Hepatic Encephalopathy

CLINICAL GASTROENTEROLOGY
Series Editor
George Y. Wu
University of Connecticut Health Center, Farmington, CT, USA
For further volumes:

http://www.springer.com/series/7672
Kevin D. Mullen
Ravi K. Prakash
Editors
Hepatic Encephalopathy
Editors
Kevin D. Mullen, MD, FRCPI Ravi K. Prakash, MD, MRCP
Division of Gastroenterology Division of Gastroenterology
MetroHealth Medical Center MetroHealth Medical Center
Cleveland, OH, USA Cleveland, OH, USA
ISBN 978-1-61779-835-1 e-ISBN 978-1-61779-836-8

DOI 10.1007/978-1-61779-836-8
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We would like dedicate this book to Sheila
Sherlock who had a major interest in hepatic
encephalopathy and also inspired many
people to join the field of hepatology.
Preface
It is with great pleasure that we write this preface. Relatively few books, especially
in recent years, have been published on the topic of Hepatic Encephalopathy
(HE). Considerable changes have occurred in this field over the last few decades so
a comprehensive update is in order. We have called upon virtually all of the leaders
in this field to contribute with their colleagues chapters on HE in their specific area
of expertise.
The first introductory chapter outlines the major changes in nomenclature and
classification in the last few decades. Specifically, a new term, Covert HE, has been
introduced to replace the less satisfactory term minimal HE.
The roles of ammonia, neural inflammation, general inflammation, oxidative
stress, and endogenous benzodiazepines in the pathogenesis of HE are discussed by
the leading authorities in these areas. This is followed by chapters on the diagnosis
of overt and covert (or minimal) HE followed by EEG changes and brain imaging
seen in HE.
The treatment section features the long-standing nonabsorbable disaccharides
for the treatment of HE. Antibiotic treatment is discussed as well as the newer agent
ornithine phenylacetate. We intended to have Gerald Kircheis contribute a chapter
on l-ornithine l-aspartate treatment, but he was incapacitated by a significant medi-
cal problem which precluded his participation.
The last section of special topics features a hodgepodge of interesting topics
within the field of HE.
We hope you enjoy this new book on Hepatic Encephalopathy as much as we
did in putting it together.
Cleveland, OH, USA Kevin D. Mullen
vii
Contents
1 Introduction, Nomenclature, and Classification of Hepatic

Encephalopathy ...................................................................................... 1
Kevin D. Mullen and Ravi K. Prakash
Part I Pathogenesis
2 Role of Ammonia in the Pathogenesis of Hepatic

Encephalopathy ...................................................................................... 7
Jan Albrecht
3 Neuroinflammation in the Pathogenesis
of Hepatic Encephalopathy ................................................................... 19
Roger F. Butterworth
4 Inflammation and Hepatic Encephalopathy ........................................ 35
Shabnam S. Shabbir, Amit Singh Seyan,
and Debbie Lindsay Shawcross
5 Oxidative Stress in Hepatic Encephalopathy ...................................... 47
Arumugam R. Jayakumar and Michael D. Norenberg
6 The Role of Natural Benzodiazepines Receptor Ligands
in Hepatic Encephalopathy ................................................................... 71
E. Anthony Jones and Kevin D. Mullen
Part II Diagnosis
7 Diagnosis of Overt Hepatic Encephalopathy ....................................... 97

Karin Weissenborn
8 Diagnosis of Minimal Hepatic Encephalopathy .................................. 103
Jennifer Y. Montgomery and Jasmohan S. Bajaj
ix
x Contents
9 The Electroencephalogram in Hepatic Encephalopathy .................... 113

Piero Amodio
10 Brain Imaging in Hepatic Encephalopathy ......................................... 123
Rita Garca-Martnez and Juan Crdoba
Part III Treatment
11 Disaccharides in the Treatment of Hepatic Encephalopathy ............. 141

Praveen Sharma and Shiv Kumar Sarin
12 Antibiotic Treatment for Hepatic Encephalopathy............................. 159
13 Ornithine Phenylacetate: A Novel Strategy for the Treatment
of Hepatic Encephalopathy ................................................................... 165
Maria Jover-Cobos, Nathan A. Davies, Yalda Sharifi,
and Rajiv Jalan
Part IV Special Topics
14 Sleep Disorders and Hepatic Encephalopathy .................................... 177

Sara Montagnese
15 Hepatic Encephalopathy and Driving .................................................. 187
Matthew R. Kappus and Jasmohan S. Bajaj
16 Nutrition and Hepatic Encephalopathy ............................................... 199
Manuela Merli, Michela Giusto, and Oliviero Riggio
17 Hepatic Encephalopathy in Patients with Transjugular
Intrahepatic Portosystemic Shunt (TIPS)............................................ 211
Martin Rssle and Wulf Euringer
18 Quality of Life in Hepatic Encephalopathy ......................................... 221
Jillian Kallman Price and Zobair M. Younossi
19 Liver Transplantation and Hepatic Encephalopathy ......................... 233
Dileep K. Atluri and Kevin D. Mullen
20 Future of Hepatic Encephalopathy ...................................................... 241
Index ................................................................................................................ 245

Contributors
Jan Albrecht, PhD Department of Neurotoxicology, M. Mossakowski

Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland
Piero Amodio, MD Department of Medicine, University Hospital of Padova,
Padova, Italy
Dileep K. Atluri, MD, MRCP (UK) Department of Gastroenterology,
Metrohealth Medical Center, Cleveland, OH, USA
Jasmohan S. Bajaj, MBBS, MD, MS Department of Gastroenterology,
Hepatology and Nutrition, Virginia Commonwealth University and McGuire
VA Medical Center, Richmond, VA, USA
Roger F. Butterworth, PhD, DSc Neuroscience Research Unit,
Hpital Saint-Luc (CHUM), Montreal, QC, Canada
Juan Crdoba, PhD Liver Unit, Department of Internal Medicine,
Vall dHebron Hospital, Barcelona, Spain
Nathan A. Davies, PhD, BSc UCL Institute of Hepatology, Royal Free Hospital,
University College of London, London, UK
Wulf Euringer, MD Department of Radiology, University Hospital Freiburg,
Freiburg, Germany
Rita Garca-Martnez, PhD Liver Unit, Department of Internal Medicine,
Vall dHebron Hospital, Barcelona, Spain
Michela Giusto, MD Department of Clinical Medicine, University Sapienza
Roma, Rome, Italy
Rajiv Jalan, MBBS, MD, PhD, FRCPE, FRCP UCL Institute of Hepatology,
Royal Free Hospital, University College of London, London, UK
xi
xii Contributors
Arumugam R. Jayakumar, PhD Department of Neuropathology,

South Florida Foundation for Research and Education Inc.,
Miami VA Medical Center, Miami, FL, USA
E. Anthony Jones, MD, DSc Winchester, Hampshire, UK
Maria Jover-Cobos, PhD UCL Institute of Hepatology, Royal Free Hospital,
University College of London, London, UK
Matthew R. Kappus, MD Department of Internal Medicine, Virginia
Commonwealth University Health Systems and Physicians, Richmond, VA, USA
Manuela Merli, MD Department of Clinical Medicine,
University Sapienza Roma, Rome, Italy
Sara Montagnese, MD, PhD Department of Medicine, University of Padova,
Padova, Italy
Jennifer Y. Montgomery, MD Department of Internal Medicine,
Virginia Commonwealth University Health System, Richmond, VA, USA
Kevin D. Mullen, MD, FRCPI Department of Internal Medicine, Division
of Gastroenterology, Metrohealth Medical Center, Cleveland, OH, USA
Michael D. Norenberg, MD Department of Pathology, Biochemistry and
Molecular Biology, Jackson Memorial Hospital, Miami VA Medical Center,
University of Miami Hospital, Miami, FL, USA
Ravi K. Prakash, MBBS, MD, MRCP (UK) Department of Internal Medicine,
Division of Gastroenterology, Metrohealth Medical Center, Cleveland, OH, USA
Jillian Kallman Price, MS Outcomes Research Program, Betty and Guy
Beatty Center for Integrated Research, Inova Fairfax Hospital,
Falls Church, VA, USA
Oliviero Riggio, MD Department of Clinical Medicine, University Sapienza
Roma, Rome, Italy
Martin Rssle, MD Department of Gastroenterology and Radiology,
University Hospital Freiburg, Freiburg, Germany
Shiv Kumar Sarin, MD, DM, FNA, FNASc Department of Hepatology,
Institute of Liver and Biliary Sciences, New Delhi, India
Amit Singh Seyan, MBBS, BSc (Hons) Kings College School of Medicine,
Kings College London, London, UK
Shabnam S. Shabbir, BSc, MBBS Kings College School of Medicine,
Kings College London, London, UK
Yalda Sharifi, MD, BAO, BCh, LRCP, SI, MRCP (UK) UCL Institute
of Hepatology, Royal Free Hospital, University College of London, London, UK
Contributors xiii
Praveen Sharma, MD, DM Department of Hepatology, Institute of Liver

and Biliary Sciences, New Delhi, India
Debbie Lindsay Shawcross, BSc, MBBS, FRCP, PhD Institute of Liver Studies,
Kings College School of Medicine at Kings College Hospital, London, UK
Karin Weissenborn, MD Department of Neurology, Hannover Medical School,
Hannover, Germany
Zobair M. Younossi, MD, MPH Beatty Liver and Obesity Research Center,
Inova Fairfax Hospital, Falls Church, VA, USA
Chapter 1
Introduction, Nomenclature,
and Classification of Hepatic Encephalopathy
Keywords Hepatic encephalopathy Terminology Overt hepatic encephalopathy

Covert hepatic encephalopathy
The area of hepatic encephalopathy (HE) has seen considerable changes in the last
decade. Nomenclature and classification of HE was formalized for the first time
with a report by the Hepatic Encephalopathy Consensus group in the World
Gastroenterology Congress meeting in Vienna in 1998 [1]. Terminology was devised
(Fig. 1.1) and in the ensuing decade we have seen virtually all publications using
this system. There was always recognition that this new system of classification
would need periodic updates and a recent meeting in Val David, Quebec has intro-
duced some changes (Fig. 1.2).
The original rationale to standardize the classification of HE was simple. Not
defining terms like acute and chronic HE was a major source of confusion [2]. The
problem was so bad we elected to totally change the terms to episodic and persistent.
Details of the rationale for change have been published [3]. In addition to the ABC
classification of the three main settings for HE, we further defined the multiaxial
classification which is evident in the enclosed figures (Figs 1.1 and 1.2).
Since that time there has been a persistent lobby to change the term minimal
HE to something better reflecting the clinical importance of this entity. We endorsed
the term covert HE in Quebec and this included in this publication from the meeting
in Val David, Quebec. The International Society for Hepatic Encephalopathy and
Nitrogen (ISHEN) is now the official authority for issuing updates on terminology
and optimum study design in this field of HE [4]. This society will be working
closely with the various international liver societies [American (AASLD), European
K.D. Mullen, MD, FRCPI (*) R.K. Prakash, MBBS, MD, MRCP (UK)
Department of Internal Medicine, Division of Gastroenterology,
Metrohealth Medical Center, 2500 Metrohealth Drive, Cleveland, OH 44109, USA
e-mail: kevin.mullen@case.edu
K.D. Mullen and R.K. Prakash (eds.), Hepatic Encephalopathy, Clinical Gastroenterology, 1
DOI 10.1007/978-1-61779-836-8_1, Springer Science+Business Media, LLC 2012
2 K.D. Mullen and R.K. Prakash
Fig. 1.1 World Congress of gastroenterology classification of hepatic encephalopathy
Fig. 1.2 Proposed changes by the International Society for Hepatic Encephalopathy and Nitrogen
(ISHEN) metabolismintroduction of the term Covert HE
(EASL) and International (IASL)] to provide periodic updates on terminology and

study design issues. One major goal is to make sure that ISHEN and other liver
societies produce guidelines that are consistent. Ideally one uniform set of guide-
lines should be produced with updates every 23 years.
The publication of the spectrum of neurocognitive impairment in cirrhosis paper
by Bajaj et al. has highlighted a new perspective on HE [5]. Instead of viewing minimal
1 Introduction, Nomenclature, and Classification of Hepatic Encephalopathy 3
HE (covert HE) and overt HE as distinct separate entities the idea now is that one
evolves into the other. Now that we are beginning to recognize that not all patients
reverse HE totally, the neurodegenerative aspect of HE is being included in the SONIC
classification. This may ultimately have a major impact on priority for liver transplan-
tation if irreversibility of neurological deficits is more formally and consistently
identified. We may reach a point where avoidance of any episodes of overt HE will be
the goal of therapy. Hence both treatment and liver transplantation may be instituted
earlier in the course of HE in the future [6]. Clearly, more effective treatment for HE
is required since organ supply for liver transplantation is limited. As these concepts
evolve, it will become more and more important to diagnose HE in its earliest steps.
Contained in the new perspective on the spectrum of HE is a difficult issue.
There is a universal concern about the accuracy of the New Haven Scale in diagnos-
ing and quantifying the severity of overt HE. The primary problem is so-called
Stage I HE of that scale [7]. The criteria for diagnosis of Stage I overt HE is subjec-
tive and is not consistently applied by physicians. Some patients have mild HE and
others do not. Attempts have been to improve measurement of Stage I HE by
including some more objective measures [8]. However, in keeping with Kircheis
and Haussinger point of view we felt a major change was needed [9]. Essentially
stage I HE as judged by the New Haven Scale has been abolished. Instead we have
combined minimal HE with what used to be stage I HE. This Covert HE will be
primarily diagnosed by psychometric tests and potentially other testing systems
such as inhibitory control test (ICT) or critical flicker fusion (CFF) (see Chap. 8).
The cut off between covert and overt HE will now be based on disorientation at least
to time. The particular element of HE diagnosis can be reliably detected by experi-
enced clinicians. The concept of covert and overt HE is essentially the same as the
low grade/high grade HE proposed by Haussinger et al. The remaining three grades
of the New Haven Scale will be kept but will feature new descriptions (moderate
overt HE, severe overt HE, and Comatose HE). It is important to note that covert HE
is not just what used to be minimal HE but with the additional aspect of what used
to be stage I HE. Purists suggest they can always find neurological abnormalities in
patients with Stage I HE but this proposed operational system is closer to reality. No
doubt there will be major debates on these proposed changes. As noted earlier it is
the intent of ISHEN and the other societies with an interest in HE to revise our
approach if new data comes to hand suggesting changes are needed.
Most of the changes proposed earlier will have a direct bearing on the conduction
of clinical treatment trials in the future. Choosing end points to evaluate for treat-
ment efficiency is crucial in the study design. We must endorse standardized meth-
ods of measuring HE and develop proper terminology to allow clear communication
at a global level. The last decade has seen progress with standardization of terminol-
ogy for HE with general use of the proposed terms in most journals. It will be a
more challenging task to agree upon standardization of diagnostic paradigm for the
detection and quantification of the entire spectrum of HE. A major impetus for this
will be the Val David accord which has addressed these issues in some detail [4].
Further progress is anticipated where working parties from upcoming liver meet-
ings delegate new guidelines.
References
1. Ferenci P, Lockwood A, Mullen K, Tarter R, Weissenborn K, Blei AT. Hepatic encephalopathy

definition, nomenclature, diagnosis, and quantification: final report of the working party at the
11th World Congresses of Gastroenterology, Vienna, 1998. Hepatology. 2002;35(3):71621.
2. Sanaka MR, Ong JP, Mullen KD. Challenges of designing hepatic encephalopathy treatment
trials. Hepatology. 2003;38(2):5278.
3. Mullen KD. Review of the final report of the 1998 Working Party on definition, nomenclature
and diagnosis of hepatic encephalopathy. Aliment Pharmacol Ther. 2007;25 Suppl 1:116.
4. Bajaj JS, Cordoba J, Mullen KD, Amodio P, Shawcross DL, Butterworth RF, et al. Review
article: the design of clinical trials in hepatic encephalopathyan International Society for
Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) consensus statement. Aliment
Pharmacol Ther. 2011;33(7):73947.
5. Bajaj JS, Wade JB, Sanyal AJ. Spectrum of neurocognitive impairment in cirrhosis: implications
for the assessment of hepatic encephalopathy. Hepatology. 2009;50(6):201421.
6. Atluri DK, Asgeri M, Mullen KD. Reversibility of hepatic encephalopathy after liver transplan-
tation. Metab Brain Dis. 2010;25(1):1113.
7. Kircheis G, Fleig WE, Gortelmeyer R, Grafe S, Haussinger D. Assessment of low-grade hepatic
encephalopathy: a critical analysis. J Hepatol. 2007;47(5):64250.
8. Hassanein TI, Hilsabeck RC, Perry W. Introduction to the Hepatic Encephalopathy Scoring
Algorithm (HESA). Dig Dis Sci. 2008;53(2):52938.
9. Haussinger D, Cordoba Cardona J, Kircheis G, Vilstrup H, Fleig WE, Jones EA. Definition and
assessment of low-grade hepatic encephalopathy. In: Haussinger D, Kircheis G, Schliess F,
editors. Hepatic encephalopathy and nitrogen metabolism. The Netherlands: Springer; 2006.
p. 42332.
Part I
Pathogenesis
Chapter 2
Role of Ammonia in the Pathogenesis
of Hepatic Encephalopathy
Jan Albrecht
Keywords Ammonia Cerebral blood ow Brain edema Glutamine Amino

acidergic neurotransmission
Introduction
There is consensus that excess of gut-derived ammonia which is not cleared from
the blood plays an important role in the pathogenesis of HE. However, as discussed
elsewhere in this book, a growing body of evidence suggests signicant contribution
of other factors, such as proinammatory cytokines and hyponatremia. Moreover,
there is a long list of gut-derived toxins that accumulate in the body when the detoxi-
fying capacity of the liver is compromised, many of which may enter the brain [1].
It thus appears worthwhile to distinguish the specic roles of ammonia in inducing
HE. This will be done in ve discrete sections. The rst issue addressed in this chap-
ter is the degree of correlation between blood ammonia levels and severity of HE as
graded by the West Haven scale (assignment to grades IIV). The impact of changes
in the rate of ammonia generation in the peripheral tissues is briey accounted for.
Next, the contribution of ammonia to the specic pathophysiological manifestations
of advanced stages of HE is analyzed. The key parameters under evaluation are
brain edema, which is the major cause of death in patients with HE accompanying
acute liver failure (ALF), and increased cerebral blood ow (CBF), which is a caus-
ative factor in brain edema. Further, the role of ammonia in the development of
cognitive and motor impairment is assessed. Wherever the net effect of ammonia
could not be directly evaluated in a clinical setting, its distinct role is demonstrated
in experimental animals with simple hyperammonemia not complicated by liver
J. Albrecht, PhD (*)

