscle branched-chain amino and keto acid
exchanges...........................................................................560
33.1.2.2.2 Muscle branched-chain amino acid metabolism.........560
33.1.2.3 Hepatosplanchnic branched-chain amino acid metabolism
during renal failure .............................................................................561
33.1.2.4 Consequences of BCAA metabolic abnormalities..........................562
33.2 Branched-chain amino and keto acid supply ................................................................562
33.2.1 Branched-chain amino and keto acid supply during chronic renal
failure.....................................................................................................................562
33.2.1.1 Rationale for the use of BCAA and BCKA in CRF patients ........563
33.2.1.2 Low-protein diets during CRF ..........................................................563
33.2.1.2.1 Metabolic adaptation to a reduction in protein
intake ..................................................................................563
33.2.1.2.2 Effect of low-protein diets on proteinuria....................564
33.2.1.2.3 Nutritional effects of low-protein diets.......................564
33.2.1.2.4 Effects of low-protein diets on the progression
of renal insufficiency........................................................566
33.2.2 Branched-chain amino and keto acid supply in dialysis patients ..............567
33.3 Conclusions.........................................................................................................................568
References .....................................................................................................................................569

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 557
1382_C33.fm Page 558 Tuesday, October 7, 2003 7:23 PM

558 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Introduction
Patients with chronic renal failure (CRF) or end-stage renal failure treated by dialysis are
characterized by multiple abnormalities of amino acid (AA) metabolism. These metabolic
disturbances, which particularly involve branched-chain amino acids (BCAAs) and their
ketoanalogs (BCKAs), are due to the lack of normal renal AA metabolism and to the impact
of renal failure on both peripheral and hepatosplanchnic nitrogen metabolism. The abnor-
malities of BCAA and BCKA metabolism result in BCAA depletion, which mainly con-
cerns valine. BCAA and BCKA supplementation during CRF aims to improve plasma AA
and nutritional status. Moreover, as protein restriction has been reported to slow the
progression of renal failure, BCAAs and BCKAs have been proposed in order to decrease
protein intake as much as possible while maintaining protein status.

33.1 The kidney and branched-chain amino metabolism


The role of kidneys in many aspects of protein metabolism, including low-molecular-
weight protein degradation1 and AA synthesis,2,3 has been recognized for a long time.
Moreover, via glutamine hydrolysis and ammonia excretion, renal AA metabolism also
plays a key role in acid–base balance regulation.4–6 Consequently, renal failure can be
responsible for altered BCAA production and overall, by alterations of BCAA metabolism,
due to metabolic acidosis.

33.1.1 Role of normal kidney in branched-chain amino acid metabolism


In man, the arteriovenous exchanges of AAs in the kidney during the absorptive phase
have not been investigated to our knowledge. In dogs given an AA load, significant
uptakes of valine, leucine, and isoleucine by the kidneys, without urinary excretion, were
observed.6 Because of the high BCAA–transferase and low BCKA–dehydrogenase activi-
ties in the renal parenchyma, it can be hypothesized that BCAAs are mainly involved in
transamination processes and/or ammoniogenesis. During the postabsorptive phase,
blood AA exchanges in the kidneys have been studied in normal man after catheterization
of a peripheral artery and a renal vein.7 It should be noted that although AAs were taken
up from arterial plasma, 50% of the AAs released in the renal veins was transported by
red cells.7 These studies of whole-blood renal AA exchanges demonstrated that the kidneys
release large amounts of leucine in the venous blood, the renal production of leucine
accounting for one third of total leucine production.8 In the same studies, no net renal
exchange of valine or isoleucine was noted. Thus, under normal conditions, BCAA metab-
olism in kidneys is characterized by an uptake of BCAAs in the absorptive phase and,
overall, by a substantial release of leucine during the postabsorptive phase. To our knowl-
edge, renal exchanges of BCKAs have not been studied.

33.1.2 Amino and keto acids during renal failure


33.1.2.1 Circulating and cellular BCAAs during uremia
Plasma concentrations of essential AAs (EAAs), except for methionine, are often decreased
during untreated CRF as well as during dialysis. The essential AA/nonessential AA ratio
is therefore decreased. Table 33.1 shows the blood, plasma, and muscle BCAA changes
observed in CRF and dialysis patients. Circulating valine concentrations are decreased in
CRF as well as in hemodialysis and peritoneal dialysis patients. After injection of a tracer
dose of L-valine-1-14C, a significant decrease in both plasma and whole free valine pools
was observed in CRF patients, compared with controls.9 The decrease in plasma valine is
1382_C33.fm Page 559 Tuesday, October 7, 2003 7:23 PM

Chapter thirty-three: Branched-chain amino and keto acids in renal failure 559

Table 33.1 Changes in Circulating and Muscle BCAAs during Chronic Renal Failure
Nondialyzed CRF Hemodialysis Peritoneal dialysis
Plasma valine Ø9,10 Ø10,11,24,28 Ø13
Blood valine Ø8
Muscle valine Ø10 Ø10,11,28 N13
Ø10
Plasma leucine Ø10 N11,24,28 Ø13
Blood leucine N8
Muscle leucine N10 N10,11,28 N13
Ø125
Plasma isoleucine Ø10 N10,11,24,28 N13
Blood isoleucine N8
Muscle isoleucine N10 N10,11,28 N13

Note: Ø = decreased concentration; N = concentrations similar to those of controls.

associated with a decrease in whole-blood and muscle concentrations of valine in CRF, as


well as in dialysis patients. It should be noted that valine depletion is constantly reported
even in patients with moderate CRF and preserved nutritional balance.8,10 Plasma leucine
and isoleucine are inconstantly altered depending on the stage of renal failure. Contrary
to valine, whole-blood and muscle concentrations of leucine and isoleucine remain normal
during uremia. Low intra- and extracellular valine, together with frequently depleted
leucine and isoleucine extracellular pools, have been described as a typical BCAA pattern
for chronic uremia.10 A preferential catabolism of valine has been proposed to explain the
depletion of valine pools during CRF.10 The abnormalities of plasma and intracellular
concentrations of BCAAs could not be corrected when BCAAs were given according to
Rose’s recommendations on EAA requirements.10 BCAA- and particularly valine-enriched
supplements were needed to normalize plasma and intracellular BCAAs in CRF patients.10
An improvement of plasma and intracellular BCAAs has been reported during hemodi-
alysis,10–12 peritoneal dialysis,13 and after the correction of acidosis.14,15 Renal transplanta-
tion can achieve a normalization of BCAA status.16,17 Data concerning the effect of eryth-
ropoietin on blood AA in uremic patients are controversial.18,19
Several authors have reported low plasma levels of BCKAs in patients with CRF20–23
as well as during hemodialysis.24 Decreases in plasma ketoisocaproate (KIC), ketoisoval-
erate (KIV), and ketomethylvalerate (KMV), respective ketoanalogs of leucine, valine, and
isoleucine, have not been found in other series.25 In these studies, plasma KIC was corre-
lated with glomerular filtration rate (GFR) and protein intake, plasma KMV with GFR
and plasma bicarbonate, and both KIC and KMV with plasma triglycerides. KIV did not
show any correlation with GFR, acidosis, protein intake, or plasma triglycerides.25 In CRF,
a reduction of whole-blood concentrations of BCKAs owing to a decrease in KIC and KIV
levels without change in KMV concentration has been reported.26 In both controls and
patients, the arterial levels of individual BCAAs were directly correlated with arterial
levels of the corresponding BCKAs. However, in CRF, the ratios of leucine to KIC and of
isoleucine to KMV were increased. In the same study, a direct correlation between whole-
blood KIC and bicarbonate levels was observed showing the role of acidosis in KIC
depletion.26
Although the abnormalities of circulating BCAAs can be worsened by undernutri-
tion,27 they are linked to renal insufficiency.7,10,28–30 During the postabsorptive phase, the
plasma AA pool is the result of AA release from muscle and kidney and AA uptake in
the hepatosplanchnic area, brain, and kidney.31,32 In addition to the suppression of renal
participation in leucine production, several factors can affect BCAA metabolism during
renal failure. Catabolic factors such as acidosis and inflammation are responsible for an
1382_C33.fm Page 560 Tuesday, October 7, 2003 7:23 PM

560 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

increase in muscle protein breakdown and BCAA degradation. Moreover, during the
absorptive phase, the profound abnormalities of hepatosplanchnic AA output alter plasma
AA composition and organ AA exchanges (see below).

33.1.2.2 Muscle branched-chain amino acid and keto acid metabolism during
renal failure
33.1.2.2.1 Muscle branched-chain amino and keto acid exchanges. Muscle AA concen-
trations result from permanent exchanges characterized by an AA uptake during the
absorptive phase and an AA release during the postabsorptive phase.31,33 In normal sub-
jects given a protein meal, valine, leucine, and isoleucine account for more than 50% of
muscle AA uptake.34 After the ingestion of an AA mixture simulating an animal protein
meal, the study of leg AA exchanges showed that total AA uptake was greater in CRF
patients than in control subjects (+71%) owing to an increase in the uptake of nonessential
AAs (NEAAs) (+156%).35 BCAA uptake by the leg was, in absolute values, similar to that
of control subjects but represented only 30% of total AA extraction, compared with 46%
in control subjects. Thus, muscle tissue faces the increased and unbalanced postprandial
supply of AAs (see below) with an augmented and unbalanced uptake.35
During the postabsorptive phase, low muscle concentrations of valine and decreased
valine release have been observed36 and were shown to be responsible for the low con-
centrations of plasma valine.36,37 Moreover, an inverse correlation between intracellular
valine and arterial bicarbonate has been reported, showing the role of acidosis in the
metabolism of this AA.11 The study of BCKA exchanges in muscle showed that during
CRF, the release of KIC and KMV was reduced, whereas KIV was neither released nor
taken up by the forearm.26 The decrease in KIC release from peripheral tissues was
associated with low circulating KIC levels and decreased KIC:leucine ratio.21,26

33.1.2.2.2 Muscle branched-chain amino acid metabolism. In well-nourished and sta-


ble CRF patients, the study of forearm protein metabolism after an arterial infusion of 3H-
phenylalanine showed an increase in both protein synthesis and degradation without
change in net proteolysis.36 Interestingly, net proteolysis and the proteolysis:proteosynthe-
sis ratio were inversely correlated with arterial bicarbonate concentrations. The proteolytic
effect of acidosis has been demonstrated in isolated-perfused muscle38 and in myocyte
cultures.39 In vivo, acidosis was similarly shown to stimulate protein degradation and
oxidation estimated from 14C-leucine kinetics.40 Conversely, the correction of acidosis by
oral bicarbonate suppressed the protein degradation due to experimental CRF.41 In healthy
volunteers, NH4Cl-induced acidosis was reported to induce protein catabolism and BCAA
oxidation.42 In CRF patients, the suppression of acidosis by oral bicarbonate was shown
to reduce protein degradation and oxidation measured after 14C-leucine infusion.43 Data
on isolated perfused-rat muscle have demonstrated that acidosis induces an irreversible
degradation of muscle BCAAs by stimulating the BCKA dehydrogenase.44 Moreover, it
was shown that the cytosolic ATP-ubiquitin-dependent proteolytic pathway is activated
by acidosis.45 Cortisol, whose secretion is stimulated by acidosis,38,46 appeared to be
necessary for metabolic acidosis to induce both BCAA oxidation47,48 and ATP-ubiquitin-
dependent proteolysis.49 In acidotic rats, it has been shown that muscle but not liver
BCKA dehydrogenase activities were stimulated.50 A parallel increase in muscle BCKA
dehydrogenase E1a and BCKA dehydrogenase E2 subunit mRNAs was demonstrated.50
Moreover, experimental acidosis is associated with an increase in muscle glutamine
synthesis51 and muscle glutamine release.52 Thus, on one hand, metabolic acidosis induces
muscle protein catabolism and BCAA breakdown together with an increase in glutamine
release. On the other hand, acidosis stimulates renal AA metabolism toward ammonium
1382_C33.fm Page 561 Tuesday, October 7, 2003 7:23 PM

Chapter thirty-three: Branched-chain amino and keto acids in renal failure 561

excretion and bicarbonate generation.5 Hence, muscle BCAA breakdown appears as an


element of an integrated regulatory mechanism for the fight against acidosis.48,52 However,
during chronic acidosis due to CRF, such a defense mechanism becomes deleterious by
inducing a progressive depletion of muscle mass.48
In addition to acidosis, abnormal insulin action has been shown to be associated with
plasma BCAA changes during CRF.10 In adolescents on dialysis, growth failure has been
reported to be associated with glucose intolerance, reduced insulin secretion, and low
plasma BCAA concentrations. It was hypothesized that the impaired utilization of con-
ventional energy sources leading to preferential oxidation of BCAAs contributed to
reduced anabolism and growth failure in uremia.53 Finally, in hemodialysis patients, it
has been reported that the abnormalities in plasma BCAAs and BCKAs were correlated
to the level of anemia, suggesting that BCAA and BCKA metabolism depends on oxygen
availability.54

33.1.2.3 Hepatosplanchnic branched-chain amino acid metabolism during


renal failure
In humans, postabsorptive hepatosplanchnic AA exchanges have been studied after arte-
rial and hepatic vein catheterization. Under normal conditions,34,55 alanine and glutamine
account for more than 50% of AA uptake from arterial plasma. Whole-blood measurements
showed an uptake of valine and leucine in the hepatosplanchnic area.55 During CRF,56 the
hepatosplanchnic exchanges of AAs have been shown to be impaired. From a quantitative
point of view, the main finding is the decrease in glutamine net uptake and a suppression
of serine, valine, and citrulline exchanges.
In normal conditions, a protein meal is followed by an enrichment of arterial blood
in EAAs and in particular in BCAAs:34,55,57,58 it was reported that the ingestion of 4 g/kg
of body weight (BW) of grilled steak induced an increase in the arterial concentrations of
EAAs (+116%) superior to that of nonessential AAs (+23%).58 The arterial blood enrichment
predominated on leucine (+216%), isoleucine (+271%), and valine (+116%). Following an
AA meal (0.8 g/kg of BW), studies of hepatosplanchnic AA exchanges showed a shift
from uptake to release.55 The release of AAs predominated on BCAAs and proline, but
also concerned arginine, lysine, threonine, tyrosine, and phenylalanine. During CRF,58 after
the ingestion of 4 g/kg of BW of grilled steak, the increase in arterial BCAAs was of similar
magnitude in CRF patients and healthy controls. However, such a meal induced a twofold
increase in arterial nonessential AA. The increase in arterial AA changes reached 260%
for glycine, 186% for cysteine, 180% for glutamine, 112% for proline, 79% for alanine, and
61% for serine. After an AA meal (0.8 g/kg of BW), hepatosplanchnic exchange study
showed an increase of whole AA release during CRF:55 135% for nonessential AAs and
65% for EAAs.
Thus, hepatosplanchnic AA exchanges are impaired during CRF, both from quantita-
tive and qualitative points of view. Experimental data have shown that in normal condi-
tions, following a protein meal, 56% of the AAs taken up by the liver were converted into
urea, 29% being used for protein synthesis and 15% released in the hepatic veins.59 Such
a rate of ureagenesis in the absorptive period makes it possible to obtain an adequate
composition of hepatic vein AAs for peripheral tissue resplenishment, i.e., enrichment in
EAAs and BCAAs: on one hand, a qualitative amelioration of AA composition is ensured,
while on the other hand, a substantial quantity of AAs is lost. During CRF, the decrease
in hepatosplanchnic retention of AAs participates in the abnormalities of arterial AAs, as
shown by the aggravation of these abnormalities after feeding.35,58 Moreover, the reduction
of hepatosplanchnic utilization of AA is ipso facto associated with a reduction of hepatocyte
protein synthesis and urea synthesis. In liver cells isolated from uremic rats, a reduction
1382_C33.fm Page 562 Tuesday, October 7, 2003 7:23 PM

562 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

of ureagenesis from alanine and glutamine was reported.60 Similarly, a decrease in ure-
agenesis was observed during enteral feeding in uremic rats.61 Acidosis may be mainly
responsible for the abnormalities of protein metabolism during the absorptive phase. In
CRF rats acidosis actually induced plasma AA changes similar to those of CRF.62 Similarly,
Garibotto and coworkers35 reported that variations in arterial concentrations and muscle
uptake of BCAAs were inversely correlated with arterial bicarbonate concentration in
absorptive CRF patients.

33.1.2.4 Consequences of BCAA metabolic abnormalities


BCAA metabolic abnormalities can alter tissue activities, particularly brain function, and
nutritional status.32,63–65 Changes in plasma BCAA concentrations due to renal failure can
influence both blood–brain barrier AA exchanges33,66 and neurotransmittor synthesis. Dur-
ing the postabsorptive phase, in normal conditions, the brain takes up numerous AAs
from arterial plasma, mainly glutamine, BCAAs, serine, cysteine, lysine, proline, ornithine,
phenylalanine, and citrulline.67–69 Glutamine accounts for 25% of this uptake, valine for
19%, and leucine together with isoleucine for 16%.68,69 During CRF, no significant brain
uptake of valine, isoleucine, and glutamine was found.68,69 The impairments of blood–brain
barrier AA exchanges during CRF are responsible for several abnormalities in cerebrospi-
nal fluid composition:70,71 increase in phosphoserine, phosphoethanolamine, glycine, orni-
thine, lysine, and 1- and 3-methylhistidine, and decrease in leucine, valine, and tyrosine
concentrations. Similarly, brain lipid and protein synthesis, which are dependent on BCAA
availability,72–76 as well as neurotransmittor synthesis from glutamine and tyrosine,77 may
be compromised. Hence, abnormal brain BCAA metabolism may participate in uremic
encephalopathy.63,78 Conversely, BCAA supplementation may improve brain functions. As
a matter of fact, in hemodialysis patients, BCAA infusion has been associated with a return
of rapid eye movement sleep to normal and a significant decrease in end-tidal CO2 during
both rapid eye movement and non-rapid eye movement sleep.79 Similarly, BCAA supple-
ments have been reported to stimulate appetite in depleted hemodialysis patients.80
The abnormal composition of arterial AAs influences organ AA uptake and compro-
mises peripheral tissue replenishment, particularly during the absorptive phase.35 In CRF
patients, nutritional indices such as body mass index, muscle mass indicators, and plasma
transthyretin have been found to be correlated with plasma leucine, isoleucine, and par-
ticularly valine.81,82 Conversely, nutritional supplementation by intradialytic parenteral
nutrition was reported to induce an improvement of nutritional status together with an
increase in plasma leucine.83
Increased protein turnover, unbalanced hepatosplanchnic AA release during the
absorptive phase, and subsequent abnormal BCAA metabolism are responsible for a
decrease in the efficacy of protein intake. Depending on the severity of renal failure, two
attitudes are currently used in clinical practice to counteract these abnormalities of AA
and protein metabolism:84–88 (1) in nondialyzed CRF patients, correction of the abnormal
plasma AA profile by the administration of EAAs or keto acids has been proposed in
order to improve protein status while limiting nitrogen load; (2) in hemodialysis patients,
a protein supply of 1.2 to 1.4 g/kg of BW/day has been recommended, and BCAA or
BCKA supplements have been used in some studies.

33.2 Branched-chain amino and keto acid supply


33.2.1 Branched-chain amino and keto acid supply during chronic renal failure
The aims of nutritional interventions in CRF can be summarized as follows:84 (1) minimize
uremic toxicity and avoid malnutrition, and (2) delay progression of kidney disease.
1382_C33.fm Page 563 Tuesday, October 7, 2003 7:23 PM

Chapter thirty-three: Branched-chain amino and keto acids in renal failure 563

Table 33.2 Recommended Protein Supply in Adults with CRF


Protein supply (g/kg/day)
ESPEN84,85 NKF adults86,88
GFR, 25–70 ml/min 0.55–0.60 (2/3 HBV) —
GFR, <25 ml/min 0.55–0.60 (2/3 HBV) 0.60
or or
0.28 + EAA or EAA + KA 0.75 (intolerance or insufficient
energy intake)

Note: ESPEN = European Society of Parenteral and Enteral Nutrition; NKF = National Kidney Foundation;
GFR = glomerular filtration rate; HBV = high biological value; KA = keto acid.

Table 33.2 gives the recommendations of the National Kidney Foundation86 and of the
European Society of Parenteral and Enteral Nutrition84 concerning protein supply during
CRF. The use of BCAAs and BCKA supplements cannot be studied separately from low-
protein diets (LPDs): these compounds are usually given in patients with severe CRF in
order to decrease further protein intake while maintaining satisfactory nutritional status.
It should be noted that in most animal and human studies, BCAAs and BCKAs were not
given solely but in association with other EAAs or their ketoanalogs.10 Therefore, the
effects of BCAA and BCKA supplementation cannot be studied separately.

33.2.1.1 Rationale for the use of BCAA and BCKA in CRF patients
BCAA-containing AA mixtures are given in association with LPDs in order to enhance
the efficiency of protein intake by ensuring suitable EAA support. Alvestrand and
coworkers10 showed that when EAAs are given in amounts corresponding to the recom-
mendations of Rose for normal man, the BCAA plasma pools and intracellular valine pool
could not be normalized. The same authors showed that increasing valine supplementa-
tion made it possible to correct the plasma and intracellular pools of valine.10
Three rationales have been proposed for the therapeutic use of BCKAs:89 (1) because
of their ability to fix amine groups and to regenerate BCAAs, BCKAs are used as amino-
free substitutes for BCAAs20,90,91; (2) BCKAs, ketoleucine in particular, have been shown
to be able to reduce muscle protein degradation89,92; and (3) BCKAs may favor a slower
progression of renal insufficiency, mainly by reducing the severity of secondary hyper-
parathyroidism.21 The intestinal absorption of BCKA treatment was shown to be unaf-
fected by CRF.25 The effects of BCKA supplementation on liver protein metabolism have
been poorly investigated. In a model of chronic protein malnutrition in rats, the addition
of BCKAs to low-protein diet markedly improved liver microsomal proteins and glu-
tathione, suggesting that BCKAs may prevent the deterioration in the nutritional state of
the liver in uremic patients.93
BCAA supplements usually include valine, leucine, isoleucine, together with phenyl-
alanine, threonine, tryptophan, lysine, methionine, and histidine. Tyrosine, considered an
EAA during CRF, was added to some preparations.10 BCKA mixtures also contain phe-
nylalanine ketoanalog, methionine hydroxyanalog, and other AAs considered essential
for CRF patients.

33.2.1.2 Low-protein diets during CRF


33.2.1.2.1 Metabolic adaptation to a reduction in protein intake. In normal adults, the
adaptation of nitrogen metabolism during a 14-day reduction of protein intake was inves-
tigated using diurnal measurements of nitrogen and leucine balance and turnover.94
Following a reduction of protein intake from 1.82 to 0.77 g of protein/kg of BW/day,
nitrogen excretion fell slowly and nitrogen balances progressively became less negative.
1382_C33.fm Page 564 Tuesday, October 7, 2003 7:23 PM

564 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

At the same time, decreased AA oxidation and protein degradation occurred together
with increased AA utilization for protein synthesis.94 During CRF, nitrogen balance and
kinetics of infused L-[15N,1-13C] leucine were measured during fasting and feeding in six
adult CRF patients and four controls given 0.6 and 1.0 g of protein/kg of BW/day diets.95
LPD similarly reduced feeding-stimulated oxidation of leucine in both groups. For both
groups, suppression of protein degradation by feeding, nitrogen balance, and protein
balance was similar.95 A similar adaptation was reported in patients with heavy proteinuria
and CRF.96 Furthermore, it has been demonstrated that following a reduction in protein
intake from 1.20 to 0.66 g/kg/day for 1 month, endogenous leucine flux decreased by 8%,
hepatic albumin synthesis decreased from 18.2 to 14.9 g/1.73 m2, and serum albumin rose
from 28.8 to 30.6 g/l.97
The metabolic effect of an LPD supplemented or not with ketoanalogs of EAA was
assessed during a longer period in a randomized, controlled study of 12 patients with
mild CRF. Protein intake in both groups was isonitrogenous. After a 4- to 6-week equilib-
rium period, patients reduced their protein intake to reach 0.71 g of protein/kg/day during
the third month. Energy intake was kept constant (31 kcal/kg of BW/day) during the
3-month period. At the end of the 3-month low-protein period, there were no changes in
patients’ body weights, serum albumin, or insulin-like growth factor (IGF)-1. Total body
flux of 13C-leucine decreased by 8% and leucine oxidation by 18%. No difference could be
attributed to the ketoanalogs themselves.98 These findings suggest a normal adaptation in
protein metabolism, similar to that reported in healthy volunteers, and confirm the safety
of protein intake of 0.7 g/kg of BW/day in CRF.98
Very low protein diets (VLPDs) have been shown to reduce AA oxidation by a greater
magnitude. In six predialysis patients, a diet providing 0.35 g of protein/kg/day supple-
mented with either keto acids or EAAs for 25 days was shown to be able to maintain
neutral nitrogen balance and body composition.99 These diets were associated with very
low oxidation rates of leucine, which were not different whether patients were supple-
mented with ketoanalogs or EAAs.99 In a 16-month follow-up of these patients, the fasting
leucine oxidation rate remained at the low level of 10.0 ± 2.2 µmol/kg of BW/h.100
These data showed that CRF patients adapt to protein restriction as normal subjects.
No advantage has been demonstrated with the use of an isonitrogenous diet containing
keto acids and EAAs. When VLPDs are given together with keto acids or EAAs, a similar
adaptation is observed with no demonstrated advantage of keto acid over EAA supple-
mentation.

33.2.1.2.2 Effect of low-protein diets on proteinuria. Most experimental and clinical


studies report a reduction in proteinuria due to protein restriction.101 Aparicio and
coworkers102 studied 15 patients with advanced renal failure (creatinine clearance
< 25 ml/min) and severe albuminuria (>1.5 g/24 h) given a very low protein (0.3 g/kg
of BW), low-phosphorus (5 to 7 mg/kg of BW) diet supplemented with EAA and ketoan-
alogs. During the 6-month follow-up, urinary albumin excretion and fractional renal
albumin clearance were reduced significantly while serum albumin concentration
increased.
Similar data have been obtained in other series97,103,104 suggesting that LPDs induce a
similar adaptation in CRF patients than in healthy subjects. Furthermore, CRF patients
may benefit from other effects of LPDs such as improved insulin sensitivity, lower hyper-
parathyroidism and reduced proteinuria.

33.2.1.2.3 Nutritional effects of low-protein diets. A major decrease in energy intake


can occur when an LPD is prescribed.101 Therefore, LPDs must be administered under
the adequate supervision of trained dietitians.86 LPDs can influence lipid metabolism:
1382_C33.fm Page 565 Tuesday, October 7, 2003 7:23 PM

Chapter thirty-three: Branched-chain amino and keto acids in renal failure 565

reducing protein in the diet increases the ratio of vegetable to animal protein, decreases
the total amount of saturated fat and cholesterol ingested, and improves the lipid profile
by increasing the Apo A1 lipoprotein and the Apo A1/B ratio.98 Insulin resistance can
be improved by protein restriction. In eight patients with advanced CRF, a VLPD
supplemented by ketoanalogs of EAAs for 3 months induced a decrease in fasting serum
glucose from 5.0 ± 0.1 to 4.7 ± 0.1 mmol/l, and plasma insulin from 82.4 ± 20.7 to 48.8 ±
8.0 pmol/l. Endogenous glucose production was reduced by 66% for comparable plasma
insulin levels. These data indicate an improved sensitivity to insulin.105
It is now admitted that malnutrition in patients entering dialysis is a key determinant
of mortality during the subsequent months.106–108 Consequently, a critical point concerning
LPDs and VLPDs given in association with keto acids or EAA supplements is to evaluate
their long-term effect on nutritional status.109 Ihle and coworkers110 conducted a prospec-
tive, randomized study of protein restriction. Sixty-four patients were randomly assigned
to follow either a regular diet or an isocaloric protein-restricted diet (0.4 g of protein per
kilogram of body weight per day) for 18 months. Body weight decreased during the first
6 months in the protein-restricted group but did not decline thereafter. Mid-arm circum-
ference, triceps skin fold, and serum albumin did not change in either group during the
18-month study. Williams and coworkers111 studied 95 CRF patients randomized to receive
either a conventional LPD (protein, 0.6 g/kg/day; phosphate, 800 mg), a low-phosphate
diet (minimum protein intake, 0.8 g/kg/day), or a control diet (minimum protein intake,
0.8 g/kg/day; no phosphate restriction). Following randomization, patients were studied
for an average of 19 ± 3 months. Changes in body weight, muscle mass, and serum
transferrin, albumin, and immunoglobulins were comparable between the groups. The
safety of dietary protein and phosphorous restriction was evaluated in the Modification
of Diet in Renal Disease (MDRD) Study. In Study A, 585 patients with a GFR of 25 to
55 ml/min/1.73 m2 were randomly assigned to a usual-protein diet (1.3 g/kg/day) or an
LPD (0.58 g/kg/day). In Study B, 255 patients with a GFR of 13 to 24 ml/min/1.73 m2
were randomly assigned to an LPD or VLPD (0.28 g/kg/day), supplemented with a keto
acid–AA mixture (0.28 g/kg/day). The mean duration of follow-up was 2.2 years in both
studies. Protein and energy intakes were lower in the LPD and VLPD groups than in the
usual-protein group. Combining patients in both diet groups in each study, a lower
achieved protein intake was not correlated with a higher rate of death, hospitalization, or
stop points, or with a progressive decline in any of the indices of nutritional status. These
analyses suggest that LPD and VLPD used in the MDRD Study are safe for periods of
2 to 3 years. Nonetheless, because protein and energy intake declined, the authors stressed
the need for carefully monitoring patients’ protein and energy intake and nutritional
status.112 Malvy and coworkers113 conducted a randomized study in 50 CRF patients in
order to compare a VLPD (0.30 g/kg/day) supplemented with a preparation of keto-
analogs, hydroxyanalogs of AAs, and AAs to an LPD (0.65 g/kg/day). Follow-up varied
from 3 to 40 months. Body weight, serum transthyretin, and albumin were unaffected in
the two groups. Plasma valine:glycine ratio decreased in the severely protein-depleted
patients and remained stable in the other group. The severe protein restriction supple-
mented by ketoanalogs improved phosphate and calcium plasma parameters. Chauveau
and coworkers114 assessed monthly the course of nutritional status for 1 year in 10 clinically
stable patients with advanced CRF (mean GFR, 13.2 ± 4.8 ml/min/1.73 m2). These patients
received 0.3 g of protein/kg/day supplemented with EAA and ketoanalogs. Conventional
nutritional markers remained unchanged after 1 year of the VLPD. During the same
period, whole-body dual-energy X-ray absorptiometry showed a significant decrease in
mean lean tissue from 46.2 to 45.0 kg; limb-trunk lean tissue ratio was reduced from 0.86
to 0.82, total body fat increased from 20.0 to 21.4 kg, and the percentage of total body fat
1382_C33.fm Page 566 Wednesday, October 8, 2003 1:40 PM

566 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

increased from 29.2 to 31.7%. These different modifications occurred abruptly during the
first 3 months and then stabilized or slightly improved thereafter.
The impact of VLPD on survival, particularly after patients began treatment by dialysis
or transplantation, has been addressed by several authors.114–118 Studies by Walser and
coworker116,117 have suggested that protein restriction and close clinical monitoring
predialysis do not worsen and may substantially improve survival during the first 2 years
on dialysis. More recently, data from 239 consecutive patients with advanced CRF were
collected.118 Patients were given 0.3 g of protein and 35 kcal/kg of BW/day, plus EAA
and ketoanalogs, calcium carbonate, iron, and multivitamins. Protein intake decreased
from 0.85 ± 0.23 to 0.43 ± 0.11 g/kg/day, and body mass index and serum albumin
concentration remained unchanged. Fourteen patients died during the follow-up, but
deaths were unrelated to nutritional parameters. Hemodialysis was initiated in 165
patients at a mean GFR of 5.8 ± 1.5 ml/min. During an average of 54 months on hemo-
dialysis, mortality was low (2.4% after 1 year) and correlated to age only, not to nutritional
parameters observed at the end of the protein-restricted regimen. Similar results were
obtained in 66 transplanted patients. The authors concluded that VLPDs supplemented
with EAAs and ketoanalogs could be safely used in patients with CRF without adverse
effects on the clinical and nutritional status of the patients.118 Although these data are
encouraging, it should be stressed that no randomized study is available to assess the
effects of long-term VLPDs on survival following dialysis or transplantation.

33.2.1.2.4 Effects of low-protein diets on the progression of renal insufficiency


33.2.1.2.4.1 Experimental data. Experimental studies on the influence of protein
diet on renal function have been recently reviewed.101 Numerous studies have shown that
elevated protein intake alters renal hemodynamics and impairs renal function and tissue
in normal animals or during experimental renal insufficiency. Experimental data have
demonstrated that dietary protein increases the glomerular filtration rate, provokes or
increases proteinuria, and induces glomerulosclerosis and renal insufficiency. The mech-
anisms of dietary protein effects on renal function involve multiple mediators such as
hormones (glucagon, insulin, IGF-1, angiotensin II), cytokines, and kinins. Moreover, the
AA load stimulates the proximal sodium/amino acid cotransporter, thus stimulating
tubulo-glomerular feedback and increasing GFR. On the contrary, reduced protein intake
lowers hyperfiltration, retards the onset of proteinuria and glomerular fibrosis, and
increases survival during experimental uremia.101
33.2.1.2.4.2 Clinical data. The effect of BCAA and keto acid supplementation on
the progression of nephropathy cannot be separated from protein restriction. The difficul-
ties in assessing the effect of LPDs come from other factors involved in the control of the
progression of nephropathy:84 hypertension, proteinuria, type of nephropathy, hyperlipo-
proteinemia, dietary phosphorus, and type of protein intake (vegetable vs. animal pro-
teins). More than 50 trials assessing the effects of protein restriction on the occurrence of
renal death (defined as the need for starting dialysis, the death of a patient, or kidney
transplant during the trial) have been reviewed by Fouque and coworkers.119 A total of
1494 patients were analyzed: 753 had received reduced protein intake and 741 a higher
protein intake. The numbers of renal deaths were collected. Two hundred and forty-two
renal deaths were recorded, 101 in the low-protein diet and 141 in the higher-protein diet
group, giving an odds ratio of 0.61 with a 95% confidence interval of 0.46 to 0.83 (p = 0.006).
The authors concluded that reducing protein intake in patients with CRF reduces the
occurrence of renal death by about 40%, compared with larger or unrestricted protein
intake.119
1382_C33.fm Page 567 Wednesday, October 8, 2003 1:42 PM

Chapter thirty-three: Branched-chain amino and keto acids in renal failure 567

Several noncontrolled studies have suggested that VLPDs associated with keto
acid–EAA mixtures were able to slow the decline in renal function. Among the seven
randomized trials selected in the meta-analysis from Fouque and coworkers, three
addressed keto acid–EAA mixtures.119 Jungers and coworkers120 studied 19 patients ran-
domly assigned to receive either 0.6 or 0.4 g of protein/kg/day in association with a mixture
of BCKA, ketophenylalanine, hydroxymethionine, L-lysine, L-threonine, L-tryptophane,
L-histidine, and L-tyrosine (Ketosteril®, 0.10 g/kg of BW/day). Mean duration of treatment
was 11.8 months in the ketoanalog group and 7.1 months in the low-protein diet group. As
far as it could be deduced from such a small number of patients, this study suggested that
VLPD supplemented with keto acids and EAAs was more effective than LPD in terms of
a decrease in blood and the urinary urea, a decrease of the 1/creatinine slope, and a
lengthened time interval until the start of dialysis. In a prospective randomized study
conducted in 50 patients by Malvy and coworkers,113 a VLPD (0.30 g/kg/day) supple-
mented with the same preparation of keto, hydroxy, and amino acids (Ketosteril®, 0.17 g/kg
of BW/day) was compared with an LPD (0.65 g/kg/day). No statistically significant dif-
ferences were found between the two dietary regimens for renal survival. Uremia decreased
significantly in the keto acid group and increased in patients under LPDs. At the end of
the study, the keto acid group showed higher calcemia and lower phosphoremia, alkaline
phosphatase, and parathormone plasma levels when compared with patients with LPDs.
Thus, Ketosteril® did not limit GFR decrease but did improve phosphocalcic plasma param-
eters.113 In MDRD Study B, 255 patients with GFRs of 13 to 24 ml/min/1.73 m2 were
randomly assigned to the LPD (0.58 g/kg/day) or the VLPD (0.28 g/kg/day) with a
keto–EAA supplement. An 18- to 45-month follow-up was planned, with monthly evalu-
ations of the patients. The VLPD group had a marginally slower decline in the GFR than
did the LPD group (p = 0.07). The authors concluded that there was no delay in the time
to the occurrence of end-stage renal disease or death.121 Since then, numerous secondary
analyses of the MDRD Study have been undertaken to clarify the effect of protein restriction
on the rate of decline in GFR and onset of end-stage renal disease.101,122 Correlation analyses
may give new insights into the efficacy of dietary intervention: when actual protein intake
was considered, independent of the group to which patients were assigned, a strong relation
was found between the magnitude of protein intake and the GFR slope (p = 0.011) or renal
death (p = 0.001).122 No additional effect of ketoanalog supplements on retardation of
progression of renal failure was found using these new analyses.

33.2.2 Branched-chain amino and keto acid supply in dialysis patients


Table 33.3 summarizes six controlled studies of EAA and keto acid supplementation in
dialysis patients conducted from 1977 to 2001. Two studies were performed in nonmal-
nourished patients given keto–EAA mixtures and, as expected, did not show any bene-
fit.123,124 The four other studies consisted of the administration of EAA mixtures, 6.6 to
15.7 g/day. All of these studies concerning malnourished patients achieved an improve-
ment of nutritional parameters. Hiroshige and coworkers80 specifically studied BCAA
supplementation. Twenty-eight anorectic patients with low plasma albumin concentration
(less than 35 g/l) were included. Fourteen patients received daily oral BCAA supplemen-
tation (12 g/day) or a placebo in random order in a crossover trial for 6 months. In BCAA-
treated malnourished patients, oral protein and caloric intakes improved within a month,
concomitant with the improvement in plasma BCAA levels. After 6 months of BCAA
supplementation, anthropometric indices showed a statistically significant increase, and
mean plasma albumin concentration increased from 33.1 to 39.3 g/l. After exchanging
BCAAs for a placebo, spontaneous oral food intake decreased, but the beneficial nutritional
status persisted for the next 6 months. In 14 patients initially treated with a placebo, no
1382_C33.fm Page 568 Tuesday, October 7, 2003 7:23 PM

568 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 33.3 Controlled Studies EAA and Keto Acid (KA) Supplementation in Dialysis Patients
Changes in
EAA and/or KA Length nutritional
Reference Study design, patients supply (days) parameters
123 Randomized EAA + histidine, 90 No change in serum
EAA, n = 10 10 g/day albumin and
EAA + KA, n = 9 transferrin
Controls, n = 11 EAA + KA,
(nonmalnourished 9.5 g/day
patients)
126 Double-blind, EAA, 15.7 g/day 90 Increase in predialysis
crossover study urea and plasma C3
n = 13 complement fraction
124 Randomized study EAA, 9.5 g/day 120 No change in
Treated, n = 10 + KA predialysis urea and
Controls , n = 9 creatinine,
(nonmalnourished serumalbumin, and
patients) transferrin
127 Double-blind EEA + histidine, 105 Increased serum
randomized study 6.6 g/day albumin and
Treated, n = 7 transferrin; improved
Controls, n = 8 bone density
128 Double-blind EAA + histidine 90 Increased serum
randomized study + tyrosine, albumin and muscle
Treated, n = 23 10.8 g/day strength
Controls, n = 24
80 Double-blind, BCAA, 12 g/day 180 Improved appetite and
crossover study plasma AA; increase
n = 14 in serum albumin

significant changes in nutritional parameters were observed during the first 6 months.
However, positive results were obtained by BCAA supplementation during the subse-
quent 6 months, and mean plasma albumin concentration increased from 32.7 to 38.1 g/l.
The authors concluded that normalization of low plasma levels of BCAAs by oral sup-
plementation can reduce anorexia and significantly improve overall nutritional status in
elderly malnourished hemodialysis patients. However, because this study did not include
a third group of patients given isonitrogenous protein supplementation, the proper role
of BCAAs in improving appetite could not be established.

33.3 Conclusions
During renal failure, BCAA status is characterized by low plasma and cellular valine
together with low plasma leucine and isoleucine, and most often decreased plasma
BCKAs. These abnormalities of circulating and cellular BCAAs are secondary to abnormal
muscle and hepatosplanchnic AA metabolism. In muscle, metabolic acidosis induces pro-
tein breakdown via an activation of both cytosolic ATP-ubiquitin-dependent proteolytic
pathway and BCKA dehydrogenase, responsible for an irreversible BCAA breakdown.
The decrease in hepatosplanchnic retention of nonessential AAs participates in the abnor-
malities of arterial AAs associated with renal insufficiency. Abnormal BCAA metabolism
can alter both tissue activities, particularly brain function, and nutritional status. In dialysis
patients, it has been reported that the normalization of plasma BCAAs by BCAA oral
supplementation was associated with an improvement of appetite and nutritional status.
1382_C33.fm Page 569 Tuesday, October 7, 2003 7:23 PM

Chapter thirty-three: Branched-chain amino and keto acids in renal failure 569

During CRF, the correction of the plasma AA profile by the administration of EAAs or
keto acids has been proposed in order to improve protein status, avoid uremic toxicity,
and delay the progression of renal disease. BCAA and BCKA supplements are integrated
in a therapeutic strategy including protein restriction and supplementation with EAAs.
Consequently, the proper effect of the use of BCAAs and BCKAs cannot be evaluated.
According to nutritional status, the data from the literature suggest that LPD and VLPD
given in association with keto acid or keto–EAA mixtures are well tolerated. Nonetheless,
the need to carefully monitor patients’ protein and energy intakes and nutritional status
has been stressed by several authors. A recent meta-analysis concluded that reducing
protein intake in patients with CRF reduces the occurrence of renal death by about 40%,
compared with larger or unrestricted protein intake.119 The optimal level of protein intake
cannot be confirmed from these studies. The additional effect of keto–EAA supplements
on retardation of progression of renal failure has not been demonstrated. VLPD together
with EAAs or keto acid–EAA mixtures have been shown to improve insulin sensitivity
and hyperparathyroidism and to reduce proteinuria.

References
1. Mogielnicki, R.P., Waldmann, T.A., and Strober, W., Renal handling of low molecular weight
proteins. I. L-chain metabolism in experimental renal disease, J. Clin. Invest., 50, 901, 1971.
2. Pitts, R.F., Damian, A.C., and Mac Leod, M.B., Synthesis of serine by rat kidney in vivo and
in vitro, Am. J. Physiol., 219, 584, 1970.
3. Guder, W.G. and Morel, F., Biochemical characterization of individual nephron segments, in
Handbook of Physiology: Section 8: Renal Physiology, Windhager, E.E., Ed., Oxford University
Press, Oxford, 1992, p. 2119.
4. Pitts, R.F., Régulation de l’équilibre acido-basique, in Physiologie du rein et du milieu intérieur,
Masson, Paris, 1973, p. 172.
5. Tannen, R.L., Ammonia metabolism, Am. J. Physiol., 235, F265, 1978.
6. Kuhlmann, M.K. and Kopple, J.D., Amino acid metabolism in the kidney, Semin. Nephrol.,
10, 445, 1990.
7. Tizianello, A., De Ferrari, G., Garibotto, G., Gurreri, G., and Robaudo, C., Renal metabolism
of amino acids and ammonia in subjects with normal renal function and in patients with
chronic renal insufficiency, J. Clin. Invest., 65, 1162, 1980.
8. Tizianello, A., Deferrari, G., Garibotto, G., Robaudo, C., Lutman, M., Passerone, G., and
Bruzzone, M., Branched-chain amino acid metabolism in chronic renal failure, Kidney Int.,
24 (Suppl. 16), S17, 1983.
9. Jones, M.R. and Kopple, J.D., Valine metabolism in normal and chronically uremic man, Am.
J. Clin. Nutr., 31, 1660, 1978.
10. Alvestrand, A., Fürst, P., and Bergström, J., Intracellular aminoacids in uremia, Kidney Int.,
24 (Suppl. 16), S9, 1983.
11. Bergström, J., Alvestrand, A., and Fürst, P., Plasma and muscle free amino acids in mainte-
nance hemodialysis patients without protein malnutrition, Kidney Int., 38, 108, 1990.
12. Raj, D.S., Ouwendyk, M., Francoeur, R., and Pierratos, A., Plasma amino acid profile on
nocturnal hemodialysis, Blood Purif., 18, 97, 2000.
13. Lindholm, B., Alvestrand, A., Fürst, P., and Bergström, J., Plasma and muscle free amino
acids during continuous ambulatory peritoneal dialysis, Kidney Int., 35, 1219, 1989.
14. Kooman, J.P., Deutz, N.E., Zijlmans, P., van den Wall Bake, A., Gerlag, P.G., van Hooff, J.P.,
and Leunissen, K.M., The influence of bicarbonate supplementation on plasma levels of
branched-chain amino acids in haemodialysis patients with metabolic acidosis, Nephrol. Dial.
Transplant., 12, 2397, 1997.
15. Kikuchi, F., Kuno, T., Nagura, Y., Takahashi, S., and Kanmatsuse, K., [The influence of
correction of acidosis on plasma level of branched-chain amino acids in chronic hemodialysis
patients], Nippon Jinzo Gakkai Shi., 40, 258, 1998.
1382_C33.fm Page 570 Tuesday, October 7, 2003 7:23 PM

570 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

16. Scolari, M.P., Stefoni, S., Mosconi, G., Coli, L., Feliciangeli, G., Baldrati, L., Buscaroli, L.,
Buscaroli, A., Prandini, R., and Bonomini, V., Effects of renal substitutive programs on amino
acid patterns in chronic uremia, Kidney Int., 24 (Suppl. 16), S77, 1983.
17. Perfumo, F., Canepa, A., Divino Filho, J.C., Nilsson, E., Carrea, A., Verrina, E., Gusmano, R.,
and Bergström, J., Muscle and plasma amino acids and nutritional status in kidney-trans-
planted children, Nephrol. Dial. Transplant., 9, 1778, 1994.
18. Riedel, E., Hampl, H., Scigalla, P., Nundel, M., and Kessel, M., Correction of amino acid
metabolism by recombinant human erythropoietin therapy in hemodialysis patients, Kidney
Int., 27, S216, 1989.
19. Garibotto, G., Gurreri, G., Robaudo, C., Saffioti, S., Magnasco, A., Sofia, A., Marchelli, M.,
and Sala, M.R., Erythropoietin treatment and amino acid metabolism in hemodialysis pa-
tients, Nephron, 65, 533, 1993.
20. Dalton, R.N. and Chantler, C., The relationship between branched-chain amino acids and
alpha-keto acids in blood in uremia, Kidney Int., 24 (Suppl. 16), S61, 1983.
21. Jones, R., Dalton, N., Turner, C., Start, K., Haycock, G., and Chantler, C., Oral essential
aminoacid and ketoacid supplements in children with chronic renal failure, Kidney Int., 24,
95, 1983.
22. Arai, J., Hara, Y., Fukuda, S., Takuma, T., and Sugino, N., Metabolic conversion of alpha-
keto valine to valine in patients with chronic renal failure, Clin. Nephrol., 23, 236, 1985.
23. Schauder, P., Matthaei, D., Henning, H.V., Scheler, F., and Langenbeck, U., Blood levels of
branched-chain amino acids and alpha-ketoacids in uremic patients given keto analogues
of essential amino acids, Am. J. Clin. Nutr., 33, 1660, 1980.
24. Riedel, E., Hampl, H., Nundel, M., and Farshidfar, G., Essential branched-chain amino acids
and alpha-ketoanalogues in haemodialysis patients, Nephrol. Dial. Transplant., 7, 117, 1992.
25. Walser, M., Jarskog, F.L., and Hill, S.B., Branched-chain-ketoacid metabolism in patients with
chronic renal failure, Am. J. Clin. Nutr., 50, 807, 1989.
26. Garibotto, G., Paoletti, E., Fiorini, F., Russo, R., Robaudo, C., Deferrari, G., and Tizianello,
A., Peripheral metabolism of branched-chain keto acids in patients with chronic renal failure,
Miner. Electrolyte Metab., 19, 25, 1993.
27. Smith, S.R., Pozefsky, T., and Chetri, M.K., Nitrogen and amino acid metabolism in adults
with protein-calorie malnutrition, Metabolism, 23, 603, 1978.
28. Smolin, L.A., Laidlaw, S.A., and Kopple, J.D., Altered plasma free and protein-bound sulfur
amino acid levels in patients undergoing maintenance hemodialysis, Am. J. Clin. Nutr., 45,
737, 1987.
29. Tizianello, A., Deferrari, G., Garibotto, G., Robaudo, C., Saffioti, S., and Pontremoli, R., Amino
acid imbalance in patients with chronic renal failure, in The Progressive Nature of Renal Disease:
Myths and Facts, Oldrizzi, L., Maschio, G., and Rugiu, C., Eds., Karger, Basel, Switzerland,
1989, p. 185.
30. Laidlaw, S.A., Berg, R.L., Kopple, J.D., Naito, H., Walker, W.G., and Walser, M., Patterns of
fasting plasma amino acid levels in chronic renal insufficiency: results from the feasibility
phase of the Modification of Diet in Renal Disease Study, Am. J. Kidney Dis., 23, 504, 1994.
31. Felig, P., Amino acid metabolism in man, Annu. Rev. Biochem., 44, 933, 1975.
32. Abumrad, M.M. and Miller, B., The physiologic and nutritional significance of plasma-free
amino acid levels, J. Parenter. Enteral Nutr., 7, 163, 1983.
33. Christensen, H.N., Interorgan amino acid nutrition, Physiol. Rev., 62, 1193, 1982.
34. Wahren, J., Felig, P., and Hagenfeld, L., Effect of protein ingestion on splanchnic and leg
metabolism in normal man and in patients with diabetes mellitus, J. Clin. Invest., 57, 987, 1976.
35. Garibotto, G., Deferrari, G., Robaudo, C., Saffioti, S., Sofia, A., Russo, R., and Tizianello, A.,
Disposal of exogenous amino acids by muscle in patients with chronic renal failure, Am. J.
Clin. Nutr., 62, 136, 1995.
36. Garibotto, G., Russo, R., Sofia, A., Sala, M.R., Robaudo, C., Moscatelli, P., Deferrari, G., and
Tizianello, A., Skeletal muscle protein synthesis and degradation in patients with chronic
renal failure, Kidney Int., 45, 1432, 1994.
1382_C33.fm Page 571 Tuesday, October 7, 2003 7:23 PM

Chapter thirty-three: Branched-chain amino and keto acids in renal failure 571

37. Deferrari, G., Garibotto, G., Robaudo, C., Canepa, A., Bagnasco, S., and Tizianello, A., Leg
metabolism of amino acids and ammonia in patients with chronic renal failure, Clin. Sci.
(Lond.), 69, 143, 1985.
38. May, R.C., Kelly, R.A., and Mitch, W.E., Metabolic acidosis stimulates protein degradation
in rat muscle by a glucocorticoid-dependent mechanism, J. Clin. Invest., 77, 614, 1986.
39. England, B.E., Chastain, J., and Mitch, W.E., Extracellular acidification changes protein syn-
thesis and degradation in BC3H-1 myocytes, Am. J. Physiol., 260, C277, 1991.
40. May, R.C., Masud, T., Logue, B., Bailey, J., and England, B., Chronic metabolic acidosis
accelerates whole body proteolysis and oxidation in awake rats, Kidney Int., 41, 1535, 1992.
41. May, R.C., Kelly, R.A., and Mitch, W.E., Mechanisms for defects in muscle protein metabolism
in rats with chronic uremia, J. Clin. Invest., 79, 1099, 1987.
42. Reaich, D., Channon, S.M., Scrimgeour, C.M., and Goodship, T.H.J., Ammonium chloride-
induced acidosis increases protein breakdown and amino acid oxidation in humans, Am. J.
Physiol., 263, E735, 1992.
43. Reaich, D., Channon, S.M., Scrimgeour, C.M., Daley, S.E., Wilkinson, R., and Goodship, T.H.J.,
Correction of acidosis in humans with CRF decreases protein degradation and amino acid
oxidation, Am. J. Physiol., 265, E230, 1993.
44. May, R.C., Hara, Y., Kelly, R.A., Block, K.P., Muse, M.G., and Mitch, W.E., Branched-chain
amino acid metabolism in rat muscle: abnormal regulation in acidosis, Am. J. Physiol., 252,
E712, 1987.
45. Mitch, W.E., Medina, R., Grieber, S., May, R.C., England, B.K., Price, S.R., Bailey, J.L., and
Goldberg, A.L., Metabolic acidosis stimulates muscle protein degradation by activating the
adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes, J. Clin.
Invest., 93, 2127, 1994.
46. Wing, S.S. and Goldberg, A.L., Glucocorticoids activate the ATP-ubiquitin-dependent pro-
teolytic system in skeletal muscle during fasting, Am. J. Physiol., 264, E668, 1993.
47. Block, K.P., Richmond, W.B., Mehard, W.B., and Buse, M.G., Glucocorticoid-mediated acti-
vation of alpha-keto acid dehydrogenase in vivo, Am. J. Physiol., 252, E396, 1987.
48. Mitch, W.E., Price, S.R., May, R.C., Jurkovitz, C., and England, B.K., Metabolic consequences
of uremia: extending the concept of adaptative responses to protein metabolism, Am. J. Kidney
Dis., 23, 224, 1994.
49. Price, S.R., England, B.K., Bailey, J.L., van Verde, K., and Mitch, W.E., Acidosis and gluco-
corticoids concomitantly increase ubiquitin and proteasome subunit mRNAs in rat muscle,
Am. J. Physiol., 267, C955, 1994.
50. Price, S.R., Wang, X., and Bailey, J.L., Tissue-specific responses of branched-chain alpha-
ketoacid dehydrogenase activity in metabolic acidosis, J. Am. Soc. Nephrol., 9, 1892, 1998.
51. King, P., Goldstein, L., and Newsholme, E.A., Glutamine synthetase activity of muscle in
acidosis, Biochem. J., 216, 523, 1983.
52. Welbourne, T.C. and Joshi, S., Interorgan glutamine metabolism during acidosis, J. Parenter.
Enteral Nutr., 14, 77S, 1990.
53. Mak, R.H., Insulin, branched-chain amino acids, and growth failure in uremia, Pediatr.
Nephrol., 12, 637, 1998.
54. Riedel, E., Nundel, M., Wendel, G., and Hampl, H., Amino acid and alpha-keto acid metab-
olism depends on oxygen availability in chronic hemodialysis patients, Clin. Nephrol., 53,
S56, 2000.
55. Deferrari, G., Garibotto, G., Robaudo, C., Sala, M., and Tizianello, A., Splanchnic exchange
of amino acids after amino acid ingestion in patients with chronic renal insufficiency, Am. J.
Clin. Nutr., 48, 72, 1988.
56. Tizianello, A., Deferrari, G., Garibotto, G., and Robaudo, C., Amino acid metabolism and the
liver in renal failure, Am. J. Clin. Nutr., 33, 1354, 1980.
57. Garibotto, G., Deferrari, G., Robaudo, C., Saffioti, S., Salvidio, G., Paoletti, E., and Tizianello,
A., Effect of amino acid ingestion on blood amino acid profile in patients with chronic renal
failure, Am. J. Clin. Nutr., 46, 949, 1987.
1382_C33.fm Page 572 Tuesday, October 7, 2003 7:23 PM

572 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

58. Garibotto, G., Deferrari, G., Robaudo, C., Saffioti, S., Paoletti, E., Pontremoli, R., and
Tizianello, A., Effects of a protein meal on blood amino acid profile in patients with chronic
renal failure, Nephron, 64, 216, 1993.
59. Elwyn, D.H., Parikh, H.C., and Shoemaker, W.C., Amino acid movements between gut, liver
and periphery in unanesthetized dogs, Am. J. Physiol., 215, 1260, 1968.
60. Cano, N., Catelloni, F., Fontaine, E., Novaretti, R., Reynier, J.P., and Leverve, X.M., Isolated
rat hepatocyte metabolism is affected by chronic renal failure, Kidney Int., 47, 1522, 1995.
61. Cano, N., Leverve, X., di Costanzo-Dufetel, J., Novaretti, R., and Reynier, J.P., Splanchnic
acid-base status and urea metabolism in uremic rats, Clin. Nutr., 14, 116, 1995.
62. Cupisti, A., Baker, F., Brown, J., Lock, C., Bevington, A., Harris, K.P.G., and Walls, J., Effects
of acid loading on serum amino acid profiles and muscle composition in normal fed rats,
Clin. Sci., 85, 445, 1993.
63. Tizianello, A., Deferrari, G., Garibotto, G., Robaudo, C., Canepa, A., and Passerone, G., Is
amino acid imbalance harmful to patients in chronic renal failure? Kidney Int., 28 (Suppl. 17),
S79, 1985.
64. Young, V.R., Bier, D.M., and Pelett, P.L., A theoretical basis for increasing current estimates
of the amino acid requirements in adult man, with experimental support, Am. J. Clin. Nutr.,
50, 80, 1989.
65. Young, V.R. and Marchini, J.S., Mechanisms and nutritional significance of metabolic re-
sponses to altered intakes of protein and amino acids with reference to nutritional adaptation
in humans, Am. J. Clin. Nutr., 51, 270, 1990.
66. Oldendorf, W.H. and Szabo, J., Amino acid assignment to one of the three blood-brain barrier
amino acid carriers, Am. J. Physiol., 230, 94, 1976.
67. Hagenfeldt, L., Eriksson, S., and Wahren, J., Influence of leucine on arterial concentrations
and regional exchanges of amino acids in healthy subjects, Clin. Sci., 59, 173, 1980.
68. Deferrari, G., Garibotto, G., Robaudo, C., Ghiggeri, G.M., and Tizianello, A., Brain metabo-
lism of amino acids and ammonia in patients with chronic renal insufficiency, Kidney Int.,
20, 505, 1981.
69. Deferrari, G., Garibotto, G., Robaudo, C., Canepa, A., Passerone, G.C., and Tizianello, A.,
Brain metabolism in uremia, in Advances in Hepatic Encephalopathy and Urea Cycle Diseases,
Kleinberger, G., Ferenci, P., Riederer, P., and Thaler, H., Karger, Basel, Switzerland, 1984,
p. 484.
70. Pye, I.F., Mac Gale, E.H.F., Stomier, C., Hutchinson, E.C., and Aben, G.M., Studies of cere-
brospinal fluid and plasma amino acids in patients with steady-state chronic renal failure,
Clin. Chim. Acta, 92, 65, 1979.
71. Gerrits, G.P., Kamphuis, S., Monnens, L.A., Trijbels, J.M., Schroder, C.H., Koster, A., and
Gabreels, F.J., Cerebrospinal fluid levels of amino acids in infants and young children with
chronic renal failure, Neuropediatrics, 29, 35, 1998.
72. Cusick, P.K., Koehler, K.M., Ferrier, B., and Haskell, B.E., The neurotoxicity of valine defi-
ciency in rats, J. Nutr., 108, 1200, 1978.
73. Polaiologos, G., Koivisto, V.A., and Felig, P., Interaction of leucine, glucose and ketone
metabolism in rat brain in vitro, J. Neurochem., 32, 67, 1979.
74. Reith, M.E.A., Schotman, P., Van Zwieten, B.J., and Gispen, W.H., The nature of amino acid
pool used for protein synthesis in rat brain slices, J. Neurochem., 32, 413, 1979.
75. Dhopeshwarkar, G.A. and Subramian, C., Lipogenesis in developing brain: utilization of
radioactive leucine, isoleucine, octanoic acid and beta-hydroxybutyrique acid, Lipids, 14, 47,
1979.
76. Roberts, S. and Morelos, B.S., Regulation of cerebral metabolism of amino acids. IV. Influence
of amino acid levels on leucine uptake, utilisation and incorporation into protein in vivo,
J. Neurochem., 12, 373, 1985.
77. Hamberger, A.C., Chiang, G.H., Nylen, E.S., Scheff, S.W., and Cotman, C.W., Glutamate as
a CNS transmitter. I. Evaluation of glucose and glutamine as precursors for the synthesis of
preferentially released glutamate, Brain Res., 168, 513, 1979.
78. Biasioli, S., D’Andrea, G., Feriani, M., Chiaramonte, S., Fabris, A., Ronco, C., and La Greca,
G., Uremic encephalopathy: an updating, Clin. Nephrol., 25, 57, 1986.
1382_C33.fm Page 573 Tuesday, October 7, 2003 7:23 PM

Chapter thirty-three: Branched-chain amino and keto acids in renal failure 573

79. Soreide, E., Skeie, B., Kirvela, O., Lynn, R., Ginsberg, N., Manner, T., Katz, D.P., and Askanazi,
J., Branched-chain amino acid in chronic renal failure patients: respiratory and sleep effects,
Kidney Int., 40, 539, 1991.
80. Hiroshige, K., Sonta, T., Suda, T., Kanegae, K., and Ohtani, A., Oral supplementation of
branched-chain amino acid improves nutritional status in elderly patients on chronic
haemodialysis, Nephrol. Dial. Transplant., 16, 1856, 2001.
81. Young, G.A., Swanepoel, C.R., Croft, M.R., Hobson, S.M., and Parsons, F.M., Anthropometry
and plasma valine, amino acids, and proteins in the nutritional assessment of hemodialysis
patients, Kidney Int., 21, 492, 1982.
82. Chazot, C., Laurent, G., Charra, B., Blanc, C., VoVan, C., Jean, G., Vanel, T., Terrat, J.C., and
Ruffet, M., Malnutrition in long-term haemodialysis survivors, Nephrol. Dial. Transplant., 16,
61, 2001.
83. Cano, N., Labastie-Coeyrehourcq, J., Lacombe, P., Stroumza, P., di Costanzo-Dufetel, J.,
Durbec, J.P., Coudray-Lucas, C., and Cynober, L., Perdialytic parenteral nutrition with lipids
and amino-acids in malnourished hemodialysis patients, Am. J. Clin. Nutr., 52, 726, 1990.
84. Toigo, G., Aparicio, M., Attman, P.-O., Cano, N., Ciancaruso, D., Fouque, D., Heidland, A.,
Howard, P., Teplan, V., and Guarnieri, G., ESPEN consensus on nutritional treatment of
patients with renal insufficiency (Part 1 of 2), Clin. Nutr., 19, 197, 2000.
85. Toigo, G., Aparicio, M., Attman, P.-O., Cano, N., Ciancaruso, D., Fouque, D., Heidland, A.,
Howard, P., Teplan, V., and Guarnieri, G., ESPEN consensus on nutritional treatment of
patients with renal insufficiency (Part 2 of 2), Clin. Nutr., 19, 281, 2000.
86. National Kidney Foundation, Kidney Disease Outcomes Quality Initiative, Clinical practice
guidelines for nutrition in chronic renal failure. I. Adult guidelines. B. Advanced chronic
renal failure without dialysis, Am. J. Kidney Dis., 35 (Suppl. 2), S56, 2000.
87. National Kidney Foundation, Kidney Disease Outcomes Quality Initiative, Clinical practice
guidelines for nutrition in chronic renal failure. II. Pediatric guidelines, Am. J. Kidney Dis.,
35 (Suppl. 2), S105, 2000.
88. National Kidney Foundation, Kidney Disease Outcomes Quality Initiative, Clinical practice
guidelines for nutrition in chronic renal failure. I. Adult guidelines. A. Maintenance dialysis,
Am. J. Kidney Dis., 35 (Suppl. 2), S17, 2000.
89. Walser, M., Rationale and indications for the use of alpha-keto analogues, J. Parenter. Enteral
Nutr. 8, 37, 1984.
90. Walser, M., Coulter, A.W., Dighe, S., and Crantz, F.R., The effects of keto-analogues of
essential amino acids in uremia, J. Clin. Invest., 52, 678, 1973.
91. Mitch, W.E., Walser, M., Steinman, T.I., Hill, S., Zeger, S., and Tungsanga, K., The effect of a
keto acid-amino acid supplement to a restricted diet on the progression of chronic renal
failure, N. Engl. J. Med., 311, 623, 1984.
92. Eriksson, L.S., Hagenfeldt, L., and Wahren, J., Intravenous infusion of alpha-oxoisocaproate:
influence on amino acid and nitrogen metabolism in patients with liver cirrhosis, Clin. Sci.
(Lond.), 62, 285, 1982.
93. Fouin-Fortunet, H., Besnier, M.O., Colin, R., Wessely, J.Y., and Rose, F., Effects of ketoacids
on liver glutathione and microsomal enzymes in malnourished rats, Kidney Int., 36 (Suppl.
27), S222, 1989.
94. Quevedo, M.R., Price, G.M., Halliday, D., Pacy, P.J., and Millward, D.J., Nitrogen homo-
eostasis in man: diurnal changes in nitrogen excretion, leucine oxidation and whole body
leucine kinetics during a reduction from a high to a moderate protein intake, Clin. Sci. (Lond.),
86, 185, 1994.
95. Goodship, T.H., Mitch, W.E., Hoerr, R.A., Wagner, D.A., Steinman, T.I., and Young, V.R.,
Adaptation to low-protein diets in renal failure: leucine turnover and nitrogen balance, J. Am.
Soc. Nephrol., 1, 66, 1990.
96. Maroni, B.J., Staffeld, C., Young, V.R., Manatunga, A., and Tom, K., Mechanisms permitting
nephrotic patients to achieve nitrogen equilibrium with a protein-restricted diet, J. Clin.
Invest., 99, 2479, 1997.
1382_C33.fm Page 574 Tuesday, October 7, 2003 7:23 PM

574 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

97. Giordano, M., De Feo, P., Lucidi, P., DePascale, E., Giordano, G., Cirillo, D., Dardo, G.,
Signorelli, S.S., and Castellino, P., Effects of dietary protein restriction on fibrinogen and
albumin metabolism in nephrotic patients, Kidney Int., 60, 235, 2001.
98. Bernhard, J., Beaufrère, B., Laville, M., and Fouque, D., Adaptive response to a low-protein
diet in predialysis chronic renal failure patients, J. Am. Soc. Nephrol., 12, 1249, 2001.
99. Masud, T., Young, V.R., Chapman, T., and Maroni, B.J., Adaptive responses to very low
protein diets: the first comparison of ketoacids to essential amino acids, Kidney Int., 45, 1182,
1994.
100. Tom, K., Young, V.R., Chapman, T., Masud, T., Akpele, L., and Maroni, B.J., Long-term
adaptive responses to dietary protein restriction in chronic renal failure, Am. J. Physiol., 268,
E668, 1995.
101. Fouque, D., Influence of dietary protein intake on the progression of chronic renal insuffi-
ciency, in Nutritional Management of Renal Disease, Kopple, J.D. and Massry, S.G., Williams &
Wilkins, Baltimore, 1997, p. 777.
102. Aparicio, M., Bouchet, J.L., Gin, H., Potaux, L., Morel, D., de Precigout, V., Lifermann, F.,
and Gonzalez, R., Effect of a low-protein diet on urinary albumin excretion in uremic patients,
Nephron, 50, 288, 1988.
103. Kaysen, G.A., Gambertoglio, J., Jimenez, I., Jones, H., and Hutchinson, F.N., Effects of dietary
protein intake on albumin homeostasis in nephrotic patients, Kidney Int., 29, 572, 1986.
104. Rosenberg, M.E., Swanson, J.E., Thomas, B.L., and Hostetter, T.H., Glomerular and hormonal
responses to dietary protein intake in human renal disease, Am. J. Physiol., 253, F1083, 1987.
105. Rigalleau, V., Combe, C., Blanchetier, V., Aubertin, J., Aparicio, M., and Gin, H., Low protein
diet in uremia: effects on glucose metabolism and energy production rate, Kidney Int., 51,
1222, 1997.
106. Goldwasser, P., Mittman, N., Antignani, A., Burell, D., Michel, M.-A., Collier, J., and Avram,
M.M., Predictors of mortality in hemodialysis patients, J. Am. Soc. Nephrol., 3, 1616, 1993.
107. Avram, M.M., Mittman, N., Bonomini, L., Chattopadhyay, J., and Fein, P., Markers for
survival in dialysis: a seven-year prospective study, Am. J. Kidney Dis., 26, 209, 1995.
108. Soucie, J.M. and McClellan, W.M., Early death in dialysis patients: risk factors and impact
on incidence and mortality rates, J. Am. Soc. Nephrol., 7, 2169, 1996.
109. Aparicio, M., Chauveau, P., and Combe, C., Are supplemented low-protein diets nutritionally
safe? Am. J. Kidney Dis., 37, S71, 2001.
110. Ihle, B.U., Becker, G.J., Whitworth, J.A., Charlwood, R.A., and Kincaid-Smith, P.S., The effect
of protein restriction on the progression of renal insufficiency, N. Engl. J. Med., 321, 1773, 1989.
111. Williams, P.S., Stevens, M.E., Fass, G., Irons, L., and Bone, J.M., Failure of dietary protein
and phosphate restriction to retard the rate of progression of chronic renal failure: a pro-
spective, randomized, controlled trial, Q. J. Med., 81, 837, 1991.
112. Kopple, J.D., Levey, A.S., Greene, T., Chumlea, W.C., Gassman, J.J., Hollinger, D.L., Maroni,
B.J., Merrill, D., Scherch, L.K., Schulman, G., Wang, S.R., and Zimmer, G.S., Effect of dietary
protein restriction on nutritional status in the Modification of Diet in Renal Disease Study,
Kidney Int., 52, 778, 1997.
113. Malvy, D., Maingourd, C., Pengloan, J., Bagros, P., and Nivet, H., Effects of severe protein
restriction with ketoanalogues in advanced renal failure, J. Am. Coll. Nutr., 18, 481, 1999.
114. Chauveau, P., Barthe, N., Rigalleau, V., Ozenne, S., Castaing, F., Delclaux, C., de Precigout,
V., Combe, C., and Aparicio, M., Outcome of nutritional status and body composition of
uremic patients on a very low protein diet, Am. J. Kidney Dis., 34, 500, 1999.
115. Coresh, J., Walser, M., and Hill, S., Survival on dialysis among chronic renal failure patients
treated with a supplemented low-protein diet before dialysis, J. Am. Soc. Nephrol., 6, 1379,
1995.
116. Walser, M., Effects of a supplemented very low protein diet in predialysis patients on the
serum albumin level, proteinuria, and subsequent survival on dialysis, Miner. Electrolyte
Metab., 24, 64, 1998.
117. Walser, M. and Hill, S., Can renal replacement be deferred by a supplemented very low
protein diet? J. Am. Soc. Nephrol., 10, 110, 1999.
1382_C33.fm Page 575 Tuesday, October 7, 2003 7:23 PM

Chapter thirty-three: Branched-chain amino and keto acids in renal failure 575

118. Aparicio, M., Chauveau, P., De Precigout, V., Bouchet, J.L., Lasseur, C., and Combe, C.,
Nutrition and outcome on renal replacement therapy of patients with chronic renal failure
treated by a supplemented very low protein diet, J. Am. Soc. Nephrol., 11, 708, 2000.
119. Fouque, D., Wang, P., Laville, M., and Boissel, J.P., Low protein diets delay end-stage renal
disease in non-diabetic adults with chronic renal failure, Nephrol. Dial. Transplant., 15, 1986,
2000.
120. Jungers, P., Chauveau, P., Ployard, F., Lebkiri, B., Ciancioni, C., and Man, N.K., Comparison
of ketoacids and low protein diet on advanced chronic renal failure progression, Kidney Int.,
32 (Suppl. 22), S67, 1987.
121. Klahr, S., Levey, A.S., Beck, G.J., Caggiula, A.W., Hunsicker, L., Kusek, J.W., and Striker, G.,
The effects of dietary protein restriction and blood-pressure control on the progression of
chronic renal disease: Modification of Diet in Renal Disease Study Group, N. Engl. J. Med.,
330, 877, 1994.
122. Levey, A.S., Greene, T., Beck, G.J., Caggiula, A.W., Kusek, J.W., Hunsicker, L.G., and Klahr,
S., Dietary protein restriction and the progression of chronic renal disease: what have all of
the results of the MDRD study shown? Modification of Diet in Renal Disease Study group,
J. Am. Soc. Nephrol., 10, 2426, 1999.
123. Neuhauser, M., Ulm, A., Leber, H.W., and Schutterle, G., Influence of essential amino acids
and keto acids on protein metabolism and the anaemia of patients on chronic intermittent
haemodialysis, Proc. Eur. Dial. Transplant. Assoc., 14, 557, 1977.
124. Ulm, A., Neuhauser, M., and Leber, H.W., Influence of essential amino acids and keto acids
on protein metabolism and anemia of patients on intermittent hemodialysis, Am. J. Clin.
Nutr., 31, 1827, 1978.
125. Kist-van Holthe tot Echten, J., Huijmans, J.G., Hop, W.C., Monnens, L.A., de Jong, M.C.,
Noordzij, C.M., Slotema, R., Nauta, J., and Wolff, E.D., Intracellular amino acid concentrations
in children with chronic renal insufficiency, Pediatr. Nephrol., 10, 46, 1996.
126. Hecking, E., Kohler, H., Zobel, R., Lemmel, E.M., Mader, H., Opferkuch, W., Prellwitz, W.,
Keim, H.J., and Muller, D., Treatment with essential amino acids in patients on chronic
hemodialysis: a double blind cross-over study, Am. J. Clin. Nutr., 31, 1821, 1978.
127. Acchiardo, S., Moore, L., and Cockrell, S., Effect of essential amino acids (EAA) on chronic
hemodialysis (CHD) patients (PTS), Trans. Am. Soc. Artif. Intern. Organs, 28, 608, 1982.
128. Eustace, J.A., Coresh, J., Kutchey, C., Te, P.L., Gimenez, L.F., Scheel, P.J., and Walser, M.,
Randomized double-blind trial of oral essential amino acids for dialysis-associated hypoal-
buminemia, Kidney Int., 57, 2527, 2000.
1382_C33.fm Page 576 Tuesday, October 7, 2003 7:23 PM
1382_C34.fm Page 577 Tuesday, October 7, 2003 7:25 PM

chapter thirty-four

Glutamine-supplemented diets
in enteral nutrition
Petra G. Boelens
VU University Medical Center, Amsterdam
Paul A.M. van Leeuwen
VU University Medical Center, Amsterdam

Contents
Introduction..................................................................................................................................577
34.1 Trauma patients .................................................................................................................578
34.2 Burn patients ......................................................................................................................581
34.3 Intensive care patients.......................................................................................................582
34.4 Patients undergoing gastrointestinal surgery ...............................................................585
34.5 Inflammatory bowel disease ............................................................................................586
34.6 Malignancy requiring bone marrow transplantation or chemotherapy....................587
34.6.1 Adults ....................................................................................................................587
34.6.2 Children with BMT and chemotherapy...........................................................589
34.7 Neonates..............................................................................................................................589
34.8 Other patient populations ................................................................................................590
34.8.1 Ischemic heart disease ........................................................................................590
34.8.2 Duchenne muscular dystrophy .........................................................................590
34.8.3 HIV/AIDS.............................................................................................................590
34.9 Conclusion ..........................................................................................................................591
References .....................................................................................................................................591

Introduction
Increasing numbers of scientific observations support the concept that glutamine is a
conditionally essential nutrient during catabolic state that can be supplemented via the
parenteral and enteral routes. This chapter handles the studies administering enteral
glutamine-enriched solutions in diverse patient populations.