Department of Neurotoxicology, M. Mossakowski Medical Research Centre,
Polish Academy of Sciences, Pawinskiego 5, Warsaw, Poland
e-mail: jalb@op.pl
8 J. Albrecht
damage or asymptomatic animals with experimentally induced chronic liver failure

subsequently given ammonia bolus. The effectiveness of therapeutic interventions
specically aimed at reducing blood ammonia level in HE patients or experimental
animals is taken as further support to the relative contribution of ammonia.
The section Cellular and Molecular Mechanisms Underlying Ammonia-Induced
Impairment of Brain Function describes the molecular and biochemical effects of
ammonia on the different cell types of the central nervous system (CNS) and on the
interactions between these cells. It focuses on the events which can be causally linked
to brain edema and to the growing imbalance between neural inhibition and excita-
tion. Progression towards neural inhibition is mainly seen in Type C HE. Distinct
contributions of ammonia itself and its direct metabolite, glutamine are emphasized.
Correlation Between Ammonia Levels in Blood and/or Its Rate

of Production in the Periphery and the Advancement of HE
Most of the studies carried out in the last few decades have demonstrated a rather
good correlation between blood ammonia and severity of HE. Occasional deviations
from this rule are now interpreted as reecting methodological inaccuracies and/or
incompatibilities of the procedures used in different medical centers [2]. One major
controversy of the past was whether arterial or venous blood should be taken for the
measurements. It has been argued that when the liver becomes dysfunctional,
detoxication of ammonia mainly occurs in the muscles, disproportionally lowering
venous blood ammonia as compared to the arterial blood ammonia. It has also been
suggested that partial pressure of ammonia correlates better with the HE grade than
blood ammonia. Recently, Ong et al. [3] compared arterial and venous ammonia con-
tent, and arterial and venous partial pressure of ammonia, in a carefully selected group
of 121 patients with liver cirrhosis, and demonstrated that blood ammonia measured
with any of these four methods correlated equally well with the severity of HE.
Ammonia delivered from the peripheral tissues to blood is mainly derived from
glutamine following its degradation by phosphate-activated glutaminase (PAG).
There is evidence that the risk of progression of cirrhotic patients to advanced HE is
associated with increased ammonia production from glutamine in the intestines [4]
and kidney [5]. Similarly, it has been demonstrated that enhanced response to oral
glutamine challenge test can identify cirrhotics with increased risk of transition to
higher grades of HE [6]. More recently, mutation in the promoter region of PAG has
been identied in in vitro tests which accelerates the transcriptional activity of this
gene, i.e., enhances production of PAG molecules [7]. Cirrhotic patients carrying
this mutation show an increased preponderance to develop symptomatic HE [7].
Although intracellular ammonia levels in the brain are not amenable to direct
testing in HE patients, it is safe to assume that the increase in blood ammonia will
lead to a proportional increase in brain ammonia. The current view is that not only
ammonia base but also ionized ammonia penetrates the bloodbrain barrier (BBB)
[8]. Experiments in an animal model of hyperammonemia revealed a substantially
increased extraction of blood ammonia by the brain [9].
2 Role of Ammonia in the Pathogenesis of Hepatic Encephalopathy 9
The Role of Ammonia in Alterations of Cerebral

Blood Flow and Development of Cerebral Edema
Associated with HE
Brain edema is a frequent complication of ALF and a major cause of death in these
patients because it leads to increased intracranial pressure (ICP) and herniation.
Both clinical and animal model studies have brought about compelling evidence
favoring a direct role of ammonia in inducing brain edema. In a retrospective study,
death of ALF patients due to cerebral herniation closely correlated with the arterial
ammonia levels [10], and a recent prospective study by the same group revealed a
good correlation in time and magnitude between arterial hyperammonemia, cere-
bral accumulation of osmotically active amino acids and ICP [11]. Brain edema
seen in ALF patients was reproduced in rats with ammonium acetate-induced hyper-
ammonemia not complicated by liver failure [12, 13] and in portacaval-shunted rats
which received an ammonia bolus [14]. Astrocytic swelling is not only associated
with ALF [15] but also with low-degree brain edema accompanying Type C HE
[16]. This phenomenon could also be induced by ammonia in cultured astrocytes
[17] and cerebral cortical slices in vitro [18].
While the effects of ALF on CBF in a clinical setting varied in different studies,
patients with increased CBF developed brain edema more frequently than those
with decreased or unchanged CBF, suggesting causal relation between the phe-
nomena [19]. The role of hyperammonemia in evoking changes in CBF and the
role of CBF changes in the development of brain edema were documented in ani-
mal model studies. While cerebral hyperemia and brain edema were found absent
in asymptomatic portacaval-shunted rats, they were precipitated by subsequent
infusion of ammonia [20, 21]. The sequence in which brain edema and hyperemia
occur has not been nally established. The current view is that the primary signals
(nitric oxide and other as yet not well-dened factors) are derived from the swollen
brain cells (astrocytes) which by inducing hyperemia elicit a self-amplifying pro-
hyperemic signaling train [20, 21]. Pharmacological decrease of CBF in hyperam-
monemic rats attenuated brain edema, bespeaking the increased CBF as a causative
factor [22].
Recent evidence suggests the role of a vasogenic component of ammonia-induced
brain edema. Studies with the magnetic resonance imaging technique revealed
stage- and brain region-dependent development of vasogenic brain edema in rats
with acute hyperammonemia [23] and ALF [24]. The above studies also have dem-
onstrated that regions with vasogenic edema show increases of BBB permeability
associated with increased activity of the matrix metalloproteinase 9 (MMP-9).
MMP-9 was earlier found to contribute to BBB dysfunction in ALF by disrupting
the brain endothelial tight junction proteins, but the specic role of ammonia was
not investigated in this study [25]. A challenging question for future investigations
is whether and to what degree the subtle BBB disruption underlying vasogenic brain
edema reects direct toxic action of ammonia on the endothelial cells of the BBB
similar to the effects of ammonia on astrocytes or neurons.
10 J. Albrecht
Ammonia and Impairment of Cognitive and Motor Functions
HE is associated with impairment of learning and memory. The complexity of the

changes makes it difcult to gauge the degree of contribution of ammonia and other
pathogenic factors to a given neuropsychological symptom. Nonetheless, in animals,
experimentally induced hyperammonemia not complicated by liver impairment
have been shown to evoke alterations in some basic learning and cognition tests
similar to those noted in animals with HE [26]. Cyclic GMP (cGMP) is a molecule
critically involved in the different aspects of learning and memory, and the activity
of NMDA receptor/NO/cGMP pathway is a marker of the cognitive functions. Both
impairment of cognitive functions coupled with decrease of cGMP in the brain, and
restoration of these functions upon pharmacological elevation of cGMP, are observed
in cirrhotic patients and animals with HE and in animals with induced hyperam-
monemia in the absence of liver failure [26, 27].
HE in cirrhotic patients is associated with impaired motor activity and coordina-
tion. These changes are due to the altered functioning of neuronal circuits involving
basal ganglia and the cerebral cortex, including altered modulation of these circuits
by the metabotropic glutamate receptor (mGluR) activity. The altered response to
mGluR activation and the motor function changes observed in rats with chronic
liver failure were mirrored in rats with induced hyperammonemia in the absence of
liver failure. For example, activation of mGluR1 by excess glutamate in the substan-
tia nigra/ventral tegmental area axis is thought to be responsible for hypokinesia in
chronic hyperammonemic rats [28].
Effectiveness of Blood Ammonia-Reducing Therapies

as an Indicator of the Role of Ammonia in HE
As discussed elsewhere in this book, nonabsorbable disaccharides (lactulose) and

antibiotics (rifaximin) are the routinely employed ammonia lowering treatment
modalities based on the principle of combating gut ora. Although the improvement
of the status of patients treated with these drugs supports the role of ammonia in the
development of HE, the effects of these drugs on specic pathophysiological mani-
festations of HE have not been assessed quantitatively. More precise information
was recently derived from the experiments with a newly invented drug, ornithine
phenylacetate (OP). OP has a two-hit mechanism of action, where l-ornithine acts
as a substrate for glutamine synthesis from ammonia in skeletal muscles, while
phenylacetate combines with ammonia-derived glutamine to form phenylacetyl glu-
tamine, which is subsequently excreted in the kidneys. Treatment of cirrhotic (bile
duct ligated) rats with OP for a few hours reduced the originally increased arterial
blood ammonia almost back to the control level, and the reduction was correlated
with an equally effective attenuation of brain edema [29].
Cellular and Molecular Mechanisms Underlying

Ammonia-Induced Impairment of Brain Function
Ammonia that enters the brain is metabolized in astrocytes to glutamine in an

ATP-consuming reaction catalyzed by glutamine synthetase (GS). Astrocytes that
are in a close topographical contact with the cerebral vascular endothelial cells
forming the BBB are the primary victim of excess ammonia. The metabolic and
molecular changes evoked by ammonia on astrocytes affect the astrocyticneuronal
interactions which impact on neuronal function. However, ammonia also affects the
neurons directly. It is beyond the scope of this chapter to discuss the multiple ways
in which ammonia affects the general metabolism of astrocytes and/or neurons: in
most general terms, astrocytic and neuronal dysfunction under excessive ammonia
load is critically coupled to decreased energy metabolism [30]. The text later focuses
on two issues: (a) the mechanisms by which ammonia specically contributes to
astrocytic swelling and subsequent brain edema and (b) how the effects of ammonia
on astrocytes and neurons are translated into the shift of balance of neurotransmis-
sion to net neural inhibition, which progresses with the advancement of HE.
Role of Ammonia in Astrocytic Swelling and Brain Edema
The current view is that the major metabolic impairments and cell membrane dys-
functions produced in astrocytes by ammonia evolve from astrocyte swelling by a
vicious cycle of oxidative/nitrosative stress (ONS) and intracellular osmotic imbalance
[16]. Swelling of cultured astrocytes treated with ammonia is invariably associated
with intracellular accumulation of reactive oxygen and nitrogen species (RONS),
including the highly toxic peroxynitrite [20]. One contributor to the increased RONS
formation in ammonia-treated astrocytes or brain slices is excessive nitric oxide
(NO) synthesis which may be associated with the overactivation of NMDA recep-
tor, in a self-amplifying mechanism involving excessive glutamate release from
astrocytes [16]. In an in vivo model of hyperammonemia, reduction of brain edema
could be achieved upon administration of an NMDA receptor antagonist, memantine
to the rat [31]. Excess of NO activates cGMP synthesis and subsequently increases
protein kinase G activity, which also contributes to ammonia-induced astrocytic
swelling [32]. The other contributing factor is the accumulation of reactive oxygen
species, mainly the superoxide anion (O2) generated by NADPH oxidase [16].
Natriuretic peptides (NPs) (atrial natriuretic peptide, C type natriuretic peptide),
which are natural components of the brain tissue, reduce RONS production in
ammonia-treated astrocytes by reducing NADPH oxidase expression and activity
[33]. This antioxidative effect is specically mediated by the natriuretic peptide
clearance receptor (NPR-C). These NPs and the NPR-C may act as targets for ther-
apy development for HE in future. Pharmacological studies demonstrated increased
activity of MAP kinases and NFk-B. These act as carriers of downstream signals
12 J. Albrecht
critical for the translation of RONS accumulation to astrocytic swelling [34].

Ammonia may also contribute to astrocytic swelling by directly interfering with the
cell membrane ion and water transport. The phosphorylation-dependent activation
and/or increased expression of NaKCl co-transporter 1 (NaKCC1) mediates
astrocytic swelling in ammonia-treated astrocytes [35] and brain edema in rats with
experimentally induced ALF [36].
There is compelling evidence for increased glutamine accumulation in ammonia-
exposed astrocytes which is a key factor mobilizing the vicious circle of ONS and
osmotic imbalance associated with HE. Early studies have shown that astrocytic
swelling and cerebral edema in rats with hyperammonemia become reduced or even
disappear upon co-administration of glutamine synthetase inhibitor, l-methionine-
d/l-sulfoximine (MSO) [13]. In the clinical setting, increased ICP in ALF patients
awaiting liver transplantation was found to correlate almost perfectly with the glu-
tamine (Gln) content measured in the cerebral microdialysates collected from the
patients at the bedside [12]. The role of glutamine in brain edema has long been
interpreted to exert exclusively by its intracellular osmotic effect. The nding that
glutamine is able to induce mitochondrial permeability transition (mPT) and swelling
in isolated mitochondria dependent on uninterrupted glutamine uptake to mitochon-
dria [37] stimulated studies in this eld. The essence of the hypothesis, nicknamed
the Trojan horse hypothesis, is that a portion of newly synthesized glutamine is
transported from astrocytic cytosol to mitochondria and is degraded back to ammo-
nia: the glutamine derived-ammonia would be responsible for astrocytic swelling
and brain edema [38]. Recently, the paradigm of directly blocking the entry of glu-
tamine to brain mitochondria (by the amino acid histidine) was successfully
employed to ameliorate brain edema in a rat model of ALF [39]. Of note, ammonia
also promotes astrocytic swelling by upregulating the peripheral benzodiazepine
receptor (PBR); recently renamed the 18-kDa translocator protein (see also section
Ammonia and the Neurotransmitter Imbalance in HE for its other roles) [40].
Since PBR is located on the outer mitochondrial membrane, it could be an easily
accessible target for the glutamine-derived, mitochondrial pool of ammonia.
In summary, it is currently accepted that both the osmotic and the Trojan horse
mode of action of glutamine contribute to its role as a mediator in ammonia-induced
astrocyte swelling and brain edema.
Ammonia and the Neurotransmitter Imbalance in HE
Progression of HE through its different stages from normality excitation to coma is

notable in ALF. In contrast, evolution to coma in chronic liver disease is much more
a gradual increase in neural inhibition. This shift from neural excitation to inhibition
mainly involves changes in the amino acid neurotransmitter systems: the excitatory
glutaminergic and the inhibitory GABAergic system, along with some evidence
implicating the serotoninergic system as swaying the balance further towards inhi-
bition. Studies in hyperammonemic models in vivo and analysis of the effects of
in vitro treatment of astrocytes or neurons strongly suggest that ammonia is largely

responsible for the neurotransmission imbalance in HE and disclosed some clues to
details of the underlying mechanisms.
The Glutamatergic Transmission
Administration of ammonia to rats results in increased activation of NMDA type of

glutamate receptor, which is the primary cause of neuronal damage in these animals
[23]. Ammonia instantly increases extracellular accumulation of glutamate, which
may reect ammonia-induced depolarization as a triggering factor for a vicious
circle of glutamate-induced NMDA-receptor-dependent glutamate release. Induced
hyperammonemia is also associated with increased glutamate exocytosis in astro-
cytes [41] and decreased astrocytic glutamate uptake [42], which may partly engage
the astrocytic NMDA receptors [16], and which further contributes to the increase
of extracellular glutamate. Extracellular gluatamate remains elevated under pro-
longed exposure to elevated ammonia levels, which eventually leads to NMDA
receptor inactivation [43]. This leads to the depression of the excitatory neurotrans-
mission in different brain regions and to the cognitive impairment associated with
the decrease of the NO/cGMP pathway. Reduction of cGMP synthesis may also be
due to excessive accumulation of glutamine, which limits the availability of arginine
for NO synthesis [44]. As mentioned earlier, hypokinesia, a typical locomotor dys-
function accompanying advanced HE, is associated with overactivation of mGluR1
by excess glutamine. The underlying mechanism appears related to altered modula-
tion of the microtubule-associated protein 2 (MAP-2) phosphorylation by mGluR1
in the neurons [28].
The GABAergic Transmission
Hyperammonemia is associated with an increased GABAergic tone. The underlying

mechanism is associated with the increased density of PBR, which are located in
astrocytes and control the synthesis of pregnenolone-derived neurosteroids, some of
which are positive modulators of the GABA (A)-benzodiazepine receptor complex.
Increase of PBR binding coupled with increased synthesis of pregnenolone and its
neuroactive derivatives were measured in hyperammonemic mice [45]. Increased
concentrations of pregnenolone and its highly active derivative allopregnenolone
were also found in the brain of cirrhotic patients who died in hepatic coma [46].
Recently, chronic hyperammonemia in rats was observed to specically increase the
GABAergic tone in cerebellum, and this effect was associated with concerted
increases of (a) extracellular GABA, (b) a neurosteroid positively modulating the
GABAA receptor activation, and (c) the amounts of relevant GABAA receptor sub-
units. Most interestingly, pharmacological blockade of GABAA receptors restored
the previously reduced ability of cerebellum to synthesize cGMP in response to
NMDA receptor stimulation and the cerebellar aspect of learning in these hyperam-
monemic rats [47]. The latter study highlights the role of imbalance between gluta-
matergic and GABAergic transmission in HE.
14 J. Albrecht
The Serotoninergic Transmission
Serotonin, the tryptophan-derived inhibitory monoamine, is involved in regulation

of sleep, circadian rhythmicity and locomotion. Increased serotoninergic tone has
been implicated in the derangement of the above parameters in HE patients and
experimental animals. In addition, increased serotonin accumulation and turnover
in the brain were positively correlated with the degree of hyperammonemia [48].
Increased serotonin synthesis in HE-affected brain is associated with increased
tryptophan uptake from the circulation, which occurs by exchange with glutamine.
Increased tryptophan/glutamine exchange was veried in the rat cerebral capillaries
treated with ammonia in vitro or derived from hyperammonemic rats [49].
Concluding Comments: Gaps in the Knowledge

of Ammonia Neurotoxicity
The data reported in this chapter strongly support the key role of ammonia in the
development of major HE symptoms and elucidate many of the underlying bio-
chemical and molecular mechanisms. In general terms, the pattern of responses to
ammonia noted in the CNS cells or brain slices in vitro and in the brain of animals
with hyperammonemia corresponds relatively well with the changes observed in
patients or experimental animals with HE. However, in light of the recent nding
that hyperammonemia evokes an inammatory response in the CNS engaging the
CNS microglia [50], data obtained with cultured astrocytes or neurons will have
to be interpreted with caution. Moreover, there may be a need for reinterpretation
of some older studies in which the brain was regarded as a homogenous entity.
As discussed in this chapter, studies of the last few years disclosed a remarkable
brain region variability of the responses to ammonia concerning edema [23] or
molecular mechanisms underlying cognition [47]. However, the contribution of
ammonia to some of the common manifestations of HE still remains to be estab-
lished beyond doubt. Events such as alterations of the dopaminergic or cholinergic
transmission, and changes in the accumulation or intercellular uxes of inhibitory
neuromodulators: sulfur amino acid taurine, and serotonin metabolites kynurenic
acid and oxindole, which frequently accompany HE, have not been analyzed in
great detail. This needs to be done under the conditions mimicking hyperammone-
mia and in the absence of other factors precipitating HE. Answers to the above
questions are needed to fully appreciate the specic role of ammonia in HE.
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Chapter 3
Neuroinflammation in the Pathogenesis
Roger F. Butterworth
Keywords Neuroinammation Hepatic encephalopathy Liver failure Cirrhosis

Ammonia Proinammatory cytokines Microglia
Introduction
Central nervous system (CNS) complications of liver failure include hepatic

encephalopathy (HE) and brain edema. Depending upon the etiology and chronicity
of the liver failure, brain edema may be low grade (cirrhosis) or high grade (acute
liver failure, ALF); the latter may result in intracranial hypertension and brain
herniation, one of the leading causes of mortality in ALF. HE is a neuropsychiatric
syndrome that occurs in both cirrhosis and in ALF and is characterized by disturbance
of both cognitive and motor function starting with personality changes and sleep
disturbances progressing through more severe cognitive and motor symptoms to
stupor and coma. In ALF, progression to severe stages of HE may occur in a matter
of days. Since the appearance of CNS symptoms in liver failure frequently heralds
a poor prognosis, potentially having a signicant impact on quality of life, on clinical
management strategies, on liver transplant priority, and on patient survival, effective
therapies are urgently needed. The design of such therapies rst requires a knowledge
of the underlying pathophysiological mechanisms.
In spite of many decades of study, the mechanisms responsible for HE and brain
edema in liver failure are still not completely understood. A great deal of attention
continues to be focused on ammonia as the culprit toxin implicated in the pathogen-
esis of these CNS complications and agents with properties aimed at reduction of
R.F. Butterworth, PhD, DSc (*)

Neuroscience Research Unit, Hpital Saint-Luc (CHUM),
1058 St. Denis, Montreal, QC, Canada H2X 3J4
e-mail: roger.butterworth@umontreal.ca
20 R.F. Butterworth
circulating ammonia remain the mainstay of clinical management and therapy.

However, recent research in both ALF patients as well as in patients with cirrhosis
in addition to studies in experimental animal models of these conditions strongly
suggests that inammation, acting alone or in concert with ammonia, may also play
an important role in the pathogenesis of the CNS complications of liver failure.
Glial Pathology in Liver Failure
The CNS is composed of two major cell types, neurons and glial cells, and the latter
consist of astrocytes, oligodendrocytes, and microglia. Liver failure results in glial
(rather than neuronal) pathology, the nature of which is dependent upon the type
(acute or chronic) and severity of liver failure. Systematic studies in material from
patients with ALF reveal swelling of astrocytes [1] leading to cytotoxic brain edema
(Fig. 3.1a).
In contrast, end-stage chronic liver failure results in pathological changes to
astrocytes known as Alzheimer type 2 astrocytosis characterized by nuclear enlarge-
ment, margination of chromatin, and glycogen depletion (Fig. 3.1b). Brain edema
may also occur in chronic liver failure but the magnitude of the edematous changes
in this case is modest and more focal in nature affecting, for example, the corticospinal
tract [2]. Neuronal pathology has also been described in end-stage chronic liver
disease consisting primarily of thalamic and cerebellar lesions due to nutritional
decits related to liver failure [3, 4]. Although neuronal degeneration in the form
of acquired non-Wilsonian neurodegeneration or postshunt myelopathy has been
reported in cirrhosis, these cases are relatively rare and it is generally concluded that
the neuronal loss in chronic liver failure is, in most cases, insufcient to account for
the symptoms of HE.
More recently, alterations of a second cell type of the glial lineage have been
reported. Activation of microglia was rst reported in 2005 in brains of experimental
animal models of ALF resulting from liver ischemia [5] and has since been
conrmed both in liver ischemia animals [6] and in animals with ALF resulting
from toxic liver injury [7]. Microglia are the immunomodulator cells of the brain
being bone marrow-derived myeloid lineage cells. In the absence of an inammatory
stimulus, microglia remain quiescent, being involved principally in surveillance
(the so-called resting phenotype). However in the presence of an inammatory stim-
ulus, these cells become reactive (the activated phenotype) with the task of pre-
vention and control of CNS damage due to altered homeostasis associated with
a wide range of insults and/or cell death. Activation of microglia is indicative of
neuroinammation and is observed in a wide range of neuroinammatory disorders
including multiple sclerosis and the AIDS-dementia complex and also in disor-
ders such as stroke and Alzheimers disease suggesting the presence of a signicant
neuroinammatory component in the pathogenesis of the neurological symptoms
in these disorders also. Subsequent to the original reports of microglial activation
in brain in experimental ALF, similar ndings have been reported in human ALF
3 Neuroinammation in the Pathogenesis of Hepatic Encephalopathy 21
Fig. 3.1 (a) Electron micrograph of cerebral cortex from a patient who died in acute liver failure
(ALF) resulting from acetaminophen hepatotoxicity. Perivascular astrocytes (A) are markedly swollen
(reprinted from Kato et al. [1], with permission from John Wiley & Sons, Inc.). (b) Alzheimer type
II astrocytosis (Alz) in prefrontal cortex of a 56-year-old cirrhotic patient who died in hepatic
coma. N normal astrocyte
brain [8] as well as in the brains of animals with bile-duct ligation [9] or end-to-side
portacaval shunts [10] suggesting that neuroinammation is a signicant component
of HE in both acute and chronic liver failure.
Neuroinflammation in Acute Liver Failure
A signicant correlation exists between the presence of the systemic inammatory

response syndrome (SIRS) and the severity of CNS complication of ALF [11].
Circulating levels of TNF-a are invariably increased in ALF patients [12] and TNF
22 R.F. Butterworth
Fig. 3.2 Microglial activation in two different experimental animal models of ALF at coma stages
of encephalopathy. (a) The hepatic devascularized rat. (b) The mouse with azoxymethane-induced
toxic liver injury. Microglial activation revealed by OX-42 immunohistochemistry in frontal cortex
of ALF animals (for further details, see refs. [6, 7, 28])
gene polymorphisms have been reported to inuence the clinical outcome in these
patients [13]. Moreover, decreases in TNF-a production have been shown to be
protective against the development of severe HE in patients with ALF resulting
from acetaminophen ingestion [13]. Increased plasma concentrations of TNF-a
are also associated with motor co-ordination decits in animals with thioacetamide-
induced ALF [14]. However, although invariably increased in human and experi-
mental ALF, plasma cytokine proles in experimental ALF resulting from toxic
liver injury show both similarities and differences that are dependent upon the nature
of the toxin [15].
Although systemic inammation has been well established for over a decade,
evidence of neuroinammation in liver failure was not provided until the publication
of a report suggestive of increased production of proinammatory cytokines in brain
in ALF patients [16] who measured TNF-a, IL-1b, and IL-6 in blood sampled from
an artery and a reverse jugular catheter in 16 patients with ALF primarily due to
acetaminophen hepatotoxicity. A signicant correlation was observed between arterial
cytokine levels and intracranial hypertension; brain cytokine efux was noted,
consistent with brain cytokine production in these patients. Unequivocal evidence
of neuroinammation was subsequently provided by studies reported by Jiang et al.
[6], who demonstrated microglial activation (see above) and concomitant increases
in expression of genes coding for proinammatory cytokines in experimental ALF
in the rat resulting from hepatic devascularization. In the study by Jiang et al. [6],
increases in expression of the major histocompatibility complex class 11 antigen
marker CD11b/c (OX-42) were observed (Fig. 3.2a, b), a feature that is characteristic
of microglial activation (Fig. 3.2a). In this experimental model of ALF, microglial
activation was found to occur early in the progression of the disorder and to increase
signicantly as HE and brain edema developed. Similar ndings were subsequently
reported in mice with ALF due to azoxymethane-induced liver injury [7] (Fig. 3.2b).
Neuroinflammation in Cirrhosis
Patients with cirrhosis are prone to infection due to their functionally immunosup-
pressed state and impaired host defense capabilities, and the presence of infection
in this patient group has the potential to complicate the clinical course leading to
encephalopathy, multiple organ failure, and death [17].
Moreover, while loss of liver function predisposes cirrhotic patients to the
development of infection, once established, sepsis has the potential to cause
deterioration of liver function resulting in a vicious cycle that invariably results in
SIRS accompanied by increased circulating levels of TNF-a, IL-1b, and IL-6.
Cytokine synthesis in cirrhosis may be triggered by a range of inammatory stimuli
including gut-derived bacterial translocation, infection, increased hepatic cytokine
production, and/or decreased renal cytokine clearance. The cells implicated include
activated phagocytic and nonphagocytic cells such as monocytes, lymphocytes,
neutrophils, and Kupffer cells of the liver.
Concentrations of circulating cytokines such as TNF-a are higher in decompensated
vs. compensated cirrhosis and the magnitude of the increases is predictive of the
severity of the encephalopathy [12]. Moreover, the presence of SIRS exacerbates
the neuropsychological effects of induced hyperammonemia in cirrhosis [18].
A major contribution of increased circulating TNF-a to the pathogenesis of HE
in cirrhosis is supported by ndings of increased serum levels of the cytokine in a wide
range of other encephalopathies including those related to the AIDS virus, cerebral
malaria, meningitis, inuenza virus, sepsis, and multiple organ failure [19].
A role for inammation in the pathogenesis of minimal hepatic encephalopathy
(MHE) was suggested by results of a study in 84 cirrhotic patients in which neurop-
sychological testing was performed before and after induction of hyperammonemia
by administration of a solution mimicking the amino acid composition of hemoglobin.
24 R.F. Butterworth
The presence and severity of MHE in these patients was found to be independent of
the severity of liver disease or serum ammonia concentrations; however, circulatory
markers of inammation were signicantly higher in patients with MHE [20]. In a
second study by the same group, ten cirrhotic patients were studied after admission
with clinical evidence of infection and following its resolution; induced hyperam-
monemia resulted in signicant worsening of neuropsychological test scores in
patients showing evidence of SIRS but not after its resolution [18]. Another study
showed that infection and systemic inammation in cirrhotic patients, but not hyper-
ammonemia, were associated with grades 3 and 4 HE [21].
In contrast to the clear evidence of a neuroinammatory response in ALF, evidence
for neuroinammation in cirrhosis is incomplete and, to date, is restricted to studies
in animal models of MHE. In a study by Cauli et al. [22], end-to-side portacaval
anastomosis in the rat was found to result in increased brain concentrations of the
proinammatory cytokine IL-6 as well as increased activities of other proinammatory
markers such as cyclooxygenese and inducible nitric oxide synthase (iNOS).
However, microglial activation was not assessed in the brains of these animals and
improvement of learning skills followed ibuprofen was found to occur without
signicant reduction in brain cytokine levels. In a more recent study by Brck et al.
[23], locomotor decits in rats following portal vein ligation were accompanied by
increases in expression of the gene coding for the proinammatory cytokine IL-6
but no evidence of microglial activation was observed in the brains of these animals.
The identity of the cell responsible for increased IL-6 gene expression was not identied
in this study. In contrast, these studies in portocaval shunted or portal vein ligated
rats in which a clear role for neuroinammation is still lacking, studies in bile duct-ligated
mice [9] or rats [10] show clear evidence of neuroinammation characterized by
microglial activation established using a range of reliable cell-specic markers and
increased brain concentrations of proinammatory cytokines. In the study of bile duct-
ligated rats, microglial activation was found to manifest brain region selectivity.
Synergy Between Ammonia and Proinflammatory Mechanisms
There is increasing evidence to support the notion that ammonia-related mechanisms

may act in concert with proinammatory mechanisms in a complex series of steps
resulting in the CNS complications of liver failure. Not only does the presence of
systemic infection/inammation have the potential to result in deterioration of liver
function and consequently increased hyperammonemia (see above), but a signicant
correlation exists between circulating levels of ammonia and TNF-a in liver failure
and both are independent predictors of HE severity [12]. Evidence for ammonia
proinammatory cytokine synergy is also emerging from studies in experimental
animal models of liver failure. For example, bile duct ligation in the mouse [9] and
rat [24] affords reproducible animal models of inammatory liver injury character-
ized by modest hyperammonemia, increased levels of circulating proinammatory
cytokines, and neurobehavioral symptoms. In one study, bile duct ligation in the rat
resulted in increased circulating levels of TNF-a and IL-6 concomitant with motor
incoordination and superimposed diet-induced hyperammonemia led to worsening
of the motor decit in these animals [24].
Based upon studies with cultured astrocytes and microglial cells, it has been
suggested that ammonia may cause increases in the production and/or release of
proinammatory cytokines. However, the effects of ammonia on cytokine release
are dependent upon the cell type and on the nature of the cytokine. For example,
exposure of human CHME-5 microglial cells to ammonia resulted in decreased
secretion of the stimulated release of IL-6 but enhanced secretion of IL-8 [25]; on
the other hand, in GL-15 astroglioma cells, stimulated release of TNF-a was
decreased by exposure to ammonia. These ndings were not conrmed in studies by
Andersson et al. [26], working with microglial-enriched and astroglial-enriched
primary cultures who were unable to nd any signicant effects of ammonia on the
release of these or other proinammatory cytokines.
An important function of astrocytes, and possibly also microglial cells, is the
rapid removal of neuronally released glutamate. This mechanism represents the
major inactivation step in the regulation of the glutamatergic neurotransmitter
system. For this purpose, astrocytes and microglia express high afnity, high capacity
glutamate transporters, the most abundant of which, EAAT-2 was previously
found to be decreased in experimental ALF [27]. Moreover, administration of
the proinammatory agent lipopolysaccharide (LPS) to rats with ALF due to liver
ischemia results in further losses in expression of EAAT-2 in brain and more rapid
progression of HE and brain edema [28]. Studies in cultured astrocytes reveal that
exposure to either ammonia [29] or proinammatory cytokines [30] causes loss of
expression of astrocytic glutamate transporters and a consequent reduction in capacity
for high afnity glutamate uptake.
More recent studies provide evidence for ammoniacytokine synergism at the
cellular/molecular level in brain [31]. Exposure of primary cultures of cortical astrocytes
to recombinant IL-1b and ammonia resulted in signicant increases in expression
of both heme oxygenase-1 (HO-1) and iNOS. Furthermore, the effects were additive
suggestive of synergism.
LiverBrain Proinflammatory Signaling
The nature of the signaling between the failing liver and the brain leading to central
neuroinammation in liver failure remains unknown. On the one hand, there is
evidence to suggest that systemic proinammatory mechanisms may initiate the
signaling process via one of several mechanisms that include (1) direct transfer
of cytokines by way of active transport, (2) interaction with receptors on circum-
ventricular organs lacking a bloodbrain barrier, or (3) by activation of afferent
neurons of the vagus nerve. In addition, it has been proposed that systemic
inammatory signals have the potential to result in increased permeability of the
bloodbrain barrier to cytokines in liver disease [21]. Direct evidence for this
26 R.F. Butterworth
intriguing possibility, however, is not currently available. More recently, using an

animal model of biliary cirrhosis, DMello et al. [9] demonstrated that activation
of cerebrovascular endothelial cells by peripherally administered TNF-a stimulated
microglia to produce monocyte chemotactic protein-1 (MCP-1) that mediates the
recruitment of monocytes into the brain with subsequent production of TFN-a.
Whether these signaling mechanisms are modied by acute or chronic liver failure
has not been established.
In addition to systemic proinammatory signals, there is evidence to suggest that
toxins generated by the failing liver (other than cytokines) may also play a role in
the pathogenesis of neuroinammation in liver failure. A wide range of molecules
with the potential to threaten the functional integrity of the brain have the capacity
to trigger the transformation of microglia from the resting to the active state. Such
molecules include ammonia, lactate, glutamate, manganese, and neurosteroids [32],
all of which have been reported to be increased in concentration in the brain in liver
failure. Despite the inconsistent ndings with respect to the effects of ammonia on
proinammatory cytokine release by microglial cells in culture [26], a recent study
clearly demonstrated that hyperammonemia in the absence of liver disease resulted
in microglia activation of a comparable magnitude and regional distribution in brain
to that observed in the bile duct-ligated rat and that hyperammonemia and bile duct
ligation led to comparable cognitive and motor impairment [10]. Together, these
ndings suggest that the ammonia molecule per se may not have been the entity
responsible for the neuroinammatory consequences of hyperammonemia.
Exposure of cultured cells to lactate in concentrations equivalent to those
described in brain in acute [33] or chronic [34] liver failure led to several-fold
increases in release of TNF-a and IL-1b [26]. Increased brain lactate in liver failure
has been attributed to an inhibitory effect of ammonia on alpha-ketoglutarate dehy-
drogenase resulting in impaired cellular oxidative metabolism [35] and increased
brain lactate synthesis signicantly correlates with severity of encephalopathy, with
the presence of brain edema and with microglial activation and cytokine production
in brain in ALF [6, 36]. A single report suggests that manganese toxicity also has the
potential to lead to microglial activation [37]. Manganese deposition is a consistent
feature of cirrhosis, deposition being greatest in basal ganglia structures of the brain
[38] but whether the concentrations of manganese reported in brain in liver failure
are sufcient to cause neuroinammation has not been ascertained.
Neuroinflammation and the CNS Complications