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 577
1382_C34.fm Page 578 Tuesday, October 7, 2003 7:25 PM

578 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

34.1 Trauma patients


The first publication on glutamine supplementation of enteral nutrition appeared by Long
et al.1 Thirty trauma patients with an injury severity score (ISS) of >20 were enrolled and
randomly assigned to receive glutamine-enriched enteral feeding (Alitraq, Ross, Abbott
Laboratories, Columbus, OH) (30.5 g of glutamine per 100 g of protein) (n = 16) or the
identical isocaloric, isonitrogenous control feeding (n = 14) for three consecutive days.
Instead of glutamine, the control feeding contained a similar nitrogen equivalent of a
combination of alanine, aspartate, glycine, proline, and serine. The study was performed
in a double-blind setting. Enteral feeding was started approximately 24 h after admission
in most of the patients by nasoduodenal route. The primary aim of this study was to
establish the efficacy of glutamine added to an enteral diet with respect to whole-body
protein synthesis after trauma as was measured by isotopic leucine, nitrogen balance, and
glucose kinetics using [U-13C]glucose and [6-3H]glucose. Patient information profiles were
similar with the exception of plasma creatinine levels on admission, which were lower in
the glutamine group. During the study the mean intakes of protein of the 3 days of feeding
and on the day of the kinetic study were lower in the glutamine group and did not reach
the protein goal set by the investigators. The explanation for a decreased enteral intake
of the glutamine formula could have been the high residuals in two of the glutamine
patients and the initiation of a slower feeding rate in one patient. No significant increase
in plasma glutamine concentrations was found in the glutamine group over the controls.
The enteral route with the gut and liver as consumers of the glutamine was considered
the main reason for the unchanged plasma glutamine levels. Other studies published later
in time did detect higher plasma glutamine levels in the group receiving enteral
glutamine.2 The mean nitrogen balance and protein kinetics were not different between
the groups, although the nitrogen intake was 14% less in the glutamine group. Exogenous
enteral glutamine did not have an effect on muscle protein breakdown, turnover, and
synthesis, which is contrary to the findings of Hammarqvist et al.3 providing a glutamine-
enriched regimen by the parenteral route. In addition, no difference was seen between
the two groups in glucose metabolism as was measured by using [U-13C]glucose and
[6-3H]glucose stable isotope tracers. The authors concluded that glutamine-enriched
enteral nutrition was well tolerated but did not provide additional nutritional advantage
over standard enteral formulas in severely injured patients in the first 4 days after trauma.
Sample sizes of the two groups were small, although from the description of the statistical
analysis, it cannot be identified on which parameter the power analysis was initially
calculated. Furthermore, the male:female ratios between both groups were not compara-
ble; the control group had 57% (8/14) females, while the glutamine group had 37.5%
(6/16) females. The authors did not comment on this in the publication. However, it is
known that sex-specific factors might play a role in glucose kinetics, energy expenditure,
survival, and immune responses.4–8
A second article by Long et al.9 on the same patient population was published with
the findings of the amino acid profiles and glutamine plasma concentrations. It was
reported that trauma patients display hypoaminoacidemia, as reflected by a reduction of
50% of the total amino acid (TAA) pool in plasma compared to normal values. Decreased
nonessential amino acids (NEAA), of which glutamine accounts for an important part,
were responsible for these changes. Suggested was that a lower glutamine intake could
account for a lower total NEAA and TAA plasma level. However, no effect on TAA or
NEAA levels was found in the glutamine-enriched group over the control group. In
addition, no increase in glutamine plasma level was found in this study in patients fed
enteral glutamine for 3 days, compared to the control fed group. Although the diets were
1382_C34.fm Page 579 Tuesday, October 7, 2003 7:25 PM

Chapter thirty-four: Glutamine-supplemented diets in enteral nutrition 579

isonitrogenous, the glutamine group was not fed an equal amount of nitrogen during the
study compared to the control group.
In the study of Houdijk et al.2 severe trauma patients with an injury severity score of
20 or more were randomized to receive an enteral nutrition enriched with glutamine
(n = 35) vs. a control enteral nutrition (n = 37). In this study the same enteral nutrition
was given as that mentioned in the study of Long et al.1 The glutamine-enriched group
received 30.5 g of glutamine per 100 g of protein. The control nutrition was made isoni-
trogenous and isocaloric, compared to the glutamine-enriched formula, by the addition
of alanine, aspartate, glycine, proline, and serine. The control feeding contained 3.5 g of
glutamine per 100 g of protein. The enteral nutrition was started within 48 h of the trauma
and was given continuously by a nasojejunal tube aiming to cover 75% of the calculated
energy expenditure. The nutrition was given for at least 5 days enterally and was supplied
until the patients were tolerating an oral food intake. None of the patients received
parenteral feeding. In the glutamine group 29 patients were fed for more than 5 days, and
in the control group 31 patients were fed for more than 5 days. The total number of days
that the patients received the enteral nutrition (glutamine group, 12.8 days; control group,
11.9 days) and the amount of calories given were not different between the groups. The
primary end point of this study was to evaluate infectious morbidity during the first
15 days following trauma, and the plasma levels of glutamine, arginine, and soluble tumor
necrosis factor (TNF) receptors were determined as secondary end points. Levels of
glutamine and arginine decreased significantly in both study groups at day 1 postinjury,
compared to healthy volunteer reference values. Arginine and glutamine levels increased
significantly from day 3 until day 7 in the glutamine group but did not differ between the
groups after day 7. Long et al.1 did not see the increase in plasma glutamine concentrations,
while they used the same enteral nutrition as Houdijk et al.2 A possible explanation could
be that Long and colleagues did not feed the patients as vigorously as the group of
Houdijk, who succeeded to reach the nutritional goal within 72 h in his patient population.
Moreover, the study of Long et al. gave the nutrition for 3 days, while in the study of
Houdijk, the enteral nutrition was given at least 5 days and in total an average of 12 days.
In addition, the number of patients in the study of Long et al. was less than that in the
study of Houdijk et al. Besides that, all patients of the Houdijk study were fed directly in
the jejunum, while in the Long study some patients had duodenal or gastric tubes. Gas-
trointestinal complications are less frequent and nutritional goals are more efficaciously
achieved in intensive care unit (ICU) patients fed via the jejunum compared to patients
fed by gastric tube.10
In the study of Houdijk et al., a significantly lower incidence of pneumonia was seen
in the glutamine-supplemented group fed for more than 5 days (5/29, 17%) compared to
the control group (14/31, 45%; p < .02). In addition, a significant reduction in the incidence
of bacteremia (2/29 (7%) of glutamine group vs. 13/31 (42%) of the controls; p < 0.005)
and sepsis (1/29 (3%) of the glutamine group compared to 8/31 (26%); p < .02) in the
control group was found. It is important to mention that the single episode of sepsis
observed in the glutamine group was caused by Staphylococcus aureus, whereas mainly
Gram-negative bacteria were cultured in the control group. Plasma-soluble TNF receptors
were significantly lower in the glutamine group, suggesting a modulated inflammatory
response in the glutamine-supplemented group over the control feeding.
Wischmeyer et al.11 did a study on the effects of glutamine given intravenously as a
pharmacologic intervention in addition to a standard enteral feeding and found signifi-
cantly less Gram-negative bacteremia in the severe burn patients in the glutamine group
than in the control patients, which is consistent with the results of the study in trauma
patients of Houdijk et al.2 Suggested from both clinical studies is that glutamine has a role
in preventing Gram-negative gut-derived bacterial translocation. On the other hand,
1382_C34.fm Page 580 Wednesday, October 8, 2003 1:43 PM

580 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

glutamine might not prevent translocation of Gram-positive bacteria, which are primarily
derived from the skin and burn eschar.11
In the same group of multitrauma patients, the effect of glutamine-enriched enteral
nutrition (EN) on human leukocyte antigen (HLA-DR) and FcgR1/CD64 expression on
CD14+ monocytes was studied.12 Trauma patients were compared to a group of age-
matched healthy volunteers (n = 17). As observed by other investigators, on day 1 the
HLA-DR expression was severely less in both trauma patient groups, compared to the
expression of the healthy volunteers. HLA-DR expression was greater on days 5, 9, and
14 in the glutamine group than in the control group. Following trauma, FcgRI/CD64
expression on monocytes was not different than the expression in healthy volunteers. It
was shown that a glutamine-enriched enteral nutrition was associated with a higher HLA-
DR expression on CD14+ monocytes of the trauma patients. No difference in monocyte
FcgRI/CD64 expression was detected between the two enteral diets and between trauma
patients and healthy volunteers.12 Similar results were obtained when glutamine was given
as a dipeptide glycine–glutamine (gly-gln) by the parenteral route.13
Boelens et al.14 investigated the proliferative response of stimulated peripheral blood
mononuclear cells (PBMCs), as determined by H3-thymidine incorporation, within the
same trauma patient population on days 1 and 14.
Therefore, PBMCs were cultured in the presence or absence of glutamine and were
stimulated with phytohemagglutinin (PHA). On day 1, stimulated PBMCs of healthy
volunteers had a greater thymidine uptake than those of trauma patients when cells were
cultured in vitro without glutamine in the medium (p < 0.05). Both patient groups enhanced
their proliferation rate (delta d1 to d14) during the study period (p < 0.005). But the
proliferation rate (delta d1 to d14) during the study of the glutamine group reached the
rate of the healthy volunteers, while that of the controls did not. With glutamine added
to the in vitro culture medium, it was seen that the control group had a greater increase
in rate of proliferation (delta d1 to d14) than healthy volunteers (p < 0.005), while a trend
(p = 0.071) was seen with glutamine-enriched nutrition. From these data, it could be
concluded that after severe trauma, PBMCs have a reduced proliferative response com-
pared to healthy volunteers; however, when glutamine was added to the culture medium,
the groups were not different.14
Severe trauma is known to lead to an immune response suppression, characterized
by a type 2 T-lymphocyte response, which is suggested to increase the susceptibility for
infectious complications. As described above, plasma concentrations of glutamine, the
preferred fuel for immunocompetent cells, severely decline after trauma. Because
glutamine-enriched enteral nutrition reduced infectious morbidity in trauma patients,
Boelens et al.15 evaluated the same group of patients in order to study the effect of
glutamine-enriched EN on the immune response of severe trauma patients toward a
primary antigen. Thirty-eight multisystem trauma patients (ISS > 20) were sensitized with
keyhole limpet hemocyanin (KLH) within 12 h of trauma. In vitro interferon-gamma
(IFN-g) production of PHA-stimulated PBMCs was significantly low after trauma on day
1 in both patient groups. On day 14, the IFN-g production increased significantly in the
glutamine group compared to in the control group. Interleukin-4 (IL-4) production was
not different following trauma. On day 14, IL-4 decreased in the control group. On day
14 no significant differences of KLH-specific immunoglobulin (Ig) G, IgM, IgA, IgG1, IgG2,
and IgG3 were measured. In conclusion, trauma caused a suppressed in vitro cellular
immune response presented by a low IFN-g production (day 1). Glutamine increased IFN-g
production (day 14), maintained a normal IL-4 production, and did not show an effect on
KLH-specific humoral response on day 14, all together suggesting a skewing toward a
more type 1 T-lymphocyte response by enteral glutamine.15
1382_C34.fm Page 581 Tuesday, October 7, 2003 7:25 PM

Chapter thirty-four: Glutamine-supplemented diets in enteral nutrition 581

In order to provide more insight in the working mechanism of glutamine, the course
of endocrine and metabolic plasma mediators after trauma was analyzed by means of
glucose, prealbumin (transthyretin), albumin, alanine, antitrypsin, C-reactive protein, com-
plement factors, cortisol, glucagon, insulin, and growth hormone concentrations. A sus-
tained hyperglycemic response in both groups was measured; it did not differ between the
glutamine and control groups during the study period. After trauma sustained hypergly-
cemia is usual and regarded as the result of increased hepatic glucose output together with
a diminished sensitivity of peripheral tissue to the action of insulin.16 The plasma levels of
the gluconeogenic amino acid alanine started at low levels in both groups. After 3 days a
rise was noticed that was not influenced by the type of feeding. Growth hormone, cortisol,
glucagon, prealbumin, and complement levels were in the normal range throughout the
study period and did not differ between the groups. In contrast, plasma insulin levels
showed a significant increase in both groups 3 days after the injury compared to day 1 (p
< 0.05), with further increases on days 7 (p < 0.01) and 10 (p < 0.01). Albumin levels were
below normal values (34 to 50 g/l) in both groups from the start until the end of the
observation period, and there were no differences between the groups. In both groups the
initial plasma activity level of antithrombin III was low but increased to near normal levels
after 3 days (days 3, 7, and 10 all p < 0.01 vs. day 0). The inflammatory marker C-reactive
protein showed a rise in plasma concentrations that reached peak levels around day 3
(p < 0.05 vs. baseline) in both groups, which was followed by a decrease to baseline values.
It was concluded from this metabolic study that enteral glutamine-enriched nutrition did
not alter the metabolic and inflammatory mediators in peripheral blood in this population.
Under consideration should be taken the fact that sample sizes were likely too small to
show a significant difference of these multifactorial modulated parameters.16,17
After severe trauma and subsequent extensive tissue damage, a trauma-related oxi-
dative stress and cellular injury is associated with retention of sodium and water and the
concomitant expansion of the extracellular fluid compartment.16,18 Scheltinga et al.18
showed that glutamine supplementation of TPN reduced extracellular fluid expansion in
surgical patients. Because enteral glutamine-enriched feeding reduced the incidence of
infectious complications and is suggested to restore body fluid distribution, Boelens et al.20
hypothesized that enteral glutamine enrichment might have an important effect on ade-
quate taurine plasma levels, which are needed to restore the osmolar disturbances as seen
following trauma.2 Moreover, since glutamine is known to serve as the preferred fuel for
neutrophils and as a precursor of the endogenous antioxidants taurine and gluthatione,
it could be hypothesized that glutamine had its beneficial effect on infectious morbidity
by increasing antioxidant availability.19 Therefore, in both patient groups taurine plasma
levels were measured.20 It was found that taurine levels were low on day 1 after trauma
in both groups. From day 3 onward the patients receiving glutamine had significantly
higher taurine levels (day 3, p < 0.005; days 4, 5, and 7, p < 0.05). Glutamine enrichment
significantly increased taurine plasma levels in trauma patients, which was also confirmed
in a stressed rat model. The reduction in morbidity by glutamine enrichment may be in
part explained by the increased taurine availability and its strong antioxidant and osmolyte
properties.20

34.2 Burn patients


Yan et al.21 enrolled 12 burn patients with total body surface area of >30% and gave them
early enteral glutamine-enriched nutrition while studying the occurrence of gastrointes-
tinal stress ulcers in these patients. All patients developed a stress ulcer on postburn day
1 or 2. Stress ulcers were not found after several wound excision operations, and about
86% of the ulcers disappeared within 2 weeks. The authors concluded that early enteral
1382_C34.fm Page 582 Tuesday, October 7, 2003 7:25 PM

582 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

glutamine was safe in major burn patients and could prevent the complications of gas-
trointestinal stress ulcer. This study did not use a control group.
Zhou et al.22 also did a study randomizing burn patients. For the first time a dipeptide
was administered enterally in burn patients. In this study the effect of enteral glutamine
on the gut barrier function on days 0, 3, 6, and 12, wound healing rate, and length of
hospital stay were investigated. Twelve patients were included; six patients received
glutamine (Ensure® enriched with glutamine dipeptide at a dose of 0.5 g/kg/day
(glutamine, 0.3 mg/kg/day)) and six a control solution (Ensure®). The feedings were
isonitrogenous. The calorie requirement was calculated by means of Curreri’s formula:
25 ¥ BW + 40 ¥ TBSA (kcal/day). It was found that the plasma glutamine levels decreased
in both groups at 12 h postburn. At 10 days postburn the glutamine plasma levels in the
glutamine group returned to normal values, while the control group still had low plasma
levels (p = 0.048). On the day of the burn injury, the lactulose:mannitol (L:M) ratio was
the highest in both groups, returning faster to lower values in the glutamine group than
in the control group on days 3 and 6. No difference between the two groups was observed
concerning the burn wound healing at day 30 and length of hospital stay. The authors
concluded that enteral nutrition enriched with glutamine dipeptide could increase the
plasma glutamine concentration and decrease the gut permeability at early stage after
burn.22
The effects of enrichment of diets with precursors of glutamine (e.g., ornithine a-keto-
glutarate) in burn patients are described in Chapter 37.

34.3 Intensive care patients


Jensen et al.23 performed a double-blind, controlled study comparing the arterial and
venous amino acid profiles in patients receiving tube feedings differing by sixfold in
glutamine content. Twenty-eight intensive care patients with an Acute Physiology and
Chronic Health Evaluation II (APACHE II) score of >10 were randomized to receive
isonitrogenous and isoenergetic nasojejunal feedings with 289 g of glutamine/kg of protein
(n = 10) or 50 g of glutamine/kg of protein (n = 9). All patients were fed within 48 h of
admission of the ICU. All tubes were placed intraoperatively or under fluoroscopic guid-
ance. Nine patients did not meet the minimum study threshold of enteral feeding at day 5
and were excluded from the analysis (two patients due to their injuries, not nutritionally
related; three patients were able to tolerate oral food again; and four were converted to
parenteral nutrition). Hence, the final glutamine group consisted of five males and five
females, and the final control group of seven males and two females. No differences with
respect to age, body mass index (BMI), therapeutic intervention scoring system (TISS),
and APACHE II score were seen. Energy and nitrogen intakes were similar in both groups.
Nitrogen balance estimations were not different between the groups. All subjects showed
hypoaminoacidemia, including glutamine levels at baseline; no difference was found in
time and between the groups. Phe:Tyr ratio was increased in both groups at baseline and
decreased significantly at day 5 in the high glutamine group. The CD4:CD8 T-cell ratios
of the high glutamine group increased from days 1 to 5 compared to the control group.
Results showed a blunting of the hyperaminoacidemia, and the aromatic amino acids were
elevated. The study was supported by Ross, although it is not clear whether the formula
used was AlitraQ.
Jones et al.24 presented results from another double-blind, randomized study concern-
ing 78 ICU patients (ISS > 11), already clinically malnourished or at risk of becoming
malnourished, and a likely incapacity to resume normal nutrition within 5 days. The study
examined the effect of enteral glutamine provision on shortening of recovery and improv-
ing survival. Fifty patients successfully received enteral feeding, of which 13 received a
1382_C34.fm Page 583 Tuesday, October 7, 2003 7:25 PM

Chapter thirty-four: Glutamine-supplemented diets in enteral nutrition 583

combination of TPN and EN (n = 4, gln; n = 9, control) and 37 patients only EN (n = 22,


glutamine; n = 15, control). The patients allocated to enteral nutrition received an iso-
caloric, isonitrogenous balanced nutrition per nasogastric route containing 5 g of
glutamine or glycine per 500-ml bottle. If patients did not tolerate enteral nutrition or the
nutritional goal of 1500 ml/48 h was not reached, TPN was given. Four groups were
evaluated with or (almost) without glutamine and with or without TPN feeding, additional
to the enteral feeding. Significantly more calories were used in the control patient groups
when compared to the glutamine groups. Long et al.1 also reported a reduced amount of
calorie intake in the glutamine group. A reduction in the median of the total costs of ICU
and post-ICU hospital costs was observed in the glutamine group when compared to
control group. The control patients stayed longer at the ICU (median, 16.5 days) and were
fed for longer (11 days) than the glutamine group (ICU stay median of 11 days and fed
for median of 8 days) (both not significant). The four patients receiving glutamine that
required extra parenteral support all died, and their median number of days fed TPN and
EN (1.5 days, n = 4) was much smaller, although not significantly different (p = 0.07) than
that of the control group requiring TPN (6.0 days, n = 9); three control patients died within
6 months of receiving both TPN and EN. No difference was seen in 6-month mortality
and survival curves between the four groups.
Barbosa et al.25 performed a prospective, randomized, controlled study in which five
critically ill children aged 9.7 ± 7.0 years received enteral 0.3 g of glutamine/kg/day and
four children aged 7.6 ± 10.0 years received 0.3 g of calcium caseinate/kg/day for 5 days.
Infants in the glutamine group were comparable to children in the control group with
respect to mean age, gender ratio, birth weight, actual weight, height, breastfeeding time,
nutrition volume, calorie intake, and protein intake. The feedings were well tolerated and
no patients needed to discontinue the program. Children in the glutamine group showed
an increase in their albumin from 32 ± 4 g/l on day 1 to 38 ± 6 on day 5, in comparison
to 31 ± 6 g/l on day 1 to 31 ± 4 on day 5 in the control group. Remarkable but not
significantly different were the erythrocyte sedimentation rate (ESR) and C-reactive pro-
tein (CRP), which were more decreased in the glutamine group than in the control group.
Septic morbidity and mortality were also tabulated, a significant decrease in septic com-
plications was recorded in the glutamine group (20%) compared to controls (75%), and
two patients died in the control group due to bacterial infections (50% vs. 0% in the
glutamine group). Due to the small sample sizes included in this study and a p value of
0.10 for significance, the results should be carefully interpreted, as a positive indication
could be formulated showing that a glutamine-enriched enteral nutrition reduces infec-
tious complications in critically ill infants.
Velasco et al.26 performed a study evaluating the effect of glutamine supplemented
polymeric enteral nutrition on the recovery of gut permeability abnormalities in 23 criti-
cally ill patients. The gut mucosal wall has an important function as a defense toward the
large bacterial load on the luminal side. Loss of this barrier function is suggested to play
a role in the gut-derived sepsis theory. In addition, glutamine is the main fuel for entero-
cytes, and an improved glutamine availability could restore loss of intestinal wall function.
In this study the L:M ratio in urine (10 g of lactulose/5 g of mannitol in 100 ml given by
the enteral route) was applied to assess the permeability of the gut at baseline, and on
day 8, the nitrogen balance was also performed. Nineteen healthy volunteers underwent
an oral L:M test as control. Patients were included if they were critically ill and were at
least 4 days on enteral fasting. In addition, patients were able to start enteral feeding by
nasoduodenal tube or jejunostomy. Patients were randomized in three groups to receive
(1) a conventional casein-based enteral formula with a supplemented 0.4 g of
casein/kg/day (A.D.N., Nutricomp, B Braun Medical SA, Santiago, Chile) (n = 8);
(2) A.D.N. plus glutamine in a dose of 0.15 g/kg/day and a supplemented casein of
1382_C34.fm Page 584 Tuesday, October 7, 2003 7:25 PM

584 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

0.2 g/kg/day (n = 7); or (3) A.D.N. plus 0.30 g of glutamine/kg/day (n = 8) for 8 days.
All three feedings were equal in calories and nitrogen. The nutritional goal was to provide
25 to 30 kcal of protein/kg/day. Supplementary parenteral nutrition was given if nutri-
tional goals were not reached. Groups 1, 2, and 3 did not differ with respect to age, body
mass index, serum albumin, serum prealbumin, and APACHE II score (near 10). However,
the distribution of diagnosis appears unequal between the groups. No difference on
admission was found for the enteral fasting period prior to the study, calorie requirements,
protein intake per day, mean days on enteral nutrition, and mean nitrogen balance, which
was negative in all three groups. Pooled data of the L:M test of all three groups of critically
ill patients showed a significant increase compared to the values of the healthy volunteers.
No significant differences were found between the three groups with regard to L:M ratios.
A power analysis was done to estimate the number of patients needed to result in a
reduction in L:M ratio with feeding policy and showed 273 patients with a power of 80%
and a significance level of 5%. In light of this calculation, the sample sizes of this study
were too small to test the authors’ hypothesis. In addition, no correlations were found
between the length of fasting and the L:M ratios. Initial vs. final L:M ratios showed a
significant reduction; however, they were not different between the three enteral nutri-
tions. No control group without enteral nutrition, with, for example, parenteral nutrition,
was incorporated in this study in order to rule out spontaneous recovery of intestinal
permeability. From this study it could not be concluded that enteral glutamine restores
intestinal permeability as measured by the lactose:mannitol test.
In a triple-blind, randomized, controlled trial,27 363 critically ill patients (APACHE II
14 (range: 1 to 37)) were enrolled to receive either a solution with 20 g of glutamine/l or
20 g of glycine/l. Both solutions were isocaloric and isonitrogenous. No differences were
seen between the two groups at baseline with respect to age, sex distribution, BMI,
diagnosis at admission, APACHE II score, and ISS. Thirty-four percent of patients in the
glutamine group underwent neurosurgery, compared to 22% in the control group, but no
information about Glascow Coma Scale was described. In the glutamine group a median
of 19 g/l (interquartile range, 11 to 27) was administered for a median duration of 10 days,
of which 91% of the patients were fed by nasogastric tube, besides a median of 10 days
of TPN. The control group received a median of 18 g of glycine/l during a median of 10
days in 76% per nasogastric route, besides receiving a median of 8 days TPN. The groups
had equivalent protein and energy intakes. There was no difference between the groups
for the main outcome events: (1) death at 6 months: 15% (27/179) glutamine group vs.
16% (30/184) control group; X2 = 0.10, p = .75, and relative risk = 0.95 (95% CI = 0.71 to
1.28); and (2) severe sepsis: 21% (38/179) glutamine group vs. 23% (43/184) control group;
X2 = 0.24, p = 0.62, and relative risk = 0.94 (95% CI = 0.72 to 1.22). There was also equivalence
for secondary end points relating to infections, febrile period, antimicrobial therapy, and
consumption of inotropes. The conclusion of this study was that glutamine supplemen-
tation did not influence the clinical outcome of critically ill patients. A couple of points
need to be considered for interpreting the results of this study. First, a control glycine was
used; it is a relatively unknown nonessential amino acid with possible strong character-
istics such as cytoprotection and being a substrate for the body’s most important antioxi-
dant glutathione.28 The same group28 published the results of the protective role of glycine
in warm ischemia-reperfusion injury in the small intestine in a rat model. Second, the
dosage of glutamine was fairly low; studies showing reduction in morbidity gave about
30 g/day instead of nearly 20 g/day. Glutamine was administered in most cases by
nasogastric route, which might not be the most preferable site of administration.10 The
overall mortality up to 6 months after ICU admittance was not very high (15%), while
other studies report that their ICU mortality is around 35%.29,30
1382_C34.fm Page 585 Tuesday, October 7, 2003 7:25 PM

Chapter thirty-four: Glutamine-supplemented diets in enteral nutrition 585

The group of Conejero31 randomly allocated 84 critically ill patients from 11 ICUs in
tertiary care hospitals to a glutamine-enriched enteral diet (AlitraQ®, Abbott Laboratories)
(n = 47) or control diet (Ensure®, Abbott Laboratories) (n = 37). The glutamine-enriched
diet provided 52.5 g of proteins per liter with 30.5 g of glutamine and a 94:1 nonprotein
calories-to-nitrogen ratio. The control diet provided 66.6 g of proteins per liter with a 90:1
nonprotein calories-to-nitrogen ratio, excluding glutamine. The nutritional goal was to
achieve the calculated caloric requirements in the first 72 h of enteral nutrition. The aim
of this prospective, randomized, double-blind study was to evaluate the effect of enteral
glutamine on intestinal permeability, nocosomial infections, and mortality in critically ill
patients who developed systemic inflammatory response syndrome (SIRS). In the
glutamine group 42 patients were fed for more than 3 days, and in the control group 29.
The groups were no different with respect to age, sex distribution, weight, APACHE II
score (mean, 18 to 20), days on mechanical ventilation, inotropics, sepsis, and shock on
admission. Patients were fed enterally for an average of 11 days and had similar caloric
intakes at days 3 and 7. Nine patients died in the control group (27%) and 14 patients
died in the glutamine group (33%) (not significant). Also, no difference was found in ICU
stay and multiple-organ failure rate. However, the number of infections in the glutamine
group was significantly less, with 11 of 43, compared to 17 of 33 control patients. Consistent
with the study of Houdijk et al.17 and Wischmeyer et al.,11 it was found that a significant
preponderance of Gram-negative bacteria was seen in the control group compared to the
glutamine group, while no increase was seen in Gram-positive microorganisms.11 Further-
more, cholesterol increased significantly in the control group (Ensure®) on day 7, and
prealbumin (transthyretin) was significantly higher in the glutamine group on day 7. L:M
ratios did not differ between the groups. However, results of this study are obscured by
the fact that the feedings were not isonitrogenous and that the control solution had a
higher lipid content than the glutamine-enriched nutrition.31

34.4 Patients undergoing gastrointestinal surgery


Fish et al.32 randomized a population of 20 consenting patients scheduled for elective
upper gastrointestinal surgery to receive glutamine-enriched enteral or glutamine-
enriched parenteral nutrition. The patients of the enteral group received nasoenteric post-
pyloric feeding tubes, which were placed during surgery. The measured glutamine content
was 10.58 g/l in the TPN formula consisting of a commercially available formulation (Ren
Amin®; Baxter) mixed with free L-glutamine and 10.0 g/l in the tube-feeding formula
(Vivonex plus®; Sandoz Nutrition). The feedings were isonenergetic but not isonitrogenous
(TPN, 7.32 g/l; EN, 7.13 g/l). Feeding was started on day 1 postsurgery and continued
for 10 days or until the patient was able to consume more than clear liquids. For final data
review, patients receiving at least 5 days of artificial feeding were evaluated, resulting in
7 patients (5 male and 2 female) receiving tube feeding and 10 patients (5 male and
5 female) receiving TPN. All patients had a significant drop in amino acid concentrations
at day 1 postoperatively. Long et al.9 and Jensen et al.23 also described this hypoami-
noacidemia in critically ill patients. By day 5 plasma concentrations of most of the amino
acids approached baseline values, as they also did for glutamine, which did not differ
between the two groups. The EN group had a significantly different amino acid profile
in time between baseline and day 5, while the TPN group did not.
Furukawa et al.33 administered L-glutamine (average of 16 g/day) enterally through
a jejunostomy to two purulent peritonitis patients. Although it was previously described
that amino acid absorption is impaired in septic patients, both case reports of this study
showed that the enteral glutamine was well tolerated, improved the patients’ glutamine
plasma levels, and spared muscle amino acid release.
1382_C34.fm Page 586 Wednesday, October 8, 2003 1:45 PM

586 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Aosasa et al.34 randomized 14 patients undergoing colorectal surgery in three groups


for preoperative nutritional management. Group 1 (n = 4) received TPN (glutamine-free)
for more than 5 days, group 2 (n = 6) received the same TPN with an oral supplement of
30 g of glutamine/day, and group 3 (n = 4) was a control group with an oral intake of
normal food. From ileal mesenteric blood samples and liver specimens, mononuclear cells
(MNCs) were isolated and stimulated with endotoxin in the culture medium. Within the
limitations of small sample sizes, the authors concluded that supplemental glutamine was
effective in preventing an overproduction of TNF-g induced by TPN.

34.5 Inflammatory bowel disease


The first study to give enteral glutamine to patients with inflammatory bowel disease
such as Crohn’s disease was performed by Akobeng et al.35 This double-blind, randomized,
controlled study included 18 children with active Crohn’s disease for a 4-week course of
either a standard polymeric diet with a low glutamine content (4% of the amino acid
composition) or a glutamine-enriched polymeric diet (42% of the amino acid composition).
Both diets were isocaloric and isonitrogenous with an identical essential amino acid profile.
The two groups were similar in age and gender. The weights of the children in the control
group had a trend (p = 0.099) to be lower (28.2 kg) than those of the children in the
glutamine group (39.6 kg). The pediatric Crohn’s disease activity index (PCDAI) tended
(p = 0.091) to be higher in the control group (37.7) than in the glutamine group (27.7). Two
children of the glutamine group were withdrawn from the study, one due to persistent
vomiting and the other to persistent abdominal pain. Except for two children in each
group who were fed by nasogastric tube, all children consumed the diet orally. No differ-
ence was observed for achieved remission at 4 weeks between the diets in an intention-
to-treat approach. The PCDAI decreased in all patients of the control group, but in only
five of seven patients from the glutamine group; adjusted for baseline differences, the
mean PCDAI was better in the control group. No other clinical parameter showed a
difference between the diets. The authors speculated that by providing glutamine to the
gut, the increased T-cell numbers present in Crohn’s disease would have been further
promoted in their inflammatory activity, thereby resulting in a worse PCDAI.
Den Hond et al.36 selected 38 patients with Crohn’s disease for a permeability test
using 51Cr-EDTA. In 14 patients an increased permeability was found (excretion of more
than 1.1% as a result of a cumulative total of 6 h), and these patients were randomized to
receive 7 g of glutamine (n = 7) three times a day dissolved in water or glycine (n = 7) in
the same dose for 4 weeks. Besides this protocol, a normal diet was sustained and med-
ications were kept at a constant dose. Some differences were seen between the two groups
that were too small to compare statistically, such as the number of patients with disease
in the terminal ileum was 6 of 7 in the glutamine group compared to two of seven in the
placebo group. In addition, in the glutamine group 4 of 7 patients received salicylates over
5 of 7 controls, while 5 of 7 gln received corticosteroids over 3 of 7 controls, and 2 of 7
immunosuppressive in the glutamine group over 1 of 7 in the control group. The mean
CDAI was not different at baseline (gln 170 ± 99 vs. gly 115 ± 69) and was not different
after treatment between the two groups (gln 163 ± 103 vs. gly 106 ± 74). This study did
not find any statistical differences between seven patients receiving glycine and seven
patients receiving glutamine.
Scolapio et al.37 studied 8 patients with short bowel syndrome who were at a mean
of 3 years past small bowel resection. This small study was performed in a randomized,
double-blind, placebo-controlled crossover manner. An 8-week treatment with oral
glutamine (0.45 g/kg/day) or control, polycose powder (Ross Laboratories, Columbus,
OH) in combination with a high-carbohydrate and low-fat (HCLF) diet, was given. No
1382_C34.fm Page 587 Tuesday, October 7, 2003 7:25 PM

Chapter thirty-four: Glutamine-supplemented diets in enteral nutrition 587

differences were found between the two groups of only four patients with regard to body
weight, small bowel morphology, gastric emptying, fecal volume, percent wet weight
absorption, percent fecal fat absorption, and percent D-xylose absorption. Scopalio et al.38
also did a small study in which glutamine was combined with growth hormone and a
HCLF diet for 3 weeks in eight short bowel patients and found merely modest improve-
ments in electrolyte absorption, a delayed gastric emptying, no improvements in small
bowel morphology, stool losses, and macronutrient absorption. Two other groups39–41
similarly administered enteral glutamine in combination with growth hormone to patients
with short bowel syndrome. Byrne et al.39 did find an enhanced nutrient absorption that
has not been confirmed by others.38,40,41

34.6 Malignancy requiring bone marrow transplantation or


chemotherapy
34.6.1 Adults
Bone marrow transplantation (BMT) is a very aggressive treatment causing a lot of chem-
ical and radiation damage resulting in an immunocompromised condition. Ziegler et al.42
already observed that TPN enriched with glutamine lowered the incidence of infection
and reduced the length of stay at the ICU. Schloerb et al.43 conducted a study in which
66 patients underwent allogeneic BMT for hematologic malignancies (He-AL, n = 18) or
autologous BMT for hematologic malignancies (He-Au, n = 25) or solid carcinoma (S-Au,
n = 23). These patients were randomized to receive 30 g of glutamine (n = 35; He-Al, n = 7;
He-Au, n = 11; S-Au, n = 10) orally per day vs. oral glycine (n = 31; He-Al, n = 5; He-Au,
n = 20; S-Au, n = 13). If patients did not tolerate oral feeding they would be given TPN
with glutamine for the glutamine group vs. standard TPN in the glycine group. In this
study no significant differences in hospital stay, duration of stay after BMT, TPN days,
neutrophil recovery, incidence of positive blood cultures, sepsis, mucositis, or diarrhea
were found.
Sixty-five patients (>70 years) with advanced breast cancer receiving doxifluridine
were evaluated in a double-blind, randomized, controlled study.44 From days 5 to 12
during each interval between chemotherapy, which was given from days 1 to 4, the patients
were randomized to take a daily dose of 30 g of glutamine dissolved in 50 ml of water
(n = 33) orally vs. nonisonitrogenous placebo (maltodextrine) (n = 32). A frequent adverse
event of doxifluridine is diarrhea, and the purpose of the study was to study the efficacy
of glutamine in preventing diarrhea and the impact on tumor growth. Incidence of diar-
rhea was recorded following the National Cancer Institute (NCI)–Bethesda criteria, and
blood was drawn for routine biochemistry and assessing hematologic toxicity. A total of
278 chemotherapy cycles in the glutamine group and 259 cycles in the placebo group were
studied. Some noncompliance was detected in both groups: two patients in both groups
claimed intolerance and discontinued taking the compounds. No differences between the
two groups at the study entry were seen with respect to age, severity of the disease,
disease-free interval, positive lymph nodes, and number of treatments for metastatic
disease. No difference was found in number of diarrhea episodes between the two groups,
and no impact of glutamine on tumor growth was detected.
Muscaritoli et al.45 gave 18 g/day divided in three portions dissolved in water during
the meals to 14 patients undergoing chemotherapy until neutrophil recovery (mean, 31
days). Eleven patients continued the supplementation and were compared to 22 unsup-
plemented patients. A significantly lower duration of diarrhea (days) was observed in the
glutamine group, as well as a reduced incidence of severe diarrhea in the glutamine group.
Moreover, the glutamine-supplemented group required significantly less systemic
1382_C34.fm Page 588 Tuesday, October 7, 2003 7:25 PM

588 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

antifungal treatment. This study was not randomized, but retrospectively controlled and
open labeled.
The first study investigating the effect of glutamine on systemic lymphocyte responses
and gut barrier function in patients with advanced esophageal cancer undergoing neoad-
juvant chemotherapy, 5-fluorouracil (FU), and cisplatin was generated by Yoshida et al.46
Seven patients received 30 g of oral glutamine/day from days 1 to 28, and six patients
received the control amino acid mix in order to obtain an isonitrogenous design (30 g/day;
Amiparin®, Otsuka Pharmaceutical, Tokushima, Japan). A reduced glutamine plasma
concentration on day 7 after chemotherapy was observed in the control group, while the
glutamine patients maintained at baseline values. Also, a significantly higher value was
found between the glutamine and control groups with respect to soluble IgA (sIgA) in
the saliva on day 14. However, the sIgA in the plasma was not different. Radiochemo-
therapy reduced lymphocyte counts in both groups, although the values in the glutamine
group were less reduced than those of the controls. In addition, the T- and B-cell counts
were greater with glutamine supplementation than without it on day 7, and the lympho-
cyte blast formation after PHA and concanavalin A (Con A) was greater in the glutamine
group than in the controls on days 7 and 14. Gut permeability as measured by amount of
phenolsulfonphthalein (PSP) excretion in the urine was less in the glutamine group on
day 7 than in the controls.
Anderson et al.47,48 conducted two studies evaluating the effect of oral glutamine on
painful stomatitis. One study included 24 patients undergoing chemotherapy who were
receiving 2 g of glutamine or glycine suspension twice a day to swish and swallow. The
duration of mouth pain was 4.5 days less in the glutamine group; and the severity of the
oral pain scored by the Modified Eastern Cooperative Oncology Grading System was
4 days less with glutamine than with the placebo.48 The other study47 included 193 patients
with painful stomatitis during bone marrow transplantation until day 28 from admission.
Patients were randomized for glutamine suspension (n = 98) or placebo (glycine) (n = 95).
Both groups were comparable with respect to type of donor, age, gender, and diagnosis
distribution. Less oral pain was reported in the glutamine group of autologous BMT
patients by self-report. The number of patients using morphine was greater in the
glutamine group. The duration of opiate usage was less in autologous BMT patients with
the glutamine suspension but higher in the matched sibling BMT patients in the glutamine
group than in the controls. Day 28 survival of allogenic patients was improved in
glutamine patients.
Coghlin et al.49 also investigated the daily oral administration of 30 g of glutamine or
the same amount of sucrose in 58 patients undergoing allogeneic or autologeous bone
marrow transplantation with or without radiation. Intakes were not isonitrogenous. If
patients could not take anything orally, the study was discontinued. In this prospective,
double-blind, randomized study no significant differences in incidence or severity of
mucositis or diarrhea, length of stay at the hospital, or usage of TPN were found.
Another study,50 on the effect of oral glutamine for oral mucositis, was done on patients
with head and neck cancer undergoing radiotherapy who were randomized to either
glutamine suspension (n = 8) (16 g in 240 ml of saline) or saline (n = 9). The duration of
oral mucositis was significantly shorter for grades 1, 2, and 3 in the glutamine group. The
mean maximum grade of objective oral mucositis was less in the glutamine group. This
study was regarded as a single-blind, randomized test since the control (saline) is visually
different from the glutamine suspension.
Cockerham et al.51 performed another study evaluating the effect of oral glutamine
on oral mucositis for patients undergoing BMT and high-dose paclitaxel and melphalan.
The first 9 patients did not receive glutamine; the subsequent 12 patients were given
glutamine mouthwash (4 g/20 ml of water) to swish and swallow every 4 h from day 7.
1382_C34.fm Page 589 Tuesday, October 7, 2003 7:25 PM

Chapter thirty-four: Glutamine-supplemented diets in enteral nutrition 589

From this study it was shown that oral glutamine around-the-clock may decrease both
the severity and duration of mucositis and subsequently reduce parenteral narcotic use.
This study was not randomized and used a retrospective analysis. It is not clear from the
article how many days glutamine was given.
Daniele et al.52 studied the effect of 18 g of oral glutamine per day (three portions in
water) (n = 29) on the 5-FU-induced intestinal toxicity in 70 patients with colorectal cancer.
A double-blind, randomized, controlled trial was done with maltodextrin as the placebo
(n = 33). Besides the registration of toxicity, a D-xylose absorption test and a cellobiose-
mannitol permeability test were performed. Chemotherapy induced a worsening of the
intestinal absorption and permeability, which were significantly improved by glutamine
treatment over the placebo. Besides that, a protective effect was reported on FU-induced
diarrhea.
A recent study53 administered an oral supplement containing beta-hydroxy-beta-
methylbutyrate (HMB) in combination with arginine and glutamine to 49 patients with
advanced solid tumors who had documented weight loss greater than 5% and found body
weight gain in the test group, compared to the control mixture consisting of alanine,
glutamic acid, glycine, and serine.

34.6.2 Children with BMT and chemotherapy


Ford et al.54 published two case reports of infants with acute nonlymphocytic leukemia
of which one child (9 months old) was fed enterally with a glutamine-enriched formula
(Vivonex Pediatric®) for 70 days and one infant (18 months old) was fed parenterally for
81 days. In this report some advantages in clinical course were seen in favor of the enterally
fed infant.
Another publication from the same group appeared some years later by Pietsch et al.55
on the feasibility of nasogastric tube feedings in children receiving intensive chemotherapy
(CTX) or bone marrow transplantation. Seventeen infants received a continuous enteral
glutamine-supplemented elemental diet during CTX and at the time of rehospitalization
for fever, neutropenia, and mucositis. Enteral nutrition was administered with a mean of
12.7 days per patient. The tubes were generally well tolerated, and no episodes of sinusitis
or epistaxis were reported. Six children received TPN in addition to enteral feedings. The
hospital costs for the enteral feedings were $25,348, compared to $112,299 for the same
number of days of TPN. Unfortunately, no control group was used in this study.

34.7 Neonates
In children, the first blinded, randomized study administering enteral nutrition with
glutamine vs. control was done by Neu et al.56 All patients were started on parenteral
nutrition on day 3. Sixty-eight very low birth weight (VLBW) neonates whose mothers
decided to formula feed were randomized to receive free glutamine at 0.08 g/kg/day,
increasing to 0.31 g/kg/day dissolved in Similac® Special Care 20 formula (10 kcal/oz),
or the control feeding without glutamine (Similac Special Care 20 formula (10 kcal/oz)).
The feeding was started on the third day of life until day 30. The test group tolerated
enteral feeding better, and analysis of T-cells showed a maintained expression of cell
surface markers.
The same group had a second publication57 that included preterm infants cared for
in neonatal intensive care units (NICUs) and reported the effects of enteral glutamine
supplementation on hospital costs in the same infant population. The median costs for
hospitalization, radiology, pharmacy, laboratory, and the NICU, and the median number
of utilization units were reduced with glutamine supplementation. This study provides
1382_C34.fm Page 590 Tuesday, October 7, 2003 7:25 PM

590 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

the first evidence for decreased hospital costs in VLBW neonates who receive enteral
glutamine supplementation.

34.8 Other patient populations


34.8.1 Ischemic heart disease
Khogali et al.58 was the first to elaborate on the effect of glutamine in ischemic heart disease
patients. Male patients (<75 year) (n = 17) with chronic (>3 months) stable angina pectoris
without a history of myocardial infarction and a previous positive test in the Standard
Bruce treadmill exercise protocol were enrolled. The test was done with an interval of
2 weeks, and patients were included if both tests were positive and within a 15% range;
10 patients met these criteria. These patients were randomized to receive 330 ml of car-
bonated water enriched with glutamine (80 mg/kg) or placebo (not clearly indicated) in
a double-blind fashion. It was seen that a single dose of glutamine significantly increased
the plasma glutamine concentration 40 min after the drink, and moreover, the time of
onset of more than 1 mm of ST depression (cardiac ischemia) was delayed from 273 to
311 sec by glutamine, suggesting cardioprotective properties. However, no difference was
measured between glutamine and placebo regarding the hemodynamic response to exer-
cise, onset of anginal symptoms, maximal workload, and total exercise time.

34.8.2 Duchenne muscular dystrophy


Hankard et al.59 performed a study investigating the anabolic effect of oral flavored water
(Kool-Aid® and 2 g of aspartame (Equal®) per 800 ml on the first day) vs. L-glutamine
added to the same mixture (0.6 g/kg on day 2) on six boys with Duchenne muscular
dystrophy (DMD). All children received an infusion of L-[1-13C]leucine and L-[2-
15N]glutamine in the postabsorptive state. The bolus of oral glutamine administration was

associated with an acute decrease in leucine release from protein degradation and a
decrease in estimates of de novo glutamine synthesis in children with DMD.

34.8.3 HIV/AIDS
Noyer et al.60 enrolled 24 patients with AIDS and an abnormal intestinal permeability,
measured by a L:M test. These patients were randomized in a double-blind fashion to
receive 6 g of placebo (table sugar) per day (n = 8), group 1; 4 g of glutamine per day
(n = 8), group 2; or 8 g or glutamine per day (n = 8), group 3. No differences were found
between the groups with respect to age distribution, CD4 counts, weight, albumin, and
cholesterol levels. No benefits were detected by this study using very low amounts of
glutamine per day for 28 days. The first study combining oral glutamine (40 g/day) with
antioxidants in a population of HIV patients (n = 26) was done by Shabert et al.61 All
patients were without active opportunistic infections and had more than 5% unintended
weight loss of usual body weight since the onset of the disease at study entry. These
patients were randomized to receive 40 g of oral glutamine with antioxidants per day
(n = 12) or 40 g of glycine per day (n = 9) for 12 weeks. The selected antioxidants consisted
of ascorbic acid (800 mg/day), alpha-tocopherol (500 IU/day), beta-carotene (27,000
IU/day), selenium (280 mg/day), and N-acetyl-cysteine (2.4 g/day) (Cambridge Nutra-
ceutical, Boston, MA). The study had a double-blind design and patients were randomized
for the following characteristics: gender, age group, antiviral treatment, nutritional status,
and i.v. vs. non-i.v. drug use. Results showed (1) a significant increase in body weight of
2.2 kg/3 months (3.2%), (2) increased intracellular water, and (3) a significantly increased
1382_C34.fm Page 591 Tuesday, October 7, 2003 7:25 PM

Chapter thirty-four: Glutamine-supplemented diets in enteral nutrition 591

body cell mass of 1.8 kg/3 months in the glutamine–antioxidant group, compared to the
glycine group. Because a mixture of several substances was given in this study, it cannot
be determined which part of the beneficial effect was rendered by glutamine. From the
study of Scheltinga et al.,18 it could be suggested that glutamine has a role in correcting
body fluid distribution disturbances, but antioxidants might be capable of correcting this
as well. Another study62 showed that a mixture of HMB in combination with arginine and
glutamine could alter the course of lean tissue loss in 43 patients with AIDS-related muscle
wasting.

34.9 Conclusion
Summarizing from the studies described in this chapter, enteral and oral glutamine sup-
plementation is well tolerated and safe in diverse catabolic states. Some studies described
in this chapter showed negative results of glutamine supplementation, which might be
attributed to low glutamine dosages, poor study design, small sample sizes (beta-prob-
lem), or the use of potentially active control substances such as amino acids as an isonitro-
genous control (glycine).
The positive results, however, are that enteral glutamine-enriched solution in trauma
patients can reduce the incidence of infectious morbidity as well as enhance immune
response and improve amino acid profiles. The meta-analysis of Novak et al.63 concludes
that in surgical patients, glutamine supplementation associates with a reduction in infec-
tious complication rates and shorter hospital stay without any adverse effect on mortality.
In critically ill patients, glutamine supplementation may be associated with a reduction
in complication and mortality rates. In catabolic states patients should be supplemented
with a glutamine-enriched solution, since their endogenous supply does not meet the
requirements. More proper studies need to be done to unravel the enteral or parenteral
route dilemma of feeding a very sick patient.

References
1. Long, C.L., Nelson, K.M., DiRienzo, D.B., et al., Glutamine supplementation of enteral
nutrition: impact on whole body protein kinetics and glucose metabolism in critically ill
patients, J. Parenter. Enteral Nutr., 19, 470, 1995.
2. Houdijk, A.P., Rijnsburger, E.R., Jansen, J., et al., Randomised trial of glutamine-enriched
enteral nutrition on infectious morbidity in patients with multiple trauma, Lancet, 352, 772,
1998.
3. Hammarqvist, F., Wernerman, J., Ali, R., et al., Addition of glutamine to total parenteral
nutrition after elective abdominal surgery spares free glutamine in muscle, counteracts the
fall in muscle protein synthesis, and improves nitrogen balance, Ann. Surg., 209, 455, 1989.
4. Paula, F.J., Pimenta, W.P., Saad, M.J., et al., Sex-related differences in peripheral glucose
metabolism in normal subjects, Diabetes Metab., 16, 234, 1990.
5. Ruby, B.C., Coggan, A.R., and Zderic, T.W., Gender differences in glucose kinetics and
substrate oxidation during exercise near the lactate threshold, J. Appl. Physiol., 92, 1125, 2002.
6. Wohltmann, C.D., Franklin, G.A., Boaz, P.W., et al., A multicenter evaluation of whether
gender dimorphism affects survival after trauma, Am. J. Surg., 181, 297, 2001.
7. Angele, M.K., Knoferl, M.W., Schwacha, M.G., et al., Sex steroids regulate pro- and anti-
inflammatory cytokine release by macrophages after trauma-hemorrhage, Am. J. Physiol.,
277, C35, 1999.
8. Wichmann, M.W., Ayala, A., and Chaudry, I.H., Male sex steroids are responsible for de-
pressing macrophage immune function after trauma-hemorrhage, Am. J. Physiol., 273, C1335,
1997.
1382_C34.fm Page 592 Tuesday, October 7, 2003 7:25 PM

592 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

9. Long, C.L., Borghesi, L., Stahl, R., et al., Impact of enteral feeding of a glutamine-supple-
mented formula on the hypoaminoacidemic response in trauma patients, J. Trauma, 40, 97,
1996.
10. Montejo, J.C., Grau, T., Acosta, J., et al., Multicenter, prospective, randomized, single-blind
study comparing the efficacy and gastrointestinal complications of early jejunal feeding with
early gastric feeding in critically ill patients, Crit. Care Med., 30, 796, 2002.
11. Wischmeyer, P.E., Lynch, J., Liedel, J., et al., Glutamine administration reduces Gram-negative
bacteremia in severely burned patients: a prospective, randomized, double-blind trial versus
isonitrogenous control, Crit. Care Med., 29, 2075, 2001.
12. Boelens, P.G., Houdijk, A.P., Fonk, J.C., et al., Glutamine-enriched enteral nutrition increases
HLA-DR expression on monocytes of trauma patients, J. Nutr., 132, 2580, 2002.
13. Spittler, A., Sautner, T., Gornikiewicz, A., et al., Postoperative glycyl-glutamine infusion
reduces immunosuppression: partial prevention of the surgery induced decrease in HLA-
DR expression on monocytes, Clin. Nutr., 20, 37, 2001.
14. Boelens, P.G., Houdijk, A.P., Fonk, J.C., et al., The proliferative response of immune cells
after trauma: the effect of glutamine, Clin. Nutr., 21, 15, 2002.
15. Boelens, P.G., Houdijk, A.P., Fonk, J.C., et al., Enteral glutamine reverses Th1/Th2 shift in
severe trauma patients: a randomised, double-blind controlled study, Eur. Surg. Res., 34, 62,
2002.
16. Black, P.R., Brooks, D.C., Bessey, P.Q., et al., Mechanisms of insulin resistance following
injury, Ann. Surg., 196, 420, 1982.
17. Houdijk, A.P., Nijveldt, R.J., and van Leeuwen, P.A., Glutamine-enriched enteral feeding in
trauma patients: reduced infectious morbidity is not related to changes in endocrine and
metabolic responses, J. Parenter. Enteral Nutr., 23, S52, 1999.
18. Scheltinga, M.R., Young, L.S., Benfell, K., et al., Glutamine-enriched intravenous feedings
attenuate extracellular fluid expansion after a standard stress, Ann. Surg., 214, 385, 1991.
19. Furukawa, S., Saito, H., Inoue, T., et al., Supplemental glutamine augments phagocytosis
and reactive oxygen intermediate production by neutrophils and monocytes from postoper-
ative patients in vitro, Nutrition, 16, 323, 2000.
20. Boelens P.G., Houdijk, A.P., Teerlink, T., et al., Plasma taurine concentrations increase after
enteral glutamine supplementation in trauma patients and stressed rats, Am. J. Clin. Nutr.,
77, 2003, 250, 2003.
21. Yan, R., Sun, Y., and Sun, R., [Early enteral feeding and supplement of glutamine prevent
occurrence of stress ulcer following severe thermal injury], Zhonghua Zheng. Xing. Shao Shang
Wai Ke. Za Zhi., 11, 189, 1995.
22. Zhou, Y.P., Liu, W., Jiang, Z.M., et al., Glutamine dipeptide enriched enteral nutrition on gut
barrier function in severe burns, J. Parenter. Enteral Nutr., 23, S160, 1999.
23. Jensen, G.L., Miller, R.H., Talabiska, D.G., et al., A double-blind, prospective, randomized
study of glutamine-enriched compared with standard peptide-based feeding in critically ill
patients, Am. J. Clin. Nutr, 64, 615, 1996.
24. Jones, C., Palmer, T.E., and Griffiths, R.D., Randomized clinical outcome study of critically
ill patients given glutamine-supplemented enteral nutrition, Nutrition, 15, 108, 1999.
25. Barbosa, E., Moreira, E.A., Goes, J.E., et al., Pilot study with a glutamine-supplemented
enteral formula in critically ill infants, Rev. Hosp. Clin. Fac. Med. Sao Paulo, 54, 21, 1999.
26. Velasco, N., Hernandez, G., Wainstein, C., et al., Influence of polymeric enteral nutrition
supplemented with different doses of glutamine on gut permeability in critically ill patients,
Nutrition, 17, 907, 2001.
27. Hall, J.C., Dobb, G., Hall, J.L., et al., A clinical trial evaluating enteral glutamine in critically
ill patients, Am. J. Clin. Nutr., 75, 415S, 2002.
28. Lee, M.A., McCauley, R.D., Kong, S.E., et al., Influence of glycine on intestinal ischemia-
reperfusion injury, J. Parenter. Enteral Nutr., 26, 130, 2002.
29. Kvale, R. and Flaatten, H., Changes in intensive care from 1987 to 1997: has outcome
improved? A single centre study, Intensive Care Med., 28, 1110, 2002.
30. Jacobs, C.J., van der Vliet, J.A., van Roozendaal, M.T., et al., Mortality and quality of life
after intensive care for critical illness, Intensive Care Med., 14, 217, 1988.
1382_C34.fm Page 593 Tuesday, October 7, 2003 7:25 PM

Chapter thirty-four: Glutamine-supplemented diets in enteral nutrition 593

31. Conejero, R., Bonet, A., Grau, T., et al., Effect of a glutamine-enriched enteral diet on intestinal
permeability and infectious morbidity at 28 days in critically ill patients with systemic
inflammatory response syndrome, Nutrition, 18, 716, 2002.
32. Fish, J., Sporay, G., Beyer, K., et al., A prospective randomized study of glutamine-enriched
parenteral compared with enteral feeding in postoperative patients, Am. J. Clin. Nutr., 65,
977, 1997.
33. Furukawa, S., Saito, H., Lin, M.T., et al., Enteral administration of glutamine in purulent
peritonitis, Nutrition, 15, 29, 1999.
34. Aosasa, S., Mochizuki, H., Yamamoto, T., et al., A clinical study of the effectiveness of oral
glutamine supplementation during total parenteral nutrition: influence on mesenteric mono-
nuclear cells, J. Parenter. Enteral Nutr., 23, S41, 1999.
35. Akobeng, A.K., Miller, V., Stanton, J., et al., Double-blind randomized controlled trial of
glutamine-enriched polymeric diet in the treatment of active Crohn’s disease, J. Pediatr.
Gastroenterol. Nutr., 30, 78, 2000.
36. Den Hond, E., Hiele, M., Peeters, M., et al., Effect of long-term oral glutamine supplements
on small intestinal permeability in patients with Crohn’s disease, J. Parenter. Enteral Nutr.,
23, 7, 1999.
37. Scolapio, J.S., McGreevy, K., Tennyson, G.S., et al., Effect of glutamine in short-bowel syn-
drome, Clin. Nutr., 20, 319, 2001.
38. Scolapio, J.S., Camilleri, M., Fleming, C.R., et al., Effect of growth hormone, glutamine, and
diet on adaptation in short-bowel syndrome: a randomized, controlled study, Gastroenter-
ology, 113, 1074, 1997.
39. Byrne, T.A., Morrissey, T.B., Nattakom, T.V., et al., Growth hormone, glutamine, and a
modified diet enhance nutrient absorption in patients with severe short bowel syndrome,
J. Parenter. Enteral Nutr., 19, 296, 1995.
40. Szkudlarek, J., Jeppesen, P.B., and Mortensen, P.B., Effect of high dose growth hormone with
glutamine and no change in diet on intestinal absorption in short bowel patients: a ran-
domised, double blind, crossover, placebo controlled study, Gut, 47, 199, 2000.
41. Jeppesen, P.B., Szkudlarek, J., Hoy, C.E., et al., Effect of high-dose growth hormone and
glutamine on body composition, urine creatinine excretion, fatty acid absorption, and essen-
tial fatty acids status in short bowel patients: a randomized, double-blind, crossover, placebo-
controlled study, Scand. J. Gastroenterol., 36, 48, 2001.
42. Ziegler, T.R., Young, L.S., Benfell, K., et al., Clinical and metabolic efficacy of glutamine-
supplemented parenteral nutrition after bone marrow transplantation: a randomized, dou-
ble-blind, controlled study, Ann. Intern. Med., 116, 821, 1992.
43. Schloerb, P.R. and Skikne, B.S., Oral and parenteral glutamine in bone marrow transplanta-
tion: a randomized, double-blind study, J. Parenter. Enteral Nutr., 23, 117, 1999.
44. Bozzetti, F., Biganzoli, L., Gavazzi, C., et al., Glutamine supplementation in cancer patients
receiving chemotherapy: a double-blind randomized study, Nutrition, 13, 748, 1997.
45. Muscaritoli, M., Micozzi, A., Conversano, L., et al., Oral glutamine in the prevention of
chemotherapy-induced gastrointestinal toxicity, Eur. J. Cancer, 33, 319, 1997.
46. Yoshida, S., Matsui, M., Shirouzu, Y., et al., Effects of glutamine supplements and radioche-
motherapy on systemic immune and gut barrier function in patients with advanced esopha-
geal cancer, Ann. Surg., 227, 485, 1998.
47. Anderson, P.M., Ramsay, N.K., Shu, X.O., et al., Effect of low-dose oral glutamine on painful
stomatitis during bone marrow transplantation, Bone Marrow Transplant., 22, 339, 1998.
48. Anderson, P.M., Schroeder, G., and Skubitz, K.M., Oral glutamine reduces the duration and
severity of stomatitis after cytotoxic cancer chemotherapy, Cancer, 83, 1433, 1998.
49. Coghlin, D.T., Wong, R.M., Offrin, R.S., et al., Effect of oral glutamine supplementation
during bone marrow transplantation, J. Parenter. Enteral Nutr., 24, 61, 2000.
50. Huang, E.Y., Leung, S.W., Wang, C.J., et al., Oral glutamine to alleviate radiation-induced
oral mucositis: a pilot randomized trial, Int. J. Radiat. Oncol. Biol. Phys., 46, 535, 2000.
51. Cockerham, M.B., Weinberger, B.B., and Lerchie, S.B., Oral glutamine for the prevention of
oral mucositis associated with high-dose paclitaxel and melphalan for autologous bone
marrow transplantation, Ann. Pharmacother., 34, 300, 2000.
1382_C34.fm Page 594 Tuesday, October 7, 2003 7:25 PM

594 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

52. Daniele, B., Perrone, F., Gallo, C., et al., Oral glutamine in the prevention of fluorouracil
induced intestinal toxicity: a double blind, placebo controlled, randomised trial, Gut, 48, 28,
2001.
53. May, P.E., Barber, A., D’Olimpio, J.T., et al., Reversal of cancer-related wasting using oral
supplementation with a combination of beta-hydroxy-beta-methylbutyrate, arginine, and
glutamine, Am. J. Surg., 183, 471, 2002.
54. Ford, C., Whitlock, J.A., and Pietsch, J.B., Glutamine-supplemented tube feedings versus
total parenteral nutrition in children receiving intensive chemotherapy, J. Pediatr. Oncol. Nurs.,
14, 68, 1997.
55. Pietsch, J.B., Ford, C., and Whitlock, J.A., Nasogastric tube feedings in children with high-
risk cancer: a pilot study, J. Pediatr. Hematol. Oncol., 21, 111, 1999.
56. Neu, J., Roig, J.C., Meetze, W.H., et al., Enteral glutamine supplementation for very low birth
weight infants decreases morbidity, J. Pediatr., 131, 691, 1997.
57. Dallas, M.J., Bowling, D., Roig, J.C., et al., Enteral glutamine supplementation for very-low-
birth-weight infants decreases hospital costs, J. Parenter. Enteral Nutr., 22, 352, 1998.
58. Khogali, S.E., Pringle, S.D., Weryk, B.V., et al., Is glutamine beneficial in ischemic heart
disease? Nutrition, 18, 123, 2002.
59. Hankard, R.G., Hammond, D., Haymond, M.W., et al., Oral glutamine slows down whole
body protein breakdown in Duchenne muscular dystrophy, Pediatr. Res., 43, 222, 1998.
60. Noyer, C.M., Simon, D., Borczuk, A., et al., A double-blind placebo-controlled pilot study of
glutamine therapy for abnormal intestinal permeability in patients with AIDS, Am. J.
Gastroenterol., 93, 972, 1998.
61. Shabert, J.K., Winslow, C., Lacey, J.M., et al., Glutamine-antioxidant supplementation increas-
es body cell mass in AIDS patients with weight loss: a randomized, double-blind controlled
trial, Nutrition, 15, 860, 1999.
62. Clark, R.H., Feleke, G., Din, M., et al., Nutritional treatment for acquired immunodeficiency
virus-associated wasting using beta-hydroxy beta-methylbutyrate, glutamine, and arginine:
a randomized, double-blind, placebo-controlled study, J. Parenter. Enteral Nutr., 24, 133, 2000.
63. Novak, F., Heyland, D.K., Avenell, A., et al., Glutamine supplementation in serious illness:
a systematic review of the evidence, Crit. Care Med., 30, 2022, 2002.
1382_C35.fm Page 595 Tuesday, October 7, 2003 7:27 PM

chapter thirty-five

The use of arginine in clinical


practice
Naji N. Abumrad
Vanderbilt University Medical Center, Nashville
Adrian Barbul
Sinai Hospital and Johns Hopkins Medical Institutions, Baltimore

Contents
Introduction..................................................................................................................................595
35.1 Arginine biochemistry and physiology..........................................................................596
35.1.1 Arginine catabolism ............................................................................................596
35.1.2 Sources of arginine ..............................................................................................597
35.1.3 Arginine kinetics in vivo .....................................................................................597
35.2 Immune functions of L-arginine ......................................................................................598
35.3 Arginine and wound healing...........................................................................................599
35.4 Arginine in combination with other nutrients ..............................................................602
35.4.1 Arginine and lysine.............................................................................................602
35.4.2 Arginine, glutamine, and b-hydroxy-b-methylbutyrate (HMB)..................602
35.5 Arginine and complete nutritional formulas.................................................................603
References .....................................................................................................................................605

Introduction
Adequate nutritional support is an essential component in the successful treatment of
the critically ill patient. In the last 3 decades the concept of nutrition support for the
hospitalized patient has evolved considerably, with the primary goal of therapy being
the maintenance of a positive nitrogen balance. Considerable advances have been made
in the understanding of the pathophysiologic events involved in sepsis, inflammation,
and multiple-organ failure. This led to the understanding of the special roles that certain
amino acids play in maintaining tissue protein homeostasis during illness. Previously,
amino acids were classified as either nonessential (dispensable) or essential (nondispens-
able). However, with better understanding of the biochemical events involving amino acid
metabolism in vivo, Young and El-Khoury1 and others2 have proposed alternate classifi-
cations, which redefine the requirements of certain amino acids as being conditionally

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 595
1382_C35.fm Page 596 Tuesday, October 7, 2003 7:27 PM

596 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

nondispensable. Such a list includes arginine,3 glutamine,4 cysteine,5 and others. This has
led many to focus on the use of these nutrients, solely or as part of a complete nutritional
regimen, to improve the nutritional outcome, as judged by either improved nitrogen
balance or improved immune response of the host. In this chapter we will focus on recent
findings regarding the utility of one of these conditionally essential amino acids, namely,
arginine, and will examine new findings regarding its role in both health and disease. The
recent observations, coupled with relative safety, make the use of arginine, solely or in
combination with other amino acids, very attractive for the care of traumatized or seriously
ill patients.

35.1 Arginine biochemistry and physiology


35.1.1 Arginine catabolism
Arginine is a dibasic amino acid,6 and its plasma and tissue concentrations are determined
by a balance between the rates of production (or intake) and breakdown (or utilization).
Arginine is a constituent of several proteins in the body, and its metabolism is intimately
tied to several metabolic pathways involved in the synthesis of urea, nitric oxide,
polyamines, agmatine, and creatine phosphate.7,8
The urea cycle represents the major metabolic pathway for ammonia detoxification
and for excretion of waste nitrogen in mammals.9 This is reviewed in more detail in
Chapter 7. L-arginine is the major substrate. L-arginine and, to a much lower degree,
homoarginine are also the primary substrates for formation of nitric oxide (NO), produced
from nitric oxide synthase (NOS) (provisionally EC 1.14.13.39),10,11 and play an important
role in the physiology and pathophysiology of the central nervous, cardiovascular, and
immune systems. NO and citrulline are formed by oxidation of one of two identical
terminal guanidino groups of L-arginine by the enzyme NOS. This reaction results in the
formation of a stable intermediate, Nw-hydroxyarginine.12–14
Nitric oxide synthase has been found in almost all tissue types examined and occurs
in all animal species, from mammals, reptiles, and birds to the horseshoe crab.9,15–17 The
crab has survived unchanged during 500 million years of evolution, indicating that the
generation of NO is one of the oldest phylogenetically preserved regulatory systems. As
discussed in Chapter 14, there are three known isoforms of NOS: two that are constitutive
(neuronal NOS (nNOS or NOS-1) and endothelial NOS (eNOS or NOS-3)) and an inducible
NOS (iNOS or NOS-2).18–20 The constitutive NOSs are cytosolic and are activated by
Ca2+/calmodulin.19 Following stimulation by various agonists, the generation of NO by
these isoforms is quantitatively smaller in both amount and duration than iNOS.10 On the
other hand, iNOS is not dependent on intracellular Ca2+ 20 and has been identified in the
brain,21,22 lung,23 retina,24 adrenal glands,25 platelets,26 endocardial cells, vascular endo-
thelial cells,27 skeletal muscle, spleen, and skin.28 Various inflammatory cytokines and
lipopolysaccharide (LPS) induce iNOS.
The most exciting new findings, however, relate to the enzyme arginase. Two isoforms
have been identified and are encoded by two different genes.29 Arginase I is a cytosolic
enzyme that is involved in urea and ornithine formation and is predominantly found in
the liver. Most of the ornithine formed is primarily channeled to successive enzymes within
the urea cycle; very little of this ornithine is involved in the synthesis of polyamines or
glutamate. On the other hand, arginase II is a mitochondrial enzyme and has more ubiq-
uitous expression, including many nonhepatic tissues, such as kidneys, small intestine,
brain, lung, leukocytes, and prostate.29–31 Both enzymes play important roles in regulating
plasma arginine levels and its availability for various catabolic pathways. In macrophages,
both arginase I and II levels are enhanced during periods of stress and hypoxia. Their
1382_C35.fm Page 597 Tuesday, October 7, 2003 7:27 PM

Chapter thirty-five: The use of arginine in clinical practice 597

activities are also increased following exposure to LPS, interleukin (IL)-4, IL-10, IL-13, or
tumor growth factor-b (TGF-b), or to glucocorticoids, catecholamines, and cAMP analogues.
The net effect is increased availability of arginine for NO synthesis,32,33 but also affecting
the synthesis of ornithine, proline, glutamate, and polyamines and their subsequent effect
on cellular proliferation.34–37 On the other hand, deficiency of arginase II enzyme (as was
recently shown in arginase II knockout mice) is associated with 1- to 2-fold elevations in
plasma arginine vs. 10- to 15-fold elevations noted with arginase I deficiency.38

35.1.2 Sources of arginine


There are two important sources for arginine, one dietary that accounts for the majority
of the arginine needed by the body and the other derived from endogenous sources,
estimated to amount to approximately 20% of the daily expenditure.39 The average daily
consumption of L-arginine in the U.S. is ~5.4 g, which is sufficient to replace the amount
utilized daily by the body.
The most important pathway for endogenous arginine biosynthesis is denoted as the
intestinal-renal axis and involves collaborative efforts between the small intestinal mucosa
and proximal renal tubular cells. Metabolism of glutamine and, to a lesser extent, proline
by the small intestine yields citrulline into the portal circulation. The liver uptake of
citrulline is almost negligible, but its uptake by the proximal tubular cells is highly efficient
where it is converted to arginine.8
A secondary and less efficient pathway for arginine synthesis, and one that has been
known for several decades, involves the recycling of citrulline to arginine by various
nonhepatic tissues.40 This pathway is denoted as the citrulline–NO cycle, where arginine
synthesis is intimately connected to NO production and appears to be co-induced with
iNOS, and also with parallel co-inductions of two urea cycle enzymes, arginosuccinate
synthase (ASS) and arginosuccinate lyase (ASL).41,42
Under normal health conditions, the quantities of arginine produced de novo are
sufficient to maintain muscle and connective tissue mass. However, in times of rapid
growth,39 immaturity, tissue repair,43 decreased dietary intake,7 and severe stress such as
sepsis, trauma, and nitrogen overload, or during conditions associated with compromised
function of either the small intestines or kidneys, arginine biosynthesis can be severely
diminished, leading to decreased availability of this amino acid. Under such conditions,
arginine becomes an indispensable amino acid for optimal growth and maintenance of
positive nitrogen balance.

35.1.3 Arginine kinetics in vivo


Several recent studies in humans have utilized stable isotopes to estimate whole-body
fluxes of arginine and citrulline and their interrelationships to whole-body fluxes of NO
synthesis in humans. In general, these studies utilized 15N2-guanidino-labeled arginine
and subsequent analysis of [15N]-nitrate in urine and the rate of conversion of the labeled
arginine to [15N]-citrulline.44–56 Studies performed in healthy adults, using these methods,
have shown (1) arginine flux in the plasma compartment to be ~80 mmol/kg/h; (2) the
rate of NO derived from arginine to be ~0.22 mmol/kg/h, accounting for about 0.5 to 1%
of arginine used for nitric oxide synthesis57; and (3) that NO production is predominantly
(>50%) derived from plasma arginine.44,45 Based on these observations and several in vitro
studies, it would be expected that the availability of plasma arginine and its uptake across
cell membranes would be limiting to NO production.58 If true, these observations would
be contradictory with enzyme kinetics of NOS (constant of affinity km = 2 to 20 mM), and
the relative availabilities of arginine from the extracellular and intracellular spaces (plasma
1382_C35.fm Page 598 Tuesday, October 7, 2003 7:27 PM

598 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

arginine = 60 to 100 vs. ~1000 mM, respectively).17 Based on this kinetics, one would expect
that the majority of the arginine required for NO synthesis would be derived from intra-
cellular sources. Rapid access to plasma arginine could occur via caveolae formed by
cytoplasmic membrane where the y+ transporter is co-located.59

35.2 Immune functions of L- arginine


Arginine, given in large doses, helps maintain immune homeostasis, particularly with
respect to T cell and macrophage functions.60 Absence of L-arginine in culture media results
in significant reduction in proliferation of T lymphocytes in response to mitogens.61 Efron
et al.62 reported the optimal L-arginine concentrations in the media necessary for ideal T
lymphocyte proliferation to be similar to those found in human plasma, 40 to 100 mM. At
in vivo conditions, this requirement for arginine can be substituted by citrulline but not
by ornithine.40 Arginine is also required for the effective induction of cytotoxic T-cell
function in vitro.63 Rodriguez et al.60 showed that Jurkat T cells cultured in media lacking
L-arginine manifest rapid reduction in the expression of the T cell receptor (TCR), CD36z,
caused by a significant short half-life of CD36z-mRNA. It is important to note that CD36z
phosphorylation is the rate-limiting step in the assembly and membrane expression of the
TCR.64 Such reduction in mRNA would consequently lower TCR internalization, leading
to a decrease in the total number of TCR expressed on the cell membrane.65,66
L-arginine is metabolized in macrophages and lymphocytes by two independent enzy-
matic pathways, the iNOS and arginase I. The former produces NO, and the latter results in
production of L-ornithine and urea.67–69 The enzymes are up-regulated in several conditions
such as trauma,70,71 sepsis,72 and liver transplantation,73,74 resulting in decreased plasma levels
of arginine, which coincide with major decreases in T cell proliferation. Supplementation
with L-arginine has been shown to increase CD4+ cells,75 suggesting that arginine may play
an important role in reversing the immunosuppression observed during periods of stress.76
A recent report by de Jonge et al.77 identified arginine as being unexpectedly important
in a specific stage of murine B cell development.78 The investigators engineered a trans-
genic mouse expressing arginase I under the control of the rat intestinal fatty acid-binding
promoter and enhancer element.79,80 The mice demonstrated 30 to 40% reduction of plasma
arginine (accompanied by hyperglycinemia) and showed a disruption in the B cell devel-
opment at the progenitor-B (pro-B) to precursor-B (pre-B) cell interface in the bone marrow.
There were also decreased B cells in secondary lymphoid organs, like spleen and Peyer’s
patches in the initial 3 weeks of the neonatal period. The defects were reversed with
arginine supplementation.79,80 These important findings could explain many of the immu-
nologic abnormalities observed in neonates during periods of stress.
It is well established that the dietary supply of arginine in milk is insufficient to meet
the minimum requirements necessary for arginine incorporation into proteins.81 Under
normal conditions, rapidly growing suckling rodents compensate for the insufficient sup-
ply of arginine in the milk by increasing endogenous arginine biosynthesis.81 During
suckling, the small intestines, not the kidneys, play a major role in arginine biosynthe-
sis.69,82 Hence, conditions associated with significant intestinal dysfunction, as occurs with
neonatal necrotizing enterocolitis,83 would be expected to have decreased plasma arginine
levels and manifest significant immune incompetence. Alternatively, conditions associated
with significant arginine catabolism could manifest multiorgan dysfunction, as has been
observed in transgenic mice overexpressing arginase I enzyme.84
Several clinical conditions, in humans and animals, have been associated with
increased activities of both arginase enzymes, leading to excessive destruction and
consequent unavailability of L-arginine. Increased arginase activity in the liver has been
implicated in the increased tolerance of the liver to organ rejection.85,86 Trauma is another
1382_C35.fm Page 599 Tuesday, October 7, 2003 7:27 PM

Chapter thirty-five: The use of arginine in clinical practice 599

condition associated with decreased plasma arginine levels, and with greater than 10-fold
increase in arginase activity.87–89 These observations led many investigators to propose the
use of arginine-based nutrients to modify the immunologic and inflammatory responses
in humans. Some investigators have proposed supplementation with as much as 100 g/kg
of formula.90
A reversal of the alteration in T cell function associated with trauma or surgery has
been demonstrated in patients fed enteral diets rich in arginine.91 Patients undergoing
major abdominal operations for gastrointestinal malignancies had increased in vitro
immune responses that correlated with decreased wound infections and decreased length
of hospital stay when supplemented with arginine.75 Additionally, moderately stressed
intensive care unit patients given an enteral diet containing large amounts of arginine
demonstrated preservation or enhancement of T lymphocyte blastogenesis.92 In children
with severe burn injuries, maintenance of normal plasma levels of arginine correlates with
parameters of enhanced host immune and nutritional status.93 Subsequently, the admin-
istration of an enteral diet supplemented with arginine was shown to result in reduced
rates of wound infections and shortened hospital stay in a small group of young burn
patients.94 In a preliminary study, patients infected with HIV showed a significant enhance-
ment of the mitogenic responses to concanavalin A (Con A) and phytohemagglutinin
(PHA) following a 2-week dietary arginine supplementation.95 Whether these effects trans-
late into an improvement in clinical outcomes in critically ill patients remains unclear.

35.3 Arginine and wound healing


Seifter et al.96 were the first to hypothesize a primary role for arginine in wound healing
and hypothesized that the amino acid requirements of the adult injured organism would
revert to those of the growing infant. Based on this postulate, it was demonstrated that
arginine-deficient animals subjected to the minor trauma of a dorsal skin incision and
closure had increased postoperative weight loss, increased mortality, and a notable
decrease in wound breaking strength and wound collagen accumulation compared to
animals fed a similarly defined diet containing arginine (Figure 35.1A). Subsequent exper-
iments revealed that non-arginine-deficient chow-fed rats given a dietary supplement of
1% arginine had enhanced wound healing responses as assessed by wound breaking
strength and collagen synthesis when compared to chow-fed controls (Figure 35.1B).
Similar findings were observed in parenterally fed rats given an amino acid mixture
containing high doses (7.5 g/l) of arginine.97 Likewise, mature or old rats fed diets sup-
plemented with a combination of arginine and glycine had enhanced wound collagen
deposition compared to controls.98
Goodson and Hunt99 described a micromodel that allows for the study of human
fibroblastic responses and wound collagen deposition. In an initial study, 36 young,
healthy human volunteers (ages 25 to 35 years) were randomized into three groups:
(1) 30-g arginine–HCl daily supplements (24.8 g of free arginine); (2) 30 g of arginine
aspartate (17 g of free arginine); and (3) placebo. Arginine supplementation at both doses
significantly increased the amount of hydroxyproline deposition at the wound site
(Figure 35.2).100 The second study evaluated 30 elderly volunteers (age, >70 years) who
were given daily supplements of 30 g of arginine aspartate (17 g of free arginine) or
placebo. Arginine supplementation significantly enhanced wound collagen accumulation
without any effect on wound DNA or total protein content. Arginine supplementation
had no effect on the rate of epithelialization of a superficial skin defect, indicating that
the predominant effect is on wound collagen deposition.101 A more recent study by
Williams et al.102 demonstrated that arginine together with other dietary supplements also
increases human wound collagen deposition (vide infra).
1382_C35.fm Page 600 Tuesday, October 7, 2003 7:27 PM

600 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

A 0.001
4000 Defined diet

+ Arginine
3000

2000
0.001
1000
0.001

FBS FxBS OHP

B Chow
4000 0.001
+Arginine
3000

0.005
2000

1000 0.01

FBS FxBS OHP

Figure 35.1 (A) Arginine-deficient animals had a notable decrease in wound breaking strength and
wound collagen accumulation when compared to animals fed a similarly defined diet containing
arginine. (B) Subsequent experiments revealed that non-arginine-deficient chow-fed rats given a
dietary supplement of 1% arginine had enhanced wound healing responses as assessed by wound
breaking strength and collagen synthesis when compared to chow-fed controls. (FBS = fresh breaking
strength, g; FxBS = formaline-fixed breaking strength, g; OHP = hydroxyproline, mg/100 mg poly
vinyl alcohol sponge.)

Several possible mechanisms have been postulated to explain the effects of arginine
on wound healing. Currently, no single theory can account for the observed effects, but
several have been useful in providing a framework for studies into the mechanisms of
action:

1. Arginine supplementation provides a deficient or necessary substrate for col-


lagen synthesis at the wound site. Although free arginine comprises a very small
amount of the collagen molecule (less than 5%), there could be utilization of
arginine as a substrate for proline through the pathway arginine Æ ornithine Æ
glutamic semialdehyde Æ proline. Arginine levels are essentially nondetectable
within the wound during the later phases of wound healing, when fibroplasia
predominates.103 While ornithine levels are higher in the wound than in the plasma,
tracer isotope studies by Albina et al.103 revealed that the rate of conversion of
ornithine to proline in the wound is actually quite low, making this mechanism of
arginine utilization unlikely.
2. Arginine induces collagen synthesis via a pituitary secretagogue mechanism.
The beneficial effects of supplemental arginine on wound healing are in many respects
similar to those of growth hormone, namely, enhanced wound breaking strength and
collagen deposition.104–106 In support for such a mechanism, it has been noted that
the effect of arginine on wound healing is abrogated in hypophysectomized
1382_C35.fm Page 601 Tuesday, October 7, 2003 7:27 PM

Chapter thirty-five: The use of arginine in clinical practice 601

(nm ol/cm graft; Mean –SEM)


30 p < 0.001

p < 0.028
20

10

Arg Asp

Arg HCl
Control

Figure 35.2 Arginine supplementation at both doses significantly increased the amount of hydroxy-
proline deposition at the wound site.

animals given growth hormone, thyroxine, and testosterone replacement.107 Fur-


ther indirect evidence is provided by data showing that arginine supplementation
to humans in doses that increase wound healing responses also induce elevations
in plasma insulin-like growth factor (IGF), the peripheral mediator of growth
hormone.101
3. Arginine has a unique effect on T cell function. Arginine stimulates T cell re-
sponses and reduces the inhibitory effect of injury and wounding on T cell func-
tion.108–111 T lymphocytes are essential for normal wound healing as evidenced by
decreased wound breaking strength in animals treated with monoclonal antibodies
against T lymphocytes.112,113 T lymphocytes are found immunohistochemically
throughout the various phases of wound healing in distinctive patterns.114 T lym-
phocyte subsets play distinctive roles and accomplish specific tasks during each
phase of healing, thus facilitating normal repair.115
4. Arginine is the unique substrate for NO. Several studies suggest that NO plays
a critical role in wound healing. Exogenous NO administration increases collagen
synthesis in cultured dermal fibroblasts.116 Inhibitors of NO have been shown to
significantly impair healing of cutaneous incisional and colonic anastomotic heal-
ing in rodents.117,118 In models of impaired healing, such as diabetes, NO wound
synthesis is impaired together with collagen accumulation, while administration
of NO restores wound healing responses toward normal.119 Transfection of iNOS
DNA into wounds results in supranormal collagen deposition.120 Conversely, mice
lacking the iNOS gene (iNOS knockout mice) have delayed closure of excisional
wounds, an impairment that is remedied by adenoviral transfer of the iNOS gene
to the wound bed.121 Strongly supporting this mechanism of action are the recent
findings that arginine does not stimulate wound healing in iNOS knockout mice,
suggesting that the iNOS pathway is at least partially responsible for the enhance-
ment of wound healing observed with the administration of arginine.122
1382_C35.fm Page 602 Tuesday, October 7, 2003 7:27 PM

602 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

35.4 Arginine in combination with other nutrients


Expanding on the numerous pharmacological actions of arginine, several clinical studies
have investigated the clinical beneficial effects of arginine combined with other nutrients
in diseases other than trauma and burns. Lysine, glutamine, and b-hydroxy, b-methyl
butyrate (HMB) have all been supplemented with arginine in an attempt to provide
additional benefit or magnify existing effects of the individual nutrients.

35.4.1 Arginine and lysine


Lysine is an essential amino acid and is considered rate limiting for protein synthesis. The
use of lysine and arginine may provide a stimulus for an increase in protein synthesis and
the necessary components for muscle growth. Daily oral administration of 1.2 g of lysine
with an equal amount of arginine to healthy men resulted in increased release of pituitary
somatotropin and insulin; supplementation of each of the amino acids alone did not have
an effect.123 Ingestion of larger amounts of lysine and arginine (6 g each) in older adult
men did not alter the secretion of either growth hormone or IGF-1.124 It should be noted
that the dose of arginine that elicits growth hormone release in humans is much higher
(30 g) and is usually given via the intravenous route over 30 min.125 Taken together, these
findings would suggest that the secretagogue activity of arginine may be more than
adequate in influencing growth hormone levels, and the addition of lysine may only
provide a necessary building block for protein synthesis.

35.4.2 Arginine, glutamine, and b-hydroxy-b-methylbutyrate (HMB)


Studies in vitro have shown additive effects of arginine and glutamine in promoting cell
proliferation by the promotion of nucleotide synthesis.126 Much like arginine, glutamine
has been designated as a conditionally essential amino acid. The biologic and physiologic
effects of glutamine have been well described (see review by Bulus et al.127).
HMB is a metabolite of the amino acid leucine and has been shown to magnify the
exercise-related positive changes in performance and increase muscle mass to a greater
extent than exercise alone.128,129 The mechanism of this effect is thought to be through
decreasing muscle protein breakdown. In addition, extensive clinical studies in animal
models indicate that HMB can nutritionally support the body’s natural immune functions,
which can become weakened during stress. HMB has also been shown to improve blood
lipid profiles, normalize blood pressure, and be safe. The working theory for HMB is that
stressed or damaged cells may not be able to make sufficient hydroxymethylglutaryl
(HMG-CoA) to support adequate membrane cholesterol synthesis. Supplemental HMB
could then be a convenient source of HMG-CoA in these cells to maintain adequate
cholesterol synthesis and support membrane function.130
The combination of arginine, glutamine, and HMB has been reported to increase lean
body mass in individuals who have experienced muscle loss.131,132 In patients with muscle
wasting from established AIDS,131 the oral administration of arginine at 14 g, glutamine
at 14 g, and HMB at 3 g per day resulted in a 3.0-kg increase in body weight, while those
supplemented with a placebo gained 0.37 kg. The gain in body weight with three nutrients
was predominantly fat-free mass compared with the placebo-supplemented group that
lost fat-free mass (Figure 35.3). Furthermore, measures of immune function were also
improved during this study. Patients supplemented with arginine, glutamine, and HMB
had increases in CD3 and CD8 cells and a decrease in the HIV viral load.
In patients with cancer-related cachexia,132 which is caused by accelerated protein
breakdown and slowed protein synthesis, supplementation of the same regimen of
1382_C35.fm Page 603 Tuesday, October 7, 2003 7:27 PM

Chapter thirty-five: The use of arginine in clinical practice 603

3
2,5 *

2
1,5
Fat-Free Mass (kg)

*
1
0,5
0
-0,5
-1
-1,5
-2
AIDS Cancer
Figure 35.3 The effect of arginine, glutamine, and HMB on fat-free mass in AIDS wasting and in
cancer patients. *, Arginine-, glutamine-, and HMB-supplemented groups were significantly different
from the placebo-supplemented groups (p = 0.003 for AIDS patients and p = 0.02 for cancer patients).
The AIDS patients received the supplements for 8 weeks, while the cancer patients received them
for 24 weeks.

arginine, glutamine, and HMB reversed the cachexia process. The primary outcomes
measured in this study were changes in body mass and fat-free mass. The patients sup-
plemented with the three nutrients gained ~1.0 kg of body mass in 4 weeks, whereas
control patients receiving an equimolar, equinitrogenous placebo lost fat-free mass (Figure
35.3). The increase in fat-free mass in both AIDS wasting and cancer cachexia is attributed
to the effects of HMB on slowing rates of protein breakdown and the improvements in
the rates of protein synthesis brought about by arginine and glutamine.
In addition to increasing fat-free mass, the combination of arginine, glutamine, and
HMB was shown to impact wound collagen deposition.102 Older adults underwent sub-
cutaneous implantation of two small, sterile polytetrafluoroethylene (PTFE) tubes. The
tubes allowed growth of fibroblasts and the deposition of matrix, an indicator of wound
healing. The tubes were removed at 7 and 14 days postimplantation and analyzed for
hydroxyproline, an index of collagen accumulation, and a-amino nitrogen, an index of
total protein deposition. Daily supplementation of the same three nutrients at the doses
already identified led to a significant increase in collagen deposition (Figure 35.4) in the
PTFE tubes without an effect on total protein accumulation. It was concluded that collagen
synthesis is significantly increased in healthy elderly volunteers by the oral administration
of the mixture of arginine, glutamine, and HMB.