of Liver Failure: The Neurosteroid Connection
A consistent nding in both acute and chronic liver failure is increased expression
in brain of the so-called translocator protein (TLP), previously known as the
peripheral-type benzodiazepine receptor. Increased TLP expression occurs in
brain in human HE [39, 40] as well as in experimental animal models of either acute
[41] or chronic [42, 43] liver failure. However, the identity of the neural cell(s)
Fig. 3.3 Structure of allopregnanolone, a neurosteroid synthesized in brain having potent neuroinhibitory
properties by virtue of its agonist action at the postsynaptic GABA-A receptor (adapted from
Ahboucha et al. [49], with permission from John Wiley & Sons, Inc.)
expressing increased TLP has not yet been denitively established. Both astrocytes
and microglia express transcripts for TLP and, prior to the discovery of microglial
activation in brain in ALF [6, 44], it had been generally assumed that increased
TLP expression was the consequence of the activation and/or proliferation of astro-
cytes. However, a review of the literature fails to provide any convincing evidence
of this. For example, expression of the astrocyte marker protein glial brillary acidic
protein (GFAP) is decreased in brain in both experimental [45] and human [46] liver
failure and morphologic studies, while showing alterations of astrocyte integrity
such as swelling or Alzheimer-type II changes [47] show no clear evidence of an
activated state. Based upon these ndings, it is unlikely that the increased TLP sig-
nal observed in brain in liver failure is a uniquely astrocytic phenomenon and there
are reasons to suspect that activations of microglia are alternatively (or additionally)
also implicated. This notion is strengthened by the observation that increased
signals in position emission tomography (PET) studies using the TLP ligand 11-C-
PK11195 in neurological disorders, such as multiple sclerosis and in AIDS-dementia
complex, have been attributed to microglial rather than astrocytic activation [48].
Activation of TLP results in increased transport of cholesterol across the
mitochondrial membrane, a step that constitutes the initial process in the synthesis
of a novel class of compounds known as neurosteroids. Neurosteroids are potent
activators of a range of neurotransmitter receptors, and one neurosteroid, allopreg-
nanolone (Fig. 3.3), is an extremely high afnity agonist for the GABA-A receptor
with consequent potent neuroinhibitory and sedative properties.
Increased concentrations of allopregnanolone have been reported in autopsied
brain tissue of cirrhotic patients who died in HE but were within normal limits in
those patients without HE (Fig. 3.4) suggestive of a role for allopregnanolone in the
pathogenesis of HE in chronic end-stage liver failure. Moreover, increased brain
concentrations of GABA-A receptor agonist neurosteroids such as allopregnano-
lone offer an alternative explanation for the phenomenon of increased GABAergic
tone in HE which had previously been attributed to increased brain concentrations
of endogenous benzodiazepines.
28 R.F. Butterworth
Fig. 3.4 Increased concentrations of allopregnanolone in autopsied brain tissue from cirrhotic
patients who died in hepatic coma (HE) compared to age-matched controls and nonencephalo-
pathic cirrhotic patients (LD). Increased brain concentrations of allopregnanolone likely form
the basis of the phenomenon of increased GABAergic tone in HE UC: Uremic coma (adapted
from Ahboucha et al. [49], with permission from John Wiley & Sons, Inc.)
Evidence of a link between microglial activation, TLP induction, and neurosteroid

formation is provided by reports of the existence of all three processes in both
experimental and clinical material. Brains of patients with chronic liver failure
express increased quantities of TLP assessed either biochemically [39] or using
PET [40] as well as increased concentrations of allopregnanolone [49]. Moreover,
portacaval anastomosis in the rat leads to increased expression levels of TLP
[42, 43], increased brain levels of neurosteroids [50], and microglial activation [51].
It has been demonstrated in studies using cultured cells that exposure to liver disease-
related toxins such as ammonia and manganese leads to upregulation of TLP sites
[52, 53]. Exposure of these cells to proinammatory cytokines also leads to upregu-
lation of TLP [54] suggesting that ammonia and proinammatory cytokines may act
synergistically to produce increased brain concentrations of inhibitory neurosteroids
leading to HE in liver failure.
Diagnostic and Therapeutic Implications
Whatever the ultimate mechanism responsible, the consistent ndings of induction

of central neuroinammatory processes in acute and chronic liver diseases have
the potential to impact signicantly on diagnostic, management, and treatment
options for the future. For example, the demonstration of microglial activation could
stimulate the use of diagnostic neuroimaging techniques such as PET. Activated
microglia express transcripts for TLP and the extent of neuroinammation is currently
assessed in a wide range of neurological disorders, including multiple sclerosis
and the AIDS-dementia complex, by PET using the TLP ligand [11C]-PK11195.
Fig. 3.5 Microglial activation in patients with acute or chronic liver failure. (a) HLA-DR (CR3/43)
immunostaining in a patient with ALF (patient material provided by Dr. Radhakrishan Dhiman,
PGIMER, Chandigarh, India) and (b) neuroinammation in a patient with mild HE by position
emission tomography. Left: T1-weighted MRI; Right: Increased [11C]-R-PK11195 binding sites in
the frontal lobe particularly in the anterior cingulate cortex (ac) consistent with microglial activation
and neuroinammation in this patient (provided by Dr. Simon Taylor-Robinson, Imperial College,
London, UK)
Increased binding sites for this PET ligand have already been reported in cirrhotic
patients with HE [40], with particular intense signals observed in anterior cingulate
cortex, a structure known to be associated with the control of attention (Fig. 3.5).
These ndings suggest a potential application of 11C-PK11195 PET for the assess-
ment of neuroinammation in brain in relation to cognitive dysfunction in end-stage
chronic liver disease.
Studies show that existing therapies for the treatment of HE in acute or chronic
liver failure that were presumed to act by lowering levels of circulating ammonia
may also act by reducing levels of proinammatory cytokines. There are several
30 R.F. Butterworth
such treatments. For example, treatment of cirrhotic patients with lactulose leads to
decreased severity of HE and reductions of both circulating ammonia and TNF-a
[12] and use of the albumen dialysis (MARS) system in patients with ALF resulted
in removal of TNF-a and in clinical improvement with better outcome [55].
Antibiotics including rifaximin that are effective in prevention of recurrence of HE
in cirrhotic patients [56] are also known to reduce circulating levels of
proinammatory cytokines [12]. In a study of cirrhotic patients with MHE treated
with synbiotics for 30 days, increased fecal content of non-urease producing species
and concomitant reductions in circulating levels of both ammonia and endotoxin
were observed along with reversal of MHE in 50% of patients compared to 13% in
the control arm of the trial [57]. Another example of an existing therapy that acts
by reduction of inammation is mild hypothermia which is increasingly being
used in the management of the CNS complications of ALF [58, 59]. Mechanisms
implicated in the mediation of the benecial effects of hypothermia in ALF involve
anti-inammatory mechanisms at both the hepatic and cerebral levels (see below).
The discovery of neuroinammation and central neuroinammatory mechanisms
in liver failure will undoubtedly provide new therapeutic targets. Already, studies in
experimental animal models of ALF or chronic liver failure have assessed the
benecial effects of known anti-inammatory agents in relation to the cerebral
complications of liver failure. Signicant improvement of locomotor impairment
following administration of indomethacin in portal vein-ligated rats was accompanied
by prevention of a rise in IL-6 mRNA [23]. Ibuprofen was also reported to improve
learning ability [22] and locomotor decits [51] in portacaval-shunted rats but, in
this case, the protective effect was independent of action on increased brain cytokine
levels. Ibuprofen has also been shown to signicantly reduce neuroinammation in
bile duct-ligated rats where it was found to inhibit microglial activation and restore
cognitive and motor function in these animals [10]. However, in this latter
study, ibuprofen was also found to normalize circulating and brain ammonia levels,
suggesting that effects on systemic inammation and improvement of hepatic func-
tion may also have contributed to the benecial effects of ibuprofen. The disparate
ndings of the effects of anti-inammatory drugs in different experimental models
of liver failure likely reect differences in the degree of systemic vs. neuroinammation
in these models.
Therapies directly targeting neuroinammatory processes include those aimed at
inhibition of microglial activation or inhibition of the actions of proinammatory
cytokines. One such example is mild hypothermia. Just two degrees of hypothermia
have been shown to delay the onset of HE, prevent brain edema, and impair both
microglial activation and the increased expression of genes coding for
proinammatory cytokines [6]. A more recent study showed that deletion (knock-
down) of the gene coding for TNF-a or IL-1b likewise delays HE onset and attenu-
ates brain edema in mice with ALF resulting from toxic liver injury [7]. Preliminary
studies demonstrate that treatment with the TNF-a receptor antagonist etanercept
likewise led to slowing in progression of HE and prevention of brain edema in
experimental ALF [31].
Fig. 3.6 Increased expression of OX-6 indicative of microglial activation in the brains of rats with
ALF due to hepatic devascularization at coma/edema stage of encephalopathy (ALF coma).
Treatment with minocycline resulted in delayed progression of HE, prevention of brain edema, and
attenuation of the increase of OX-6 expression (ALF mino) (reprinted by permission from
Macmillan Publishers Ltd: Jiang et al. [6] 2009)
An interesting new dimension in the search for novel anti-inammatory agents

for potential application in the treatment of the CNS complications of liver failure
was recently provided by the report that minocycline, an agent with well-established
potent inhibitory properties on microglial activation that are independent of its
antimicrobial properties [60], inhibits proinammatory cytokine production in
brain, delays progression of HE, and attenuates brain edema in experimental ALF
[44] (Fig. 3.6).
Translation of these interesting leads to the clinic has the potential to provide
novel, targeted strategies for the management and treatment of the CNS complications
of acute and chronic liver failure in the near future.
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Chapter 4
Inflammation and Hepatic Encephalopathy
Shabnam S. Shabbir, Amit Singh Seyan, and Debbie Lindsay Shawcross
Keywords Inammation Infection Hepatic encephalopathy Ammonia

Oxidative stress Neutrophils
Introduction
Following the seminal studies on portacaval shunted dogs by Nencki, Pavlov and
Zaleski in the 1890s, it is widely agreed that ammonia plays an important role in the
pathogenesis of hepatic encephalopathy (HE) [1]. In acute liver failure, arterial
ammonia concentrations of >150 mmol/L predict a greater likelihood of dying from
brain herniation [2], and intracranial hypertension develops in 55% of patients with
an arterial ammonia concentration of >200 mmol/L [3]. However in patients with
cirrhosis, the relationship is less clear cut. There is conicting evidence regarding
the relationship between blood ammonia concentration and the development of
covert (minimal) and overt HE. Clinically, it is not unusual to nd patients with
cirrhosis presenting with overt HE with normal or only mildly elevated blood
ammonia concentrations [4, 5]. Indeed, studies have shown single blood ammonia
The authors Shabnam S. Shabbir and Amit Singh Seyan contributed equally to this chapter.
S.S. Shabbir, BSc, MBBS A.S. Seyan, MBBS, BSc (Hons)
Kings College School of Medicine, Kings College London,
London, UK
D.L. Shawcross, BSc, MBBS, FRCP, PhD (*)
Institute of Liver Studies, Kings College School of Medicine at Kings College Hospital,
Denmark Hill, London SE5 9RS, UK
e-mail: debbie.shawcross@kcl.ac.uk
36 S.S. Shabbir et al.
concentrations to correlate unreliably with the severity of HE [6]. It is therefore

reasonable to postulate a key role for other factors in the pathogenesis of HE. The
idea that the pathogenesis of HE might involve synergism between several toxins
was rst suggested by Zieve et al. [7], and this hypothesis has since evolved to
include the complex role of inammation and oxidative stress in the presence and
absence of infection.
Infection Versus Inflammation in Acute and Chronic Liver

Failure
In both acute and chronic liver failure, patients are functionally immunosuppressed
because of a signicant reduction in liver synthetic function and impairment of host
defence mechanisms. This makes them highly susceptible to developing infections,
which can complicate their clinical course leading to the development of organ failure
and death [8, 9]. Rolando et al. have demonstrated evidence of infection in up to
90% of patients early in the course of acute liver failure [10]. Other indicators of the
presence of inammation and infection in acute liver failure come from studies
demonstrating raised proinammatory cytokines, including TNF-a, IL-1b and IL-6
which have been associated with poorer outcomes and the development of cerebral
edema and intracranial hypertension [11, 12].
In patients with cirrhosis, the increased susceptibility to infection is thought to be
multifactorial. One important contributing factor is neutrophil dysfunction.
Neutrophils play a key role in the early innate immune response of the body by
engulng foreign microbes and debris by a process known as phagocytosis. They
then eliminate the engulfed foreign bodies through the generation of an oxidative
burst, whereby reactive oxygen species are released into the phagosomes. These
reactive oxygen species not only kill invading micro-organisms but may also cause
damage to nearby tissues causing local inammation, tissue destruction and organ
failure. Neutrophil dysfunction is prevalent in patients with cirrhosis [13], particu-
larly where alcohol is implicated in the aetiology [14, 15] and is associated with a
signicantly greater risk of infection, organ failure and mortality.
The terms inammation and infection are frequently used interchangeably.
Although functionally related, they should be treated as separate entities. The sys-
temic inammatory response syndrome (SIRS) results from the release into the cir-
culation of proinammatory mediators and cytokines that can arise directly from
hepatocyte injury, e.g. acetaminophen hepatotoxicity, or can arise peripherally from
the production of reactive oxygen species and concomitant tissue injury, e.g. isch-
emia or burns. Alternatively, this will occur as sequelae from local or systemic
infection. Frequently, it is impossible to delineate these phenomena in patients with
liver failure, especially those with cirrhosis who may have low grade endotoxemia
resulting from bacterial translocation across the gut into the portal circulation [16].
4 Inammation and Hepatic Encephalopathy 37
This population is prone to developing infection which is difcult to conrm with

microbial cultures which have a low yield.
Inammation results from the activation of circulating immune cells, interaction
with the endothelium and multiple mediator cascades balanced by an anti-
inammatory system which include the cytokines IL-4, IL-10 and IL-13. Following
injury, proinammatory mediators are released locally to combat foreign antigens
and promote wound healing. This is balanced by the release of anti-inammatory
mediators which downregulate and prevent excess inammation (compensatory
anti-inammatory responseCARS). If the inammatory response is not con-
trolled, the proinammatory mediators enter the systemic circulation leading to
neutrophil recruitment and activation. When homeostasis is disturbed resulting in
an exaggerated SIRS or CARS, then immunological dissonance occurs which can
lead to cellular immune depression, multiorgan dysfunction and death [17]. This is
frequently seen in those with acute or chronic liver failure, particularly in the con-
text of severe sepsis. The extent of inammation is also dependent on the aetiology
of the liver injury, e.g. alcoholic hepatitis, and the severity of the underlying liver
disease. Infection is a frequent precipitant of HE and it is not unusual for changes in
mental state to be the sole manifestation of infection in this cirrhotic cohort.
Infection and the BloodBrain Barrier
In the absence of liver disease, it is widely accepted that sepsis can cause agitation
and delirium. This can progress to a condition known as sepsis-associated enceph-
alopathy, which encompasses a range of changes in motor activity and mental status,
ranging from delirium to coma. Asterixis, paratonic rigidity, tremor and myoclonus
may even be observed. It is thought that these changes occur due to a reduction in
cerebral blood ow, changes in brain metabolites and amino acids, and disruption to
the bloodbrain barrier resulting from the direct effect of inammatory cytokines
on the endothelium of the bloodbrain barrier [18]. Although sepsis-associated
encephalopathy is distinctly different to HE from a pathophysiological standpoint,
it is not inconceivable that infection may induce changes in mental status in patients
both with and without liver disease.
Inammatory mediators are able to signal the brain through activation of afferent
neurons of the vagus nerve, interaction of cytokines with circumventricular organs
or via the direct effect of active transport across the bloodbrain barrier [19].
Furthermore, endothelial cells and astrocytes, integral parts of the bloodbrain
barrier, can be stimulated to release a full repertoire of immune mediators into the
brain activating neurons and microglial cells. Endothelial cells have receptors for
IL-1b and TNF-a, which can alter the integrity of the bloodbrain barrier and
activate signalling pathways leading to the intracerebral synthesis of nitric oxide
and prostanoids [20].
Infection and Inflammation Modulate Hepatic Encephalopathy
Over the past decade there has been a growing evidence base implicating infection
as being important in the manifestation of HE in acute and chronic liver failure. It
was rst shown by Nancy Rolando at Kings that patients with acute liver failure
progress more quickly to severe HE if they have signs of systemic inammation
[21], and in a study by the US Liver Failure Group, in patients with acute liver fail-
ure induced by acetaminophen, the consequent systemic inammatory response
was a signicant contributor to the severity of HE [22]. Liver-derived proinammatory
cytokines are important in driving cerebral edema and intracranial hypertension in
acute liver failure [11]. Furthermore, the brain itself produces a number of
proinammatory cytokines in patients with acute liver failure and advanced cere-
bral edema [12]. When interventions such as hypothermia are utilised, a reduction
in intracranial hypertension can be seen resulting from a reduction in cerebral blood
ow, brain ammonia uptake, oxidative stress and systemic inammation [23, 24].
In patients with cirrhosis, the role of systemic inammation in exacerbating HE
has also become evident. Studies have shown that those patients with minimal HE
have elevated plasma levels of inammatory markers, and the severity of the HE is
not indicative of the liver disease severity nor of plasma ammonia levels [25]. The
synergistic effect of inammation and ammonia has been demonstrated in a cir-
rhotic population admitted with infection and given an amino acid load to temporar-
ily and reversibly induce hyperammonemia. Patients had deterioration in
neuropsychological tests scores during infection but not after its resolution, provid-
ing evidence in support of infection modulating the effects of ammonia on the brain
[26]. In a large study of patients with cirrhosis from Kings College Hospital admit-
ted to intensive care with the primary indication of severe HE (grades 3 and 4),
almost 50% of patients were found to have culture-positive infection and a further
22% had sterile SIRS. Arterial ammonia concentration and blood biochemistry
were found not to correlate with the severity of HE supporting the theory that infec-
tion and inammation, not hyperammonemia, have the more pivotal role in increas-
ing the severity of HE [5].
Infection and Inflammation Act Synergistically with Ammonia
The notion of the existence of a synergistic relationship between inammation,

infection and ammonia has been examined in several studies. A mouse model with
chronic hyperammonemia was shown not only to have an increased sensitivity to
inammation but signicant cognitive defects when exposed to an inammatory
stimulus [27]. Jover et al. used a rat model to demonstrate neurological changes
following bile duct ligation and were fed a hyperammonemic diet. These rats had
increased levels of cerebral ammonia and type II Alzheimer astrocytosis, similar to
that seen in patients with HE and cirrhosis. These rats also had signs of systemic
inammation and low grade brain edema. All these changes contributed to impaired
motor activity on co-ordination tests [28]. Wright et al. in another bile duct-ligated
rat model were able to show that lipopolysaccharide administration increased brain
water in ammonia-fed, bile duct-ligated and sham-operated animals signicantly,
but this was associated with progression to pre-coma only in the bile duct-ligated
animals. Lipopolysaccharide induced cytotoxic brain edema but the bloodbrain
barrier remained intact. Nitrosation of brain proteins was seen in the lipopolysac-
charide-treated, bile duct-ligated animals only suggesting subliminal inammation
may be a pre-requisite to the development of HE [29]. In a portacaval-shunted rat
model mimicking minimal HE, Cauli et al. showed that administrating a high dose
of ibuprofen, a non-steroidal anti-inammatory, resulted in improved ability to
learn. This was thought to occur through normalisation of the glutamatenitric
oxidecyclic GMP pathway in the cerebral cortex, and so supports the fact that
inammation is pivotal to the development of cognitive impairment in HE [30]. The
non-selective cyclo-oxygenase (COX) inhibitor indomethacin has been demon-
strated to be effective in reducing intracranial hypertension in patients with acute
liver failure [31, 32] and in a portacaval-shunted rat model [33].
Immune Dysfunction and Oxidative Stress in Hepatic

Encephalopathy
Systemic immune dysfunction in acute and chronic liver failure and the resultant
oxidative stress response play an irrefutable role in the development of HE particu-
larly in the context of elevated blood ammonia concentrations [34]. In a proof of
concept study, ammonia was shown to lead to signicant neutrophil malfunction.
This led to a reduced capacity to engulf opsonised Escherichia coli and high spon-
taneous oxidative burst. These observations were replicated in ammonia-fed rats
and ex vivo in patients with cirrhosis given a simulated upper gastrointestinal bleed
inducing hyperammonemia compared to controls [35]. The mechanism underlying
this neutrophil malfunction was shown to be related to the development of ammonia-
induced cell swelling resulting from an inability of a key osmoregulator p38MAPK to
regulate neutrophil volume. This has interestingly also been replicated in hepato-
cytes [36] and astrocytes [37].
Small bowel overgrowth and increased bacterial translocation from the gut due
to breakdown in mucosal barrier function can result in bacterial burden being deliv-
ered to the liver via the portal vein. The presence of porto-systemic shunting results
in the bypassing of the reticuloendothelial system and delivery of low-grade endotoxin
to the systemic circulation. Bacteria and bacterial by-products such as endotoxin
can activate various immune cells, either directly through pattern-recognition recep-
tors such as Toll-like receptors (TLRs) or through the generation of proinammatory
and anti-inammatory cytokines. Priming of circulating neutrophils through such
mechanisms can lead to changes in surface receptors, conformational changes in
Fig. 4.1 Pictorial representation of the interface between the systemic inammatory response and
the bloodbrain barrier in acute and chronic liver failure. A cytotoxic soup of ammonia (NH3),
lipopolysaccharide (LPS), chemokines and cytokines and bacteria or bacterial peptides can lead to
endothelial interaction, neutrophil activation and degranulation at the bloodbrain barrier. Granules
(containing substances such as myeloperoxidase) and chemokines can induce astrocyte and micro-
glial activation and neuronal dysfunction. In patients with overt sepsis resulting in an overlap
between hepatic encephalopathy and sepsis-related encephalopathy, neutrophils and monocytes
may even be able to directly pass across the bloodbrain barrier
binding ligands and increased metabolic demand. This ultimately leads to alterations
in phagocytic capacity and bacteriocidal function [14, 38]. Thus, in a patient with
cirrhosis, hyperammonemia and chronic endotoxemia pre-primed neutrophils may
enhance endothelialneutrophil interaction within the cerebral microcirculation
(Fig. 4.1). The cerebral effects of ammonia will therefore potentially have their
greatest impact in this inammatory milieu. This may be exacerbated by astrocytes
producing chemokines that may attract and recruit neutrophils and other immune
cells [39].
Targeting Inflammation in the Treatment of Hepatic

Encephalopathy
In patients with acute liver failure, the main therapeutic goal along with liver trans-
plantation is to lower arterial ammonia. Hemoltration of the blood is highly
efcacious in removing ammonia and is now a standard of care [40]. However in
patients with cirrhosis, treatments that focus on lowering arterial ammonia and
modulating interorgan ammonia metabolism are less effective. The main therapeu-
tic target will depend on the nature of the HE but reversing any precipitating factor
should always be considered as a priority; infection being the most common precipi-
tant particularly in those presenting with the severest grades of HE [5].
The use of absorbable and non-absorbable antibiotics has become well estab-
lished in the treatment of HE in patients with cirrhosis [41]. However, some antibi-
otics, such as neomycin, vancomycin and metronidazole that have been used to
effectively reduce the production of ammonia by gut bacterial ora, have nephro-
toxic and ototoxic effects as well as the potential to cause a peripheral neuropathy.
Rifaximin is a minimally absorbed antibiotic, with broad spectrum activity and is
concentrated in the gastrointestinal tract. Bass et al. performed a randomised, dou-
ble-blind, placebo-controlled trial enrolling 299 patients with cirrhosis who were
currently in remission from HE. Rifaximin was signicant in reducing the risk of
developing an episode of HE when compared to a placebo, over a period of 6
months; not only did rifaximin maintain remission from HE, but also reduced the
risk of hospitalisation [42]. Bajaj et al. assessed whether patients with minimal HE
had an improved driving performance after treatment with rifaximin. Patients were
either assigned to placebo or rifaximin for 8 weeks, undertaking driving simulation
at the beginning and end of the 8-week study period. Patients taking rifaximin had
fewer total driving errors than the placebo group. Ninety one per cent of patients on
rifaximin improved their cognitive performance compared to 61% of patients on
placebo. Patients taking rifaximin had an improved sickness impact prole and
increased interleukin-10 levels suggesting that rifaximin may be more than a modu-
lator of gut ora but may lead to reduced bacterial translocation across the gut and
systemic inammation [43].
As the role of infection and inammation in mediating HE has become estab-
lished, therapies that target inammation and modulate the immune system have
been of interest to hepatologists. However in doing this, one must also remember
that augmenting immune function can lead to damage of normal healthy tissue and
organs. The use of granulocyte colony-stimulating factor [44], leucodepletion, [45]
antagonism of proinammatory cytokines or their receptors, anti-inammatory
(COX inhibitors) [31], antioxidants (N-acetylcysteine [46] and albumin), probiotics
[47] and hypothermia [23] all hold potential. Inducing a hypothermic state has the
benet of decreasing brain ammonia, cerebral blood ow as well as inammatory
mediators and oxidative stress, particularly in those with the severest grades of HE
[23]. Moderate hypothermia abolishes ammonia-induced neutrophil spontaneous
oxidative burst without impairing phagocytic capacity, suggesting that hypothermia
could be a valuable tool not only in patients with acute liver failure, but also those
with cirrhosis and grade 3/4 HE [48].
Patients with end-stage cirrhosis have alterations in the functional capacity of
albumin which can act as an endotoxin scavenger and may explain the benecial
effects of albumin infusion and dialysis on HE [49].
Jiang et al. showed that treatment with the antibiotic minocycline in rats with
acute liver failure prevented central microglial activation and upregulation of many
proinammatory mediators including IL-1b, IL-6, TNF-a, haeme-oxygenase-1,

eNOS, iNOS mRNA and protein expression, slowing progression of HE, in part due
to a reduction in nitrosative and oxidative stress [50]. This supports minocycline as
being a promising new candidate drug in HE and being taken forward into a ran-
domised, placebo-controlled trial in patients with acute and acute-on-chronic liver
failure and severe HE.
The use of TLR-2, TLR-4 and TLR-9 inhibitors and molecules involved in
TLR-4 signalling could downregulate overactive neutrophil responses. Another
therapeutic option could be to modulate the microbiota of the intestine, in turn pre-
venting bacterial translocation of lipopolysaccharide and bacteria that activate
TLRs. Probiotics have been shown to improve liver function, reduce infection and
the development of minimal HE in cirrhosis [47]. Patients with alcohol-related
cirrhosis given probiotics had improved neutrophil phagocytic activity possibly
resulting from reduced interleukin-10 and TLR-4 expression [51].
Summary
This chapter has highlighted the fundamental role that infection and inammation
plays in the development of HE in acute and chronic liver failure. No longer can
ammonia be thought of as the sole perpetrator of HE but instead there is a synergistic
relationship between inammation in modulating the cerebral effects of ammonia.
It has been shown that astrocytes and endothelial cells at the bloodbrain barrier
respond to a systemic inammatory stimulus and play a role in eliciting an
inammatory response which incorporates a number of close knit proinammatory
and neurotransmitter pathways.
Ammonia is not only directly toxic to astrocytes but induces immune dysfunc-
tion leading to the release of reactive oxygen species which contributes to systemic
inammation and an increased vulnerability to ghting microbial invasion. Increased
neutrophil and endothelial cell interaction at the bloodbrain barrier may even play
a direct pathogenic role analogous to that seen in sepsis-related encephalopathy.
In addition to direct ammonia-lowering strategies, targeting systemic inammation
and infection is therefore key in developing effective treatments for HE. Furthermore,
the neutrophil and other components of the innate and adaptive immune systems
should be considered as legitimate novel pharmacotherapeutic targets for drug
development in the future.
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Chapter 5
Oxidative Stress in Hepatic Encephalopathy
Arumugam R. Jayakumar and Michael D. Norenberg
Keywords Astrocytes Brain edema Hepatic encephalopathy Inammation

Intracellular signaling systems Oxidative/nitrative stress
Introduction
Hepatic encephalopathy (HE) occurs in both acute and chronic liver disease. Chronic
(or Type C) HE usually occurs in patients with underlying cirrhosis. It is character-
ized by impaired neurological function, including changes in personality, altered
mood, diminished intellectual capacity, and abnormal muscle tone and tremor [1].
HE in acute liver failure (fulminant hepatic failure) occurs following massive liver
necrosis due to viral hepatitis (hepatitis B and C), hepatic neoplasm, vascular causes,
acetaminophen toxicity, or exposure to various hepatotoxins. ALF is associated
with the abrupt onset of delirium, seizures, and coma. Cerebral edema with increased
intracranial pressure and brain herniation occurs in up to 80% of patients with ALF
and represents the most frequent cause of death in these patients [2, 3].
To date, the precise mechanism responsible for the development of both acute
and chronic HE is not known. Increased blood and brain ammonia has been consid-
ered an important pathogenetic factor and astrocytes appear to be the major cell type
involved in its pathogenesis [4]. While the precise means by which ammonia causes
A.R. Jayakumar, PhD

Department of Neuropathology, South Florida Foundation for Research
and Education Inc., Miami VA Medical Center, Miami, FL, USA
M.D. Norenberg, MD (*)
Department of Pathology, Biochemistry and Molecular Biology, Jackson Memorial Hospital,
Miami VA Medical Center, University of Miami Hospital, 1611 NW,
12th Avenue, Miami, FL 33136, USA
e-mail: mnorenbe@med.miami.edu
48 A.R. Jayakumar and M.D. Norenberg
neurotoxicity in HE are not clear, ammonia has been shown to impair bioenergetics,
alter neurotransmission, cause electrophysiologic derangements, promote glutamate-
mediated excitotoxicity, and stimulate various intracellular signaling pathways [5].
More recently, oxidative/nitrative stress (ONS) has been viewed as an important
pathogenetic factor in HE. This chapter will summarize the involvement of ONS in
the mechanism of HE, its consequences and potential role in therapy.
Oxidative/Nitrative Stress
Studies in Experimental Animals
Oxidative Stress
Evidence for the involvement of oxidative stress (OS) in HE initially arose from the
observation that Alzheimer type II astrocytes, a prominent neuropathological
component of HE, contain large amounts of lipofuscin pigment (indication of
peroxidized lipids) [6] (Fig. 5.1). Excessive amounts of lipofuscin pigment were
also detected in ammonia-treated astrocyte cultures [7, 8]. Subsequently, OConnor
and Costell [9] documented the presence of lipid peroxidation, a marker of OS,
Fig. 5.1 An Alzheimer type II astrocyte showing an enlarged and vacuolated nucleus containing
a prominent nucleolus that is adherent to the nuclear membrane. No well-dened cytoplasm is
evident, except for the presence of lipofuscin pigment granules (arrows). Two normal-sized astro-
cyte nuclei are present below the Alzheimer type II cell that also contain lipofuscin pigment
5 Oxidative Stress in Hepatic Encephalopathy 49
Fig. 5.2 Oxidation of brain proteins in TAA-treated rats. Oxidized proteins were detected by
Western blot analysis with 2-DNPH. Proteins ranging in molecular weight from 90 to 40 kDa, as
well as 3224 kDa were highly oxidized in TAA-treated rats (lanes T14 from four separate
animals) as compared to sham controls (C13 are controls from three separate animals)
in brains of hyperammonemic mice. These ndings were further elaborated by

Kosenko et al. [1013], who showed an increase in superoxide production and lipid
peroxidation, as well as a decrease in the activity of various antioxidant enzymes
(glutathione peroxidase, manganese superoxide dismutase, and catalase) in rat brain
cerebral cortex after acute hyperammonemia. Similar ndings were also observed
in the cerebellum of rat after an acute ammonia infusion [14], as well as in cerebral
cortex of rats with thioacetamide (TAA)-induced ALF [1517].
Increased hydrogen peroxide production [15, 18], elevated levels of oxidized
proteins [19], and a decreased level of reduced glutathione as compared to oxidized
glutathione (GSH/GSSG ratio) were identied in cerebral cortex of rats with TAA-
induced ALF [15]. Decreased GSH/GSSG ratio was also detected in cerebral cortex
of mice with azoxymethane-induced ALF [20, 21]. Hemeoxygenase-1 (HO-1), an
enzyme that catalyzes the degradation of heme to iron and carbon monoxide and a
marker of OS, was upregulated in hepatic devascularized rats [22, 23], in mice with
azoxymethane-induced ALF [24], as well as in TAA-induced acute liver failure in
rats [25]. Infusion of ammonia into the striatum of rats was shown to produce
hydroxyl radicals [26], while an increase in oxidized proteins was found in cerebral
cortex of rats after TAA-induced acute liver failure (Fig. 5.2).
HO-1 upregulation was also observed in rat brain in a chronic model of HE
(portacaval-shunted rat) [27], and recently, Carbonero-Aguilar et al. [28] documented
increased levels of malondialdehyde and hydroxynonenal in brains of portacaval-
shunted rats. These studies strongly suggest that oxy-radicals and their derivatives
are also produced in chronic HE. However, Yang et al. [29] found no changes in the
level of oxidative stress markers in portacaval-shunted rats. Potential explanations
for these conicting results may be the time of study selected (4 weeks vs. 6 weeks)
and/or the sensitivity of the methods used to detect oxidative stress markers.
Nitrative Stress
Similar to OS, nitrative stress can also alter protein structure and potentially inter-
fere with their cellular function. Nitrative stress has been documented in brains of
Fig. 5.3 Protein tyrosine

nitration from brains of
TAA-treated rats. Protein
tyrosine nitration was detected
by western blot analysis with
an antibody raised against
3-nitrotyrosine. Only two
proteins are observed to be
nitrated in sham treatment
(control lane). By contrast,
many proteins (between 85
and 40 kDa as well as
17.5 kDa) are observed to be
highly nitrated (TAA-1 and 2
lane). Increase in protein
nitration is also observed in
TAA-treated rat brain at 132
and 2030 kDa
acute and chronic liver failure. Increased nitric oxide synthase (NOS) activity,
inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS)
protein expression, along with increased protein tyrosine nitration were observed in
portacaval-shunted rats [3032]. Increased protein tyrosine nitration was also
observed in brains of rats with chronic liver failure produced by a low dose admin-
istration of TAA [33].
Increased brain nitric oxide (NO) production was observed in portacaval-shunted
rats given an ammonia infusion, a model of acute liver failure [34]. Subsequently,
elevated level of iNOS protein expression was demonstrated in rat brain astrocytes
after acute ammonia infusion [35]. Increased endothelial nitric oxide synthase
(eNOS) and iNOS protein expression were identied in brains of hepatic devascu-
larized rats [22, 23] as well as following ischemic liver damage in rats [36]. Likewise,
increased iNOS protein expression [37] and NO production were detected in brains
of mice and rats, respectively, in TAA-induced acute liver failure [15]. Additionally,
we found an increase in protein tyrosine nitration in cerebral cortex of rats after
TAA-induced acute liver failure (Fig. 5.3).
Studies in Astrocyte Cultures
Oxidative Stress
While changes in OS markers have been demonstrated in different animal models

of liver failure, much of the evidence for a role of OS in ammonia neurotoxicity
has been derived from cell culture studies (for review, see Norenberg et al. [5]).
Fig. 5.4 Time-dependent

changes in free radical
production following
treatment with ammonia
(5 mM NH4Cl) in cultured
astrocytes. *p < 0.05 vs.
control
A signicant decrease in cellular glutathione (GSH) level was rst detected in

ammonia-treated astrocyte cultures [38]. Since increased GSH is a major free
radical scavenging system [3941], we investigated whether ammonia produces
free radicals in cultured astrocytes. Cultured astrocytes exposed to a pathophysio-
logical concentration of ammonia (5 mM NH4Cl) were found to stimulate the
production of free radicals. Ammonia-induced free radical generation, including
the activation of NADPH oxidase, was observed in cultured astrocytes [4244]
(Fig. 5.4), while increased HO-1 expression was identied in cultured astrocytes
after ammonia treatment [45].
In addition to free radical production, a variety of morphological abnormalities,
including enhanced stellation, a highly basophilic cytoplasm, prominent vacuoles
and dense bodies were demonstrated in astrocyte cultures that had been exposed to
ammonia [7, 8, 46], and such effects were diminished by the antioxidants SOD and
catalase [46]. Furthermore, natriuretic peptides, which are known to attenuate the
production of reactive oxygen species (ROS) in other systems [47, 48], were shown
to reduce the accumulation of ROS in ammonia-treated cultured astrocytes [49].
Altogether, these studies suggest that oxy-radicals are produced by astrocytes in
conditions associated with hyperammonemia.
Nitrative Stress
Astrocyte cultures exposed to ammonia caused an increase in iNOS protein expres-

sion as well as NO production [35, 43]. Ammonia was shown to increase soluble
guanylyl cyclase (a source of NO) in cultured astrocytes [50]. Additionally, stimula-
tion of natriuretic peptide receptor C attenuated NOS activity in ammonia-treated
astrocytes [49].
Studies in Humans
While considerable evidence indicates the presence of oxidative stress markers

in experimental models of HE, documentation of OS in humans is limited.
Increased amount of lipofuscin pigment was found in brains of patients with HE