35.5 Arginine and complete nutritional formulas


In a meta-analysis published between 1990 and 2000, a total of 22 randomized trials (2419
patients) were conducted on critically ill or surgical patients that received some form of
enteral nutrition supplemented (immunonutrition) with a combination of either arginine,
1382_C35.fm Page 604 Tuesday, October 7, 2003 7:27 PM

604 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

80
*
70

Hydroxy -Proline (nmol /cm )


60

50

40

30

20

10

0
Control Arginine,
Glutamine
and HMB

Figure 35.4 Hydroxyproline levels measured in tubes removed after 14 days of implantation. Values
are in nmol/cm of tubing. *, Arginine-, glutamine-, and HMB-supplemented group was significantly
different from the control unsupplemented group (p < 0.03).

glutamine, nucleotides, or omega-3 fatty acids to determine infectious complication and


mortality rates.133,134 The meta-analysis distinguished patients in the intensive care unit,
receiving arginine-rich diets, and scored studies as being of good vs. poor quality. The
results showed that patients receiving commercial formulas with high arginine content
manifested significant reduction in infectious complications compared with other
immune-enhancing diets. The most beneficial effects were seen in surgical patients over
nonsurgical critically ill patients. Indeed, the latter group showed a tendency toward
increased mortality with immunonutrition. The authors concluded that immunonutrition
may decrease infectious complication rates but could be associated with an overall mor-
tality disadvantage.
Arginine possesses numerous pharmacological actions, which can have great potential
benefit in clinical practice. This was the subject of a whole supplement of the Journal of
Parenteral and Enteral Nutrition,135 with many favoring its use, while others issued more
cautionary notes. Although the animal experimental data are compelling, there is need
for continued clinical studies in order to better define the role of arginine in the care of
patients. Since arginine is relatively safe, is well tolerated, and has thus far shown only
mild or no untoward effects after administration of large doses, there is always the danger
that it will be applied indiscriminately in a variety of conditions where it may be ineffec-
tual, at best, or harmful, at worst. The need to define its role and use for wound healing,
for states of diminished immune function, or as part of anticancer therapy is critical. Even
issues such as minimal effective dosage in human clinical practice have not been fully
defined. It is conceivable that in conditions where iNOS activity is markedly increased,
such as in sepsis, the use of arginine-based nutrients, at high doses, could lead to detri-
mental effects. This plea for careful studies is not meant to hinder the application of
arginine in the clinic, but it is meant as a call for judicious and scientific usage. Such
application should result in arginine playing an important role in the nutritional and
pharmacological management of patients.
1382_C35.fm Page 605 Tuesday, October 7, 2003 7:27 PM

Chapter thirty-five: The use of arginine in clinical practice 605

References
1. Young, V.R. and EI-Khoury, A., The notion of the nutritional essentiality of amino acids,
revisited, with a note on the indispensable amino acid requirements in adults, in Amino Acid
Metabolism and Therapy in Health and Disease, Cynober, L.A., Ed., CRC Press, Boca Raton, FL,
1995, pp. 267–308.
2. Laidlaw, S.A. and Kopple, J.D., Newer concepts of the indispensable amino acids, Am. J.
Clin. Nutr., 46, 593–605, 1987.
3. Curran, R.D., Ferrai, F.K., Kispert, P.H., Stadler, J., Stuehr, D.J., Simmons, R.L., and Billiar,
T.R., Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthe-
sis, FASEB J., 5, 2085–2092, 1991.
4. Wilmore, D.W. and Rombeau, J.L., Eds., Glutamine metabolism: nutritional and clinical
significance, J. Nutr., 131, 2447s–2602s, 2001.
5. Meister, A. and Anderson, M.E., Glutathione, Annu. Rev. Biochem., 52, 711–760, 1983.
6. Rose, W.C., The nutritive significance of the amino acids and certain related compounds,
Science, 86, 298–300, 1937.
7. Morris, S.M., Jr., Kepka-Lenhart, D., and Chen, L.C., Differential regulation of arginases and
inducible nitric oxide synthase in murine macrophage cells, Am. J. Physiol., 275, E740–E747,
1998.
8. Morris, S.M., Jr., Regulation of enzymes of urea cycle and arginine metabolism, Annu. Rev.
Nutr., 22, 87–105, 2002.
9. Fahey, J.L., Toxicity and blood ammonia rise resulting from intravenous amino acid admin-
istration in man: the protective effect of L-arginine, J. Clin. Invest., 36, 1647–1655, 1957.
10. Moncada, S. and Higgs, A., The L-arginine-nitric oxide pathway, N. Engl. J. Med., 329,
2002–2012, 1993.
11. Nathan, C., Inducible nitric oxide synthase: what difference does it make? J. Clin. Invest.,
100, 2417–2423, 1997.
12. Stuehr, D.J., Kwon, N.S., Nathan, C.F., Griffith, O.W., Feldman, P.L., and Wiseman, J.,
Nw-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from arginine,
J. Biol. Chem., 266, 6259–6263, 1991.
13. Stuehr, D., Cho, H.J., Kwon, N.S., Weise, M.F., and Nathan, C.F., Purification and character-
ization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-
containing flavoprotein, Proc. Natl. Acad. Sci. U.S.A., 88, 7773–7777, 1991.
14. Stuehr, D.J., Fasehun, O.A., Kwon, N.S., et al., Inhibition of macrophage and endothelial cell
nitric oxide synthase by diphenyleneiodonium and its analogs, FASEB J., 5, 98–103, 1991.
15. Miller, V. and Vanhoutte, P., Endothelium-dependent relaxation in isolated blood vessels of
lower vertebrates, Blood Vessels, 23, 411–425, 1986.
16. Radomski, M.M., Martin, J.F., and Moncada, S., Synthesis of nitric oxide by the haemocytes
of the American horseshoe crab (Limulus polyphemus), Phil. Trans. R. Soc. Lond. B, 334,
129–133, 1991.
17. Sung, Y.J., Hotchkiss, J.H., Auistic, R.E., and Dretert, R.R., L-arginine-dependent production
of a reactive nitrogen intermediate by macrophages of a uricotelic species, J. Leukoc. Biol.,
50, 49–56, 1991.
18. Hevel, J.M., White, K.A., and Marletta, M.A., Purification of the inducible murine macroph-
age nitric oxide synthase, J. Biol. Chem., 266, 22789–22791, 1991.
19. Forstermann, U., Closs, E.I., Pollock, J.S., Nakane, M., Schwartz, P., Gath, I., et al., Nitric
oxide synthase isozymes: characterization, purification, molecular cloning, and functions,
Hypertension, 23, 1121–1131, 1994.
20. Knowles, R.G. and Moncada, S., Nitric oxide synthases in mammals, Biochem. J., 298, 249–258,
1994.
21. Knowles, R.G., Palacios, M., Palmer, R.M.J., and Moncada, S., Formation of nitric oxide from
L-arginine in the central nervous system: a transduction mechanism for stimulation of the
soluble guanylate cyclase, Proc. Natl. Acad. Sci. U.S.A., 86, 5159–5162, 1989.
22. Bredt, D.S. and Snyder, S.H., Isolation of nitric oxide synthetase, a calmodulin-requiring
enzyme, Proc. Natl. Acad. Sci. U.S.A., 87, 682–685, 1990.
1382_C35.fm Page 606 Tuesday, October 7, 2003 7:27 PM

606 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

23. Mayer, B. and Böhme, E., Ca2+-dependent formation of an L-arginine-derived activator of


soluble guanylate cyclase in bovine lung, FEBS Lett., 256, 211–214, 1989.
24. Venturini, C.M., Knowles, R.G., Palmer, R.M.J., and Moncada, S., Synthesis of nitric oxide
in the bovine retina, Biochem. Biophys. Res. Commun., 180, 920–925, 1991.
25. Palacios, M., Knowles, R.G., Palmer, R.M.J., and Moncada, S., Nitric oxide from L-arginine
stimulates the soluble guanylate cyclase in adrenal glands, Biochem. Biophys. Res. Commun.,
165, 802–809, 1989.
26. Radomski, M.W., Palmer, R.M., and Moncada, S., An L-arginine/nitric oxide pathway present
in human platelets regulates aggregation, Proc. Natl. Acad. Sci. U.S.A., 87, 5193–5197, 1990.
27. Palmer, R.M. and Moncada, S., A novel citrulline-forming enzyme implicated in the forma-
tion of nitric oxide by vascular endothelial cells, Biochem. Biophys. Res. Commun., 159, 348–352,
1989.
28. Salter, M., Knowles, R.G., and Moncada, S., Widespread tissue distribution, species distri-
bution and changes in activity of Ca2+-dependent and Ca2+-independent nitric oxide syn-
thases, FEBS Lett., 291, 145–149, 1991.
29. Morris, S.M., Jr., Kepka-Lenhart, D., and Chen, L.C., Differential regulation of arginases and
inducible nitric oxide synthase in murine macrophage cells, Am. J. Physiol., 275, E740–E747,
1998.
30. Iyer, R., Jenkinson, C.P., Vockley, J.G., Kern, R.M., Grody, W.W., and Cederbaum, S., The
human arginases and arginase deficiency, J. Metab. Dis., 21, 86–100, 1998.
31. Gotoh, T., Sonoki, T., Nagasaki, A., Terada, K., Takiguchi, M., and Mori, M., Molecular cloning
of cDNA for nonhepatic mitochondrial arginase (arginase II) and comparison of its induction
with nitric oxide synthase in a murine macrophage-like cell line, FEBS Lett., 395, 119–122,
1995.
32. Boucher, J.L., Moali, C., and Tenu, J.P., Nitric oxide biosynthesis, nitric oxide synthase inhib-
itors and arginase competition of L-arginine utilization, Cell. Mol. Life Sci., 55, 1015–1028, 1999.
33. Mori, M. and Gotoh, T., Regulation of nitric oxide production by arginine metabolic enzymes,
Biochem. Biophys. Res. Commun., 275, 715–719, 2000.
34. Kepka-Lenhart, D., Mistry, S.K., Wu, G., and Morris, S.M., Jr., Arginase I: a limiting factor
for nitric oxide and polyamine synthesis by activated macrophages? Am. J. Physiol., 279,
R2237–R2242, 2000.
35. Li, H., Meininger, C.J., Hawker, J.R., Jr., Kelly, K.A., Morris, S.M., Jr., and Wu, G., Activities
of arginase I and II are limiting for endothelial cell proliferation, Am. J. Physiol., 282, R64–R69,
2002.
36. Singh, R., Pervin, S., Karimi, A., Cederbaum, S., and Chaudhuri, G., Arginase activity in
human breast cancer cell lines: N_hydroxy-L-arginine selectively inhibits cell proliferation
and induces apoptosis in MDA-MB-468 cells, Cancer Res., 60, 3305–3312, 2000.
37. Wei, L.H., Wu, G., Morris, S.M., Jr., and Ignarro, L.J., Elevated arginase I expression in rat
smooth muscle cells increases cell proliferation, Proc. Natl. Acad. Sci. U.S.A., 98, 9260–9264,
2001.
38. Shi, O., Morris, S.M., Jr., Zoghbi, H., Porter, C.W., and O’Brien, W.E., Generation of a mouse
model of arginase II deficiency by targeted disruption of the arginase II gene, Mol. Cell. Biol.,
21, 811–813, 2001.
39. Visek, W.J., Arginine needs physiological state and usual diets: a reevaluation, J. Nutr., 116,
36–46, 1986.
40. Eagle, H., Amino acid metabolism in mammalian cell cultures, Science, 130, 432–437, 1959.
41. Hecker, M., Sessa, W.C., Harris, H.J., Anggard, E.E., and Vane, J.R., The metabolism of
L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor:
cultured endothelial cells recycle L-citrulline to L-arginine, Proc. Natl. Acad. Sci. U.S.A., 87,
8612–8616, 1990.
42. Nussler, A.K., Billiar, T.R., Liu, Z.Z., and Morris, S.M., Jr., Coinduction of nitric oxide synthase
and arginosuccinate synthetase in a murine macrophage cell line: implications for regulation
of nitric oxide production, J. Biol. Chem., 269, 1257–1261, 1994.
43. Thornton, F.J., Schaffer, M.R., and Barbul, A., Wound healing and thymotropic effects of
arginine: a pituitary mechanism of action, Am. J. Clin. Nutr., 37, 786–794, 1997.
1382_C35.fm Page 607 Tuesday, October 7, 2003 7:27 PM

Chapter thirty-five: The use of arginine in clinical practice 607

44. Castillo, L., Beaumier, L., Ajami, A.M., and Young, V.R., Whole body nitric oxide synthesis
in healthy men determined from [15N]arginine-to-[15N]citrulline labeling, Proc. Natl. Acad.
Sci. U.S.A., 93, 11460–11465, 1996.
45. Forte, P., Smith, L.M., Milne, E., and Benjamin, N., Measurement of nitric oxide synthesis in
humans using L-[15N2]arginine, Meth. Enzymol., 301, 92–98, 1999.
46. De Jonge, W.J. et al., Overexpression of arginase I alters circulating and tissue amino acids
and guanidine compounds and affects neuromotor behavior in mice, J. Nutr., 131, 2732–2740,
1994.
47. Castillo, L., Chapman, T.E., Yu, Y.M., et al., Dietary arginine uptake by the splanchnic region
in adult humans, Am. J. Physiol. Endocrinol. Metab., 265, E532–E539, 1993.
48. Castillo, L., deRojas, T.C., Chapman, T.E., et al., Splanchnic metabolism of dietary arginine
in relation to nitric oxide synthesis in normal adult man, Proc. Natl. Acad. Sci. U.S.A., 90,
193–197, 1993.
49. Castillo, L., DeRojas-Walker, T., Yu, Y.M., et al., Whole body arginine metabolism and nitric
oxide synthesis in newborns with persistent pulmonary hypertension, Pediatr. Res., 38, 17–24,
1995.
50. Castillo, L., Sanchez, M., Vogt, J., et al., Plasma arginine, citrulline, and ornithine kinetics in
adults, with observations on nitric oxide synthesis, Am. J. Physiol., 268, E360–E367, 1995.
51. Forte, P., Dykhuizen, R.S., Milne, E., et al., Nitric oxide synthesis in patients with infective
gastroenteritis, Gut, 45, 355–361, 1999.
52. Hallemeesch, M.M., Soeters, P.B., and Deutz, N.E., Renal arginine and protein synthesis are
increased during early endotoxemia in mice, Am. J. Physiol. Renal Physiol., 282, F316–F323,
2002.
53. Lau, T., Owen, W., Yu, Y.M., et al., Arginine, citrulline, and nitric oxide metabolism in end-
stage renal disease patients, J. Clin. Invest., 105, 1217–1225, 2000.
54. Macallan, D.C., Smith, L.M., Ferber, J., et al., Measurement of NO synthesis in humans by
L-[15N2]arginine: application to the response to vaccination, Am. J. Physiol., 272, R1888–R1896,
1997.
55. Santak, B., Radermacher, P., Iber, T., et al., In vivo quantification of endotoxin-induced nitric
oxide production in pigs from Na15NO3-infusion, Br. J. Pharmacol., 122, 1605–1610, 1997.
56. Wever, R., Boer, P., Hijmering, M., et al., Nitric oxide production is reduced in patients with
chronic renal failure, Arterioscler. Thromb. Vasc. Biol., 19, 1168–1172, 1999.
57. Luiking, Y.C. and Deutz, N.E., Isotopic investigation of nitric oxide metabolism in disease,
Curr. Opin. Clin. Nutr. Metab. Care, 6, 103–108, 2003.
58. Simmon, W.W., Closs, E.J., Cunningham, J.M., Smith, T.W., and Kelly, R.A., Cytokines and
insulin induce cationic transporter (CAT) expression in cardiac myocytes, J. Biol. Chem., 271,
11694–11702, 1996.
59. McDonald, K.K., Zharikov, S., Block, E.R., and Kilberg, M.S., A caveolar complex between
the cationic amino acid transporter 1 and endothelial nitric-oxide synthase may explain the
“arginine paradox,” J. Biol. Chem., 272, 31213–31216, 1997.
60. Rodriguez, P.C. et al., Regulation of T cell receptor CD3– chain expression by L-arginine,
J. Biol. Chem., 227, 21123–21129, 2002.
61. Brittenden, J., Heys, S.D., Ros, J., et al., Nutritional pharmacology: effects of L-arginine on
host defenses, response to trauma and tumor growth, Clin. Sci., 86, 123–132, 1994.
62. Efron, D., Kirk, S.J., Regan, M.C., Wasserkrug, H.L., and Barbul, A., Nitric oxide generation
from L-arginine is required for optimal peripheral blood lymphocyte DNA synthesis, Surgery,
110, 327–334, 1991.
63. Moriguchi, A., Mukai, K., Hiroaka, I., and Kishino, Y., Functional changes in human lym-
phocytes and monocytes after in vitro incubation with arginine, Nutr. Res., 7, 719–729, 1987.
64. Minami, Y., Weissman, A.M., Samelson, L.E., and Klausner, R.D., Building a multichain
receptor: synthesis, degradation, and assembly of the T-cell antigen receptor, Proc. Natl. Acad.
Sci. U.S.A., 84, 2688–2692, 1987.
65. Valitutti, S., Muller, S., Salio, M., and Lazavecchia, A., Degradation of T cell receptor (TCR)-
CD3-zeta complexes after antigenic stimulation, J. Exp. Med., 185, 1859–1864, 1997.
1382_C35.fm Page 608 Tuesday, October 7, 2003 7:27 PM

608 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

66. Bronstein-Sitton, N., Wang, L., Cohen, L., and Baniyash, M., Expression of the T cell antigen
receptor zeta chain following activation is controlled at distinct checkpoints: implications
for cell surface receptor down-modulation and re-expression, J. Biol. Chem., 274, 23659–23665,
1999.
67. Crawford, D.H., Chen, S., and Boyd, C.A., Cationic amino acid transport in human T lym-
phocytes is markedly increased in the CD45RA CD8+ population after activation, Immunology,
82, 357–600, 1994.
68. Ochoa, J.B., Strange, J., Kearney, P., Gellin, G., Endean, E., and Fitzpatrick, E., Effects of
L-arginine on the proliferation of T-lymphocyte subpopulation, J. Parenter. Enteral Nutr., 25,
23–29, 2001.
69. Wu, G. and Morris, S.M., Jr., Arginine metabolism: nitric oxide and beyond, Biochem. J., 336,
1–17, 1998.
70. Munder, M., Eichmann, K. and Modolell, M., Alternative metabolic states in murine mac-
rophages reflected by the nitric oxide synthase/arginase balance: competitive regulation by
CD4+ T cells correlates with Th1/Th2 phenotype, J. Immunol., 160, 5347–5354, 1998.
71. Bernard, A.C., Mistry, S.K., Morris, S.M., Jr., O’Brien, W.E., Tsuei, B.J., Maley, M.E., Shirly,
L.A., Kearny, P.A., Boulanger, B.R., and Ochoa, J.B., Alterations in arginine metabolic en-
zymes in trauma, Shock, 15, 215–219, 2001.
72. Caraway, M.S., Piantadosi, C.A., Jenkinson, C.P., and Huang, Y.C., Differential expression of
arginase and iNOS in the lung in sepsis, Exp. Lung Res., 24, 253–268, 1998.
73. Ikemoto, M., Tsunekawa, S., Tanaka, K., Tanaka, A., Yamaoka, Y., Ozawa, K., Fukuda, Y.,
Moriyasu, F., Totani, M., Kasa, Y., Mori, T., and Ueda, K., Liver-type arginase in serum during
and after liver transplantation: a novel index in monitoring conditions of the liver graft and
its clinical significance, Clin. Chim. Acta, 271, 11–23, 1998.
74. Langle, F., Steininger, R., Roth, R., Winkler, S., Andel, H., Acimovic, S., Fugger, R., and
Mulbacher, F., L-arginine deficiency and hemodynamic changes as a result of arginase efflux
following orthotopic liver transplantation, Transplant. Proc., 27, 2872–2873, 1995.
75. Kirk, S.J., Regan, M.C., Wasserkrug, H.L., Sodeyama, M., and Barbul, A., Arginine enhances
T cell responses in athymic nude mice, J. Parenter. Enteral Nutr., 16, 429–432, 1992.
76. Bernstein, L.H., The impact of immunonutrition, Gastroenterology, 110, 1677–1678, 1996.
77. De Jonge, W.J. et al., Overexpression of arginase I in enterocytes of transgenic mice elicit a
selective arginine deficiency and affects skin, muscle, and lymphoid development, Am. J.
Nutr., 76, 128–140, 2002.
78. De Jonge, W.J., Kwikkers, K.L., te Velde, A.A., van Deventer, S.J.H., Nolte, M.A., Mebius,
R.E., Ruijter, J.M., Lamers, M.C., and Lamers, W.H., Arginine deficiency affects early B cell
maturation and lymphoid organ development in transgenic mice, J. Clin. Invest., 110,
1539–1548, 2002.
79. Fafournoux, P., Bruhat, A., and Jousse, C., Amino acid regulation of gene expression, Biochem.
J., 351, 1–12, 2000.
80. LeBien, T.W., Arginine: an unusual dietary requirement of pre-B lymphocytes? J. Clin. Invest.,
110, 1411–1413, 2002.
81. Davis, T.A., Fiorotto, M.L., and Reeds, P.J., Amino acid composition of body and milk protein
change during the suckling period in rats, J. Nutr., 123, 947–956, 1993.
82. Herzfeld, A. and Raper, S.M., Enzymes of ornithine metabolism in adult and developing rat
intestine, Biochim. Biophys. Acta, 428, 600–610, 1976.
83. Zamora, S.A. et al., Plasma arginine concentration in premature infants with necrotizing
enterocolitis, J. Pediatr., 131, 226–232, 1997.
84. De Jonge, W.J. et al., Overexpression of arginase I alters circulating and tissue amino acids
and guanidine compounds and affects neuromotor behavior in mice, J. Nutr., 131, 2732–2740,
1994.
85. Callery, M.P., Mangino, M.J., and Fly, M.W., Arginine-specific suppression of mixed lympho-
cyte culture reactivity by Kupffer cells: a basis of portal venous tolerance, Transplantation,
51, 1076–1080, 1991.
86. Schrempf-Decker, G.E., Baron, D.P., Brattige, N.W. et al., Biological and immunological
characterization of a human liver immuno-regulatory protein, Hepatology, 3, 939–946, 1983.
1382_C35.fm Page 609 Tuesday, October 7, 2003 7:27 PM

Chapter thirty-five: The use of arginine in clinical practice 609

87. Bernard, A., Griffen, M.M., Rockich, A., et al., Trauma increases arginase activity in human
peripheral leucocytes, Surg. Forum, 50, 266–269, 1999.
88. Figert, P., Wray, C., Maley, M., et al., Induced nitric oxide synthesis is modulated by increased
arginase activity after trauma, Surg. Forum, 49, 58–59, 1998.
89. Ochoa, J.B., Udekwo, A.O., Billiar, T.R., et al., Nitrogen oxide levels in patients after trauma
and during sepsis, Ann. Surg., 214, 621–626, 1991.
90. Alexander, J.W., Ogle, C.K., and Nelson, J.L., Diets and infection: composition and conse-
quences, World J. Surg., 22, 209–212, 1998.
91. Daly, J., Reynolds, J., Thom, A., et al., Immune and metabolic effects of arginine in the surgical
patient, Ann. Surg., 208, 512–523, 1988.
92. Barbul, A., Rettura, G., Levenson, S.M., Seifter, E., Thymotropic actions of arginine, ornithine
and growth hormone, Fed. Proc., 37, 264, 1978 (abstract 282).
93. Kirk, S.J., Regan, M.C., Wasserkrug, H.L., and Barbul, A., Inhibition of prolactin secretion
reduces the T cell immunostimulatory effects of arginine, Surg. Forum, 42, 3–5, 1991.
94. Kiess, W. and Butenandt, O., Specific growth hormone receptors on human peripheral
mononuclear cells: reexpression, identification, and characterization, J. Clin. Endocrinol.
Metab., 60, 740–746, 1985.
95. Davila, D.R., Breif, S., Simon, J., et al., Role of growth hormone in regulating T-dependent
immune events in aged, nude, and transgenic rodents, J. Neurosci. Res., 18, 108–116, 1987.
96. Seifter, E., Rettura, G., Barbul, A., and Levenson, S.M., Arginine: an essential amino acid for
injured rats, Surgery, 84, 224–230, 1978.
97. Barbul, A., Fishel, R.S., Shimazu, S., Wasserkrug, H.L., Yoshimura, N.N., and Efron, G.,
Intravenous hyperalimentation with high arginine levels improves wound healing and im-
mune function, J. Surg. Res., 38, 328–334, 1985.
98. Chyun, J. and Griminger, P., Improvement of nitrogen retention by arginine and glycine
supplementation and its relation to collagen synthesis, J. Nutr., 114, 1697–1704, 1984.
99. Goodson, W.H. and Hunt, T.K., Development of a new miniature method for the study of
wound healing in human subjects, J. Surg. Res., 33, 394–401, 1982.
100. Barbul, A., Lazarou, S., Efron, D.T., Wasserkrug, H.L., and Efron, G., Arginine enhances
wound healing in humans, Surgery, 108, 331–337, 1990.
101. Kirk, S.J., Hurson, M., Regan, M.C., Holt, D.R., Wasserkrug, H.L., and Barbul, A., Arginine
stimulates wound healing and immune function in aged humans, Surgery, 114, 155–160, 1993.
102. Williams, J.Z., Abumrad, N., and Barbul, A., Effect of a specialized amino acid mixture on
human collagen deposition, Ann. Surg., 236, 369–375, 2002.
103. Albina, J.E., Mills, C.D., Barbul, A., et al., Arginine metabolism in wounds, Am. J. Phys., 254,
E459–E467, 1988.
104. Herndon, D.N., Barrow, R.E., Kunkel, K.R., et al., Effects of recombinant human growth
hormone on donor site healing in severely burned children, Ann. Surg., 212, 424–429, 1990.
105. Jorgensen, P.H. and Andreassen, T.T., Influence of biosynthetic human growth hormone on
biochemical properties of rat skin incisional wounds, Acta Chir. Scand., 154, 623–626, 1988.
106. Kowalewski, K. and Yong, S., Effect of growth hormone an anabolic steroid on hydroxypro-
line in healing dermal wounds in rats, Acta Endocrinol., 59, 53–66, 1968.
107. Barbul, A., Rettura, G., Levenson, S.M., and Seifter, E., Wound healing and thymotropic
effects of arginine: a pituitary mechanism of action, Am. J. Clin. Nutr., 37, 786–794, 1983.
108. Barbul, A., Rettura, G., Levenson, S.M., and Seifter, E., Arginine: a thymotropic and wound
healing promoting agent, Surg. Forum, 28, 101–103, 1977.
109. Barbul, A., Wasserkrug, H.L., Seifter, E., et al., Immunostimulatory effects of arginine in
normal and injured rats, J. Surg. Res., 29, 228–235, 1980.
110. Barbul, A., Wasserkrug, H.L., Sisto, D.A., et al., Thymic and immune stimulatory actions of
arginine, J. Parenter. Enteral Nutr., 4, 446–449, 1980.
111. Fabris, N. and Mocchegiani, E., Arginine-containing compounds and thymic endocrine ac-
tivity, Thymus, 19, S21–S30, 1992.
1382_C35.fm Page 610 Tuesday, October 7, 2003 7:27 PM

610 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

112. Barbul, A., Role of T cell-dependent immune system in wound healing, in Growth Factors
and Other Aspects of Wound Healing: Biological and Clinical Implications, Progress in Clinical
and Biological Research, Vol. 266, Barbul, A., Pines, E., Caldwell, M.D., and Hunt, T.K., Eds.,
Alan R. Liss Publishers, New York, 1988, pp. 161–175.
113. Peterson, J.M., Barbul, A., Breslin, R.J., et al., Significance of T lymphocytes in wound healing,
Surgery, 102, 300–305, 1987.
114. Fishel, R.S., Barbul, A., Beschorner, W.E., et al., Lymphocyte participation in wound healing:
morphologic assessment using monoclonal antibodies, Ann. Surg., 206, 25–29, 1987.
115. Agaiby, A.D. and Dyson, M., Immuno-inflammatory cell dynamics during cutaneous wound
healing, J. Anat., 195, 531–542, 1999.
116. Schaffer, M.R. et al., Nitric oxide, an autocrine regulator of wound fibroblast synthetic
function, J. Immunol., 158, 2375–2381, 1997.
117. Schaffer, M.R. et al., Inhibition of nitric oxide synthesis in wounds: pharmacology and effect
on accumulation of collagen in wounds in mice, Eur. J. Surg., 165, 262–267, 1999.
118. Efron, D.T. et al., Expression and function of inducible nitric oxide synthase during rat colon
anastomotic healing, J. Gastrointest. Surg., 3, 592–601, 1999.
119. Witte, M.B., Thornton, F.J., Kiyama, T., Tantry, U., and Barbul, A., Nitric oxide enhances
wound collagen deposition in diabetic rats, Surg. Forum, 48, 665–667, 1997.
120. Thornton, F.J., Schaffer, M.R., et al., Enhanced collagen accumulation following direct trans-
fection of the inducible nitric oxide synthase gene in cutaneous wounds, Biochem. Biophys.
Res. Commun., 246, 654–659, 1998.
121. Yamasaki, K., Edington, H.D., et al., Reversal of impaired wounds repair in iNOS deficient
mice by topical adenoviral-mediated iNOS gene transfer, J. Clin. Invest., 101, 967–971, 1998.
122. Shi, H.P., Efron, D.T., Tantry, U., and Barbul, A., Supplemental dietary arginine enhances
wound healing in normal but not in inducible nitric oxide synthase knockout mice, Surgery,
128, 374–378, 2000.
123. Isidori, A., Monaco, A.L., and Cappa, M., A study of growth hormone release in man after
oral administration of amino acids, Curr. Med. Res. Opin., 7, 475–481, 1981.
124. Corpas, E., Blackman, M.R., Roberson, R., Scholfied, D., and Harman, S.M., Oral arginine-
lysine does not increase growth hormone or insulin-like growth factor-I in old men,
J. Gerontol., 48, M128–M133, 1993.
125. Barbul, A., Arginine, biochemistry, physiology and therapeutic implications, J. Parenter. En-
teral Nutr., 10, 227–238, 1986.
126. Yamauchi, K., Komatsu, T., Kularni, A.D., Ohmori, Y., Minami, H., Ushiyama, Y., Nakayama,
M., and Yamamoto, S., Glutamine and arginine affect Caco-2 cell proliferation by promotion
of nucleotide synthesis, Nutrition, 18, 329–333, 2002.
127. Bulus, N., Cersosimo, E., Ghishan, F., and Abumrad, N.N., Physiologic importance of
glutamine, Metab. Clin. Exp., 38, 1–5, 1989.
128. Panton, L.B., Rathmacher, J.A., Baier, S., and Nissen, S., Nutritional supplementation of the
leucine metabolite b-hydroxy, b-methyl butyrate (HMB) during resistance training, Nutrition,
16, 734–739, 2000.
129. Nissen, S., Sharp, R., Ray, M., Rathmacher, J.A., Rice, J., Fuller, J.C., Jr., Connely, A.S., and
Abumrad, N.N., The effect of leucine metabolite b-hydroxy, b-methyl butyrate on muscle
metabolism during resistance-exercise training, J. Appl. Physiol., 81, 2095–2104, 1996.
130. Nissen, S. and Abumrad, N.N., Nutritional role of the leucine metabolite hydroxy-methyl-
butyrate (HMB) in stress, Nutr. Biochem., 8, 300–311, 1997.
131. Clark, R.H., Feleke, G., Din, M., Yasmin, T., Singh, G., Khan, F., and Rathmacher, J.A.,
Nutritional treatment for acquired immunodeficiency virus-associated wasting using b-hy-
droxy, b-methyl butyrate, glutamine and arginine: a randomized double blind, placebo-
controlled study, J. Parenter. Enteral Nutr., 24, 133–139, 2000.
132. May, P.E., Barber, A., D’Olimpio, J.T., Hourihane, A., and Abumrad, N.N., Reversal of cancer-
related wasting using oral supplementation with a combination of beta-hydroxy-beta-
methylbutyrate, arginine, and glutamine, Am. J. Surg., 183, 471–479, 2002.
133. Heyland, D.K., Novak, F., Drover, J.W., et al., Should immunonutrition become routine in
critically ill patients? JAMA, 286, 22–29, 2001.
1382_C35.fm Page 611 Tuesday, October 7, 2003 7:27 PM

Chapter thirty-five: The use of arginine in clinical practice 611

134. Heyland, D.K. and Novak, F., Immunonutrition in the critically ill patients: more harm than
good? J. Parenter. Enteral Nutr., 25 (Suppl.), S51–S55, 2001.
135. Journal of Parenteral and Enteral Nutrition, Vol. 25 (Suppl.), 2001.
1382_C35.fm Page 612 Tuesday, October 7, 2003 7:27 PM
1382_C36.fm Page 613 Tuesday, October 7, 2003 7:29 PM

chapter thirty-six

Glutamine and glutamine-


containing dipeptides
Peter Fürst
University of Bonn
Peter Stehle
University of Bonn

Contents
Introduction..................................................................................................................................613
36.1 Glutamine: the major intracellular amino acid constituent.........................................614
36.1.1 The history of glutamine ....................................................................................614
36.1.2 The intracellular glutamine pool in man.........................................................615
36.2 The physiologic functions of glutamine in health and disease ...................................616
36.3 Provision of glutamine:free amino acid .........................................................................617
36.3.1 Stability of free glutamine: a critical issue ......................................................617
36.3.2 Use of parenteral glutamine: clinical studies..................................................617
36.4 Provision of glutamine: the dipeptide concept .............................................................619
36.4.1 Animal studies .....................................................................................................619
36.4.2 Implications in healty volunteers .....................................................................620
36.4.3 Clinical studies .....................................................................................................620
36.5 Glutamine nutrition: mechanisms, outcome, and cost–benefit calculations.............624
36.6 Future perspectives ...........................................................................................................624
References .....................................................................................................................................625

Introduction
The general approach to the nutritional care of the catabolic, malnourished, or critically
ill patient involves delivery of a balanced diet, including an adequate amount of protein
or suitable amino acid preparation.1,2 The present direction of clinical nutrition considers
two main approaches: (1) Provision of tailor-made formulas assumes increased benefits
for specific patient groups like children and renal and liver disease patients. These kinds
of formulas have been designed to improve tolerance of nitrogen load rather than to
provide an appropriate composition for individual organs or tissues. (2) An important

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 613
1382_C36.fm Page 614 Tuesday, October 7, 2003 7:29 PM

614 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

alternative proposal is therefore the consideration of individual amino acids as tissue- or


organ-specific single nutrients. Accordingly, certain classic nonessential amino acids like
histidine, serine, arginine, tyrosine, cyst(e)ine, and especially glutamine have to be recon-
sidered as conditionally indispensable substrates.
With the recognition that during episodes of catabolic stress intracellular glutamine
depletion occurs,3 various motions and proposals suggesting modalities of glutamine
nutrition received revitalized interest.4 It has been convincingly demonstrated that sup-
plementation with glutamine or glutamine dipeptides improves nitrogen balance,
enhances the rate of protein synthesis, supports immune cells, maintains integrity of the
mucosa, and reduces morbidity.5 It is also postulated that provision of glutamine prevents
translocation of bacteria and toxins in the intestinal tract to the general circulation. Finally,
in a recent meta-analysis, evidence has been brought forward that parenteral glutamine
supplementation is associated with significantly reduced mortality.6
This chapter briefly reviews the remarkable portrayal of the amino acid glutamine as
a nutritional substrate, with emphasis upon its particular physiologic role. The potential
use of glutamine and glutamine-containing dipeptides in experimental and clinical set-
tings will be recapitulated, and the obtained results will be critically scrutinized. Finally,
the prospective importance of the developing field of glutamine nutrition, especially
concerning practicability and the need of future studies, will be discussed.

36.1 Glutamine: the major intracellular amino acid constituent


36.1.1 The history of glutamine
In a letter to the editor of the Journal für praktische Chemie, Ritthausen reported in 1866
about a newly discovered substance7 found in the proteins from lupin and almond
(conglutin):

The new body which is formed during boiling of gluten in the presence of
sulfuric acid, apart from tyrosine and leucine, is a monobasic nitrogen contain-
ing acid; its formula after analysis of the free acid being C10H6NO8. I name it
glutamic acid by considering the material it has been gained of.

Some years later Hlasiwetz and Habermann8 observed that during decomposition of
casein not only the typical amino acids like glutamic acid, aspartic acid, leucine, and
tyrosine were formed, but also ammonia was liberated: “this nitrogen must correspond
to NH2 groups of substances like asparagine and glutamine released as ammonia during
the formation of aspartic acid and glutamic acid.”
The first proper description of glutamine was given by Schulze and Bosshard9 in their
historical paper “Über das Glutamin” in 1883: “beetroots contain an amide of glutamic
acid which decomposes during heating in the presence of acid to ammonia and glutamic
acid. It is conceivable to assume that this body in question is homologues with asparagine
— thus it would be glutamine = C5H10N2O3.” Indeed, the results of this important paper
enabled subsequent substrate isolation and were followed by numerous reports dealing
with analyses and chemical characterization of glutamine.
The first hint about the presence and function of glutamine in the human body came
from Thierfelder and Sherwin in 1914.10 Twenty years later Krebs and coworkers succeeded
with in vitro glutamine synthesis in an incubation model employing glutamic acid and
ammonium ions with liver specimens.11 Simultaneously, the enzyme glutaminase catalyz-
ing deamidation of glutamine could be isolated from liver, kidney, retina, and brain tissues.
These findings were very soon followed by the definition of the amide–nitrogen cycle and
1382_C36.fm Page 615 Tuesday, October 7, 2003 7:29 PM

Chapter thirty-six: Glutamine and glutamine-containing dipeptides 615

the conclusion that glutamine serves as an ammonia donor and glutamic acid as an
ammonia acceptor.12
Free amino acids were found in all body fluids and tissues. The central position of
intracellular free amino acids in protein metabolism was established already in 1912 by
van Slyke and Meyer.13 They showed that amino acids are concentrated within tissues
prior to being metabolized. Various analytical procedures (enzymatic, microbiologic, col-
orimetric) have been employed to assess the content of glutamine in different biological
fluids. The introduction of an automated ion exchange chromatographic method enabled
a flood of work relative to occurrence and distribution of glutamine in animal organisms
and human tissues (for references cf. Benson et al.14). In the 1950s, Eagle and co-workers15
found that glutamine is an essential substrate to support dividing cells in culture. More
than 35 years ago, Munro16 extensively reviewed the available literature concerning free
amino acid pools. In animal experiments the concentration of several free amino acids
was found to be considerably higher in cells than in extracellular fluid, free glutamine
being the major amino acid constituent in many tissues of the various species investigated.

36.1.2 The intracellular glutamine pool in man


The first reliable data on intracellular concentrations of free amino acids in human muscle
tissue were reported in 1974.17 As skeletal muscle contains the largest pool of intracellular
free amino acids, it is of interest to estimate the size of this pool. Direct determinations in
man showed that 1 kg of skeletal muscle contains 230 g of dry solids, 120 g of extracellular
water, and 650 g of intracellular water. The total free amino acid concentration in muscle
was found to be approximately 35 mmol/L of intracellular water; the free glutamine is
present at a concentration of 19.5 mmol/L. Taurine is additionally present at a concentra-
tion of 15 mmol/L and free carnosine at 6 mmol/L. Of the total pool, the eight essential
amino acids represent only 8.4%. For a normal man with a body weight of 70 kg and a
muscle mass of 400 g/kg of body weight, the total volume of intracellular muscle water
is 18.2 L; thus, the total intracellular amino acid content can be estimated to be 86.5 g, of
which 51.8 g is glutamine. This share corresponds to 60% of the total estimated intracellular
amino acid pool.17
The transmembrane gradient over the muscle cell membrane is high for glutamine
(33.8 ± 11.8). The existence of this marked concentration gradient requires that free diffu-
sion through the muscle–fiber membrane must be restricted. It is known that glutamine
is actively transported into cells by a Na+-dependent system, which requires the expend-
iture of metabolic energy.18 Individual glutamine transporters are operating on functional
characteristics like substrate specificity, ion dependence, and regulatory properties. Within
the past 3 years, several genes encoding for proteins with these defined activities (termed
systems) have been isolated from human and rodent cDNA libraries and found to be
distributed among four distinct gene families (see Chapter 4 for details). Most importantly,
these newly isolated transporter genes provide the long-awaited tools necessary to study
their molecular regulation during the catabolic states, in which glutamine is considered
to be conditionally indispensable.
Thus, the steady-state concentration observed in intracellular fluid and the concen-
tration gradient across the cell membrane should be the combined effect of the affinity to
the transport system, the influence of other amino acids competing for carrier molecules,
the intracellular rate of production and utilization, the extracellular supply, and the leakage
rate across the cell membrane. Nevertheless, the fact that the tissue-specific intracellular
glutamine concentration is reproducible from one individual to another suggests that the
concentration of glutamine in the cell is precisely regulated by biophysical and biochemical
mechanisms.17
1382_C36.fm Page 616 Tuesday, October 7, 2003 7:29 PM

616 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Free glutamine differs widely in the size of its tissue pools and there are distinct
species differences.16 An examination of the nonessential pools of muscle and liver in rat
and human reveals that glutamine, glutamic acid, alanine, and glycine represent the bulk
of the intracellular free amino acids. It might be pertinent to point out that the plasma
nonessential pool is very similar in rat and man; about 60% of these pools comprise the
amino acids glutamine, alanine, and glycine, while glutamic acid is practically absent. In
contrast, the content of glutamine in human muscle is higher than the contents of all the
other amino acids combined, whereas in rat muscle, glycine is the major intracellular
amino acid constituent. An entirely different pattern can be observed in the liver tissue.
In the rat, once again, glycine appears to be present with the highest concentration. In
human liver, in contrast to muscle tissue, glutamic acid, not glutamine, is the principal
nonessential amino acid constituent, whereas glutamine, alanine, and glycine are more
evenly allocated.19
It can be concluded that, first, glutamine concentrations diverge distinctly in various
tissue pools. Second, there are considerable species differences in tissue free glutamine
concentrations. While it is frequently possible to make qualitative conjectures about human
metabolism acquired from animal studies, a quantitative understanding may require direct
measurements of glutamine in man. Third, plasma contains only a very small proportion
of the free glutamine pool. Therefore, plasma concentrations do not necessarily reflect
intracellular concentrations and are not representative for possible changes occurring in
the free amino acid pool as a whole.

36.2 The physiologic functions of glutamine in health and disease


Glutamine not only acts as a precursor for protein synthesis but also is an important
intermediate in a large number of metabolic pathways.20 It is a precursor that donates
nitrogen for the synthesis of purines, pyrimidines, nucleotides, and amino sugars.
Glutamine is the most important substrate for renal ammoniagenesis and thus takes part
in the regulation of the acid–base balance. As the highest concentrated amino acid in the
bloodstream, glutamine serves as a nitrogen transporter between various tissues. Due to
its diverse participation in transamination reactions, glutamine can be classified as a true
regulator of amino acid homeostasis.20–22
It is well known that glutamine represents an important metabolic fuel for the cells
of the gastrointestinal tract (enterocytes, colonocytes).4,23 Recently, evidence has been
brought forward that rapidly proliferating cells, mainly those of the immune system,
strictly depend on the availability of glutamine as an energy (carbon, nitrogen) source.24
There are interesting data showing that despite the extensive metabolism of glutamine
by the intestine, intestinal tissue is not compellingly related to its intermediary metabo-
lism.25 In fact, it is claimed that glutamate and proline, especially derived from the diet,
can readily substitute for many of the metabolic roles of glutamine, including energy
generation and amino acid synthesis.26,27 On the other hand, mucosal cells not only utilize
extracellular glutamine but also synthesize it. Given that inhibition of glutamine synthesis
impairs both proliferation and differentiation of mucosal cell cultures, it seems that
glutamine may be performing some more subtle regulatory functions. This notion is
supported by the demonstration that glutamine will activate a number of genes associated
with cell cycle progression in the mucosal cells.25,28
Interestingly, in vivo nuclear magnetic resonance (NMR) studies support a tight linkage
between glucose and glutamate neurotransmitter metabolism in the cerebral cortex. Stud-
ies have shown that the glutamate–glutamine cycle, in which neuronal glutamate is
released by neurotransmission from the nerve terminal and taken up and converted to
glutamine by the astrocyte, is a major pathway of glutamate and glutamine metabolism.29
1382_C36.fm Page 617 Tuesday, October 7, 2003 7:29 PM

Chapter thirty-six: Glutamine and glutamine-containing dipeptides 617

There are numerous data available that hypercatabolic and hypermetabolic situations
are accompanied with a glutamine deprivation. During prolonged starvation30 and after
elective operations, major injury, burns, infections,3 and pancreatitis,31 intramuscular
glutamine concentrations declined considerably irrespective of nutritional efforts. This
reduction of the muscle free glutamine pool (about 50% of normal) can be seen as a typical
feature of injury and malnutrition, extent and duration of the depletion being proportional
to the severity of the illness.20 Recent studies underlined that the glutamine deprivation
is mainly caused by trauma-induced alterations in the interorgan glutamine flow.4 Muscle
and, as postulated, lung glutamine efflux are accelerated to provide substrate for the gut,
immune cells, and the kidneys,4,32 explaining the profound decline in muscle free
glutamine concentration.
Two early observations suggest that glutamine is involved in the regulation of muscle
protein balance: the striking direct correlation between muscle glutamine and the rate of
protein synthesis, and the positive effect of maintaining intracellular glutamine content
on protein anabolic processes in vitro.33,34 If maintenance of the intracellular glutamine
pool promotes conservation of muscle protein, there is a theoretical case for glutamine
supplements in the frame of clinical nutrition in stressed and malnourished patients.
Numerous studies in experimental animals support this notion. Glutamine-supple-
mented enteral or parenteral nutrition was associated with increased intestinal mucosal
thickness, DNA, and protein content35 ; reduced bacterial translocation following
radiation36; weakened adverse effects of experimentally induced enterocolitis37; preserved
intestinal mucosa during parenteral nutrition38–42; enhanced rat mucosal hyperplasia after
small bowel resection43; and improved glutamine metabolism in the small bowel of septic
rats.44 In vitro glutamine promoted protein sythesis in isolated intestinal epithelial cells.45

36.3 Provision of glutamine:free amino acid


36.3.1 Stability of free glutamine: a critical issue
Two unfavorable chemical properties of free glutamine hamper its use as a nutrition
substrate in the routine clinical setting:20 (1) instability, especially during heat sterilization
and prolonged storage; and (2) limited solubility (~3 g/100 mL at 20˚C). The rate of
breakdown of free glutamine depends on temperature, pH, and anion concentration.
Indeed, this decomposition of free glutamine is quantitative and yields the cyclic product
pyroglutamic acid and ammonia.46 Regardless of this subject of dispute, several studies
have shown that free glutamine may be provided by adding the crystalline amino acid to
a commercially available amino acid solution before administration. However, appropriate
preparation of such a solution requires a daily procedure at +4˚C under strict aseptic
conditions in a local pharmacy and subsequent laborious sterilization by membrane fil-
tration.47 In addition, to diminish the risk of precipitation, the glutamine concentrations
in such solutions should not exceed 1 to 1.5%. Consequently, provision of adequate
amounts of glutamine to injured or critically ill patients represents a severe burden,
especially in volume-restricted situations. Thus, the parenteral use of free glutamine for
the time being is reserved for controlled and well-conducted clinical trials.

36.3.2 Use of parenteral glutamine: clinical studies (Table 36.1)


Pharmacokinetics and clinical/metabolic effects of intravenous administration of free
glutamine have been investigated in healthy humans48 as well as in select patient popula-
tions (Table 36.1).49,50 Hammarqvist et al.49 evaluated nitrogen balance, plasma, and intra-
cellular muscle amino acids in patients following elective cholecystectomy receiving
1382_C36.fm Page 618 Tuesday, October 7, 2003 7:29 PM

618 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 36.1 Clinical Effects of Free Glutamine-Supplemented Parenteral Nutrition


End point Observation Reference
Intra-/extracellular glutamine pools Increased 49, 50
Not influenced 51
Nitrogen balance and muscle protein synthesis Improved 49
Duration of TPN and ventilator dependency in VLBWIs Decreased 60
Length of hospital stay Decreased 52, 53, 58
Not influenced 57
Survival/6-month outcome Improved 51, 55, 56
Patient’s mood Improved 54

glutamine-supplemented total parenteral nutrition (TPN). In the glutamine group, the


postoperative decrease in the intracellular glutamine concentration as well as the cumula-
tive nitrogen loss was less pronounced than in the control group. The effect of free
glutamine-enriched TPN on muscle glutamine content in critically ill patients was studied
by Palmer et al.51 Glutamine was infused (25 g/day) beginning at day 3 after ICU admission
with a median of 3 days of administration. Muscle biopsy before feeding revealed very
low muscle glutamine concentrations; the patterns were not changed after 5 days.
In patients undergoing allogenic bone marrow transplantation (BMT), glutamine-
enriched TPN (0.57 g of glutamine/kg/day) resulted in an improved nitrogen balance
and a significant reduced 3-methylhistidine:creatinine excretion ratio compared to controls
receiving isoenergetic and isonitrogenous TPN.52 More importantly, post-BMT morbidity
was diminished with glutamine supplementation: the incidence of clinical infection, total
and site-specific microbial colonization, and length of hospital stay were reduced com-
pared with controls. In contrast, by using a similar protocol in patients with both hema-
tological malignancies and solid tumors, and with both allogenic and autologous BMTs,
the incidence of positive bacterial cultures, clinical infections, and mortality did not differ
between control and glutamine-supplemented groups.53 In agreement with the previous
study, however, the length of hospital stay after BMT was less in patients receiving
glutamine. In a current study, patients receiving glutamine-supplemented intravenous
feedings revealed an improvement in mood as assessed by the profile of mood states
questionnaire, quantifying the degree of tension, depression, anger, vigor, fatigue, and
confusion. It is postulated that glutamine may influence patients’ feelings of well-being
either directly by affecting central nervous system (CNS) neurotransmitters or via its
effects on the protein status.54
The first study to describe a reduced 6-month mortality rate in critically ill patients
was conducted by Griffiths et al.55 in 84 ICU patients. In 42 critically ill patients, glutamine-
containing parenteral nutrition was associated with significantly reduced mortality, com-
pared with 42 control patients receiving conventional TPN, 6 months after admission.
Although the pattern of early deaths in the ICU was similar between the two groups, the
number of late deaths in the ICU was greater in the patients receiving standard nutrition
than in those receiving glutamine–TPN.55
In a more recent study in a more heterogenous group of ICU patients able to tolerate
enteral feeding (many of whom were already infected on admission), there was no sug-
gestion of reduced mortality, but total postintervention hospital costs were significantly
reduced in both enteral and parenteral glutamine recipients.56 In another randomized,
double-blind study, oral and parenteral glutamine supplementation was evaluated in 66
BMT patients. Unfortunately, the authors57 did not distinguish between enteral (oral) and
TPN treatments. Nevertheless, possible improved long-term survival is suggested by the
results in the mixed material.
1382_C36.fm Page 619 Tuesday, October 7, 2003 7:29 PM

Chapter thirty-six: Glutamine and glutamine-containing dipeptides 619

Powell-Tuck et al.58 designed a study with the intent to reduce mortality in ICU
patients. One hundred sixty-eight patients were investigated. Eighty-three patients
received glutamine-free, standard TPN, and 85 received 20 g of L-glutamine-enriched TPN
for an average of 8 days. No differences were seen between the groups regarding infectious
complications and length of hospital stay. Mortality in the glutamine group (16.9%) was
less, but not significantly different from the control group (24%). Nevertheless, in surgical
patients a considerably reduced length of hospital stay was observed (vide infra). In a
neonatal intensive care unit, very low birth weight infants (VLBWIs) receiving the standard
formula had a threefold higher incidence of sepsis than the glutamine-supplemented
infants (30.6 vs. 11.4%).59 Indeed, glutamine supplementation may be of particular benefit
to preterm infants receiving parenteral nutrition. Importantly, glutamine enhances growth,
development, and function of the immune system. Accordingly, glutamine-supplemented
premature infants at high risk for necrotizing enterocolitis required fewer days on TPN,
had a shorter length of time to full feeds, needed less time on the ventilator, and had a
tendency toward a shorter length of stay in the ICU.60

36.4 Provision of glutamine: the dipeptide concept


The obvious limitations to use of free glutamine in routine clinical settings started off an
intensive search for alternative new substrates. The implication of stable and highly soluble
synthetic dipeptides shows great promise as an effective route for the provision of amino
acids otherwise difficult to deliver.61 Dipeptides with a glutamine residue at the C-terminal
position reveal high solubility in water (glycyl-L-glutamine (Gly-Gln), 154 g/L; L-alanyl-
L-glutamine (Ala-Gln), 568 g/L) and sufficient stability during heat sterilization and pro-
longed storage. These properties qualify the dipeptides to be approved by the authorities
as suitable constituents of liquid nutritional preparations. In a current review, new devel-
opments in glutamine delivery are described.62

36.4.1 Animal studies


Basic studies with various synthetic glutamine-containing short-chain peptides provide
convincing evidence that these new substrates are rapidly cleared from plasma after
parenteral administration without being accumulated in tissues and with inconsequential
losses in urine. Considerable hydrolase activity in extra- and intracellular tissue
compartments63–66 ensures a quantitative peptide hydrolysis, the liberated amino acids
being available for protein synthesis and generation of energy.
Following a bolus injection or under conditions of continuous TPN, these peptides
provide glutamine for maintenance of the intra- and extracellular glutamine pools.40,63,67–69
Parenteral dipeptide nutrition promotes growth and nitrogen retention.39,40,70 Interestingly,
intravenous provision of Ala-Gln reduces muscle loss of glutamine during stress.71 Addi-
tion of Ala-Gln as a stable glutamine source to standard TPN solution preserves or even
enhances mucosal cellularity and function in parenterally feds rats with or without sys-
temic septic complications.40,71–74 In a recent investigation the beneficial effect of parenteral
glutamine nutrition on gut barrier function and mucosal immunity could not be con-
firmed.75
Monosaccharide transport, water absorption, and mucosal morphology are preserved
with Ala-Gln-enriched TPN following an experimental two-step small bowel transplan-
tation procedure. It is concluded that glutamine is essential for physiological absorptive
and barrier function of the intestinal graft.76 Direct intraluminal infusion of glutamine into
the graft (segmental small bowel autotransplantation) improved mucosal structure and
absorption of D-xylose (Li et al., personal communication). In a series of experimental
1382_C36.fm Page 620 Tuesday, October 7, 2003 7:29 PM

620 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

studies, Rombeau and coworkers77,78 reported beneficial effects of glutamine in protecting


morphological structure of small bowel grafts from cold preservation injury. In this context
it is notable that glutamine apparently induces synthesis of heat shock protein (HSP) 70
and its RNA transcription in epithelial cells (see Chapter 11 for details). This would mean
that glutamine protects intestinal mucosa during critical illness against exogenous (che-
motherapy, radiation) or endogenous (oxygen-free radicals, endotoxinemia) insults.79
Parenteral glutamine dipeptide supplementation reversed TPN-induced gut-associated
lymphoid tissue (GALT) atrophy and attenuated TPN-associated reduction of intestinal
immunoglobulin (Ig) A. Interestingly, parenteral glutamine improved IgA-mediated pro-
tection in the upper respiratory tract.80,81 In a rat model of protracted peritonitis, protein
synthesis in liver and skeletal muscle was enhanced, the morphology of the gastrointes-
tinal tract was protected, and survival improved with supplemental Ala-Gln, which thus
may be beneficial in sepsis.82
Recent reports emphasize that supplemental glutamine preserves hepatic and intes-
tinal stores of glutathione and maintains plasma concentrations.83–85 Experimental feeding
with glutamine resulted in considerable increase in gut fractional uptake and a marked
increase in intestinal glutathione fractional release.86 This means increased intestinal glu-
tathione production. The biochemical explanation for this finding rests in the fact that the
highly charged glutamic acid molecule, one of the direct precursors of glutathione, is
poorly transported across the cell membrane. Glutamine is, however, readily taken up by
the cell and is then deaminated and thus can serve as a glutamic acid precursor.87

36.4.2 Implications in healty volunteers (Table 36.2)


Human studies in healthy volunteers demonstrated88,89 that Ala-Gln is readily hydrolyzed
after its bolus injection, the elimination t1/2 ranging between 3 and 4 min. Continuous
infusion of a commercial amino acid solution supplemented with Ala-Gln or Gly-Gln was
not accompanied by any side effects, and no complaints were reported.90,91 Infusion of the
peptide-supplemented solutions resulted in a prompt increase in alanine, glutamine, and
glycine concentrations. During the entire infusion period, only trace amounts of the dipep-
tides could be measured in plasma, the values being just at the detection limit. Since
urinary losses of dipeptides were only inconsequential, the results suggest a nearly quan-
titative hydrolysis of the infused peptide and indeed indicate subsequent utilization of
the constituent free amino acids. Lochs et al.92,93 studied the organ clearance of glutamine-
containing dipeptides in postabsorptive and starved humans. Peptides were effectively
cleared by the kidney, other splanchnic organs, and skeletal muscle.

36.4.3 Clinical studies (Table 36.2)


In patients undergoing major elective surgery, infusion of Ala-Gln-supplemented TPN over
5 days resulted in an improvement of the nitrogen balance on each postoperative day
compared with controls receiving isonitrogenous and isoenergetic TPN without peptide.94
The improved net nitrogen balance was associated with maintenance of the intracellular
glutamine pool, whereas in patients receiving the control solution glutamine levels were
markedly decreased compared to preoperative values. The peptide was not detectable in
plasma and muscle, and the plasma concentrations of the constituent amino acids did not
differ between the treatment groups. The infusion of the solutions was free of any side
effects, and postoperative recovery was normal for each patient. In patients undergoing
major abdominal operations, the beneficial effects of glutamine dipeptide-supplemented
TPN on nitrogen economy, lymphocyte count, and maintainance of plasma free glutamine
1382_C36.fm Page 621 Tuesday, October 7, 2003 7:29 PM

Chapter thirty-six: Glutamine and glutamine-containing dipeptides 621

Table 36.2 Clinical Effects of Glutamine Peptide-Supplemented Parenteral Nutrition


End Point Observation Reference

Healthy Volunteers
Extracellular glutamine pools Increased 88–93

Patients
Intra-/extracellular glutamine concentrations Maintained/improved 94–96, 101, 102
Not influenced 99
Nitrogen balance Improved 94, 95, 101, 124
Protein synthesis Enhanced 96–98
Weight gain in nonselected hematological Improved 100
patients
Trauma-related gastrointestinal dysfunctions Avoided 113–115, 124
Immunity/host defense Improved 95, 103, 106, 107, 111,
112
Length of hospital stay Reduced 95, 101, 102, 124
Six-month survival Improved 102

concentrations could be confirmed, and additionally, shortened hospital stays were


demonstrated.95
In good agreement with these results, intravenous supply of Ala-Gln following chole-
cystectomy preserved the intracellular glutamine pool (91% of preoperative value), and
the characteristic postoperative change in muscle ribosome profile was abolished.96
Petersson et al.97 studied the long-term effect of postoperative TPN supplemented with
Gly-Gln on protein synthesis in skeletal muscle. In the glutamine group, the decrease in
protein synthesis (assessed by ribosome profiles) was less pronounced than in the controls.
Beneficial effects of short-term infusion of Ala-Gln on muscle protein synthesis assessed
by [13C]leucine incorporation were reported by Barua et al.98 in postsurgical patients
receiving glutamine-free parenteral nutrition. A very high dose of Ala-Gln (40 to 60 g/day)
was given to patients with acute pancreatitis over a relatively short period without an
apparent positive influence of the supplementation.99 In nonselected hematological
patients with intensive chemotherapy, no differences in neutrogenic period, fever, extra-
antibiotics, and toxicity scores, except for gain in body weight, were observed with Ala-
Gln supplementation (40 g/day) compared with isonitrogenous control patients.100
A recent multicenter study performed between 1997 and 1998 in 11 centers in European
countries with a total of 126 patients showed better daily and cumulative N balances in
the glutamine dipeptide group than in the control group.101 Despite the heterogenous
material, the length of hospital stay was 2.0 days shorter in the test group (21%), compared
with the controls. Plasma free glutamine was in the low normal range before operation
and showed significantly higher concentration with supplemental Ala-Gln, while in the
controls plasma glutamine levels remained unchanged. Length of hospital stay could be
calculated in one of the centers with similar degrees of illness showing significantly
reduced hospitalization with glutamine peptide.101 The time of hospitalization for both
test and control groups is also depicted by the Kaplan-Meier probability diagram, clearly
demonstrating a reduction in hospital stay with Ala-Gln. Group stratification, length of
operation, intraoperative blood loss, and type of operation were considered independent
variables for entry into the model of the Cox regression. This analysis clearly revealed
that hospital stay was significantly dependent on TPN regimen and was not influenced
by type of surgery, length of operation, or intraoperative blood loss.
1382_C36.fm Page 622 Tuesday, October 7, 2003 7:29 PM

622 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Probability to survive
1.0

0.8

0.6 Ala-Gln * Cox-Regression


* p < 0.05

0.4
Control

0.2

40 80 120 160
days

Figure 36.1 Survival plot of a subgroup of parenterally fed patients treated for 9 days and longer
under standardized conditions. Ala-Gln: 1.2 g of standard amino acid solution per kilogram of body
weight plus 0.3 g of alanyl-glutamine per kilogram of body weight per day. Control: 1.5 g of standard
amino acid solution per kilogram of body weight. (Reproduced with permission from Goeters, C.
et al., Crit. Care Med., 30, 2032, 2002.)

In a current study, 95 patients were randomized and treated for more than 5 days,
and 68 patients for 9 days or more, to receive either standard parenteral nutrition or
supplemented TPN with Ala-Gln (0.3 g/kg of body weight). Forty-six patients received
the dipeptide supplement and 49 served as controls. Six-month survival was improved
for patients treated for 9 days or longer with glutamine supplementation (66.7%) vs.
patients receiving standard TPN (40%) (Figure 36.1). In the dipeptide-treated group,
plasma free glutamine concentrations increased after 6 to 9 days. These results support
the notion that replacement of glutamine deficiency may correct excess mortality in ICU
patients due to inadequate parenteral nutrition.102
A novel finding is the striking influence of supplemental glutamine dipeptide on
cysteinyl-leukotriene (Cys-LT) metabolism. Cys-LTs are potent lipid mediators. It has been
emphasized that diminished release of these mediators is accompanied by an attenuated
endogenous host defense.103 After surgery, the low Cys-LT concentration in isolated poly-
morphonuclear leucocytes was completely restored with supplemental dipeptide, while
it remained low with conventional TPN.104 In line with these observations, a current study
in critically ill (sepsis, systemic inflammatory response syndrome (SIRS), sepsis syndrome),
nonsurviving patients revealed low LTC4 generation, while in surviving patients LTC4
generation was normalized during convalescence. In these latter patients LTC4 correlated
to sepsis severity score, whereas in nonsurviving patients the high sepsis severity score
showed no correlation with LTC4 generation.104 Reduced LTC4 generation in the critically
ill might be due to the anergic state caused by the underlying illness or lack of available
fatty acid precursor at the site of the membrane. However, the likely explanation is a
decrease in antioxidant capacity during critical illness. It should be remembered that
1382_C36.fm Page 623 Tuesday, October 7, 2003 7:29 PM

Chapter thirty-six: Glutamine and glutamine-containing dipeptides 623

intracellular stores of both glutathione and glutamine are depleted in these situations.
Thus, a major question is whether the combined deficiencies are intrinsically related to
LTC4-synthesizing capacity of the sick cell. Accordingly, we propose that the capacity of
cysteinyl-leukotriene generation might be a biomarker for survival in the critically ill, and
the system related to LTC4 and glutathione might be normalized with supplemental
glutamine.104
Many data emphasize the immunostimulatory role of supplemental glutamine dipep-
tides.105,106 Importantly, an increase in glutamine concentration is associated with enhanced
bacterial killing in postoperative state, suggesting promotion of bactericide function of
neutrophils.107 Increased counts of circulating total lymphocytes and enhanced T-cell lym-
phocyte synthesis are consistently found in stressed patients following provision of
glutamine or glutamine dipeptide-containing nutrition.108 Selected components of lym-
phocyte activation like interleukin (IL)-2 production, IL-2 use, IL-2 receptor expression,
and transferrin receptor expression are dependent on glutamine concentration.109 Cell
surface activation markers like CD25, CD45RO, and CD71, and the production of interferon-
gamma (IFN-g) and tumor necrosis factor-a (TNF-a) require an exogenous supply of
glutamine.110 In the presence of Ala-Gln or Gly-Gln, release of pro-inflammatory cytokines
(IL-8, TNF-a) by polymorphonuclear leukocytes (PMNs) was decreased, while the ability
to express the anti-inflammatory IL-10 was enhanced,111,112 suggesting that glutamine selec-
tively influences the generation of certain cytokines.
In similarity to animal experiments, it could be demonstrated in clinical studies that
glutamine dipeptide-containing TPN may avoid trauma-related intestinal atrophy, known
to be associated with glutamine-free TPN. In patients with inflammatory bowel disease
and neoplastic disease, intestinal permeability could be maintained and villus height
preserved with Gly-Gln supplementation.113 In another study, Ala-Gln-supplemented TPN
maintained absorptive capacity (assessed by D-xylose absorption test) in the proximal
portion of the small intestine in critically ill patients, compared with patients receiving
conventional glutamine-free TPN.114 In patients undergoing bone marrow transplantation,
the results with high doses of glutamine dipeptide supplementation (50 g of Gly-Gln
corresponding to 38 g of glutamine) were compared with those receiving standard TPN.
Significant improvement in the lower gastrointestinal score and improved small intestinal
permeability were found with glutamine. There were also fewer episodes of fever in the
glutamine group compared with controls. Quantitation of the effect of glutamine on the
gastrointestinal tract was an important contribution made by these investigators.115 Since
the large intestine harbors far more bacteria than the duodenum, jejunum, or ileum,
maintainance of an intact colonic barrier may be crucial. The postulate that glutamine or
glutamine dipeptides exert beneficial effects on the mucosa is strongly supported by the
results of a current study in which biopsies from normal human ileum, proximal colon,
and rectosigmoid were incubated with glutamine, Ala-Gln, and saline. Glutamine and
Ala-Gln equally stimulated crypt cell proliferation; the tropic effect was mainly confined
to the basal crypt compartments.116 Table 36.2 summarizes the effects of glutamine dipep-
tide therapy in various clinical situations.
Importantly, glutamine stimulates induction of ornithine decarboxylase (ODC), which
is rate limiting in the synthesis of polyamine-implicated proliferation.117 The proposed
mechanism is that glutamine is additive to epidermal growth factor (EGF) and insulin-
like growth factor-1 (IGF-1) in stimulating DNA synthesis and also activates both extra-
cellular signal-regulated kinases (ERK5). This results in a fourfold increase in activating
protein 1 (AP1)-dependent gene transcription. It should also be noted that glutamine is
required for EGF signaling via ERK5, and cJun mRNA respond to glutamine in cell
cultures.118,119 The conclusion is that glutamine may be a unique nutrient for enterocytes
1382_C36.fm Page 624 Tuesday, October 7, 2003 7:29 PM

624 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

capable of dual signaling and augmenting the effects of growth factors that govern cellular
proliferation and repair.

36.5 Glutamine nutrition: mechanisms, outcome, and cost–benefit


calculations
Recently the question was raised: “Glutamine saves lives! What does it mean?”120 The
underlying mechanism of supplemental glutamine (dipeptide) in causing reversal of
severe illness might be due to support of the mucosa, the immune system, and the hepatic
biosynthesis of glutathione. According to another proposal, glutamine plays a more global
regulatory role by modifying the endogenous inflammatory responses. These mechanisms
might be due to attenuation of the elaboration of pro-inflammatory mediators and up-
regulation of anti-inflammatory factors.121 The contributions of glutamine to protein anab-
olism and through acid–base homeostasis may elicit a further defense mechanism of the
host, but the specific role of these functions to the host defense have not been quantitated.
In numerous studies, calculation of cost–benefit revealed considerable cost reduction
with glutamine (dipeptide) nutrition. Hospital stay was reduced by about 5 to 7 days in
the two BMT studies and in surgical investigation.5,58,95,101 Consequently, hospital costs
were markedly diminished, primarily as a function of reduced charges for room and
board.122 A decreased length of hospital stay of the magnitude seen in these studies thus
has not only significant benefits for patient care but also considerable economic implica-
tions. In a standard university hospital, treating 30 (BMT) patients per year at a cost of
$1000 per day per patient, conservative estimating of glutamine use would amount to a
savings of $180,000 considering the observed 5.8-day average decrease of hospitalization.
Taking into account the abundant number of surgical patients, the savings with supple-
mental glutamine dipeptides are considerable, amounting to about $3000 per patient.5,95
In very low birth weight infants, glutamine supplementation accounted for an overall
reduction in expenses of about $20,000 per baby. For glutamine recipients at the ICU, the
supplementation resulted in a 15% reduction of the total costs of hospital care ($6373 per
patient), which, when expressed as cost per survivor, was 50% less than that seen with
conventional TPN ($46,403 vs. $94,077).55 The costs of adding glutamine (dipeptides) to
TPN is marginal, about 0.2% compared with the estimated total costs at the ICU following
BMT (about 2 to 3%) or after operations (about 4 to 5%).5
In a current comprehensive study, Novak and co-workers6 examined the relationship
between glutamine supplementation and hospital length of stay, morbidity, and mortality
in patients undergoing surgery and experiencing critical illness. They reviewed 550 titles,
abstracts, and papers. There were 14 randomized trials showing a lower risk ratio with
glutamine supplementation; the rate of infectious complications was also lower and the
hospital stay shorter with glutamine nutrition. With respect to mortality, the treatment
benefit was observed in studies of parenteral glutamine and high-dose glutamine, com-
pared to studies of enteral glutamine and low-dose glutamine, respectively. With respect
to hospital length of stay, all of the treatment benefit was observed in surgical patients
compared to critically ill patients (–3.5 days vs. 0.9 day).

36.6 Future perspectives


Future implications of glutamine (dipeptide) therapy are full of promise. A consistent
observation is that glutamine-enriched parenteral feeding attenuates the expansion of
extracellular and total body water.53,95,123,124 This interesting finding suggests that provision
of glutamine (dipeptides) may influence stress-induced accumulation of extracellular fluid
1382_C36.fm Page 625 Tuesday, October 7, 2003 7:29 PM

Chapter thirty-six: Glutamine and glutamine-containing dipeptides 625

by affecting membrane function and thereby changing the cellular hydration state125 —
indeed, an encouraging future therapy in situations with extracellular edema. Experimen-
tal studies suggest that glutamine availability is an essential factor during conditions
associated with glucose intolerance. During hyperinsulemic euglycemia, increased
glutamine availability blunted insulin action on glucose production and enhanced insulin-
mediated glucose utilization. Thus, glutamine (dipeptide) appears to possess a future
potential to be of benefit as a nutrient adjuvant during clinical situations associated with
insulin resistance, such as diabetes mellitus, sepsis, trauma, and others.126,127
A further fascinating approach proposes glutamine (dipeptides) as a suitable cardio-
protective and rescue agent.128,129 The mechanism through which glutamine exerts its
beneficial effects may involve maintainance of myocardial glutamate, and thus glutathione
as well as myocardial high-energy phosphates, and prevention of myocardial lactate
accumulation. The implication of these results might include the possible use of glutamine
to support the heart during reperfusion initiated by thrombolysis or coronary angioplasty
in patients with acute myocardial infarction. Furthermore, glutamine enrichment of car-
dioplegia during cardiac surgery might improve the postoperative ventricular function
and the postoperative survival in patients with coronary artery disease.
In a new analysis of clinical and microbiological data of the Griffiths et al. study,130
the authors report the nature and overall rates of intensive care-acquired infections (ICAIs)
during the outcome study.131 It appears that the improved survival is mostly related to
reduced mortality for multiple-organ failure (MOF) in the ICU. Glutamine-treated patients
developed fewer Candida infections after longer stay, and importantly, none of those
infected died, whereas many control patients on standard TPN not only became infected
sooner but also had more infections and died from MOF (p ≥ 0.02). Overall, only 38%
ICAIs were in glutamine groups who died, compared with 74% in controls. These new
data show that glutamine is significantly associated with improved survival.132 Glutamine
therapy not only may reduce the overall incidence of ICAIs but also is demonstrably safe.
Indeed, preventing nosocomial infections is a valuable goal resulting in reduced ICU stays,
duration of mechanical ventilation, and antibiotic pressure; fewer side effects; lower costs;
and less emergence of resistant microorganisms.133 As a consequence of the available
evidence, Wilmore defines glutamine in a current editorial “as a highly unique and impor-
tant nutrient, one that serves as both an important metabolite and a metabolic switch or
regulator, essential to human health and survival.”133

References
1. Wretlind, A., Parenteral nutrition, Nutr. Rev., 39, 257, 1981.
2. Wilmore, D.W., The practice of clinical nutrition: how to prepare for the future, J. Parenter.
Enteral Nutr., 13, 337, 1989.
3. Fürst, P., Intracellular muscle free amino acids: their measurements and function, Proc. Nutr.
Soc., 42, 451, 1983.
4. Souba, W.W., Glutamine: a key substrate for the splanchnic bed, Annu. Rev. Nutr., 11, 285,
1991.
5. Fürst, P., Pogan, K., and Stehle, P., Glutamine dipeptides in clinical nutrition, Nutrition, 13,
439, 1997.
6. Novak, F., Heyland, D.K., Avenell, A., Drover, J.W., and Xiangyao, S., Glutamine supplemen-
tation in serious illness: a systematic review of the evidence, Crit. Care Med., 30, 2022, 2002.
7. Ritthausen, H., Über die Glutaminsäure, J. Prakt. Chem., 99, 454, 1883.
8. Hlasiwetz, H. and Habermann, J., Über die Proteinstoffe, Ann. Chem. Pharm., 169, 150, 1873.
9. Schulze E. and Bosshard E., Über das Glutamin, Landw. Vers. Sta., 29, 295, 1883.
10. Thierfelder, H. and Sherwin, C.P., Phenylacetyl-glutamin, ein Stoffwechsel-Produkt des men-
schlichen Körpers nach Eingabe von Phenylessigsäure, Ber. Dtsch. Chem. Ges., 47, 2630, 1914.
1382_C36.fm Page 626 Tuesday, October 7, 2003 7:29 PM

626 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

11. Krebs, H.A., Metabolism of amino acids. IV. The synthesis of glutamine from glutamic acid
and ammonia, and the enzymatic hydrolysis of glutamine in animal tissue, Biochem. J., 29,
1951, 1935.
12. Leuthardt, F. and Glasson, B., Le sort des acides aminés lors de leur absorption, Helv. Chim.
Acta, 29, 1344, 1946.
13. Van Slyke, D.D. and Meyer, G.M., The fate of protein digestion products in the body, J. Biol.
Chem., 16, 197, 1913.
14. Benson J.V., Jr., Gordon, M.J., and Patterson, J.A., Accelerated chromatographic analysis of
amino acids in physiological fluids containing glutamine and asparagines, Anal. Biochem.,
18, 228, 1937.
15. Eagle, H., Oyama, V.L., Lery, M., Horton, C.L., and Fleischman, R., The growth response of
mammalian cells in tissue culture to L-glutamine and L-glutamic acid, J. Biol. Chem., 218, 607,
1956.
16. Munro, H.N., Free amino acid pools and their role in regulation, in Mammalian Protein
Metabolism, Vol. 4, Munro, H.N., Ed., Academic Press, New York, 1970, p. 299.
17. Bergström, J., Fürst, P., Norree, L.-O., and Vinnars E., Intracellular free amino acid concen-
tration in human muscle tissue, J. Appl. Physiol., 36, 693, 1974.
18. Rennie, M.J., MacLennan, P., Hundal, H.S., Weryk, B., Smith, K., Taylor, P.M., Egan, C., and
Watt, P.W., Skeletal muscle glutamine transport, intramuscular glutamine concentration and
muscle-protein turnover, Metabolism, 38, 47, 1989.
19. Fürst, P., Regulation of intracellular metabolism of amino acids, in Nutrition in Trauma and
Cancer Sepsis, Bozetti, F. and Dionigi, R., Eds., Karger, Basel, Switzerland, 1985, p. 21.
20. Meister, A., Metabolism of glutamine, Physiol. Rev., 36, 103, 1956.
21. Phromphet-Charat, V., Jackson, A., Dass, P.D., and Welbourne, T.C., Ammonia partitioning
between glutamine and urea: interorgan participation in metabolic acidosis, Kidney Int., 20,
598, 1981.
22. Sies, H. and Häussinger, D., Hepatic glutamine and ammonia metabolism, in Glutamine
Metabolism in Mammalian Tissues, Häussinger, D. and Sies, H., Eds., Springer-Verlag, Berlin,
1984, p. 78.
23. Windmueller, H.G. and Spaeth, A.E., Respiratory fuels and nitrogen metabolism in vivo in
small intestine of fed rats: quantitative importance of glutamine, glutamate and aspartate,
J. Biol. Chem., 255, 107, 1980.
24. Newsholme, E.A., Newsholme, P., Curi, R., Challoner, E., and Ardawi, M.S.M., A role for
muscle in the immune system and its importance in surgery, trauma, sepsis and burns,
Nutrition, 4, 261, 1988.
25. Reeds, P.J. and Burrin, D.G., Glutamine and the bowel, J. Nutr., 131, 2505, 2001.
26. Brunton, J.A., Bertolom, R.F., Pencharz, P.B., and Ball, R.O., Proline ameliorates arginine
deficiency during enteral but not parenteral feeding in neonatal piglets, Am. J. Physiol., 277,
E223, 1999.
27. Reeds, P.J., Burrin, D.G., Stoll, B., Jahoor, F., Wykes, L., Henry, J., and Frazer, M.E., Enteral
glutamate is the preferential source for mucosal glutathione synthesis in fed piglets, Am. J.
Physiol., 273, E408, 1997.
28. Rhoads, J.M., Argenzio, R.A., Chen, W., Graves, L.M., Licato, L.L., Blikslager, A.T., Smith, J.,
Gatzy, J., and Brenner, D.A., Glutamine metabolism stimulates intestinal cell MAPKs by a
cAMP in inhibitable rat independent mechanism, Gastroenterology, 118, 90, 2000.
29. Sibson, N.R., Mason, G.F., Shen, S., Cline, G.W., Herskovitz, A.Z., Wall, J.E.M., Behar, K.L.,
Rothman, D.L., and Shulman, R.G., In vivo 13C-NMR measurement of neurotransmitter
glutamate cycling, anaplerosis and TCA cycle Hux in rat brain during [2-13C]glucose infusion
in rat brain, J. Neurochem., 76,975, 2001.
30. Elwyn, D.H., Fürst, P., Askanazi, J., and Kinney, J.M., Effect of fasting on muscle concentra-
tions of branched-chain amino acids, in Metabolism and Clinical Implications of Branched-Chain
Amino-Ketoacids, Walser, M. and Williamson, P., Eds., Elsevier/North-Holland, New York,
1981, p. 547.
31. Karner, J. and Roth, E., Alanylglutamine infusions to patients with acute pancreatitis, Clin.
Nutr., 9, 43, 1990.
1382_C36.fm Page 627 Tuesday, October 7, 2003 7:29 PM

Chapter thirty-six: Glutamine and glutamine-containing dipeptides 627

32. Souba, W.W., Herskowitz, K., and Plumley, D.A., Lung glutamine metabolism, J. Parenter.
Enteral Nutr., 14, 68S, 1990.
33. Jepson, M.M., Bates, P.C., Broadbent, P., Pell, J.M., and Millward, D.J., Relationship between
glutamine concentration and protein synthesis in rat skeletal muscle, Am. J. Physiol., 255,
E166, 1988.
34. MacLennan, P.A., Brown, R.A., and Rennie, M.J., A positive relationship between protein
synthetic rate and intracellular glutamine concentration in perfused rat skeletal muscle, FEBS
Lett., 215, 187, 1987.
35. Wilmore, D.W., Smith, R.J., O’Dwyer, S.T., Jacobs, D.O., Ziegler, T.R., and Wang, X.-D., The
gut: a central organ after surgical stress, Surgery, 104, 917, 1988.
36. Souba, W.W., Klimberg, V.S., Hautamaki, R.D., Mendenhall, W.H., Bova, F.C., Howard, R.J.,
Bland, K.I., and Copeland, E.M., III, Oral glutamine reduces bacterial translocation following
abdominal radiation, J. Surg. Res., 48, 1, 1990.
37. Rombeau, J.L., A review of the effects of glutamine-enriched diets on experimentally induced
enterocolitis, J. Parenter. Enteral Nutr., 14, 100S, 1998.
38. Hwang, T.L., O’Dwyer, S.T., Smith, R.J., and Wilmore, D.W., Preservation of the small bowel
mucosa using glutamine-enriched parenteral nutrition, Surg. Forum, 37, 56, 1986.
39. Babst, R., Hörig, H., Stehle, P., Brand, O., Filgueira, L., Marti, W., Fischer, M., Oberholzer,
M., Gudat, F., Fürst, P., and Heberer, M., Glutamine peptide-supplemented long-term total
parenteral nutrition: effects on intracellular and extracellular amino acid patterns, nitrogen
economy, and tissue morphology in growing rats, J. Parenter. Enteral Nutr., 17, 566, 1993.
40. Jiang, Z.-M., Wang, L.-J., Qi, Y., Liu, T.-H., Qui, M.-R., Yang, N.-F., and Wilmore, D.W.,
Comparison of parenteral nutrition supplemented with L-glutamine or glutamine dipeptides,
J. Parenter. Enteral Nutr., 17, 134, 1993.
41. Inoue, Y., Grant, J.P., and Snyder, P.J., Effect of glutamine-supplemented total parenteral
nutrition on recovery of the small intestine after starvation atrophy, J. Parenter. Enteral Nutr.,
17, 165, 1993.
42. Platell, C., McCauley, R., McCulloch, R., and Hall, J., Influence of glutamine and branched-
chain amino acids on the jejunal atrophy associated with parenteral nutrition, J. Gastroenterol.
Hepatol., 6, 345, 1991.
43. Klimberg, V.S., Souba, W.W., and Salloum, R.M., Intestinal glutamine metabolism after mas-
sive small bowel resection, Am. J. Surg., 159, 27, 1990.
44. Ardawi, M.S.M., Effects of epidermal growth factor and glutamine-supplemented parenteral
nutrition on the small bowel of septic rats, Clin. Sci., 82, 573, 1992.
45. Higashiguchi, T., Hasselgren, P.-O., Wagner, K., and Fischer, J.-E., Effect of glutamine on
protein synthesis in isolated intestinal epithelial cells, J. Parenter. Enteral Nutr., 17, 307, 1993.
46. Dimarchi, R.D., Tam, J.P., Kent, S.B.H., and Merrifield, R.B., Weak acid-catalyzed pyrrolidone
carboxylic acid formation from glutamine during solid phase peptide synthesis, Int. J. Pept.
Protein Res., 19, 88, 1982.
47. Khan, K., Hardy, G., McElroy, B., and Elia, M., The stability of L-glutamine in total parenteral
nutrition, Clin. Nutr., 10, 193, 1991.
48. Lowe, D.K., Benfell, K., Smith, R.J., Jacobs, D.O., Murawski, B., Ziegler, T.R., and Wilmore,
D.W., Safety of glutamine-enriched parenteral nutrient solutions in humans, Am. J. Clin.
Nutr., 52, 1101, 1990.
49. Hammarqvist, F., Wernerman, J., Ali, R., von der Decken, A., and Vinnars, E., Addition of
glutamine to total parenteral nutrition after elective abdominal surgery spares free glutamine
in muscle, counteracts the fall in muscle protein synthesis and improves nitrogen balance,
Ann. Surg., 209, 455, 1989.
50. van der Hulst, R.R.W.J., von Meyenfeldt, M.F., Deutz, N.E.P., Stockbrügger, R.W., and Soeters,
P., The effect of glutamine administration on intestinal glutamine content, J. Surg. Res., 61,
30, 1996.
51. Palmer, T.E., Griffiths, R.D., and Jones C., Effect of parenteral L-glutamine on muscle in the
very severely ill, Nutrition, 12, 316, 1996.
1382_C36.fm Page 628 Tuesday, October 7, 2003 7:29 PM

628 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

52. Ziegler, T.R., Young, L.S., Benfell, K., Scheltinga, M., Hortos, K., Bye, R., Morrow, F.D., Jacobs,
D.O., Smith, R.J., Antin, J.H., and Wilmore, D.W., Clinical and metabolic efficacy of
glutamine-supplemented parenteral nutrition after bone marrow transplantation: a rand-
omized, double-blind controlled study, Ann. Intern. Med., 116, 821, 1992.
53. Schloerb, P.A. and Amare, M., Total parenteral nutrition with glutamine in bone marrow
transplantation and other clinical applications (a randomized, double-blind study), J. Parent-
er. Enteral Nutr., 17, 407, 1993.
54. Young, L.S., Bye, R., Scheltinga, M., Ziegler, T.R., Jacobs, D.O., and Wilmore, D.W., Patients
receiving glutamine-supplemented intravenous feedings report an improvement in mood,
J. Parenter. Enteral Nutr., 17, 422, 1993.
55. Griffiths, R.D., Jones, C., and Palmer T.E., Six-month outcome of critically ill patients given
glutamine-supplemented parenteral nutrition, Nutrition, 13, 295, 1997.
56. Jones, C., Palmer, T.E., and Griffiths, R.D., Randomized clinical outcome study of critically
ill patients given glutamine supplemented enteral nutrition, Nutrition, 15, 108, 1999.
57. Schloerb, P.R. and Skikne, B.S., Oral and parenteral glutamine in bone marrow transplanta-
tion: a randomized double-blind study, J. Parenter. Enteral Nutr., 23, 117, 1999.
58. Powell-Tuck, J., Jamieson, C.P., Bettany, G.E., Obeid, O., Fawcett, H.V., Archer, C., and
Murphy, D.L., A double-blind, randomized, controlled trial of glutamine supplementation
in parenteral nutrition, Gut, 45, 82, 1999.
59. Neu, J., Roig, J.C., Meetze, W.H., Veerman, M., Carter, C., Millsaps, M., Bowling, D., Dallas,
M.J., Sleasman, J., Knight, T., and Auestad, N., Enteral glutamine supplementation for very
low birth weight infants decreases morbidity, J. Pediatr., 131, 691, 1997.
60. Lacey, J.M., Crouch, J.B., Benfell, K., Ringer, S.A., Wilmore, C.K., Maguire, D., and Wilmore,
D.W., The effects of glutamine-supplemented parenteral nutrition in premature infants,
J. Parenter. Enteral Nutr., 20, 74, 1996.
61. Fürst, P., Peptides in clinical nutrition, Clin. Nutr., 10 (Suppl. 1), 19, 1991.
62. Fürst, P., New developments in glutamine delivery, J. Nutr., 131, 2562S, 2001.
63. Stehle, P. and Fürst, P., In vitro hydrolysis of glutamine-, tyrosine- and cystine-containing
short-chain peptides, Clin. Nutr., 9, 37, 1990.
64. Lochs, H., Williams, P.E., Morse, E.L., Abumrad, N.N., and Adibi, S.A., Metabolism of
dipeptides and their constituent amino acids by liver, gut, kidney and muscle, Am. J. Physiol.,
254, E588, 1988.
65. Hundal, H.S. and Rennie, M.J., Skeletal muscle tissue contains extracellular aminopeptidase
activity against Ala-Gln but no peptide transporter, Eur. J. Clin. Invest., 16, 163, 1988.
66. Herzog, B., Frey, B., Stehle, P., and Fürst, P., In vitro peptidase activity of different cell fractions
of rat mucosa: kinetic studies using glutamine-containing dipeptides, Clin. Nutr., 10, 32, 1991.
67. Abumrad, N.N., Morse, E.L., Lochs, H., Williams, P.E., and Adibi, S.A., Possible sources of
glutamine for parenteral nutrition: impact on glutamine metabolism, Am. J. Physiol., 257,
E228, 1998.
68. Stehle, P., Ratz, I., and Fürst, P., In vivo utilization of intravenously supplied L-alanyl-
L-glutamine in various tissues of the rat, Nutrition, 5, 411, 1989.
69. Stehle, P., Ratz, I., and Fürst, P., Whole-body autoradiography in the rat after intravenous
bolus injection of L-alanyl-L-[U-14C]glutamine, Ann. Nutr. Metab., 35, 213, 1991.
70. Karner, J., Roth, E., Ollenschläger, G., Fürst, P., and Simmel, A., Glutamine-containing dipep-
tides as infusion substrates in the septic state, Surgery, 106, 893, 1989.
71. Roth, E., Karner, J., Ollenschläger, G., Simmel, A., Fürst, P., and Funovics, J., Alanylglutamine
reduces muscle loss of alanine and glutamine in postoperative anaesthetized dogs, Clin. Sci.,
75, 641, 1988.
72. Yoshida, S., Leskiw, M.J., Schluter, M.D., Bush, K.T., Nagele, R.G., Lanza-Jacoby, S., and Stein,
T.P., Effect of total parenteral nutrition, systemic sepsis, and glutamine on gut mucosa in
rats, Am. J. Physiol. Endocrinol. Metab., 263, E368, 1992.
73. Tamada, H., Nezu, R., Imamura, I., Matsuo, Y., Takagi, Y., Kamata, S., and Okada, A., The
dipeptide alanyl-glutamine prevents intestinal mucosal atrophy in parenterally fed rats,
J. Parenter. Enteral Nutr., 16, 110, 1992.
1382_C36.fm Page 629 Tuesday, October 7, 2003 7:29 PM

Chapter thirty-six: Glutamine and glutamine-containing dipeptides 629

74. Tamada, H., Nezu, R., Imamura, I., Matsuo, Y., Takagi, Y., and Okada, A., Alanyl-glutamine
enriched total parenteral nutrition restores intestinal adaption after either proximal or distal
massive resection in rats, J. Parenter. Enteral Nutr., 17, 236, 1993.
75. Spaeth, G., Gottwald, T., Haas, W., and Holmer, M., Glutamine peptide does not improve
gut barrier function and mucosal immunity in total parenteral nutrition, J. Parenter. Enteral
Nutr., 17, 317, 1993.
76. Schroeder, P., Schweizer, E., Blömer, A., and Deltz, E., Glutamine prevents mucosal injury
after small bowel transplantation, Transplant. Proc., 24, 1104, 1992.
77. Sasaki, K., Hirata, K., Zou, X.M., Sakawaki, T., Yagihashi, A., Tsuruma, T., Katsuramaki, T.,
Koide, S., Zhang, W., and Rombeau, J.L., Optimum small bowel preservation solutions and
conditions: comparison of UW solution and saline with or without glutamine, Transplant.
Proc., 28, 2620, 1996.
78. Lew, J.I., Zhang, W., Koide, S., Smith, R.J., and Rombeau, J.L., Glutamine improves cold-
preserved small bowel graft structure and function following ischemia and reperfusion,
Transplant. Proc., 28, 2605, 1996.
79. Wischmeyer, P.E., Musch, M.W., Madonna, M.B., Thisted, R., and Chang, E.B., Glutamine
protects intestinal epithelial cells: role of inducible HSP70, Am. J. Physiol., 272, G879, 1997.
80. Li, J., King, B.K., Janu, P.G., Renegar, K.B., and Kudsk, K.A., Glycyl-L-glutamine-enriched
total parenteral nutrition maintains small intestine gut-associated lymphoid tissue and upper
respiratory tract immunity, J. Parenter. Enteral Nutr., 22, 31, 1998.
81. Li, J., Kudsk, K.A., Janu, P.G., and Renegar, K.B., Effect of glutamine-enriched total parenteral
nutrition on small intestinal gut-associated lymphoid tissue and upper respiratory tract
immunity, Surgery, 121, 542, 1997.
82. Naka, S., Saito, H., Hashiguchi, Y., Lin, M.T., Furukawa, S., Inaba, T., Fukushima, R., Wada,
N., and Muto, T., Alanyl-glutamine-supplemented total parenteral nutrition improves sur-
vival and protein metabolism in rat protracted peritonitis model, J. Parenter. Enteral Nutr.,
20, 417, 1996.
83. Harward, T.R., Coe, D., Souba, W.W., Klingman, N., and Seeger, J.M., Glutamine preserves
gut glutathione levels during intestinal ischemia/reperfusion, J. Surg. Res., 56, 351, 1994.
84. Yun, J.C., Jiang, Z.M., Li, D.M., Yang, N.F., and Bai, M.X., Alanyl-glutamine preserves hepatic
glutathione stores after 5-FU treatment, Clin. Nutr., 15, 261, 1996.
85. Denno, R., Rounds, J.D., Paris, R., Holejko, L.B., and Wilmore, D.W., Glutamine-enriched
total parenteral nutrition enhances plasma glutathione in the resting state, J. Surg. Res., 67,
35, 1996.
86. Cao, Y., Feng, Z., Hoos, A., and Klimberg, V.S., Glutamine enhances gut glutathione produc-
tion, J. Parenter. Enteral Nutr., 22, 224, 1998.
87. Hong, R.W., Rounds, J.D., Helton, W.S., Robinson, M.K., and Wilmore, D.W., Glutamine
preserves liver glutathione after lethal hepatic injury, Ann. Surg., 215, 114, 1992.
88. Albers, S., Wernerman, J., Stehle, P., Vinnars, E., and Fürst, P., Availability of amino acids
supplied intravenously as synthetic dipeptides: kinetic evaluation of L-alanyl-L-glutamine
and glycyl-L-tyrosine, Clin. Sci., 75, 643, 1988.
89. Matthews, D.E., Battezzati, A., and Fürst, P., Alanylglutamine kinetics in humans, Clin. Nutr.,
12, 57, 1993.
90. Albers, S., Wernerman, J., Stehle, P., Vinnars, E., and Fürst, P., Availability of amino acids
supplied by constant intravenous infusion of synthetic dipeptides in healthy man, Clin. Sci.,
76, 643, 1989.
91. Brandl, M., Sailer, D., Langer, K., Engelhardt, A., Kleinhenz, H., Adibi, S.A., and Fekl, W.,
Parenteral nutrition with an amino acid solution containing a mixture of dipeptides in man,
Contrib. Infusion Ther. Clin. Nutr., 17, 103, 1987.
92. Lochs, H., Hübl, W., Gasic, S., Roth, E., Morse, E.L., and Adibi, S.A, Glycylglutamine:
metabolism and effects on organ balances of amino acids in postabsorptive and starved
subjects, Am. J. Physiol. Endocrinol. Metab., 262, E155, 1992.
93. Lochs, H., Roth, E., Gasic, S., Hübl, W., Morse, E.L., and Adibi, S.A., Splanchnic, renal and
muscle clearance of alanylglutamine in man and organ fluxes of alanine and glutamine when
infused in free and peptide forms, Metabolism, 39, 833, 1990.
1382_C36.fm Page 630 Tuesday, October 7, 2003 7:29 PM

630 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

94. Stehle, P., Zander, J., Mertes, N., Albers, S., Puchstein, C., Lawin, P., and Fürst, P., Effect of
parenteral glutamine peptide supplements on muscle glutamine loss and nitrogen balance
after major surgery, Lancet, i, 231, 1989.
95. Morlion, B.J., Stehle, P., Wachtler, P., Siedhoff, H.P. Köller, M., König, W., Fürst, P., and
Puchstein, C., Total parenteral nutrition with glutamine dipeptide after major abdominal
surgery, Ann. Surg., 227, 302, 1998.
96. Hammarqvist, F., Wernerman, J., von der Decken, A., and Vinnars, E., Alanyl-glutamine
counteracts the depletion of free glutamine and the postoperative decline in protein synthesis
in skeletal muscle, Ann. Surg., 212, 637, 1990.
97. Petersson, B., von der Decken, A., Vinnars, E., and Wernerman, J., Long term effects of
postoperative total parenteral nutrition supplemented with glycyl-glutamine on subjective
fatigue and muscle protein synthesis, Br. J. Surg., 81, 1520, 1994.
98. Barua, J.M., Wilson, E., Downie, S., Weryk, B., Cuschiere, A., and Rennie, M.J., The effect of
alanyl-glutamine peptide supplementation on muscle protein synthesis in post-surgical pa-
tients receiving glutamine-free amino-acid intravenously, Proc. Nutr. Soc., 51, 104a, 1992.
99. Karner, J. and Roth, E., Alanylglutamine infusions to patients with acute pancreatitis, Clin.
Nutr., 9, 43, 1990.
100. Van Zaanen, H.C.T., Lelie, H., Timmer, J.G., Fürst, P., and Sauerwein, H., Parenteral glutamine
dipeptide does not ameliorate chemotherapy-induced toxicity, Cancer, 74, 2879, 1994.
101. Mertes, N., Schulzki, C., Goeters, C., Winde, G., Benzing, S., Kuhn, K.S., Van Aken, H., Stehle,
P., and Fürst, P., Cost containment through L-alanyl-L-glutamine supplemented total
parenteral nutrition after major abdominal surgery: a prospective randomized double-blind
controlled study, Clin. Nutr., 19, 395, 2000.
102. Goeters, C., Wenn, A., Mertes, N., Wempe, C., Van Aken, H., Stehle, P., and Bone, H.-G.,
Parenteral L-alanyl-L-glutamine improves 6-month outcome in critically ill patients, Crit. Care
Med., 30, 2032, 2002.
103. Köller, M., König, W., Brom, J., Raulff, M., Gross-Weege, W., Erbs, G., and Müller, F.E.,
Generation of leukotrienes from human polymorphonuclear granulocytes of severely burned
patients, J. Trauma, 28, 733, 1988.
104. Morlion, B.J., Torwesten, E., Kuhn, K.S., Puchstein, C., and Fürst, P., Cysteinylleukotriene
generation as a biomarker for survival in the critically ill, Crit. Care Med., 28, 3655, 2000.
105. Calder, P.C., Glutamine and the immune system, Clin. Nutr., 13, 2, 1994.
106. O’Riordain, M., Fearon, K.C., Ross, J.A., Rogers, P., Falconer, J.S., Bartolo, D.C., Garden, O.J.,
and Carter, D.C., Glutamine supplemented total parenteral nutrition enhances T-lymphocyte
response in surgical patients undergoing colorectal resection, Ann. Surg., 220, 212, 1994.
107. Furukawa, S., Saito, H., Inoue, T., Matsuda, T., Fakatsu, K., Han, I., Ikeda, S., and Hidemura,
A., Supplemental glutamine augments phagocytosis and reactive oxygen intermediate pro-
duction by neutrophils and monocytes from postoperative patiens in vitro, Nutrition, 16, 323,
2000.
108. Ziegler, T.R., Bye, R.L., Persinger, R.L., Young, L.S., Antin, J.H., and Wilmore, D.W., Effects
of glutamine supplementation in circulating lymphocytes after bone marrow transplantation:
a pilot study, Am. J. Med. Sci., 315, 4, 1998.
109. Yacoob, P. and Calder, P.C., Glutamine requirement of proliferating T-lymphocytes, Nutrition,
13, 646, 1997.
110. Heberer, M., Babst, R., Juretic, A., Gross, T., Hörig, H., Harder, F., and Spagnoli, G.C., Role
of glutamine in the immune response in critical illness, Nutrition, 12, S71, 1996.
111. de Beaux, A.C., O’Riordain, M.G., Ross, J.A., Jodozi, L., Carter, D.C., and Fearon, K.C.,
Glutamine supplemented total parenteral nutrition reduces blood mononuclear cell inter-
leukin-8-release in severe acute pancreatitis, Nutrition, 14, 261, 1998.
112. Morlion, B.J., Köller, M., Wachtler, P., et al., Influence of L-alanyl-L-glutamine (ala-gln) Dipep-
tide on the Synthesis of Leukotrienes and Cytokines in vitro. Paper presented at the 4th
International Congress on the Immune Consequences of Trauma, Shock and Sepsis, Munich,
1997, p. 269.
1382_C36.fm Page 631 Tuesday, October 7, 2003 7:29 PM

Chapter thirty-six: Glutamine and glutamine-containing dipeptides 631

113. Van der Hulst, R.R.W.J., van Kreel, B.K., von Meyenfeldt, M.F., Brummer, R.-J.M., Arends,
J.-W., Deutz, N.E.P., and Soeters, P.B., Glutamine and the preservation of gut integrity, Lancet,
341, 1363, 1993.
114. Tremel, H., Kienle, B., Weilemann, L.S., Stehle, P., and Fürst, P., Glutamine dipeptide sup-
plemented parenteral nutrition maintains intestinal function in critically ill, Gastroenterology,
107, 1595, 1994.
115. Poynton, C.H., Maughan, T., and Elia, M., Glycyl-L-glutamine reduces gut toxicity in bone
marrow transplantation, Blood, 86, 586, 1995.
116. Scheppach, W., Loges, C., Bartram, P., Christl, S.U., Richter, F., Dusel, G., Stehle, P., Fürst, P.,
and Kasper, H., Effect of free glutamine and alanyl-glutamine dipeptide on mucosal prolif-
eration of the human ileum and colon, Gastroenterology, 107, 429, 1994.
117. Kandil, H.M., Argenzio, R.A., Chen, W., Berschneider, H.M., Stiles, A.D., Westwick, J.K.,
Rippe, R.A., Brenner, D.A., and Rhoads, J.M., L-glutamine and L-asparagine stimulate ODC
activity and proliferation in a porcine jejunal enterocyte line, Am. J. Physiol., 269, G591, 1995.
118. Simmons, J.G., Hoyt, E.C., Westwick, J.K., Brenner, D.A., Pucilowska, J.B., and Lund, P.K.,
Insulin-like growth factor-I and epidermal growth factor interact to regulate growth and
gene expression in IEC-6 intestinal epithelial cells, Mol. Endocrinol., 9, 1157, 1995.
119. Rhoads, J.M., Argenzio, R.A., Chen, W., Rippe, R.A., Westwick, J.K., Cox, A.D., Berschneider,
H.M., and Brenner, D.A., L-glutamine stimulates intestinal cell proliferation and activates
mitogen-activated protein kinases, Am. J. Physiol., 2, G943, 1997.
120. Wilmore, D.W., Glutamine saves lives! What does it mean? Nutrition, 13, 375, 1997.
121. Fürst, P., Jonathan E. Rhoads Lecture: a thirty-year odyssey in nitrogen metabolism: from
ammonium to dipeptides, J. Parenter. Enteral Nutr., 24, 197, 2000.
122. McBurney, M., Young, L.S., Ziegler, T.R., and Wilmore, D.W., A cost-evaluation of glutamine-
supplemented parenteral nutrition in adult bone marrow transplantation, J. Am. Diet. Assoc.,
94, 1263, 1994.
123. Scheltinga, M.R., Young, L.S., Benfell, K., Bye, R.L., Ziegler, T.R., Santos, A.A., Antin, J.H.,
Schloerb, P.R., and Wilmore, D.W., Glutamine-enriched intravenous feedings attenuate ex-
tracellular fluid expansion after a standard stress, Ann. Surg., 214, 385, 1991.
124. Jiang, Z.M., Cao, J.D., Zhu, X.G., Zhaos, W.X., Yu, J.C., Ma, E.L., Wang, X.R., Zhu, M.W., Shu,
H., and Liu, Y.W., The impact of alanyl-glutamine on clinical safety, nitrogen balance, intes-
tinal permeability and clinical outcome in postoperative patients: a randomized, double-
blind, controlled study of 120 patients, J. Parenter. Enteral Nutr., 23, S62, 1999.
125. Häussinger, D., Roth, E., Lang, F., and Gerok, W., Cellular hydration state: an important
determinant of protein catabolism in health and disease, Lancet, 341, 1330, 1993.
126. Ballard, T.C., Farag, A., Branum, G.D., Akwardi, O.E., and Opara, E.C., Effect of L-glutamine
supplementation on impaired glucose regulation during intravenous lipid administration,
Nutrition, 12, 349, 1996.
127. Borel, M.J., Williams, P.E., Jabbour, K., Levenhagen, D., Kaizer, E., and Flakoll, P.J., Parenteral
glutamine infusion alters insulin-mediated glucose metabolism, J. Parenter. Enteral Nutr., 22,
280, 1998.
128. Khogali, S.O., Harper, A.A., Lyall, J.A., and Rennie, M.J., Effects of L-glutamine on postis-
chaemic cardiac function: protection and rescue, J. Mol. Cell. Cardiol., 30, 819, 1998.
129. Khogali, S.O., Lyall, J.A., Harper, A.A., and Rennie, M.J., Glutamine is superior to glutamate
in enhancing post ischaemic recovery of the isolated perfused working rat heart, J. Physiol.,
501P, 127, 1997.
130. Griffiths, R.D., Allen, K.D., Andrews, F.J., and Jones, C., Infection, multiple organ failure,
and survival in the intensive care unit: influence of glutamine-supplemented parenteral
nutrition on acquired infection, Nutrition, 18, 456, 2002.
131. Hardy, G., Does glutamine enable severely ill intensive care patients to cope better with
infection and increase their chance of survival? Nutrition, 18, 712, 2002.
132. Vincent, J.L., Nosocomial infection and outcome, Nutrition, 18, 713, 2002.
133. Wilmore, D.W., Why should a single nutrient reduce mortality? Crit. Care Med., 30, 2153, 2002.
1382_C36.fm Page 632 Tuesday, October 7, 2003 7:29 PM
1382_C37.fm Page 633 Tuesday, October 7, 2003 7:31 PM

chapter thirty-seven

Ornithine a-ketoglutarate
Luc Cynober
Hôtel-Dieu Hospital and
Paris 5 University

Contents
Introduction..................................................................................................................................634
37.1 Background.........................................................................................................................634
37.2 Physical and chemical properties of OKG .....................................................................634
37.3 Action of OKG in surgical and trauma patients............................................................634
37.3.1 Burn injury............................................................................................................634
37.3.2 Trauma and sepsis ...............................................................................................635
37.3.3 Surgical patients...................................................................................................636
37.3.4 Cancer ....................................................................................................................636
37.4 OKG and chronic malnutrition........................................................................................636
37.4.1 Elderly patients ....................................................................................................636
37.4.2 Children.................................................................................................................637
37.4.3 Renal failure..........................................................................................................637
37.5 Effect of OKG on gut structure and functions...............................................................637
37.6 OKG, wound healing, and immunity .............................................................................637
37.7 Mechanism of OKG action: a puzzle...............................................................................638
37.7.1 Stimulation of insulin and growth hormone secretion .................................638
37.7.2 Involvement of OKG metabolites .....................................................................639
37.7.2.1 Evidence of a metabolic interaction between ornithine and
a-ketoglutarate .....................................................................................639
37.7.2.2 The possible role of glutamate (Glu) in OKG action ....................639
37.7.2.3 Glutamine .............................................................................................640
37.7.2.4 Polyamines............................................................................................641
37.7.2.5 Proline....................................................................................................641
37.7.2.6 Arginine.................................................................................................641
37.7.2.7 Keto acids..............................................................................................641
37.8 Conclusion ..........................................................................................................................642
Acknowledgment ........................................................................................................................642
References .....................................................................................................................................642

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 633
1382_C37.fm Page 634 Tuesday, October 7, 2003 7:31 PM

634 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Introduction
Besides their importance as building blocks for protein synthesis and as energy substrates,
some amino acids are also vital metabolic regulators. The supplementation of
enteral/parenteral nutrition with these amino acids takes us into a new area — that of
pharmacological nutrition.1 The best-known representatives of this category of amino acids
are arginine (Arg) and glutamine (Gln), which are discussed in other chapters of this book.
Ornithine a-ketoglutarate (OKG) is an interesting molecule because it is a precursor
not only of glutamine and arginine, but also of some other amino and keto acids (such as
proline (Pro) and a-ketoisocaproate) that are probably important in the control of protein
metabolism. OKG also has a potent secretagogic effect on hormones such as insulin and
growth hormone (GH).

37.1 Background
OKG supplementation was conceived during the 1960s for improving the neurological
status of patients with hepatic encephalopathy at a time when it was thought that ammonia
played a major role in coma pathogenesis. The a-ketoglutarate rationale was to trap
ammonia and form glutamate (Glu), which in turn is degraded in ureagenesis, this path-
way being activated by ornithine. OKG is, as might be expected, a potent antihyperam-
moniemic agent,2,3 but unfortunately, clinical results were inconclusive and no correlation
was found between normalization of ammonia levels and coma status.4 However, when
OKG was administered to patients with liver failure, an improvement in their nutritional
status was observed.5,6 Consequently, in the early 1980s the possible benefit of OKG
therapy was evaluated in other situations characterized by malnutrition. The first results
for OKG administered to trauma patients were published in the mid-1980s.7–9

37.2 Physical and chemical properties of OKG


OKG is a salt formed from one molecule of a-ketoglutarate (a-KG) and two molecules of
ornithine (Orn). The pKs of a-KG (1.9 and 1.4) and Orn (10.8) give a pH of ~7 in aqueous
solution over a wide range of OKG concentrations (up to 5 mM).
The stability of OKG in solution (containing glucose, electrolytes, and trace elements)
was studied at 4 and 24˚C over 21 days, a period far exceeding the usual storage times
of parenteral solutions containing OKG. Orn was found to be stable at both temperatures
for 21 days. a-KG was also stable at 4˚C, but less so at 24˚C (98.9 of initial value on day
15, nonsignificant; 93.7 on day 21, p < 0.05 vs. initial values).10

37.3 Action of OKG in surgical and trauma patients


37.3.1 Burn injury
The trauma situation in which the action of OKG has been best defined is burn injury.
Several prospective studies and one retrospective study have been published, all giving
similar results. The results of these studies have been detailed recently in other reviews.11,12
The most significant findings concerning the action of OKG on the nutritional status of
burn patients are reported in Table 37.1. In addition to this biological data, Donati et al.15
have shown that OKG administration has a positive effect on the rate of wound healing,
the quality of the graft, and the reepithelization of donor sites. These findings are further
supported by the recent study of Coudray-Lucas et al.16 These authors found that OKG
1382_C37.fm Page 635 Tuesday, October 7, 2003 7:31 PM

Chapter thirty-seven: Ornithine a-ketoglutarate 635

Table 37.1 Effect of Enterally Administered OKG on Nutritional Status of Burn Patients
Number
(control/ BSA OKG dose Plasma N Nutritional
Ref. treated) (%) (g/day) PHE balance proteins 3MH
7 6/8 21–60 2 ¥ 10 Ø
13, 14 7/7 16–31 10 Ø ≠ ≠ =
15 19/21 20–60 2 ¥ 10 ≠ ≠
16 24/23 25–95 2 ¥ 10 Ø ≠ Ø
17 16/32 20–50 10–30 = ≠ Ø

Note: PHE = phenylalanine; Ø or ≠ = decreased or increased compared with control; and equal sign = not
significantly different from control.

administration reduced the time required for wound healing (61.5 ± 6.2 vs. 93.2 ± 9.1
days). The time required for the last graft was significantly shortened compared with an
isonitrogenously fed group of patients.17 A retrospective study has also been performed.18
One hundred and thirty-six patients met the criteria for inclusion in the study: admission
to the burn unit within 48 h of injury, survival at least 5 days, and application of enteral
nutrition. Forty-three percent of these patients received OKG. The two groups had com-
parable burn surface areas (BSAs). The results indicated an average reduction of 16 days
in the length of hospital stay in the OKG-treated subgroup of severely burned patients
(BSA > 20) (statistically nonsignificant due to wide dispersion of the data). Mortality in
the OKG-treated group showed a trend (p <.09) toward a reduction.
To address questions that cannot, for obvious ethical reasons, be solved by human
studies, a burned rat model was developed. Hypercatabolism was obtained with a double
injury: immersion of the dorsum for 12 sec in water at 95˚C, followed by 24 h of starva-
tion.19 OKG (5 g/kg/day) was tested in a mixture with a low-calorie hyponitrogenous
diet (1.2 g of N/kg/day, 210 kcal/kg/day). OKG administration prevented increased
muscle catabolism (measured ex vivo), muscle loss, and depletion of muscle glutamine
pool induced by burn injury. In addition, OKG treatment increased the Gln pool in the
proximal intestine.19 Using the same protocol, a further study confirmed that OKG inhib-
ited muscle hypercatabolism and indicated that OKG treatment improved protein synthe-
sis in the liver and intestine.20
In summary, in burn injury, a highly catabolic state, the action of OKG involves both
muscle and splanchnic areas, with decreased muscle protein catabolism and increased
liver and intestine anabolism.21

37.3.2 Trauma and sepsis


Four randomized clinical studies are reported in the literature,22–25 one of them a crossover
study. OKG was administered at 20 to 25 g/day by the parenteral22,23 or enteral route.22,24,25
In these four trials, nitrogen balance was significantly better in the OKG group22,23,25 or
during the OKG administration phase in the crossover study.24 Transthyretin was found
to be improved in the OKG group by Demarcq et al.,22 but not by Mertes et al.23 OKG also
limited the trauma-induced drop in plasma glutamine.25
In a rat model with bilateral femur fracture, it has been shown26 that OKG adminis-
tration (mixed in a liquid diet) increases food intake and nitrogen balance and abrogates
the decrease in muscle glutamine content induced by trauma. In a study on endotoxemic
rats (by intraperitoneal administration of lipopolysaccharide (LPS) from Escherichia coli),
we also demonstrated27 that OKG counteracts muscle glutamine depletion.
1382_C37.fm Page 636 Tuesday, October 7, 2003 7:31 PM

636 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

37.3.3 Surgical patients


Vinnars and co-workers have conducted a series of important clinical studies. In the first
one, patients were studied for 5 days after colorectal surgery.8,9 Some patients received
total parenteral nutrition (TPN) (165 kJ/day/kg, 0.15 g of N/kg/day), whereas others
received an isocaloric, isonitrogenous diet including 25 g of OKG to replace 2.7 g of N in
the TPN. Nitrogen balance, urinary 3-methylhistidine (3MH), creatinine, and ammonia
levels were improved in the OKG-treated patients. Arginine was increased and branched-
chain amino acids were decreased in muscle. Muscle Gln levels were slightly (but not
significantly) higher in the OKG group.
In a second study with a similar protocol involving cholecystectomized patients receiv-
ing more nitrogen (0.2 g of N/kg/day) but fewer calories (135 kJ/kg/day), the efficacy of
OKG (0.35 g/kg/day) on nitrogen balance was confirmed.28,29 This effect was probably
even more favorable than reported because the investigators underestimated the amount
of nitrogen in OKG. Furthermore, the usual postoperative decreases in muscle polyribo-
some percentage and Gln levels were abolished in the OKG-treated group. OKG was as
efficient as Gln perfusion itself regarding repletion of muscle Gln.30
The same group also showed that intravenous administration of a-KG alone had an
action on ribosome profiles and muscle Gln content similar to that of OKG.31 However,
patients treated with a-KG received 194 mg of a-KG per kilogram per day, whereas those
treated with OKG received only 126 mg of a-KG per kilogram per day (i.e., 350 mg of
OKG, given that OKG contains two Orn per a-KG). Because a-KG uptake by muscle after
intravenous administration is dose dependent,32 we cannot conclude from these data that
the effects of OKG are solely mediated by a-KG (see below). Finally, a-KG administration
(0.28 g/kg/day) was shown to effectively attenuate the decrease in muscle glutamine
content 24 h after total hip replacement.33
When given enterally to postoperative patients, a-ketoglutarate does not modify
nutritional status.34 This suggests that the administration of a-KG is not as efficient as
OKG (see below).

37.3.4 Cancer
In hypercatabolic rats bearing Yoshida ascites hepatoma AH130, OKG decreased muscle
protein catabolism (measured ex vivo) by 33%.35 In another model (Hepatoma Morris 7777)
OKG did not improve nutritional status in tumor-bearing animals but significantly
improved nitrogen balance and intestine protein synthesis after tumor removal.36 It is
interesting to note that tumor growth was not stimulated by OKG.
In summary, all studies performed in acute situations indicate a beneficial effect of
OKG on nitrogen metabolism. Whether this effect is due to an anabolic20,29 or anticatabolic
action19,20,35 is controversial and probably depends on the tissue and the predominant
underlying mechanism of malnutrition (increased muscle catabolism in severe trauma and
sepsis vs. decreased protein synthesis in less severe injury).

37.4 OKG and chronic malnutrition


37.4.1 Elderly patients
The effects of OKG in elderly patients have been reviewed recently.37 Briefly, OKG
improves the nutritional status of elderly patients at least in part by increasing their food
intake. Such an effect on appetite has also been noticed in normally growing rats fed ad
libitum with an oral liquid diet for 7 days.38 Notably, a study39 indicates that OKG treatment
1382_C37.fm Page 637 Tuesday, October 7, 2003 7:31 PM

Chapter thirty-seven: Ornithine a-ketoglutarate 637

improves the quality of life of malnourished elderly patients. Also, OKG displays a potent
effect in the treatment of bedsores in elderly patients (see Blondé-Cynober et al.37 for a
review).

37.4.2 Children
Six prepubescent children receiving TPN for short bowel syndrome or Crohn’s disease
were studied.40 All were 2 to 4 standard deviations below the 50th height percentile. The
children were studied for two consecutive 5-month periods. OKG (15 g/day) was added
to TPN during the first period and deleted during the second. Height velocity increased
significantly during the OKG period (157 ± 22%) and decreased in the second period in
three subjects. Also, insulin-like growth factor-1/somatomedin C (IGF-1/Sm-C) plasma
levels increased with OKG, and there was a relationship between IGF-1/Sm-C and height
gain velocity.

37.4.3 Renal failure


Hemodialyzed patients with chronic renal failure have been studied in a pharmacokinetic
and clinical study.41. Ten grams of OKG was given orally to fasting hemodialyzed patients.
Compared with healthy subjects, OKG metabolism appeared to be impaired in hemodi-
alyzed patients; peak orinithinemia was shifted to 90 min and remained level up to
210 min. There was no appearance of glutamate or arginine in the plasma after the OKG
load. This probably explains why OKG administration (10 g/day) for 3 months had almost
no effect (except on serum albumin, which increased in OKG-treated patients) on the
nutritional status of patients suffering from chronic renal failure, and points, as discussed
later, to the possible role of the kidney in OKG metabolism and action.

37.5 Effect of OKG on gut structure and functions


After 3 days of starvation, enteral refeeding of rats with OKG (1 g/kg/day) results in a
higher villus height and higher sucrase and aminopeptidase (but not lactase) contents in
the mucosa than in iso-N controls.42
Two studies report the effects of OKG in rats after a 5043 or 80%44 resection of the
proximal small intestine, OKG being used at 1 and 5 g/kg/day, respectively. Adaptative
ileum hypertrophy (in terms of villus height, putrescine content, and ornithine decarbox-
ylase mRNA) was amplified compared with iso-N.
When OKG (1 g/kg/day) was administered preventively to rats before transient
ischemia (by occluding the upper mesenteric artery for 90 min), unlike iso-N controls,
there was a rapid restoration of villi (in 4 h), which was complete in 24 h.45
Finally, OKG administration prevents or limits bacterial translocation after orthotopic
small bowel transplantation,46 after abdominal radiations,47 and after decontamination/E. coli
contamination of the gut followed by LPS administration.48
In summary, these findings suggest that OKG is as efficient as GLN (see Chapter 42)
on gut structure and functions. These promising results justify clinical trials in situations
where gut functions are compromised.

37.6 OKG, wound healing, and immunity


Impairment of wound healing and immunity is a classical feature of injury and is impli-
cated in trauma-associated morbidity and mortality. Deficiency of key nutrients (such as
glutamine) or mediators (such as nitric oxide (NO) or polyamines) may be involved in
1382_C37.fm Page 638 Tuesday, October 7, 2003 7:31 PM

638 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

this pathological process (see other chapters in this book for references). Several studies
provide evidence that OKG improves both wound healing and immunity. In a double-
blind trial, 25 of 52 patients who had undergone reconstructive surgery received 10 to 15 g
of OKG per day orally with their oral nutrition. The treated patients showed more rapid
wound healing (18 ± 1 vs. 26 ± 3 days, p <.01) and a significantly lower rate of complica-
tions.49 Similar results were obtained in patients undergoing head and neck surgery: orally
administered OKG reduced the healing period and the number of complications.50 Con-
sidering also the effect of OKG on healing of burn patients and on bedsores (see above),
it is clear that OKG exerts a potent effect on the healing process.
Studies in rats27,51–56 and man57 indicate an action of OKG on immunity, with an
increase in the lymphocyte count, response to mitogens, synthesis of immunoglobulins,
and polymorphonuclear (PMN) cell and macrophage functions. In addition, OKG admin-
istration to endotoxemic27 or burn58 rats suppresses injury-induced thymus involution.
Interestingly, variations of this last parameter were correlated to glutamine and arginine
muscle contents.27,58

37.7 Mechanism of OKG action: a puzzle


The mechanism of action of OKG is probably multifactorial, linked to the stimulation of
anabolic hormone secretion and the production of Orn and a-KG metabolites. In addition,
several recent studies support the suggestion that most, if not all, OKG action results from
the specific interaction between Orn and a-KG.

37.7.1 Stimulation of insulin and growth hormone secretion


Insulin and GH are potent anabolic hormones, and as their secretion is increased by OKG,
both in healthy subjects and in various pathological states (see Cynober and Coudray-
Lucas12 for a review), they are probably involved in the mechanism of the action of this
compound.
An intriguing feature is that the action of OKG on insulinemia observed after oral
administration to healthy subjects who have first received a standard breakfast59 is much
higher than in 12-h fasted healthy subjects.60 The reasons for this discrepancy are unclear.
However, because pancreatic intracellular a-KG levels appear to be crucial for amino
acid-stimulated insulin release,61 it may be that in fasted subjects, excessive consumption
of a-KG as an energy substrate prevents full expression of Orn-mediated insulin
secretion.
A series of experiments was conducted recently by Schneid et al.62 that clarifies the
action of OKG on insulin secretion at the cellular level. These experiments were performed
on incubated or perfused rat Langherans islets, and the results indicate that OKG stimu-
lates insulin secretion dose dependently, that this effect is additive to those mediated by
glucose, and that it is not reproduced when islets are incubated with either a-KG or Orn
alone. In addition, the use of metabolic blockers (see below) suggests that OKG action is
related to the generation of nitric oxide (NO) and is modulated by glutamine production.
The same group63 conducted the same experiments in vivo in rats (i.e., perfusing OKG)
and reached the same conclusions.
In burn patients, who classically present hyperglycemia together with hyperinsuline-
mia and peripheral insulin resistance, another type of OKG action is observed: high insulin
levels are not modified, but OKG improves glucose tolerance.64
1382_C37.fm Page 639 Tuesday, October 7, 2003 7:31 PM

Chapter thirty-seven: Ornithine a-ketoglutarate 639

37.7.2 Involvement of OKG metabolites


Vaudourdolle et al.65,66 characterized the tissue distribution and metabolism following
enteral or parenteral administration of labeled OKG in healthy rats. OKG and metabolites
concentrate in the splanchnic area, muscles, and salivary glands. As expected, metabolites
are glutamate, glutamine, proline, arginine, and polyamines.

37.7.2.1 Evidence of a metabolic interaction between ornithine and a-ketoglutarate


Since the first report suggesting a possible reciprocal influence of a-KG and Orn on their
respective metabolisms,60 several studies have provided supporting evidence. The bio-
chemical explanation of the metabolic interaction between a-KG and Orn is that these two
molecules possess a common metabolic pathway with a-KG as the end product of Orn,
and Orn the end product of a-KG (Figure 37.1). As the reactions involved are nearly in
equilibrium, providing a-KG and Orn together, these molecules and their direct metabo-
lites are diverted toward other pathways generating proline, glutamine, polyamines, and
arginine, all of which play an important role in maintaining protein homeostasis.
Administration of OKG to healthy subjects (10 g in one bolus) increased plasma
arginine and proline and increased urinary citrulline, effects not seen when subjects
received only Orn or only a-KG.60 When OKG is administered to burn patients, an increase
in plasma glutamine, arginine, and proline levels is observed that is dependent on the
rate of infusion.67
A study in anesthetized catheterized dogs indicated that the major site of a-KG/Orn
interaction was the intestine.68 A bolus injection of OKG (500 mg), but not of a-KG or
ornithine, increased glutamine portal concentration and glutamine uptake by the liver. It
is suggested that providing a-KG and ornithine together decreases the consumption of
glutamine at the venous pole of enterocytes, leading to an increase in glutamine concen-
tration in portal blood. Another possible site for the a-KG/ornithine interaction could be
the kidney.69 The sparing effect of OKG on glutamine utilization would account for the
increase in glutamine in peripheral blood, explaining the decrease in muscle glutamine
output and the increase in free glutamine content in muscle.
In addition, this interaction appears to be specific to ornithine and a-KG, because
replacing ornithine by arginine70 or a-ketoglutarate by a-ketoisocaproate71 does not result
in a similar pattern (i.e., no increase in Arg, Gln, and Pro plasma levels).
Finally, it is likely that the rate of administration is important in the metabolic pattern
observed after OKG administration: increasing the rate of enteral administration (bolus
vs. continuous infusion) increases the production of arginine and glutamine in both
healthy subjects72 and burn patients.67

37.7.2.2 The possible role of glutamate (Glu) in OKG action


There is no proof that Glu per se can affect protein metabolism. However, synthesis of
Glu from a-KG means that one nitrogen molecule is recovered and escapes ureagenesis
and ammoniagenesis, i.e., excretion. This nitrogen economy has been implicated in the
nitrogen-sparing effect of OKG,9 particularly because early studies suggested that Orn
inhibits ureagenesis.73 However, in a study on isolated perfused rat liver, we showed that
Orn, with or without a-KG, increases urea flux moderately.74 Plasma urea decreases after
a bolus of OKG in healthy fasting subjects,60 but increases when OKG is administered at
the same time as enteral nutrition.72 This emphasizes how a molecule such as OKG may
exert varied effects on intermediary metabolism depending on mode of administration
and clinical status.
1382_C37.fm Page 640 Tuesday, October 7, 2003 7:31 PM

640 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

glucose
CO2

Krebs cycle
gluconeogenesis

α-ketoglutarate

NH3
Amino acid
6 7
ketoacid
9
glutamate glutamine
8

Glutamyl-P

Glutamate P5C Proline


semialdehye

Polyamines 1

5
ornithine
urea 2
4
3
Arginine Citrulline
NO

Urea cycle

Figure 37.1 Ornithine and a-ketoglutarate metabolic pathways. Key enzymes are (1) ornithine
aminotransferase, (2) ornithine carbamoyltransferase, (3) NO synthase, (4) arginase, (5) ornithine
decarboxylase, (6) transaminases, (7) glutamate deshydrogenase, (8) glutamine synthetase, and
(9) glutaminase.

37.7.2.3 Glutamine
In addition to the sparing effect of OKG on Gln consumption described above, Gln is
produced from both a-KG and Orn via Glu, which is converted into Gln by the action of
Gln synthetase in muscle, brain, and perivenous hepatocytes. In the latter cells, the rapid
1382_C37.fm Page 641 Tuesday, October 7, 2003 7:31 PM

Chapter thirty-seven: Ornithine a-ketoglutarate 641

metabolism of a-KG into Glu and Gln has been highlighted by studies on isolated perfused
rat liver.74,75
The key role of Gln in the control of protein metabolism, especially in muscle, as an
energy substrate in rapidly dividing cells, and as a precursor of purines and pyrimidines
is the subject of specific chapters in this book and so will not be further discussed here.

37.7.2.4 Polyamines
OKG increases the production of aliphatic polyamines (putrescine, spermine, and sper-
midine).42,76 These molecules produced from Orn are potent inducers of protein synthesis
and cell differentiation.77 Their implication in certain effects of OKG is supported by the
fact that the action of OKG on the synthesis and secretion of albumin by isolated rat
hepatocytes is inhibited by difluoromethylornithine, an inhibitor of Orn decarboxylase
and thus of polyamine formation.78,79 In the same way, difluoromethylornithine inhibits
the OKG-induced proliferation of fibroblasts in culture.80
Lastly, full activation of T lymphocytes depends on polyamine synthesis, and OKG
action on immune function seems to be dependent, at least in part, on the polyamine
pathway.55,56 Conversely, polyamines seem to have no role in OKG-mediated insulin
secretion.62,63

37.7.2.5 Proline
Pro has two important anabolic functions. First, it is the precursor of hydroxyproline, a
qualitatively and quantitatively important amino acid in collagen that has a fundamental
role in tissue repair and wound healing.81 Second, Pro has a stimulating action on hepa-
tocyte DNA and protein synthesis.82

37.7.2.6 Arginine
Arg is the end product of Orn metabolism in the urea cycle and gives rise to Orn again
via the reaction catalyzed by arginase.
Arg has many metabolic effects in common with Orn: both have hormonal secretagogic
effects (stimulating the secretion of growth hormone, insulin, and glucagon), prevent
protein hypercatabolism and weight loss in trauma situations, maintain normal growth,
and accelerate wound healing and display immunological action (see Chapter 35 for more
details). However, arginase is a widely distributed enzyme, and the question is whether
administered Arg acts via Orn formation and the resulting Orn metabolites.83 To date,
there is no answer to this question, but data from Daly et al.84 are significant. Postoperative
patients supplemented enterally with Arg (25 g/day) exhibited equivalent increases in
plasma Orn and Arg. An increase in ornithinemia during Arg-supplemented nutrition has
also been observed in burned rats.85 Finally, after perfusing isolated rat livers with 2 mM
Arg, we observed a marked release of Orn into the perfusion medium.74
Alternatively, OKG action could partly be mediated by the formation of Arg that is
subsequently metabolized into citrulline; the nitric oxide formed from OKG appears to
play a key role in PMN function56 and in OKG-mediated insulin secretion.62,63

37.7.2.7 Keto acids


a-KG stimulates branched-chain keto acid (BCKA) synthesis. This is an indirect process
since it results from transamination reactions, the direction of which depends on the
relative quantities of the substrates and reaction products. It has been shown that the
administration of OKG to healthy subjects results in an increase in the plasma concentra-
tion of Glu and a reduction in that of leucine.60
1382_C37.fm Page 642 Tuesday, October 7, 2003 7:31 PM

642 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

The action of BCKA on protein metabolism has been the subject of numerous studies
(see other chapters in this book). In postoperative patients, OKG and BCKA have the same
effects on nitrogen metabolism, including a decrease in muscle protein catabolism and an
increase in hepatic protein synthesis.

37.8 Conclusion
OKG is a molecule of interest, not only owing to its efficacy in various malnutrition states
and its ability to generate anabolic signals (hormonal and metabolic), but also because
a-KG and ornithine interact at the intermediary metabolism level. This underlines the
importance of carefully evaluating the behavior of infused or perfused nutrients, because
in vivo they can follow metabolic pathways that are not indicated in biochemistry text-
books.

Acknowledgment
The author thanks S. Ngon for her expert secretarial assistance.

References
1. Cerra, F.B., Role of nutrition in the management of malnutrition and immune dysfunction
of trauma, J. Am. Coll. Nutr., 11, 512, 1992.
2. Molimard, R., Charpentier, C., and Lemonnier, F., Modifications de l’amino acidémie des
cirrhotiques sous l’influence de sels d’ornithine, Ann. Nutr. Metab., 26, 25, 1982.
3. James, I.M., Dorf, G., Hall, S., Michel, H., Dojcinov, D., Gravache, G., et al., Effect of ornithine
a-ketoglutarate on disturbances of brain metabolism caused by high blood ammonia, Gut,
13, 551, 1972.
4. Yousfi, A., Bories, P., and Michel, H., L’encéphalopathie hépatique, Presse Méd., 17, 1385, 1988.
5. Tremolieres, J., Scheggia, E., and Flament, C., Effets de l’a-cétoglutarate d’ornithine sur le
bilan azoté et sur la vitesse d’oxydation de l’éthanol, Cah. Nutr. Diet., 7, 2, 1972.
6. Gay, G., Villaume, C., Beaufrand, M.J., Felber, J.P., and Debry, G., Effects of ornithine a-keto-
glutarate on blood insulin, glucagon and amino acids in alcoholic cirrhosis, Biomedicine, 30,
173, 1979.
7. Cynober, L., Saizy, R., N’Guyen Dinh, F., Lioret, N., and Giboudeau, J., Effect of enterally
administered ornithine a-ketoglutarate on plasma and urinary amino acid levels after burn
injury, J. Trauma, 24, 590, 1984.
8. Leander, U., Fürst, P., Vesterberg, K., and Vinnars, E., Nitrogen sparing effect of Ornicetil®
in the immediate post-operative state. I. Clinical biochemistry and nitrogen balance, Clin.
Nutr., 4, 43, 1985.
9. Vesterberg, K., Vinnars, E., Leander, U., and Fürst, P., Nitrogen sparing effect of Ornicetil®
in the immediate post-operative state. II. Plasma and muscle amino acids, Clin. Nutr., 6, 213,
1987.
10. Anglade, P., Arnaud, P.H., Briond, F., and Postaire, E., Stabilité de l’a-cétoglularate d’orni-
thine dans les solutions pour nutrition parentérale, Gastroentérol. Clin. Biol., 14, 370, 1990
(abstract).
11. Cardenas, D., Le Bricon, T., and Cynober, L., a-Cétoglutarate d’ornithine: mécanismes d’ac-
tion et place actuelle en nutrition artificielle, Nutr. Clin. Metabol., 2002, 16:151.
12. Cynober, L. and Coudray-Lucas, C., Ornithine alpha-ketoglutarate administration in surgi-
cal, trauma and cancer-bearing patients, in Nutritional Support in Cancer and Transplant
Patients, Latifi, R. and Marrell, R.C., Eds., R.G. Landes Co., Austin, TX, 2001, p. 145.
13. Cynober, L., Blondé, F., Lioret, N., Coudray-Lucas, C., Saizy, R., and Giboudeau, J., Arterio-
venous differences in amino acids, glucose, lactate and fatty acids in burn patients: effect of
ornithine a-ketoglutarate, Clin Nutr., 5, 221, 1986.
1382_C37.fm Page 643 Tuesday, October 7, 2003 7:31 PM

Chapter thirty-seven: Ornithine a-ketoglutarate 643

14. Cynober, L., Lioret, N., Coudray-Lucas, C., Aussel, C., Ziegler, F., Baudin, B., Saizy, R., and
Giboudeau, J., Action of ornithine a-ketoglutarate on protein metabolism in burn patients,
Nutrition, 3, 187, 1987.
15. Donati, L., Ziegler, F., Pongelli, G., and Signorini, M.S., Nutritional and clinical efficacy of
ornithine a-ketoglutarate in severe burn patients, Clin. Nutr., 18, 307, 1999.
16. Coudray-Lucas, C., Le Bever, H., Cynober, L., De Bandt, J.P., and Carsin, H., Ornithine
a-ketoglutarate improves wound healing in severe burn patients: a prospective randomized
double-blind trial versus isonitrogenous controls, Crit. Care Med., 28, 1772, 2000.
17. De Bandt, J.P., Coudray-Lucas, C., Lioret, N., and Cynober, L., Effect of enterally administered
ornithine a-ketoglutarate in burn patients is dependent upon its mode of administration,
J. Nutr., 128, 563, 1998.
18. Cynober, L., Amino acid metabolism in thermal bums, J. Parenter. Enteral Nutr., 13, 193, 1989.
19. Vaubourdolle, M., Coudray-Lucas, C., Jardel, A., Ziegler, F., Ekindjian, O.G., and Cynober,
L., Action of enterally-administered ornithine alpha-ketoglutarate on protein breakdown in
skeletal muscle and liver of the burned rat, J. Parenter. Enteral Nutr., 15, 517, 1991.
20. Le Boucher, J., Obled, C., Farges, M.C., and Cynober, L., Ornithine a-ketoglutarate modulates
tissue protein metabolism in burn-injured rats, Am. J. Physiol., 273, E557, 1997.
21. Cynober, L., The use of a-ketoglutarate salts in clinical nutrition and metabolic care, Curr.
Opin. Clin. Nutr. Metab. Care, 2, 33, 1999.
22. Demarcq, J.M., Delbar, M., Trochu, G., and Crignon, J.J., Effets de l’a-cétoglutarate d’ornithine
sur l’état nutritionnel des malades de réanimation, Cah. Anesthésiol., 32, 3, 1984.
23. Mertes, N., Mollmann, M., Pfisterer, M., Fürst, P., Puchstein, C., Nolte, C., and Winde, G.,
Nitrogen sparing effect of ornicetil supplemented TPN in hypercatabolic septic or polytrau-
matized patients, in Advances in Ammonia Metabolism and Hepatic Encephalopathy, Soeters, P.B.,
Wilson, J.H.P., and Holm, E., Eds., Elsevier, Amsterdam, 1988, p. 141.
24. Nicolas, F. and Rodineau, P., Essai contrôlé croisé de l’a-cétoglutarate d’ornithine en alimen-
tation entérale, Ouest Med., 35, 711, 1982.
25. Jeevanandam, M., Omithine a-ketoglutarate in trauma, Clin. Nutr., 12, 70, 1993.
26. Jeevanandam, M. and Ali, M.R., Altered tissue amino acid levels in traumatized, growing
rats due to ornithine a-ketoglutarate supplemented oral diet, J. Clin. Nutr. Gastroenterol., 6,
23, 1991.
27. Lasnier, E., Coudray-Lucas, C., Le Boucher, J., Jardel, A., and Cynober, L., Ornithine alpha-
ketoglutarate counteracts thymus involution and glutamine depletion in endotoxemic rats,
Clin. Nutr., 15, 197, 1996.
28. Wernerman, J., Hammarqvist, F., Ali, M.R., and Vinnars, E., Glutamine and ornithine a-keto-
glutarate but not branched-chain amino acids reduce the loss of muscle glutamine after
surgical trauma, Metabolism, 38 (Suppl.), 63, 1989.
29. Wernerman, J., Hammarqvist, F., Von der Decken, A., and Vinnars, E., Ornithine a-ketoglut-
arate improves skeletal muscle protein synthesis as assessed by ribosome analysis and
nitrogen balance post-operatively, Ann. Surg., 206, 674, 1987.
30. Vinnars, E., Hammarqvist, F., Von der Decken, A., and Wernerman, J., Role of glutamine and
its analogs in posttraumatic muscle protein and amino acid metabolism, J. Parenter. Enteral
Nutr., 14, 125S, 1990.
31. Wernerman, J., Hammarqvist, F., and Vinnars, E., a-Ketoglutarate and postoperative muscle
catabolism, Lancet, 335, 701, 1990.
32. Roth, E., Karner, J., Roth-Merten, A., Winkler, S., Valentini, L., and Schaupp, K., Effect of
a-ketoglutarate infusions on organ balances of glutamine and glutamate in anaesthetized
dogs in the catabolic state, Clin. Sci., 80, 625, 1991.
33. Blomqvist, B.J., Hammarqvist, F., Von der Decken, A., and Wernerman, J., Glutamine and
alpha-ketoglutarate attenuate the fall in muscle free glutamine concentration after total hip
replacement, Clin. Nutr., 12 (Suppl.), 12, 1993 (abstract).
34. Wiren, M., Permet, J., and Larsson, J., a-Ketoglutarate supplemented enteral nutrition: effects
on postoperative nitrogen balance and muscle catabolism, Nutrition, 18, 725, 2002.
1382_C37.fm Page 644 Tuesday, October 7, 2003 7:31 PM

644 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

35. Le Bricon, T., Cynober, L., and Baracos, V.E., Ornithine a-ketoglutarate limits muscle protein
breakdown without stimulating tumor growth in rats bearing yoshida ascites hepatoma,
Metabolism, 43, 899, 1994.
36. Le Bricon, T., Cynober, L., Field, C., and Baracos V.E., Supplemental nutrition with ornithine
a-ketoglutarate in rats with cancer-associated cachexia: surgical treatment of the tumor
improves efficacy of nutritional support, J. Nutr., 125, 2999, 1995.
37. Blondé-Cynober, F., Aussel, C., and Cynober, L., Use of ornithine a-ketoglutarate in clinical
nutrition of elderly patients, Nutrition, 2002, 19, 73, 2003.
38. Jeevanandam, M., Ali, M.R., Ramias, L., and Schiller, W.R., Efficacy of ornithine-alpha-
ketoglutarate (OKGA) as a dietary supplement in growing rats, Clin. Nutr., 10, 155, 1991.
39. Brocker, P., Vellas, B., Albarède, J.L., and Poynard, T. A two-center, randomized, double-blind
trial of ornithine oxoglutarate in 194 elderly, ambulatory, convalescent subjects, Age Ageing,
23, 303, 1994.
40. Moukarzel, A., Goulet, O., Solas, J., Marti-Henneberg, C., Buchman, A., Cynober, L., et al.,
Growth retardation in children receiving long-term total parenteral nutrition: effects of
ornithine a-ketoglutarate, Am. J. Clin. Nutr., 60, 408, 1994.
41. Cano, N., Coudray-Lucas, C., Cynober, L., Lacombe, P., Labastie-Coeyreboucq, J., Durbec,
J.P., and Costanzo-Dufetel, J., Ornithine a-ketoglutarate in hemodialyzed patients: metabo-
lism and nutritional effects, Clin. Nutr., 7 (Suppl.), 93, 1988 (abstract).
42. Raul, F., Gosse, F., Galluser, M., Hasselmann, M., and Seiler, N., Functional and metabolic
changes in intestinal mucosa of rats after enteral administration of ornithine a-ketoglutarate
salt, J. Parenter. Enteral Nutr., 19, 145, 1995.
43. Czernichow, B., Nsi-Emvo, E., Galluser, M., Gosse, F., and Raul, F., Enteral supplementation
with ornithine a-ketoglutarate improves the early adaptative response to resection, Gut, 40,
67, 1997.
44. Dumas, F., De Bandt, J.P., Colomb, V., Le Boucher, J., Coudray-Lucas, C., Lavie, S., et al.,
Enteral ornithine a-ketoglutarate enhances intestinal adaptation to massive resection in rats,
Metabolism, 47, 1366, 1998.
45. Duranton, B., Schleiffer, R., Gosse, F., and Raul, F., Preventive administration of ornithine
a-ketoglutarate improves intestinal mucosal repair after transient ischemia in rats, Crit. Care
Med., 26, 120, 1998.
46. De Oca, J., Bettonica, C., Cuadrado, S., Vallet, J., Martin, E., Garcia, A., et al., Effect of oral
supplementation of ornithine a-ketoglutarate on the intestinal barrier after orthotopic small
bowel transplantation, Transplantation, 63, 636, 1997.
47. Kalfarentzos, F., Spiliotis, J., Melachrinou, M., Katsarou, C.H., Spiliopoulou, I., Panagopoulos,
C., et al., Oral ornithine a-ketoglutarate accelerates healing of the small intestine and reduces
bacterial translocation after abdominal radiation, Clin. Nutr., 15, 29, 1996.
48. Schlegel, L., Coudray-Lucas, C., Barbut, F., Le Boucher, J., Jardel, A., Zarrabian, S., and
Cynober, L., Bacterial dissemination and metabolic changes in rats induced by endotoxemia
following E. coli overgrowth are reduced by ornithine a-ketoglutarate administration,
J. Nutr., 130, 2897, 2000.
49. Bouchon, Y. and Merle, M., L’a-cétoglutarate d’ornithine per os dans la prévention des
complications locales de la chirurgie plastique, Ann. Chir. Plast. Esthet., 29, 385, 1984.
50. Pradoura, J.P., Carcassonne, Y., and Spitalier, J.M., Incidence de l’oxoglutarate d’ornithine
(Cétornan) sur la réparation cutanée des malades de carcinologie cervico-faciale opérés, Cah.
ORL, 25, 61, 1990.
51. Albina, J.E., Ornithine a-ketoglutarate enhances macrophages tumor cytotoxicity, Clin. Nutr.,
12 (Suppl.), 2, 1993.
52. Roch-Arveiller, M., Tissot, M., Coudray-Lucas, C., Fontagne, J., Le Boucher, J., Giroud, J.P.,
et al., Immunomodulatory effects of ornithine a-ketoglutarate in rats with burn injuries,
Arch. Surg., 131, 718, 1996.
53. Robinson, L.E., Bussière, F.I., Le Boucher, J., Farges, M.C., Le Boucher, J., Cynober, L.A., et al.,
Amino acid nutrition and immune function in tumour-bearing rats: a comparison of
glutamine-, arginine- and ornithine 2-oxoglutarate-supplemented diets, Clin. Sci., 97, 657,
1999.
1382_C37.fm Page 645 Tuesday, October 7, 2003 7:31 PM

Chapter thirty-seven: Ornithine a-ketoglutarate 645

54. Moinard, C., Chauveau, B., Walrand, S., Felgines, C., Chassagne, J., Caldefie, F., et al.,
Phagocyte functions in stressed rats: comparison of modulation by glutamine, arginine and
ornithine 2-oxoglutarate, Clin. Sci., 97, 59, 1999.
55. Moinard, C., Caldefie, F., Walrand, S., Felgines, C., Vasson, M.P., and Cynober, L., Involve-
ment of glutamine, arginine, and polyamines in the action of ornithine a-ketoglutarate on
macrophage functions in stressed rats, J. Leukoc. Biol., 67, 834, 2000.
56. Moinard, C., Caldefie, F., Walrand, S., Tridon, A., Chassagne, J., Vasson, M.P., et al., Effects
of ornithine 2-oxoglutarate on neutrophils in stressed rats: evidence for the involvement of
nitric oxide and polyamines, Clin. Sci., 102, 287, 2002.
57. Pasquali, J.L., La stimulation lymphocytaire in vitro par le pokeweed mitogène chez les sujets
normaux et les sujets dénutris: influence de sels d’ornithine, Pathol. Biol., 31, 191, 1983.
58. Le Boucher, J., Farges, M.C., Minet, R., Vasson, M.P., and Cynober, L., Modulation of immune
response with ornithine a-ketoglutarate in burn injury: an arginine or glutamine dependen-
cy? Nutrition, 15, 773, 1999.
59. Cynober, L., Vaubourdolle, M., Dore, A., and Giboudeau, J., Kinetics and metabolic effects
of orally administered ornithine a-ketoglutarate in healthy subjects fed with a standardized
regimen, Am. J. Clin. Nutr., 39, 514, 1984.
60. Cynober, L., Coudray-Lucas, C., De Bandt, J.P., Guéchot, J., Aussel, C., Salvucci, M., and
Giboudeau, J., Action of ornithine a-ketoglutarate, ornithine hydrochloride and calcium
a-ketoglutarate on plasma amino acid and hormonal patterns in healthy subjects, J. Am. Coll.
Nutr., 9, 2, 1990.
61. Lenzen, S., Schmidt, W., Rustenbeck, I., and Panten, U., 2-Ketoglutarate generation in pan-
creatic B-cell mitochondria regulates insulin secretory action of amino acids and 2-ketoacids,
Biosci. Rep., 6, 163, 1986.
62. Schneid, C., Darquy, S., Cynober, L., Reach, G., and De Bandt, J.P., Effects of ornithine
a-ketoglutarate on insulin secretion in rat pancreatic islets: implication of nitric oxide syn-
thase and glutamine synthetase pathways, Brit. J. Nutr., 89, 249, 2003.
63. Schneid, C., De Bandt, J.P., Cynober, L., Torres, E., Reach, G., and Darquy, S., In vivo induction
of insulin secretion by ornithine a-ketoglultarate: involvement of nitric oxide and glutamine,
Metabolism, 2003, 52, 344.
64. Vaubourdolle, M., Cynober, L., Lioret, N., Coudray-Lucas, C., Aussel, C., Saizy, R., and
Giboudeau, J., Influence of enterally administered ornithine a-ketoglutarate on hormonal
patterns in burn patients, Burns, 13, 349, 1987.
65. Vaubourdolle, M., Jardel, A., Coudray-Lucas, C., Ekindjian, O.G., Agneray, J., and Cynober,
L., Metabolism and kinetics of parenterally administered ornithine and a-ketoglutarate in
healthy and burn animals, Clin. Nutr., 7, 105, 1988.
66. Vaubourdolle, M., Jardel, A., Coudray-Lucas, C., Ekindjian, O.G., Agneray, J., and Cynober,
L., Fate of enterally administered ornithine in healthy animals: interactions with a-ketoglu-
tarate, Nutrition, 5, 183, 1989.
67. Le Bricon, T., Coudray-Lucas, C., Lioret, N., Lim, S.K., Plassart, F., Schlegel, L., et al., Orni-
thine a-ketoglutarate metabolism after enteral administration in burn patients: bolus com-
pared with continuous infusion, Am. J. Clin. Nutr., 65, 512, 1997.
68. Winkler, S., Hotzenbein, Th., Karner, J., and Roth, E., Kinetics of organ specific metabolism
of a bolus injection into the jejunum of glutamine, a-ketoglutarate, ornithine and ornithine
a-ketoglutarate, Clin. Nutr., 12, 57, 1993.
69. Welbourne, T., a-Ketoglutarate, ornithine and growth hormone displace glutamine depen-
dent for ammoniagenesis and enhance renal base regeneration and function, Clin. Nutr., 12,
49, 1993.
70. Le Boucher, J., Coudray-Lucas, C., Lasnier, E., Jardel, A., Ekindjian, O.G., and Cynober, L.,
Enteral administration of ornithine a-ketoglutarate or arginine a-ketoglutarate: a compara-
tive study of their effects on glutamine pools in burn-injured rats, Crit. Care Med., 25, 293,
1997.
71. Jeevanandam, M., Holaday, N.J., Ali, R., and Petersen, S.R., Ornithine a-ketoglutarate sup-
plementation is more effective than its component salts in traumatized rats, J. Nutr., 126,
2141, 1996.
1382_C37.fm Page 646 Tuesday, October 7, 2003 7:31 PM

646 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

72. Payne-James, J., Grimble, G., Cahill, E., and Silk, D.B.A., Enteral administration of ornithine-
oxoglutarate (OKGA) in man: effects on hormone profiles and nitrogen (N) metabolism,
J. Parenter. Enteral Nutr., 13 (Suppl.), 1, 36, 1989 (abstract).
73. Hunter, A. and Downs, C. E., The inhibition of arginase by amino acids, J. Biol. Chem., 157,
427, 1945.
74. De Bandt, J.P., Cynober, L., Lim, S.K., Coudray-Lucas, C., Poupon, R., and Giboudeau, J.,
Metabolism of ornithine, a-ketoglutarate and arginine in perfused rat liver, Br. J. Nutr., 73,
227, 1995.
75. Stoll, B. and Haussinger, D., Functional hepatocyte heterogeneity: vascular 2-oxoglutarate is
almost exclusively taken up by perivenous, glutamine synthetase-containing hepatocytes,
Eur. J. Biochem., 181, 709, 1989.
76. Jeevanandam, M., Holaday, N.J., and Sli, M.R., Altered tissue polyamine levels due to
ornithine-alpha-ketoglutarate in traumatized growing rats, Metabolism, 41, 1204, 1992.
77. Grillo, M.A., Metabolism and function of polyamines, Int. J. Biochem., 17, 943, 1985.
78. Lescoat, G., Theze, N., Fraslin, J.M., Pasdeloup, N., Kneip, B., and Guguen-Guillouzo, C.,
Influence of ornithine on albumin synthesis by fetal and neonatal hepatocytes maintained
in culture, Cell. Differ., 21, 21, 1987.
79. Lescoat, G., Desvergne, B., Pasdeloup, N., Glaise, D., Gazeau, J.P., Molimard, R., et al., Effect
of ornithine a-ketoglutarate on albumin secretion by adult rat hepatocyte co-cultures, in
Liver Cells and Drugs, Guillouzo, A., Ed., INSERM/John Libbey Eurotext, Paris, 1988, pp. 164,
431.
80. Vaubourdolle, M., Salvucci, M., Coudray-Lucas, C., Cynober, L., and Ekindjian, O.G., Action
of ornithine a-ketoglutarate on DNA synthesis by human fibroblasts in vitro, Cell. Dev. Biol.,
26, 187, 1990.
81. Adams, E., Metabolism of proline and of hydroxyproline, Int. Rev. Connect. Tissue Res., 5, 1,
1970.
82. Perez-Sala, D., Parilla, R., and Ayuso, M.S., Key role of L-alanine in the control of hepatic
protein synthesis, Biochem. J., 241, 491, 1987.
83. Cynober, L., The role of arginine and related compounds in intestinal functions, in Organ
Metabolism and Nutrition: Ideas for Future Critical Care, Kinney, J.M. and Tucker, H.N., Eds.,
Raven Press, New York, 1994, p. 245.
84. Daly, J.M., Reynolds, J., Thom, A., Kinsley, L., Dietrick-Gallagher, M., Shou, J., and Ruggieri,
B., Immune and metabolic effects of arginine in the surgical patient, Ann. Surg., 208, 512, 1988.
85. Saito, H., Trocki, O., Wang, S.L., Gonce, S.J., Joffe, S.N., and Alexander, J.W., Metabolic and
immune effects of dietary arginine supplementation after burn, Arch. Surg., 122, 784, 1987.
1382_C38.fm Page 647 Tuesday, October 7, 2003 7:34 PM

section B

Formulas devoted to specific situations


1382_C38.fm Page 648 Tuesday, October 7, 2003 7:34 PM
1382_C38.fm Page 649 Tuesday, October 7, 2003 7:34 PM

chapter thirty-eight

Amino acid support in patients


with catabolic illness
Nicole M. Daignault
Emory University School of Medicine
Daniel P. Griffith
Emory University School of Medicine
Thomas V. Nattakom
Memorial Medical Center
Thomas R. Ziegler
Emory University School of Medicine

Contents
Introduction..................................................................................................................................649
38.1 Protein requirements and administration during catabolic states...........................650
38.2 Influence of energy intake on protein utilization .......................................................651
38.3 Are current amino acid solutions unbalanced? ..........................................................652
38.4 Amino acid administration in renal or hepatic insufficiency ...................................654
38.5 Use of modified amino acid formulations in renal or hepatic dysfunction............655
38.6 Protein needs and amino acid support in pediatric patients ....................................656
38.7 Use of glutamine in nutrition support .........................................................................658
38.8 Clinical efficacy of arginine supplementation in catabolic illness ...........................659
38.9 The use of cysteine in catabolic illness .........................................................................660
38.10 Summary...........................................................................................................................660
References .....................................................................................................................................661

Introduction
Erosion of body protein occurs as a component of the hypermetabolic response to stress,
during such states as trauma, burn injury, major operations, severe inflammation, and
sepsis. In severe illness, kinetic measurements indicate that net body protein catabolism
(negative nitrogen balance) occurs due to increased rates of muscle protein breakdown
that exceed increased rates of whole-body protein synthesis.1–2 The increased protein
synthesis is due, in part, to increased muscle intracellular amino acid availability due to

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 649
1382_C38.fm Page 650 Tuesday, October 7, 2003 7:34 PM

650 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

accelerated protein catabolism.3 With lesser degrees of stress, such as following minor
operations, the rate of protein synthesis may be reduced, while protein breakdown rates
remain unchanged or are slightly increased, again resulting in net loss of body protein.4
Skeletal muscle supplies endogenous amino acids to be utilized by other body tissues
during stress, and these amino acids (a large proportion of which are alanine and
glutamine (GLN)) may be released by muscle at rates three to four times the normal rate.4
Plasma amino acids are utilized for such diverse processes as gluconeogenesis, synthesis
of circulating and tissue proteins and immunologic cells, wound healing, maintenance of
organ structure and function, and direct fuel sources.1–4 The magnitude of the protein
catabolic responses to stress is directly proportional to the severity of illness and is influ-
enced by the amount and composition of dietary energy and protein.1–4
The mechanisms regulating amino acid flux and other aspects of protein metabolism
during catabolic stress are incompletely understood but clearly are influenced by coun-
terregulatory hormones (especially cortisol and catecholamines) and levels of proinflam-
matory cytokines (e.g., tumor necrosis factor and the interleukins).1,5 These hormonal
signals interrelate with other clinical factors, including control of infectious and inflam-
matory foci, dietary intake, acid–base status, and activity level, to determine the net protein
catabolic effect during hypercatabolic states.2,4–5
Erosion of lean tissue during catabolic stress is associated with poor clinical out-
comes.1–5 However, the quality and quantity of nutrition support (including amino acid
support) and the timing of these interventions are uncertain in many clinical situations.6–10
An important caveat is that stress-induced skeletal muscle protein breakdown represents
a homeostatic mechanism to provide critical amino acids for use by gut mucosa, immune
cells, wounds, and other tissues (e.g., glutamine, discussed in Chapters 5, 19, 34, and 36).
Thus, use of anabolic agents, such as growth hormone, in critically ill patients may worsen
clinical outcomes, in part, due to decreased net release of muscle glutamine and other
amino acids from muscle, and result in diminished availability of endogenous amino acids
for systemic uses.11,12

38.1 Protein requirements and administration during catabolic states


A recent detailed reevaluation of the existing published literature suggests that the rec-
ommended dietary allowance (RDA), designed to cover needs of 97.5% of the healthy
population for protein in healthy adults is 0.83 g of good-quality protein/kg/day13 (see
Chapter 27 for more details). Requirements for protein and amino acids in children and
adults with catabolic stress remain uncertain but are undoubtedly widely variable depend-
ing on clinical circumstances. Several studies in adult humans evaluated the relationship
between the amount of protein intake and the degree of nitrogen retention and changes
in protein kinetics (rates of whole-body breakdown and synthesis) in hypercatabolic states.
Wolfe et al.14 studied the effect of two levels of protein intake (1.4 and 2.2 g/kg/day)
combined with adequate energy intake (25% above resting energy expenditure) on leucine
kinetics in six severely burned adult patients. Protein intake of 1.4 g/kg/day resulted in
net balance between protein synthesis and catabolism; however, no further beneficial effect
on net body protein synthesis was observed when protein intake was increased to
2.2 g/kg/day.14
Shaw et al.15 studied protein kinetics in septic adult patients receiving standard
parenteral nutrition (PN) providing adequate nonprotein energy (50% as glucose and 50%
lipid emulsion) with three levels of amino acid intake (1.1, 1.5, and 2.2 g of protein
equivalents per kilogram per day). A significant reduction in net body protein catabolism
(~50%) occurred when the protein dose was increased from 1.1 to 1.5 g/kg/day. However,
when the protein load was increased (to 2.2 g/kg/day), net protein catabolism increased
1382_C38.fm Page 651 Tuesday, October 7, 2003 7:34 PM

Chapter thirty-eight: Amino acid support in patients with catabolic illness 651

to levels with infusion of the 1.1 g/kg/day dose. The authors concluded that protein
intake of 1.5 g/kg/day was optimal for protein utilization in these patients; larger protein
intakes resulted in increased urea generation but no further improvement in protein
anabolism.15
The previous data are consistent with results in other patients where diets providing
greater than 2 g of protein per kilogram per day (and adequate calories) were unable to
induce net nitrogen retention in septic patients,9 or were unable to reduce net protein
catabolism compared to effects of a lower-protein dose (1.75 g/kg/day).16 Therefore, the
available data (much of it from studies performed in the 1980s) suggest that the optimal
protein intake for most hypercatabolic adult patients should be in the range of 1.5 to
1.75 g/kg/day, in the absence of renal or hepatic failure.17 However, recent data suggest
that lower doses of protein provision (1.0 to 1.2 g/kg/day) during the first 2 weeks of
critical illness are adequate to minimize loss of body protein during the first few weeks
of critical illness.17,18 Others suggest that a reasonable intake goal is 1.2 g/kg/day based
upon premorbid or dry body weight, or 1.0 g/kg/day of measured body weight when
other information is unavailable.6 Finally, some patients such as burned individuals or
clinically stable, convalescing patients may be able to efficiently utilize increased intakes
(generally, £2.0 g/kg/day).4 Although it is important in clinical practice to correct for
fluid-induced weight alterations, additional randomized, prospective, controlled trials are
indicated prior to the universal application of these recommendations in critically ill
patients.19 It is not difficult to meet protein requirements using available intravenous amino
acid formulations; however, it may be necessary to add protein supplements (e.g., hydro-
lyzed whey or casein powder) to some diets or standard tube feeding solutions in order
to meet these protein intake goals. Patients with mild to moderate malnutrition, milder
catabolic illnesses, or in the convalescent phase after illness may require lower protein
intakes (1.0 to 1.5 g/kg/day) for lean tissue maintenance or repletion. Individual amino
acid requirements are doubtlessly influenced by a number of diverse factors, including
organ dysfunction, severity and type of injury, previous nutritional status, age, sex, and
other variables (see below).

38.2 Influence of energy intake on protein utilization


The recommendations for protein intake are based on simultaneous provision of adequate
energy to enable efficient utilization of administered protein in synthetic pathways. A
linear relationship exists between nitrogen balance and nitrogen intake when energy is
not limiting.2,4 When nitrogen intake is fixed, increasing the dietary calories will improve
nitrogen balance in a curvilinear fashion.1,4 Insufficient provision of nonprotein energy
results in utilization of administered amino acids (which may be oxidized in energy-
generating pathways with waste nitrogen excreted as urea). Increasing the amount of
nonprotein calories (especially carbohydrate) in such settings enhances utilization effi-
ciency of dietary amino acids and reduces urea generation.
Current recommendations for energy (and protein) intake in stable and in critically
ill patients are considerably lower (by 30 to 50%) than the amounts commonly used in
the 1970s and 1980s.2,7,18 It is apparent that overfeeding (hyperalimentation) contributed
to the reported rates of respiratory insufficiency, liver dysfunction, azotemia, hyperglyce-
mia, and electrolyte disturbances associated with the delivery of specialized nutrition
support, particularly in critically ill patients.2,7,18 Studies have demonstrated energy
requirements in some critically ill patients of ª25 kcal/kg during the first week of illness
or total energy expenditure that closely approximates the total measured resting energy
expenditure.18,20 However, energy requirements in trauma and septic patients may rise
considerably after that period, although they are quite variable depending on clinical
1382_C38.fm Page 652 Tuesday, October 7, 2003 7:34 PM

652 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

circumstances.20 It is unclear whether provision of energy at rates of >25 to 30 kcal/kg/day


benefits catabolic patients metabolically or clinically, as limited dose–response data are
available.6,7,18 Requirements clearly vary considerably between and within individual cat-
abolic patients, depending on complications and the overall severity of underlying illness
or injury.18 Lower-than-maintenance caloric intakes during nutritional support may be
indicated, based on the metabolic milieu (e.g., in the presence of hyperglycemia) and other
factors, until the patient has stabilized and enters the convalescent phase of illness; at this
point the hormonal/cytokine signals that inhibit protein repletion have abated, and the
patient’s activity level has increased, both of which favor protein anabolism.5
Some studies suggest that carbohydrate exerts superior protein-sparing effects com-
pared to effects of isocaloric standard lipid emulsions, although controversy exists on this
point.4 Many nutrition support specialists typically provide 70 to 80% of nonprotein
calories as glucose, with the remainder as fat emulsion in parenteral nutrition support.
The amount of carbohydrate is decreased or insulin is administered to maintain blood
glucose concentrations at levels of <5.5 mmol/l, a level of glucose control recently shown
to markedly decrease morbidity and mortality in ICU patients.21
In normal subjects, nitrogen equilibrium may be achieved with a calorie-to-nitrogen
ratio of approximately 350:1. During critical illness, protein economy decreases, and this
may affect the optimal ratio between protein and calorie intake. Most clinicians recom-
mend total calorie-to-nitrogen ratios of between 100:1 and 150:1 in hospitalized patients
with catabolic illness or those requiring nutritional repletion, but these ratios may need
to be increased with renal or hepatic dysfunction, which inhibit protein utilization (see
below).
The clinical and nutritional efficacy of hypocaloric, high-protein feeding regimens
in critically ill obese patients has been documented. Dickerson et al.22 demonstrated a
net protein anabolism and wound healing in mildly to moderately stressed obese subjects
receiving hypocaloric, high-dose amino acid (2.13 ± 0.59 g/kg of ideal body weight/day)
parenteral feedings.22 Consistently low respiratory quotients derived from the indirect
calorimetry results confirmed an increased oxidation of endogenous fat stores as an
energy source during administration of the low-calorie feeding regimen. The data indi-
cated that the level of nitrogen intake may be the most significant parameter influencing
nitrogen balance. Likewise, Choban et al.23 demonstrated no difference in nitrogen bal-
ance in acutely ill obese patients receiving hypocaloric vs. normoenergetic parenteral
feedings when a high-dose amino acid regimen (2.0 g/kg of ideal body weight/day)
was provided.
A recent retrospective analysis examined the potential benefits of hypocaloric, high-
protein enteral feedings (<20 kcal/kg of adjusted body weight/day vs. >20 kcal/kg/day;
each regimen providing 2.0 g of protein/kg of ideal body weight/day) in critically ill
obese trauma and surgical patients.24 Patients who were administered the hypocaloric
regimen had decreased length of ICU stay, decreased antibiotic days, and a trend toward
decreased mechanical ventilation days. There was no difference in nitrogen balance iden-
tified between the two groups; however, patients were determined to be in negative
nitrogen balance during the first 2 weeks of the trial period.24 Further research to better
clarify protein and amino acid requirements in critically ill obese patients is indicated.