[6, 51]. Elevated blood levels of free radicals were also identied in patients with
HE resulting from chronic alcohol abuse which was associated with diminished
antioxidative capacity [52]. Increased SOD activity, thiobarbituric acid reactive
substances, and decreased catalase activities were observed in cirrhotic children
[53]. Increased NO after transjugular intrahepatic portosystemic shunt (TIPS) inser-
tion in patients with cirrhosis [54], as well as elevated levels of tyrosine-nitrated
proteins, heat shock protein-27, and 8-hydroxyguanosine (markers of RNA oxida-
tion), was described in the cerebral cortex in patients with HE [55].
Mechanisms of ONS Formation
Intracellular Calcium
While the precise mechanism by which ammonia generates free radicals is not clear,
the elevation of intracellular Ca2+ ([Ca2+]) is likely an important factor as Ca2+ has
been shown to stimulate the production of RONS in other conditions [5658]. It is
noteworthy that a rise in [Ca2+]i was shown to be an early event following ammonia
exposure to cultured astrocytes [35, 59, 60]. Consistent with these ndings, we
recently reported that treatment of astrocyte cultures with the Ca2+ chelator, 1,2-bis-
(o-aminophenoxy)-ethane-N,N,-N,N-tetraacetic acid tetraacetoxy-methyl ester
(BAPTA), signicantly blocked the ammonia-induced production of free radicals
[44]. The ammonia-induced increase in [Ca2+]i is likely due to a rise in intracellular
pH since trimethylamine, a weak base, also increased [Ca2+]i concentration in
cultured astrocytes [60].
Ca2+ generates RONS likely through the activation of various Ca2+-dependent
enzymes, including constitutive nitric oxide synthase (cNOS) [61], the cytosolic
form of phospholipase A2 (cPLA2) [62] whose product, arachidonic acid (AA), is
known to produce free radicals [63] and NADPH oxidase (NOX) [44, 64], all of
which generate superoxides. We recently reported that astrocytes exposed to ammo-
nia showed increased activities of cNOS, NOX, and PLA2 and that pretreatment of
cultures with their respective inhibitors blocked free radical production [44].
Additionally, ammonia-induced increase in cNOS, and PLA2 and NOX activities
were blocked by BAPTA [44].
The Mitochondrial Permeability Transition in ONS Production
One factor that is known to induce free radicals is the mitochondrial permeability
transition (mPT), a Ca2+-dependent process associated with a collapse of the inner
mitochondrial membrane potential due to a sudden opening of the permeability
transition pore (PTP) in the inner mitochondrial membrane [6567]. Opening of the
pore increases the permeability of the inner mitochondrial membrane to protons,
ions, and other small solutes (<1,500 Da) resulting in a collapse of the inner
mitochondrial membrane potential which then leads to mitochondrial dysfunc-
tion and enhanced free radical production [68, 69].
While OS is a consequence of the mPT, it can also be a major cause of the mPT
[70, 71]. This may occur due to the oxidation of pyridine nucleotides [72], which
diminishes glutathione levels, thereby decreasing the activity of glutathione
peroxidase resulting in free radical production and induction of the mPT [71].
Additionally, ROS-mediated oxidation of thiol groups on mitochondrial proteins
can result in pore opening [70].
Electron Transport Chain
Another pathway by which ammonia may generate free radicals is through inhibition
of the mitochondrial electron transport chain (ETC). Hyperammonemia has been
shown to inhibit the ETC in brain [73, 74]. It was recently demonstrated that rats
treated with the liver toxin carbon tetrachloride signicantly inhibited complexes I, II,
and IV in brain. Such effects were reversed by treatment of animals with the antioxi-
dant N-acetylcysteine (NAC) or with the iron chelator desferrioxamine (DFX) which
inhibits lipid peroxidation and hydroxyl radical production created by the Fenton
reaction [75, 76]. Similar ndings were observed in rat brain after acetaminophen-
induced liver failure [77]. Ammonia is also known to inhibit the activity of
a-ketoglutarate dehydrogenase (a-KGDH). Such inhibition is known to diminish
FADH2 and NADH formation in the Krebs cycle, leading to NAD hyperoxidation and
subsequent inhibition of the ETC, ultimately resulting in free radical formation [78].
Nuclear Factor-Kappa B
Nuclear factor-kappa B (NF-kB) is a major transcription factor known to activate

many genes, including NOX, PLA2, and iNOS that cause excessive oxy-nitro
radical formation [79, 80]. It has been shown that cultured astrocytes exposed to
ammonia caused the activation of NF-kB [35, 43] and inhibition of such activation
attenuated ammonia-induced upregulation of iNOS protein expression and the
subsequent generation of NO [35, 43]. It was also recently demonstrated that astro-
cyte cultures from transgenic (Tg) mice with a functional inactivation of astrocytic
NF-kB exhibit a lesser increment in iNOS and NADPH oxidase activity after
ammonia treatment as compared to astrocytes derived from WT mice [37].
N-Methyl D-Aspartate Receptors
Activation of N-methyl d-aspartate (NMDA) receptors can be an additional source

of free radicals in HE. Acute ammonia intoxication was shown to activate NMDA
Fig. 5.5 Metabolism of ammonia (NH4+) in astrocytes resulting in the mitochondrial production
of reactive oxygen species (ROS). Glutamine, synthesized by glutamine synthatase (GS), enters
mitochondria through the glutamine transporter (GLN-Tx). Glutamine is then hydrolyzed by phosphate-
activated glutaminase. The unloading of ammonia from glutamine led to the so-called Trojan
horse hypothesis of ammonia neurotoxicity. GLU glutamate
receptors in rat brain [13], and inhibition of the receptor with MK-801 reversed the
ammonia-induced decrease in the activity of various antioxidant enzymes (glutathi-
one peroxidase, manganese superoxide dismutase, and catalase) in rat brain [13, 81,
82]. These studies indicate that ammonia-induced oxidative stress in brain was
mediated, in part, by excessive activation of NMDA receptors. It should be noted
that in addition to their neuronal localization, NMDA receptors are also expressed
in astrocytes [8385]. Activation of these receptors is well known to increase
intracellular calcium [86]. It is thus possible that increased intracellular calcium,
by activation of NMDA receptors, may also contribute to the formation of free
radicals in HE.
Glutamine
The synthesis of glutamine in astrocytes has generally been viewed as the principal
means of ammonia detoxication in brain. However, recent studies suggest that
some, if not most, of the deleterious effects of ammonia may actually be mediated
by glutamine rather than ammonia per se. Studies have shown that many of the
ammonia effects on astrocytes can be inhibited by interference with the synthesis of
glutamine, by blocking the entry of glutamine into mitochondria, or by inhibition of
mitochondrial glutamine hydrolysis [87, 88]. These ndings indicate that glutamine
hydrolysis in mitochondria and the subsequent increase in mitochondrial ammonia
content constitute a major pathway by which ammonia neurotoxicity occurs (the
Trojan horse hypothesis) (Fig. 5.5).
Treatment of cultured astrocytes with 4.5 mM glutamine was shown to increase
free radical production [89], which was blocked by cyclosporine A (CsA), an inhibi-
tor of the mPT. It was also blocked by 6-diazo-5-oxo-l-norleucine (DON), an inhib-
itor of phosphate-activated glutaminase, suggesting that mitochondrial ammonia
released by glutamine hydrolysis is responsible for the generation of free radicals.
Additionally, it was recently shown that l-histidine, an inhibitor of mitochondrial

glutamine transport, mitigated oxidative stress, the mPT, as well as cell swelling in
cultured astrocytes treated with ammonia [90]. l-histidine was subsequently found
to attenuate oxidative stress, the mPT, as well as brain edema in a rat model of acute
liver failure [25]. The above ndings indicate that astrocytes generate free radicals
following glutamine exposure and that glutamine-induced oxidative and/or nitrative
stress, likely through mitochondrial ammonia production, represents a fundamental
mechanism in ammonia neurotoxicity.
Peripheral Benzodiazepine Receptor
A prominent feature of HE is the upregulation of the peripheral benzodiazepine

receptor (PBR) [91, 92], which has recently been renamed as the 18-kDa translocator
protein (TSPO) [93]. The TSPO is distinct from the central benzodiazepine
receptor in its molecular structure, pharmacology, anatomical distribution, subcel-
lular localization, and physiological functions [94]. While astrocytes and microglia
are considered the predominant cell populations expressing the TSPO in the CNS
[9597], they are also expressed in neurons, although at much lower levels as
compared to astrocytes or microglia [98].
Increased TSPOs have been reported in acute hyperammonemic mice [99, 100]
and in portacaval-shunted rats [101], as well as in postmortem brain specimens from
patients with HE [102]. Furthermore, a signicant increase of TSPOs was identied
in cultured astrocytes after treatment with ammonia [103]. The signicance of this
upregulation relative to the pathogenesis of HE or hyperammonemia remains poorly
understood. It is of interest that activation of the TSPO may also contribute to oxida-
tive stress. In support of this possibility, ligands of the TSPO (PK11195, Ro5-4864,
and protophorphyrin IX) were shown to induce free radicals in cultured astrocytes,
microglia, and neurons [98]. On the other hand, TSPO gene knockdown was shown
to protect against oxidative stress in a human glioblastoma cell line [104].
Additionally, Grg et al. [105] showed that other TSPO ligands (diazepam, PK11195,
Ro5-4864, and diazepam binding inhibitor) induce protein tyrosine nitration in cul-
tured astrocytes, as well as in rat brain in vivo. Some of these effects were additive
to those produced by ammonia. In aggregate, these studies suggest that upregulation
of TSPO may contribute to the pathogenesis of HE by also increasing the level of
ONS.
The means by which TSPO produces free radical in neural cells is not known.
Hirsch et al. [106] reported that TSPO ligands inhibited mitochondrial respiration.
It is therefore possible that free radical production after exposure of cells to TSPO
ligands may be mediated by inhibition of mitochondrial respiratory complexes.
Additionally, TSPO is believed to be a component of the mPT pore [107111]. We
previously showed that the TSPO ligand Ro5-4864 at nanomolar concentration
induced the mPT [112]. Using a gene knockdown approach, it was recently demon-
strated that inhibition of TSPO protein synthesis signicantly prevented the mPT [113].
As the mPT is a known source of free radicals (noted above), it is possible that a
TSPO-mediated mPT may be another source of free radical formation in HE.
Manganese
Manganese is an essential trace element. At low levels, manganese binds with

superoxide dismutase to form manganese-superoxide dismutase (Mn-SOD), an
important antioxidant enzyme in mitochondria [114]. However, when excessive,
manganese contributes to neurological abnormalities such as parkinsonism and
dystonia [115]. Chronic exposure of various cell types to manganese was shown to
induce oxidative stress [116118]. Exposure of cultured astrocytes to manganese
decreases energy production and antioxidant capacity, as well as stimulates the
synthesis of glutamine [118]. It is possible that the manganese-induced activation
of glutamine synthesis may also contribute to ONS.
Manganese has also been implicated in the pathogenesis of HE [119]. Manganese
is elevated in plasma and brains of patients with cirrhosis or in individuals who had
surgically created portal-systemic shunts. Selective neuronal loss in the basal ganglia
(especially in the globus pallidus) and reactive gliosis are prominent features of
manganese neurotoxicity in HE. T1-weighted magnetic resonance imaging (MRI)
intensity of the globus pallidus suggested the possible accumulation of manganese
in this region [120122]. Subsequent studies indeed disclosed elevated manganese
levels in the globus pallidus obtained at autopsy from patients with chronic liver
disease [123, 124]. A concomitant loss of dopamine D2 binding sites was also
identied in these specimens [125].
Morphologic and functional changes after exposure of astrocytes to manganese
are similar to those observed after ammonia treatment. Cultured astrocytes exposed
to 5 mM ammonia or 100 mM manganese acetate were shown to increase both
free radical production and l-arginine uptake (a precursor of NO), and such effects
were synergized when manganese was co-treated with ammonia [126, 127]. Similarly,
exposure of primary cortical astrocytes to a low concentration of manganese (10 mM)
was shown to potentiate interferon-gamma (IFN-g) and tumor necrosis factor-alpha
(TNF-a)-induced expression of iNOS mRNA and protein along with an increased
production of NO [128]. The potentiating effect was a consequence of the activation
of soluble guanylate cyclase and MAPK signaling pathways [128]. Cultured astro-
cyte exposed to manganese was also shown to induce the mPT [129] and to inhibit
glutamate uptake by a process involving oxidative stress [130, 131]. Additionally,
Hazell et al. [132] demonstrated that treatment of rats with manganese chloride led
to an increase in manganese level in brain that was accompanied by the development
of pathological changes similar to those seen in HE (Alzheimer type II astrocyto-
sis), and such changes were signicantly reduced when rats were treated with the
antioxidant NAC. These studies suggest that manganese contributes to oxidative
stress in HE and that such effect is exacerbated in the presence of ammonia.
Inflammation
Recent studies have suggested that inammation plays a signicant role in the
pathogenesis of ALF [133137]. Patients with ALF frequently develop infections
and sepsis, and when present, the severity of encephalopathy is greatly exacerbated
[138]. Further, induction of endotoxemia with lipopolysaccharide (LPS) in rats was
shown to aggravate the brain edema and encephalopathy associated with ALF [138].
Consistent with a role of inammation, blood levels of TNF-a, IL-1b, and IL-6 are
elevated in patients with ALF [139141]. High ammonia levels in brain may also
contribute to the production of cytokines as a recent study showed increased levels
of IL-1b, and other inammatory mediators in brains of hyperammonemic rats
[22, 142, 143]. Additionally, astrocyte cultures exposed to cytokines (TNF-a, IL-1b,
IL-6, and IFN-g) were recently shown to activate NF-kB and blocking this activa-
tion prevented astrocyte swelling [144]. Since cytokines are well-known inducers
of oxidative stress and activators of NF-kB [145], it is likely that, in addition to
ammonia, cytokines also contribute to the ONS in HE.
Additional Cellular Sources of Free Radicals
Microglia
As noted earlier, the available data indicate that astrocytes are a major source of free
radicals in brain in HE. However, other neural cells may also contribute to RONS
formation in HE. Recent studies have demonstrated activation of microglial cells in
ALF as well as in a rat model of hyperammonemia [22, 143]. It is likely that microglia,
the principal inammatory cell in brain [146], play a role in the production of free
radicals in HE since activated microglia are well known to induce free radical for-
mation [147]. Once activated by stimulation of cell surface receptors [148], micro-
glial cells produce proinammatory cytokines and other inammatory mediators,
including prostaglandins and arachidonic acid that are well known to induce free
radical formation in the CNS [149]. Additionally, ammonia and glutamine were
shown to induce free radical production in cultured BV2 microglia cells [150].
These studies suggest that microglial cells contribute to the ONS observed in HE.
Endothelial Cells
Another cell type that may potentially be involved in the production of free radicals
in HE are brain endothelial cells (ECs). ECs perform a variety of functions, including
provision of a barrier against potentially toxic substances, transport of nutrients, and
leukocyte trafcking. ECs are the rst resident brain cells that could be impacted by
blood-derived toxins (e.g., ammonia, cytokines). This may trigger an inammatory
response, including the production of free radicals [151]. It is well known that LPS
and systemic cytokines activate brain ECs [152] that subsequently produce free
radicals [153]. In unpublished observations, we found free radical formation is
increased in endothelial cells that are exposed to ammonia.
Consequences of ONS
Astrocyte Swelling/Brain Edema
Brain edema and the associated increase in intracranial pressure and brain
herniation are major complications in patients with ALF [154]. Astrocyte swelling
represents the principal alteration of this condition [91, 155, 156]. Ammonia plays
a key role in the development of astrocyte swelling and brain edema in ALF [157,
158]. While mechanisms responsible for astrocyte swelling/brain edema in ALF
remain poorly understood, recent studies have demonstrated an important role of
ONS in this process. Exposure of cultured astrocytes to ammonia or oxidants caused
cell swelling [159, 160], which was attenuated by antioxidants or NO synthase inhib-
itors. Additionally, exposure of astrocytes to cytokines (IL-1b, IL-6, TNF-a, and
IFN-g) resulted in signicant cell swelling, a process that was markedly potentiated
when cultures were previously treated (sensitized) with ammonia [144].
While the means by which ONS contribute to astrocyte swelling in HE is not
well understood, ONS was shown to activate various signaling pathways, including
activation of mitogen-activated protein kinases (MAPKs), and the transcription
factors p53 and NF-kB. These signaling factors ultimately activate membrane ion
channels/transporters/exchangers (ion transporting systems, ITSs), and such activa-
tion leads to disturbances in cell volume homoeostasis [5]. These ITSs include the
Na+-K+-2Cl cotransporter (NKCC), volume-sensitive osmolyte anion channels
(VSOAC), Na+/Ca2+ exchanger (NCX), and the Na+/H+ exchanger (NHE) that is
functionally coupled to the Cl/HCO3 exchanger, as well as the nonselective cation
channel (NCCa-ATP channel). For reviews on these ion transport systems, see
Kahle et al. [161], Jayakumar and Norenberg [162], Simard et al. [163].
It was recently demonstrated that exposure of cultured astrocytes to ammonia
caused an increase in nitrated/carbonylated proteins [164, 165]. Additionally,
cultured astrocytes exposed to ammonia or oxidants/NO donors signicantly
increased oxidation and/or nitration as well as the activation of NKCC1 [166], and
antioxidants, including Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a
cell permeant superoxide dismutase mimetic, dimethylthiourea (a hydroxyl radical
scavenger), Tempol (a cell permeable superoxide scavenger), catalase (a hydrogen
peroxide decomposer), a-tocopherol (a lipid-soluble antioxidant), or the NOS
inhibitor L-NAME as well as the peroxynitrite scavenger uric acid signicantly
reduced NKCC activity as well as cell swelling [166]. An increase in the activity of
NCX in ammonia-treated astrocytes was also observed and the antioxidants
MnTBAP and Tempol signicantly diminished ammonia-induced NCX activity
Fig. 5.6 Schematic representation of mechanisms by which ammonia leads to ONS and cell
swelling in Type A HE. (1) Mobilization of intracellular calcium; (2) activation of Ca2+-dependent
RONS producing enzymes (PLA2, cNOS, NOX; not shown); (3) activation of NF-kB by MAPKs,
p53, TSPO, and the mPT; (4) activation of RONS producing factors by NF-kB activation (iNOS,
NOX, and cytokines; not shown); (5) ROS generation via the mPT and TSPO; (6) activation of
ITSs and AQP4 by ONS, resulting in astrocyte swelling/brain edema
(unpublished observations). Ammonia also increased the activity of NHE1, and this
activity was diminished by antioxidants (PBN and catalase), and the NOS inhibitor
L-NAME. These studies suggest that ammonia-induced ONS inuences various
signaling pathways that ultimately activate membrane ion transporting systems.
Activation of ion transporting system will increase the intracellular ionic
concentration. To maintain osmo-neutrality, water will enter the cell resulting in
astrocyte swelling/brain edema. Such water entry is facilitated by the presence of
the water channel aquaporin-4 (AQP4). Increased AQP4 expression in the plasma
membrane of ammonia and manganese-treated astrocytes was recently implicated
in the development of astrocyte swelling/brain edema in ALF. Cultured astrocytes
exposed to a pathophysiological concentration of ammonia and manganese were
shown to increase the AQP4 content in the plasma membrane [167, 168], and such
effect was blocked by antioxidants (PBN, Tempol) or with the NOS inhibitor
L-NAME [168], suggesting that ONS, in addition to increasing NKCC activity, also
caused the overexpression of AQP4. Additionally, rats treated with TAA showed an
increase in AQP4 protein in the plasma membrane of cortical astrocytes [169].
Treatment of rats with l-histidine (a potent antioxidant, as well as an inhibitor of
glutamine transport into mitochondria) diminished TAA-induced AQP4 accumulation
in the plasma membrane of cortical astrocytes and blocked TAA-induced brain
edema [169]. Altogether, these ndings suggest that the accumulation of AQP4 in
astrocytic plasma membranes is a consequence of ONS and a factor in the astrocyte
swelling/brain edema in ALF (Fig. 5.6).
Neurobehavioral Defects
While ONS contributes to astrocyte swelling/brain edema in acute HE, the consequences
of ONS in chronic HE are unclear. Only a few studies have examined the role of
ONS in the neurobehavioral abnormalities associated with chronic HE. Rats that
underwent portal vein ligation were shown to increase protein tyrosine nitration,
RNA oxidation, IL-6 mRNA increase in brains, which may have impaired locomotor
activity [170]. Further, these authors reported that prevention of protein tyrosine
nitration and RNA oxidation with indomethacin, a nonspecic cyclooxygenase-2
inhibitor that also has antioxidant properties, prevented brain protein tyrosine
nitration, RNA oxidation, as well as disturbances in locomotor activity associated
with chronic HE [170].
As noted in ONS, one consequence of ammonia neurotoxicity is the activation of
NMDA receptors. Activation of these receptors was shown to induce behavioral
changes such as impairment in active and passive avoidance behavior, conditional
discrimination learning, as well as in long-term potentiation [171]. Such effects were
mediated through the glutamatenitric oxidecyclic GMP pathway [172]. Ammonia
was also shown to inhibit the induction and maintenance of long-term potentiation
and these effects were blocked by l-carnitine, which has antioxidant properties, as
well as by DL-APV, an antagonist of the NMDA receptor [173]. Since activation of
NMDA receptor is well known to induce an increase in intracellular calcium and
subsequent production of free radicals [86], it is possible that NMDA receptor-
mediated ONS in brain may contribute to the observed behavioral abnormalities.
Therapy of HE with Antioxidants
Treatment of experimental animals with HE/hyperammonemia with antioxidants