38.3 Are current amino acid solutions unbalanced?


Synthetic L-amino acid solutions available for intravenous use should provide estimated
daily requirements of the eight classical essential amino acids: isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan, and valine25 (Table 38.1). Commercially
available amino acid solutions in the U.S. also contain L-histidine, which is essential for
1382_C38.fm Page 653 Tuesday, October 7, 2003 7:34 PM

Chapter thirty-eight: Amino acid support in patients with catabolic illness 653

Table 38.1 Composition of Some Representative Amino Acid Injection Products


15% 10% 10% FreAmine 6%
Clinisol Aminosyn III Trophamine
Amino acid product (Baxter) II (Abbott) (B. Braun) (B. Braun)
Nitrogen content (g/dl) 2.37 1.53 1.53 0.93
EAA content (% of total) 51% 43% 46% 57%
BCAA content (% of total) 18% 26% 23% 33%
Aromatic AA + MET content 14% 7% 12% 11%
(% of total)

Note: None of these products contain GLN. Aromatic amino acids include tryptophan and phenylalanine.
MET = methionine.

infants and may be semiessential for older children and adults.25 Standard amino acid
solutions typically provide 43 to 57% of the total amino acid nitrogen as a mixture of all
eight essential amino acids (EAA) (Table 38.1). The proportion of EAA is greater in the
solutions modified for pediatrics and patients with hepatic or renal failure (primarily due
to greater percentage of branched-chain amino acids (BCAA))25 (Table 38.2). Commercially
available amino acid solutions for adults typically provide 19 to 26% of amino acids as
BCAA, and the BCAA-enriched formulations provide 36 to 46% of total protein as BCAA.
A mixture of seven to nine nonessential amino acids (NEAA) make up the remainder of
the solutions; however, the largest amount of NEAA is usually represented by alanine,
arginine, and proline.25 Of interest, arginine may have beneficial effects in clinical nutrition
(see below and elsewhere in this volume), and this potentially conditionally essential
amino acid is present at 7 to 14% of the total amino acid content in most solutions. A
variety of newer amino acid formulations containing glutamine dipeptides and with
different concentrations of both EAA and NEAA have been introduced commercially
outside of the U.S. and are also undergoing active clinical investigation.26,27 In addition,
use of single amino acids (e.g., GLN, cysteine) or combinations of amino acids given
intravenously or enterally are undergoing active clinical investigation.28

Table 38.2 Some Representative Disease-Specific Amino Acid Injection Products


Condition Commercial product Amino acid characteristic
Stress/sepsis Aminosyn–HBC, 7% Similar to standard AA products except
increased BCAA
FreAmine–HBC Similar to standard AA products except
increased BCAA
BranchAmin, 4% BCAA-only supplement
Liver disease HepatAmine, 8% Increased BCAA; decreased aromatic AA
Renal failure Aminosyn–RF, 5.2% EAA only, plus histidine and arginine
NephrAmine, 5.4% EAA only, plus histidine
RenAmin, 6.5% EAA and NEAA; increased proportion of
EAA (60%)
Pediatric Trophamine, 6%, 10% Both products similar to standard AA
Aminosyn–PF, 7%, 10% products but with lower concentrations
of glycine, methionine, and
phenylalanine; also contain histidine and
tyrosine

Note: Aromatic amino acids include tryptophan and phenylalanine.


1382_C38.fm Page 654 Tuesday, October 7, 2003 7:34 PM

654 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

In enteral solutions used as meal supplements or for tube feeding, amino acids are
provided in the form of intact proteins (e.g., milk, egg white solids), partially hydrolyzed
proteins (e.g., casein, lactalbumin), dipeptides, or crystalline amino acids. A mixture of
these protein sources is provided in many complete enteral formulas. These formulations
provide known adequate amounts of EAA for stressed patients; however, because the
amino acid composition is usually derived directly from high biologic value proteins,
such as egg or milk, the overall amino acid pattern (especially the NEAA pattern) of
enteral formulas may be more complete or balanced relative to parenteral amino acid
solutions. The possible clinical implications of these differences between parenteral and
enteral amino acid patterns are unclear at the present time and require further clinical
investigation.
Current intravenous amino acid formulas may provide limiting or excessive amounts
of certain amino acids and therefore may be unbalanced in certain patient subgroups.26 It
is difficult to determine amino acid requirements or to diagnose amino acid deficiency as
measured by plasma levels or free amino acid levels in tissues. Amino acid levels are in
a dynamic state of flux in many clinical situations and are influenced by prior nutritional
status, amino acid and energy intake, underlying diseases, severity of illness, and patient
age and sex.26 For example, low plasma or intracellular free histidine, serine, taurine,
tyrosine, valine, and threonine levels occur in uremic patients, and low cysteine, taurine,
and tyrosine levels have been documented in cirrhotic patients receiving PN, suggesting
possible conditional deficiency of these amino acids.26,29
Of interest, some amino acids are currently totally lacking in most parenteral amino
acid formulations (taurine, cysteine, and glutamine) or are present in very small amounts
(tyrosine). Poor solubility (cysteine, tyrosine, and glutamine) or instability in solution
(cysteine conversion to cystine) may limit their use in the free form in parenteral diets.25
However, supplementation with these amino acids may be beneficial in selected clinical
conditions, in the form of dipeptides, which are much more soluble and stable in solution.26
Newer amino acid formulations are being investigated that provide increased amounts of
certain amino acids such as arginine, cysteine, cystine, glutamate, and glutamine (see
below) and less of amino acids potentially provided in excess (e.g., glycine, alanine,
proline, phenylalanine).26 For example, Bérard et al.27 recently investigated the metabolic
effects of a parenteral nutrition formulation containing supplemental arginine and
glutamate vs. an isonitrogenous, conventional control PN solution in postoperative
patients. The investigators found that the experimental amino acid formulation promoted
improved nitrogen balance, limited myofibrillar protein catabolism, and increased
glutamine levels vs. the controls.27 Further clinical study is needed on the efficacy of these
new amino acid formulations in organ failure and other clinical conditions.

38.4 Amino acid administration in renal or hepatic insufficiency


Patients with liver or kidney insufficiency often do not efficiently utilize enteral or
parenteral amino acids. Intolerance to dietary protein in these states may be manifested
by worsening azotemia or increasing levels of serum transaminases, bilirubin, and alkaline
phosphatase. When organ insufficiency is severe, altered mental status and encephalopathy
may occur with protein infusion. For PN use, modified amino acid solutions have been
developed for these conditions (Table 38.2); however, evidence for the superiority of these
products compared to standard amino acid formulations on clinical outcome is lacking.25
Based upon the recently published guidelines by the Board of Directors of the Amer-
ican Society for Parenteral and Enteral Nutrition (ASPEN) and data derived from stable
home patients, dietary protein restriction of 0.6 to 0.8 g/kg/day in hospital patients with
chronic renal failure and patients not requiring dialysis is advised.18,30,31 The process of
1382_C38.fm Page 655 Tuesday, October 7, 2003 7:34 PM

Chapter thirty-eight: Amino acid support in patients with catabolic illness 655

various dialysis techniques has been shown to lead to increased amino acid elimination
from the bloodstream. Thus, the prescribed protein intake for patients who are receiving
these therapies should be adjusted accordingly. A protein intake goal of approximately
1.2 g/kg/day for hospitalized patients receiving hemodialysis and 1.2 to 1.3 g/kg/day
for patients undergoing chronic ambulatory peritoneal dialysis is suggested.18 Likewise,
the dietary protein (or amino acid) intake of patients who are receiving continuous renal
replacement therapies should be increased by approximately 0.2 g/kg/day in order to
compensate for the additional losses.31 The ability to achieve a positive nitrogen balance
in hypermetabolic patients with acute renal failure (ARF) with protein intakes of greater
than or equal to 1.0 g/kg/day has been demonstrated. Based upon these results, the
current ASPEN guidelines suggest that protein intakes of 1.5 to 1.8 g/kg/day for severely
malnourished or hypercatabolic patients with ARF may be indicated, as tolerated based
on the azotemic response.18 Protein requirements are increased in patients with hepatic
failure due to altered protein metabolism and increased catabolism that varies with the
stage of disease and the degree of physiological stress.32 Traditionally, patients who suf-
fered from hepatic encephalopathy were treated with protein (or amino acid) restrictions.
The starvation response generated was found to lead to an amino acid profile similar to
that observed in patients receiving a high-protein diet. These findings may be attributed
to the quicker onset of gluconeogenesis as a result of diminished glycogen stores and a
subsequent increase in skeletal muscle catabolism. Thus, provision of adequate protein in
this patient population is advocated. According to the recently published ASPEN guide-
lines,18 a protein (or amino acid) restriction of 0.5 to 0.6 g/kg/day is advocated only in
patients with acute, overt encephalopathy and can be increased as tolerated by 0.25 to
0.5 g/kg/day until the goal intake of 1.0 to 1.5 g/kg is obtained.18 Vegetable sources of
protein may be better tolerated than those derived from animal origin, although the data
in this regard are inconclusive. Possible etiologies for the efficacy of this dietary modifica-
tion include reduced aromatic amino acid and methionine provision, a higher fiber content
to facilitate bacterial and nitrogen fecal excretion, and a more effective use of nitrogen.33
In estimating protein requirements in patients with hepatic or renal failure, it is
important to correct for body weight alterations due to ascites, edema, or anasarca. When
possible to obtain, a usual body weight (prior to fluid accumulation) or a dry weight
(i.e., postdialysis or parencentisis) is useful.34 As a general guideline, the amino acid or
protein intake should be decreased or increased in proportion to the degree of azotemia
or hepatic dysfunction and the tempo of change in these organ systems.

38.5 Use of modified amino acid formulations in renal or hepatic


dysfunction
The administration of specially modified amino acid formulations in hepatic dysfunction
remains controversial. Patients with chronic hepatic failure may develop elevated plasma
levels of methionine and aromatic amino acids (tryptophan and phenylalanine) with
correspondingly reduced plasma levels of the BCAA leucine, valine, and isoleucine. The
data suggest that the hyperammonemia often associated with liver disease may lead to
an increased utilization of BCAA and stimulation of glutamine synthesis, with a subse-
quent reduction in plasma BCAA levels.35 Thus, the use of BCAA-enriched enteral and
parenteral solutions with lowered amounts of aromatic amino acids and methionine has
been advocated in patients with liver failure and encephalopathy.36,37 Although few well-
designed prospective studies comparing amino acid solutions with isonitrogenous, isoca-
loric, BCAA-enriched diets in matched catabolic patients have been published, available
data suggest that BCAA-enriched solutions (with adequate carbohydrate energy) may
1382_C38.fm Page 656 Tuesday, October 7, 2003 7:34 PM

656 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

improve the degree of encephalopathy in some patients with hepatic failure.18,36,37 A case
report documented the ability of 45 g/day of an oral BCAA-enriched feeding to induce
a rapid improvement in grade 4 hepatic encephalopathy, that was sustained for at least
12 months.38 The author’s view is that the current data are insufficient to support the
routine use of BCAA supplementation in this patient population. Of interest, BCAA-
enriched solutions have not been consistently shown to improve nitrogen balance com-
pared to isonitrogenous solutions in critical illness and other catabolic states.18 Further
study of BCAA-enriched solutions in catabolic states is warranted.
In an older controlled trial in acute alcoholic hepatitis, reduced mortality was noted
with provision of a standard non-BCAA-enriched parenteral amino acid mixture provid-
ing ~80 g of protein per day as a supplement to oral diet.39 Administration of a standard
complete tube feeding in another study of acute alcoholic hepatitis improved encephal-
opathy and liver function tests compared to ad libitum-fed control patients.40 Therefore,
early nutritional support in acute alcoholic liver disease appears to have a clinical benefit.18
Malnutrition is a major contributor to morbidity and mortality in patients with chronic
renal failure, and intradialytic parenteral nutrition (IDPN) has been considered to be useful
in some of these individuals, especially when hospitalized for a catabolic illness.41 How-
ever, the clinical efficacy of this approach has not been established.41 Thus, IDPN should
be potentially used only in malnourished chronic renal failure patients with gastro-
intestinal dysfunction or venous access problems who are otherwise poor candidates for
enteral or parenteral support.18,41
Early studies suggested that PN solutions containing only EAA and glucose were
clinically beneficial in the setting of acute renal failure. However, subsequent studies failed
to confirm beneficial effects of amino acid solutions providing only EAA.18,42,43 These
formulations are potentially deleterious, as they provide insufficient amino acids (arginine,
citrulline, ornithine) to support the Krebs urea cycle, and subsequent hyperammonemia
and encephalopathy may develop.42,43 Thus, in renal failure, provision of standard amino
acid solutions containing adequate EAA and a mixture of NEAA is recommended, using
usual guidelines for protein dosage and responses to azotemia.18 It is particularly impor-
tant to provide adequate nonprotein energy in this patient population. Dialysis should
not be initiated in azotemic patients solely to enable provision of large amino acid loads.
When patients are being dialyzed as part of routine clinical care, standard amino acid
formulations are used when specialized parenteral or enteral nutrition is deemed neces-
sary. Some clinicians provide amino acid-containing parenteral nutrient solutions at the
time of dialysis to supplement oral intake; however, the clinical efficacy of this approach
requires further study.

38.6 Protein needs and amino acid support in pediatric patients


A detailed discussion of amino acid administration in pediatric patients is beyond the
scope of this chapter, but the recent ASPEN guidelines provide detailed recommendations
for pediatric nutrition support.18 These guidelines recommend protein intakes of 2.0 to
4.0 g/kg/day in low-birth-weight (<1500 g) neonates and 2.0 to 3.0 g/kg/day in full-term
neonates.18 Infants and children (1 to 10 years of age) require protein at approximately
1.0 to 1.2 g/kg/day for optimal growth. Current common clinical practice is to give hos-
pitalized children approximately 1.5 g/kg/day protein (or amino acid, in children requir-
ing PN).18 During the acute phase of critical illness, protein intakes of 1.0 to 1.5 g/kg/day
in older children and doses of 2.0 to 3.0 g/kg/day in toddlers and infants are suggested,
with the same caveats regarding renal and liver dysfunction outlined above for adults.18
Higher-protein doses were well tolerated in a study by Alexander et al.44 who fed burned
children high-protein enteral/parenteral feedings. Patients receiving the higher-protein
1382_C38.fm Page 657 Wednesday, October 8, 2003 1:48 PM

Chapter thirty-eight: Amino acid support in patients with catabolic illness 657

doses (4.9 g/kg/day; calorie-to-nitrogen ratio, 110:1) demonstrated improved immune


function and nitrogen balance and reduced infection and mortality compared to children
on a lower-protein diet (3.8 g/kg/day; calorie-to-nitrogen ratio, 150:1).44
The amino acid and protein requirements of children are more complex than in adults,
as they are altered as a function of age and growth phase (e.g., infancy, puberty) as well
as injury severity. For example, neonates appear to require cysteine and taurine and infants
require additional histidine and tyrosine; thus, pediatric amino acid formulations used in
PN are supplemented with histidine and tyrosine, while cysteine hydrochloride is typically
added as a single amino acid admixture on the day of PN infusion. The evidence in the
present literature suggests that the catabolic state in pediatric patients is associated with
increased lipolysis, proteolysis, and loss of cell mass.45,46 Coss-Bu et al.47 conducted a cross-
sectional study in critically ill, mechanically ventilated pediatric patients who were receiv-
ing PN support. Their objective was to examine the relationship of the patient’s metabolic
state and nutrient consumption, substrate utilization, and nitrogen balance. The study
confirmed earlier findings of the existence of a hypermetabolic state in physiologically
stressed pediatric patients.46 A negative nitrogen balance was determined to be associated
with increased protein oxidation rates, indicating a stress-induced higher-protein utiliza-
tion.47 Likewise, a positive nitrogen balance was only achieved in subjects with a protein
intake that on average exceeded the rate of expenditure.47 This study thus demonstrates
the importance of adequate protein and energy intake in the maintenance of nitrogen
balance in children.
Some studies have investigated the use of specialized amino acids or amino acid
formulations to improve clinical outcome in critically ill pediatric patients. Very low birth
weight (VLBW) infants who received enteral glutamine supplementation were found to
have a decreased incidence of hospital-acquired sepsis and improved tolerance to enteral
feedings, as compared to controls.48 Likewise, stable isotope kinetic studies with leucine
have been used to demonstrate a protein-sparing effect to glutamine supplementation in
VLBW infants, as evidenced by a reduction in endogenous protein breakdown.49 It has
also been established that glutamine and glutamate are major constituents of breast milk
and increase 20-fold and by a factor of 2.5, respectively, as lactation progresses past
3 months.49 Glutamine and glutamate are thought to serve as potential sources for neu-
rotransmitters and gut mucosal protection.49 The presently available infant formulas con-
tain little or no added glutamine or glutamate. The potential benefits of supplementation
of glutamine in infant formulas, therefore, warrants further investigation. Furthermore,
infants diagnosed with necrotizing enterocolitis (NEC) were found to have lower plasma
levels of both glutamine and arginine, suggesting that specific amino acid deficiencies
may predispose low-birth-weight and premature infants to this condition.50,51 In a recent
double-blind controlled study, 152 premature infants received supplemental L-Arg
(1.5 mmol/kg/day) or placebo with oral feeds and/or parenteral nutrition during the
first days of life.52 NEC developed only in 5 infants in the Arg supplemented group,
compared with 21 infants in the placebo group (p < 0.001). The authors did not observe
any significant adverse effects, including hypotension, with Arg supplemention. Plasma
Arg levels increased significantly at 14 and 28 days with supplementation and were lower
in those infants with the diagnosis of NEC.52 Glutamine levels in cerebrospinal fluid of
children with suspected viral or bacterial meningitis have also been determined to be
lower than normal and improve in correspondence to improvements with the child’s
clinical condition.49
Gazzaniga et al. 45 studied the effects of a pediatric amino acid formula
(6% Trophamine, B. Braun) administered as part of balanced PN on plasma amino acids
and nitrogen balance in stable adult patients requiring PN for a variety of illnesses. This
pediatric formula contains a concentration of 33% BCAA (intermediate between adult
1382_C38.fm Page 658 Tuesday, October 7, 2003 7:34 PM

658 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

standard and BCAA-enriched solutions); increased amounts of histidine, tyrosine, and


arginine; and glutamate, aspartate, and taurine. Patients received Trophamine with or
without added cysteine HCl (0.5 mmol/kg/day) at a protein dose of 1.5 g/kg/day for at
least 6 days. Positive nitrogen balance was achieved in each group of adult patients
(cysteine supplemented, +3.21 ± 0.70 g/day, vs. no cysteine, +1.75 ± 0.70 g/day; no
significance), and plasma tyrosine levels normalized in each group. A significant positive
correlation between the increase in taurine levels in plasma and improved nitrogen balance
was noted only in the group receiving cysteine supplementation.45 Positive correlations
were also noted between the improvement in nitrogen balance and levels of cystine, total
cysteine + cystine, tyrosine, and ornithine.45 Although a control group receiving standard
adult solutions was not studied, these findings suggest possible benefits in adult patients
of amino acid solutions tailored initially for infants and children.

38.7 Use of glutamine in nutrition support


Perhaps no single amino acid has received as much attention in the clinical nutrition
research community in recent years as GLN.53–63 The interest in GLN derives from many
studies performed in animals and a growing number of clinical investigations demon-
strating clinical and metabolic efficacy of GLN supplementation, particularly during cat-
abolic states.53–57 These data are largely reviewed in detail elsewhere in this book (see
Chapters 34 and 36). The initial double-blind, randomized clinical outcome studies on
GLN-supplemented PN were performed in patients undergoing bone marrow transplan-
tation (BMT) in whom decreased infection rates59 and decreased length of hospital stay
were documented.59,60 It is important to note, however, that these studies were done at a
time in which BMT regimens utilized higher doses of chemotherapy and radiation and
before the era of routine use of peripheral blood stem cells, colony-stimulating growth
factors, and other improvements in care. The subjects undergoing BMT in the late 1980s
to early 1990s were more catabolic and had a higher rate of hospital infection than
individuals undergoing BMT with current regimens. Thus, additional studies of GLN
efficacy in PN (and of PN itself) need to be conducted under current BMT regimens before
general recommendations for use of this amino acid in BMT can be made. Recent studies
on use of oral or enteral GLN as a strategy to prevent mucositis in patients undergoing
high-dose chemotherapy have shown mixed results.54,64 Also, two recent studies have been
published examining use of PN supplemented with alanine–GLN dipeptide in mixed
groups of autologous BMT recipients.65,66 One study suggested that GLN supplementation
was not beneficial and, in fact, may have worsened clinical outcomes,65 while the other
indicated that GLN supplementation safely improved immune reconstitution and
decreased mucositis.66 Of interest, a recent meta-analysis64 of the available data concluded
that GLN-supplemented PN appears to be of clinical benefit in BMT patients; however,
the authors suggested that randomized, controlled trials comparing routes of GLN admin-
istration (enteral vs. parenteral) and including control groups not receiving aggressive
nutritional support are indicated.
The mechanisms of GLN-induced beneficial effects in catabolic patients remain unclear
but are likely multifactorial. Reduced infection and microbial colonization may be due in
part to a reduction in net protein catabolism,57–59 increased blood lymphocytes,54,66 main-
tained intestinal mucosal integrity, or other aspects of improved gut mucosal function,53,61
by maintenance of antioxidant capacity via glutathione production67,68 or other factors.
Recent studies have also explored the concept of providing growth factors with GLN-
supplemented nutrition support as a method to induce benefical additive or synergistic
metabolic and clinical effects.53,69 Other studies have demonstrated the important meta-
bolic relationship between GLN and BCAA.70
1382_C38.fm Page 659 Tuesday, October 7, 2003 7:34 PM

Chapter thirty-eight: Amino acid support in patients with catabolic illness 659

As summarized elsewhere in this volume (see Chapters 34 and 36), GLN administra-
tion has been well tolerated clinically; however, dose–response data are still relatively
limited.53,54,71 Several studies have demonstrated that free L-GLN is stable in solution when
heat sterilization techniques are not employed and standard precautions are taken in
manufacturing.54,72 GLN-containing dipeptides have also been developed for parenteral
use and are soluble and heat stable.26

38.8 Clinical efficacy of arginine supplementation in catabolic illness


As outlined elsewhere in this book (see Chapter 35), the beneficial role of arginine sup-
plementation in critically ill patients is being increasingly investigated, sometimes as an
intravenous agent27,52 or as a single enterally supplied agent,73,74 but most commonly in
combination with other immunostimulatory nutrients in enteral tube feedings.75–78 Argi-
nine has been shown to exhibit anabolic and immunostimulatory properties and also
appears to facilitate wound healing.79 L-arginine is the substrate for synthesis of nitric
oxide (NO) that may mediate some of its effects in catabolic states.79,80 Kinetic studies in
burned and septic pediatric patients suggest that arginine oxidation increases with these
forms of severe catabolic stress; this and other evidence suggest that arginine is a condi-
tionally indispensable amino acid for maintaining body protein homeostasis and nutrition
after burn injury or sepsis,81–83 but metabolic and clinical outcome data with arginine-
supplemented feedings in various types of catabolic illness have been variable to
date.27,52,75–78,84
Arginine supplementation to enteral feedings has been shown to be readily absorbed
and metabolized. Preiser et al.75 attributed the observed rise in plasma ornithine concen-
tration in response to provision of arginine-enriched feedings to the enzymatic conversion
by arginase in the gut and the liver. Others have observed significant reductions in
catheter-related sepsis in critically ill patients receiving arginine-enriched enteral feed-
ings.76 The administration of immune-enhancing formulas containing arginine as com-
pared with a high-protein control feeding was reported to significantly reduce mortality
and infection rates in septic ICU patients.77 However, this observed improvement in clinical
outcome is likely multifactorial and may be partially due to the other immune-enhancing
agents provided, rather than being specifically arginine induced.77 PN supplemented with
arginine and glutamate was found to improve nitrogen balance and attenuate protein
myofibrillar catabolism in postsurgical, moderately stressed subjects.27 The supplemental
arginine provided was thought to be the primary stimulus for glutamine generation, with
ornithine as its intermediate product. During periods of catabolic stress these three amino
acids play important metabolic functions, including stimulation of proteolysis, improved
nitrogen balance, and polyamine synthesis.27 A recent randomized controlled trial showed
that preoperative or perioperative oral supplementation with a specialized formula
enriched in arginine, n-3 fatty acids, and RNA significantly decreased infectious compli-
cations and length of hospital stay in patients undergoing elective surgery for gastro-
intestinal cancer.78 Some concerns have been raised, however, with regards to the potential
for immune-enhancing substrates such as arginine to aggravate the systemic inflammatory
response or induce vasodilation (via NO) in hemodynamically unstable patients and
thereby worsen clinical outcome.79,80 Recently, studies have appeared suggesting that
enteral products containing a combination of beta-hydroxy beta-methylbutyrate (a leucine
metabolite), GLN, and arginine have anabolic effects in patients with wasting due to cancer
or acquired immunodeficiency syndrome (AIDS).85,86 Further research is clearly needed
on the safety and efficacy of arginine-enriched enteral products in catabolic states, as these
are readily available to clinicians.
1382_C38.fm Page 660 Tuesday, October 7, 2003 7:34 PM

660 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

38.9 The use of cysteine in catabolic illness


As noted above, an injectible cysteine hydrochloride product is available for use in children
as an admixture to PN. Cysteine is a critical and rate-limiting constituent of the tripeptide
antioxidant GSH, and there appears to be an increased requirement for cysteine during
periods of catabolic illness.87,88 Thus, there is an increased interest in supplementing this
amino acid in catabolic states. In a recent study, standard cysteine and cystine-free PN
given after BMT were associated with significant oxidation of both the GSH and the
cysteine–cystine redox pools in plasma, as well as a decrease in blood alpha- and gamma-
tocopherol concentrations.87 Thus, standard PN does not appear to support systemic
antioxidant status. The excessive production of oxygen free radicals and diminished
endogenous antioxidant mechanisms have been implicated as contributors to multiple-
organ failure in septic shock. Ortolani et al.89 studied whether intravenous GSH or
N-acetyl-L-cysteine (NAC) + GSH were protective in patients with early septic shock. All
30 patients enrolled received standard septic shock therapy, including PN. One group
received 70 mg of intravenous GSH/kg/day; a second group received 70 mg of intra-
venous GSH/kg/day and 75 mg of intravenous NAC/kg/day. Compared to controls not
receiving GSH or NAC, significant decreases in indices of peroxidative stress were
observed at day 5 of treatment in the GSH-supplemented group, which were even more
marked in the subjects receiving GSH + NAC.89
The amino acid requirements of the parenterally fed neonate remain poorly defined,
and several lines of evidence suggest that intake of sulfur amino acids (cysteine and
methionine) is inadequate in conventionally fed ill neonates and children.90–92 For exam-
ple, in children with N-dependent short gut syndrome, cysteine addition to PN solutions
formulated with a pediatric amino acid product increased low plasma taurine concen-
trations to within the normal reference range.91 In a recent study of children with
kwashiorkor (known to exhibit oxidative stress and low GSH and cysteine concentra-
tions in plasma), early dietary supplementation with cysteine (NAC) significantly
increased the GSH synthesis rate in association with an increased erythrocyte cysteine
concentration.92 Several studies on the use of GSH precursors, including NAC (given
enterally and parenterally), are currently in progress in critically ill and other groups of
catabolic patients.

38.10 Summary
Amino acid support in catabolic states is evolving rapidly. The amounts of protein, amino
acid, and energy prescribed by physicians have decreased, enteral feeding is being increas-
ingly emphasized, and newer formulations for clinical use containing additional amounts
of conditionally essential amino acids such as glutamine and arginine are undergoing
clinical trials. In addition, administration of recombinant growth factors, modified lipid
products, and increased amounts of certain vitamins and antioxidant substrates are also
being evaluated in the clinical setting, and these approaches will be combined with new
amino acid formulations in clinical investigation.
Increasing recognition of the important metabolic roles of certain amino acids and
their dietary requirements with organ dysfunction and catabolic illness will doubtless
lead to a growing number of modified amino acid products for clinical care. For example,
the potential anabolic and immunomodulatory role of GLN and arginine in critically ill
patients has been described briefly in this chapter and will be outlined in more detail in
other chapters of this book. Additional trials have investigated the efficacy of specific
combinations of nutrients known to individually attenuate proteolysis, in order to
1382_C38.fm Page 661 Tuesday, October 7, 2003 7:34 PM

Chapter thirty-eight: Amino acid support in patients with catabolic illness 661

ascertain their potential synergistic response. The utility of other amino acid-related agents
in catabolic illness are increasingly being investigated, including beta-hydroxy beta-
methylbutyrate, a-ketoglutarate, and ornithine a-ketoglutarate (a known GLN and Arg
precursor — see elsewhere in this volume). Further research is needed to better define the
patients with catabolic stress who may benefit from these newer strategies for amino acid
support, as well the cost:benefit ratio of these approaches.

References
1. Ziegler, T.R., Gatzen, C., and Wilmore, D.W., Strategies for attenuating protein-catabolic
responses in the critically ill, Annu. Rev. Med., 45, 459, 1994.
2. Ziegler, T.R., Fuel metabolism and nutrient delivery in critical illness, in Principles and Practice
of Endocrinology and Metabolism, Becker, K.L., Ed., J.B. Lippincott Co., Philadelphia, 2001,
p. 2102.
3. Biolo, G., Fleming, R.Y., Maggi, S.P., et al., Inverse regulation of protein turnover and amino
acid transport in skeletal muscle of hypercatabolic patients, J. Clin. Endocrinol. Metab., 87,
3378, 2002.
4. Souba, W.W. and Wilmore, D.W., Diet and nutrition in the care of the patient with surgery,
trauma, and sepsis, in Modern Nutrition in Health and Disease, 8th ed., Shils, M.E., Olson, J.A.,
and Shike, M., Eds., Lea and Febiger, Philadelphia, 1994, p. 1207.
5. Estívariz, C.F. and Ziegler, T.R., Endocrinology of critical illness, in The Oxford Textbook of
Endocrinology and Diabetes, Wass, J.A.H. and Shalet, S.M., Eds., Oxford University Press,
Oxford, 2002, p. 1457.
6. Bistrian, B. and Babineau, T., Optimal protein intake in critical illness? Crit. Care Med., 26,
1476, 1998.
7. Jeejeebhoy, K.N., Total parenteral nutrition: potion or poison, Am. J. Clin. Nutr., 74, 160, 2001.
8. Streat, S.J., Beddoe, A.H., and Hill, G.L., Aggressive nutritional support does not prevent
protein loss despite fat gain in septic intensive care patients, J. Trauma, 27, 262, 1987.
9. Warnold, I., Eden, E., and Lundholm, K., The inefficiency of total parenteral nutrition to
stimulate protein synthesis in moderately malnourished patients, Ann. Surg., 208, 143, 1988.
10. Biolo, G., Fleming, R.Y., Maggi, S.P., et al., Inhibition of muscle glutamine formation in
hypercatabolic patients, Clin. Sci., 99, 189, 2000.
11. Ruokonen, E. and Takala, J., Dangers of growth hormone therapy in critically ill patients,
Curr. Opin. Clin. Nutr. Metab. Care, 5, 199, 2002.
12. Biolo, G., Iscra, F., Bosutti, A., et al., Growth hormone decreases muscle glutamine production
and stimulates protein synthesis in hypercatabolic patients, Am. J. Physiol., 279, E323, 2000.
13. Rand, W.M., Pellett, P.L., and Young, V.R., Meta-analysis of nitrogen balance studies for
estimating protein requirements in healthy adults, Am. J. Clin. Nutr., 77, 109, 2003.
14. Wolfe, R.R., Goodenough, R.D., Burke, J.F., and Wolfe, M.H., Response of protein and urea
kinetics in burn patients to different levels of protein intake, Ann. Surg., 197, 163, 1983.
15. Shaw, J.H.F., Wildbor, M., and Wolfe, R.R., Whole body protein kinetics in severely septic
patients: the response to glucose infusion and total parenteral nutrition, Ann. Surg., 205, 288,
1987.
16. Shaw, J.H.F. and Wolfe, R.R., Whole-body protein kinetics in patients with early and ad-
vanced gastrointestinal cancer: the response to glucose infusion and total parenteral nutri-
tion, Surgery, 103, 148, 1988.
17. Ishibashi, N., Plank, L., Sando, K., and Hill, G., Optimal protein requirements during the
first 2 weeks after the onset of critical illness, Crit. Care Med., 26, 1529, 1998.
18. American Society for Parenteral and Enteral Nutrition Board of Directors, Guidelines for the
use of parenteral and enteral nutrition in adult and pediatric patients, J. Parenter. Enteral
Nutr., 26 (Suppl. 1), 1SA, 2002.
19. Obled, C., Papet, I., and Breuille, D., Metabolic basis of amino acid requirements in acute
diseases, Curr. Opin. Clin. Nutr. Metab. Care, 5, 189, 2002.
1382_C38.fm Page 662 Tuesday, October 7, 2003 7:34 PM

662 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

20. Uehara, M., Plank, L.D., and Hill, G.L., Components of energy expenditure in patients with
severe sepsis and major trauma: a basis for clinical care, Crit. Care Med., 27, 1295, 1999.
21. Van den Berghe, G., Wouters, P., Weekers, F., et al., Intensive insulin therapy in critically ill
patients, N. Engl. J. Med., 345, 1359, 2001.
22. Dickerson, R.N., Rosato, E., and Mullen, J.M., Net protein anabolism with hypocaloric
parenteral nutrition in obese stressed patients, Am. J. Clin. Nutr., 44, 747, 1986.
23. Choban, P., Burge, J., and Flancbaum, L., Hypoenergetic nutrition support in hospitalized
obese patients: a simplified method for clinical application, Am. J. Clin. Nutr., 66, 546, 1997.
24. Dickerson, R.N., Boschert, K.J., Kudsk, K.A., and Brown, R.O., Hypocaloric enteral tube
feeding in critically ill obese patients, Nutrition, 18, 241, 2002.
25. Mirtallo, J.M., Parenteral formulas, in Clinical Nutrition, Vol. 3, Parenteral Nutrition, Rombeau,
J.L. and Rolandelli, R.H., Eds., W.B. Saunders, Philadelphia, 2001, p. 118.
26. Fürst, P., Old and new substrates in clinical nutrition, J. Nutr., 128, 789, 1998.
27. Bérard, M., Zazzo, J., Condat, P., et al., Total parenteral nutrition enriched with arginine and
glutamate generates and limits protein catabolism in surgical patients hospitalized in inten-
sive care units, Crit. Care Med., 28, 3637, 2000.
28. Wischmeyer, P., Lynch, J., Liedel, J., et al., Glutamine administration reduces gram-negative
bacteremia in severely burned patients: a prospective, randomized, double-blind trial versus
isonitrogenous control, Crit. Care Med., 29, 2075, 2001.
29. Rudman, D. and Williams, P.J., Nutrient deficiencies during total parenteral nutrition, Nutr.
Rev., 43, 1, 1985.
30. Levey A., Adler, S., Caggiula, A., et al., Effects of dietary protein restriction on the progression
of advanced renal disease in the modification of diet in disease study, Am. J. Kidney Dis., 27,
652, 1996.
31. Druml, W., Metabolic aspects of continuous renal replacement therapies, Kidney Int., 56
(Suppl. 72), S56, 1999.
32. Patton, K. and Aranda-Michel, J., Nutritional aspects in liver disease and liver transplanta-
tion, Nutr. Clin. Pract., 17, 332, 2002.
33. Mizock, B., Nutritional support in hepatic encephalopathy, Nutrition, 15, 220, 1999.
34. Pomposelli, J. and Burns, D., Nutrition support in the liver transplant patient, Nutr. Clin.
Pract., 17, 341, 2002.
35. James, H., Branched chain amino acids in hepatic encephalopathy, Am. J. Surg., 183, 424, 2002
36. Fischer, J.E., Branched-chain enriched amino acid solutions in patients with liver failure: an
early example of nutritional pharmacology, J. Parenter. Enteral Nutr., 14, 249S, 1990.
37. Freund, H. and Hanani, M., The metabolic role of branched-chain amino acids, Nutrition,
18, 287, 2002.
38. Chalasani, N. and Gitlin, N., Severe recurrent hepatic encephalopathy that responded to oral
branched chain amino acids, Am. J. Gastroenterol., 91, 1266, 1996.
39. Nasrallah, S.M. and Galambos, J.T., Amino acid therapy for alcoholic hepatitis, Lancet, ii,
1276, 1980.
40. Kearns, P.J., Young, H., Garcia, G., et al., Accelerated improvement of alcoholic liver disease
with enteral nutrition, Gastroenterology, 102, 200, 1992.
41. Kopple, J., Therapeutic approaches to malnutrition in chronic dialysis patients: the different
modalities of nutritional support, Am. J. Kidney Dis., 33, 180, 1999.
42. Mirtallo, J., Schneider, P., Mavko, K., et al., A comparison of essential and general amino
acid infusions in the nutritional support of patients with compromised renal function,
J. Parenter. Enteral Nutr., 6, 109, 1982.
43. Freund, H., Atamian, S., and Fischer, J.E., Comparative study of parenteral nutrition in renal
failure using essential and nonessential amino acid containing solutions, Surg. Gynecol.
Obstet., 151, 652, 1980.
44. Alexander, J.W., MacMillan, B.G., Stinnett, J.D., Ogle, C.K., Bozian, R.C., Fischer, J.E., Cakes,
J.B., Morris, M.J., and Krummel, R., Beneficial effects of aggressive protein feeding in severely
burned children, Ann. Surg., 192, 505, 1980.
45. Gazzaniga, A.B., Waxman, K., Day, A.T., et al., Nitrogen balance in adult hospitalized patients
with the use of a pediatric amino acid model, Arch. Surg., 123, 1275, 1988.
1382_C38.fm Page 663 Tuesday, October 7, 2003 7:34 PM

Chapter thirty-eight: Amino acid support in patients with catabolic illness 663

46. Bodamer, O., Leonard, J., Tasker, R., et al., Protein turnover in critically ill children, Eur. J.
Ped., 156 (Suppl. 1), S59, 1997.
47. Coss-Bu, J., Klish, W., Walding, D., et al., Energy metabolism, nitrogen balance, and substrate
utilization in critically ill children, Am. J. Clin. Nutr., 74, 664, 2001.
48. Neu, J., Roig, J., Meetze, W., Veerman, M., et al., Enteral glutamine supplementation for very
low birth weight infants decreases morbidity, J. Pediatr., 131, 691, 1997.
49. Ball, P. and Hardy, G., Glutamine in pediatrics: where next, Nutrition, 18, 451, 2002.
50. Zamora, S.A., Amin, H.J., McMillan, D.D., et al., Plasma L-arginine concentrations in prema-
ture infants with necrotizing enterocolitis, J. Pediatr., 131, 226, 1997.
51. Becker, R.M., Wu, G., Galanko, J.A., et al., Reduced serum amino acid concentrations in
infants with necrotizing enterocolitis, J. Pediatr., 137, 785, 2000.
52. Amin, H.J., Zamora, S.A., McMillan, D.D., et al., Arginine supplementation prevents necro-
tizing enterocolitis in the premature infant, J. Pediatr., 140, 425, 2002.
53. Ziegler, T.R., Bazargan, N., Leader, L.M., et al., Glutamine and the gastrointestinal tract, Curr.
Opin. Clin. Nutr. Metab. Care, 3, 355, 2000.
54. Ziegler, T.R., Glutamine supplementation in cancer patients receiving bone marrow trans-
plantation and high-dose chemotherapy, J. Nutr., 131, 2578S, 2001.
55. Novak, F., Heyland, D.K., Avenell, A., et al., Glutamine supplementation in serious illness:
a systematic review of the evidence, Crit. Care Med., 30, 2002.
56. Neu, J., DeMarco, V., and Li, N., Glutamine: clinical applications and mechanisms of action,
Curr. Opin. Clin. Nutr. Metab. Care, 5, 69, 2002, 2002.
57. Goeters, C., Wenn, A., Mertes, N., et al., Parenteral L-alanyl-L-glutamine improves 6-month
outcome in critically ill patients, Crit. Care Med., 30, 2032, 2002.
58. Stehle, P., Mertes, N., Puchstein, Ch., and Furst, P., Effect of parenteral glutamine peptide
supplements on muscle glutamine loss and nitrogen balance after major surgery, Lancet, i,
231, 1989.
59. Ziegler, T.R., Young, L.S., Benfell, K., et al., Clinical and metabolic efficacy of glutamine-
supplemented parenteral nutrition after bone marrow transplantation: a randomized, dou-
ble-blind, controlled study, Ann. Intern. Med., 116, 821, 1992.
60. Schloerb, P.R. and Amare, M., Total parenteral nutrition with glutamine in bone marrow
transplantation and other clinical applications (a randomized, double-blind study), J. Parent-
er. Enteral Nutr., 17, 407, 1993.
61. van der Hulst, R.R.W., van Kreel, B.K., von Meyenfeldt, M.F., et al., Glutamine and the
preservation of gut integrity, Lancet, 341, 1363, 1993.
62. Walser, M., Misinterpretation of nitrogen balances when glutamine stores fall or are replen-
ished, Am. J. Clin. Nutr., 53, 1337, 1991.
63. Haussinger, D., Roth, E., Lang, F., et al., Cellular hydration state: an important determinant
of protein catabolism in health and disease, Lancet, 341, 1330, 1993.
64. Murray, S.M. and Pindoria, S., Nutrition support for bone marrow transplant patients,
Cochrane Database Syst. Rev., 2, CD002920, 2002.
65. Pytlik, R., Benes, P., Patorkova, M., et al., Standardized parenteral alanyl-glutamine dipeptide
supplementation is not beneficial in autologous transplant patients: a randomized, double-
blind, placebo controlled study, Bone Marrow Transplant., 30, 953, 2002.
66. Piccirillo, N., De Matteis, S., Laurenti, L., et al., Glutamine-enriched parenteral nutrition after
autologous peripheral blood stem cell transplantation: effects on immune reconstitution and
mucositis, Haematologica, 88, 192, 2003.
67. Hong, R.W., Rounds, J.D., Helton, W.S., et al., Glutamine protects liver glutathione after
lethal hepatic injury, Ann. Surg., 215, 114, 1992.
68. Flaring, U.B., Rooyackers, O.E., Wernerman, J., et al., Glutamine attenuates post-traumatic
glutathione depletion in human muscle, Clin. Sci. (Lond.), 104, 275, 2003.
69. Hammarqvist, F., Sandgren, A., Andersson, K., et al., Growth hormone together with
glutamine-containing total parenteral nutrition maintains muscle glutamine levels and re-
sults in a less negative nitrogen balance after surgical trauma, Surgery, 129, 576, 2001.
70. Holecek, M., Relation between glutamine, branched-chain amino acids, and protein metab-
olism, Nutrition, 18, 130, 2002.
1382_C38.fm Page 664 Tuesday, October 7, 2003 7:34 PM

664 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

71. Poindexter, B., Ehrenkranz, E.A., Stoll, B.J., et al., Effect of parenteral glutamine supplemen-
tation on plasma amino acid concentrations in extremely low-birth-weight infants, Am. J.
Clin. Nutr., 77, 737, 2003.
72. Khan, K., Hardy, G., McElroy, B., et al., The stability of L-glutamine in total parenteral
solutions, Clin. Nutr., 10, 193, 1991.
73. Daly, J.M., Reynolds, J., Thom, A., et al., Immune and metabolic effects of arginine in the
surgical patient, Ann. Surg., 208, 512, 1988.
74. Daly, J.M., Lieberman, M.D., Goldfine, J., et al., Enteral nutrition with supplemental arginine,
RNA, and omega-3 fatty acids in patients after operation: immunologic, metabolic, and
clinical outcome, Surgery, 112, 56, 1992.
75. Preiser, J., Berre, J., Van Gossum, A., Cynober, L., Vray, B., Carpentier, Y., and Vincent, J.,
Metabolic effects of arginine addition to the enteral feeding of critically ill patients, J. Parenter.
Enteral Nutr., 25, 182, 2001.
76. Caparros, T., Lopez, J., and Grau, T., Early enteral nutrition in critically ill patients with a
high-protein diet enriched with arginine, fiber, and antioxidants compared with a standard
high-protein diet: the effect on nosocomial infections and outcome, J. Parenter. Enteral Nutr.,
25, 308, 2001.
77. Galban, C., Montejo, J., Mesejo, A., et al., An immune-enhancing enteral diet reduces mor-
tality rate and episodes of bacteremia in septic intensive care unit patients, Crit. Care Med.,
28, 643, 2000.
78. Giannotti, L., Braga, M., Nespoli, L., et al., A randomized controlled trial of preoperative
oral supplementation with a specialized diet in patients with gastrointestinal cancer, Gastro-
enterology, 122, 1763, 2002.
79. Suchner, U., Heyland, D., and Peter, K., Immune-modulatory actions of arginine in the
critically ill patient, Br. J. Nutr., 87 (Suppl. 1), S121, 2002.
80. Heyland, D.K., Novak, F., Drover, J.W., et al., Should immunonutrition become routine in
critically ill patients? A systematic review of the evidence, JAMA, 286, 944, 2001.
81 Yu, Y.M., Sheridan, R.L., Burke, J.F., et al., Kinetics of plasma arginine and leucine in pediatric
burn patients, Am. J. Clin. Nutr., 64, 60, 1996.
82. Yu, Y.M., Ryan, C.M., Castillo, L., et al., Arginine and ornithine kinetics in severely burned
patients: increased rate of arginine disposal, Am. J. Physiol., 280, E509, 2001.
83. Argaman, Z., Young, V.R., Noviski, N., et al., Arginine and nitric oxide metabolism in
critically ill septic pediatric patients, Crit. Care Med., 31, 591, 2003.
84. van Bokhorst-De Van Der Schueren, M.A., Quak, J.J., et al., Effect of perioperative nutrition,
with and without arginine supplementation, on nutritional status, immune function, post-
operative morbidity, and survival in severely malnourished head and neck cancer patients,
Am. J. Clin. Nutr., 73, 323, 2001.
85. Clarke, R., Feleke, G., Din, M., et al., Nutritional treatment for acquired immunodeficiency
virus-associated wasting using beta-hydroxy beta-methylbutyrate, glutamine, and arginine:
a randomized, double-blind, placebo-controlled study, J. Parenter. Enteral Nutr., 24, 133, 2000.
86. May, P., Barber, A., D’Olimpio, J., et al., Reversal of cancer-related wasting using oral sup-
plementation with a combination of beta-hydroxy-beta-methylbutyrate, arginine, and
glutamine, Am. J. Surg., 183, 471, 2002.
87. Jonas, C.R., Puckett, A.B., Jones, D.P., et al., Plasma antioxidant status after high-dose
chemotherapy: a randomized trial of parenteral nutrition in bone marrow transplantation
patients, Am. J. Clin. Nutr., 72, 181, 2000.
88. Yu, Y.M., Ryan, C.M., Fei, Z.W., Lu, X.M., et al., Plasma L-5-oxoproline kinetics and whole
blood glutathione synthesis rates in severely burned adult humans, Am. J. Physiol., 282, E247,
2002.
89. Ortolani, O., Conti, A., and De Gaudio, A.R., The effect of glutathione and N-acetylcysteine
on lipoperoxidative damage in patients with early septic shock, Am. J. Respir. Crit. Care Med.,
161, 1907, 2000.
90. Brunton, J.A., Ball, R.O., and Pencharz, P.B., Current total parenteral nutrition solutions for
the neonate are inadequate, Curr. Opin. Clin. Nutr. Metab. Care, 3, 299, 2000.
1382_C38.fm Page 665 Tuesday, October 7, 2003 7:34 PM

Chapter thirty-eight: Amino acid support in patients with catabolic illness 665

91. Helms, R.A., Storm, M.C., and Christensen, M.L., Cysteine supplementation results in nor-
malization of plasma taurine concentrations in children receiving home parenteral nutrition,
J. Pediatr., 134, 358, 1999.
92. Badaloo, A., Reid, M., Forrester, T., et al., Cysteine supplementation improves the erythrocyte
glutathione synthesis rate in children with severe edematous malnutrition, Am. J. Clin. Nutr.,
76, 495, 2002.
1382_C38.fm Page 666 Tuesday, October 7, 2003 7:34 PM
1382_C39.fm Page 667 Tuesday, October 7, 2003 7:36 PM

chapter thirty-nine

Sulfur-containing amino acids


and glutathione in diseases
Christiane Obled
Unité de Nutrition et Métabolisme Protéique, INRA, Theix
Isabelle Papet
Unité de Nutrition et Métabolisme Protéique, INRA, Theix
Denis Breuillé
Nestlé Research Center, Vers-Chez-Les-Blanc, Switzerland

Contents
Introduction..................................................................................................................................668
39.1 Sulfur amino acids in healthy conditions.......................................................................668
39.1.1 Metabolism: metabolic pathways and nutritional regulation......................668
39.1.1.1 Methionine............................................................................................668
39.1.1.2 Cysteine .................................................................................................670
39.1.1.3 Glutathione ...........................................................................................670
39.1.2 Plasma and tissue concentrations .....................................................................671
39.2 Roles of sulfur compounds...............................................................................................672
39.3 Sulfur amino acids in diseases .........................................................................................673
39.3.1 Plasma and tissue concentrations in diseases ................................................673
39.3.2 Metabolism ...........................................................................................................674
39.3.2.1 Metabolism of methionine and cysteine..........................................674
39.3.2.2 GSH synthesis ......................................................................................675
39.3.2.3 Taurine, metallothioneins, and acute phase proteins....................675
39.4 Sulfur-containing diets in diseases..................................................................................675
39.4.1 Forms of cysteine supply ...................................................................................676
39.4.2 Effects of sulfur amino acid supplementation of the diet ............................677
39.4.2.1 Glutathione levels and synthesis ......................................................677
39.4.2.2 Amino acid and protein metabolism ...............................................677
39.4.2.3 Cytokine production and transcription factor activity .................679
39.4.2.4 Modulation of the immune response...............................................679
39.4.2.5 Clinical outcomes ................................................................................680
39.5 Conclusion ..........................................................................................................................680
References .....................................................................................................................................681

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 667
1382_C39.fm Page 668 Tuesday, October 7, 2003 7:36 PM

668 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Introduction
Sulfur-containing amino acids constitute a large group of compounds whose metabolisms
are closely interconnected. Methionine, cysteine, and taurine are the most important. In
mammals, only methionine is a nutritionally indispensable amino acid; all other sulfur
amino acids, especially cysteine, are produced through its metabolism. Therefore, cysteine,
which like methionine is used for protein synthesis, is considered to be a dispensable
amino acid or a conditionally indispensable amino acid. However cysteine is indispensable
in immature infants due to cystathionase deficiency (Figure 39.1).1 The purpose of this
chapter is to examine whether the requirement of sulfur amino acids and especially
cysteine is increased in pathological situations. Since no other dedicated chapter is devoted
to sulfur-containing amino acids in this book (except taurine, see Chapter 44), we first
summarize the knowledge about their metabolism in health and diseases before focusing
on their supply in diseases.

39.1 Sulfur amino acids in healthy conditions


39.1.1 Metabolism: metabolic pathways and nutritional regulation
39.1.1.1 Methionine
Methionine is mainly used for protein synthesis like other indispensable amino acids. In
humans fed adequate diets, methionine used for protein synthesis accounts for 75 to 85%
of its total utilization in the postprandial state.2,3 As most amino acids, methionine can be
catabolized via a transamination–decarboxylation route. However, this route seems to be
of minor importance, at least in humans,4,5 and methionine is mainly metabolized in the
liver through the transmethylation–transsulfuration pathway (Figure 39.1). In other tis-
sues, the pathway may be incomplete.5 Methionine is adenylated by methionine adenosyl
transferase to form S-adenosylmethionine, an important biological methyl donor, the
methyl groups being used for synthesis of choline, creatinine, etc. Numerous methyltrans-
ferases catalyze the transfer of a methyl group from S-adenosylmethionine to a methyl
acceptor, producing a methylated product and S-adenosylhomocysteine, which is subse-
quently hydrolyzed to form homocysteine.5,6
Homocysteine follows two different pathways. It can be remethylated to form
methionine via either the cobalamin-dependent methionine synthase (using N5-methyltet-
rahydrofolate as a methyl donor) or betaïne:homocysteine methyl transferase (using
betaïne as a methyl donor). Homocysteine can also be catabolized via the transsulfuration
pathway, which ultimately forms cysteine (Figure 39.1). Two vitamin B6-dependent
enzymes constitute the transsulfuration pathway: cystathionine b-synthase, which con-
denses homocysteine with serine to form cystathionine, and cystathionase, which cleaves
cystathionine to cysteine and a-ketobutyrate. Therefore, the carbon skeleton of cysteine
is provided by serine, and methionine gives only its sulfur to cysteine.5,6 The flux through
remethylation or transsulfuration pathways can be altered as a result of impaired vitamin
status leading to increased homocysteinemia, which has been associated with an increased
risk of cardiovascular diseases.7
Moreover, the distribution of homocysteine between the remethylation and irrevers-
ible transsulfuration provides a major regulatory locus for methionine metabolism. Under
adequate supply of proteins, it has been shown that the percentage of homocysteine
utilized for cystathionine synthesis was 46% in rat liver. This percentage decreased to 34%
in rats fed a 3.5% casein diet but increased to 89% in rats fed a 55% casein diet.8 In humans,
the proportion of homocysteine entering the transsulfuration pathway decreased from
56% under normal protein intake to 21% under protein-free diets.3,9,10 Moreover, it is well
L-METHIONINE
N,N-
DIMETHYL
Remethylation THF methyl Transmethylation
GLYCINE
CH 3- THF S-ADENOSYL-L-HOMOCYSTEINE
BETAINE
L-HOMOCYSTEINE
L-SERINE 1
Chapter thirty-nine:

L-CYSTATHIONINE Glu
Transsulfuration
α-ketobutyrate 2
5-oxoproline
O2 L-CYSTEINE
4 AA
1382_C39.fm Page 669 Tuesday, October 7, 2003 7:36 PM

3
Cysteine sulfinate CYSTEINE
SULFINATE
γ-Glutamyl cycle
pathway α-keto acid γ-Glu-CysH γ-Glu-Cys 2
CysH-Gly γ-Glu-AA
CO 2 Amino acid
HYPOTAURINE β-SULFINYL 5 Gly
PYRUVATE 6

X
GLUTATHIONE
γ-Glu-CysH- Gly Cys 2
TAURINE PYRUVATE + SO 42 - AA
9
8 7
γ-Glu-Cys-Gly
X GSSG
Mercapturate pathway
Sulfur-containing amino acids and glutathione in diseases

N-acetyl-cysteine-X

Figure 39.1 Schematic representation of metabolic pathways for methionine, cysteine, and glutathione metabolism. The numbers represent the following
enzymes: 1, cystathionine-b-synthase; 2, cystathionase; 3, cysteine dioxygenase; 4, g-glutamylcysteine synthetase; 5, glutathione synthetase; 6, g-glutamyl
transpeptidase; 7, glutathione peroxidase; 8, glutathione reductase; 9, glutathione transferase.
669
1382_C39.fm Page 670 Tuesday, October 7, 2003 7:36 PM

670 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

known that dietary cystine spares methionine by lowering transsulfuration, and that
cystine can replace part of the dietary requirement of methionine in the growing rat.11
Similar findings were reported more recently in humans since transsulfuration was
reduced when cystine was added to a methionine-free diet.12

39.1.1.2 Cysteine
Transsulfuration constitutes a significant source of cysteine since it accounts for 5 to 14%
of cysteine flux in humans.2,9 Cysteine is mainly catabolized by the cysteine sulfinate
pathway to yield either taurine or sulfate and pyruvate6 (Figure 39.1). A cysteine sulfinate-
independent pathway leads also to sulfate and pyruvate. A lot of tissues are able to
metabolize cysteine, but taurine is produced mainly by the liver.13 In vitro studies on rat
hepatocytes have shown that the production of glutathione, sulfate, and taurine increased
as cysteine or methionine concentrations in the medium were increased. However, the
synthesis of glutathione is favored at low concentrations, and sulfate and taurine are
produced predominantly at high concentrations.14 Therefore, in case of low availability of
sulfur amino acids, cysteine is efficiently used for glutathione synthesis rather than being
catabolized to taurine and sulfate. Hyperproteic diets favored the catabolism of cysteine
into sulfate,15 but the supplementation of low-protein diets with excess methionine
increased the proportion of cysteine catabolism to taurine.16

39.1.1.3 Glutathione
Apart from protein synthesis, glutathione (GSH) synthesis is the main pathway of cysteine
utilization. In healthy humans, more than 50% of the plasma cysteine flux may be
explained by glutathione synthesis.9 Glutathione synthesis is a two-step reaction that takes
place mainly in the liver17,18 (Figure 39.1). In the initial rate-limiting step, g-glutamylcys-
teine is formed from glutamate and cysteine. The g-glutamylcysteine synthetase is regu-
lated by feedback inhibition by glutathione. In extrahepatic tissues, the g-glutamyl
transpeptidase reaction, which transfers a g-glutamyl moiety from extracellular glu-
tathione to an amino acid, provides an additional source of cysteine for glutathione
synthesis (Figure 39.1). Therefore, amino acid precursors for glutathione are transported
into the cell either directly or indirectly via the reaction catalyzed by g-glutamyl transpep-
tidase, an enzyme present on the cell membrane.18 In the second step of glutathione
synthesis, glutathione synthetase catalyzes the reaction between glycine and g-glutamyl-
cysteine to form glutathione. The synthesis of tissue glutathione is mainly limited by
availability of cysteine.19 The degradation of glutathione occurs extracellularly through
the action of the membrane-bound g-glutamyl transpeptidase (cf. above).
All the various reactions involved in glutathione synthesis and degradation constitute
the g-glutamyl cycle, which must be viewed at the whole-body level17,18 (Figure 39.1). The
liver is both a producer and exporter of GSH, since GSH efflux accounts for about 90%
of the hepatic turnover of GSH20 and constitutes the major source of plasma GSH. The
kidneys, the lungs, and the gut are the major consumers of glutathione. GSH uptake by
kidneys accounts for up to 70% of the hepatic production of glutathione. In intestine, the
transpeptidation reaction allows possible reabsorption of GSH present in the lumen, due
to exportation from the liver into the bile conduct. In the kidneys, intestine, and lung,
GSH is also able to enter cells as a whole, without any degradation.18
Glutathione can be oxidized leading to a glutathione dimer (GSSG) through the action
of glutathione peroxidase (Figure 39.1). This reaction is very important for the defense of
the organism against oxidative stress (cf. Hiramatsu et al.2). For this reason the GSH:GSSG
ratio is often used as an index of the oxidative stress. However, it cannot be considered
as a universal biomarker since the dimer generally does not accumulate but is either
1382_C39.fm Page 671 Tuesday, October 7, 2003 7:36 PM

Chapter thirty-nine: Sulfur-containing amino acids and glutathione in diseases 671

recycled to GSH by glutathione reductase or exported from the cell.18 Glutathione also
reacts with toxic compounds to form products that are removed from the cell.21 GSH is
the substrate of various GSH S-transferases and enzymes occurring in liver and other
tissues such as kidneys that catalyze the conjugation of electrophilic agents. The GSH
S-conjugates follow the mercapturic pathway with formation of S-conjugates of N-acetyl-
cysteine and elimination in bile and urine17,22 (Figure 39.1). Therefore, oxidation of glu-
tathione and complex formation lead to depletion of the intracellular pool of glutathione.
The availability of substrates for GSH synthesis, especially cysteine, is a major deter-
minant of GSH tissue concentration.18,23,24 Starvation results in a decrease of glutathione
levels in liver, intestine, and muscle of rats, which is corrected by refeeding.25 However,
it seems that glutathione levels are tightly regulated since high-protein diets do not further
enhance glutathione concentrations.26 GSH concentration in various tissues falls when
food or protein intake is inadequate.26–28 In children with edematous protein–energy mal-
nutrition and in protein-deficient pigs, erythrocyte glutathione deficiency is due to
impaired synthesis.27–29 Supplementation of low-protein diets with methionine or cysteine
increases the hepatic glutathione concentration and synthesis.28 The efficiencies of different
sulfur amino acids for glutathione synthesis in the liver have been compared, but both
in vivo and in vitro studies gave contradictory results, methionine being superior,28 infe-
rior,14,30 or equal28,31 to cysteine in restoring glutathione levels or supporting glutathione
synthesis. Therefore, in healthy conditions, methionine and cysteine seem to be equal in
regard to their bioavailability for glutathione synthesis.
The blood or erythrocyte glutathione fractional synthesis rate varied from 48 to
75%/day in humans.32–35 However, the values reported by Yu et al.32 and Lyons et al.,33 73
and 65%/day, respectively, are surprisingly high, since they used the plasma enrichment
of the tracer amino acid as enrichment of the precursor pool, whereas Jahoor et al.34 and
Faber et al.35 used the enrichment in the erythrocyte, which is less than half the plasma
value.27,35 Using the plasma enrichment as precursor, Capitan et al.36 reported a value of
22.5%/day, which would be more than twice that if calculated from erythrocyte enrich-
ment, and is therefore in agreement with the data of Faber et al.35 and Jahoor et al.34 High
synthesis rate was found in rat liver, about 440%/day.31 The apparent turnover rates were
the most rapid in liver and kidneys, while those for muscle and blood were the lowest,
and intestine had an intermediate turnover rate.37

39.1.2 Plasma and tissue concentrations


As sulhydryl compounds are easily oxidized, they exist as a variety of disulfide forms in
vivo. For example, cysteine is found in plasma in one of three forms: free or cysteine,
oxidized or cystine, and protein-bound mainly with albumin. Cysteine is found also in
dipeptides such as cysteinylglycine. The total amount of the various cysteine species was
reported to be 236 mM in healthy women and 264 mM in healthy men.38 The protein-bound
form represents 65% of total cysteine, 32 to 34% of cystine, and only 3 to 4% of free
cysteine.38 Cysteine is not readily analyzed by amino acid analyzer technology, which
provides only cystine. However, the value obtained can tend more or less toward the sum
cysteine plus cystine according to the degree of oxidation of cysteine during sample
processing. The protein-bound amount is generally not measured and requires a reducing
step before sample deproteinization. A fraction of glutathione is also linked to proteins,
about 20% in plasma and 16% in whole blood.38,39 Glutathione is present at a low concen-
tration in plasma, about 10 mM. The conditions of sampling and of sample storage and
processing can markedly alter the repartition between the various forms and, for example,
increase the proportion of protein-bound cysteine or oxidized glutathione. Rossi et al.40
have concluded that GSSG is present at very low concentrations in human blood, less
1382_C39.fm Page 672 Tuesday, October 7, 2003 7:36 PM

672 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

than 0.5%. Therefore, the conditions of analysis have to be strictly controlled if the ratio
of the reduced and oxidized forms has to be used as an index of the in vivo redox status.
Cysteine concentration in tissues is poorly documented. Values between 30 and 100
nmol/g for free cysteine and between 70 and 400 nmol/g for total free cysteine (free
cysteine and cystine) have been reported in liver, muscle, and intestinal mucosa of mice
and humans.41–43 By contrast, very low concentrations were found in erythrocytes
(5 nmol/g).34,39 Glutathione concentrations are far greater than those of cysteine and have
been reported in most tissues. The highest concentration is found in the liver (6 to
10 mmol/g) and the lowest in blood and muscle (about 1 mmol/g). Intermediate values
(2 to 3 mmol/g) have been reported in other tissues such as spleen, intestine, kidneys, and
lung.25,28,44

39.2 Roles of sulfur compounds


Cysteine residues are important in the catalytic site of many enzymes. The thiol group of
cysteine allows formation of disulfide bonds that are important for the structure of
proteins.
Models of GSH depletion have been developed to investigate the functions of this
compound. Rats fed a low-protein diet had decreased lung GSH concentrations associated
with a lower tolerance to hyperoxic stress.45 In healthy animals, GSH deficiency leads to
mitochondrial lesions19 and damage to the intestinal mucosa.46 Depletion of GSH sensitizes
cells to the effects of various toxic substances. However, this effect can be beneficial, such
as in the treatment of cancer cells to lower the resistance of tumors to chemotherapy and
radiation.19 Glutathione depletion impairs the immune function.47 The treatment of T-cells
with inhibitors of GSH synthesis decreased their proliferation48,49 and their production of
cytokines.49
The main characteristic of sulfur compounds, cysteine, taurine, and glutathione is
their ability to modulate the redox status of the cell, and glutathione is the most important
intracellular antioxidant of the body. GSH and cysteine can function as direct scavengers
of reactive oxygen species, or they can protect proteins from oxidation through the for-
mation of mixed disulfides.
Reactive oxygen species and hydrogen peroxide are formed extensively in biological
systems as a consequence of aerobic metabolism and may produce deleterious effects,
including macromolecule oxidation and alteration of metabolic processes. GSH peroxidase
catalyzes GSH-dependent reduction of hydrogen peroxide. As a consequence, GSH is
oxidized to GSSG, which is either rapidly reduced back to GSH by GSSG reductase or
eliminated from the cell17,18 (Figure 39.1). The GSH/GSSG ratio and the level of GSH within
the cell determine the thiol redox status of the cell, which in turn can regulate metabolic
pathways, by activating or inactivating key enzymes or cellular processes such as apop-
tosis, signal transduction, and gene transcription.50,51 For example, the transsulfuration
and GSH synthesis pathways are under redox control.52,53 Nuclear factor kappa B (NF-kB)
plays a pivotal role in the regulation of many genes involved in inflammation such as
pro-inflammatory cytokines, and therefore in modulating immune function.54 This factor
is present in an inactive form in the cytosol since it is linked to an inhibitor kappa B (IkB).
Oxidative stress or depletion of reduced glutathione induces the phosphorylation and
proteolysis of the inhibitor and the liberation of NF-kB, which can migrate into the nucleus,
bind to specific DNA sequences, and induce the transcription of genes, including IkB, to
down-regulate the system.55 By contrast, under reducing conditions, the activation of
NF-kB is inhibited.56,57 However, reducing conditions are required in the nucleus for the
transcriptional activity.58
1382_C39.fm Page 673 Tuesday, October 7, 2003 7:36 PM

Chapter thirty-nine: Sulfur-containing amino acids and glutathione in diseases 673

The interaction between glutathione and proteins constitutes another important mech-
anism of regulation of cell activity. Protein S-thiolation is the formation of mixed disulfides
between protein sulfhydryls and low molecular thiols such as cysteine and glutathione.
As glutathione is present in cells at a much higher concentration than other thiols, glu-
tathiolated (or glutathionylated) proteins (protein–SSG) are likely to be the major form of
S-thiolated proteins. Protein S-glutathiolation has been proposed to have many conse-
quences on cellular metabolism. Three points can be mentioned. Protein S-glutathiolation
may serve to protect proteins against irreversible oxidative damage and loss.59 Indeed,
proteins contain a variety of reactive cysteines, which are associated with the function of
these proteins. Thus, irreversible oxidation of cysteine residues is particularly threatening
to these functions that require a restrictive reduced state.60 Protein S-glutathiolation may
constitute a mechanism of regulation of protein activity associated with either an increase
or, more often, an inhibition of activity. A number of examples have been reported. For
instance, glutathione transferase is activated through S-thiolation in rat liver microsomes
incubated with GSSG.61 Various pathways of proteolysis have been described. The activity
of the ATP/ubiquitin-dependent 26S proteasome pathway was reduced under oxidative
stress, probably due to the glutathiolation and inactivation of some enzymes involved in
this pathway.62 By contrast, the ubiquitin-independent 20S proteasome appears to be more
resistant to oxidative inactivation and could be responsible for the degradation of oxidized
proteins.63 These findings raise the possibility that changes in the level of glutathione and
the GSSG/GSH ratio may be important physiological regulators of proteasome functions.
Finally, protein glutathiolation can modulate signal transduction and gene expression. The
binding of NF-kB to its DNA-binding domain depends on the integrity of a cysteine
residue (cys62), which must be in a reduced state. It has been shown that the glutathiolation
of the p50 subunit of NF-kB can reversibly inhibit its DNA-binding activity.64
Another important role of glutathione is the detoxification of xenobiotic compounds
through the formation of conjugates, which are converted to mercapturic acids excreted
into bile and urine22 (Figure 39.1). GSH has many other physiological functions, including
the storage of cysteine,25 the transport of cysteine and other amino acids,18 leukotrien
synthesis,65 metal transport, storage, and metabolism.50

39.3 Sulfur amino acids in diseases


39.3.1 Plasma and tissue concentrations in diseases
In trauma or sepsis, the plasma concentration of methionine is either unchanged,66–69
increased,70,71 or decreased.72 Contradictory results were also reported for cyst(e)ine.
Plasma concentrations were reported increased in septic patients69,73,74 but unchanged after
trauma, surgery, or inflammation.66,68,71 Cystine plasma concentration was decreased in
Crohn’s disease and ulcerative colitis but rapidly restored after removal of the inflamed
bowel.43 In chronic colitis in rats, we have recently observed lower plasma total cysteine
and methionine concentrations in treated animals than in their pair-fed controls.75 In
tissues, cysteine concentrations were generally unchanged.43,70,76 The reasons for these
conflicting results are unclear. Most studies describing a decreased concentration of these
amino acids concern patients with chronic diseases or moderately severe injury. On the
other hand, increased concentration is generally described in cases of dramatic diseases
such as septic shock.73 A decreased concentration could reflect an increased sulfur amino
acid utilization in response to the disease and associated oxidative stress. Very acute
diseases could induce liver dysfunction and impaired glutathione synthesis resulting in
accumulation of sulfur amino acids. Indeed, a large increase of cyst(e)ine concentration
was reported in nonsurviving septic patients, but normal concentrations in surviving
1382_C39.fm Page 674 Tuesday, October 7, 2003 7:36 PM

674 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

patients.73 However, the plasma cyst(e)ine level is not always increased in liver
diseases.77–79
Glutathione is lost either by the excretion of glutathione conjugates or by the extra-
cellular export and catabolism of oxidized glutathione.80,81 Therefore, catabolic states and
chronic diseases are generally associated with depletion of glutathione pools, with few
modifications of the oxidized form.76,82,83 However, 4 h after zymosan injection in rats,
glutathione was reduced to the benefit of the oxidized form in liver and lung.84 In patients
with intestinal bowel disease (IBD), GSSG was unchanged in noninflamed mucosa but
increased in inflamed mucosa.43
Total glutathione concentrations in plasma, blood, or erythrocytes are generally
decreased in chronic diseases34,82 and after acute injury.32,74 Nevertheless, unchanged levels
are observed in mild injury such as surgery in humans or after turpentine injection in
pigs.27,76 By contrast, glutathione is always depleted in muscle after surgery or in critically
ill patients,76,85 and in the intestinal mucosa of patients with IBD.43,86,87 Therefore, the
depletion of glutathione in tissues is not always associated with a decrease in blood or
plasma. In animal models, different results can be observed. In chronic intestinal inflam-
mation, glutathione was maintained or increased in liver and intestine but decreased in
muscle, suggesting a preferential synthesis at the inflammatory site.75 In acute injury,
glutathione is depressed in comparison to well-fed animals,28,88,89 but the feature can be
different if the comparison is made with pair-fed animals. This is particularly true during
the early period after induction of the stress, when food intake is markedly affected. During
the first days following infection or injection of tumor necrosis factor alpha (TNF-a) or
lipopolysaccharide (LPS), liver glutathione levels are better maintained in infected animals
than in pair-fed animals28,44,88,89 but depleted later on.88 Nevertheless, an early depletion
of glutathione status has been reported in the first hours following the stress.90,91 Thus,
glutathione status may follow a rebound kinetic: the increased consumption of glutathione
due to the early oxidative stress associated with injury would rapidly induce depletion
of glutathione stores. Between 12 and 24 h after the initial stress, a rebound effect occurs,
resulting in a net increase in organ levels of glutathione.91 This rebound effect could reflect
a stimulation of glutathione synthesis, which would be a mechanism whereby cellular
adjustment to stress occurs. Such adaptation is possible if substrate availability is sufficient.
After prolonged stress or in chronic diseases, the low availability of nutrients linked to
anorexia and persistent oxidative stress leads to the net consumption of glutathione in
excess of the ability of the organism to synthesize the molecule. This results in depletion
of GSH concentrations.

39.3.2 Metabolism
39.3.2.1 Metabolism of methionine and cysteine
Whole-body methionine and cysteine fluxes are greatly increased after injury.70,74,92 How-
ever, whole-body amino acid utilization is generally increased in these situations due to
the hypermetabolic state,93 and it is not clear if the metabolic demand for methionine and
cysteine is greater than that for the other amino acids. In burn patients, the contribution
of the transsulfuration pathway to the methionine flux is 40%, compared to 24% in healthy
controls.92 The synthesis of cysteine from methionine has also been found to be increased
2.7-fold 2 days after infection in rats.70 Methionine synthase and cystathionine b-synthase
have revealed reciprocal sensivity to oxidative stress. The activity of the first is reduced
under oxidizing conditions, whereas the activity of the second is enhanced under the same
conditions.52 The regulation of the transsulfuration pathway by redox changes allows the
increase of glutathione synthesis under oxidative conditions.52 Taken together, these results
indicate an increased metabolic demand for cysteine in diseases. However, cysteine
1382_C39.fm Page 675 Tuesday, October 7, 2003 7:36 PM

Chapter thirty-nine: Sulfur-containing amino acids and glutathione in diseases 675

synthesis alone is not likely sufficient to meet the increased cysteine requirement since
the elevated methionine flux through the transsulfuration pathway in septic rats is less
than the cysteine flux increase.70 By contrast, methionine utilization and cysteine biosyn-
thesis are impaired in cirrhotics, probably due to liver dysfunction.78,94
The modification of cysteine catabolism in response to injury can be another indicator
of increased cysteine requirement. Unlike other amino acids such as leucine, whose catab-
olism strongly increased,93 cysteine oxidation seems to be unchanged in septic pediatric
patients.10,74 A reduction of cysteine catabolism was found after infection in rats.95,96 These
results suggest that cysteine is spared in order to synthesize important compounds for
protection against the oxidative stress associated with sepsis, namely, taurine and mainly
glutathione.

39.3.2.2 GSH synthesis


In humans, only blood and erythrocyte glutathione synthesis is documented. GSH defi-
ciency is always associated with an impaired absolute synthesis, but the fractional syn-
thesis rate was reported either decreased29,60,74 or not.34 The decreased glutathione concen-
tration was correlated with a low glutathione synthetase activity in muscle after surgical
stress and with a decreased activity of g-glutamylcysteine synthetase76 and g-glutamyl
transferase in the intestinal mucosa of patients with IBD.43
In animal models, increased glutathione concentrations observed 1 or 2 days after
infection or an inflammatory challenge were associated with increased synthesis in all
tissues except blood and small intestine.28,44 Therefore, glutathione concentration changes
are generally correlated with changes in synthesis, suggesting similar variations in sub-
strate availability, especially cysteine availability. Indeed, pigs fed a protein-deficient diet
were unable to maintain either glutathione levels or rate of synthesis 2 days after turpen-
tine injection.27

39.3.2.3 Taurine, metallothioneins, and acute phase proteins


Infection induces an increased production of taurine in the liver.96 It can be excreted in
the plasma13 and removed by tissues such as the lungs or cells such as lymphocytes to
fight against the oxidative injury.13,97 Indeed, taurine is the free amino acid present at the
highest concentration in lymphocytes where it plays different functions that are exacer-
bated in case of infection.
Metallothioneins are high-cysteine-containing proteins (about 30%) of low molecular
weight, present in most tissues, that are involved in heavy metal homeostasis and detox-
ification, and in the protection of tissues against various forms of oxidative injury.98 Even
if metallothioneins are induced after endotoxin injection99,100 or after infection in rats
(Papet, unpublished results), their concentration in tissues is rather low (a few micrograms
per gram99), and it is unlikely that their synthesis contributes to a great extent to the
utilization of cysteine.
The increase in the synthesis of acute phase proteins is far greater than the change in
their concentration in inflammatory states.83 Moreover, the synthesis of albumin, the
plasma concentration that falls in stress conditions, is also enhanced.93 The cysteine content
of acute phase proteins is not generally high.101 By contrast, albumin contains about 6%
cysteine and its synthesis can have an important effect on cysteine utilization.

39.4 Sulfur-containing diets in diseases


Metabolic data indicate that cysteine becomes indispensable in diseases. It is spared,
probably to sustain the synthesis of important compounds for the defense of the organism,
1382_C39.fm Page 676 Tuesday, October 7, 2003 7:36 PM

676 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

such as acute phase proteins, taurine, and probably, more importantly, glutathione. Low
tissue content of glutathione can exacerbate injury or predispose the subject to multisystem
organ failure in sepsis or shock. For example, rats rendered GSH deficient by the injection
of diethyl maleate developed a higher incidence of organ dysfunction when subjected to
hemorrhagic shock.102 Moreover, glutathione and cysteine deficiencies may partly impair
the immune response of the body.47 Because depletion of glutathione is observed in many
diseases, treatments that help maintain glutathione levels may improve the ability of
patients to recover. For this reason, it is important to determine which level of cysteine is
required in the diet in disease states and how to supply this amino acid.

39.4.1 Forms of cysteine supply


Cysteine in solution is readily oxidized to cystine, which has a low solubility. It can also
form derivatives such as D-glucocysteine. However, despite such high cysteine conversion,
its utilization seems normal during parenteral administration.103 Cystine is currently used
to supplement oral diets, and it allows normal growth of animals. However, cystine uptake
by isolated hepatocytes is too slow to support a maximal rate of glutathione and taurine
production.104 By contrast to the number of other amino acids, cysteine is poorly preserved
during the industrial processes used for the preparation of parenteral or enteral solutions.
Therefore, alternative sources of cysteine or glutathione are required.
Small peptides containing cysteine can overcome the problem of stability of cysteine
in solution and can provide a good alternative to supply cysteine, especially by the
parenteral route.105 Whey proteins contain a rather high cysteine content, about 4 to 5%,
but few studies have explored the usefulness of oral supplementation with these proteins.106
Hagen and Jones107 have established the existence of a transepithelial transport for
intact glutathione in rat isolated small intestine loops. However, in vivo studies have
provided conflicting results on the ability of oral glutathione administration to increase
plasma or tissue cysteine and glutathione concentrations both in rats and humans.107–109
By contrast, the intravenous administration of glutathione can increase the plasma con-
centration of cysteine and glutathione,110 but not GSH tissue levels.111 Glutathione esters
are transported into cells more easily than glutathione and converted intracellularly in
glutathione. Therefore, the oral administration of these compounds results in an increased
glutathione concentration in tissues.111,112
Two cysteine prodrugs have been extensively used in both animals and humans: L-2-
oxothiazolidine-4-carboxylate (OTC) and N-acetyl cysteine (NAC). OTC is well trans-
ported into many cells and is converted to cysteine by 5-oxoprolinase, an enzyme present
in most tissues.113 The oral administration of 0.15 mmol OTC/kg to healthy volunteers
increases the concentration of free cysteine and glutathione in plasma and lymphocytes.114
The level of glutathione in the liver increases after the administration of OTC to fasted
mice.113 The supplementation of a sulfur amino acid-deficient diet with OTC restores
growth rate and tissue glutathione concentrations in rats.115 NAC appears to be deacylated
in liver, lung, intestine, and kidneys of various species, including man.116–118 However, the
mechanism by which cysteine is provided by NAC is not clear, either by the reduction of
cystine to cysteine due to the liaison between NAC and cystine or by the deacylation of
NAC.23,119 NAC seems to be equivalent to cysteine for the synthesis of glutathione in
isolated rat hepatocytes.120 The oral availability seems to be low.116,121 However, NAC can
be metabolized in the intestine and provide cysteine to other tissues. Indeed, plasma
cysteine levels are increased after ingestion of NAC by human volunteers.122 By contrast,
NAC given parenterally supported weight gain in rats.123 Nevertheless, the bioavailability
of NAC is not entirely clear in humans.124
1382_C39.fm Page 677 Tuesday, October 7, 2003 7:36 PM

Chapter thirty-nine: Sulfur-containing amino acids and glutathione in diseases 677

The safety of cysteine and cysteine precursors has been reviewed.125 Addition of
cyst(e)ine to diets at levels up to about 1.5% does not reduce the growth rate of rats.
Mortality was observed for levels greater than 2.5%.126 These levels are far above the
amount required to have beneficial effects.28 In humans, no adverse effect was reported
after the administration of single doses of cysteine up to 7 g/day. Higher doses produce
nausea and bad feeling.125 No adverse events have been reported in several clinical trials
with daily supplementation of NAC or OTC at doses up to 440 mg of NAC/kg·day and
190 mg of OTC/kg·day (Table 39.1). However, attention must be paid to patients with
liver diseases since in these circumstances, as already mentioned, the metabolism of sulfur
amino acids can be altered.78,94

39.4.2 Effects of sulfur amino acid supplementation of the diet


39.4.2.1 Glutathione levels and synthesis
The ability of GSH to provide protection against ongoing oxidative stress can only be
restored by glutathione synthesis since diseases lead to a consumption of glutathione (cf.
paragraph 39.1.11). After intravenous administration of TNF-a in rats fed a protein-defi-
cient diet, supplementation with methionine or cysteine (but not with alanine) increased
the glutathione content of liver and lung.28 After endotoxin administration, this effect has
been demonstrated to be associated with increased glutathione synthesis rate in liver.28 In
this study, methionine appeared more efficient to restore glutathione than cysteine sup-
plementation. By contrast, Breuillé et al.127 have observed that cysteine but neither
methionine nor cystine supplementation of the diet allowed the normalization of glu-
tathione content in the liver of septic rats. During pancreatitis in mice, the administration
of OTC attenuated the decrease in pancreatic glutathione.128
In patients with chronic diseases, cysteine and glutathione depletion was the basis of
a rationale for the treatment with N-acetyl-cysteine or cysteine prodrugs.129 A single oral
dose of NAC given to HIV patients increased cysteine concentration in plasma and mono-
nuclear cells, but only moderately modified glutathione concentration.130 Most studies
with repeated administration have shown that NAC or OTC treatment replenished eryth-
rocyte, whole blood, or plasma glutathione (Table 39.1), but one study failed to show any
effect.131 Cysteine was generally increased in erythrocytes or whole blood but not always
in plasma.34,133,134,136,138 The increase in plasma and erythrocyte GSH levels is associated
with an increase of GSH synthesis rate in most patients, suggesting that the glutathione
deficiency in HIV or in edematous malnourished patients is partly due to a reduced
synthesis rate secondary to a shortage in cysteine availability.34,136 Moreover, the replen-
ishment of blood glutathione is associated with improved survival.139 Supplementation
with cysteine or cysteine precursors is poorly documented in acute injury. Results obtained
in infected rats (see above) suggest that such supplementation would be beneficial in acute
diseases characterized by disturbances of cysteine and glutathione status such as trauma
or sepsis.

39.4.2.2 Amino acid and protein metabolism


We have studied140 methionine and cysteine metabolism in polytrauma patients receiv-
ing a total enteral nutrition supplemented or not with cysteine. The cysteine supple-
mentation induced an increased cysteine utilization, which could reflect an increased
glutathione synthesis. Indeed, the sulfur balance indicated at the same time a decreased
cysteine catabolism in the supplemented group. Cysteine supplementation also limited
the increased cysteine production from methionine observed in patients. Together, these
data demonstrate an increased cysteine requirement in trauma patients and a need for
supplementation of this amino acid in hypercatabolic patients.140 Moreover, cysteine
678

Table 39.1 Effect of Diet Supplementation with Cysteine Precursors on Glutathione Levels in Patients (mM)
Length of Tissue or fluid Pretreatment or
Patient type Supplement treatment Route assessed controls Posttreatment Reference
HIV NAC, 1.2 g/day 1 week Per os Erythrocytes 1.45 1.60a 34
1382_C39.fm Page 678 Tuesday, October 7, 2003 7:36 PM

Plasma 9 19a
HIV NAC, 8 g/day 8 weeks Per os Blood 0.88 0.98a 132
HIV Whey proteins, 45 g/d 2 weeks Per os Plasma 1.92 2.79 106
1.98 2.51a
HIV OTC, 12 g/day 4 weeks Per os Blood 0.86 32%a 133
ARDS NAC, 210 mg·kg·day 10 days IV Erythrocytes 1.76 2.37a 134
OTC, 189 mg/kg·day 10 days 1.44 2.04a
IPF NAC, 1.8 g One dose IV Pulmonary epithelial 0.99 1.79a 135
lining fluid
Edematous NAC, 81.6 mg/kg·day 1 week Per os Erythrocytes 1.8 2.35a 136
malnourished
children
Cirrhosis OTC, 210 mg/kg·day 9 days IV Blood 0.38 0.51a 137

Note: HIV = human immunodeficiency virus; ARDS = adult respiratory distress syndrome; IPF = idiopathic pulmonary fibrosis; IV = intravenous; per os = orally.
a Significant effect.
Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition
1382_C39.fm Page 679 Tuesday, October 7, 2003 7:36 PM

Chapter thirty-nine: Sulfur-containing amino acids and glutathione in diseases 679

supplementation maintained blood GSH status and improved muscle protein synthe-
sis.141 In septic rats, cysteine supplementation decreased nitrogen excretion and limited
body weight loss and muscle atrophy.142 During injury, muscles serve as an amino acid
reservoir that delivers substrates for anabolic processes participating in the defense of
the organism. A cysteine-rich amino acid supplementation could be able to better cover
the specific amino requirements in acute trauma, thus resulting in improved muscle
protein synthesis. However, an active mechanism on proteolysis may also explain the
benefit of cysteine supplementation on muscle proteins. Indeed, cysteine administration
to tail-suspended rats for 2 weeks significantly reduced the loss of hindlimb muscle
mass, the protein ubiquitination, and major histocompatibility complex (MHC) frag-
mentation in muscles by comparison to alanine administration.143

39.4.2.3 Cytokine production and transcription factor activity


During the recent years, a lot of in vitro studies have clearly demonstrated that cysteine
and glutathione can modulate gene expression. Exposure of various cell lines or cells of
various tissues in culture to hydrogen peroxide, LPS, or proinflammatory cytokines
induced in a time- and dose-dependent manner NF-kB activation, and increased cytokine
mRNA expression and release, such as TNF-a or interleukin 6 (IL-6).56,57,144–147 It is generally
well demonstrated that the addition of sulfur compounds in the culture medium prevents
the activation of NF-kB and the production of cytokines induced by an oxidative stress.
When cells were treated with cysteine, NAC, or glutathione mono-ethylester, the activation
of NF-kB and the subsequent increase in cytokine release were inhibited.144,145,148 The effect
of NAC on TNF-a release was unaltered by concomitant inhibition of glutathione synthe-
sis, suggesting that the effect is independent of glutathione metabolism.145,149 This effect
could be due to an inhibition of the proteasome activity.150 The depletion of cellular
glutathione increases the cytokine biosynthesis without NF-kB activation in primary cell
cultures of alveolar epithelia, suggesting another pathway for the regulation of cytokine
production.147 However, contradictory results were found in L6 cells.57
In vivo studies have provided some evidence that cysteine prodrugs could regulate
cytokine production in injury. The administration of a bolus of NAC prior to LPS injection
in mice inhibits the increase of plasma TNF-a but not IL-1 and IL-6. The administration
of an inhibitor of GSH synthesis increases the effect of LPS on TNF-a production, but this
effect is prevented by the administration of NAC.151 Similar results were found in endotoxic
shock models.152,153 NAC (150 mg/kg followed by a continuous infusion of 50 mg/kg over
4 h) administered early after a septic shock in patients had no effect on plasma TNF-a,
IL-6, and IL-10, but decreased IL-8 and sTNFR-p55 levels over 24 h.154 OTC was admin-
istered during 9 days (210 mg/kg·day) to patients with liver injury. The treatment signif-
icantly decreased the monocyte production of TNF-a, IL-6, and IL-8, but no change in
biomarkers of liver function was seen at the end of the treatment.137 Lower ceruloplasmin
and a1-acid glycoprotein levels but not a2-macroglobulin were observed in TNF-a-
injected rats receiving low-protein diets supplemented with cysteine by comparison with
diets supplemented with alanine.155

39.4.2.4 Modulation of the immune response


Taurine reduces IL-2-induced lung injury in rats by attenuating neutrophil endothelial
adhesion and migration.156 Similar results were found in LPS-treated rats receiving NAC.157
Intravenous administration of NAC decreased the lung leak and the neutrophil influx that
occur in rats given intratracheal IL-1.158 By contrast, in sepsis induced by cecal ligature
and puncture in mice, NAC augmented the migration of polymorphonuclear leukocytes
to the site of infection and decreased bacterial colonies in the peritoneal cavity.159
1382_C39.fm Page 680 Tuesday, October 7, 2003 7:36 PM

680 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

In vitro studies have shown that GSH and NAC inhibit reverse transcriptase activity
and HIV expression in chronically infected cells.148,160 In HIV patients, NAC administration
did not increase CD4 count, but it did prevent a steep decrease in CD4 cells.138 In a recent
double-bind randomized trial, HIV patients received a NAC treatment for 7 months. The
treatment improves immune function without any change in the viral load.161

39.4.2.5 Clinical outcomes


Most studies in animals have been devoted to the early period of sepsis with shock. In
an endotoxic shock model in the dog, the intravenous administration of NAC improved
oxygen extraction capabilities and myocardial function.152 By contrast, no effect of NAC
treatment at similar doses was found on hemodynamics in a porcine bacterial shock162 or
after endotoxemia in rats.157 In that study, macromolecular leakage was reduced, suggest-
ing a decrease in endothelial damage.157 GSH depletion is associated with an increased
mortality in endotoxemia and sepsis, and NAC treatment significantly reduced the
mortality.151,159
Few clinical trials have explored the potential beneficial effect of cysteine prodrug
supplementation in patients. Spapen et al.154 demonstrated that infusion of NAC for 4 h
(50 mg/kg preceded by a 150 mg/kg bolus dose) to patients with early septic shock
improved respiratory function and shortened the length of stay of survivors in the
intensive care unit. This effect was associated with an attenuated production of IL-8. In
patients with adult respiratory distress syndrome (ARDS), administration of NAC at
100 mg/kg·day and OTC at 190 mg/kg·day resulted in repletion of glutathione in eryth-
rocytes as well as a shortening of acute lung injury and an increase in cardiac index.134
By contrast, no difference in oxygenation and mortality was found in another study on
patients with ARDS.163 No difference in multiple-organ failure score and in mortality
following administration of up to 150 mg of NAC/kg was found in ventilated patients
with at least one organ failure.164
An open study demonstrated that glutathione replenishment with doses ranging from
3.2 to 8 g of NAC/day for up to 8 months is associated with increased survival in HIV
patients with low CD4 count. Chances of survival doubled after 2 years from 35 to 70%
in the NAC group.139

39.5 Conclusion
The data presented in this review suggest that cysteine becomes indispensable in diseases.
Most of the increased cysteine requirement associated with diseases is probably related
to an increased demand for glutathione synthesis. Cysteine and glutathione may also
exhibit a number of effects through the control of the redox status of the cell. The discrim-
ination of the complex mechanisms that would allow understanding of the efficiency and
role of cysteine and glutathione in diseases is probably one of the coming challenges for
nutrition research.
Cysteine and glutathione exert numerous functions that are potentially important for
the defense of the organism, and the glutathione depletion observed in diseases can greatly
impair the recovery of patients. The supplementation with cysteine or cysteine precursors
generally improves glutathione levels. Clinical studies suggest that the improvement in
glutathione status is correlated with some improved clinical outcomes. However, addi-
tional studies are required to determine the best qualitative and quantitative supply for
different pathological situations. Clinical trials with a larger number of patients are needed
to confirm potential benefits in term of clinical outcomes.
1382_C39.fm Page 681 Tuesday, October 7, 2003 7:36 PM

Chapter thirty-nine: Sulfur-containing amino acids and glutathione in diseases 681

References
1. Vina, J. et al., L-cysteine and glutathione metabolism are impaired in premature infants due
to cystathionase deficiency, Am. J. Clin. Nutr., 61, 1067, 1995.
2. Hiramatsu, T. et al., Methionine and cysteine kinetics at different intakes of cystine in healthy
adult men, Am. J. Clin. Nutr., 60, 525, 1994.
3. MacCoss, M.J., Fukagawa, N.K., and Matthews, D.E., Measurement of intracellular sulfur
amino acid metabolism in humans, Am. J. Physiol., 280, E947, 2001.
4. Blom, H.J., et al., Transamination of methionine in humans, Clin. Sci., 76, 43, 1989.
5. Finkelstein, J.D., Methionine metabolism in mammals, J. Nutr. Biochem., 1, 228, 1990.
6. Stipanuk, M.H., Metabolism of sulfur-containing amino acids, Annu. Rev. Nutr., 6, 179, 1986.
7. Malinow, M.R., Plasma homocyst(e)ine: a risk factor for arterial occlusive diseases, J. Nutr.,
126, 1238S, 1996.
8. Finkelstein, J.D. and Martin, J.J., Methionine metabolism in mammals: distribution of
homocysteine between competing pathways, J. Biol. Chem., 259, 9508, 1984.
9. Fukagawa, N.K., Ajami, A.M., and Young, V.R., Plasma methionine and cysteine kinetics in
response to an intravenous glutathione infusion in adult humans, Am. J. Physiol., 270, E209,
1996.
10. Raguso, C.A., Regan, M.M., and Young, V.R., Cysteine kinetics and oxidation at different
intakes of methionine and cystine in young adults, Am. J. Clin. Nutr., 71, 491, 2000.
11. Stipanuk, M.H. and Benevenga, N.J., Effect of cystine on the metabolism of methionine in
rats, J. Nutr., 107, 1455, 1977.
12. Storch, K.J. et al., [1-13C, methyl-2H3]methionine kinetics in humans: methionine conserva-
tion and cystine sparing, Am. J. Physiol., 258, E790, 1990.
13. Garcia, R.A.G. and Stipanuk, M.H., The splanchnic organs, liver and kidney have unique
roles in the metabolism of sulfur amino acids and their metabolites in rats, J. Nutr., 122, 1693,
1992.
14. Stipanuk, M.H. et al., Cysteine concentration regulates cysteine metabolism to glutathione,
sulfate and taurine in rat hepatocyte, J. Nutr., 122, 420, 1992.
15. Bella, D.L., Kwon, Y.H., and Stipanuk, M.H., Variations in dietary protein but not in dietary
fat plus cellulose or carbohydrate levels affect cysteine metabolism in rat isolated hepat-
ocytes, J. Nutr., 126, 2179, 1996.
16. Bella, D.L. and Stipanuk, M.H., Effects of protein, methionine, or chloride on acid-base
balance and on cysteine catabolism, Am. J Physiol., 269, E910, 1995.
17. Meister, A. and Anderson, M.E., Glutathione, Annu. Rev. Biochem., 52, 711, 1983.
18. Deneke, S.M. and Fanburg, B.L., Regulation of cellular glutathione, Am. J. Physiol., 257, L163,
1989.
19. Meister, A., Glutathione deficiency produced by inhibition of its synthesis, and its reversal:
applications in research and therapy, Pharmacol. Ther., 51, 155, 1991.
20. Lauterburg, B.H., Adams, J.D., and Mitchell, J.R., Hepatic glutathione homeostasis in the rat:
efflux accounts for glutathione turnover, Hepatology, 4, 586, 1984.
21. Pullar, J.M., Vissers, M.C.M., and Winterbourn, C.C., Glutathione oxidation by hypochlorous
acid in endothelial cells produces glutathione sulfonamide as a major product but not
glutathione disulfide, J. Biol. Chem., 276, 22120, 2001.
22. Hinchman, C.A. and Ballatori, N., Glutathione conjugation and conversion to mercapturic
acids can occur as an intrahepatic process, J. Toxicol. Environ. Health, 41, 387, 1994.
23. White, A.C., Thannickal, V.J., and Fanburg, B.L., Glutathione deficiency in human disease,
J. Nutr. Biochem., 5, 218, 1994.
24. Taylor, C.G., Nagy, L.E., and Bray, T.M., Nutritional and hormonal regulation of glutathione
homeostasis, in Current Topics in Cellular Regulation, Stadtman, E.R. and Chock, P.B., Eds.,
Academic Press, New York, 1996, p. 189.
25. Cho, E.S., Sahyoun, N., and Stegink, L.D., Tissue glutathione as a cyst(e)ine reservoir during
fasting and refeeding of rats, J. Nutr., 111, 914, 1981.
26. Hum, S., Koski, K.G., and Hoffer, L.J., Varied protein intake alters glutathione metabolism
in rats, J. Nutr., 122, 2010, 1992.
1382_C39.fm Page 682 Tuesday, October 7, 2003 7:36 PM

682 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

27. Jahoor, F. et al., Protein-deficient pigs cannot maintain reduced glutathione homeostasis
when subjected to the stress of inflammation, J. Nutr., 125, 1462, 1995.
28. Hunter, E.A.L. and Grimble, R.F., Dietary sulfur amino acid adequacy influences glutathione
synthesis and glutathione dependent enzymes during the inflammatory response to endo-
toxin and tumour necrosis factor-alpha in rats, Clin. Sci., 92, 297, 1997.
29. Reid, M. et al., In vivo rates of erythrocyte glutathione synthesis in children with severe
protein-energy malnutrition, Am. J. Physiol., 278, E405, 2000.
30. Tateishi, N. et al., Relative contributions of sulfur atoms of dietary cysteine and methionine
to rat liver glutathione and proteins, J. Biochem., 90, 1603, 1981.
31. Coloso, R.M., Hirschberger, L.L., and Stipanuk, M.H., Uptake and metabolism of L-2-oxo-
[35S]thiazolidine-4-carboxylate by rat cells is slower than that of L-[35S]cysteine or
L-[35S]methionine, J. Nutr., 121, 1341, 1991.
32. Yu, Y.M. et al., Plasma L-5-oxoproline kinetics and whole blood glutathione synthesis rates
in severely burned adult humans, Am. J. Physiol., 282, E247, 2002.
33. Lyons, J. et al., Blood glutathione synthesis rates in healthy adults receiving a sulfur amino
acid-free diet, Proc. Natl. Acad. Sci. U.S.A., 97, 5071, 2000.
34. Jahoor, F. et al., Erythrocyte glutathione deficiency in symptom-free HIV infection is associ-
ated with decreased synthesis rate, Am. J. Physiol., 276, E205, 1999.
35. Faber, P. et al., The effect of rate of weight loss on erythrocyte glutathione concentration and
synthesis in healthy obese men, Clin. Sci., 102, 569, 2002.
36. Capitan, P. et al., Gas chromatographic-mass spectrometric analysis of stable isotopes of
cysteine and glutathione in biological samples, J. Chromatogr. B, 732, 127, 1999.
37. Potter, D.W. and Tran, T.B., Apparent rates of glutathione turnover in rat tissues, Toxicol.
Appl. Pharmacol., 120, 186, 1993.
38. Mansoor, M.A., Svardal, A.M., and Ueland, P.M., Determination of the in vivo redox status
of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma, Anal. Bio-
chem., 200, 218, 1992.
39. Mills, B.J. and Lang, C.A., Differential distribution of free and bound glutathione and
cyst(e)ine in human blood, Biochem. Pharmacol., 52, 401, 1996.
40. Rossi, R. et al., Blood glutathione disulfide: in vivo factor or in vitro artifact? Clin. Chem., 48,
742, 2002.
41. Luo, J.L. et al., Skeletal muscle glutathione after surgical trauma, Ann. Surg., 223, 420, 1996.
42. Neuschwander-Tetri, B.A. and Rozin, T., Diurnal variability of cysteine and glutathione
content in the pancreas and liver of the mouse, Comp. Biochem. Physiol. B., 114, 91, 1996.
43. Sido, B. et al., Impairment of intestinal glutathione synthesis in patients with inflammatory
bowel disease, Gut, 42, 485, 1998.
44. Malmezat, T. et al., Glutathione turnover is increased during the acute phase of sepsis in
rats, J. Nutr., 130, 1239, 2000.
45. Deneke, S.M., Lynch, B.A., and Fanburg, B.L., Effects of low protein diets or feed restriction
on rat lung glutathione and oxygen toxicity, J. Nutr., 115, 726, 1985.
46. Märtensson, J., Jain, A., and Meister, A., Glutathione is required for intestinal function, Proc.
Natl. Acad. Sci. U.S.A., 87, 1715, 1990.
47. Dröge, W. and Breitkreutz, R., Glutathione and immune function, Proc. Nutr. Soc., 59, 595,
2000.
48. Robinson, M.K. et al., Glutathione depletion in rats impairs T-cell and macrophage immune
function, Arch. Surg., 128, 29, 1993.
49. Suthanthiran, M. et al., Glutathione regulates activation-dependent DNA synthesis in highly
purified normal human T lymphocytes stimulated via the CD2 and CD3 antigen, Proc. Natl.
Acad. Sci. U.S.A., 87, 3343, 1990.
50. Hammond, C.L., Lee, T. ., and Ballatori, N., Novel roles for glutathione in gene expression,
cell death, and membrane transport of organic solutes, J. Hepatol., 34, 946, 2001.
51. Lander, H.M., An essential role for free radicals and derived species in signal transduction,
FASEB J., 11, 118, 1997.
1382_C39.fm Page 683 Tuesday, October 7, 2003 7:36 PM

Chapter thirty-nine: Sulfur-containing amino acids and glutathione in diseases 683

52. Mosharov, E., Cranford, M.R., and Banerjee, R., The quantitatively important relationship
between homocysteine metabolism and glutathione synthesis by the transsulfuration path-
way and its regulation by redox changes, Biochemistry, 39, 13005, 2000.
53. Deplancke, B. and Gaskins, H.R., Redox control of the transsulfuration and glutathione
biosynthesis pathways, Curr. Opin. Clin. Nutr. Metab. Care, 5, 85, 2002.
54. Ginn-Pease, M.E. and Whisler, R.L., Redox signals and NF-kappa B activation in T cells, Free
Radic. Biol. Med., 25, 346, 1998.
55. May, M.J. and Ghosh, S., Signal transduction through NF-kappa B, Immunol. Today, 19, 80,
1998.
56. Renard, P. et al., Effects of antioxidant enzyme modulations on interleukin-1-induced nuclear
factor kappa B activation, Biochem. Pharmacol., 53, 149, 1997.
57. Sen, C.K. et al., Glutathione regulation of tumor necrosis factor-alpha induced NF-kappa B
activation in skeletal muscle derived L6 cells, Biochem. Biophys. Res. Commun., 237, 645, 1997.
58. Hirota, K. et al., Distinct roles of thioredoxin in the cytoplasm and in the nucleus A two-
step mechanism of redox regulation of transcription factor NF-KappaB, J. Biol. Chem., 274,
27891, 1999.
59. Mallis, R.J. et al., Irreversible thiol oxidation in carbonic anhydrase III: protection by S-glu-
tathiolation and detection in aging rats. Biol. Chem., 383, 649, 2002.
60. Thomas, J.A. and Mallis, R.J., Aging and oxidation of reactive protein sulfhydryls, Exp.
Gerontol., 36, 1519, 2001.
61. Dafré, A.L., Sies, H., and Akerboom, T., Protein S-thiolation and regulation of microsomal
glutathione transferase activity by the glutathione redox couple, Arch. Biochem. Biophys., 332,
288, 1996.
62. Jahngen-Hodge, J. et al., Regulation of ubiquitin-conjugating enzymes by glutathione fol-
lowing oxidative stress, J. Biol. Chem., 272, 28218, 1997.
63. Davies, K.J.A., Degradation of oxidized proteins by the 20S proteasome, Biochimie, 83, 301,
2001.
64. Pineda-Molina, E. et al., Glutathionylation of the p50 subunit of NF-kappaB: a mechanism
for redox-induced inhibition of DNA binding, Biochemistry, 40, 14134, 2001.
65. Rouzer, C.A. et al., Arachidonic acid metabolism in glutathione-deficient macrophages, Proc.
Natl. Acad. Sci. U.S.A., 79, 1621, 1982.
66. Dale, G. et al., The effect of surgical operation on venous plasma free amino acids, Surgery,
81, 295, 1977.
67. Sax, H.C. et al., Amino acid uptake in isolated, perfused liver: effect of trauma and sepsis
J. Surg. Res., 45, 50, 1988.
68. Jeevanandam, M. et al., Aminoaciduria of severe trauma, Am. J. Clin. Nutr., 49, 814, 1989.
69. Vente, J.P. et al., Plasma-amino acid profiles in sepsis and stress, Ann. Surg., 209, 57, 1989.
70. Malmezat, T. et al., Methionine transsulfuration is increased during sepsis in rats, Am. J.
Physiol., 279, E1391, 2000.
71. Vina, J.R. et al., Blood sulfur-amino acid concentration reflects an impairment of liver trans-
sulfuration pathway in patients with acute abdominal inflammatory processes, Br. J. Nutr.,
85, 173, 2001.
72. Rooyackers, O.E. et al., Prolonged changes in protein and amino acid metabolism after
zymosan treatment in rats, Clin. Sci., 87, 619, 1994.
73. Freund, H. et al., Plasma amino acid as predictors of the severity and outcome of sepsis,
Ann. Surg., 190, 571, 1979.
74. Lyons, J. et al., Cysteine metabolism and whole blood glutathione synthesis in septic pediatric
patients, Crit. Care Med., 29, 870, 2001.
75. Mercier, S. et al., Chronic inflammation alters protein metabolism in several organs of adult
rats, J. Nutr., 132, 1921, 2002.
76. Luo, J.L. et al., Surgical trauma decreases glutathione synthetic capacity in human skeletal
muscle tissue, Am. J. Physiol., 275, E359, 1998.
77. Chawla, R.K. et al., Plasma concentrations of transsulfuration pathway products during
nasoenteral and intravenous hyperalimentation of malnourished patient, Am. J. Clin. Nutr.,
42, 577, 1985.
1382_C39.fm Page 684 Tuesday, October 7, 2003 7:36 PM

684 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

78. Tribble, D.L. et al., Hypercysteinemia and delayed sulfur excretion in cirrhotics after oral
cysteine loads, Am. J. Clin. Nutr., 50, 1401, 1989.
79. Holecek, M., Mraz, J., and Tilser, I., Plasma amino acids in four models of experimental liver
injury in rats, Amino Acids, 10, 229, 1996.
80. Miura, K. et al., Cystine uptake and glutathione level in endothelial cells exposed to oxidative
stress, Am. J. Physiol., 262, C50, 1992.
81. Rahman, I. et al., Glutathione homeostasis in alveolar epithelial cells in vitro and lung in
vivo under oxidative stress, Am. J. Physiol., 269, L285, 1995.
82. Lang, C.A. et al., Blood glutathione decreases in chronic diseases, J. Lab. Clin. Med., 135, 402,
2000.
83. Obled, C., Papet, I., and Breuillé, D., Metabolic bases of amino acid requirements in acute
diseases, Curr. Opin. Clin. Nutr. Metab. Care, 5, 189, 2002.
84. Ikegami, K. et al., Comparison of plasma reduced glutathione and oxidized glutathione with
lung and liver tissue oxidant and antioxidant activity during acute inflammation, Shock, 1,
307, 1994.
85. Hammarqvist, F. et al., Skeletal muscle glutathione is depleted in critically ill patients, Crit.
Care Med., 25, 78, 1997.
86. Ruan, E.A. et al., Glutathione levels in chronic inflammatory disorders of the human colon,
Nutr. Res., 17, 463, 1997.
87. Miralles-Barrachina, O. et al., Low levels of glutathione in endoscopic biopsies of patients
with Crohn’s colitis: the role of malnutrition, Clin. Nutr., 18, 313, 1999.
88. Breuillé, D. et al., Assessment of tissue glutathione status during experimental sepsis, Clin.
Nutr., 13, 5, 1994.
89. Colomb, V. et al., Influence of antibiotics and food intake on liver glutathione and cytochrome
P-450 in septic rats, Br. J. Nutr., 73, 99, 1995.
90. Keller, G.A. et al., Decreased hepatic glutathione levels in septic shock, Arch. Surg., 120, 941,
1985.
91. Reichard, S.M. and Bailey, N.M., Alterations in tissue glutathione levels following traumatic
shock, Adv. Shock Res., 5, 37, 1981.
92. Yu, Y.M., Burke, J.F., and Young, V.R., A kinetic study of L-2H3-methyl-1-13C-methionine in
patients with severe burn injury, J. Trauma, 35, 1, 1993.
93. Mansoor, O. et al., Albumin and fibrinogen syntheses increase while muscle protein synthesis
decreases in head-injured patients, Am. J. Physiol., 273, E898, 1997.
94. Bianchi, G. et al., Synthesis of glutathione in response to methionine load in control subjects
and in patients with cirrhosis, Metabolism, 49, 1434, 2000.
95. Breuillé, D. et al., Increased cysteine requirement induced by sepsis, Clin. Nutr., 15, 21, 1996.
96. Malmezat, T. et al., Metabolism of cysteine is modified during the acute phase of sepsis in
rats. J. Nutr., 128, 97, 1998.
97. Wright, C.E. et al., Taurine: biological update, Annu. Rev. Biochem., 55, 427, 1986.
98. Bremner, I. and Beattie, J.H., Copper and zinc metabolism in health and disease: speciation
and interactions, Proc. Nutr. Soc., 54, 489, 1995.
99. Bremner, I. et al., Effects of changes in dietary zinc, copper and selenium supply and of
endotoxin administration on metallothionein I concentrations in blood cells and urine in the
rat, J. Nutr., 117, 1595, 1987.
100. Abe, S. et al., Metallothionein and zinc metabolism in endotoxin shock rats, Experientia,
Suppl. 52, 587, 1987.
101. Reeds, P.J., Fjeld, C.R., and Jahoor, F., Do the differences between the amino acid compositions
of acute-phase and muscle proteins have a bearing on nitrogen loss in traumatic states?
J. Nutr., 124, 906, 1994.
102. Robinson, M.K. et al., Glutathione deficiency increases organ dysfunction after hemorrhagic
shock, Surgery, 112, 140, 1992.
103. Bjelton, L. and Fransson, G.B., Availability of cysteine and of L-2-oxo-thiazolidine-4-carbox-
ylic acid as a source of cysteine in intravenous nutrition, J. Parenter. Enteral. Nutr., 14, 177,
1990.
1382_C39.fm Page 685 Tuesday, October 7, 2003 7:36 PM

Chapter thirty-nine: Sulfur-containing amino acids and glutathione in diseases 685

104. Coloso, R.M., Drake, M.R., and Stipanuk, M.H., Effect of bathocuproine disulfonate, a copper
chelator, on cyst(e)ine metabolism by freshly isolated rat hepatocytes, Am. J. Physiol., 259,
E443, 1990.
105. Stehle, P. et al., In vivo utilization of cystine-containing synthetic short-chain peptides after
intravenous bolus injection in the rat, J. Nutr., 118, 1470, 1988.
106. Micke, P. et al., Oral supplementation with whey proteins increases plasma glutathione levels
of HIV-infected patients, Eur. J. Clin. Invest., 31, 171, 2001.
107. Hagen, T.M. and Jones, D.P., Transepithelial transport of glutathione in vascularly perfused
small intestine of rat, Am. J. Physiol., 252, G607, 1987.
108. Witschi, A. et al., The systemic availability of oral glutathione, Eur. J. Clin. Pharmacol., 43,
667, 1992.
109. Favilli, F. et al., Effect of orally administered glutathione on glutathione levels in some organs
of rats: role of specific transporters, Br. J. Nutr., 78, 293, 1997.
110. Aebi, S., Assereto, R., and Lauterburg, B.H., High-dose intravenous glutathione in man:
pharmacokinetics and effects on cyst(e)ine in plasma and urine, Eur. J. Clin. Invest., 21, 103,
1991.
111. Robinson, M.K., et al., Parenteral glutathione monoester enhances tissue antioxidant stores,
J. Parenter. Enteral Nutr., 16, 413, 1992.
112. Puri, R.N. and Meister, A., Transport of glutathione, as gamma-glutamylcysteinylglycyl ester,
into liver and kidney, Proc. Natl. Acad. Sci. U.S.A., 80, 5258, 1983.
113. Meister, A., Anderson, M.E., and Hwang, O., Intracellular cysteine and glutathione delivery
systems, J. Am. Coll. Nutr., 5, 137, 1986.
114. Porta, P. et al., L-2-oxothiazolidine-4-carboxylic acid, a cysteine prodrug: pharmacokinetics
and effects on thiols in plasma and lymphocytes in human, J. Pharmacol. Exp. Ther., 257, 331,
1991.
115. Jain, A. et al., L-2-oxothiazolidine-4-carboxylate, a cysteine precursor, stimulates growth and
normalizes tissue glutathione concentrations in rats fed a sulfur amino acid-deficient diet,
J. Nutr., 125, 851, 1995.
116. Cotgreave, I.A. et al., Gastrointestinal metabolism of N-acetylcysteine in the rat, including
an assay for sulfite in biological systems, Biopharm. Drug Dispos., 8, 377, 1987.
117. Sjodin, K. et al., Metabolism of N-acetyl-L-cysteine: some structural requirements for the
deacetylation and consequences for the oral bioavailability, Biochem. Pharmacol., 38, 3981,
1989.
118. Yamauchi, A. et al., Tissue distribution of and species differences in deacetylation of N-acetyl-
L-cysteine and immunohistochemical localization of acylase I in the primate kidney, J. Pharm.
Pharmacol., 54, 205, 2002.
119. Phelps, D.T. et al., Elevation of glutathione levels in bovine pulmonary artery endothelial
cells by N-acetylcysteine, Am. J. Respir. Cell Mol. Biol., 7, 293, 1992.
120. Banks, M.F. and Stipanuk, M.H., The utilization of N-acetylcysteine and 2-oxothiazolidine-
4-carboxylate by rat hepatocytes is limited by their rate of uptake and conversion to cysteine,
J. Nutr., 124, 378, 1994.
121. Olsson, B. et al., Pharmacokinetics and bioavailability of reduced and oxidized N-acetyl-
cysteine, Eur. J. Clin. Pharmacol., 34, 77, 1988.
122. Burgunder, J.M., Varriale, A., and Lauterburg, B.H., Effect of N-acetylcysteine on plasma
cysteine and glutathione following paracetamol administration, Eur. J. Clin. Pharmacol., 36,
127, 1989.
123. Neuhauser, M. et al., Utilization of methionine and N-acetyl-L-cysteine during long-term
parenteral nutrition in the growing rat, Metabolism, 35, 869, 1986.
124. Dröge, W., Cysteine and glutathione in catabolic conditions and immunological dysfunction,
Curr. Opin. Clin. Nutr. Metab. Care, 2, 227, 1999.
125. Anderson, S.A. and Raiten, D.J., Eds., Safety of Amino Acids Used as Dietary Supplements, FDA
Report, Life Sciences Research Office, FASEB, Bethesda, MD, 1992, p. 140.
126. Harper, A.E., Benevenga, N.J., and Wohlhueter, R.M., Effects of ingestion of disproportionate
amounts of amino acids, Physiol. Rev., 50, 428, 1970.
1382_C39.fm Page 686 Wednesday, October 8, 2003 1:50 PM

686 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

127. Breuillé, D. et al., Differential effect of dietary supplementation with cysteine or cystine or
methionine on liver glutathione concentration in septic rats, Clin. Nutr., 17, 66, 1998.
128. Luthen, R. et al., Beneficial effects of L-2-oxothiazolidine-4-carboxylate on cerulein pancre-
atitis in mice, Gastroenterology, 112, 1681, 1997.
129. Dröge, W. and Holm, E., Role of cysteine and glutathione in HIV infection and other diseases
associated with muscle wasting and immunological dysfunction, FASEB J., 11, 1077, 1997.
130. De Quay, B., Malinverni, R., and Lauterburg, B.H., Glutathione depletion in HIV-infected
patients: role of cysteine deficiency and effect of oral N-acetylcysteine, AIDS, 6, 815, 1992.
131. Witschi, A. et al., Supplementation of N-acetylcysteine fails to increase glutathione in lym-
phocytes and plasma of patients with AIDS, AIDS Res. Hum. Retroviruses, 11, 141, 1995.
132. De Rosa, S.C. et al., N-acetylcysteine replenishes glutathione in HIV infection, Eur. J. Clin.
Invest., 30, 915, 2000.
133. Barditch-Crovo, P. et al., A phase I/II evaluation of oral L-2-oxothiazolidine-4-carboxylic
acid in asymptomatic patients infected with human immunodeficiency virus, J. Clin. Phar-
macol., 38, 357, 1998.
134. Bernard, G.R. et al., A trial of antioxidants N-acetylcysteine and procysteine in ARDS: the
Antioxidant in ARDS Study Group, Chest, 112, 164, 1997.
135. Meyer, A. et al., Intravenous N-acetylcysteine and lung glutathione of patients with pulmo-
nary fibrosis and normals, Am. J. Respir. Crit. Care Med., 152, 1055, 1995.
136. Badaloo, A. et al., Cysteine supplementation improves the erythrocyte glutathione synthesis
rate in children with severe edematous malnutrition, Am. J. Clin. Nutr., 76, 646, 2002.
137. Pena, L.R., Hill, D.B., and McClain, C.J., Treatment with glutathione precursor decreases
cytokine activity, J. Parenter. Enter. Nutr., 23, 1, 1999.
138. Akerlund, B. et al., Effect of N-acetylcysteine (NAC) treatment on HIV-1 infection: a double-
blind placebo-controlled trial, Eur. J. Clin. Pharmacol., 50, 457, 1996.
139. Herzenberg, L.A. et al., Glutathione deficiency is associated with impaired survival in HIV
disease, Proc. Natl. Acad. Sci. U.S.A., 94, 1967, 1997.
140. Breuillé, D. et al., Cysteine requirement is increased in ICU patients, Clin. Nutr., 20, 43, 2001.
141. Mansoor, O. et al., Amino acid supplementation improves muscle protein synthesis in ICU
patients, Clin. Nutr., 20, 53, 2001.
142. Breuillé, D. et al., Beneficial effect of cysteine supplementation in response to sepsis, Clin.
Nutr., 16, 17, 1997.
143. Ikemoto, M. et al., Cysteine supplementation prevents unweighting-induced ubiquitination
in association with redox regulation in rat skeletal muscle, Biol. Chem., 383, 715, 2002.
144. Shibanuma, M., Kuroki, T., and Nose, K., Inhibition by N-acetyl-L-cysteine of interleukin-6
mRNA induction and activation of NF kappa B by tumor necrosis factor alpha in a mouse
fibroblastic cell line, balb/3T3, FEBS Lett., 353, 62, 1994.
145. Neuschwander-Tetri, B.A. et al., Thiol regulation of endotoxin-induced release of tumour
necrosis factor alpha from isolated rat Kupffer, Biochem. J., 320, 1005, 1996.
146. Antonicelli, F. et al., Nacystelyn inhibits oxidant-mediated interleukin-8 expression and NF-
kappa B nuclear binding in alveolar epithelial cells, Free Radic. Biol. Med., 32, 492, 2002.
147. Haddad, J.J., Saade, N.E., and Safieh-Garabedian, B., Redox regulation of TNF-alpha biosyn-
thesis: augmentation by irreversible inhibition of gamma-glutamylcysteine synthetase and
the involvement of an I kappa B-alpha/NF-kappa B-independent pathway in alveolar epi-
thelial cells, Cell. Signal., 14, 211, 2002.
148. Mihm, S. et al., Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and
cysteine derivatives, AIDS, 5, 497, 1991.
149. Gosset, P. et al., Thiol regulation of the production of TNF-alpha, IL-6 and IL-8 by human
alveolar macrophages, Eur. Respir. J., 14, 98, 1999.
150. Pajonk, F. et al., N-acetyl-L-cysteine inhibits 26s proteasome function: implications for effects
on NF-kappa B activation, Free Radic. Biol. Med., 32, 536, 2002.
151. Peristeris, P. et al., N-acetylcysteine and glutathione as inhibitors of tumor necrosis factor
production, Cell. Immunol., 140, 390, 1992.
152. Zhang, H. et al., Protective effects of N-acetyl-L-cysteine in endotoxemia, Am. J. Physiol., 266,
H1746, 1994.
1382_C39.fm Page 687 Wednesday, October 8, 2003 1:50 PM

Chapter thirty-nine: Sulfur-containing amino acids and glutathione in diseases 687

153. Bakker, J. et al., Effects of N-acetyl-cysteine in endotoxic shock, J. Crit. Care, 9, 236, 1994.
154. Spapen H., et al., Does N-acetyl-L-cysteine influence cytokine response during early human
septic shock? Chest, 113, 1616, 1998.
155. Grimble, R.F. et al., Cysteine and glycine supplementation modulate the metabolic response
to tumor necrosis factor alpha in rats fed a low protein diet, J. Nutr., 122, 2066, 1992.
156. Abdih, H. et al., Taurine prevents interleukin-2-induced acute lung injury in rats, Eur. Surg.
Res., 32, 347, 2000.
157. Schmidt, H. et al., N-acetylcysteine attenuates endotoxin-induced leukocyte-endothelial cell
adhesion and macromolecular leakage in vivo, Crit. Care Med., 25, 858, 1997.
158. Leff, J.A. et al., Postinsult treatment with N-acetyl-L-cysteine decreases IL-1-induced neu-
trophil influx and lung leak in rats, Am. J. Physiol., 265, L501, 1993.
159. Villa, P. et al., Glutathione protects mice from lethal sepsis by limiting inflammation and
potentiating host defense, J. Infect. Dis., 185, 1115, 2002.
160. Kalebic, T. et al., Suppression of human immunodeficiency virus expression in chronically
infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine, Proc. Natl.
Acad. Sci. U.S.A., 88, 986, 1991.
161. Breitkreutz, R. et al., Improvement of immune functions in HIV infection by sulfur supple-
mentation: two randomized trials, J. Mol. Med., 78, 55, 2000.
162. Groeneveld, A.B.J. et al., Effects of N-acetylcysteine and terbutaline treatment on hemody-
namics and regional albumin extravasation in porcine septic shock, Circ. Shock, 30, 185, 1990.
163. Jepsen, S. et al., Antioxidant treatment with N-acetylcysteine during adult respiratory distress
syndrome: a prospective, randomized, placebo-controlled study, Crit. Care Med., 20, 918, 1992.
164. Molnar, Z. et al., The effect of N-acetylcysteine on total serum anti-oxidant potential and
urinary albumin excretion in critically ill patients, Intensive Care Med., 24, 230, 1998.
1382_C39.fm Page 688 Tuesday, October 7, 2003 7:36 PM
1382_C40.fm Page 689 Tuesday, October 7, 2003 7:38 PM

chapter forty

Amino acid requirement in cancer


Maurizio Muscaritoli
University ‘La Sapienza’ Rome, Italy
Filippo Rossi Fanelli
University ‘La Sapienza’ Rome, Italy
Michael M. Meguid
SUNY Upstate Medical University, Syracuse, NY
Antonio C.L. Campos
Federal University of Parana, Brazil

Contents
Introduction..................................................................................................................................690
40.1 Does tumor growth affect plasma amino acid profile?................................................690
40.2 Do different tumors induce different plasma amino acid changes? ..........................691
40.2.1 Solid tumors .........................................................................................................691
40.2.2 Hematologic malignancies .................................................................................693
40.3 Altered plasma amino acid profile and cancer anorexia: a role for free
tryptophan? ........................................................................................................................694
40.4 Indications for nutritional therapy in cancer patients..................................................696
40.4.1 Nutritional status.................................................................................................696
40.4.2 Type and location of the tumor.........................................................................696
40.4.3 Type of medical intervention.............................................................................697
40.4.3.1 Chemotherapy......................................................................................697
40.4.3.2 Radiation therapy ................................................................................697
40.4.3.3 Surgery ..................................................................................................698
40.4.3.3.1 Perioperative parenteral nutrition .................................698
40.4.3.3.2 Perioperative enteral nutrition.......................................698
40.4.3.4 Home nutritional therapy in cancer patients .................................699
40.4.3.5 Impact of nutrition therapy on tumor growth ...............................699
40.5 Conclusions.........................................................................................................................700
References .....................................................................................................................................700

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 689
1382_C40.fm Page 690 Tuesday, October 7, 2003 7:38 PM

690 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Introduction
Changes in the metabolism of body protein, carbohydrate, and fat stores characterize
cancer.1,2 The progression of cancer growth leads to disturbances in protein metabolism,
which is mainly represented by decreased muscle protein synthesis,3 a well-documented
increased activity of proteolytic systems at the muscular level,4–6 increased hepatic protein
synthesis,3 increased whole-body protein turnover, and increased gluconeogenesis from
amino acids (reviewed in Kurzer and Meguid3). Muscle wasting and protein loss are main
characteristics of cancer cachexia. The skeletal muscle compartment appears to be the most
compromised. Other authors7 have shown in advanced gastric cancer patients a significant
decrease in skeletal muscle protein synthesis, compared to controls, with no difference in
breakdown, resulting in decreased muscular mass.7 The muscle mass depletion observed
in their study was estimated to range between 50 and 80% of the normal. Intestinal protein
turnover is also affected by cancer. In rats, altered proliferation and differentiation of
enterocytes were shown to such an extent that they resulted in decreased barrier function
of the gut.8 In the late stages, when the cachexia syndrome is clinically evident, such
metabolic disturbances may also depend on the severe protein–calorie malnutrition result-
ing from reduced food intake, secondary to anorexia and antineoplastic therapy rather
than to the presence of the tumor per se. However, at the time of making the diagnosis
of cancer or in the early stages before treatment is started, the changes in protein metab-
olism are tumor dependent and, as such, are indicators of the tumor’s effects on the host’s
metabolism.
Changes in protein synthesis may contribute to much of the increased energy expen-
diture and elevated metabolic rate seen in some cancer patients. Increases in the activity
of enzymes involved in muscle protein degradation, along with decreases in the activity
of several enzymes involved in muscle protein synthesis, were observed in biopsies of
cancer patients.9 The same activity was not found in matched control patients.10 In par-
ticular, the activities of cathepsin-D and glucuronidase were increased, while the activities
of hexokinase, phosphofructokinase, lactate dehydrogenase, and cytochrome-c oxidase
were decreased.
Among the several amino acids in the body, glutamine is the most abundant and has
several biological functions. One important aspect of glutamine is that it is avidly con-
sumed by rapidly dividing cells. Tumor cells are major glutamine consumers and compete
with the host for circulating glutamine. In several animal models the tumor acts as a
glutamine trap. The presence of cancer is associated with glutamine depletion, in both the
arterial and muscle pools. This can be explained in part by the tumor consumption of
glutamine and by a reduced capacity of the muscle tissue in producing glutamine. The
depletion of glutamine results in decreased functioning of several other glutamine-con-
suming organs. In particular, an increased gut permeability was reported in tumor-bearing
rats.11

40.1 Does tumor growth affect plasma amino acid profile?


Changes in plasma amino acid patterns reliably reflect the quantitative and qualitative
changes in protein metabolism that occur with different pathological conditions. These
include chronic liver failure,12 renal failure,13 sepsis,14 diabetes,15 and pure malnutrition.16
Similarly, alterations of the plasma amino acid pattern also occur in cancer patients and
reflect the consequences of altered protein metabolism. The potential role of these changes
in the diagnostic assessment of patients with malignant diseases has been previously
discussed.17 The nutritional relevance of the altered amino acid profiles in the cancer
patient and their possible implications in clinical practice continuously challenge those
1382_C40.fm Page 691 Tuesday, October 7, 2003 7:38 PM

Chapter forty: Amino acid requirement in cancer 691

involved in the field of clinical nutrition. Progressively gaining insights into the metabolic
alterations leading to the altered amino acid profile in cancer would eventually lead to
the devising of amino acid formulas specifically tailored for the nutritional needs of
neoplastic patients.
Several studies have been performed in the past in which plasma amino acid concen-
trations were measured in patients with cancers of different origin.2,18–21 Although, taken
together, these studies consistently show a reduction in gluconeogenic amino acids (GAA)
and normal or even increased concentration of the branched-chain amino acids (BCAA),
some differences exist relative to some individual amino acids, which may be due to the
nonhomogeneity of the patients studied and the different sites of tumor origin. In a more
recent study, only sulfur amino acids were measured in 63 patients with mixed types of
cancer, and plasma taurine (TAU) was 50% lower than in controls.22 Based on available
data, it seems likely that in cancer patients, even in the presence of malnutrition, BCAA
are maintained within normal range despite marked reduction in total amino acid (TAA)
concentrations. In contrast, in pure chronic malnutrition, BCAA are characteristically
decreased.23–25) (See also Chapters 8 and 23.)
The widely reported reduction in GAA concentration in cancer patients is usually
attributed to increased hepatic gluconeogenesis induced by the tumor to assure adequate
provision of glucose to the tumor itself. But other factors, including impaired tolerance to
carbohydrates, may affect plasma amino acid concentrations.15 Since glucose intolerance
is a common feature in cancer patients, we hypothesized that some of the changes in a
plasma amino acid profile could be secondary to altered metabolism. This hypothesis was
tested by studying the plasma amino acid pattern in a series of untreated cancer patients
in whom the degree of glucose tolerance had been carefully evaluated.26 In this series of
miscellaneous cancer patients, plasma acid pattern was characterized by a significant rise
in phenylalanine, tyrosine, free tryptophan, methionine (MET), proline, glutamic acid, and
ornithine (ORN). When patients were subdivided according to glucose tolerance, no
differences were found between normo-tolerant, glucose intolerant, and diabetics, indi-
cating that the modifications in carbohydrate utilization do not play a significant role in
the observed changes in plasma amino acids. Another variable that might affect the
concentrations of plasma amino acids, particularly of GAA, is the tumor burden. It is
conceivable that a larger tumor mass has a greater need for fuel substrates essential for
intrinsic tumor metabolism. However, dividing cancer patients according to their tumor
stages (stage I/II and stage III), no significant difference in plasma free amino acid profile
could be demonstrated. This finding is in keeping with a selective influence of the tumor
on host protein and amino acid metabolism. The lack of correlation also suggests that the
observed plasma amino acid patterns may not be due to a disturbed amino acid metab-
olism within the tumor tissue itself but instead to the effects of the tumor on host tissues.

40.2 Do different tumors induce different plasma amino acid changes?


40.2.1 Solid tumors
It is well documented that human tumors, originating in different organs, may greatly
differ in terms of rate of proliferation and ability to metastasize, thereby influencing the
host’s metabolism.27 It is thus conceivable that they may also cause different and specific
alterations in the plasma amino acid profile. To test this hypothesis, patients with newly
diagnosed and histologically proven lung or breast cancer were studied.28 Patients with
lung cancer showed a significant reduction of GAA alanine, threonine, serine (SER), and
glycine (GLY) and a significant increase of free tryptophan and glutamic acid. Breast cancer
patients showed normal GAA and significantly higher free tryptophan, glutamic acid, and
1382_C40.fm Page 692 Tuesday, October 7, 2003 7:38 PM

692 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

ornithine. A twofold increase in mean taurine concentrations was found with respect to
controls, although the difference did not reach statistical significance. From these data, it
is apparent that lung and breast cancer have profoundly different systemic effects on the
host’s metabolism. Thus, the plasma amino acid imbalance associated with lung cancer
is probably due to tumor-induced systemic metabolic alterations. In contrast, the increase
in specific amino acids in the presence of breast cancer probably reflects the tumor metab-
olism itself and would not be related to systemic metabolic changes induced by the tumor.
Elevations in plasma aromatic amino acids and methionine occur in the presence of
cirrhosis,12 without hepatocellular carcinoma. These plasma amino acid changes do not
occur in cirrhotic patients with hepatocellular carcinoma.29 Furthermore, plasma methion-
ine, tyrosine, and phenylalanine concentrations in patients with hepatocellular carcinoma
are much higher than in those with cirrhosis without hepatocellular carcinoma or with
normal liver. These findings suggest that there are characteristic plasma amino acid pat-
terns in patients who have cirrhosis with hepatocellular carcinoma that might have clinical
importance in the diagnostic assessment of this disease state.
In a subsequent study, we measured the concentrations of 28 plasma free amino acids
in three different groups of cancer patients to confirm the hypothesis that patients with
different cancers may have different amino acid patterns.30 Twenty-two well-nourished
breast cancer patients, 24 with gastrointestinal tract cancer, and 12 with tumors of the
head and neck were studied. Patients from the gastrointestinal group and the head and
neck group were malnourished, as indicated by recent weight loss of more than 10% and
serum albumin concentrations of less than 35 g/l. Fasting plasma amino acid concentra-
tions in cancer patients were compared with those obtained in 11 healthy well-nourished
controls. For data analysis, amino acids were grouped into TAA, essential amino acids
(EAA), BCAA, aromatic amino acids (AAA), and GAA. Patients’ data were also tabulated
for individual diagnostic group and evaluated by discriminant analysis as assessed by
computing Mahalanobis distances, and used to classify the patient into a diagnostic
group.31 The discriminant function identified the amino acids that most closely represented
a diagnosis consistent with either control subjects or gastrointestinal, head and neck, or
breast cancer. The data showed that in breast cancer, TAA concentrations were significantly
higher than those in female controls, even though both were in good nutritional state. The
TAA concentrations in the gastrointestinal and the head and neck groups did not differ
from controls, even though both patient groups were malnourished and controls were
well nourished. Of the 28 amino acids analyzed, there were 7 that were identified as
correlating highly with diagnosis. These were glutamine, threonine, histidine, cysteine,
alanine, arginine, and ornithine. Despite comparable well-nourished status of patients
with breast cancer and female controls, the plasma concentrations of TAA, EAA, BCAA,
AAA, and GAA, and the GAA:TAA ratio in breast cancer patients were all significantly
higher than those found in control subjects. Moreover, even if patients in the gastrointes-
tinal and the head and neck groups were similarly malnourished, plasma GAA:TAA ratios
in gastrointestinal patients were significantly higher than those in the head and neck
group.
Scioscia et al.32 measured the amino acid profile in squamous cell carcinoma of the
head and neck patients. In comparison to the control group, patients with squamous cell
carcinoma had significantly decreased preoperative serum concentrations of alanine,
asparagine, aspartic acid, glycine, histidine, 3-methylhistidine, ornithine, phenylalanine,
serine, taurine, and threonine. Concentrations of cystine were significantly elevated in the
group of cancer patients. No significant differences were noted on the basis of T stage, N
stage, or nutritional status. Serum concentrations increased postoperatively for the major-
ity of the amino acids tested. Postoperative histidine concentrations were associated with
1382_C40.fm Page 693 Tuesday, October 7, 2003 7:38 PM

Chapter forty: Amino acid requirement in cancer 693

tumor recurrence. The authors concluded that serum amino acid concentrations may prove
to be useful markers of disease status and provide prognostic information.

40.2.2 Hematologic malignancies


The first quantitative studies of plasma amino acid changes in cancer were initially per-
formed in patients with acute and chronic leukemias.33–35 Increased plasma glutamine,
histidine, alanine, threonine, methionine, and proline were found in acute leukemia.35
However, no conclusions regarding the pathophysiologic mechanisms of these alterations
or possible practical implications were provided. Recently, we reappraised the issue of
plasma amino acid profile in nonsolid hematologic malignancies, in order to compare the
plasma amino acid profiles of solid tumors with those of leukemias. To this aim, plasma
FAA were measured by ion exchange chromatography in 40 well-nourished patients with
acute myeloid leukemia (AML) and in 24 healthy volunteers.36 Plasma concentrations of
glutamate (GLU), ORN, GLY, and free tryptophan were significantly higher in AML,
whereas SER, MET, and TAU were significantly lower in AML than in controls and tended
to be even lower in patients who had not responded to chemotherapy or had relapsed
within 18 months of enrollment in the study. Patients with non-M3 subtype AML showed
significantly higher GLU concentrations with respect to M3 AML patients. Such changes
were unrelated to age, gender, and white blood cell (WBC) count. The data obtained in
this study confirmed that in adult, well-nourished AML patients, the plasma amino acid
pattern is altered, suggesting that hematologic malignancies may indeed affect the host’s
protein metabolism.
Of particular interest in this clinical setting is the significant reduction of SER, MET,
and TAU, which might be interrelated. It is in fact known that adult mammalian TAU
may be synthesized through different metabolic pathways using mainly MET but also
SER as a precursor amino acid.37,38 MET is essential in the methylation process in DNA
and RNA synthesis.39 Although the reasons for SER decrease are not entirely clear, MET
in AML patients might be reduced because of abnormalities in DNA synthesis. Both SER
and MET deficiency might eventually result in a reduction in plasma TAU. It cannot be
excluded, however, that TAU deficiency in AML patients might also be secondary to a
reduction in the activity of the enzyme cysteine sulfinate decarboxylase, which is thought
to be the rate-limiting step in TAU synthesis.37 Whatever the cause of TAU reduction in
leukemic patients, however, the present data would indicate that TAU endogenous syn-
thesis from precursors is insufficient to maintain normal plasma TAU concentrations. This
observation might have some relevance in the clinical outcome of AML patients. TAU is
in fact the most abundant free amino acid in WBC, and its concentrations drop significantly
after chemotherapy for hematologic malignancies, while its intracellular content was
demonstrated to correlate directly to the chemosensitivity of a leukemia cell line. Moreover,
TAU supplementation has been shown to accelerate recovery from neutropenia in irradi-
ated mice. Therefore, the finding that AML patients with poorer outcome tend to have
lower concentrations of TAU and its precursors led us to hypothesize that TAU deficiency
might be detrimental in leukemic patients.
The concept that TAU deficiency might negatively affect the outcome of hematologic
malignancies was indeed subsequently supported by two elegant studies. In the first,
Finnegan et al.40 analyzed the effects of taurine on recombinant interleukin-2 (rIL-2)-
activated, lymphocyte-mediated endothelial cell (EC) and tumor cell cytotoxicity. IL-2-
activated cytotoxicity, mediated by peripheral blood mononuclear cells, against susceptible
tumor cell lines and against EC in the presence of taurine was assessed. The authors found
that the addition of taurine significantly reduced rIL-2-activated EC cytotoxicity mediated
by natural killer cells, without reducing antitumor response. Taurine was also shown to
1382_C40.fm Page 694 Tuesday, October 7, 2003 7:38 PM

694 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

reduce significantly EC lysis mediated by lymphokine-activated killer (LAK) cells, while


also significantly increasing tumor cytotoxicity. They concluded that taurine may play a
dual role in rIL-2 immunotherapy, due to its ability to reduce the vascular injury associated
with this therapy while enhancing its antineoplastic activity.
In the second study, Ozgen et al.41 investigated the mechanisms of inherent resistance
of myeloblasts to vincristine (VCR), which has been related to the activity of myeloper-
oxidase (MPO), which can degrade VCR in the presence of hydrogen peroxide (H2O2).
The authors investigated the relationship between VCR degradation and hypochlorous
acid (HOCl) generation from the reaction of H2O2 with chlorine (Cl), as catalyzed by MPO
in a cell-free system, in 3 human leukemia cell lines (CEM/CCRF, HL-60, U937) and 15
bone marrow samples from children with acute myeloid leukemia. VCR was degraded
by increasing concentrations of HOCl in cell-free systems, and this activity was inhibited
by taurine, which is known to block HOCl activity. This finding was confirmed by the
VCR cytotoxicity studies on cell lines.
These results suggest that oxidation by HOCl may be the final step in VCR degradation
catalyzed by MPO through its action on intracellular H2O2 and Cl, and that taurine avail-
ability may significantly affect this process.
In summary, changes in the plasma free amino acid profile in AML patients are only
in part similar to those observed in the presence of solid tumors (i.e., the increase of GLU,
ORN, and FTRP), while the reduction of TAU appears to be a typical feature of AML and
might be secondary to the deficiency of its biosynthetic precursors. Whether TAU defi-
ciency is common to other hematologic malignancies and whether its supplementation
may be of any benefit during or after chemotherapy for acute leukemias and other hema-
tological malignancies, although extremely likely, still remains to be tested in prospective
randomized trials.
Taken together, all these data would suggest that the differences between controls and
patients with primary sites of cancer in some plasma amino acid concentrations are
independent of nutritional status, and that some cancers induce characteristic changes in
the plasma amino acid profile.
A single amino acid that has received particular attention in the setting of hematologic
malignancies is glutamine. The rationale for use and benefits and controversies of clinical
efficacy of glutamine in patients undergoing bone marrow transplantation has been
recently reviewed.42,43 See also Chapters 11, 34, and 36 in this book for further details on
metabolism and clinical uses of glutamine.

40.3 Altered plasma amino acid profile and cancer anorexia:


a role for free tryptophan?
When considering plasma amino acid imbalance in the presence of cancer, tryptophan
behavior deserves to be discussed separately. Tryptophan is unique among the plasma
amino acids since it circulates in blood in two forms. Under physiological conditions,
about 90% of plasma tryptophan is bound to its natural carrier, albumin; the remainder
circulates unbound as free tryptophan.44 Plasma free tryptophan concentrations are influ-
enced by the concentrations of albumin and also by the concentration of circulating free
fatty acids. Free fatty acids displace tryptophan from albumin by competing for the same
binding sites. High plasma and brain concentrations of free tryptophan have been reported
in cancer-bearing animals.45,46 Interestingly enough, our previous studies have also docu-
mented an increase in plasma free tryptophan concentration in untreated cancer patients
with both solid tumors and leukemias26,28,36 and that both plasma47 and CSF 48 tryptophan
concentrations significantly correlate with the presence of cancer anorexia. More recently,
1382_C40.fm Page 695 Tuesday, October 7, 2003 7:38 PM

Chapter forty: Amino acid requirement in cancer 695

we have shown that the increase in plasma free tryptophan occurs early during the growth
of an experimental tumor,49 and that this increase is paralleled by increased brain tryp-
tophan availability and inversely correlates with food intake.50
The presence of cancer anorexia in both animal models and humans has been attrib-
uted to the increased synthesis of serotonin in the brain secondary to increased brain
availability of its precursor, tryptophan.16,51 A peculiarity of the biochemical pathway
transforming tryptophan into serotonin is that the final product does not inhibit the
reaction. Thus, the more tryptophan that reaches the brain, the more serotonin is pro-
duced.52,53 By inference, it is concluded that the increased brain tryptophan entry would
result in increased serotonergic activity at the hypothalamic level. In order to confirm this
postulate, hypothalamic serotonin concentrations were measured in the living cancer-
bearing rat before and after surgical removal of the tumor.54 To this aim the microdialysis
technique was employed, which exploits the characteristics of semipermeable membranes.
The results showed increased hypothalamic serotonin concentrations in tumor-bearing
anorectic rats that normalize after surgical tumor ablation. In line with the given hypoth-
esis, this change in hypothalamic neurochemistry is paralleled by an improvement in food
intake. To strengthen the link between the anorexia of cancer and hypothalamic seroton-
ergic neurotransmission, the effect on food intake of the hypothalamic serotonergic block
during cancer anorexia was investigated. In animals, this was achieved via stereotaxically
located intrahypothalamic microinjection of the serotonin antagonist mianserin.55
Mianserin injection resulted in a significant rise in food intake when compared with
placebo-treated control rats.
In humans the inhibition of hypothalamic serotonergic neurotransmission can be
indirectly achieved. As mentioned above, cancer anorexia is associated with increased
availability to the brain of serotonin precursor, tryptophan. To reach the brain, tryptophan
crosses the blood–brain barrier by competing with the other large neutral amino acids
(namely, aromatic and branched-chain amino acids) for the same carrier.52,53 During tumor
growth plasma tryptophan concentrations are increased, while the concentrations of
competitors are normal26,28,36,47,48 or decreased.50 Thus, tryptophan is facilitated in the
competition with the large neutral amino acids for crossing blood–brain barrier and more
tryptophan reaches the brain where it is converted into serotonin, eventually mediating
the development of anorexia. Among large neutral amino acids, branched-chain amino
acid administration has long been proven safe in humans. A mixture of these amino acids
was therefore orally supplemented in a group of anorectic cancer patients whose food
intake significantly improved when compared to controls receiving an isonitrogenous
supplementation.56 With the same approach, the tryptophan-mediated serotonergic
hypothesis of cancer anorexia was further strengthened in rats bearing the methylcholan-
threne-induced sarcoma. In this model we recently demonstrated that a branched-chain
amino acid-enriched chow may significantly delay the onset of tumor-related anorexia.57
Taken together, all these data strongly support the concept that the increase in free
tryptophan plasma concentrations may play a significant role in the pathogenesis of cancer
anorexia.
Why is plasma free tryptophan increased in the presence of cancer? Plasma free
tryptophan concentrations do not correlate with either albumin or free fatty acid concen-
trations in untreated, well-nourished cancer patients.47 This has led us to postulate that
the free tryptophan increase in the tumor-bearing host may be due to a direct effect of the
tumor on the binding of tryptophan to albumin. To verify this hypothesis, free tryptophan,
albumin, and free fatty acid concentrations were assayed in 12 lung and 16 breast cancer
patients before and 15 days after complete surgical resection of the tumor.58 Eight subjects
undergoing operation for nonneoplastic disease served as a control. Free tryptophan
1382_C40.fm Page 696 Tuesday, October 7, 2003 7:38 PM

696 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

concentrations significantly decreased after tumor ablation but did not change in controls.
Since no correlation was found between free tryptophan and fluctuations in albumin and
free fatty acid, it is conceivable that the tumor itself may be responsible for free tryptophan
elevation. The mechanism(s) whereby this occurs, however, remains largely unknown,
although recent evidence suggests that it could include cytokines. Administration of both
IL-1 and tumor necrosis factor-a (TNF-a) has in fact been shown to increase plasma free
tryptophan, while decreasing food intake in normal rats.59 Also, serum TNF-a concentra-
tions are higher in gastric cancer patients with anorexia and weight loss.60
In summary, the increase of plasma free tryptophan concentrations is a common
feature of both human solid and hematologic malignancies and some experimental tumors.
Increased plasma tryptophan facilitates transport into the brain. Moreover, although the
pathogenesis of cancer anorexia is certainly multifactorial, involving neuropeptides, hor-
mones, and pro-inflammatory cytokines, hypothalamic serotonergic activity is in fact
stimulated in the presence of anorexia of cancer. The modulation of brain tryptophan entry
and brain serotonin synthesis might prove a safe and effective therapeutic approach to
improve food intake in anorectic cancer patients.

40.4 Indications for nutritional therapy in cancer patients


For the indication of nutritional therapy in cancer patients, three factors are important
when evaluating patient needs and selecting the most adequate form of nutrition therapy:
(1) overall clinical and nutritional status of the patient, (2) type and location of the tumor,
and (3) type of medical intervention used in the treatment.

40.4.1 Nutritional status


The clinical landmark of cancer is malnutrition. It is present in 30 to 50% of the patients
by the time of diagnosis, and virtually all patients who die of cancer will have weight loss
by the time of death. In addition, the presence of weight loss by the time of diagnosis is
a poor prognostic sign. Therefore, aggressive nutritional support of patients with cancer
received widespread clinical application. However, recent evidence has questioned this
practice. The clinical efficacy of nutrition therapy in patients with cancer receiving radi-
ation or chemotherapy has been reviewed. It appears from many studies that parenteral
nutrition is not helpful, and some studies even suggest that it may be detrimental. There-
fore, the demonstration by nutrition assessment techniques that the patient undergoing
radiation or chemotherapy is malnourished should not be per se an indication for nutrition
therapy.
Nutrition therapy can be effective in reversing malnutrition but not in combating
cancer. That means that nutrition therapy should be indicated preferentially when an
antitumor therapy is being successfully used. It is unlikely that nutritional therapy will
be of any benefit in well-nourished or minimally malnourished cancer patients. Nutritional
therapy should not be indicated in patients who have terminal cancer and no realistic
anticancer treatment options.61

40.4.2 Type and location of the tumor


The type and location of the tumor is another important variable when deciding to start
nutritional therapy. For example, a head and neck or esophago-gastric tumor causing
obstruction may have a mechanical component contributing to the weight loss. Bypassing
the obstruction with a feeding tube allows partial reversal of the malnourished state
before surgery. When efforts to promote oral intake are ineffective or inappropriate, more
1382_C40.fm Page 697 Tuesday, October 7, 2003 7:38 PM

Chapter forty: Amino acid requirement in cancer 697

aggressive measures must be used. If the gut is functional, enteral feeding is recom-
mended over parenteral feeding. Short-term nutritional therapy should be supplied via
a nasogastric or nasoenteric tube. Patients needing support for more than 6 weeks will
require a permanent tube, which can be placed into the stomach, duodenum, or jejunum
using laparotomy, laparoscopy, fluoroscopy, or endoscopy.62 The placement of tubes
allows feeding distal to metastatic or obstructing tumors or surgical resection.63

40.4.3 Type of medical intervention


40.4.3.1 Chemotherapy
The effects of chemotherapy may directly interfere with metabolism or may indirectly
affect the patient by producing nausea, vomiting, diarrhea, changes in taste sensation,
anorexia, and learned food aversions.64–66 The two most common acute toxicities with
chemotherapy are nausea and vomiting. Onset of symptoms can be immediate or delayed;
duration can range from several hours to days. If not controlled, nausea and vomiting can
result in electrolyte imbalance, weight loss, and weakness.64,67
In an intersociety communication paper published by Klein et al.68 in 1997, including
a careful review of published data on nutritional support in cancer patients, 18 prospective
randomized trials were identified evaluating the use of parenteral nutrition in patients
receiving cancer chemotherapy. The use of parenteral nutrition was usually associated
with weight gain. In general, the use of parenteral nutrition did not affect survival and
did not decrease hematologic or gastrointestinal toxicity from chemotherapy. Infectious
complications were higher in patients receiving parenteral nutrition. The use of enteral
nutrition was evaluated in seven prospective randomized trials. Again, no obvious benefit
was observed in terms of survival, tumor response, or chemotherapy toxicity. The authors
have pointed out several criticisms of the articles reviewed, such as the inclusion of
heterogeneous populations, exclusion of severely malnourished patients in many studies,
inadequate end points, and insufficient sample sizes. Also, the composition, time, and
duration of the nutrition therapy varied widely. These results do not mean that nutrition
therapy should not be used in such patients. For example, if a patient develops severe
therapy-related toxicity that precludes normal food intake for long periods, parenteral
nutrition may be necessary and appropriate.
In children with solid tumors or relapsing leukemia or lymphoma who underwent
intensive combined therapy, an average weight loss of 16% of body weight was observed
during the first month of treatment while receiving oral diet.69 The Wilms’ tumor subgroup
lost an average of 22% of body weight during this period. Vigorous attempts at maintain-
ing adequate enteral nutrition have been shown to be very successful, even in advanced-
stage neuroblastoma patients undergoing chemotherapy.69

40.4.3.2 Radiation therapy


The relationship between radiation therapy and nutritional therapy depends upon tumor
location, type of radiation used, size of irradiation field, patient status, and dose duration
of therapy.70 The dose of radiation and anatomic site or field of radiation are major
determinants of potential side effects. Tumors located in the area of the head, neck, and
abdomen have the greatest implications for nutritional therapy in cancer patients receiving
radiotherapy. Radiation treatments to the head and neck are known to cause inflammation,
pain, decreased salivation, caries, altered taste perceptions, stomatitis, oral infections,
trismus (tetany of the jaw muscles), dysphagia, and odynophagia (painful swallowing).70,71
Food aversions often develop as the patient loses the ability to differentiate between bitter
and sour tastes. (This condition usually returns to normal within 12 months after therapy.)
The nutritional consequences of abdominal and pelvic radiation are a direct result of
1382_C40.fm Page 698 Tuesday, October 7, 2003 7:38 PM

698 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

irradiation to organs of the GI tract. At low radiation doses, anorexia and nausea may
occur and cause less severe nutrition problems. At higher doses, GI ulcers, bleeding,
nausea, vomiting, diarrhea, and weight loss may occur.
The small bowel is also highly sensitive to radiation damage, and therapy in the small
intestine may cause extensive nausea, vomiting, abdominal cramps, and watery diarrhea.
These symptoms, accompanied by characteristic functional and morphologic changes, are
collectively termed acute radiation enteritis.72
In Klein et al.’s68 intersociety communication paper, referred to previously, four pro-
spective randomized controlled trials were identified evaluating the clinical efficacy of
parenteral nutrition in patients receiving radiation therapy for abdominal or pelvic cancer.
Overall, there were no differences in survival or in radiation-induced side effects between
groups receiving parenteral nutrition and controls. In contrast, oral or enteral nutrition,
evaluated by seven prospective randomized trials, was associated with decreased weight
loss during the treatment in most studies. Furthermore, most studies reported fewer
hematological or gastrointestinal side effects of radiation therapy in patients receiving
enteral nutrition. Survival was not affected by enteral nutrition.

40.4.3.3 Surgery
40.4.3.3.1 Perioperative parenteral nutrition. The value of parenteral nutrition as an
adjuvant to operation for malignancy has been examined in a number of studies in either
the pre- or postoperative periods. The rationale for the use of parenteral nutrition prior
to operations is that in such patients nutritional support may increase the level of serum
albumin, enhance immune function, and increase body weight, reducing the incidence of
anastomotic breakdown and wound complications.
In 1992 Campos and Meguid73 reviewed the published articles on perioperative nutri-
tional support and concluded that perioperative parenteral nutrition was effective in
reducing complications when given to malnourished patients for at least 7 to 15 days
before major gastrointestinal surgery in severely malnourished patients. The well-known
huge Veterans Administration parenteral nutrition cooperative study74 on perioperative
parenteral nutrition published in 1991 has shown that parenteral nutrition was able to
reduce postoperative complications in the subset of severely malnourished patients only.
The intersociety communication paper by Klein et al.68 concluded, based on 13 pro-
spective randomized trials involving 1250 patients, that parenteral nutrition given for 7 to
10 days before surgery decreases postoperative complications by approximately 10%. In
contrast, postoperative parenteral nutrition did not yield similar results. Indeed, a 10%
increase in postoperative complications was reported by the same study with postopera-
tive parenteral nutrition.
Recently, Morlion et al.75 evaluated the use of postoperative parenteral nutrition
enriched with glutamine dipeptide following elective colorectal resections for carcinoma.
The rationale for this study is detailed in Chapters 11 and 36 of this book. In this study
an improved nitrogen balance was noted in the group receiving glutamine dipeptide. The
incidence of postoperative complications was not reported, but the postoperative hospital
stay was 6.2 days shorter in the glutamine dipeptide-supplemented group. Since conven-
tional parenteral nutrition solutions do not contain glutamine, the authors considered the
glutamine supplementation a replacement of a deficiency rather than a supplementation.

40.4.3.3.2 Perioperative enteral nutrition. In recent years several studies have eval-
uated the use of enteral nutrition following operations for malignancy. Daly et al.76 rand-
omized 60 malnourished cancer patients subjected to upper gastrointestinal and pancreatic
resection for cancer to receive or not receive enteral nutrition via jejunostomy, beginning
1382_C40.fm Page 699 Tuesday, October 7, 2003 7:38 PM

Chapter forty: Amino acid requirement in cancer 699

on the first postoperative day. Enteral feeding patients were further randomized to receive
standard diet or an enteral formula supplemented with immuno-enhancing nutrients.
Infectious or wound complications occurred in 43% of the patients receiving the standard
diet and in only 10% of the patients of the supplemented group (p < 0.05). Similarly, the
length of hospital stay was reduced from 22 ± 2.9 to 16 ± 0.9 days in the supplemented
diet group (p < 0.02).
Kenler et al.77 randomized 50 patients with upper gastrointestinal malignancies to
receive a standard diet or a fish oil structured lipid formula through a feeding jejunostomy
starting in the immediate postoperative period. Patients receiving the fish oil formula had
fewer infectious complications and a 50% decline in the number of gastrointestinal com-
plications.
Recently, Braga et al.78 conducted a prospective randomized trial in patients under-
going cancer surgery. A total of 206 consecutive patients were enrolled in the study.
Patients were randomized to receive either a standard enteral formula or a supplemented
liquid diet. The administration of the supplemented diet allowed a more rapid recovery
of the postsurgical immune depression than the standard diet. The administration of the
supplemented diet significantly reduced the rate of postoperative infections regardless of
the baseline nutrition status. A reduction in the total number of infectious complications
from 30 to 14% was noted with the supplemented formula (p < 0.009).

40.4.3.4 Home nutritional therapy in cancer patients


Finally, cancer patients may be selected to receive nutritional therapy at home during or
after their cancer treatment. Campos et al.79 demonstrated that home enteral nutrition via
gastrostomy was able to maintain the nutritional status and to improve comfort and
quality of life in advanced head and neck cancer patients. Recently, Cozzaglio et al.80
demonstrated similar benefits in a group of cancer patients receiving home parenteral
nutrition who survived longer than 3 months. Quality of life improved in 68% of these
patients. In contrast, only 9% of those who survived less than 3 months presented a positive
effect of home parenteral nutrition.

40.4.3.5 Impact of nutrition therapy on tumor growth


Many studies have found that protein–calorie deprivation is related to a general decline
in the rate of establishment of transplanted tumors in animals. Since the early 1950s,
investigators have consistently found a relationship between protein and calorie restric-
tions and the inhibition of spontaneous tumorigenesis in rats.81,82
In a number of animal models tumor growth rates have been shown to correlate
positively with nutrition status and exogenous nutrient delivery, raising the concern that
nutrition therapy might promote tumor growth and thus worsen the outcome. In contrast,
the overall effectiveness of chemotherapy on reducing tumor growth in rats may actually
be enhanced by nutrient administration. In a number of animal studies, the stimulation
of tumor growth associated with nutritional supplementation appears to improve tumor
response to chemotherapy.83,84 These investigators postulated that continuous substrate
administration in rats stimulated tumor cell proliferation, which increased activity of the
tumor and enhanced the response to chemotherapy. Nutrition therapy restores carcass
weight and immunocompetence without adversely stimulating tumor growth out of pro-
portion to the growth of the host.
Despite the observation of significant increases in the ratio of tumor weight to host
body weight when replenishing nutrients in rats,85 there are no human studies substanti-
ating a preferential growth of tumors associated with total parenteral nutrition (TPN). In
a 1992 review, Torosian noted three characteristics of animal and human models that may
1382_C40.fm Page 700 Tuesday, October 7, 2003 7:38 PM

700 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

account for this discrepancy in findings: (1) Rates of tumor growth in animals and humans
are dramatically different. Animal tumors double as rapidly as every 2 to 7 days, whereas
human tumors double in a minimum of 30 days. (2) Animal tumors may account for as
much as 60% of overall body weight; the human tumors studied accounted for a relatively
small proportion of total body weight. (3) The range of tumor immunogenicity in animals
is much broader than the range found in humans.

40.5 Conclusions
During the last 30 years, several research groups have provided valid contributions that
helped to clarify the existing relationships between the presence of a tumor and the
changes in the plasma amino acid profile. The sum of these clinical studies clearly indicates
that during all stages of cancer growth studied so far, disturbances in the host’s protein
metabolism are present, which are closely reflected by changes in plasma amino acid
concentrations. It can be concluded that specific alterations in plasma amino acid patterns
characterize cancer states independently of concomitant malnutrition, glucose intolerance,
and tumor stage.
In the case of renal failure, liver failure, and sepsis (see relevant chapters in this book),
the imbalanced plasma amino acid profile can be normalized by the administration of
specifically tailored amino acid mixtures to positively influence clinical management. By
contrast, in the cancer patient the nutritional relevance of the altered amino acid profiles
continuously challenges those involved in the field of clinical nutrition in the attempt to
realize an amino acid formula, the composition of which may well fit the metabolic
disturbances and the nutritional needs of cancer patients. To date, however, despite the
vast array of clinical and experimental studies documenting an imbalance in circulating
amino acids in cancer, no specific “cancer formulas” have been elaborated. Nonetheless,
some of the evidence derived from the study of cancer patients and animal models of
cancer suggests that modulation in supply of some specific amino acids might prove
beneficial in improving symptoms secondary to the presence of the tumor, as in the cases
of cancer anorexia; ameliorating tolerance to antineoplastic treatments, such as mucositis
and diarrhea caused by radiochemotherapy; and increasing sensitivity to chemotherapy
regimens. From this perspective, BCAA, taurine, and glutamine would appear to be the
most promising substrates to ameliorate the metabolic and nutritional support for the
cancer patient. The patient’s clinical condition and tumor type affect nutritional therapy
decisions. Chemotherapy, radiation, surgery, and bone marrow transplantation also have
unique implications for nutritional therapy. Appropriate nutrition therapy may attenuate
the adverse effects of the tumor and its treatment, thus increasing tolerance to treatment
and decreasing morbidity and mortality.