(e.g., ascorbate, a-tocopherol, desferrioxamine, butylated-hydroxyanisole, dimethyl-
sulfoxide, and dimethylthiourea) was shown to have benecial effects by improving
antioxidant status as well as their clinical condition [174, 175]. The antioxidant
melatonin was shown to reduce blood and brain ammonia level as well as attenuate
brain lipid peroxidation in rats after TAA injection [16]. Additionally, increased
malondialdehyde levels and decreased glutathione peroxidase, catalase, and super-
oxide dismutase activities were found in the hippocampal tissue of rats with portal
hypertension (a model of low-grade HE), and such effects were reversed when rats
were treated with curcumin, a known antioxidant, as well as an anti-inammatory
agent [176].
Rats treated with morin (3,5,7,2,4-pentahydroxyavone), a avonol, were
shown to be protected against oxidative stress in brains of chronic hyperammone-
mic rats [177]. The therapeutic potential of antioxidants, including PBN, catalase,
and the NOS inhibitor L-NAME in TAA-induced acute liver failure in rats was
recently shown by Norenberg et al. [19]. Additionally, increased NF-kB activation,
a source of nitro-radicals, was found in TAA-treated rat brain and BAY 11-7082, an
inhibitor of NF-kB, signicantly reduced the brain edema (unpublished observations).

Likewise, transgenic (Tg) mice that have a functional inactivation of astrocytic
NF-kB are resistant to TAA-induced iNOS protein expression and brain edema in
acute liver failure [37].
The antioxidant NAC has proven useful in reducing the brain edema in acute liver
failure [20] and in the management of patients with ALF [178181]. Additionally,
NAC was shown to delay the progression of encephalopathy in azoxymethane-induced
ALF in mice, as well as to reduce the brain water content and proinammatory
cytokine levels [21]. Mannitol, which is used for the treatment of the brain edema in
ALF due to its osmotic effect, also has antioxidant properties [182, 183]. Additionally,
hypothermia which has been shown to improve the brain edema in animals and
humans with ALF is also known to reduce free radical production [184].
Conclusions
ONS has evolved in recent years as a major pathogenetic factor in HE/ALF. A growing
body of evidence indicates the presence of ONS in brain in experimental models of
acute and chronic liver failure. While the factors responsible for ONS formation in HE
remain incompletely understood, it appears that ammonia-induced increase in intra-
cellular calcium is an early event responsible for the production of free radicals. Such
free radical formation occurs likely through activation of various Ca2+-dependent
enzymes, including cNOS, PLA2, and NOX, the induction of the mPT, and activation
of the major inammatory transcription factor NF-kB.
While increased ONS in chronic HE has been demonstrated by some groups, its
consequences are not well established. Additional studies on the role of ONS in
chronic HE are clearly needed. On the other hand, the role of ONS in acute HE is
relatively well established. Increased ONS has been documented in numerous
studies, and antioxidants were shown to be protective against ammonia-induced
astrocyte swelling and brain edema in acute liver failure. The antioxidant NAC is
already in clinical trials. We anticipate that further recognition of ONS as a major
factor in the pathogenesis of HE/ALF will be exploited into novel therapies for the
treatment of patients aficted with HE.
Acknowledgments This work was supported by a Merit Review from the Department of Veterans
Affairs and by a grant from the National Institutes of Health (DK063311).
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Chapter 6
The Role of Natural Benzodiazepines Receptor
Ligands in Hepatic Encephalopathy
E. Anthony Jones and Kevin D. Mullen
Keywords Natural benzodiazepine Benzodiazepine receptor Benzodiazepine

agonist Hepatic encephalopathy Gamma-aminobutyric acid Benzodiazepine
antagonist Flumazenil
Benzodiazepine Ligands and GABA-Mediated

Inhibitory Neurotransmission
The GABAA/Benzodiazepine Receptor Complex
Benzodiazepine (BZ) receptor ligands comprise a diverse class of compounds that

include pharmaceutical BZs. These ligands bind to central BZ receptors, which are
an integral component of the GABAA/benzodiazepine receptor supramolecular
complex in synaptic neural membranes within the central nervous system. The other
components of this complex are a receptor for GABA (the GABAA receptor) and a
chloride channel (ionophore) [1, 2] (Fig. 6.1). Central BZ receptors are distinct
from peripheral BZ receptors, which are located on mitochondrial outer membranes
in nonneuronal tissues [3]. After its release from presynaptic neurons, GABA binds
to GABAA receptors on postsynaptic neurons. This binding triggers the opening of
the chloride channel, which allows passage of chloride ions into the neuron and
results in hyperpolarization of its surface membrane. These events are the basis of
E.A. Jones, MD, DSc

Winchester, Hampshire, UK
K.D. Mullen, MD, FRCPI ()
Department of Internal Medicine, Division of Gastroenterology, Metrohealth Medical Center,
2500 Metrohealth Drive, Cleveland, OH 44109, USA
72 E.A. Jones and K.D. Mullen
Closed GABA receptor

Cl
Outside
Picrotoxin
site
Chloride
channel BZ receptor
Inside
Bicuculline
Muscimol
Barbiturates GABA Benzodiazepine ligands
Open
Cl O O H2 High Affinity
C N+
CH2 CH2
CH2 BZ receptor
Fig. 6.1 Diagrammatic representation of the GABAA/benzodiazepine (BZ) chloride ionophore

supramolecular receptor complex in the surface membrane of a postsynaptic neuron. There are three
components to these complexes: chloride channels, GABAA receptors, and central BZ receptors.
The complex is depicted in the inactivated state with the chloride cannel closed. Activation of the
complex with associated conformational changes. Opening of the chloride channel occurs in
response to the binding of GABA to its receptor and this phenomenon is potentiated if a BZ agonist
binds to the BZ receptor. Passage of chloride from the synaptic cleft to the interior of the neuron,
as a consequence of opening of the chloride channel, is associated with hyperpolarization of the
neural membrane. These phenomena are the basis of GABAergic inhibitory neurotransmission
GABA-mediated inhibitory neurotransmission [4]. Gating of the chloride channel

by the GABAA receptor is allosterically modulated by the central BZ receptor,
which may increase or decrease the efficacy of GABA-gated chloride conductance,
depending on the nature of ligands occupying the BZ receptor [57].
The Spectrum on Intrinsic Activities of BZ Receptor Ligands
Three main classes of BZ receptor ligands are recognized: agonists, inverse agonists,
and antagonists. Full agonists include the classical pharmaceutical 1,4-substituted
BZs, such as diazepam. Occupation of the BZ receptor by an agonist induces con-
formational changes in the receptor that increase the affinity of GABA for its recep-
tor and, consequently, the frequency of GABA-gated chloride channel openings [8].
6 The Role of Natural Benzodiazepines Receptor Ligands 73
The resulting increase in GABAergic tone is the molecular basis of the ability of
BZ agonists, including pharmaceutical BZs, to decrease anxiety, muscle tone,
and vigilance and to mediate amnesia, sedation, and anticonvulsant effects [1, 2].
The effects of BZ agonist-induced augmentation of GABAergic tone include
decreased consciousness and impaired motor function [1, 2], which are two of
the major manifestations of the syndrome of hepatic encephalopathy (HE) [9].
In contrast, full inverse agonists, such as beta carbolines (e.g., methyl-6,7-dimethoxy-
4-ethyl-beta-carboline-3-carboxylate (DMCM)), induce conformational changes in
the BZ receptor that decrease the synaptic response to GABA and, consequently,
reduce GABAergic tone. The pharmacological effects of inverse agonists can be
regarded as opposite to those of agonists and include anxiety, increased muscle
tension, and proconvulsant and convulsant effects. Other BZ receptor ligands
include partial agonists and partial inverse agonists. Pure antagonists have minimal
(agonist or inverse agonist) intrinsic activity and, consequently, when occupying
central BZ receptors, do not alter neuronal electrical activity, neuronal responsiveness
to GABA, or behavior over a wide range of concentrations in normal animals.
Antagonists, however, competitively antagonize the binding of other BZ receptor
ligands. Consequently, antagonists tend to normalize changes in GABAergic tone
induced by agonists or inverse agonists [57, 10]. Thus, the intrinsic activities of
different BZ receptor ligands are diverse; they can be classified with respect to their
placement on a continuum or spectrum of intrinsic activities [57, 1012] (Fig. 6.2).
The properties of a pure antagonist would place it at the central (GABA neutral)
point of this spectrum. However, many compounds classified in the central region,
which have weak partial agonist or weak partial inverse agonist properties, act
predominantly as antagonists [57, 11].
Origin of the Concept of a Role for BZ Receptor Ligands in HE
GABAergic Tone and HE
That increased GABAergic tone may contribute to HE was first suggested in the
early 1980s, when the abnormal patterns of visual evoked potentials associated with
HE in animal models were shown to be similar to those induced in normal animals
by administering drugs which induce augmentation of GABAergic tone, such as
pentobarbital [1315]. Subsequently, other findings also provided support for the
concept that increased GABAergic tone contributes to the manifestations of HE [16].
BZ Receptor Antagonism and HE
If augmentation of GABAergic tone induced by BZ receptor agonist ligands

contributes to HE, then, theoretically, pharmacologically induced displace-
ment of such ligands from central BZ receptors might induce an amelioration of HE.
Full Inverse Partial Inverse Partial Full

Agonist Agonist Antagonist Agonist Agonist
Anxiogenic No effect Anxiolytic

Increased muscle tension Myorelaxant
Proconvulsant Amnestic
Convulsant Decreased vigilance
Hypnotic
Anticonvulsant
Cumulative Use of Treatment
Diazepam
Flumazenil
Increase
Triazolam
(Ro 15-1788)
Midazolam
0
Decrease
Ro 14-7437
Ro 15-3505
Ro 15-4523
DMCM
Fig. 6.2 The spectrum of intrinsic activities mediated by central BZ receptor ligands when they
binding to central BZ receptors. Full agonist ligands, including pharmaceutical BZs, increase
GABAergic tone. In contrast, full inverse agonists, such as DMCM, decrease GABAergic tone.
A pure BZ antagonist, such as Ro 14-7437, has no intrinsic activity; thus on binding to central
BZ receptors it neither increases nor decreases GABAergic tone. Consequently, such a ligand is
classified at the GABA-neutral mid-point on the spectrum. Flumazenil (Ro 15-1788) is classified
as a weak partial agonist, and Ro 15-4513 and Ro 15-3505 (sarmazenil) as partial inverse agonists
This concept was originally expressed in 1984 by Anderson: If there exists an

endogenous modulator of the BZ receptor (in HE) that functions in an agonistic
manner then blocking its action with an antagonist should remove this tonic facilitation
and indirectly decrease GABAergic tone and, consequently, decrease the severity
of HE [17]. Subsequently, when the first prototypic benzodiazepine receptor antag-
onist became available for experimental use in patients, Bansky postulated in 1985
that: since BZs stimulate the neuroinhibitory effect of GABA, a BZ antagonist
might improve the level of consciousness and EEG appearing in patients with
hepatic coma [18]. This prediction was initially supported in the mid-1980s by the
first report of reversal of CNS changes in a rat model of HE by a BZ antagonist
(CGS-8216) [19] and by anecdotal reports of ameliorations of hepatic coma in
patients with cirrhosis or fulminant hepatic failure (FHF) following the intravenous
administration of the newly characterized BZ receptor antagonist, Ro 15-1788
[18, 20], i.e., flumazenil [10]. Thus, by the late 1980s there was a basis for postulat-
ing that BZ receptor agonist ligands may be involved in the mediation of HE [21].
However, it should be noted that, if the action of such ligands is a mechanism that
contributes to increased GABAergic tone in HE, this mechanism would appear to
be only one of several mechanisms that have been implicated in contributing to

increased GABAergic tone in HE [16].
To evaluate the validity of the hypothesis that central BZ receptor agonist ligands
contribute to HE, it became necessary (1) to adopt a critical approach to evaluating
the validity of quantitative methods designed to measure such ligands; (2) to obtain
measurements of the concentrations of such ligands in animals and humans in the
presence and absence of HE, and to determine whether such concentrations corre-
late with the severity of HE; (3) to assess potential sources of nonpharmaceutical
BZ receptor ligand activity in vivo in the presence and absence of HE; and, finally,
(4) to interpret results of studies of the effects of BZ receptor ligands with different
intrinsic activities on the manifestations of HE.
Some Issues Relating to Measurements of BZ Receptor Ligands
Detection and measurement of BZs in biological specimens are dependent on the

type of initial extraction procedure applied. BZs are both lipophylic and highly
protein bound [22]. Failure to make proper allowance for these properties is likely
to lead to major inaccuracies in their detection and measurement. Standardized
laboratory or toxicological procedures, such as deproteinization, may affect the
detection of BZs unpredictably. Furthermore, a major problem with most commer-
cially available toxicology assays for BZs is the unknown cross reactivity of the
antibenzodiazepine antibody with heterogenous BZ ligands [23].
Many compounds may contribute to BZ activity found in blood or other tissues.
Indeed, it is possible that a large proportion of putative BZ ligands that may be pres-
ent in biological systems could be novel compounds [22]. Distinguishing accurately
between BZ activity due to occult ingestion of pharmaceutical BZs and that attribut-
able to nonpharmaceutical BZs is a recurrent and important analytical challenge.
Major difficulties have been encountered in identifying all of the individual BZ
compounds responsible for BZ activity detected in biological specimens. While
minute quantities of known BZs can be measured accurately by gas chromatography/
mass spectrometry techniques using specimens of conventional size, the quantities
of material available after extensive purification procedures may be insufficient
for accurate quantitation of novel BZ compounds. Moreover, in applying the
radioreceptor assay, problems in quantitation arise as a consequence of the differing
avidity/affinity of individual BZs and the BZ (e.g., diazepam) used as a calibration
standard. For example, the absolute amounts of compounds with high affinities for
BZ receptors (e.g., picomolar) compared to that of diazepam (low nanomolar) are
overestimated considerably by the radioreceptor assay when diazepam is used as
the standard. The opposite is the case for compounds with low affinities for BZ
receptors. Using samples of conventional size, many BZ compounds may tend to
disappear during purification procedures. Consequently, it may be difficult to obtain
blood or tissue samples of sufficient size to provide enough material for definitive
identification of many BZ compounds.
In the subsequent discussion of studies in which concentrations of BZs were

measured in humans or animals in the presence or absence of HE, the specific methods
used are mentioned only to draw attention to a particular methodological issue or to
illuminate a particular point in the text.
Concentrations of BZs in Animals and Humans