References
1. Lundholm, K., Edstrom, S., Ekman, L., et al., Metabolism in peripheral tissues in cancer
patients, Cancer Treat. Rep., 65 (Suppl. 5), 79, 1981.
2. Heber, D., Byerly, L.O., and Chlebowski, R.T., Metabolic abnormalities in the cancer patients,
Cancer, 55, 225, 1985.
3. Kurzer, M. and Meguid, M.M., Cancer and protein metabolism, Surg. Clin. North Am., 66,
969, 1986.
4. Costelli, P. and Baccino, F.M., Cancer cachexia: from experimental models to patient man-
agement, Curr. Opin. Clin. Nutr. Metab. Care, 3, 177, 2000.
5. Bossola, M., Muscaritoli, M., Costelli, P., et al., Increased muscle ubiquitin m-RNA levels in
gastric cancer patients, Am. J. Physiol., 280, R1518, 2001.
1382_C40.fm Page 701 Tuesday, October 7, 2003 7:38 PM

Chapter forty: Amino acid requirement in cancer 701

6. Bossola, M., Muscaritoli, M., Costelli, P., et al., Muscle proteasome activity is increased in
gastric cancer patients and correlates with disease stage and weight loss, Ann. Surg., 237,
384, 2003.
7. Dworzak, F., Ferrai, P., Gavazzi, C., Maiorana, C., and Bozzetti, F., Effects of cachexia due to
cancer on whole body and skeletal muscle protein turnover, Cancer, 82, 42, 1998.
8. Blaauw, I., Deutz, N.E.P., and Von Meyenfeldt, M.F., Metabolic changes in cancer cachexia:
first of two parts, Clin. Nutr., 16, 169, 1997.
9. Blaauw, I., Heeneman, S., Deutz, N.E.P., and Von Meyenfeldt, M.F., Increased whole-body
protein and glutamine turnover in advanced cancer is not matched by an increased muscle
protein and glutamine turnover, J. Surg. Res., 68, 44, 1997.
10. Lundholm, K., Bylund, A.C., Holm, J., et al., Skeletal muscle metabolism in patients with
malignant tumor, Eur. J. Cancer, 12, 465, 1976.
11. Blaauw, I., Van der Hulst, R.R.W.J., Deutz, N.E.P., and Von Meyenfeldt, M.F., Glutamine
depletion and increased gut permeability in non-anorectic, non weight-losing tumor bearing
rats, Gastroenterology, 112, 118, 1997.
12. Cascino, A., Cangiano, C., Fiaccadori, F., et al., Plasma and cerebro-spinal fluid amino acid
patterns in hepatic encephalopathy, Dig. Dis. Sci., 27, 828, 1982.
13. Kopple, J.D., Jones, M., Fukuda, S., et al., Amino acid and protein metabolism in renal failure,
Am. J. Clin. Nutr., 31, 1532, 1978.
14. Freund, H.R., Ryan, J.A., and Fischer, J.B., Amino acid derangements in patients with sepsis:
treatment with branched-chain amino acid rich, Am. J. Surg., 188, 423, 1978.
15. Wahren, J., Felig, P., Cerasi, B., et al., Splanchnic and peripheral glucose and amino acid
metabolism in diabetes mellitus, J. Clin. Invest., 51, 1870, 1972.
16. Felig, P., Owen, O.B., Wahren, J., et al., Amino acid metabolism during prolonged starvation,
J. Clin. Invest., 48, 584, 1969.
17. Meguid, M.M., Muscaritoli, M., Cascino, A., and Rossi Fanelli, F., Plasma amino acid profile
in cancer patients: a novel diagnostic tool comes of age, in Amino Acids in Critical Care and
Cancer, R. Latifi, Ed., Austin, TX, RG Landes Company, 1994, chap. 13, p. 159.
18. Clarke, B.F., Lewis, A.M., and Waterhouse, C., Peripheral amino acid levels in patients with
cancer, Cancer, 42, 2909, 1978.
19. Levin, L., Gebers, W., Jardine, L., et al., Serum amino acids in weight losing patients with
cancer and tuberculosis, Eur. J. Cancer Clin. Oncol., 19, 711, 1983.
20. Naini, A.B., Dickerson, J.W.T, and Brown, M.M., Preoperative and postoperative levels of
plasma protein and amino acid in esophageal and lung cancer patients, Cancer, 62, 355, 1988.
21. Tayek, J.A. and Chlebowski, R.T., Metabolic response to chemotherapy in colon cancer
patients, J. Parenter. Enteral Nutr., 16 (Suppl. 6), 65S, 1992.
22. Gray, G.E., Landel, A.M., and Meguid, M.M., Taurine-supplemenred TPN and taurine status
of malnourished cancer patients, Nutrition, 10, 11, 1994.
23. Arroyave, G., Wilson, D., Funes, C.D., et al., The free amino acids in blood plasma of children
with kwashiorkor and marasmus, Am. J. Clin. Nutr., 11, 517, 1962.
24. Holt, L.E., Synderman, S.E., Norton, P.M., et al., The plasma aminogram in kwashiorkor,
Lancet, 2, 1343, 1963.
25. Smith, S.R., Pozefsky, T., and Chetri, M.K., Nitrogen and amino acid metabolism in adults
with protein-calorie malnutrition, Metabolism, 23, 603, 1974.
26. Cascino, A., Cangiano, C., Ceci, F., et al., Plasma amino acids in human cancer: the individual
role of tumour, malnutrition and glucose tolerance, Clin. Nutr., 7, 213, 1988.
27. Kern, K.A. and Norton, J.A., Cancer cachexia, J. Parenter. Enteral Nutr., 12, 286, 1988.
28. Cascino, A., Muscaritoli, M., Cangiano, C., et al., Plasma amino acid imbalance in patients
with lung and breast cancer, Anticancer Res., 15, 507, 1995.
29. Watanabe, A., Higashi, T., Sakata, T., et al., Serum amino acid levels in patients with hepa-
tocellular carcinoma, Cancer, 54, 1875, 1984.
30. Kubota, A., Meguid, M.M., and Hitch, D.C., Amino acid profiles correlate diagnostically with
organ site in three kinds of malignant tumors, Cancer, 69, 2343, 1992.
31. Wilkinson, L., SYSTAT: The System for Statistics, SYSTAT, Inc., Evanston, IL, 1988, p. 532.
1382_C40.fm Page 702 Tuesday, October 7, 2003 7:38 PM

702 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

32. Scioscia, K.A., Snyderman, C.H., and Wagner, R., Altered serum amino acid profiles in head
and neck cancer, Nutr. Cancer, 30, 144, 1998.
33. Kelley, J.J. and Waisman, H.A., Quantitative plasma amino acid values in leukemic blood,
Blood, 12, 635, 1957.
34. Waisman, H.A., Pastel, R.A., and Poncher, H.G., Amino acid metabolism in patients with
acute leukemia, Pediatrics, 10, 653, 1952.
35. Rudman, D., Vogler, W.R., Howard, C.H., et al., Observations on the plasma amino acids of
patients with acute leukemia, Cancer Res., 31, 1159, 1971.
36. Muscaritoli, M., Conversano, L., Petti, M.C., Cascino, A., Mecarocci, M., Annicchiarico, M.A.,
and Rossi Fanelli, F., Plasma amino acid concentrations in patients with acute myelogenous
leukemia, Nutrition, 15, 195, 1999.
37. Huxtable, R.J., Physiological action of taurine, Physiol. Rev., 72, 101, 1992.
38. Wright, C.E., Tallan, H.H., and Lin, Y.Y., Taurine: biological update, Annu. Rev. Biochem., 55,
427, 1986.
39. Ashe, H., Clar, B.R., Hardy, B.N., et al., N-Methyltetrahydrofolate homocysteine methyltrans-
ferase activity in extracts from normal, malignant and embryonic tissue culture cells, Biochem.
Biophys. Res. Commun., T57, 417, 1974.
40. Finnegan, N.M., Redmond, H.P., and Bouchier-Hayes, D.J., Taurine attenuates recombinant
interleukin-2-activated, lymphocyte-mediated endothelial cell injury, Cancer, 82, 186, 1998.
41. Ozgen, U., Savasan, S., Stout, M., Buck, S., and Ravindranath, Y., Further elucidation of
mechanism of resistance to vincristine in myeloid cells: role of hypochlorous acid in degra-
dation of vincristine by myeloperoxidase, Leukemia, 14, 47, 2000.
42. Ziegler, T.R., Glutamine supplementation in cancer patients receiving bone marrow trans-
plantation and high-dose chemotherapy, J. Nutr., 131, 2578S, 2001.
43. Muscaritoli, M., Grieco, G., Capria, S., Iori, A.P., and Fanelli, F.R., Nutritional and metabolic
support in patients undergoing bone marrow transplantation, Am. J. Clin. Nutr., 75, 183, 2002.
44. McMenamy, R.H. and Oncley, J.L., The specific binding of L-tryprophan to serum albumin,
J. Biol. Chem., 233, 1436, 1958.
45. Krause, R., James, J.H., Ziparo, V., et al., Brain tryptophan and the neoplastic anorexia-
cachexia syndrome, Cancer, 44, 1003, 1979.
46. Krause, R., Humphrey, C., von Meyenfeld, M., et al., A central mechanism for anorexia in
cancer: a hypothesis, Cancer Treat. Rep., 65, 15, 1981.
47. Rossi-Fanelli, F., Cangiano, C., Ceci, F., et al., Plasma tryptophan and anorexia in human
cancer, Eur. J. Cancer Clin. Oncol., 22, 89, 1986.
48. Cangiano, C., Cascino, A., Ceci, F., et al., Plasma and CSF tryptophan in cancer anorexia,
J. Neural Transm., 81, 225, 1990.
49. Meguid, M.M., Muscaritoli, M., Beverly, J.L., et al., The early cancer anorexia paradigm:
changes in plasma free tryptophan and feeding indexes, J. Parenter. Enteral Nutr., 16 (Suppl.
6), 56S, 1992.
50. Muscaritoli, M., Meguid, M.M., Beverly, J.L., Yang, Z.J., Cangiano, C., and Rossi Fanelli, F.,
Mechanisms of early tumor anorexia, J. Surg. Res., 60, 389, 1996.
51. Rossi-Fanelli, F. and Cangiano, C., Increased availability of tryptophan in brain as common
pathogenic mechanism for anorexia associated with different diseases, Nutrition, 7, 364, 1991.
52. Fernstrom, J.D. and Wurtman, R.J., Brain serotonin content: physiological dependence on
plasma tryptophan levels, Science, 173, 149, 1971.
53. Tagliamone, A., Bigio, G., Vargiu, L., and Gessa, G.L., Free tryptophan in serum controls
brain tryptophan levels and serotonin synthesis, Life Sci., 12, 277, 1973.
54. Blaha, V., Yang, Z.J., Meguid, M.M., Laviano, A., Zadak, Z., Rossi Fanelli, F., Cancer anorexia
is modulated by interaction of hypothalamic-VMN dopamine and serotonin and not solely
serotonin as currently thought, Surg. Forum, 47, 517, 1996.
55. Laviano, A., Gleason, J.R., Meguid, M.M., et al., Effects of intra-VMN miansein and IL-1ra
on meal number in anorectic tumor bearing rats, J. Invest. Med., 48, 40, 2000.
56. Cangiano, C., Laviano, A., Meguid, M.M., et al., Effects of administration of oral branched-
chain amino acids on anorexia and caloric intake in cancer patients, J. Natl. Cancer Inst., 88,
550, 1996.
1382_C40.fm Page 703 Tuesday, October 7, 2003 7:38 PM

Chapter forty: Amino acid requirement in cancer 703

57. Fanfarillo, F., Laviano, A., Muscaritoli, M., et al., Branched-chain amino acids (BCAA) delay
the onset of cancer anorexia in tumor-bearing (TB) rats, Clin. Nutr., 20, 20, 2001 (abstract).
58. Cascino, A., Cangiano, C., Ceci, F., et al., Increased plasma free tryptophan levels in human
cancer: a tumor related effect? Anticancer Res., 11, 1313, 1991.
59. Sato, T., Laviano, A., Meguid, M.M., Chen, C., and Rossi Fanelli, F., Involvement of plasma
leptin, insulin and free tryptophan in cytokine induced anorexia, Clin Nutr., 22, 139, 2003.
60. Bossola, M., Muscaritoli, M., Bellantone, R., et al., Serum tumor necrosis factor-a levels in
cancer patients are discontinuous and correlate with weight loss, Eur. J. Clin. Invest., 30, 1107,
2000.
61. Torelli, G.F., Campos, A.C.L., and Meguid, M.M., Use of TPN in terminally ill cancer patients,
Nutrition, 15, 665, 1999.
62. Campos, A.C.L. and Marchesini, J.B., Recent advances in the placement of tubes for enteral
nutrition, Curr. Opin. Clin. Nutr. Metab. Care, 2, 265, 1999.
63. Ellis, L.M., Evans, D.B., Martin, D., et al., Laparoscopic feeding jejunostomy tube in oncology
patients, Surg. Oncol., 1, 245, 1992.
64. Laszlo, J., Stevenson, D., and Lucas, V.S., Chemotherapy, in Physician’s Guide to Cancer Care
Complications: Prevention and Management, Laszlo, J., Ed., Marcel Dekker, New York, 1986,
p. 61.
65. Bernstein, I.L., Webster, M.M., and Bernstein, I.D., Food aversions in children receiving
chemotherapy for cancer, Cancer, 50, 2961, 1982.
66. Stoudemire, A., Cotanch, P., and Laszlo, J., Recent advances in the pharmacologic and
behavioral management of chemotherapy-induced emesis, Arch. Intern. Med., 144, 1029, 1984.
67. Dennis, V.W., Fluid and electrolyte changes after vomiting, in Antiernetics and Cancer Chemo-
therapy, Laszlo, J., Ed., Williams & Wilkins, Baltimore, 1983, p. 34.
68. Klein, S., Kinney, J., Jeejeebhoy, K., Alpers, D., Ellerstein, M., Murray, M., and Twomey, P.,
Nutrition support in clinical practice: review of published data and recommendations for
future research directions, Am. J. Clin. Nutr., 66, 683, 1997.
69. Andrassy, R.J. and Chwals, W.J., Nutritional support of the pediatric oncology patient,
Nutrition, 14, 124, 1998.
70. Kouba, J., Nutritional care of the individual with cancer, Nutr. Clin. Pract., 3, 175, 1988.
71. Goodwin, W.J. and Byers, P.M., Nutritional management of the head and neck cancer patient,
Med. Clin. North Am., 77, 597, 1993.
72. Knox, L.S., Nutrition and cancer, Nursing Clin. North Am., 18, 97, 1993.
73. Campos, A.C.L. and Meguid, M.M., A critical appraisal of the usefulness of perioperative
nutritional support, Am. J. Clin. Nutr., 55, 117, 1992.
74. VA TPN Cooperative Study: perioperative total parenteral nutrition in surgical patients,
N. Engl. J. Med., 325, 525, 1991.
75. Molion, B.J., Stehle, P., Wachtler, P., et al., Total parenteral nutrition with glutamine dipeptide
after major abdominal surgery, Ann. Surg., 227, 302, 1998.
76. Daly, J.M., Weintraub, F.N., Shou, J., Rosato, E.F., and Lucia, M., Enteral nutrition during
multimodality therapy in upper gastrointestinal cancer patients, Ann. Surg., 221, 327, 1995.
77. Kenler, A.S., Swails, W.S., Driscoll, D.F., et al., Early enteral feeding in postsurgical cancer
patients: fish oil structured lipid-based polymeric formula versus a standard polymeric
formula, Ann. Surg., 223, 316, 1996.
78. Braga, M., Gianotti, L., Radaelli, G., Vignali, A., Mari, G., Gentilini, O., and Di Carlo, V.,
Perioperative immunonutrition in patients undergoing cancer surgery: results of a random-
ized double-blind phase 3 trial, Arch. Surg., 134, 428, 1999.
79. Campos, A.C.L., Butters, M., and Meguid, M.M., Home enteral nutrition via gastrostomy in
advanced head and neck cancer patients, Head Neck, 2, 137, 1990.
80. Cozzaglio, L. et al., Outcome of cancer patients receiving home parenteral nutrition,
J. Parenter. Enteral Nutr., 21, 339, 1997.
81. Tannenbaum, A. and Silverstone, H., Nutrition in relation to cancer, Adv. Cancer Res., 1, 451,
1953.
82. Green, J.W., Benditt, E.D., and Humphreys, E.M., The effect of protein depletion on the host
response to transplantable rat tumor Walker 256, Cancer Res., 10, 769, 1950.
1382_C40.fm Page 704 Tuesday, October 7, 2003 7:38 PM

704 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

83. Daly, J.M., Reynolds, H.M., Copeland, E.M., et al., Effects of enteral and parenteral nutrition
on tumor response to chemotherapy in experimental animals, J. Surg. Oncol., 16, 79, 1981.
84. Reynolds, H.M., Daly, J.M., Rowland, B.J., et al., Effects of nutritional repletion on host and
tumor response to chemotherapy, Cancer, 45, 3069, 1980.
85. Cameron, I.L., Effect of total parenteral nutrition on tumor-host responses in rats, Cancer
Treat. Rep., 65 (Suppl.), 93, 1981.
86. Campos, A.C.L., Waitzberg, D., and Meguid, M.M., A comparison of the changes in carbo-
hydrate, fat and protein metabolism occurring with malignant and benign tumors and the
impact of nutritional support, in Human Nutrition: A Comprehensive Treatise, Vol. 7, Alfin-
Slaler, R. and Kritchevsky, D., Eds., Plenum, New York, 1991, p. 69.
1382_C41.fm Page 705 Tuesday, October 7, 2003 7:40 PM

chapter forty-one

Amino acid solutions for acute


renal failure
Charles J. Foulks
Texas A&M University Health Science Center

Contents
Introduction..................................................................................................................................705
41.1 Protein metabolism in ARF ..............................................................................................706
41.2 Protein and energy requirements in ARF ......................................................................707
41.3 Clinical studies utilizing amino acid-based TPN in ARF patients .............................708
41.4 Impact of newer continuous renal replacement therapy techniques for the
treatment of ARF................................................................................................................710
41.5 Conclusions.........................................................................................................................712
Acknowledgment ........................................................................................................................713
References .....................................................................................................................................713

Introduction
Although many improvements have been made in the delivery of dialysis services to
patients with acute renal failure (ARF), mortality remains high.1,2 The explanation for this
involves many factors, including dialysis offered to elderly patients who previously would
have been denied dialysis, as well as improved acute resuscitation therapies that permit
patient survival for a period of time sufficient to allow dialysis. Most nephrologists have
noted that the improvement in surgical procedures and techniques and better pharmaco-
logic management of nephrotoxic drugs have changed the face of ARF. The patients who
now develop ARF sufficient to require dialysis are older, have more comorbid diseases,
and have a greater severity of illness. Many of these patients are malnourished at the time
that they suffer ARF because of the prevalence of malnutrition in elderly patients with
chronic diseases and the improved medical and surgical techniques that prolong their
lives in the hospital such that they are at risk of ARF, as opposed to the same patient who
in the past would have died. Although early intervention with nutritional support is
advocated, few patients receive it unless they are in centers with nutrition support services,
trauma or burn centers, or intensive care units staffed by trained intensivists.

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 705
1382_C41.fm Page 706 Tuesday, October 7, 2003 7:40 PM

706 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Prior to the development of total parenteral nutrition (TPN), patients with ARF were
managed with very low protein oral diets. Those who survived exhibited severe wasting
and had a marked decrease in lean body mass.3 Schuberth4 considered ARF to be a
contrindication to the use of TPN; however, TPN administration to ARF patients was
considered by others to be feasible.3 The report by Abel et al.5 in 1973 changed the way
in which ARF patients were managed by demonstrating a decrease in mortality in those
patients who received TPN. Unfortunately, those results have never been duplicated by
other investigators.6–8 The standard of practice for many years following Abel et al.’s report
was to use TPN with an essential amino acid (EAA) formulation; however, following the
report of Feinstein and co-workers,8 a general amino acid (GAA)-based TPN that included
EAA and nonessential amino acids (NEAA) became the standard of care. Although nutri-
tion experts recommend the use of enteral feedings when feasible and most nephrologists
follow this recommendation, there is no body of literature that exists to guide the practi-
tioner in the use of enteral feedings to support ARF patients. As recognized for many
years by intensivists and nephrologists, malnutrition is an independent risk factor for
mortality in ARF patients.9

41.1 Protein metabolism in ARF


There are multiple alterations in protein metabolism in chronic renal failure (CRF).8,10–16
Decreases in the concentrations of albumin, transferrin, valine, leucine, isoleucine, lysine,
and tyrosine are found, and the metabolic clearances of threonine, phenylalanine, valine,
leucine, and isoleucine are decreased.13 Plasma concentrations of aspartic acid and 1- and
3-methylhistidine (3MH) are elevated,10 and increases in aspartic acid and 3MH have been
confirmed.11 Cysteine is elevated presumably secondary to methionine supplementation.11
The increase in 3MH is associated with muscle wasting.10–12 These findings in chronic renal
failure differ from those in ARF.
Abel and co-workers12 have demonstrated that patients with ARF have normal levels
of amino acids with the exceptions of valine, methionine, isoleucine, phenylalanine, and
ornithine, which are low. They also demonstrated that infusions of EAA were well toler-
ated and did not cause significant elevations of EAA concentrations.12 Additionally, they
demonstrated that plasma amino acid levels in ARF are not different from normal and
that intravenously administered amino acids are rapidly cleared from the circulation.12
Pelosi et al.16 have shown that the intraerythrocytic concentrations of EAA are decreased
in ARF patients treated by hemo- and peritoneal dialysis.
Both the alanine turnover rate and glucose production are markedly increased in
patients with chronic renal failure. These abnormalities improve but do not normalize
after hemodialysis. Glucose production is increased threefold, with the percentage of
alanine used for glucose production being increased by 60%.14 It is not known if these
alterations are seen in patients with ARF.
The protein catabolic rate (PCR) is increased in CRF patients as a consequence of
impaired protein synthesis and enhanced protein degradation.15 A dialyzable substance is
thought to be responsible for the decreased protein synthesis since protein synthesis
improves after dialysis.17,18 Additionally, various proteinases have been described in chronic
uremia that are thought to play an important role in the enhanced protein catabolism of
uremia. Enhanced cytokine and prostaglandin production are also thought to be involved
in the increased PCR seen in uremics.15,19 These same factors are probably seen in ARF
patients. Hemodialysis is associated with an increase in the PCR, and blood–dialyzer
membrane interactions have been noted that result in increased amino acid release by
muscle. These alterations are not seen with polyacrylonitrile or polysulfone membranes.20
1382_C41.fm Page 707 Tuesday, October 7, 2003 7:40 PM

Chapter forty-one: Amino acid solutions for acute renal failure 707

Kopple et al.21 have shown that 5 to 8 g of free amino acids (one third EAA) and 4 to
5 g of peptide-bound amino acids are lost during hemodialysis using cuprophane low-
efficiency dialyzers. However, when modern high-solute flux dialyzers were studied in
patients with ARF who received TPN, 7.3 ± 1.8 g of amino acids were lost per dialysis
compared to 5.2 ± 0.6 g in the patient dialyzed with a non-high-flux cellulosic dialyzer
(p < 0.05).22 Although the dialyzer clearances for amino acids were significantly higher in
the high-flux dialyzers, even the modern cellulosic dialyzers represent an improvement
when compared to the older dialyzers used by Kopple et al.21 Peritoneal dialysis is asso-
ciated with the loss of albumin, total protein, and gamma globulin; during episodes of
peritonitis, these losses may be very high.23

41.2 Protein and energy requirements in ARF


Patients with CRF have normal energy expenditure and requirements compared to non-
uremic persons with like physical activity (0.94 ± 0.24 vs. 0.91 ± 0.20 kcal/kg/min).24 Latos
et al.25 have made similar observations. It is not known if these calculations apply to ARF
patients.
Spreiter and colleagues26 studied 14 ARF patients requiring hemodialysis. Thirteen of
the 14 patients received TPN that contained 6 to 71 kcal/kg/day (mean ± SEM,
30 ± 5 kcal/kg/day) and 0.1 to 1.6 g of GAA/kg/day (mean ± SEM, 0.53 ± 0.09 g/kg/day).
The generation of urea nitrogen (Gun) was calculated from a single-pool urea kinetic
model. Urea nitrogen and nonprotein urea nitrogen were calculated from measurements
in plasma pre- and postdialysis, dialysis ultrafiltrate, and in all body fluids removed by
drainage. The nitrogen balance was calculated using the Gun (–5.6 ± 1.0 g of N per day)
and the generation of nonprotein nitrogen (–9.9 ± 2.3 g of N per day). Regression analysis
indicated that positive nitrogen balance correlated with >50 kcal of energy intake/kg/day
(r = 0.50, p < .01) or >1.03 g of protein intake/kg/day (r = 0.64, p < .01). No clinical details
were provided, and no severity of illness score was presented. It is therefore difficult to
know if these findings can be extrapolated to other patients with ARF.
Nephrologists are familiar with dialyzing ARF patients in the intensive care unit who
require mechanical ventilation. Buthard and co-workers27 measured the resting energy
expenditure (REE) by indirect calorimetry in 20 mechanically ventilated ARF patients in
the intensive care unit. Data were presented as mean ± SEM. The REE was 119 ± 3% of
the predicted basal energy expenditure (BEE), which was calculated by the Harris–Bene-
dict equation. When septic patients were compared with nonseptic patients, a difference
in energy requirements was seen (131 ± 3% vs. 114 ± 2%, p < .05). The measured glucose
oxidation rate was 4.65 mg/kg/min, which exceeded the glucose intake of
2.6 mg/kg/min. The nitrogen loss was –17.3 ± 1.7 g/day, and the nitrogen balance was
–11.9 ± 1.9 g/day. A large interindividual variation in nitrogen balance ranging from –40
to +5 g/day was noted. Of interest was the finding of a respiratory quotient (RQ) of 1.02
± 0.01 in the setting of modest energy intake. Studying three ARF patients, Braun et al.28
found similar results: 150% of the BEE. Using a Douglas bag method of calculating energy
requirements, Miller et al.29 estimated the energy requirements as being 250% of the BEE.
Because of the discontinuous method of sampling inherent in the Douglas bag technique,
these data may be somewhat questionable.
What is the message in these data? Mechanically ventilated patients may have an REE
that is not greatly increased from that calculated by the Harris–Benedict equation. How-
ever, septic ARF patients have higher energy expenditure than nonseptic patients, and the
N balance is quite variable. The finding of an RQ of 1.02 with a glucose oxidation rate
higher than the amount delivered suggests that the delivery of increased calories may not
be beneficial.27 When Spreiter and co-workers’ data26 are carefully examined, only 6 of 32
1382_C41.fm Page 708 Tuesday, October 7, 2003 7:40 PM

708 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

data collection periods in 14 patients indicated positive N balance as a function of caloric


intake: one each at 28, 33, 38, 53, 68, and 71 kcal/kg/day, indicating that only 3 of 32
collection periods benefited from a caloric intake of >50 kcal/kg/day. The effect of such
high caloric intake on lipogenesis was not measured in this study. Seven of 32 collection
periods revealed positive N balance as a function of protein intake: one each at 0.17, 0.72,
0.9, 0.97, 1.10, and 1.62 and two at 0.77 g of amino acids/kg/day. The collection periods
depicting positive N balance differ when N balance is plotted as a function of N intake
or caloric intake. Therefore, the regression equations are of doubtful benefit in assisting
clinical decision making. In addition, there was also no severity of illness scale so that one
could be satisfied that the degree of stress was similar in all of these patients.

41.3 Clinical studies utilizing amino acid-based TPN in ARF patients


Prior to the article by Abel and co-workers5 in 1973, the only previous observations on
the use of TPN in the therapy of ARF consisted of uncontrolled trials similar to phase one
studies of antineoplastic drugs.30,31 In 1970, Dudrick and co-workers30 reported the suc-
cessful maintenance of acute and chronic renal failure patients using TPN consisting of
hypertonic dextrose and EAA. Clinical details were sparse, and morbidity and mortality
were not reported. Nevertheless, the authors claimed successful management of these
patients without dialytic intervention. Others in which a similar solution was used to treat
ARF patients followed this report.32,33 These were uncontrolled, nonblinded, nonrandom-
ized observations on the utility of TPN in the management of ARF.
In 1973, the landmark article of Abel and co-investigators was published.5 Utilizing a
“renal failure fluid,” patients with ARF were randomized to receive the renal failure fluid
or dextrose alone in addition to other therapies required by the clinical situation. The TPN
solutions used consisted of 375 g of dextrose and vitamins as the control fluid (D) vs. an
experimental fluid (D/EAA) of 350 g of dextrose, 13.1 g of EAA, and vitamins (renal
failure fluid). Each patient received one bottle daily containing either D or D/EAA. The
control and experimental groups were not different with respect to demographic data,
presence of oliguria, the numbers or types of surgical procedures, the requirement for
dialysis, or the cause of ARF. Seventy-five percent of the group receiving the D/EAA
survived compared to 44% of the D group (p = 0.02). However, when discharge from the
hospital was compared, 61% of the D/EAA group were discharged alive compared to
40% of the D group (p > 0.05). Based upon this report, the use of D/EAA in the manage-
ment of ARF became widespread in the U.S.
Unfortunately, three controlled studies have not duplicated these results.6–8 Leonard
et al.6 found no difference in survival in ARF patients treated with D vs. D/EAA. All
patients were in negative N balance (p > 0.05, D vs. D/EAA). The rate of rise of the blood
urea nitrogen (BUN) was less in the D group, but the dialysis requirement was the same.
Mirtallo and co-investigators8 randomly assigned ARF patients to receive either D/EAA
or D/GAA (EAA + NEAA) therapy. No difference was noted in clinical factors associated
with the ARF. N intake, Gun, and N balance were not different, although the mean N
balance was positive in both groups (D/EAA, 0.84 ± 1.02 g of N per day; D/GAA,
1.13 ± 1.06 g of N per day). No difference in morbidity or mortality was reported. The
authors concluded that an EAA-based TPN solution was not necessary. In a well-designed
study, Feinstein et al.7 randomized ARF patients to treatment with D, D/EAA, or D/GAA.
There was no difference in morbidity, mortality, demographic data, and clinical course or
characteristics. However, patients who received D/EAA had a significantly lower Gun
(p < 0.05). The authors felt that a D/GAA TPN solution was optimal. In addition, there
are several nonrandomized studies, some of which are retrospective, that claim the supe-
riority of D/EAA-based TPN over D/GAA-based TPN.31,34–39
1382_C41.fm Page 709 Tuesday, October 7, 2003 7:40 PM

Chapter forty-one: Amino acid solutions for acute renal failure 709

Table 41.1

Randomized Studies (5–8)


D D/EAA D/GAA
Mortality 23/41 (56%) 27/66 (41%)a 27/64 (44%) X2 = 2.59, p = 0.27

All Studies (2,5-8,31,34-39)


Mortality 69/107 (64%) 68/137 (50%)b 7/128 (45%) X2 = 9.85, p = 0.007

Randomized Studies (5–8)


D D/EAA + D/GAA
Mortality 23/41 (56%) 54/129 (42%) X2 = 2.54, p = 0.13

All Studies (2,5–8,31,34–39)


Mortality 92/148 (62%) 179/394 (45%) X2 = 51.6, p < .0001
a D/EAA vs. D/GAA, X2 = 0.05, p > .5.
b D/EAA vs. D/GAA, X2 = 0.6, p > .5.

To resolve this issue, we have completed a meta-analysis of the available data utilizing
both randomized studies alone and all studies grouped together. This analysis makes the
assumption that all studies were appropriately randomized, which is clearly not the case.
In particular, the outcome(s) from combining different studies is hampered by nonunifor-
mity of entry and treatment criteria. Outcome variables are diverse. This analysis should
be viewed with great caution. The MEDLINE system was used for the search, the bibli-
ography of each reference was reviewed, and pertinent citations were also collected. All
references in English were accepted, and those not in English, which did not have an
English abstract sufficient to establish the details of the study, were rejected. Only those
studies that explicitly stated that randomization had occurred were accepted as random-
ized studies.5–8 All others were considered to be nonrandomized.31,34–39 Only those studies
with data sufficient to confirm a diagnosis of ARF were utilized. No distinction was made
between nonoliguric or oliguric ARF. Survival was compared by chi-square analysis
(Table 41.1). The Gun (also termed urea nitrogen appearance (UNA)) and N balances are
reported in Table 41.2; analysis was by analysis of variance (ANOVA). The number of
dialyses was also compared by ANOVA (Table 41.3). Table 41.4 is a summary table of
these studies.
The randomized studies indicate no difference in mortality between patients treated
with D/EAA and those treated with D/GAA.5–8 When all studies are examined, the
difference in mortality between D, D/EAA, and D/GAA is significant (p = .007). However,
subgroup analysis strongly suggests no difference between D/EAA and D/GAA (p > 0.50).
The true decrease in mortality is between patients treated with an AA-containing TPN
(D/EAA and D/GAA) and those who only received D. EAA-based TPN showed no
superiority over GAA-based TPN.
The study by Mirtallo et al.8 showed a significant difference in Gun, while Feinstein
et al.’s study7 approached significance. However, both D/GAA groups received signifi-
cantly more protein than the D/EAA groups. This did not appear to be clinically important
since there was no difference in the number of dialysis treatments per patient in both
studies. This may be quite misleading since data (blood and dialysate flow rates, dialyzer
surface area, length of treatment, dialyzer clearance characteristics), which are necessary
to determine the amount of dialysis delivered, were not given.
1382_C41.fm Page 710 Tuesday, October 7, 2003 7:40 PM

710 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 41.2 Nutritional Indices


D D/EAA D/GAA
Mirtallo et al.8 — 3.2 ± 1.1a 4.2 ± 1.1a t = 3.173, p = .003
Feinstein et al.7 10.4 ± 5.9 6.7 ± 7.2 b 14.0 ± 8.0b F = 2.885, p = .07

N Balance (g/day)
Leonard et al. 6 –10.8 ± 5.2 –9.8 ± 5.8 — t = 0.402, p = .69
Mirtallo et al.8 — 0.84 ± 1.02 1.13 ± 1.06 t = 0.934, p = .36
Pelosi et al.37 –0.22 ± 0.05 0.16 ± 0.05 –0.1 ± 0.05 F = 70.79, p < .0001

D/GAA D/GAA + BCAAc TF/BCAAc


Proietti et al.38 –0.16 ± 0.05 –0.08 ± 0.06 –0.07 ± 0.04 F = 13.68, p < .0001
a The D/EAA group received 4.57 ± 0.39 g of N/day; the D/GAA group received 5.35 ± 0.43 g of N/day; t =
4.105, p < .0001.
b The D/EAA group received 2.28 ± 0.46 g of N/day; the D/GAA group received 5.34 ± 0.66 g of N/day; t =
33.64, p < .0001.
c The middle group (D/GAA + BCAA) received a GAA-based TPN with enrichment with BCAA. The last group
received no TPN and was fed with a tube feeding (TF) enriched with BCAA.

Table 41.3 Dialysis Requirements


Number
of dialyses D D/EAA D/GAA
Leonard et al.6 a 5.2 4.7 —
Feinstein et al.7 4.1 ± 3.5 1.7 ± 5.7 3.8 ± 3.1 F = 0.922, p = .41
a Only the mean was reported.

The study by Mirtallo et al.8 is intriguing since it is the only study to demonstrate the
attainment of positive N balance. The reason for this is not clear. Nevertheless, there was
no difference in N balance between patients who received EAA and those who received
GAA. Pelosi et al.37 have shown the superiority of EAA and GAA TPN over D alone, with
a GAA TPN being superior to EAA TPN. Adequate details of the clinical results were not
reported, which would allow confirmation of their conclusions. Using a novel therapy,
Proietti et al.38 demonstrated better N balance using a branched-chain amino acid (BCAA)-
enriched GAA than a standard GAA-based solution. Also of interest was the observation
that an enterally delivered feeding enriched with BCAA was the equal of BCAA-enriched
TPN. One must be cautious in interpreting improved N balance as representing either
repair or improvement in lean body mass. The preservation of lean body mass is the goal
of enteral/parenteral nutrition therapy, and positive N balance does not necessarily equate
with this.

41.4 Impact of newer continuous renal replacement therapy


techniques for the treatment of ARF
Intermittent hemodialysis has been the preferred method for treating patients with ARF
for many years. Peritoneal dialysis has been used in hemodynamically unstable patients
or those in whom vascular access was difficult or no longer possible, but it is rarely
used anymore. Continuous renal replacement therapies (CRRTs) such as continuous
veno-venous hemofiltration (CVVH), continuous arteriovenous hemofiltration (CAVH),
Table 41.4 Effects of Type of Amino Acid Used in TPN Therapy of ARF
Chapter forty-one:

Author Year Random Prospective D D/E GAA Mortality N balance


Lee et al. 3 1967 N N Fructose/ethanol/lipid/GAA, n = 42
Abel et al.31 1972 N N n = 17
Abel et al.5 1973 Y Y n = 25 n = 28 Ya
Abel et al.32 1974 N N n = 41 n = 98b
Baek et al.34 1975 N Y n = 66 n = 63 N
1382_C41.fm Page 711 Tuesday, October 7, 2003 7:40 PM

Leonard et al.6 1975 N N n=9 n = 11 N p = NS


McMurray et al.2 1978 N N Yc Yc Yd
Freund et al.35 1980 N N n = 22
Motil et al.36 1980 N N 2
Pelosi et al.37 1981 N Y n = 30 n = 11 n=5 e

Feinstein et al.7 1981 Y Y 7 11 12 N


Mirtallo et al.8 1982 Y Y 24 21 N p = NS
Proetti et al.38 1983 N Y n = 32f N BCAAg
a p < 0.02. There was no difference in hospitalization survival to discharge in either group, p = 0.11.
b Includes 28 patients from Abel et al.5
Amino acid solutions for acute renal failure

c The number of patients in each group was not given.


d A decrease in mortality was seen only in those TPN patients with peritonitis (p < 0.05) or who had >3 complications (p < 0.01). The type of TPN solution was
not given.
e Less negative with D/GAA > D/E > D. Significance not given.
f Twenty-two patients received D/GAA and 10 received D/GAA enriched with BCAA.
g Improved with BCAA.
711
1382_C41.fm Page 712 Tuesday, October 7, 2003 7:40 PM

712 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

continuous AV or VV hemodiafiltration (CAVHD, CVVHD), and combinations of both


HF and HD (CAVHDF, CVVHDF) have been developed and have become popular
therapies in ICU patients with ARF.40,41 The advantages of these therapies are that they
have less hemodynamic instability than conventional hemodialysis and larger amounts
of fluids, medications, and nutrition may be given, since these are continuous therapies.
Unfortunately, these benefits have not translated into decreased mortality in ARF
patients treated with these therapies, when compared to conventional hemodialysis.42
Tremblay et al.43 treated 12 burn patients with a variety of CRRT techniques. None
were compared to standard intermittent hemodialysis. Nine of the 12 patients were fed
with enteral and parenteral methods and three with parenteral only. The study is unclear
about whether the reported results are mean ± SD or mean ± SEM. The details of the
feedings were not reported. Total caloric intake was 31.5 ± 7 kcal/kg/day, protein intake
was 1.8 ± 0.4 g/kg/day, and the normalized protein catabolic rate (nPCR) was 2.28 ± 0.78
g/kg/day. The nPCR was higher than previously reported in nonburned ARF patients
treated with CRRT (1.7 to 1.9 g/kg/day).44–46 It is more appropriate to view the nPCR in
ARF patients as a marker of severity of illness rather than being a surrogate of protein
intake. Nevertheless, it may be appropriate to use it as a rough guide for the amount of
protein needed to attain neutral or positive nitrogen balance, although these studies have
not been done in ARF patients.
Maxvold et al.47 compared AA losses and nitrogen balance in 12 children with ARF
treated with either CVVH or CVVHD. All of the children received TPN that provided
caloric intake at 120 to 130% of their measured resting energy expenditure (indirect
calorimetry) and 1.5 g of protein/kg/day. They found that AA clearances were greater
with CVVH than CVVHD (with the exception of glutamine); urea clearance was similar
as was the loss of AA (12 to 13% of that infused by TPN). They also noted that N balance
ranged from positive to negative in patients receiving either type of CRRT.

41.5 Conclusions
There is little doubt that the supportive therapy of patients with ARF should include TPN
when appropriate. It is certainly intuitive that ill patients will have an improved outcome
if they are properly supported nutritionally. However, data do not exist that show clearly
that the use of an EAA- or GAA-based formula results in a decrease in mortality. In fact,
it may well be that no nutritional intervention can reverse the course of an acute illness
severe enough to result in ARF. Although there are data that show improved renal protein
metabolism and renal tubular regeneration in ARF animals treated with amino acid infu-
sions,48,49 no studies in humans exist. Unfortunately, such therapy could be harmful.50,51
Given our current state of knowledge, a final recommendation favoring either EAA-
or GAA-based TPN cannot be made. Considering the marked cost of EAA compared to
GAA, it is reasonable to initiate TPN with a GAA solution. EAA-based solutions should
be utilized in those patients in whom dialytic intervention should be avoided or decreased
in frequency. If EAA are used, one must closely follow the Gun and N balance of the
patient. If the nutritional status deteriorates while an EAA-based TPN is being utilized,
one must seriously consider either an increase in nonprotein kilocalories,7,22,28,29 an increase
in the amount of AA being delivered (although there is some hazard to this approach
with amino acid imbalance occurring), or a change to a GAA formula. Solutions enriched
with BCAA might be considered.
Patients receiving GAA-based TPN should be monitored closely and the caloric and
amino acid intakes adjusted by following the REE, RQ, Gun, and N balance. Although
not definitively known, an increase in the nonprotein kilocalorie intake may be useful.21,28,29
Clearly, this area of renal nutrition needs further study. The possibility of enhancing renal
1382_C41.fm Page 713 Tuesday, October 7, 2003 7:40 PM

Chapter forty-one: Amino acid solutions for acute renal failure 713

repair and shortening the course of ARF or preventing it by altering the balance or type
of AA administered to patients is an exciting prospect for future evaluation.47,48

Acknowledgment
The author expresses his gratitude to Mrs. Lyna Young for her expert secretarial support
and for keeping him organized.

References
1. Rosenfeld, J.B., Shohat, J., Grosskopf, I., and Boner, G., Acute renal failure: a disease of the
elderly, in Advances in Nephrology, Griinfeld, J.-P., Bach, J.-F., Crosnier, J., Funch-Bretano, J.-L.,
and Maxwell, M.-H., Eds., YearBook Medical Publishers, Chicago, 1987, p. 159.
2. McMurray, S.T., Luft, F.C., Maxwell, D.R., Hamburger, R.J., Futty, D., Szwed, J.J., Lavelle,
K.J., and Kleit, S.A., Prevailing patterns and predictor variables in patients with acute tubular
necrosis, Arch. Intern. Med., 138, 950, 1978.
3. Lee, H.A., Sharpstone, P., and Ames, A.C., Parenteral nutrition in renal failure, Postgrad. Med.
J., 43, 81, 1967.
4. Schuberth, O., Clinical experience with fat emulsions for intravenous use, Acta Chir. Scand.,
43 (Suppl.), 325, 1964.
5. Abel, R.M., Beck, C.H., Jr., Abbott, W.M., Ryan, J.A., Jr., Barnett, G.O., and Fischer, J.E.,
Improved survival from acute renal failure after treatment with intravenous essential L-ami-
no acids and glucose: results of a prospective, double-blind study, N. Engl. J. Med., 288, 695,
1973.
6. Leonard, C.D., Luke, R.G., and Siegel, R.R., Parenteral essential amino acids in acute renal
failure, Urology, 6, 154, 1975.
7. Feinstein, E.I., Blumenkrantz, M.J., Healy, M., Komer, A., Silberman, H., Massry, S.G., and
Kopple, J.D., Clinical and metabolic responses to parenteral nutrition in acute renal failure:
a controlled double-blind study, Medicine, 60, 124, 1981.
8. Mirtallo, J.M., Schneider, P.J., Mavko, K., Ruberg, R.L., and Fabri, P.J., A comparison of
essential and general amino acid infusions in the nutritional support of patients with com-
promised renal function, J. Parenter. Enteral Nutr., 6, 109, 1982.
9. Fiaccadori, E., Lombardi, M., Sabina, L., Rotelli, C.F., Tortorells, G., and Borghetti, A., Prev-
alence and clinical outcome associated with preexisting malnutrition in acute renal failure:
a prospective cohort study, J. Am. Soc. Nephrol., 10, 581, 1999.
10. Peters, J.H., Gulyassy, P.F., Lin, S.C., Ryan, P.M., Berridge, B.J., Jr., Chao, W.R., and Cummings,
J.G., Amino acid patterns in uremia: comparative effects of hemodialysis and transplantation,
Trans. Am. Soc. Artif. Intern. Organs, 14, 405, 1968.
11. Condon, J.R. and Asatoor, A.M., Amino acid metabolism in uraemic patients, Clin. Chim.
Acta, 32, 333, 1971.
12. Abel, R.M., Shih, V.E., Abbott, W.M., Beck, C.H., and Fischer, J.E., Amino acid metabolism
in acute renal failure: influence of intravenous essential L-amino acid hyperalimentation
therapy, Ann. Surg., 180, 350, 1973.
13. Chami, J., Reidenberg, M.M., Wellner, D., David, D.S., Rubin, A.L., and Stenzel, K.H., Phar-
maco-kinetics of essential amino acids in chronic dialysis-patients, Am. J. Clin. Nutr., 31, 1652,
1978.
14. Maillet, C. and Garber, A.J., Skeletal muscle amino acid metabolism in chronic uremia, Am.
J. Surg., 33, 1343, 1980.
15. Alvestrand, A., Protein metabolism and nutrition in hemodialysis patients, Contrib. Nephrol.,
78, 102, 1990.
16. Pelosi, G., Proietti, R., Ranieri, R., Bondoli, A., Gagliardia, A., Scrascia, E., and Magalini, S.I.,
Amino acid loss in acute renal failure: comparative effects of peritoneal dialysis and haemo-
dialysis, Resuscitation, 5, 217, 1977.
1382_C41.fm Page 714 Tuesday, October 7, 2003 7:40 PM

714 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

17. Delaprote, C., Gros, F., and Aragnostopoulos, T., Inhibitory effect of plasma dialysate on
protein synthesis in vitro: influence of dialysis and transplantation, Am. J. Clin. Nutr., 33,
1407, 1980.
18. Cernacek, P., Sputsova, V., and Dzurik, R., Inhibitor(s) of protein synthesis in uremic serum
and urine: partial purification and relationship to amino acid transport, Biochem. Med., 27,
305, 1982.
19. Jahoor, J. and Wolfe, R.R., Regulation of protein catabolism, Kidney Int., 32, 581, 1987.
20. Guiterrez, A., Bergstrom, J., and Alvestrand, A., Protein catabolism in sham-hemodialysis:
the effect of different membranes, Clin. Nephrol., 38, 20, 1992.
21. Kopple, J.D., Swendseid, M.E., Shinaberger, J.H., and Umezawa, C.Y., The free and bound
amino acids removed by hemodialysis, Trans. Am. Soc. Artif. Intern. Organs, 14, 309, 1973.
22. Hynote, E.D., McCamish, M.A., Depner, T.A., and Davis, P.A., Amino acid losses during
hemodialysis: effects of high-solute flux and parenteral nutrition in acute renal failure,
J. Parenter. Enteral Nutr., 19, 15, 1995.
23. Dulaney, J.T. and Hatch, F.E., Jr., Peritoneal dialysis and loss of proteins: a review, Kidney
Int., 26, 253, 1984.
24. Monteon, F.J., Laidlaw, S.A., Shaib, J.K., and Kopple, J.D., Energy expenditure in patients
with chronic renal failure, Kidney Int., 30, 741, 1986.
25. Latos, D., Strimel, D., and Boring, W., Resting energy expenditure in uremic patients, Kidney
Int., 27, 144, 1985 (abstract).
26. Spreiter, S.C., Myers, B.D., and Swenson, R.S., Protein-energy requirements in subjects with
acute renal failure receiving intermittent hemodialysis, Am. J. Clin. Nutr., 33, 1433, 1980.
27. Bouthard, Y., Viale, J.P., Annat, G., Delafosse, B., Guillaume, C., and Motin, J., Energy expen-
diture in the acute renal failure patient mechanically ventilated, Intensive Care Med., 13, 401,
1987.
28. Braun, V., Berger, C., Kunzl, E., Martell, J., Schwarzkopf, V., Trapp, V., and Kramer, P., Daily
energy and nitrogen balance in acute catabolic renal failure, in Continuous Arteriovenous
Hemofiltration, International Conference on CAVH, S. Karger, Basel, Switzerland, 1984, p. 219.
29. Miller, R.L., Taylor, W.R., Gentry, W., Day, A.T., and Gazzaniga, A.B., Indirect calorimetry in
post operative patients with acute renal failure, Ann. Surg., 49, 494, 1983.
30. Dudrick, S.J., Steiger, E., and Long, J.M., Renal failure in surgical patients: treatment with
intravenous essential amino acids and hypertonic glucose, Surgery, 68, 180, 1970.
31. Abel, R.M., Abbott, W.M., Beck, C.H., Jr., Ryan, J.A., Jr., and Fischer, J.E., Essential L-amino
acids and hyperalimentation in patients with disordered nitrogen metabolism, Am. J. Surg.,
128, 317, 1974.
32. Abel, R.M., Abott, W.M., and Fischer, J.E., Acute renal failure: treatment without dialysis by
total parenteral nutrition, Arch. Surg., 103, 513, 1971.
33. Abbott, W.M., Abel, R.M., and Fischer, J.E., Treatment of acute renal insufficiency after
aortoiliac surgery, Arch. Surg., 103, 590, 1971.
34. Baek, S.M., Makabali, G.G., Bryan-Brown, C.W., and Kuseck, J., The influence of parenteral
nutrition on the course of acute renal failure, Surg. Gynecol. Obstet., 141, 405, 1975.
35. Freund, H., Atamian, S., and Fischer, J.E., Comparative study of parenteral nutrition in renal
failure using essential and nonessential amino acid containing solutions, Surg. Gynecol.
Obstet., 151, 652, 1980.
36. Motil, K.J., Harmon, W.E., and Grupe, W.E., Complications of essential amino acid hyper-
alimentation in children with acute renal failure, J. Parenter. Enteral Nutr., 4, 32, 1980.
37. Pelosi, G., Proietti, R., Arcangeli, A., Magalini, S.I., and Bondoli, A., Total parenteral nutrition
infusate: an approach to its optimal composition in post-trauma acute renal failure, Resus-
citation, 9, 45, 1981.
38. Proietti, R., Pelosi, G., Santori, R., Giammaria, A., Arcangeli, A., Sciarra, M., and Zanghi, F.,
Nutrition in acute renal failure, Resuscitation, 10, 159, 1983.
39. Dudrick, S.J., Steiger, E., and Long, J.M., Renal failure in surgical patients: treatment with
intravenous essential amino acids and hypertonic glucose, Surgery, 68, 180, 1970.
40. Murray, P. and Hall, J., Renal replacement therapy for acute renal failure, Am. J. Respir. Crit.
Care Med., 162, 777, 2000.
1382_C41.fm Page 715 Tuesday, October 7, 2003 7:40 PM

Chapter forty-one: Amino acid solutions for acute renal failure 715

41. Thadhani, R., Pascual, M., and Bonventre, J.V., Acute renal failure, N. Engl. J. Med., 334, 1448,
1996.
42. Mehta, R.L., McDonald, B., Gabbai, F.B., Pahl, M., Pascual, M.T., Farkas, A., and Kaplan, R.,
A randomized clinical trial of continuous versus intermittent dialysis for acute renal failure,
Kidney Int., 60, 1154, 2001.
43. Tremblay, R., Ethier, J., Querin, S., Beroniade, V., Falardeau, P., and LeBlanc, M., Veno-venous
continuous renal replacement therapy for burned patients with acute renal failure, Burns,
26, 638, 2000.
44. LeBlanc, M., Garred, L.J., Cardinal, J., Pichette, V., Nolin, L., and Ouimet, D., Catabolism in
critical illness: estimation from urea nitrogen appearance and creatinine production during
continuous renal replacement therapy, Am. J. Kidney Dis., 32, 444, 1998.
45. Chima, C.S., Meyer, L., Hummell, A.C., Bosworth, C., Heyka, R., Paganini, E.P., and Werinshi,
A., Protein catabolic rate in patients with acute renal failure on continuous arteriovenous
hemofiltration and total parenteral nutrition, J. Am. Soc. Nephrol., 3, 1516, 1993.
46. Macias, W.L., Alaka, K.J., Murphy, M.H., Miller, M.E., Clark, W.R., and Mueller, B.A., Impact
of the nutritional regimen on protein catabolism and nitrogen balance in patients with acute
renal failure, J. Parenter. Enteral Nutr., 20, 56, 1996.
47. Maxvold, N.J., Smoyer, W.E., Custer, J.R., and Bunchman, T.E., Amino acid loss and nitrogen
balance in critically ill children with acute renal failure: a prospective comparison between
classic hemofiltration and hemofiltration with dialysis, Crit. Care Med., 28, 1161, 2000.
48. Toback, F.G., Dodd, R.C., Maier, E.R., and Havener, L.J., Amino acid administration enhances
renal protein metabolism after acute tubular necrosis, Nephron, 33, 238, 1983.
49. Toback, F.G., Amino acid enhancement of renal regeneration after acute tubular necrosis,
Kidney Int., 12, 193, 1977.
50. Zager, R.A., Johannes, G., Tuttle, S.E., and Sharma, H.M., Acute amino acid nephrotoxicity,
J. Lab. Clin. Med., 101, 130, 1983.
51. Zager, R.A. and Venkatachalam M.A., Potentiation of ischemic renal injury by amino acid
infusion, Kidney Int., 24, 620, 1983.
1382_C41.fm Page 716 Tuesday, October 7, 2003 7:40 PM
1382_C42.fm Page 717 Tuesday, October 7, 2003 7:42 PM

chapter forty-two

Amino acids to support gut


function and morphology
Gordon S. Sacks
The University of Wisconsin–Madison
Kenneth A. Kudsk
The University of Wisconsin–Madison

Contents
Introduction..................................................................................................................................717
42.1 Gut morphology and function: the mucosa...................................................................718
42.2 Gut morphology and function: the immune system ....................................................719
42.3 Gut morphology and function: the vasculature............................................................721
References .....................................................................................................................................723

Introduction
Over the past decade, the gut has become recognized as a central organ of metabolic
activity during critical illness and stress rather than a quiescent organ as previously
thought. In particular, the small intestine regulates terminal digestion and absorption of
protein and amino acids. The significance of intestinal amino acid metabolism lies in the
provision of major fuels for maintenance of structural function and integrity. For instance,
dietary glutamine (GLN) and glutamate (GLU) are essential for metabolic processes such
as nutrient transport and protein turnover.1 GLN is also an important precursor for a
number of metabolic pathways, especially those involved in the synthesis of ornithine,
arginine, and glutathione (GSH). Ornithine is an intermediate precursor for polyamine
synthesis and is essential for repair of epithelial cells.2 Arginine is an important substrate
for nitric oxide (NO) synthesis, which is a key factor in controlling intestinal blood flow
and integrity.3 GSH, an important antioxidant and scavenger, protects the intestinal
mucosa from toxic and peroxidative damage.4 Lack of dietary amino acids via the gas-
trointestinal tract (e.g., as with parenteral nutrition (PN)) causes intestinal atrophy as well
as impairment of the gut-associated lymphoid tissue (GALT). The following discussion
focuses on the effects of dietary amino acids on gut morphology, the mucosal immune
system, and mucosal vasculature.

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 717
1382_C42.fm Page 718 Tuesday, October 7, 2003 7:42 PM

718 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

42.1 Gut morphology and function: the mucosa


Amino acids play a vital role in regulating intestinal integrity and function. Windmueller
and Spaeth5 were the first to recognize that the small intestine removed as much as 25%
of the systemic flux of GLN, identifying this as a key amino acid for maintenance of
intestinal morphology. With the identification of GLN as an important fuel source for the
intestinal tract, investigators were stimulated to evaluate the impact of GLN deprivation.
Significant reductions in total mucosal thickness, jejunal villus height, and villus cell count
were observed in normal volunteers after receiving standard GLN-free PN for 14 days as
the sole source of nutrition.6 Urinary excretion of lactulose and mannitol revealed an
increased intestinal permeability in these patients despite unaltered plasma GLN concen-
trations. Van der Hulst et al.7 compared standard GLN-free PN vs. GLN-supplemented
PN (0.23 g of free GLN/kg/day) in hospitalized patients. Two weeks of GLN-free PN
significantly decreased duodenal villus height and increased intestinal permeability (via
urinary lactulose and mannitol ratio). Intestinal villus height and permeability remained
unchanged in the GLN group. The effect of GLN on intestinal function and morphology
during critical illness was investigated in 12 intensive care unit (ICU) patients requiring
PN.8 Patients were randomized to receive isonitrogenous and isocaloric GLN-free or GLN-
dipeptide PN (~0.15 g of free GLN/kg/day) for 9 days. Small bowel malabsorption
occurred in patients receiving GLN-free PN compared to GLN-PN, as suggested by serum
and urine D-xylose concentrations.8
Overall, IV-GLN appears to play a supportive role in maintaining gut integrity during
restriction of luminal nutrition in health and critical illness. However, increased intestinal
permeability to these macromolecules due to lack of GLN or gut “starvation” does not
imply an increased permeability to bacteria.9 Bacteria and endotoxins are severalfold larger
than the macromolecules (i.e., lactulose, mannitol, D-xylose) used to evaluate mucosal
structure. Furthermore, human studies have only documented a reduction in cell size
(hypoplasia) rather than a disruption in the tight junctions of villus epithelium that would
be necessary to facilitate the passage of large bacteria. Thus, other measures of intestinal
changes in barrier integrity are needed in order to interpret the clinical significance of
improved intestinal permeability with GLN-PN.
GLN’s influence on the structural integrity of mucosal enterocytes can be attributed
to its role in GSH biosynthesis during periods of metabolic stress. Intestinal synthesis of
the tripeptide GSH depends on the presence of the precursor amino acids GLU, cysteine,
and glycine. Through deamination, dietary GLN contributes significantly to the mucosal
GLU pool in order to preserve tissue GSH levels. However, Reeds et al.10 reported that in
fed piglets, luminal GLU utilized for GSH synthesis was derived primarily from direct
metabolism of enteral GLU (i.e., from the diet) rather than from metabolism within the
mucosa or deamination from GLN. Based upon these results, the altered gut mucosal
barrier function observed with PN may be a manifestation of the lack of enteral amino
acid precursors for GSH synthesis. Synthesis of GSH is catalyzed by g-glutamyl-cysteine
synthetase and glutathione synthetase. Using a murine model, Martensson et al.11 induced
a GSH-deficient state with the administration of L-buthionine-SR-sulfoximine (BSO), an
inhibitor of g-glutamyl-cysteine synthetase. BSO produced severe damage of the epithelial
layer in the jejunal and colonic mucosa. Specifically, electron micrographs showed a
marked loss of microvilli, epithelial height, mitochondrial degeneration, and mitochondria
with vacuolization. Provision of GSH esters (orally or via the intraperitoneal route) pre-
vented mitochondrial and other cellular damage by maintaining GSH levels. Advantages
of GSH monoesters include effective transport and conversion to GSH intracellularly
compared with poor transport of GSH into cells. However, oral administration of GSH
improved jejunal and colonic levels of GSH and prevented BSO-induced GSH depletion.
1382_C42.fm Page 719 Tuesday, October 7, 2003 7:42 PM

Chapter forty-two: Amino acids to support gut function and morphology 719

Thus, oral GSH may promote intracellular GSH synthesis and facilitate protection of the
gastrointestinal epithelium.

42.2 Gut morphology and function: the immune system


In health, a strategic collection of immunocytes referred to as mucosal-associated lym-
phoid tissue (MALT) provides immunologic protection for both the gastrointestinal and
respiratory tract against microbial flora and infectious pathogens.12 Naïve B and T cells
are sensitized to antigens processed within the Peyer’s patches (PP) of the small intestine
and travel via the bloodstream to the lamina propria (LP). Secretory immunoglobulin A
(sIgA), the principal mediator of this system, is produced through the interaction of
sensitized B and T cells in the LP below the mucosa.13 The sIgA is immediately transported
to mucosal surfaces to prevent bacterial and viral adherence to epithelial cell layers and
mucosal penetration. Data from rat studies showed that nutrients delivered parenterally
result in a marked deficiency in biliary IgA- and gut LP IgA-producing plasma cells
compared to enterally fed animals.16–18 However, the mouse more closely resembles the
structural and functional changes in mucosal anatomy and the immune system that occur
in health and during gut starvation in humans. While rat IgA is transported into the
intestine primarily by the hepatobiliary route after release of IgA from the LP into the
portal system, humans and mice transport most IgA directly across the mucosa with bile
serving as a minor contributor. Atrophy of GALT as a result of PN impairs the production
of GALT B and T cells within the LP mucosa, damaging sIgA-dependent host defenses
against intraluminal infectious agents.19 At least one amino acid, GLN, has direct GALT
effects.
sIgA production is directed by Th1 and Th2 cells, two distinct cytokine-producing
T cell subsets located within the GALT. Th1 type cells produce cytokines such as interleu-
kin-2 (IL-2), interferon-gamma (IFN-g), and tumor necrosis factor-b (TNF-b) (lympho-
toxin), which down-regulate sIgA release, whereas Th2 type cells produce IL-4, IL-5, IL-6,
and IL-10, which appear to up-regulate sIgA production.14,15 A balance between Th1 and
Th2 type cells is thought to be necessary for IgA-mediated preservation of normal mucosal
immunity; the reduction in intestinal IgA associated with lack of enteral stimulation during
parenteral feeding appears to be associated with an imbalance of gut Th1 and Th2 cytok-
ines. Prior work had shown that IV-PN changed the LP CD4:CD8 ratio from 2:1 in normal
mice to 1:1 to suggest altered cytokine production.19 After feeding mice chow, IV-PN,
intragastric (IG) PN, or a complex enteral diet (CED) containing fat, proteins, and complex
carbohydrates for 5 days, the mice were sacrificed and supernatants from samples of
intestine were harvested, homogenized, and assayed for Th1 and Th2 cytokines by
ELISA.20 Intestinal IL-4 and IL-10 were particularly sensitive to the route and type of
nutrition, with IL-4 falling significantly in IV-PN mice, compared with the chow or CED
animals (Table 42.1). IL-10 concentrations also decreased only with IV-PN.20 No significant
changes were observed in IL-5 and IL-6 (Th2 cytokines) or in IFN-g (Th1 cytokine).
Preservation of IFN-g, an important Th1 type IgA-inhibiting cytokine, may exert an
increased inhibitory effect upon IgA-producing cells due to lowered IL-4 and IL-10. Intes-
tinal IgA concentrations decreased significantly in both the IV-PN and IG-PN mice when
compared with the chow group20 (Table 42.1).
These intestinal changes have implications on extraintestinal mucosal sites as well.
Because IgA-producing cells released from PP also travel to extraintestinal sites such as
the respiratory tract, the concept of a common mucosal immune system (MALT) has been
proposed. The impact of nutrient delivery on changes in small intestinal GALT and
established respiratory immunity was first demonstrated in a murine infection model.21
The A/PR8 (H1N1) influenza virus induces an IgA-mediated immunity and serves as the
1382_C42.fm Page 720 Tuesday, October 7, 2003 7:42 PM

720 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 42.1 The Influence of Nutrient and Administration Route on Intestinal Mediators
Cytokine Effect of PN PN + GLN PN + BBS
IL-4 Decreaseda Unchangeda/Increasedb Unchangeda/Increasedb
IL-10 Decreaseda Unchangeda,b Decreaseda/Unchangedb
IFN-g Unchanged c c

Intestinal IgA Decreased Increased Increased


Respiratory IgA Decreased Increased Increased
Intestinal ICAM-1 Increaseda Unchangeda/Decreasedb Increaseda/Unchangedb
Lung ICAM-1 Increaseda Increaseda/Unchangedb Unchangeda/Decreasedb
Kidney ICAM-1 Increaseda Increaseda/Unchangedb Increaseda/Unchangedb
MAdCAM-1 Decreaseda c c

a Compared to chow.
b Compared to PN alone.
c Unknown.

test for evaluating nutritional manipulation of IgA-dependent host defenses. Once immu-
nized by an initial dose of virus, mice quickly clear and sterilize subsequent viral inocu-
lations from the nasal passages within hours. To examine immunologic integrity after
nutrient manipulation, mice were randomized to chow, IV-PN, or a CED 3 weeks after
immunization. All animals receiving nutrients via the gastrointestinal tract maintained
normal immunity against the virus and cleared it from the nasal passages, while 50%
(5 of 10) of the IV-PN animals continued to shed virus in nasal washes and failed to sterilize
the nasal passages. Continued viral shedding in the form of continued viral proliferation
within the respiratory tract represented a loss of this IgA-mediated defense system. Thus,
the lack of enteral stimulation of the gut interfered with established IgA-mediated
defenses, creating a susceptibility to infectious complications.
Reductions in sIgA concentrations also correlate with functional changes in antibac-
terial immunity. Production of Pseudomonas-specific IgA can be stimulated within the
respiratory secretions of animals upon immunization with Pseudomonas aeruginosa antigens
in liposomes. Immunization induces a protective defense against a subsequent lethal dose
of Pseudomonas in animals receiving chow or CED. This protection is completely lost in
animals receiving IV-PN for 5 days.22 Thus, these data provide support for enteral stim-
ulation to maintain normal mucosal antibacterial and antiviral defenses.
Sometimes the gut cannot be utilized for feeding due to dysfunction or inaccessibility,
and parenteral feeding is necessary to prevent malnutrition. Under these conditions, the
amino acid GLN may support the mucosal immune defenses noted above. GLN can
maintain luminal sIgA levels. Rats receiving GLN-supplemented PN preserved intestinal
IgA levels and displayed an incidence of bacterial translocation to mesenteric lymph nodes
similar to that of controls eating chow.18 However, rats receiving standard GLN-free PN
exhibit a 50% reduction in biliary sIgA and increased bacterial adherence to cecal mucosa,
with increased bacterial translocation to mesenteric lymph nodes.18 In the mouse, GLN
supplementation of PN prevents GALT atrophy and at least partially maintains upper
respiratory tract immunity.23,24 Using the same models described above, mice were ran-
domized to chow, standard IV-PN, or an isonitrogenous 2% GLN-supplemented PN for-
mulation (GLN-PN). Standard IV-PN significantly lowered the total cell yield in the PP,
intraepithelial layer (IEL), and LP compared with chow or GLN-PN groups. The
CD4+:CD8+ ratio was preserved in all areas except the LP, where it fell significantly with
standard IV-PN but remained normal with chow and GLN-PN. Further work showed that
GLN supplementation normalized intestinal and respiratory IgA as well as intestinal Th2
cytokines (IL-4 and IL-10).25 For these experiments, mice received chow, standard IV-PN,
1382_C42.fm Page 721 Tuesday, October 7, 2003 7:42 PM

Chapter forty-two: Amino acids to support gut function and morphology 721

or an isonitrogenous 2% GLN-PN formulation. Standard IV-PN significantly reduced


intestinal and respiratory IgA concentrations compared with chow-fed animals and GLN-
PN. GLN significantly improved respiratory and intestinal IgA concentrations, preserved
IL-4 concentrations, and maintained IL-10 concentrations midway between chow and
standard IV-PN animals, lending further support to the importance of Th2 IgA-stimulating
cytokines within the intestine and the ability of intravenous GLN to contribute to mucosal
immunity during the lack of enteral stimulation. GLN administration was further shown
to influence established antibacterial and antiviral immunity. After immunization and
dietary manipulation with chow, IV-PN, or GLN-PN, 87% (13 of 15) of immunized mice
receiving standard IV-PN failed to clear virus from their upper respiratory tracts vs. 38.5%
of mice on GLN-PN (p < 0.05 vs. IV-PN) and 7.1% of mice fed chow (p < 0.002 vs. IV-PN).
After Pseudomonas immunization, GLN-TPN significantly improved survival from 22% in
IV-PN-fed mice to 65%, but not to the levels of chow-fed mice (90% survived).24
Although the exact effects of GLN on immune cells need to be clarified, alterations
in immune function may arise from deficiencies of the intracellular GSH pool. Inducing
GSH depletion with BSO impairs a variety of lymphocyte functions mediated by IL-2,
such as T cell proliferation, cytotoxic T cell activity, and natural killer cell activity. Decreases
in intracellular GSH stores promote the production of IL-2 and IFN-g, thus affecting IgA
synthesis and mucosal protection, as mentioned above.26 Reductions in plasma cysteine
concentrations parallel decreases in plasma GLN as well. The agent N-acetyl-cysteine acts
as both a source of cysteine and a precursor of GSH biosynthesis. Randomized, controlled
clinical trials have documented significant improvement in several immunologic param-
eters in patients infected with human immunodeficiency virus (HIV) when given N-acetyl-
cysteine compared to placebo.27 Whether the cysteine and GSH deficiency associated with
HIV is similar to that observed with a lack of enteral intake remains to be determined.

42.3 Gut morphology and function: the vasculature


Diet induces changes in intestinal IgA levels.23 The IgA levels correlate with both the IL-4
(r = 0.65) and IL-10 (r = 0.54) levels in tissue. Although significant, these correlation
coefficients suggest that other mediators are involved in GALT maintenance. While the
reduction in T and B cell mass in GALT almost certainly accounts for some IgA reduction,
parameters that signal Th1/Th2 actions or play a role in Th1/Th2 cell regulation such as
chemokines, chemokine receptors, and adhesion molecules may play a role. Chemokines
act as signal lamps to recruit cells into GALT and direct their migration.28
Little is known about chemokine and chemokine receptor alterations during malnu-
trition or gut starvation, but expression of specific adhesion molecules on the endothelium
such as intracellular adhesion molecule-1 (ICAM-1) has drawn attention. Expression of
ICAM-1 is responsible for firm adhesion between endothelium and circulatory polymor-
phonuclear neutrophils (PMNs) in various tissues, including the GI tract. Experimentally,
this produces deleterious immunologic effects during reperfusion following ischemia since
the gut serves as a priming bed for circulating neutrophils. This priming alters their
response to subsequent injury and increases their ability to injure tissue.29 Since IL-10 and
IL-4 normally inhibit ICAM-1 expression and both of these Th2 cytokines decrease in
response to IV-PN, we suspected that route of nutrition would alter ICAM-1 expression.
After pretreatment with chow or standard IV-PN, mice were injected with 125I-labeled anti-
ICAM-1 antibody and 131I-labeled nonbonding antibody to quantify ICAM-1 expression
in the lungs, liver, and kidney.30 Myeloperoxidase (MPO), an enzyme found primarily in
PMNs, was used to assess PMN accumulation in various tissues. Expression of intestinal
ICAM-1 significantly increased with IV-PN simultaneously with increases in MPO levels.
This reversed quickly with chow refeeding.
1382_C42.fm Page 722 Tuesday, October 7, 2003 7:42 PM

722 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Since GLN reduces PN-associated impairment of mucosal immunity, the effect of GLN
on ICAM-1 expression was examined.31 Standard IV-PN significantly increased ICAM-1
expression in the small intestine, lung, and kidney. GLN-PN maintained normal intestinal
ICAM-1 expression but failed to lower pulmonary and renal ICAM-1 to chow levels. GLN
maintained intestinal concentrations of IL-4, but did not increase IL-10 to concentrations
observed in chow-fed mice. GLN supplementation of PN modified the vascular endothe-
lium of the unfed gastrointestinal tract but did not return it completely to normal.
These ICAM-1 alterations appeared to have functional significance. Gut vascular
endothelial changes influence the local and systemic responses to ischemia/reperfusion.
Other investigators demonstrated that gut ischemia/reperfusion (I/R) induces acute lung
and liver injury.29 Shock induces a disproportionate splanchnic hypoperfusion. The pos-
tischemic gut provokes PMN-mediated tissue injury leading to failure of remote organs,
such as the lung, kidney, and liver. The effect occurs at least partially through circulating
PMNs, which are primed by shock and subsequently accumulate in remote organs during
reperfusion. This priming augments their biochemical inflammatory response to continued
— or delayed — insults. Speculating that standard IV-PN and enteral nutrition would
influence organ injury after gut I/R, mice were randomized to chow, standard IV-PN, or
CED feeding for 5 days.32 Accumulation of 125I-labeled albumin was used to measure
vascular permeability in various tissues, and lung PMN accumulation was assessed with
MPO. IV-PN induced a significantly higher vascular permeability index in the lung and
liver, but no significant differences in MPO of the lung were observed between any groups.
However, myeloid cells in pulmonary tissues demonstrated dramatic increases in expres-
sion of CD18, a marker of PMN cell priming. This increase in CD18 expression occurred
only in the lungs of IV-PN-fed mice with no increases in enterally fed mice, i.e., the number
of pulmonary PMNs were similar with IV-PN, but the PMNs appeared to be primed to
induce more injury.
These changes affected survival after gut I/R. Survival rates after 15 min of superior
mesenteric artery occlusion in the enterally fed groups (chow or CED) were significantly
greater than in mice given IV-PN. GLN supplementation of IV-PN affected the response.33
After 5 days of chow, standard IV-PN, or 2% GLN-PN, mice were exposed to gut I/R and
resuscitated; survival was noted for the next 72 h. Survival at the study end point was
significantly lower in the IV-PN group (15%) than in the chow (83%) or GLN-PN (62%)
groups. We suspect that the survival benefits of GLN relate to the reduction of ICAM-1
expression, preservation of IL-4 levels in the gut, minimal PMN priming, and a blunting
of the subsequent PMN-induced tissue injury.
The specific mechanisms for these GLN vascular alterations are unclear. Van der Hulst
et al.34 examined intestinal biopsies obtained before and 10 days after patients received
either GLN-PN or standard IV-PN. Intraepithelial lymphocytes dropped significantly in
the GLN group with no changes observed in the standard IV-PN group. They speculated
that a reduction in these lymphocytes resulted in decreased IFN-g, which prevented an
opening of tight junctions between epithelial cells and decreased production of oxygen
free radicals by PMNs. An antioxidant effect of GLN was implicated as a means to abrogate
increased intestinal permeability. Administration of GLN is known to enhance intracellular
GSH concentrations and lower the activity of redox-sensitive protein kinases, thereby
inhibiting the synthesis of nuclear factor kappa B (NFkB).4 Experimental studies linked
I/R in the heart and kidney to activation of protein kinase cascades that regulate the
expression of pro-inflammatory genes. Excessive gene activation can lead to decreased
GALT and, ultimately, multiple-organ failure.35
Some benefits of GLN on I/R may result from GLN’s influence on arginine production.
Arginine is generated from the combination of circulating citrulline with aspartic acid
forming arginosuccinate, which is cleaved to form arginine. The primary source for
1382_C42.fm Page 723 Tuesday, October 7, 2003 7:42 PM

Chapter forty-two: Amino acids to support gut function and morphology 723

citrulline is the intestinal metabolism of GLN.36 Houdijk et al.37 demonstrated that an


enteral diet supplemented with GLN yielded higher arterial plasma concentrations of
arginine due to renal arginine production from citrulline. Rats were randomized to receive
a 12.5% GLN-supplemented enteral diet or an isocaloric, isonitrogenous diet for 14 days,
both of which contained arginine but lacked citrulline. Arginine release by the kidney was
38% greater in the GLN-fed animals than in the controls.
Arginine may work through several mechanisms. Through its role in nitric oxide (NO)
production, arginine can increase local blood flow through its vasodilatory effects. Argi-
nine is the primary substrate for NO, which is converted to citrulline by nitric oxide
synthase (NOS) to produce NO. Rat perfusion studies demonstrated that intestinal
ischemia inhibits NOS. Schleiffer et al.38 demonstrated that pretreatment with L-arginine
(0.8 g/kg) increased mesenteric venous blood flow compared with control rats given casein
hydrolysate. L-arginine reduces intestinal barrier permeability compared to the control
group as measured by 14C-PEG absorption for the integrated first 10-min period (4.0 ± 0.9
vs. 11.2 ± 1.6 14C-PEG pmol/segment, p < .05), implying improved intestinal recovery with
more successful reperfusion of the postischemic intestine. The authors suggested that
L-arginine facilitates mucosal recovery by preventing the reduction in mucosal blood flow
induced by the I/R event; thus impaired NO synthesis via arginine metabolic pathways
may contribute to I/R-induced mucosal injury.
Future research will be required to unravel the exact mechanisms by which dietary
amino acids can influence gut morphology, mucosal immunity, and mucosal vasculature.
GLN is particularly intriguing as it appears to have effects on the gut in each of these
three areas. By improving intestinal permeability or serving as a precursor for GSH
biosynthesis, GLN appears promising for maintaining gut barrier function. Immunologic
defects induced by lack of enteral feeding have also been reversed by supplementation
of PN with GLN. The current understanding for this preservation of intestinal and extra-
intestinal mucosal immunity lies within GLN effects on chemokine and cytokine produc-
tion and modulation of Th-2 cytokines, including IL-4 and IL-10. Finally, GLN has positive
effects on the vascular endothelium of the gastrointestinal tract, owing to some inherent
antioxidant properties or by promoting NO production via arginine synthesis. Hopefully,
future manipulation of dietary amino acids will allow for the development of nutritional
therapies that can preserve gut integrity, immunological function, and vasculature in
patients unable to be fed via the gastrointestinal tract.

References
1. Wu, G., Intestinal mucosal amino acid catabolism, J. Nutr., 128, 1249, 1998.
2. Luk, G.D., Marton, L.J., and Baylin, S.B., Ornithine decarboxylase is important in intestinal
mucosal maturation and recovery from injury in rats, Science, 210, 195, 1980.
3. Alican, I. and Kubes, P., A critical role for nitric oxide in intestinal barrier function and
dysfunction, Am. J. Physiol., 270, G225, 1996.
4. Roth, E., Oehler, R., Manhart, N., et al., Regulative potential of glutamine: relation to glu-
tathione metabolism, Nutrition, 18, 217, 2002.
5. Windmueller, H.G. and Spaeth, A.E., Uptake of and metabolism of plasma glutamine by the
small intestine, J. Biol. Chem., 249, 5070, 1974.
6. Buchman, A.L., Moukarzel A.A., Bhuta, S., et al., Parenteral nutrition is associated with
intestinal morphologic and functional changes in humans, J. Parenter. Enteral Nutr., 19, 453,
1995.
7. Van der Hulst, R.R.W.J., Van Kreel, B.K., Von Meyenfeldt, M.F., et al., Glutamine and the
preservation of gut integrity, Lancet, 341, 1363, 1993.
8. Tremel, H., Kienle B., Weilemann, L.S., et al., Glutamine dipeptide-supplemented parenteral
nutrition maintains intestinal function in the critically ill, Gastroenterology, 107, 1595, 1994.
1382_C42.fm Page 724 Tuesday, October 7, 2003 7:42 PM

724 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

9. Buchman A.L., Glutamine: a conditionally required nutrient for the human intestine? Nutri-
tion, 13, 240, 1997.
10. Reeds, P.J., Burrin, D.G., Stoll B., et al., Enteral glutamate is the preferential source for mucosal
glutathione synthesis in fed piglets, Am. J. Physiol., 273, E408, 1997.
11. Martensson, J., Jain, A., and Meister A., Glutathione is required for intestinal function, Proc.
Natl. Acad. Sci. U.S.A., 87, 1715, 1990.
12. Salmi, M. and Jalkanen, S., Regulation of lymphocytic traffic to mucosa-associated lymphatic
tissues, Gastroenterol. Clin. North Am., 20, 495, 1991.
13. Lamm, M.E., Mazaneca, M.B., Nedrud, J.G., and Kaetzel, C.S., New functions for mucosal
IgA, in Advances in Mucosal Immunology, Mestecky, J., Russell, M.W., Jackson, S., et al., Eds.,
Plenum Press, New York, 1995, p. 647.
14. Kramer, D.R., Sutherland, R.M., Bao, S., et al., Cytokine-mediated effects in mucosal immu-
nity, Immunol. Cell Biol., 73, 389, 1995.
15. Ramsay, A.J., Genetic approaches to the study of cytokine regulation of mucosal immunity,
Immunol. Cell Biol., 73, 484, 1995.
16. Alverdy, J.A., Chi, H.S., and Sheldon, G.S., The effect of parenteral nutrition on gastro-
intestinal immunity: the importance of enteral stimulation, Ann. Surg., 202, 681, 1985.
17. Burke, D.J., Alverdy, J.C., Aoys, E., et al., Glutamine-supplemented total parenteral nutrition
improves gut immune function, Arch. Surg., 124, 1396, 1989.
18. Alverdy, J.C., Chi, H.S., and Sheldon, G.S., The effect of parenteral nutrition on gastro-
intestinal immunity, the importance of enteral stimulation, Ann. Surg., 202, 681, 1985.
19. Li, J., Kudsk, K.A., Gocinski, B., et al., Effects of parenteral and enteral nutrition on gut-
associated lymphoid tissue, J. Trauma, 39, 44, 1995.
20. Kudsk, K.A., Li, J., and Renegar, K.B., Loss of upper respiratory tract immunity with parenter-
al feeding, Ann. Surg., 223, 629, 1996.
21. Li, J., Kudsk, K.A., Janu, P.G., et al., Effect of glutamine-enriched total parenteral nutrition
on small gut-associated lymphoid tissue and upper respiratory tract immunity, Surgery, 121,
542, 1997.
22. DeWitt, R.C., Wu, Y., Renegar, K.B., et al., Glutamine-enriched total parenteral nutrition
preserves respiratory immunity and improves survival to Pseudomonas pneumonia, J. Surg.
Res., 84, 13, 1999.
23. Yong, W., Kudsk, K.A., DeWitt, R.C., et al., Route and type of nutrition influence IgA-
mediating intestinal cytokines, Ann. Surg., 229, 662, 1999.
24. King, B.K., Kudsk, K.A., Li, J., et al., Route and type of nutrition influence mucosal immunity
to bacterial pneumonia, Ann. Surg., 229, 272, 1999.
25. Kudsk, K.A., Yong, W., Fukatsu, K., et al., Glutamine-enriched total parenteral nutrition
maintains intestinal interleukin-4 and mucosal immunoglobulin A levels, J. Parenter. Enteral
Nutr., 24, 270, 2000.
26. Dröge, W., Schulze-Osthoff, K., Mihm, S., et al., Function of glutathione and glutathione
disulfide in immunology and immunopathology, FASEB, 8, 1131, 1994.
27. Dröge, W. and Breitkreutz R., Glutathione and immune function, Proc. Nutr. Soc., 59, 595,
2000.
28. Moser, B. and Loetscher, P., Lymphocyte traffic control by chemokines, Nat. Immunol., 2, 123,
2001.
29. Moore, E.E., Moore, F.A., Franciose, R.J., et al., The ischemic gut serves as a priming bed for
circulating neutrophils that provoke multiple organ failure, J. Trauma, 37, 881, 1994.
30. Fukatsu, K., Lundberg, A.H., Hanna, M.K., et al., Route of nutrition influences ICAM-1
expression and neutrophil accumulation in the intestine, Arch. Surg., 134, 1055, 1999.
31. Fukatsu, K., Lundberg, A.H., Kudsk, K.A., et al., Modulation of organ ICAM-1 expression
during IV-TPN with glutamine and bombesin, Shock, 15, 1, 2001.
32. Fukatsu, K., Zarzaur, B.L., Johnson, C.D., et al., Enteral nutrition prevents remote organ
injury and death after a gut ischemic insult, Ann. Surg., 233, 660, 2001.
33. Ikeda, S., Zarzaur, B.L., Johnson, C.D., et al., TPN supplementation with glutamine improves
survival after gut ischemia/reperfusion, J. Parenter. Enteral Nutr., 263, 169, 2002.
1382_C42.fm Page 725 Tuesday, October 7, 2003 7:42 PM

Chapter forty-two: Amino acids to support gut function and morphology 725

34. van der Hulst, R.R.W.J., von Meyenfeldt, M.F., Tiebosch, A., et al., Glutamine and intestinal
immune cells in humans, J. Parenter. Enteral Nutr., 21, 310, 1997.
35. Hassoun, H.T., Kone, B.C., Mercer, D.W., et al., Post-injury multiple organ failure: the role
of the gut, Shock, 15, 1, 2001.
36. Mourad, F.H., O’Donnell, L.J., Andre, E.A., et al., L-arginine, nitric oxide, and intestinal
secretion: studies in rat jejunum in vivo, Gut, 39, 539, 1996.
37. Houdijk, A.P.J., van Leeuwen, P.A.M., Teerlink, T., et al., Glutamine-enriched enteral diet
increases renal arginine production, J. Parenter. Enteral Nutr., 18, 422, 1994.
38. Schleiffer, R., Raul, F., et al., Prophylactic administration of L-arginine improves the intestinal
barrier function after mesenteric ischaemia, Gut, 39, 194, 1996.
1382_C42.fm Page 726 Tuesday, October 7, 2003 7:42 PM
1382_C43.fm Page 727 Tuesday, October 7, 2003 7:43 PM

section C

Nutraceutics
1382_C43.fm Page 728 Tuesday, October 7, 2003 7:43 PM
1382_C43.fm Page 729 Tuesday, October 7, 2003 7:43 PM

chapter forty-three

L-arginine-enriched diets
in cardiovascular diseases
Marika Collin
University of Helsinki
Heikki Vapaatalo
University of Helsinki

Contents
Introduction..................................................................................................................................729
43.1 Biochemical aspects ...........................................................................................................730
43.2 Nonpharmacological treatment of cardiovascular diseases........................................731
43.3 Effect of L-arginine on cardiovascular risk factors........................................................731
43.3.1 Hyperlipidemia ....................................................................................................731
43.3.2 Diabetes .................................................................................................................732
43.3.3 Hypertension ........................................................................................................732
43.3.4 Menopause............................................................................................................732
43.4 Effect of L-arginine in cardiovascular disorders............................................................732
43.4.1 Ischemic heart disease ........................................................................................732
43.4.2 Heart failure..........................................................................................................733
43.5 Effect of L-arginine in healthy volunteers ......................................................................733
43.6 Adverse effects ...................................................................................................................733
43.7 Mechanism of action..........................................................................................................733
43.8 Discussion ...........................................................................................................................736
References .....................................................................................................................................737

Introduction
In 1980, Furchgott and Zawadski1 described the finding that rabbit arterial rings in vitro
did not relax in response to acetylcholine when the endothelium was destroyed. They
called the factor(s) responsible for the relaxation an endothelium-dependent relaxing factor
(EDRF). A few years later, different research groups identified the factor(s) as a small
gaseous molecule — nitric oxide (NO).2,3 Soon, it was confirmed that physiologically NO
is formed stereospecifically from an amino acid L-arginine. No other amino acids have
been shown to release NO either in vivo or in vitro. After the discovery of NO, a great
number of reports on its role in cardiovascular diseases have been published, and the
possibility of using its precursor, L-arginine, in the prevention and treatment of these
diseases has recently been evaluated and reviewed.4–8
0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 729
1382_C43.fm Page 730 Tuesday, October 7, 2003 7:43 PM

730 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Arginine was isolated from animal horn tissue and also from lupine seedlings in the
late 19th century. In addition to its unique role as the substrate to NO synthesis, L-arginine
has many important biochemical functions: immunoreactivity, release of growth hormones
glucagon and insulin, amino acid detoxification, synthesis of creatinine and polyamines,
etc. The crucial role of L-arginine as the precursor of NO formation has stimulated the
basic and clinical research to answer the question of whether an increase in the daily intake
of L-arginine could have beneficial effects on the vascular endothelial function in diseases
such as atherosclerosis, hypertension, and renal diseases, in which it has been shown to
be impaired.
This chapter aims to summarize and give a critical opinion on the effects of oral intake
of L-arginine in the prevention or treatment of different cardiovascular diseases based on
the clinical studies on humans published during the last few years. Studies investigating
the effects of intravenously administered L-arginine in humans are not reviewed, as the
relevance of a route of administration other than oral in the setting of long-term health
maintenance is limited. Studies on experimental models are not reviewed either. Readers
interested in these topics are advised to see a recent excellent review.9

43.1 Biochemical aspects


As mentioned above, the most interesting and important L-arginine-related question is its
role in the formation of the most important endothelium-derived relaxing factor, NO, which
regulates the endothelium-dependent vasodilation and antithrombosis in concert with pros-
tacyclin (PGI2), an arachidonic acid-derived compound, and endothelium-derived hyperpo-
larizing factor (EDHF), whose chemical nature has not been fully established yet.
Many different enzymes have been characterized to catabolize arginine or convert it
into bioactive compounds in addition to NO synthases (NOSs) constitutive endothelial
(eNOS) and neuronal (nNOS) as well as inducible (iNOS), which form NO and L-citrulline.
Other enzymes include arginase to produce ornithine and concomitantly urea, and argi-
nine, glycine transaminase produces guanidine acetic acid, which is the precursor of
cecatine and major source of high-energy phosphate for regeneration of adenosine triph-
osphate (ATP). Kyotorphine synthase produces kyotorphine, and arginine decarboxylase
catalyzes the conversion of L-arginine into agmatine, an endogenous agonist of adrenergic
a2-receptors in the brain (for reviews see Cheng and Baldwin8 and Nakaki and Kato10).
Arginine is a semiessential amino acid for infants and growing children, whose livers
are not capable of producing arginine rapidly enough for growth, and which is required
for the urea cycle in protein metabolism. It has been estimated that a normal diet provides
about 5 g L-arginine daily. In many clinical trials much higher doses have been administered.
The theoretical calculations integrating an adequate amount of L-arginine in the diet
with plasma concentration and intracellular concentration in the endothelial cells that would
produce positive cardiovascular effects are complicated. The average plasma concentrations
of L-arginine in subjects on a “normal Western diet” range from 50 to 130 mmol/l.11 The
intracellular concentrations of L-arginine are in the range of 500 to 1200 mmol/l,12 while
the concentration of L-arginine needed to produce NO in healthy endothelial cells is only
3 mmol/l. These calculations raise questions on the NO-related cardiovascular benefits
shown in clinical trials with high doses of arginine administered acutely intravenously or
in short-term trials (orally for a few weeks). Furthermore, it is possible that the strongest
effects can only be seen in L-arginine-depleted subjects. This may explain why most trials
have not shown any real benefits.
Based on the positive findings on high acute intravenous doses of L-arginine that
have improved the brachial or coronary artery dilation in response to acetylcholine or
other endothelial-dependent vasodilators, a question regarding other, NO-independent
1382_C43.fm Page 731 Tuesday, October 7, 2003 7:43 PM

Chapter forty-three: L-arginine-enriched diets in cardiovascular diseases 731

mechanisms is raised. The issue is discussed in an excellent recent review.8 L-arginine may
inhibit the release of endothelial vasoconstrictors such as endothelin; counteract the inhibitory
effect of glutamine on L-arginine bioavailability; decrease the formation of asymmetric
dimethyl arginine (ADMA), a naturally existing inhibitor of NOS; posses antioxidative action;
and stimulate vasodilatory insulin formation/release; and participate in nonenzymatic, non-
stereospecific formation of NO as a result of chemical reaction with hydrogen superoxide.

43.2 Nonpharmacological treatment of cardiovascular diseases


Lifestyle factors play an important role in the prevention and treatment of cardiovascular
diseases. Nonpharmacological therapy should also be considered the basis for the treat-
ment of hypertensive patients receiving antihypertensive medication. Lifestyle factors that
should be acknowledged include diminished use of salt (sodium chloride), saturated fats,
and alcohol; decrease in weight; etc. Increased intake of certain dietary components, such
as potassium, magnesium, calcium, and polyunsaturated fats, may also be advantageous.
This chapter evaluates the role of L-arginine supplementation in cardiovascular diseases
in the light of current knowledge and reviews the available human studies on the field.
The possible beneficial effects of oral L-arginine administration have been evaluated in
numerous published clinical trials. In these studies, however, some rather moderate evi-
dence has been reported for the antiatherogenic, antiischemic, platelet aggregation inhibi-
tory and antithrombotic properties of this semiessential amino acid. Impaired endothelial
function in these studies has been related to hypercholesterolemia, heart failure, ischemic
heart disease, diabetes, hypertension, and smoking. However, in most of the studies the
number of subjects has been small, and the L-arginine doses given have been fairly high.
The treatment period has often been short, ranging from a few days to a few weeks. In
some cases, the study design has not fulfilled the criteria of good clinical trial practice
(GCP), as the studies have not been placebo controlled or blinded. The changes seen, though
sometimes statistically significant, have been only marginal and therefore clinically not
significant. More convincing evidence, even though possibly not related to increased NO
formation but to other nonspecific mechanisms, arises from studies investigating the effect
of acute intravenous L-arginine administration. In these studies, high doses of L-arginine
have improved peripheral vascular dilation in response to acetylcholine or increased pain-
free walking distance. These studies are not included in the present overview. Readers are
advised to consult recent comprehensive reviews on the topic.7–9

43.3 Effect of L-arginine on cardiovascular risk factors


43.3.1 Hyperlipidemia
Stimulation of endogenous NO production may inhibit atherogenesis, and oral L-arginine
supplementation may therefore be of benefit in patients with risk factors for atherosclerosis.
Plasma concentrations of L-arginine are known to be lower in hypercholesterolemic
patients than in healthy subjects.13 Dietary supplementation with oral L-arginine has
slightly improved an endothelium-dependent relaxation of the vascular wall smooth
muscle (vasodilation) in small placebo-controlled crossover studies in men with coronary
artery disease14 and in young hypercholesterolemic adults.15 The beneficial effect was
observed within 3 days to 4 weeks after the beginning of the treatment. Monocyte adhesion
to endothelial cells was also reduced in an in vitro setting using human umbilical vein
endothelial cells (HUVECs).14 Dietary supplementation with L-arginine has also modestly
reduced platelet aggregability in hypercholesterolemic patients.16 Platelet aggregability
plays an important role in the development of atherosclerotic complications.
1382_C43.fm Page 732 Tuesday, October 7, 2003 7:43 PM

732 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Thus, L-arginine supplementation seems to provide some benefit in hyperlipidemic


patients. However, the observed effects have been fairly modest, and although the studies
were randomized and double-blinded, a question remains whether oral L-arginine sup-
plementation is beneficial in hyperlipidemia.

43.3.2 Diabetes
Patients with diabetes are particularly vulnerable to developing atherosclerotic disease
due to metabolic abnormalities, hypercoagulable states, and inherent endothelial dysfunc-
tion in this population. In young normocholesterolemic adults with type I diabetes mel-
litus, oral L-arginine supplementation for 6 weeks elicited no effects on endothelial function
measured as unchanged endothelium-dependent vasodilation in a high dose (14 g/day),
which improved endothelial function in patients with hyperlipidemia.17 Therefore, L-argi-
nine supplementation does not seem to provide health benefits in diabetics. However,
more studies are needed before solid conclusions can be drawn.

43.3.3 Hypertension
Since abnormalities in endothelium-dependent vasodilation may be involved in the patho-
genesis of hypertension, L-arginine, as a vasodilatory compound, may have a blood pres-
sure-lowering effect. The effect of oral L-arginine supplementation was studied in 20
patients, who were hypertensive despite 3 months of therapy with antihypertensive med-
ication. In this study, oral L-arginine (6 g/day) lowered blood pressure within 6 weeks.18
The authors suggested that the beneficial effect on blood pressure was due to improved
endothelial function. Unfortunately, vasodilation was not measured in this study, leaving
the question of mechanism of action unanswered. Nevertheless, in addition to other
beneficial dietary factors, L-arginine may turn out to be useful in hypertensive patients.

43.3.4 Menopause
In healthy postmenopausal women, 1 month of therapy with L-arginine induced no
changes in endothelium-dependent vasodilation.19 L-arginine also did not lower serum
levels of inflammation markers, such as intercellular adhesion molecule-1 (ICAM-1), vas-
cular cell adhesion molecule-1 (VCAM-1), and L-selectin, which are increased in patients
with atherosclerosis.20,21 The authors concluded that L-arginine is therefore unlikely to
protect healthy postmenopausal women from developing atherosclerosis. This may be a
fair conclusion, even though longer-term clinical trials are needed to establish the associ-
ation or lack of it.

43.4 Effect of L-arginine in cardiovascular disorders


43.4.1 Ischemic heart disease
The effect of oral L-arginine on exercise capacity has been evaluated in patients with stable
angina pectoris. L-arginine supplementation significantly prolonged mean exercise time
and increased the maximum workload in a group of 20 patients.22 In 26 subjects who had
been referred for coronary angiography because of recurrent chest pain, oral L-arginine
supplementation (3 g/day) for 6 months increased coronary blood flow and improved
patients’ symptoms compared with the placebo group.23 In contrast, no effect on vascular
function or inflammation markers (E-selectin, P-selectin, ICAM-1, VCAM-1) was observed
in elderly patients with coronary artery disease, who had been supplemented with L-argi-
nine (9 g/day) for 4 weeks.24
1382_C43.fm Page 733 Tuesday, October 7, 2003 7:43 PM

Chapter forty-three: L-arginine-enriched diets in cardiovascular diseases 733

Recently, a small intervention study was carried out to examine the effects of L-argi-
nine-enriched food in patients with coronary artery disease.25 The study found that con-
sumption of two nutrient bars containing L-arginine (3.3 g each, 6.6 g/day) for 2 weeks
increased exercise time and improved endothelium-dependent vasodilation in patients
with chronic stable angina pectoris.25 Two-week consumption of the nutrient bar also
increased total walking distance by 66% in patients with claudication from atherosclerotic
peripheral arterial disease.26 However, in addition to L-arginine, the nutrient bar contained,
for example, vitamins C and E, folate, and soy isoflavones, which may have contributed
to the observed beneficial effects. The benefit provided by the consumption of the nutrient
bars may therefore not be solely attributed to L-arginine, particularly because plasma levels
of L-arginine were not measured in this study. Nonetheless, this approach shows that
improvement in health status can be achieved by changes in diet, by adding a healthy or
removing a nonhealthy component.

43.4.2 Heart failure


The potential of L-arginine in the treatment of congestive heart failure has also been of
interest to investigate, because these patients demonstrate a subnormal peripheral vaso-
dilation in response to endothelium-dependent vasodilators.27–30 Supplemental oral L-argi-
nine (6 to 13 g/day) for 6 weeks increased forearm blood flow during forearm exercises
in patients with heart failure.31 Therefore, supplemental oral L-arginine may be beneficial
in this population, but the therapeutic potential needs to be established.

43.5 Effect of L-arginine in healthy volunteers


The effect of L-arginine-enriched diet on blood pressure, renal function, plasma glucose
levels, and plasma cholesterol levels has also been investigated in healthy volunteers.32
Compared to control diet, which provided 4 g of L-arginine daily, an L-arginine-enriched
diet (10 g/day) lowered blood pressure, lowered fasting glucose level, and increased crea-
tinine clearance. It made no difference whether the higher level of L-arginine intake origi-
nated from foods rich in the amino acid or from synthetic capsules.32 Thus, since the intake
of L-arginine during the control diet was below average intake (5 g/day), these results may
reflect the effects of L-arginine in subjects deficient in this semiessential amino acid.

43.6 Adverse effects


Oral L-arginine seems to cause some adverse effects, of which the most common ones are
nausea and diarrhea. Other adverse effects observed during L-arginine-enriched diet reg-
imens have included flatulence, bowel habit change, bloating, colitis, headache, increased
fatigue, and rash. However, the safety of long-term L-arginine supplementation has not
been investigated systematically.

43.7 Mechanism of action


The mechanism of action underlying the putative beneficial effect of supplemental L-argi-
nine needs to be established. One possible explanation is an enhanced formation of the
vasodilatory NO from its precursor L-arginine. The observed benefits in patients would
consequently derive from the more abundant availability of NO. However, this assump-
tion is controversial from an enzyme–biochemical point of view, because NOS should be
saturated with L-arginine, its substrate, at physiological levels and not be dependent on
extracellular supply.33 Nevertheless, L-arginine has improved endothelium-dependent
vasodilation in many studies (Table 43.1). There may be several explanations for this.
734

Table 43.1 Studies of Effects of Oral L-arginine (L-Arg) Supplementation in Cardiovascular Diseases in Humans
Subjects Design Dose Results Reference

Coronary artery disease (CAD)


28 M, 8 F; 66 y Randomized, double-blind 2 ¥ nutrient bar containing 6.6 P-L-arginine not reported; total 25
crossover, placebo-controlled g L-Arg/d; 14 d, 30-d washout exercise time ≠; endothelium-
1382_C43.fm Page 734 Tuesday, October 7, 2003 7:43 PM

dependent vasodilation of brachial


artery ≠
29 M, 1 F; 67 y Randomized, double-blind L-Arg 3 ¥ 3 g/d; 4 wk, 4-wk P-L-arginine ≠; no effect on vascular 24
crossover, placebo-controlled washout function
13 M, 13 F; 49 y Randomized, single-blind, L-Arg 3 g/d; 6 mo P-L-arginine ≠; coronary blood flow in 23
placebo-controlled response to acetylcholine ≠
10 M; 41 y Randomized, double-blind L-Arg3 ¥ 7 g/d; 3 d, 10-d P-L-arginine ≠; endothelium- 14
crossover, placebo-controlled washout dependent vasodilation of brachial
artery ≠
20 M, 2 F; 57 y, on Randomized, double-blind, L-Arg 3 ¥ 2 g/d; 3 d No change in P-L-arginine; mean 22
medication placebo-controlled exercise time ≠, maximum work load

Peripheral arterial disease (PAD)


31 M, 10 F; 68 y, on Randomized, double-blind, 2 ¥ nutrient bar containing 6.6 P-L-arginine not reported; pain-free 26
medication placebo-controlled g L-Arg/d; 14 d walking distance ≠, total walking
distance ≠

Hypercholesterolemia
27 patients; 29 y Randomized, double-blind L-Arg3 ¥ 7 g/d; 4 wk, 4-wk P-L-arginine ≠; endothelium- 15
crossover, placebo-controlled washout dependent vasodilation of brachial
artery ≠
Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition
30 M, 7 F; age not Randomized, double-blind, L-Arg 8.4 g/d; 2 wk P-L-arginine ≠; platelet aggregability Ø 16
reported placebo-controlled

Heart failure (HF)


14 M, 1 F; 56 y, on Randomized, double-blind, L-Arg 6-13 g/d; 6 wk P-L-arginine ≠; forearm blood flow 31
medication placebo-controlled during exercise ≠
Chapter forty-three:

Hypertension (HT)
8 M, 12 F; 48 y, anti-HT Randomized, placebo- L-Arg 3 ¥ 3 g/d; 6 wk P-L-arginine not reported; blood 18
medication for 3 mo controlled pressure Ø

Diabetes (Type I)
53 M, 31 F; 34 y Randomized, double-blind, L-Arg 2 ¥ 7 g/d; 6 wk P-L-arginine ≠; no effect on endothelial 17
1382_C43.fm Page 735 Tuesday, October 7, 2003 7:43 PM

placebo-controlled function

Healthy Volunteers
L-arginine-enriched

6 subjects; 39 y Single-blind crossover, Control diet L-Arg 4 g/d; P-L-arginine not reported; blood 32
controlled; 1 wk each diet L-Arg-enriched foods 10 g/d; pressure Ø, CCL ≠, fasting glucose Ø
L-Arg supplement 10 g/d vs. control; S-cholesterol Ø, S-HDL ≠
after enriched foods
10 F menopausal; 55 y Randomized, double-blind L-Arg3 ¥ 3 g/d; 4 wk, 4-wk P-L-arginine ≠; no effect on vascular 19
crossover, placebo-controlled washout function or inflammation markers

Note: M = male; F = female; y = years; d = days; wk = weeks; mo = months; P = plasma; CCL = creatinine clearance; ≠ = increased, improved; Ø = decreased.
diets in cardiovascular diseases
735
1382_C43.fm Page 736 Tuesday, October 7, 2003 7:43 PM

736 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

There is evidence demonstrating that local concentrations in the vicinity of endothelial


NOS may be lower than expected based on in vitro studies, and that L-arginine may thus
be preferentially utilized by NOS.34
Another possible explanation underlying the beneficial effect of L-arginine may be the
presence of endogenous NOS inhibitors. Elevated concentrations of endogenous asym-
metric dimethyl arginine (ADMA) have been shown in patients with vascular diseases,35
thus resulting in diminished NOS activity and therefore impaired vasodilation. Supple-
mental L-arginine may be able to overcome the effects of ADMA competitively.
Yet another possible mechanism by which L-arginine exerts its vasodilatory action is
via its metabolite agmatine. Agmatine is a ligand at central a2- and imidazoline receptors,36
where it induces clonidine-like effects,37 i.e., lowers peripheral sympathetic tone and
therefore lowers blood pressure and induces vasodilation.
Endocrine mechanisms have also been suggested to underlie the beneficial cardiovas-
cular effects of L-arginine. The amino acid stimulates growth hormone secretion, as well
as the release of insulin and glucagon.38–40 These hormones can induce vasodilation by
mechanisms that remain to be established. However, at least in the case of growth hor-
mone, the effect seems to be mediated via NO.41
Antioxidant effects of L-arginine may also play a role. In cholesterol-fed rabbits, super-
oxide radical release in isolated aortic rings has been diminished after L-arginine supple-
mentation.42 In addition, following high-dose administration, unspecific effects cannot be
ruled out.

43.8 Discussion
Data on the effects of oral L-arginine supplementation in cardiovascular diseases in
humans are limited. Only one published epidemiological study has evaluated the associ-
ation between dietary L-arginine intake and the risk of coronary heart disease mortality.43
The findings of the study did not support the hypothesis that L-arginine lowers the risk.
The clinical and experimental studies referred to in this chapter demonstrate that
L-arginine has elicited moderate beneficial effects in certain conditions, such as hypercho-
lesterolemia or coronary artery disease, but not in others.
Some questions arise concerning the methodology used in some studies assessing the
role of L-arginine in cardiovascular diseases. Most of the human studies available at present
have been small and did not consistently measure clinical end points, such as improvement
in symptoms. The results of improved endothelium-dependent vasodilation are, in turn,
not easily clinically applicable. Forearm blood flow is a widely used method of measuring
the effects on vascular function in humans (for a review see West44). It is a relatively easily
determinable parameter, but its relevance remains questionable, since when it comes to
the regulation of the systemic circulation, the forearm blood flow is of fairly limited
importance.
The optimal dosage regimen for each disease state is yet to be established. Most
published studies have measured changes in plasma L-arginine concentrations following
the supplementation. Unfortunately, this is not the case in every study. L-arginine concen-
trations need to be measured routinely, since effects may only be observed in patients
deficient in L-arginine. The studies referred to above have examined the effect of oral
L-arginine supplementation using doses ranging from 3 to 21 g/day. The average diet
provides about 5 g of L-arginine, mostly from red meat, fish, poultry, cereals, and milk
products.5 Therefore, vegetarians may be at higher risk of developing L-arginine deficiency
and have a greater need for L-arginine supplementation.
Most of the studies performed so far have been relatively short-term experiments, the
duration of intervention ranging from 3 days to 6 weeks. Thus, the beneficial effects
1382_C43.fm Page 737 Tuesday, October 7, 2003 7:43 PM

Chapter forty-three: L-arginine-enriched diets in cardiovascular diseases 737

observed may only be valid for a limited period of time. Whether the beneficial effects
persist remains to be evaluated in longer-term settings. However, the study by Lerman
et al.,23 which investigated the effect of oral L-arginine supplementation in patients with
recurrent chest pain episodes, observed a beneficial effect after relatively low dose of
supplemental L-arginine (3 g/day) administered over a fairly long period (6 months).
Nevertheless, even longer-term data are needed to clarify the persistency of the effect.
In conclusion, L-arginine may play a positive role in the prevention of cardiovascular
diseases. However, evidence of the beneficial effects of L-arginine supplementation in
humans is insufficient. Further studies are required to evaluate the role of L-arginine in
the treatment of cardiovascular diseases, especially as an adjuvant therapy.

References
1. Furchgott, R.F. and Zawadski, J.V., The obligatory role of endothelial cells in the relaxation
of arterial smooth muscle by acetylcholine, Nature, 288, 373, 1980.
2. Ignarro, L.J. et al., Endothelium-derived relaxing factor produced and released from artery
and vein is nitric oxide, Proc. Acad. Natl. Sci. U.S.A., 84, 9265, 1987.
3. Palmer, R.M. et al., Nitric oxide release accounts for the biological activity of endothelium-
derived relaxing factor, Nature, 327, 524, 1987.
4. Cooke, J.P. and Tsao, P.S., Is NO an endogenous antiatherogenic molecule? Arterioscler.
Thromb., 14, 653, 1994.
5. Niittynen, L. et al., Role of arginine, taurine and homocysteine in cardiovascular diseases,
Ann. Med., 31, 318, 1999.
6. Tentolouris, C. et al., L- arginine in coronary atherosclerosis, Int. J. Cardiol., 75, 123, 2000.
7. Brown, A.A. and Hu, F.B., Dietary modulation of endothelial function: implications for
cardiovascular disease, Am. J. Clin. Nutr., 73, 673, 2001.
8. Cheng, J.W.M. and Balwin, S.N., L- arginine in the management of cardiovascular diseases,
Ann. Pharmacother., 35, 755, 2001.
9. Preli, R.B. et al., Vascular effects of dietary L- arginine supplementation, Atherosclerosis, 162,
1, 2002.
10. Nakaki, T. and Kato, R., Beneficial circulatory effect of L- arginine, Jpn. J. Pharmacol., 66, 167,
1994.
11. Bode-Boger, S.M. et al., Elevated L- arginine/dimethylarginine ratio contributes to enhanced
systemic NO production by dietary L- arginine in hypercholesterolemic rabbits, Biochem.
Biophys. Res. Commun., 19, 598, 1996.
12. Bredt, D.S. and Snyder, S.H., Isolation of nitric oxide synthetase, a calmodulin-requiring
system, Proc. Natl. Acad. Sci. U.S.A., 87, 682, 1990.
13. Jeserich, M. et al., Reduced plasma L- arginine in hypercholesterolaemia, Lancet, 339, 561, 1992.
14. Adams, M.R. et al., Oral L- arginine improves endothelium-dependent dilatation and reduces
monocyte adhesion to endothelial cells in young men with coronary artery disease, Athero-
sclerosis, 129, 261, 1997.
15. Clarkson, P. et al., Oral L- arginine improves endothelium-dependent dilation in hyper-
cholesterolemic young adults, J. Clin. Invest., 97, 1989, 1996.
16. Wolf, A. et al., Dietary L- arginine supplementation normalizes platelet aggregation in hyper-
cholesterolemic humans, J. Am. Coll. Cardiol., 29, 479, 1997.
17. Mullen, M.J. et al., Atorvastatin but not L- arginine improves endothelial function in type I
diabetes mellitus: a double-blind study, J. Am. Coll. Cardiol., 36, 410, 2000.
18. Pezza, V. et al., Study of supplemental oral L- arginine in hypertensives treated with enalapril
+ hydrochlorothiazide, Am. J. Hypertens., 11, 1267, 1998.
19. Blum, A. et al., Effects of oral L- arginine on endothelium-dependent vasodilation and markers
of inflammation in healthy postmenopausal women, J. Am. Coll. Cardiol., 35, 271, 2000.
20. Nakai, K. et al., Concentration of soluble vascular cell adhesion molecule-1 (VCAM-1) cor-
related with expression of VCAM-1 mRNA in the human atherosclerotic aorta, Coron. Artery
Dis., 6, 497, 1995.
1382_C43.fm Page 738 Tuesday, October 7, 2003 7:43 PM

738 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

21. Haught, W.H. et al., Alterations in circulating intercellular adhesion molecule-1 and L-selec-
tin: further evidence for chronic inflammation in ischemic heart disease, Am. Heart J., 132,
1, 1996.
22. Ceremuzynski, L., Chamiec, T., and Herbaczynska-Cedro, K., Effect of supplemental oral
L-arginine on exercise capacity in patients with stable angina pectoris, Am. J. Cardiol., 80,
331, 1997.
23. Lerman, A. et al., Long-term L- arginine supplementation improves small-vessel coronary
endothelial function in humans, Circulation, 97, 2123, 1998.
24. Blum, A. et al., Oral L- arginine in patients with coronary artery disease on medical manage-
ment, Circulation, 101, 2160, 2000.
25. Maxwell, A.J. et al., Randomised trial of a medical food for the dietary management of
chronic stable angina, J. Am. Coll. Cardiol., 39, 37, 2002.
26. Maxwell, A. et al., Nutritional therapy for peripheral arterial disease: a double-blind, placebo-
controlled, randomised trial of HeartBar“, Vasc. Med., 5, 11, 2000.
27. Kubo, S.H. et al., Endothelium-dependent vasodilation is attenuated in patients with heart
failure, Circulation, 84, 1589, 1991.
28. Drexler, H. et al., Endothelial function in chronic congestive heart failure, Am. J. Cardiol., 69,
1596, 1992.
29. Katz, S.D. et al., Impaired endothelium-mediated vasodilation in the peripheral vasculature
of patients with congestive heart failure, J. Am. Coll. Cardiol., 19, 918, 1992.
30. Nakamura, M. et al., Attenuated endothelium-dependent peripheral vasodilation and clinical
characteristics in patients with chronic heart failure, Am. Heart J., 128, 1164, 1994.
31. Rector, T.S. et al., Randomised, double-blind, placebo-controlled study of supplemental oral
L- arginine in patients with heart failure, Circulation, 93, 2135, 1996.
32. Siani, A. et al., Blood pressure and metabolic changes during dietary L- arginine supplemen-
tation in humans, Am. J. Hypertens., 13, 547, 2000.
33. Boger, R.H. and Bode-Boger, S.M., The clinical pharmacology of L- arginine, Annu. Rev. Phar-
macol. Toxicol., 41, 79, 2001.
34. McDonald, K.K. et al., A caveolar complex between the cationic amino acid transporter 1
and endothelial nitric-oxide synthase may explain the “arginine paradox,” J. Biol. Chem., 272,
31213, 1997.
35. Vallance, P. et al., Accumulation of an endogenous inhibitor of NO synthesis in chronic renal
failure, Lancet, 339, 572, 1992.
36. Li, G. et al., Agmatine: an endogenous clonidine-displacing substance in the brain, Science,
263, 966, 1994.
37. Sun, M.K. et al., Cardiovascular responses to agmatine, a clonidine-displacing substance, in
anaesthetized rat, Clin. Exp. Hypertens., 17, 115, 1995.
38. Merimee, T.J. et al., Plasma growth hormone after arginine injection, N. Engl. J. Med., 276,
434, 1967.
39. Schmidt, H.H.H.W. et al., Insulin secretion from pancreatic B cells caused by L- arginine-
derived nitrogen oxides, Science, 255, 721, 1992.
40. Gerich, J.E. et al., Inhibition of pancreatic glucagon responses to arginine by somatostatin in
normal man and in insulin-dependent diabetics, Diabetes, 23, 876, 1974.
41. Bode-Boger, S.M. et al., L- arginine stimulates NO-dependent vasodilation in humans: effect
of somatostatin pretreatment, J. Invest. Med., 47, 43, 1999.
42. Bode-Boger, R.H. et al., Supplementation of hypercholesterolaemic rabbits with L- arginine
reduces the vascular release of superoxide anions and restores NO production, Atherosclerosis,
117, 273, 1995.
43. Oomen, C. et al., Arginine intake and risk of coronary heart disease mortality in elderly men,
Arterioscler. Thromb. Vasc. Biol., 20, 2134, 2000.
44. West, S.G., Effect of diet on vascular reactivity: an emerging marker for vascular risk, Curr.
Atheroscler. Rep., 3, 446, 2001.
1382_C44.fm Page 739 Tuesday, October 7, 2003 7:45 PM

chapter forty-four

Taurine homeostasis and its


importance for physiological
functions
Svend Høime Hansen
Rigshospitalet, Copenhagen University Hospital

Contents
44.1 Taurine: Background.........................................................................................................739
44.1.1 Physiological functions of taurine ....................................................................740
44.1.2 Physical–chemical properties.............................................................................740
44.1.3 Taurine transporter..............................................................................................740
44.1.4 Osmolyte ...............................................................................................................740
44.2 Taurine homeostasis..........................................................................................................741
44.2.1 Taurine metabolism: biosynthesis, degradation, and excretion...................741
44.2.2 Conjugation reactions with taurine ..................................................................741
44.2.3 Taurine and nutrition..........................................................................................742
44.3 Animal models ...................................................................................................................742
44.3.1 The cat ...................................................................................................................742
44.3.2 The C57BL/6 mouse strain ................................................................................743
44.4 Taurine supplementation as treatment...........................................................................743
44.4.1 Taurine and cardiomyopathy ............................................................................743
44.4.2 Diabetes mellitus and taurine............................................................................744
44.4.3 Taurine and pancreatic islets .............................................................................744
Acknowledgments ......................................................................................................................744
References .....................................................................................................................................744

44.1 Taurine: Background


When performing amino acid analysis on biological samples, results are normally obtained
not only for the well-known alpha-amino acids, but also for all other compounds with
free amino groups. One of these compounds is taurine, 2-amino-ethane sulfonic acid. Due
to the sulfonic acid group, taurine cannot form peptide bonds and thus cannot be part of
proteins. Nevertheless, taurine seems to be a very important compound as it is found as

0-8493-1382-1/04/$0.00+$1.50
© 2004 by CRC Press LLC 739
1382_C44.fm Page 740 Tuesday, October 7, 2003 7:45 PM

740 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Table 44.1 The Most Important Physiological Roles of Taurine

Conjugation with organic acids in the liver, e.g., formation of bile acids
Osmolyte in all cells and organs, including heart and kidney
Involved in ion transport, e.g., Na+, Ca2+, Cl–
Myeloperoxidase system in neutrophils
Growth and fetal development
Involvement in the photoreceptor system in the retina

a free compound intracellularly in all animal cells and normally in millimolar concentra-
tions, i.e., in tissue often in the range of 5 to 30 mmol/g. Taurine is not found in any plants
except some algae; it could thus be reasonable to assume that taurine has formed an
important role in the evolutionary difference between plants and animals. However, such
hypotheses are difficult to verify, as the importance of taurine for cellular and physiological
functions in animals is not fully clear yet.

44.1.1 Physiological functions of taurine


Although ubiquitously found in animal cells, the general biochemical knowledge and
awareness of taurine seem to be very low, as it is hardly mentioned in most biochemical
and physiological textbooks. Often taurine is only mentioned as the end point in sulfur
metabolism and is connected to cholesterol metabolism and excretion due to the formation
of the bile acid taurocholic acid. As summarized in Table 44.1, taurine is very important
for correct physiological action in several organs and cell types. This chapter presents
information on the important actions of taurine with regard to mammalian and human
nutrition and health. Further information on taurine can be found in a number of
reviews1–16 and several monographs with conference proceedings.17,18

44.1.2 Physical–chemical properties


The sulfonic acid group in taurine has a very low pKa value at about 2, whereas the pKa
value for the amino group is about 9. Consequently, the sulfonic acid group is negatively
charged and the amino group predominantly positively charged at all physiological pH
values. As a consequence, taurine is found as a zwitterion, making it impossible for the
taurine to pass cellular membranes without being actively transported. However, it should
be noted that due to the zwitterionic nature, transport of taurine does not represent any
transfer of ionic charge.

44.1.3 Taurine transporter


The high intracellular concentrations in the millimolar range compared to 10- to 100-fold
lower extracellular concentrations demonstrate a very active transporter system. A taurine
transporter protein and its function have been characterized in several different human
tissues.19–21 A recent gene knockout study in mice has demonstrated the importance of the
taurine transporter, as the mice were blind due to development of a retinopathy similar
to retinitis pigmentosa.22

44.1.4 Osmolyte
On of the most important physiological functions of taurine is as intracellular osmolyte,
i.e., a low-molecular-mass compound whose primary task is to maintain osmotic equilib-
rium across cellular membranes and thus regulate the cell volume.7,9,23
1382_C44.fm Page 741 Tuesday, October 7, 2003 7:45 PM

Chapter forty-four: Taurine homeostasis and its importance for physiological functions 741

The cell physiological function as osmolyte is easily demonstrated by reducing the


osmolarity in a cell culture medium; the osmolytes are among the first compounds released
in considerable amounts as observed in neurons and astrocytes, for example.23,24 Com-
pounds like taurine, betaine, or myo-inositol are ideal osmolytes as the compounds do not
carry any total ion charge and do not participate in any energy-producing metabolic
processes. The importance of osmolytes has recently been demonstrated in endothelial
cells, as these cells are capable of accumulating the osmolytes intracellularly and thus
preventing apoptosis when subjected to hypertonic growth conditions.25

44.2 Taurine homeostasis


44.2.1 Taurine metabolism: biosynthesis, degradation, and excretion
Taurine is considered an end product for the sulfur metabolism in animal cells. The
primary biosynthetic pathway of taurine is from cysteine by oxidation to cysteine sulfinic
acid with a subsequent decarboxylation to hypotaurine and final oxidation to taurine.
Cysteine sulfinic acid decarboxylase is considered the rate-limiting enzyme in the biosyn-
thesis of taurine.26 The biosynthesis occurs mainly in the liver.27 However, biosynthesis
can also be observed in epithelial cells like glia cells, but not in cells like endothelial cells25
or neurons.28 The biosynthetic capacity of taurine depends strongly on species, with a high
capacity in rodents like the rat and the mouse,29,30 whereas it is limited in man,31 especially
babies,29 and almost absent in the cat (see Section 44.3.1).
The myeloperoxidase system in neutrophils is capable of performing the conversion
of taurine into the long-lived N-chlorotaurine that will decompose to sulfoacetaldehyde,
which, most likely, is reduced to 2-hydroxy-ethanesulfonate subsequently.32 Alternately,
several bacteria, including some intestinal bacteria, can degrade taurine. The structure of
the enzyme responsible for conversion of taurine to sulfite and aminoacetaldehyde in
Escherichia coli has recently been elucidated by application of X-ray crystallography.33
Taurine metabolism has been studied in man using 35S-labeled taurine.31 It was
shown that taurine is predominantly excreted in the urine as taurine (about 70%) or due
to bacterial degradation in the intestine as sulfate (about 25%). A minor part (less than
5%) was detected as 2-hydroxy-ethanesulfonate due to myeloperoxidase-induced
degradation.

44.2.2 Conjugation reactions with taurine


From biochemical textbooks it is well known that bile acids are formed by conjugation of
cholic acid and either taurine or glycine. The single enzyme bile acid–CoA:amino acid
N-acyltransferase (hBAT) is responsible for these conjugation reactions.34 Taurine conju-
gation should probably be considered a more general hepatic scavenging reaction for
carboxylic compounds, as taurine adducts have been characterized with drugs,35 for exam-
ple, and prostaglandine-like products.36
In addition to the enzyme-catalyzed conjugation with carboxylic groups, taurine has
been shown to form Schiff bases with aldehydes and ketones.37 The amino group in taurine
has been reported to be more reactive, forming imines, than the amino group in other
amino acids. Consequently, taurine could be part of the intracellular protection toward
formation of cross-linking of proteins due to glycation of proteins. Such glycation processes
are considered to be part of general aging reactions, although they occur much more
rapidly in diabetic patients.
1382_C44.fm Page 742 Tuesday, October 7, 2003 7:45 PM

742 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

Nutritional
sources: Taurine Other sulfur amino acids
Taurine
absorption Biosynthesis

Taurine body pool


Myeloperioxidase Cholic acid conjugation

N-chloro-taurine Bile acids


(neutrophils) (taurine conjugated)

Urinary excretion- Excretion - primarily as Bacterial degradation


primarily as taurine hydroxy-ethanesulfonate and fecal excretion -
primarily as sulfate

Figure 44.1 Taurine homeostasis: The taurine body pool can only be supplemented through nutri-
tional sources of either taurine or sulfur-containing amino acids. Excretion of taurine occurs either
through the urine, through degradation of N-chloro-taurine formed in the neutrophils, or through
taurine-conjugated bile acids, which are subsequently either reabsorbed or bacterially degraded in
the intestine.

44.2.3 Taurine and nutrition


Taurine can obviously only be supplied to the body through a nutritional source, for
example, by food with a high content of taurine, like fish, meat, or breast milk for babies.
Taurine is found in high concentrations in the millimolar range in seafood and animal
meat. Alternatively, taurine can be produced from methionine or cysteine in accordance
with the biosynthetic pathway for taurine.
A recent epidemiological study uses urinary taurine excretion as a marker of intake
of seafood38 in populations from several different countries. The study demonstrates a
strong inverse correlation between levels of urinary taurine excretion and ischemic heart
mortality. When evaluating the results of such a study, either intake of seafood or taurine
itself is responsible for beneficial results demonstrated by the strong inverse correlation.
Based on the information presented above, a model for taurine homeostasis of the
taurine body pool can easily be summarized as shown in Figure 44.1.

44.3 Animal models


Although taurine is found in all animal cells, defects in taurine biosynthesis or homeostasis
have been found in a few types of animals. The cat lacks one of the biosynthetic enzymes,
cysteine sulfinic acid decarboxylase, and the mouse strain C57BL/6 has a defect in the
renal reabsorption of taurine. In addition to these two animals, presented below, several
reports exist in the veterinarian literature on taurine deficiency in dogs and the fox, with
the main focus on development of myocardial problems due to the taurine deficiency.
Such pieces of information are even included in standard veterinarian textbooks on small
animals.39

44.3.1 The cat


The cat represents the best characterized model for defects in taurine homeostasis and its
physiological consequences. In the mid-1970s it was observed that taurine deficiency in
cats leads to a retinal degeneration (feline central retinal degeneration (FCRD)).40,41 In
1382_C44.fm Page 743 Tuesday, October 7, 2003 7:45 PM

Chapter forty-four: Taurine homeostasis and its importance for physiological functions 743

addition, several other physiological problems develop in the taurine-deficient cat, e.g.,
dilated cardiomyopathy,42 reproductive failure and growth retardation,43,44 platelet aggre-
gation,45 dysfunction of the central nervous system, and impaired immune function.46
Studies of the taurine metabolism in the cat revealed that the taurine biosynthesis is very
limited or can be considered absent.3,46–49 Instead, the cat depends solely on taurine from
a nutritional source, i.e., raw meat or fish. In commercially available cat feed, taurine is
added to obtain a content of about 0.05%.

44.3.2 The C57BL/6 mouse strain


Back in 1950 a study was performed comparing the excretion of taurine from different
mouse strains. It was found that the C57BL/6 mouse strain had a much higher excretion
of taurine than any of the other mouse strains studied.50 Thirty years later, in the 1970s
and 1980s, the increased taurine excretion was ascribed to a defect in the renal reabsorption
of taurine.51,52 In humans, taurine depletion has been observed in infants with very low
birth weight and thus poorly developed renal function,53 and in patients with renal failure,
low plasma and muscle tissue levels of taurine have been reported.54,55 However, as excess
taurine is cleared by urinary excretion, patients with renal failure risk accumulating taurine
in case of intake of large taurine amounts.56
Several studies on cholesterol metabolism have been performed in different mouse
strains fed a high-cholesterol diet. Compared with other mouse strains, the C57BL/6 strain
has been shown to be very susceptible to accumulation of cholesterol in the vascular
system, and thus development of a condition comparable to atherosclerosis. Studies have
shown that addition of taurine to drinking water can improve the cholesterol metabolism
for C57BL/6 animals given a high-fat diet.57,58 Prevention of the development of athero-
sclerotic lesions was observed in C57BL/6 mice as well as in apolipoprotein-E-deficient
mice.58 Although not mentioned in the paper, the apolipoprotein-E-deficient mice (received
from Jackson Laboratory) are most likely based on the C57BL/6 strain. In general, this
mouse strain is by far the most used when performing gene knockouts in mice, e.g., almost
all mouse studies with relation to diabetes or lipid metabolism have been performed in
C57BL/6 mice or strains very closely related.
Although taurine is very closely related to cholesterol metabolism due to the formation
of taurocholate, no experimental studies on the C57BL/6 mouse strain (or knockouts) have
been performed with the aim of elucidating the possible relation between the defect in
taurine reabsorption and enhanced cholesterol accumulation with subsequent develop-
ment of atherosclerosis. Actually, a number of the clinical observations related to athero-
sclerosis can be linked to taurine depletion.15

44.4 Taurine supplementation as treatment


44.4.1 Taurine and cardiomyopathy
As mentioned above, the physiological function of taurine in the heart is not fully under-
stood (for reviews, see Chapman et al.11 and Militante and Lombardini16). However, taurine
deficiency or depletion has been reported in cats and dogs as a possible cause for cardio-
myopathy.39,42,59 As already mentioned, an epidemiological study has shown a strong
inverse correlation between levels of urinary taurine excretion and ischemic heart mor-
tality,38 and several clinical studies have reported successful results using taurine as treat-
ment for myocardial problems60–62 or in association with cardiac bypass surgery.63,64
1382_C44.fm Page 744 Tuesday, October 7, 2003 7:45 PM

744 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

44.4.2 Diabetes mellitus and taurine


The high plasma glucose concentrations in diabetic patients represent an obvious challenge
for the osmoregulation systems in the body.65 The immediate involvement of taurine as
an osmolyte has been demonstrated in rat models using streptozotocin to induce diabetes.
In a 48-h time study,66 as well as in a longer 55-day study,67 taurine was redistributed in
the organs with increased taurine concentrations in the heart, whereas taurine was
depleted from several other organs, like the kidney.
To interpret these results, it is necessary to introduce the aldose reductase system —
also known as the polyol pathway — which allows some cell types to convert intracellular
glucose to intracellular sorbitol: sorbitol cannot be transported across cellular membranes
before it is converted to fructose; however, this oxidation process is rather slow. Conse-
quently, sorbitol-producing cells will accumulate sorbitol when exposed to high levels of
glucose and are only capable of performing rapid osmoregulation by releasing transport-
able osmolytes like taurine, betaine, or myo-inositol. In vitro studies have demonstrated
that taurine is gradually depleted in sorbitol-producing cells and that aldose reductase
inhibitors can prevent the depletion.68 The polyol pathway with its subsequent osmolyte
and taurine depletion can be linked to the development of diabetic late complications, as
many of the tissues affected by the clinical manifestations can be characterized as having
high concentrations of taurine in combination with cells with an active aldose reductase
system. A more thorough presentation of the hypothesis that taurine depletion could be
causal for diabetic late complications can be found in a recent review.15
A clinical study with taurine supplementation for diabetics has been performed,69
reporting normalization of platelet hyperaggregation, whereas other clinical manifesta-
tions of diabetic complications were not monitored. Perhaps the unsuccessful treatments
in man with aldose reductase inhibitor should be combined with taurine supplementation
due to the low biosynthetic capacity of taurine in man.15

44.4.3 Taurine and pancreatic islets


Several recent papers have presented results showing that taurine is very important for
the fetal and early postnatal development of islets in order to obtain normal insulin
secretion from the islets.70,71 The importance of taurine in the early postnatal life is empha-
sized by the very high concentrations of taurine in breast milk,10,72–74 reflecting that the
enzymes for taurine biosynthesis are not fully developed at the time of birth.29 These
observations could be linked to the intrauterine environment hypothesis on the importance
of the prenatal development for development of non-insulin-dependent diabetes mellitus
(NIDDM) later in life.75,76
Furthermore, recent studies have demonstrated very high taurine localized concen-
trations in pancreatic islets, as taurine is found in high concentrations in alpha-cells and
considerably lower concentrations in beta-cells.77,78

Acknowledgments
The review presented here is part of a research program on taurine supported by the
Danish Ministry of Health through the VIFAB program.

References
1. Jacobsen, J.G. and Smith, L.H., Biochemistry and physiology of taurine and taurine deriva-
tives, Physiol. Rev., 48, 424, 1968.
1382_C44.fm Page 745 Tuesday, October 7, 2003 7:45 PM

Chapter forty-four: Taurine homeostasis and its importance for physiological functions 745

2. Sturman, J.A., Minireview: taurine in development, Life Sci., 21, 1, 1977.


3. Hayes, K.C. and Sturman, J.A., Taurine in metabolism, Annu. Rev. Nutr., 1, 401, 1981.
4. Hayes, K.C., Taurine requirement in primates, Nutr. Rev., 43, 65, 1985.
5. Wright, C.E. et al., Taurine: biological update, Annu. Rev. Biochem., 55, 427, 1986.
6. Gaull, G.E., Taurine in pediatric nutrition: review and update, Pediatrics, 83, 433, 1989.
7. Huxtable, R.J., Taurine in the central nervous system and the mammalian actions of taurine,
Prog. Neurobiol., 32, 471, 1989.
8. Kendler, B.S., Taurine: an overview of its role in preventive medicine, Prev. Med., 18, 79, 1989.
9. Huxtable, R.J., Physiological actions of taurine, Physiol. Rev., 72, 101, 1992.
10. Sturman, J.A., Taurine in development, Physiol. Rev., 73, 119, 1993.
11. Chapman, R.A., Suleiman, M.-S., and Earm, Y.E., Taurine and the heart, Cardiovasc. Res., 27,
358, 1993.
12. O’Flaherty, L. et al., Intestinal taurine transport: a review, Eur. J. Clin. Invest., 27, 873, 1997.
13. Stapleton, P.P. et al., Taurine and human nutrition, Clin. Nutr., 16, 103, 1997.
14. Stapleton, P.P. et al., Host defense: a role for the amino acid taurine? J. Parenter. Enteral Nutr.,
22, 42, 1998.
15. Hansen, S.H., The role of taurine in diabetes and the development of diabetic complications,
Diabetes Metab. Res. Rev., 17, 330, 2001.
16. Militante, J.D. and Lombardini, J.B., Increased cardiac levels of taurine in cardiomyopathy:
the paradoxical benefits of oral taurine treatment, Nutr. Res., 21, 93, 2001.
17. Lombardini, J.B., Schaffer, S.W., and Azuma, J., Eds., Taurine: nutritional value and mecha-
nisms of action, Adv. Exp. Med. Biol., 315, 1992.
18. Huxtable, R.J. and Michalk, D., Eds., Taurine in health and disease, Adv. Exp. Med. Biol., 359,
1994.
19. Jhiang, S.M. et al., Cloning of the human taurine transporter and characterization of taurine
uptake in thyroid cells, FEBS Lett., 318, 139, 1993.
20. Ramamoorthy, S. et al., Functional characterization and chromosomal localization of a cloned
taurine transporter from human placenta, Biochem. J., 300, 893, 1994.
21. Miyamoto, Y., Liou, G.I., and Sprinkle, T.J., Isolation of a cDNA encoding a taurine trans-
porter in the human retinal pigment epithelium, Curr. Eye Res., 15, 345, 1996.
22. Heller-Stilb, B. et al., Disruption of the taurine transporter gene (taut) leads to retinal degen-
eration in mice, FASEB J., 16, 231, 2002 (full paper can be found at http://www.fasebj.org).
23. Pasentes-Morales, H. and Schousboe, A., Role of taurine in osmoregulation in brain cells:
mechanisms and functional implications, Amino Acids, 12, 281, 1997.
24. Olson, J.E., Osmolyte contents of cultured astrocytes grown in hypoosmotic medium, Bio-
chim. Biophys. Acta., 1453, 175, 1999.
25. Alfieri, R.R. et al., Compatible osmolytes modulate the response of porcine endothelial cells
to hypertonicity and protect them from apoptosis, J. Physiol., 540, 499, 2002.
26. De La Rosa, J. and Stipanuk, M.H., Evidence for a rate-limiting role of cysteinesulfinate
decarboxylase activity in taurine biosynthesis in vivo, Comp. Biochem. Physiol. B, 81B, 565,
1985.
27. Spaeth, D.G. and Schneider, D.L., Taurine synthesis, concentration, and bile salt conjugation
in rat, guinea pig, and rabbit, Proc. Soc. Exp. Biol. Med., 147, 855, 1974.
28. Brand, A. et al., Metabolism of cysteine in astroglial cells: synthesis of hypotaurine and
taurine, J. Neurochem., 71, 827, 1998.
29. Gaull, G.E. et al., Milk protein quantity and quality in low-birth-weight infants, J. Pediatr.,
90, 348, 1977.
30. Huxtable, R.J. and Lippincott, S.E., Diet and biosynthesis as sources of taurine in the mouse,
J. Nutr., 112, 1003, 1982.
31. Sturman, J.A. et al., Metabolism of [35S] in man, J. Nutr., 105, 1206, 1975.
32. Cunningham, C., Tipton, K.F., and Dixon, H.B.F., Conversion of taurine into N-chlorotaurine
and sulphoacetaldehyde in response to oxidative stress, Biochem. J., 330, 939, 1998.
33. Elkins, J.M. et al., X-ray crystal structure of Escherichia coli taurine/a-ketoglutarate dioxy-
genase complexed to ferrous iron and substrates, Biochemistry, 41, 5185, 2002.
1382_C44.fm Page 746 Tuesday, October 7, 2003 7:45 PM

746 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

34. Falany, C.N. et al., Glycine and taurine conjugation of bile acids by a single enzyme, J. Biol.
Chem., 269, 19375, 1994.
35. Boberg, M. et al., Biotransformation of cerivastatin in mice, rats, and dogs in vivo, Drug
Metab. Dispos., 26, 640, 1998.
36. Chiabrando, C. et al., Identification of metabolites from type III F2-isoprostane diastereoi-
somers by mass spectrometry, J. Lipid Res., 43, 495, 2002.
37. Ogasawara, M. et al., Reactivity of taurine with aldehydes and its physiological role, Chem.
Pharm. Bull., 41, 2172, 1993.
38. Yamori, Y. et al., Distribution of twenty-four hour urinary taurine excretion and association
with ischemic heart disease mortality in 24 populations of 16 countries: results from the
WHO-CARDIAC study, Hypertens. Res., 24, 453, 2001.
39. Ettinger, S.J. and Feldman, E.C., Eds., Textbook of Veterinary Internal Medicine: Diseases of the
Dog and Cat, 5th Ed., W.B. Saunders Company, Philadelphia, 2000.
40. Hayes, K.C., Carey, R.E., and Schmidt, S.Y., Retinal degeneration associated with taurine
deficiency in the cat, Science, 188, 949, 1975.
41. Barnett, K.C. and Burger, I.H., Taurine deficiency retinopathy in the cat, J. Small Anim. Pract.,
21, 521, 1980.
42. Pion, P.D. et al., Myocardial failure in cats associated with low plasma taurine: a reversible
cardiomyopathy, Science, 237, 764, 1987.
43. Sturman, J.A. et al., Feline maternal taurine deficiency: effect on mother and offspring,
J. Nutr., 116, 655, 1986.
44. Sturman J.A. and Messing, J.M., Dietary taurine content and feline reproduction and out-
come, J. Nutr., 121, 1195, 1991.
45. Welles, E.G., Boudreaux, M.K., and Tyler, J.W., Platelet, antithrombin, and fibrinolytic activ-
ities in taurine-deficient and taurine-replete cats, Am. J. Vet. Res., 54, 1235, 1993.
46. Hayes, K.C. and Trautwein, E.A., Taurine deficiency syndrome in cats, Vet. Clin. North Am.
Small Anim. Pract., 19, 403, 1989.
47. Knopf, K. et al., Taurine: an essential nutrient for the cat, J. Nutr., 108, 773, 1978.
48. Ryan, J.A., Taurine deficiency in cats, Companion Anim. Pract., 19, 28, 1989.
49. Markwell, P.J. and Earle, K.E., Taurine: an essential nutrient for the cat: a brief review of the
biochemistry of its requirement and the clinical consequences of deficiency, Nutr. Res., 15,
53, 1995.
50. Harris, H. and Searle, A.G., Urinary amino acids in mice of different genotypes, Ann.
Eugenics, 17, 165, 1953.
51. Chesney, R.W., Scriver, C.R., and Mohyuddin, F., Localization of the membrane defect in
transepithelial transport of taurine by parallel studies in vivo and in vitro in hypertaurinuric
mice, J. Clin. Invest., 57, 183, 1976.
52. Mandla, S., Scriver, C.R., and Tenenhouse, H.S., Decreased transport in renal basolateral
membrane vesicles from hypertaurinuric mice, Am. J. Physiol., 255, F88, 1988.
53. Zelikovic, I. et al., Taurine depletion in very low birth weight infants receiving prolonged
total parenteral nutrition: role of renal immaturity, J. Pediatr., 116, 301, 1990.
54. Bergström, J. et al., Sulfur amino acids in plasma and muscle in patients with chronic renal
failure: evidence for taurine depletion, J. Intern. Med., 226, 189, 1989.
55. Suliman, M.E., Anderstam, B., and Bergström, J., Evidence of taurine depletion and accu-
mulation of cysteine sulfinic acid in chronic dialysis patients, Kidney. Int., 50, 1713, 1996.
56. Suliman, M.E. et al., Accumulation of taurine in patients with renal failure, Nephrol. Dial.
Transplant., 17, 528, 2002.
57. Murakami, S., Kondo-Ohta, Y., and Tomisawa, K., Improvement in cholesterol metabolism
in mice given chronic treatment of taurine and fed a high-fat diet, Life Sci., 64, 83, 1999.
58. Murakami, S. et al., Prevention of atherosclerotic lesion development in mice by taurine,
Drugs Exp. Clin. Res. 25, 227, 1999.
59. Alroy, J. et al., Inherited infantile dilated cardiomyopathy in dogs: genetic, clinical, biochem-
ical, and morphologic findings, Am. J. Med. Gen., 95, 57, 2000.
60. Azuma, J. et al., Therapy of congestive heart failure with orally administered taurine, Clin.
Ther., 5, 398, 1983.
1382_C44.fm Page 747 Tuesday, October 7, 2003 7:45 PM

Chapter forty-four: Taurine homeostasis and its importance for physiological functions 747

61. Azuma, J. et al., Therapeutic effect of taurine in congestive heart failure: a double-blind
crossover trial, Clin. Cardiol., 8, 276, 1985.
62. Azuma, J., Sawamura, A., and Awata, N., Usefulness of taurine in chronic congestive heart
failure and its prospective application, Jpn. Circ. J., 56, 95, 1992.
63. Milei, J. et al., Reduction of reperfusion injury with preoperative intravenous infusion of
taurine during myocardial revascularization, Am. Heart J., 123, 339, 1992.
64. Suleiman, M.-S. et al., A loss of taurine and other amino acids from ventricles of patients
undergoing bypass surgery, Br. Heart J., 69, 241, 1993.
65. McManus, M.L., Churchwell, K.B., and Strange, K., Regulation of cell volume in health and
disease, N. Engl. J. Med., 333, 1260, 1995.
66. Reibel, D.K. et al., Changes in taurine content in heart and other organs of diabetic rats.
J. Mol. Cell. Cardiol., 11, 827, 1979.
67. Goodman, H.O. and Shihabi, Z.K., Supplemental taurine in diabetic rats: effects on plasma
glucose and triglycerides, Biochem. Med. Metab. Biol., 43, 1, 1990.
68. Stevens, M.J. et al., Osmotically-induced nerve taurine depletion and the compatible os-
molyte hypothesis in experimental diabetic neuropathy in the rat, Diabetologia, 36, 608, 1993.
69. Franconi, F. et al., Plasma and platelet taurine are reduced in subjects with insulin-dependent
diabetes mellitus: effects of taurine supplementation, Am. J. Clin. Nutr., 61, 1115, 1995.
70. Cherif, H. et al., Effects of taurine on the insulin secretion of rat islets from dams fed a low-
protein diet, J. Endocrinol., 159, 341, 1998.
71. Boujendar, S. et al., Taurine supplementation to a low protein diet during foetal and early
postnatal life restores a normal proliferation and apoptosis of rat pancratic islets, Diabetologia,
45, 856, 2002.
72. Rassin, D.K., Sturman, J.A., and Gaull, G.E., Taurine and other free amino acids in milk of
man and other mammals, Early Hum. Dev., 2, 1, 1978.
73. Sarwar, G. et al., Free amino acids in milks of human subjects, other primates and non-
primates, Br. J. Nutr., 79, 129, 1998.
74. Agostoni, C. et al., Free amino acid content in standard infant formulas: comparison with
human milk, J. Am. Coll. Nutr., 19, 434, 2000.
75. Hales, C.N. and Barker D.J.P., Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty
phenotype hypothesis, Diabetologia, 35, 595, 1992.
76. Barker, D.J.P., Mothers, Babies and Health in Later Life, 2nd ed., Churchill Livingstone, London,
1998.
77. Bustamante, J. et al., Taurine levels and localization in pancreatic islets, Adv. Exp. Med. Biol.,
442, 65, 1998.
78. Bustamante, J. et al., An osmotic-sensitive taurine pool is localized in rat pancreatic islet cells
containing glucagon and somatostatin, Am. J. Physiol., 281, E1275, 2001.
1382_C44.fm Page 748 Tuesday, October 7, 2003 7:45 PM
1382__Index.fm Page 749 Wednesday, October 8, 2003 2:44 PM

Index
A insulin secretion, 194, 325–327, 329–330, 332,
502
Aceto-acetate see Ketogenesis intestinal metabolism, 409–410
Acidosis, 426, 559–560, 568 ischemic heart disease, 732
ADMA, 9, 160, 736 kinetics, 597–598
Adrenaline, 203 metabolism, 138, 143, 155, 597, 730
Age metabolite of ornithine a-ketoglutarate, 641
insulin sensitivity, 359 milk content, 598
protein turnover, 484 requirements, 444, 523
splanchnic sequestration of amino acids, 391, safety, 604, 733
397, 414 supplementation in critically ill patients,
AIDS, 590, 602, 677 658–659
Agmatine, 159–160, 736 supplementation in sport practice, 502–503
Alanine supplementation, 145
cancer, 346 synthesis, 154–156
turnover, 55
cell transport, 68, 72, 73
wound healing, 599–601, 603
counter regulatory hormones (effect of), 202
Asparagine
glucose-alanine cycle see gluconeogenesis
anti-proteolytic action, 278
gluconeogenesis, 84–87, 89, 128–129, 346
cell transport, 68, 71, 72
insulin secretion, 330-331
insulin secretion, 324–325
intestinal metabolism, 408–410
muscle metabolism, 125
kidney metabolism, 425 supplementation in sport practice, 503
muscle metabolism, 125, 128–129,204 Aspartate
plasma concentration, 48, 202 cell transport, 69, 114
requirement, 523 in urea cycle, 114, 125
transamination, 130 muscle metabolism, 125
Alanylglutamine, 314, 619–623 supplementation in sport practice, 503
Albumin Autophagy see Protein breakdown
synthesis, 205–206
Ammonia
brain metabolism, 424–425 B
intestinal metabolism, 421–422
Bacterial translocation, 579, 623
kidney metabolism, 425–428
Bone marrow transplantation, 314, 587–589, 618,
liver metabolism, 422–423
623, 658
metabolism, 172, 378–380, 408
Brain
muscle metabolism, 423–424
amino acid uptake, 69, 72, 73, 562
role in fatigue, 503 ketone bodies metabolism, 103
Apoptosis, 178–179, 478 renal failure, 562
Arginase, 157–158, 596–597 Branched-chain amino acids
interaction with nitric oxide synthase, 214, 597 cancer, 346
Arginine content in parenteral solutions, 654
cancer, 345 counter regulatory hormones (effect of), 202
catabolism, 156–160 during exercise, 126–127, 129–131, 505–508
cell transport, 70, 71, 156, 596–597 insulin secretion, 322–323
essentiality, 155, 156 isoleucine intake and metabolism, 411
growth hormone secretion, 502, 600–601 metabolism, 123–126, 409, 558
hyperlipemia (effect on), 731–732 metabolism in renal failure, 552–561
hypertension (effect on), 732 protein turnover, 506
immune function, 309–311, 598–599, 658, 723 requirement, 445, 458–460, 463, 489

749
1382__Index.fm Page 750 Wednesday, October 8, 2003 2:44 PM

750 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

supplementation in liver failure, 656 NF-IL6, 215


supplementation in renal failure, 561–568, 653, peptide chain initiation process, 191–193
656 PI3-kinase, 186, 256–257, 259, 282–283, 397
surgery, 202 protein kinase C, 69, 216
transamination, 101, 124, 128 S6 protein, 255, 283, 295, 297, 397
Branched-chain keto acids Chemotherapy, 587–589, 658, 697
as precursors of ketone bodies, 101 Citrin, 114
dehydrogenase, 101, 189, 244, 346, 362 Citrulline
elderly, 397 citrulline-NO cycle, 156, 597
immunological effects, 508 Immune functions, 598
insulin secretion, 322 in urea cycle, 114
metabolites of ornithine a-ketoglutarate, intestinal diseases, 48, 146, 147, 155
641–642 intestinal metabolism, 144, 155, 409–410
muscle metabolism, 204 kidney metabolism, 144, 145
renal failure, 558–562 plasma concentration, 48
supplementation in renal failure, 562–568 renal failure, 145
Burn supplementation, 145
amino acid fluxes, 203 Cortisol
arginine-enriched diets, 599 gluconeogenesis (effect on), 88
branched-chain amino acid metabolism, 103 protein synthesis (effect on), 204–205
gluconeogenesis, 90 Creatine, 158
glutamine-enriched diets, 581–582 Critically ill patients
ornithine a-ketoglutarate administration, glutamine-enriched diet, 582–585, 618–619,
634–635 621–623
glutathione, 660
ornithine a-ketoglutarate administration, 635
C requirements, 523
Cystathionase, 474, 668
Cancer
Cysteine
amino acid metabolism, 342–347
cancer, 346, 348
arginine and glutamine supplementation,
cell transport, 68
602–603
immune cells, 314
arginine supplementation, 310–311, 348
immune function, 315
experimental models, 340–341
requirement in infants, 477
glutamine supplementation, 347, 587–589
supplementation in critically ill patients, 660
nitric oxide, 224–225
stability, 477, 676
ornithine a-ketoglutarate administration, 636
Cystine
plasma amino acid pattern, 690–696
cancer, 346
tumor protein synthesis, 344
cell transport, 73
Carbamoylphosphate synthase, 112, 140
Cytokines
Caveolae, 215, 598
action on amino acid transport, 71
Cell hydration
action on arginine metabolism, 157
amino acids involved, 254, 279–281, 292
action on ketogenesis, 106
apoptosis, 176
action on nitric oxide synthesis, 159
glucogenesis, 292
cancer, 343
glutamine-mediated swelling, 176, 178, 179, 292
modulation by cysteine and glutathione, 679
insulin (regulation by), 189
modulation by glutamine, 623, 722–723
protein breakdown, 255, 271–282
modulation by nutrition, 719
taurine-mediated swelling, 740–741
sport practice, 508
ureagenesis, 118
sulfur amino acids, 315
Cell signalling
trauma, 580
aMPK, 126, 176, 292–293, 297
AP-1, 215, 623
ERK, 186, 623
IRS, 186
D
MAPK, 187, 216, 281 Diabetes
mTOR see mTOR arginine supplementation, 732
NFkB, 176, 215, 217, 672 ketogenesis, 105–106
1382__Index.fm Page 751 Wednesday, October 8, 2003 2:44 PM

Index 751

muscle metabolism, 363 surgery for cancer, 698–699


protein turnover, 359–362, 364, 366 versus parenteral nutrition, 478–479, 585, 589,
taurine, 744 624, 653, 710, 719
Digestive diseases Exercise
glutamine-enriched diets, 586–587 branched-chain amino acid metabolism,
nitrogen absorption, 545 126–127, 129–131
Dosage gluconeogenesis, 89
3-methylhistidine, 35
arterio-venous difference, 48–50, 520
blood, 19 G
calibration, 24
GABA, 174
cerebrospinal fluid, 20
Glucagon
dansyl derivatives, 30, 32, 33
gluconeogenesis (effect on), 87
deproteinization, 19, 20
protein breakdown, 255, 279, 367
factors affecting normal values, 37, 38
Glucocorticoids
FMOC derivatives, 30–32
glutamine synthase, 141
glutamine (protein bound), 36
Gluconeogenesis
glutathione, 33
cancer (in), 342
HPLC, 29–44
hormonal control, 87–88, 203, 356
hydroxyproline, 35
pathological states (in), 90
interferences, 24–25
pathways, 84–87, 90–91
internal standard, 24
Glutamate
ion-exchange chromatography, 17–28
acidosis, 382–384
muscle biopsies, 24
cell transport, 69, 171–172
neurotransmetters, 20
dehydrogenase, 322–323, 376, 378–380, 383
ninhidrin, 18
intestinal metabolism, 143, 532
normal values, 39–41
kidney metabolism, 376, 425
OPA derivatives, 30, 31
metabolism, 72–73, 135–152, 204
orthophtalaldehyde (opa), 24
metabolite of ornithine a-ketoglutarate, 639
PITC derivatives, 30
neurotoxicity, 477
plasma amino acids, 39–41, 46–49
requirement, 143
quality control, 36
safety, 532
SBD-F derivatives, 30, 33
stability to heat, 532
sensitivity, 18, 24
Glutaminase, 384, 421, 422, 425
storage, 19, 20
Glutamine
sulfur amino acids, 671
acidosis, 376–378, 382–385
urine, 19, 34, 35
adrenaline (response to), 203
Duchenne muscular dystrophy, 590
aging, 398–399
apoptosis, 384–385
E brain metabolism, 424,616
catabolism, 173–174
Eicosanoids, 343 cell transport, 68, 71, 72, 284, 376, 615
Elderly patients cost-benefit calculations, 624
ornithine a-ketoglutarate administration, dipeptides see Alanylglutamine and
636–637 Glycylglutamine
Epinephrine, 204–205 functions, 616–617, 623
Endothelial cells gluconeogenesis, 84–87, 89
amino acid transport, 71 glutamine synthase, 171, 398–399, 423
Enteral nutrition immunological actions, 8, 177, 179–180,
amino acids versus peptides versus proteins, 312–314, 504–505, 622–623
541–546 insulin secretion, 323–324
critically ill patients, 545 interspecies differences, 616
digestive diseases, 545 intestinal metabolism, 143, 173, 406–409,
liver failure, 654–656 421–422, 532, 718
pediatric critically ill patients, 656–658 intestinal permeability, 623
peptide digestion, 538 intracellular pool, 615–616
renal failure, 654–656 kidney metabolism, 376–378, 425–428
1382__Index.fm Page 752 Wednesday, October 8, 2003 2:44 PM

752 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

liver metabolism, 173, 422–423 mucosa protein turnover, 364


lung metabolism, 428 permeability, 583, 718
metabolism in B-cells, 324
metabolism in immune cells, 173, 312
metabolism in tumor, 345, 408, 690 H
metabolism, 72–73, 115–116, 174–175
Heat shock proteins, 177
metabolite of ornithine a-ketoglutarate,
Hepatic encephalopathy
640–641
role of ammonia, 421–422, 425, 429
muscle function, 504
History, 1–2, 440–441, 530, 614–615, 634
muscle metabolism, 128–129, 407, 423–424
Histidine
pharmacokinetic, 620
cell transport, 72
plasma concentration, 48, 202, 399, 615
insulin secretion, 329
protein breakdown (effect on), 256, 278–280, 284
protein syntheis (effect on), 617 requirement, 445, 462, 463, 489
regulation of glycogenesis, 292–294 Homocysteine, 668
regulation of lipogenesis, 292–294 Home artificial nutrition, 699
requirement, 444
safety, 505, 617, 620
solubility, 617
I
sport practice (effect of supplementation), IGF-1, 309
504–505 Immune Cells
stability, 617 amino acid transport, 71
supplementation, 347, 658–659 trauma, 580
synthesis, 170–171 Immune-enhancing diets, 604, 658–659, 699
turnover, 54 Infants and neonates
Glutathione cysteine intake, 474
anti-apoptotie properties, 179 glutamine-enriched diets, 583, 586, 589–590,
anti-oxidant properties, 178 619, 657
cancer, 346 nitrogen absorption, 546
critically ill patients, 660 ornithine a-ketoglutarate administration, 637
immune functions, 315 plasma amino acid levels, 476
infants, 478 Insulin
metabolism, 174, 312, 620, 670–671, 718 amino acid mediated-secretion, 263–264, 638,
metabolism in diseases, 674 744
supplementation, 676 cell transport (effect on), 189, 356
Glycated amino acids, 485 effect on protein synthesis, 259, 262, 357–358
Glycine effect on protein turnover, 3–5, 189–195, 243
cell swelling, 279 gene transcription (effect on), 188, 357
cell transport, 69 intestine, 7–8
gluconeogenesis, 89 leucine metabolism (effect on), 190
intestinal metabolism, 410 mechanisms of action, 186–187
isonitrogenous placebo, 584 protein breakdown (effect on), 279–358
requirement, 463 Integrins, 281–283
Glycylglutamine, 314, 619–623 Interleukin-1, 308
Growth hormone nitric oxide synthesis, 214
action of, 48 Interleukin-6, 308
effect on cell transport, 69
Ischemic heart disease, 590, 732–733
immune function, 309
Isoleucine see branched-chain amino acids
muscular performance, 502
Stimulation by ornithine a-ketoglutarate, 639
Gut K
effect of arginine-enriched diet, 723
effect of glutamine-enriched parenteral Ketogenesis
nutrition, 619, 718–721 from Leucine, 101–103
effect of ornithine a-ketoglutarate in sepsis, 105,
administration, 637 in starvation, 104–105, 246
immunology, 180, 313 pathways, 98–103
1382__Index.fm Page 753 Wednesday, October 8, 2003 2:44 PM

Index 753

Ketoglutaric acid Muscle


gluconeogenesis, 86 alanine metabolism, 204
insulin secretion, 323 amino acid transport, 72, 356
intestinal metabolism, 532 branched-chain amino acid metabolism,
metabolism, 171–172, 425 101–102, 124, 363, 560–561
Ketoisocaproic acid elderly, 396–397
determination, 52 glutamate metabolism, 204
insulin secretion, 323 protein turnover, 392–396
Kidney TCA cycle, 129–131
ammoniagenesis, 376–378
gluconeogenesis, 84, 85
ornithine a-ketoglutarate, 637 N
uptake, 69, 70 N-acetylcysteine, 676, 677
N-acetylglutamate synthase, 113
NFkB see Cell signalling
L Nitric oxide
Leucine apoptosis, 223–224
diabetes, 363, 366–368 cancer, 224–225, 345
elderly, 397, 486 cell localization see caveolae
glycogenesis (effect on), 294 cGMP mediated effects, 216
insulin secretion, 194, 322–323 fibroblasts, 220
glucose metabolism, 227
liver metabolism, 299
glutathione (interaction with), 217
protein breakdown (effect on), 255–257, 278
insulin secretion, 326
protein synthesis, 359
keratinocytes, 220
requirement, 445, 458–459, 463, 489, 491
macrophages, 221, 228
Lymphocytes
metabolism, 157, 160
arginine, 310–311, 598
nervous system, 226–227
glutamine, 312–314
pharmacology, 9, 215–217
protein turnover, 205
platelet (effect on), 219
Lipopolysaccharide
sepsis, 223
nitric oxide synthesis, 214
vasculature, 217–220
Liver Nitric oxide synthase
amino acid uptake, 72 cofactors, 212–213
gluconeogenesis, 84–87, 89 gene expression, 215
hepatic amino acid metabolism in renal failure, isoforms, 159, 212, 215, 596
561–562 structure, 212
proteolysis, 282 Nitrogen balance
branched-chain amino acid metabolism, diabetes, 360–361
102–103 effect of carbohydrate, 242–244
Lysine effect of fat, 242–246
cell transport, 70, 71 effect of ketone bodies, 246
insulin secretion, 328 elderly, 485–486
Requirement, 445, 454–455, 463, 489–491 injury, 520
significance, 46, 520
sport practice, 500
M tool for requirement determination, 445–447,
Macrophages, 158, 221–222, 228, 309–311, 598 487
Mc Ardle’s disease, 131–132 Nosocomial infection, 579
Methionine
immune cells, 314
requirement, 445, 460–462, 463, 489
O
Methionine sulfoximine, 427 Obesity, 246, 652
Mitochondrial transport, see ureagenesis Ornithine
Monocytes, 221–222 cell transport, 70, 71
mTOR, 126, 176–177, 255, 258, 261–263, 282–283, in urea cycle, 114
295–297, 397 insulin secretion, 325–327, 329, 332
1382__Index.fm Page 754 Wednesday, October 8, 2003 2:44 PM

754 Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition, Second Edition

liver metabolism, 116 metabolism, 143


metabolism through OAT, 138, 143, 158 metabolite of ornithine a-ketoglutarate, 641
sport practice, 508 requirement, 143, 441, 444
supplementation, 145, 508 turnover, 55
wound healing, 600 Protein
Ornithine decarboxylase see Polyamines amino acid composition, 475, 478
Ornithine a-ketoglutarate breakdown, 254–258, 278, 397, 485
cancer, 348, 636 calpain mediated degradation, 394
effect on immunity, 637–638 digestion, 313–314, 534–536, 540
interaction between a-ketoglutarate and macro-autophagy, 277–278, 394
ornithine, 639 metabolism in acute exercise, 498–499
metabolic action, 48 metabolism in exercise training, 499–500
pharmacokinetic, 639 synthesis, 3–6, 258–266, 297–299, 397
stability, 634 turnover, 127, 284, 342, 390–392, 412
Oxidative stress, 660, 670–672 ubiquitin-proteasone mediated degradation,
394
Prolactine, 309
P metabolism in acute exercise, 498–499
metabolism in acute renal failure, 707
Parenteral nutrition metabolism in exercise training, 499–500
acute renal failure, 708–710
amino acid pattern, 477, 522
arginine content, 474 R
liver failure, 654–656
pediatric critically ill patients, 656–658 Radiation therapy, 697–698
renal failure, 654–656 Requirements
surgery for cancer, 698 acute renal failure (in), 707–708
Pharmacokinetic, 349, 447–448, 487–488, 522, 620 cancer (in), 348–349
Phenylalanine injury (in), 650–651
cancer, 347 methods of determination, 445–454
cell transport, 71
insulin secretion, 329
plasma concentration, 47, 203
S
requirement, 445, 455–458, 463, 489 Sepsis
surgery (in), 203 glutamine metabolism, 204, 284, 407–408
Phenylalanine hydroxylase, 189 glutathione metabolism, 674
Phosphoenolpyruvate carboxykinase nitric oxide, 223
gluconeogenesis, 84 nitrogen absorption, 540
insulin sensitivity, 187 ornithine a-ketoglutarate administration, 635
Polyamines Serine
cancer (in), 158, 344 cell transport, 68
immunological properties, 309 Serotonin see Tryptophan
insulin secretion, 327, 332 Short bowel syndrome
metabolite of ornithine a-ketoglutarate, 641 citrulline, 409
stimmulation by glutamine, 623 Stable isotopes
synthesis, 155 glycine, 51
Polymorphonuclear cells, 221 leucine, 51–55
response to glutamine enriched-diets, 580, methodology, 51–57
721–722 phenylalanine, 54–57
PPARa, 117 precursor pool, 52
PPARg, 195, 383 protein turnover, 51–57
Preterm infants tool for requirement determination, 448–453,
phenylalanine/tyrosine intake, 478–479 488
requirements, 314, 472 urea, 51
sulfur amino acids, 474 Starvation, 128–129, 175–176
urea cycle, 473 amino acid mediated control of proteolysis, 278
Proline glutamine metabolism, 204
cell transport, 73 Sulfur amino acids
1382__Index.fm Page 755 Wednesday, October 8, 2003 2:44 PM

Index 755

metabolism, 668–672 sodium-dependant, 64, 68, 71–72, 254, 279, 284,


functions, 672–673 292
metabolism in diseases, 673–675 Trauma
requirements, 490–491 glutamine enriched diets, 578–581
Superoxide, 222 glutamine metabolism, 407–408, 489–491
Surgery plasma amino acids, 520
arginine-enriched diets, 599 Tryptophan
glutamine-enriched diets, 585–586, 620–621 cancer, 344, 347, 694–696
immunological response, 205 fatigue, 506, 509
ornithine a-ketoglutarate administration, 636 insulin secretion, 330
requirement, 445, 460, 463
supplementation in sport practice, 509
T Tumor necrosis factor, 178, 179, 308
3-methylhistidine nitric oxide synthesis (effect on), 214
elderly, 394–395 Tyrosine
metabolism, 50 cell transport, 71
Taurine requirement, 445, 455–458, 463, 489
cell transport, 70, 740 solubility, 477
comparative biochemistry, 742–743 supplementation in sport practice, 509
elderly, 398
immune functions, 315–316, 693–694
infants, 474 U
insulin secretion, 333
intestinal metabolism, 410 Ubiquitin, 5, 358
metabolism, 741–742 Ureagenesis
metabolism in cancer, 693 interaction with fatty acids, 117
pharmacology, 508–509 interaction with glucose, 117–118
requirement, 444 pathways, 112, 157
retinal function, 479 regulation by insulin, 188
Threonine role in pH homeostasis, 115, 173, 378
requirement, 445, 458, 463, 489–490, 492
Transmethylation-transsulfuration pathway,
668–669 V
Transplantation
Valine see Branched chain amino acids
glutamine supplementation, 619
Transport, 63–78
CAT family, 70, 71, 142 W
classification of systems, 64–68
insulin mediated, 188, 356 Wound healing
intestinal, 536–540 nitric oxide, 220, 601
peptide, 536–537, 539–540 ornithine a-ketoglutarate administration,
SLC family systems, 68–73 634–635, 637–638
1382__Index.fm Page 756 Wednesday, October 8, 2003 2:44 PM

Anda mungkin juga menyukai