with and Without HE
As the brain is the end organ of HE, the optimal site for measuring the concentrations
of BZ ligands in studies of their potential role in HE would be the brain. However,
it would also be relevant to make measurements in cerebrospinal fluid or plasma.
In general, BZ receptor ligands are not only lipid soluble but they also rapidly
traverse the bloodbrain barrier. Thus, BZ receptor ligands present in increased
concentrations in plasma could readily contribute to the manifestations of HE [22].
Factors that would tend to result in increased concentrations of free BZs in plasma
in liver failure would include low protein binding potentiated by decreased hepatic
synthesis of albumin, decreased hepatic metabolism, and increased portal-systemic
shunting [24].
Further evidence supporting a relationship between BZs and HE was provided
by the demonstration of a BZ receptor binding substance in the cerebrospinal
fluid of a rabbit model of HE as a result of applying an assay to measure BZs
directly [2527]. This study was followed by the demonstration of BZ activity in
body fluids of patients with HE, in whom there was no evidence of recent ingestion
of pharmaceutical BZs [27, 28]. Subsequently, activity that reversibly and competi-
tively inhibited radiolabeled BZ receptor ligand binding to normal brain membranes
was demonstrated in brain, plasma, and several peripheral organs of animal models
of HE [2933].
In an autoradiographic study, the binding of a radiolabeled BZ receptor ligand to
unwashed brain sections was found to be reduced in a model of HE. This decrease
could be eliminated by prewashing brain sections, indicating the presence in HE of
ligands that reversibly bind to BZ receptors. In this autoradiographic study, the
distribution of the BZ receptor ligands was not uniform throughout the brain [30].
In two studies, the potency of the BZ receptor binding inhibitory activity in a model
of HE was enhanced by GABA [29, 30]. This phenomenon, known as a positive
GABA shift, is attributable to an increase in the affinities of the BZ receptor ligands
present and indicates that they include ligands with agonist properties [3436].
High performance liquid chromatographic analysis of whole brain extracts from
two animal models of HE and control animals revealed peaks of inhibitory activity
with retention times similar to those of known 1,4-BZs [31, 33]. 1,4-BZs were found
in normal brain, but total brain levels of these ligands were significantly greater in
the models of HE [31, 33].
Both BZ receptor binding activity and 1,4-BZ immunoreactivity were found to

be increased in cerebrospinal fluid, plasma, and urine of patients with decompen-
sated cirrhosis in whom there was no evidence of ingestion of pharmaceutical BZs
during the preceding 3 months [28, 32, 37].
A study that showed predominantly unchanged levels of BZ receptor ligands in
a presumed model of HE raises questions whether the animal model used adequately
reflects the syndrome of HE in humans; it also raises issues relating to the validity
of the methods used [38, 39].
Increased levels of total BZ receptor binding activity and of diazepam and
N-desmethyldiazepam have been demonstrated in about 60% of brains obtained at
autopsy from patients who died from acetaminophen-induced FHF [40, 41].
Total brain levels of BZ receptor ligands are insufficient to account for all of the
manifestations of HE [22, 31, 33]. Brain levels of total BZ receptor ligands are
lower in animal models of FHF than in humans with FHF. Specifically, the mean
brain level of total BZ receptor ligands in rats with thioacetamide-induced FHF
was 71 ng/g [31], and in rabbits with galactosamine-induced FHF it was 21 ng/g [33];
in contrast, the corresponding mean for humans with acetaminophen-induced FHF
was 300 ng/g [40].
Brain levels of total BZ receptor ligands were not elevated in rats with a porta-
caval shunt [32], but the rat with a portacaval shunt does not fulfill criteria that are
necessary for a model of HE [42].
Brain levels of BZ receptor ligands in an animal model of FHF [43] and plasma
levels of BZ receptor ligands in humans with FHF [44] or decompensated cirrhosis
[28] have been shown to correlate directly with the severity of HE.
Nomenclature and the Nature of BZ Receptor Ligands in HE
Only a proportion of the BZ receptor binding activity present in the brain in animal
models of FHF and humans with FHF has been shown to be due to the presence of
classical 1,4-BZs; 1755% of this activity appears to be due to diazepam and
N-desmethyldiazepam [31, 33, 40]. The chemical and functional nature (agonist,
antagonist, inverse agonist) of a large proportion of the BZ receptor ligands present
in the brain in HE is currently unknown [22, 31, 40]. The presence of antagonist
ligands in HE might influence the responsiveness of the encephalopathy to an
administered antagonist, and the presence of ligands with inverse agonist properties
would tend to counteract the effects of agonist ligands on the encephalopathy.
Originally, when BZ activity was first found in animals and humans with HE, the
term endogenous BZs was used [21, 28]. Endogenous BZs are distinct from
endozepines and diazepine-binding inhibitor, which were discovered at about the
same time [4548]. Increased levels of diazepam-binding inhibitor, a BZ receptor
ligand with partial inverse agonist properties, have been demonstrated in the cerebrospinal
fluid of cirrhotic patients with HE [49]. However, the relevance of endozepines to
the pathogenesis of HE is uncertain. High molecular weight endozepines and their

cleavage products are not detected by the radioreceptor assay for BZs [50].
Application of the word endogenous to BZs would appear to imply that BZs are
synthesized by mammalian cells. However, evidence that mammalian cells can
synthesize BZs is lacking. While the thyroid gland can halogenate a tyrosine ring,
there is considerable uncertainty whether any mammalian tissue can halogenate
(e.g., 7-chlorination) the A ring of a 1,4-BZ [51], an essential step for the biosynthesis
of BZs. If the BZs present in patients with HE come from food or are synthesized
and released within the gut lumen, it would be inappropriate to classify them as
endogenous. Thus, it is currently uncertain whether the term endogenous is appropriate
for the BZs present in patients with HE. Until the source of the BZs present in
patients with HE has been clarified unequivocally, it is suggested that an appropriate
interim term for these compounds be natural BZs.
Possible Sources of Natural BZs in HE
The origin of increased BZ receptor ligand levels in HE is uncertain [52]. Possibilities

include the food cycle [5356], precursor compounds in the food cycle, direct
synthesis or synthesis of precursors by intestinal bacteria [57], and occult inges-
tion of pharmaceutical BZs. BZs, including diazepam and N-desmethyldiazepam,
have been found in low concentrations in a variety of human tissues from subjects
without liver disease [5355, 5861], and in foods such as wheat and potatoes
[55, 56], milk [53], soy, beans, rice, and mushrooms [54]. BZ levels have been
shown to increase five- to eightfold during germination of wheat and potatoes,
suggesting that biosynthesis of BZs occurs in these plants [56]. Even if subnormal
metabolism of BZ receptor ligands by a failing liver is assumed, the concentra-
tions of preformed BZ receptor ligands in food seem to be too low to account
for the levels of BZ receptor ligands found in animal models of HE and patients
with HE. The term natural BZs has been applied to BZs found in the food cycle
that are not attributable to industrial contamination with BZs [5356]. Most of
these BZs that have been identified are identical to commercially synthesized
BZs, such as lorazepam and N-desmethyldiazepam. Commercial BZs, which are
clearly an important potential source of BZs in patients with HE, cannot account for
the elevated levels of BZs found in most patients with HE. Brains of human subjects
without liver disease, which were preserved at a time before BZs had become
commercially available, have been shown to contain 1,4-BZs [51, 58]. One pos-
sible source for such natural BZs might be ingested BZ precursors, which are
converted into biologically active BZs in the gut or after absorption. Diazepam
and N-desmethyldiazepam may be synthesized by prokaryotes [62]. Finally, gut
bacteria have been shown to be a potential source of precursors of BZ receptor
ligands in a rat model of HE [57].
Effects of BZ Receptor Antagonist Ligands on HE
Flumazenil
The 1,4-imidazobenzodiazepine, flumazenil (Ro 15-1788), binds competitively,

reversibly, and with high specificity and high affinity to central BZ receptors [10, 63],
at which it exhibits very weak, dose-dependent, partial agonist effects [64]. Its location
on the spectrum of intrinsic activities of BZ receptor ligands, slightly on the agonist
side of the central, GABA-neutral, point [12] (Fig. 6.2), implies that it acts predomi-
nantly as a BZ receptor antagonist. Furthermore, its precise location on this spectrum
contributes significantly to its safety profile, since it is devoid of the convulsive
potential associated with ligands that possess inverse agonist intrinsic activity. Thus,
an overdose of flumazenil is likely to induce only weak diazepam-like effects, such
as mild sedation [10]. Flumazenil antagonizes the actions of BZs, beta-carbolines,
and other compounds with significant intrinsic activity that bind directly to central
BZ receptors. It blocks all of the specific behavioral and pharmacological effects of
classical BZ receptor agonists, such as diazepam [10, 63].
The efficacy of flumazenil depends not only on its precise placement on the spectrum
of intrinsic activities of BZ receptor ligands (Fig. 6.2), but also on its occupancy of
central BZ receptors and its rate of metabolism [10, 22, 24, 63, 65]. Plasma clearance
of flumazenil is rapid in normal subjects; it is slower in patients with impaired
hepatocellular function [66]. Positron emission tomography after the intravenous
injection of 11C-labeled flumazenil to normal subjects has demonstrated prompt
binding of the radioligand to BZ receptors in the brain. Cerebral levels of the radio-
ligand decrease with a half-life of 2538 min, which is due to displacement rather
than metabolism; the rate of decline of cerebral radioactivity may exceed that of
plasma radioactivity. The radioligand is rapidly removed from BZ receptors in the
brain after the intravenous administration of unlabeled flumazenil [67]. Cerebral
retention of 11C-labeled flumazenil is prolonged in patients with advanced hepato-
cellular disease [68], probably due to impaired hepatic metabolism of flumazenil
[66]. Only small doses of flumazenil (e.g., 0.30.5 mg) administered intravenously
appear to be necessary to occupy a large proportion of central BZ receptors, and,
hence to mediate BZ receptor antagonist effects [10, 22, 63, 65, 69]. Doses for clinical
administration as intravenous bolus injections are usually less than 2 mg.
Like many other BZ receptor ligands, flumazenil is lipid soluble and, following
its intravenous administration, it rapidly traverses the bloodbrain barrier and gains
access to central BZ receptors. Its effects become apparent less than 4 min after
intravenous administration [10, 22, 63]. The drug also mediates BZ antagonist
effects when given by mouth, but its oral bioavailability is low mainly due to its
high first-pass hepatic extraction [10, 22, 24]. Thus, flumazenil is effective at lower
doses after intravenous than oral administration. Its duration of action, which lasts
23 h in man after an intravenous bolus injection [10], is dependent on its rapid rate
of metabolism. In patients with impaired hepatocellular function, its slower rate of
metabolism [66] may contribute to a more sustained action.
Flumazenil for parenteral use is currently the only BZ receptor antagonist preparation
approved for clinical use.
Uncontrolled Studies of Flumazenil in Humans
Normal Subjects
When low doses of flumazenil (15 mg IV or 30 mg orally) were administered to

normal subjects, phenomena indicative of central neuronal activation were observed,
including anxiety, autonomic arousal, sleep disturbances, and increased neuronal
electrical activity [70]. At higher doses (25 mg IV or 100400 mg orally) flumazenil
exhibited agonist properties; specifically, it acted as an anticonvulsant, impaired
motor function, suppressed neuronal electrical activity, and induced mild sedation
[24, 71]. Because flumazenil is devoid of inverse agonist intrinsic activity [64], the
anxiogenic and CNS-activating effects of low doses can be explained by postulating
that it displaces natural BZ receptor ligands with agonist properties from central BZ
receptors, thereby inducing activation of the CNS as a consequence of neuronal
disinhibition [70]. This interpretation of the effects of low doses of flumazenil in
normal subjects is consistent with the existence of low levels of natural BZ agonist
ligands in the brain under physiological conditions.
Patients with HE
Anecdotal reports have described clinical and electrophysiological ameliorations

of HE following intravenous bolus injections of flumazenil to patients with FHF or
cirrhosis [22, 72, 73]. These observations, although uncontrolled, were usually
made in patients with stable clinical and electrophysiological indices of HE before
drug administration [72, 73], and the ameliorations that occurred were often repro-
ducible [22]. Thus, the responses seemed to be genuine, especially as basic research
on the pathogenesis of HE [22, 65, 74] provided a logical explanation for flumazenil-
induced ameliorations of HE. These reports have documented certain characteristics
of ameliorations of HE following intravenous bolus injections of fl umazenil:
(1) readily detectable ameliorations are inconsistent, occurring in about 60% of
patients with HE secondary to either acute (FHF) or chronic liver disease (cirrhosis)
[10, 22, 65, 74]; (2) responses occur rapidly, usually within 4 min of drug adminis-
tration [72, 73], the range being 28 s to 30 min [22, 75]; such responses are unlikely
to be spontaneous, but are consistent with flumazenil rapidly crossing the bloodbrain
barrier and gaining access to central BZ receptors [10, 63]; (3) substantial ameliora-
tions of HE occur after low doses, e.g., 0.30.5 mg, supporting the concept that only
small amounts of the drug are necessary to occupy a large proportion of central BZ
receptors and, hence, mediate BZ antagonist effects [22, 65, 69]; (4) consistent with
its rapid rate of catabolism, which includes hepatic metabolism and the action of
plasma esterases [10, 22, 24], ameliorations of HE have a short duration (0.64 h)
[22, 72, 73]; and (5) ameliorations are usually partial (e.g., one to two of the classical
clinical stages of HE [9]); typically only some neurologic deficits are reversed and
improvements in motor function tend to be limited, but, occasionally, a patient
becomes conscious and starts talking with dramatic rapidity [22, 72, 73, 75].
In addition, an intravenous infusion of flumazenil (0.2 mg) has been shown to
improve the cognitive component of a reaction time task in patients with subclinical
HE [9, 76]. Furthermore, administration of flumazenil to patients in hepatic coma
was shown, not only to induce clinical ameliorations of encephalopathy, but also to
induce improvements in associated abnormal patterns of visual evoked potentials
[75], which are an index of brain electrophysiologic function that does not depend
on cognitive function. In contrast, flumazenil did not induce an improvement in
visual event-related potentials, an index of cognitive function, in cirrhotic patients
without overt encephalopathy [77], but the patients studied were not shown to fill
criteria necessary for a diagnosis of subclinical HE [9].
In contrast to the intravenous route of administration, orally administered flumazenil,
25 mg twice daily, has been reported to induce consistently a complete and sustained
amelioration of intractable portal-systemic encephalopathy in a middle-aged woman.
While taking the drug, a normal or supranormal dietary intake of protein was well
tolerated [78]. Furthermore, 2540 min after administration of each dose of flumazenil,
this patient regularly experienced a feeling of anxiety, which subsided after 3060 min
[78]. This observation can be explained by postulating that the drug displaced
agonist ligands from central BZ receptors, thereby precipitating a clinically overt
manifestation of neuronal disinhibition.
Controlled Studies of Flumazenil
Animal Models of HE
Flumazenil markedly excited the spontaneous neuronal activity of isolated Purkinje

neurons from the cerebellum of a rabbit model of FHF, but partially suppressed the
spontaneous activity of the same neurons from control animals [64]. The effect of
flumazenil on the control neurons is a direct demonstration of its very weak partial
agonist intrinsic activity, whereas its effect on neurons from the model of HE can be
explained by their disinhibition as a consequence of displacement of ligands with
agonist intrinsic activity from central BZ receptors.
Controlled data on the effects of flumazenil on behavioral and electrophysiologi-
cal indices of HE have been generated using animal models of FHF. Both positive
[79, 80] and negative [8185] results have been obtained. In the light of extensive
positive findings in humans [22, 86], negative results must always raise concerns
about the adequacy of the animal model studied. For example, the syndrome of
acute hepatic ischemia in animals [81, 82, 87] differs in important respects from
the typical syndrome of FHF in humans [9, 88], including the probability that
factors other than uncomplicated hepatocellular failure contribute significantly to

encephalopathy at an early stage in the ischemic model; such factors may include
other metabolic encephalopathies (e.g., hypoglycemia) and cerebral edema. Other
potential explanations for a lack of amelioration of encephalopathy following
administration of flumazenil to animal models of HE include pharmacokinetic
factors (e.g., effects of dissolving the drug in gum arabic as vehicle), and, possibly,
the weak partial agonist properties of the drug. In two animal studies in which
positive data were generated, at least three factors enhance the significance of the
results: (1) the models used had been extensively characterized and validated
[80, 89, 90]; (2) increased levels of natural BZs in the brain had been demonstrated
in both of the models [31, 33]; and (3) the animals studied had not been exposed to
pharmaceutical BZs.
Patients with HE
Thirteen randomized controlled trials of the effects of flumazenil on HE complicating

cirrhosis have been reviewed by Als-Nielsen et al. [91]. The number of patients
entered into these trials was 805. All of them were double blind and assessed the
effects of flumazenil vs. those of placebo. Eight of the trials had a crossover design.
Flumazenil administration was associated with a significant increase in short-term
ameliorations of HE, predominantly in patients who had a favorable outcome.
Certain issues of concern may be applicable to the design of some of the trials
included in this review; these include: (1) poorly defined criteria for making a diag-
nosis of HE; (2) selection of suboptimal stages of encephalopathy as inclusion criteria;
(3) failure to ensure adequately that patients had not recently received pharmaceutical
BZs; and (4) inappropriate interpretation of screening tests for BZs in body fluids.
Comments on Studies of Flumazenil in HE
None of the traditional treatments for HE, such as lactulose and neomycin, induce
such substantial ameliorations of HE, so rapidly and so frequently, as those that
have been documented to occur after administering flumazenil intravenously
[22, 72, 73]. This finding highlights that a fundamental shift of approach to treating
HE has been initiated [92, 93]. Traditional approaches have typically involved
administering agents that are believed to decrease formation and/or absorption of
encephalopathogenic substances in the colon. A more efficacious approach may be
to administer therapies that have the potential of directly reversing relevant
pathophysiological mechanisms in the brain, the end organ of HE [92, 93]. The first
example of such a new approach to treating HE is the administration of flumazenil,
which acts directly as an antagonist at central BZ receptors in the brain.
Although subject to the limitations inherent in all human autopsy studies, the
percentage of patients with FHF in whom brain levels of BZ receptor ligands were
found to be increased at autopsy was found to correspond closely to the percentage

of patients with FHF in whom ameliorations of HE occur following a bolus intrave-
nous injection of flumazenil. This finding is consistent with elevated brain levels
of BZ receptor ligands being a necessary prerequisite for a flumazenil-induced ame-
lioration of HE to occur [22, 40, 41, 65, 74, 94]. Because of the specificity of the
action of flumazenil for central BZ receptors and its very weak partial agonist
properties at this receptor, the most logical explanation for a flumazenil-induced
amelioration of HE is that the drug reduces increased GABAergic tone that occurs
in HE [16] by displacing natural agonist ligands from central BZ receptors (Fig. 6.1).
The efficacy of flumazenil in ameliorating HE implies that levels of BZ agonist
ligands in the brain in liver failure are sufficiently high to mediate at least some of
the behavioral manifestations of HE. Flumazenil-induced displacement of BZ ago-
nist ligands from central BZ receptors would lead to a disinhibition of neurons
and, hence, an increase in their spontaneous activity. Furthermore, the transient
anxiety that occurred consistently soon after the oral administration of flumazenil to
a patient with intractable chronic portal-systemic encephalopathy can be explained
by the same mechanism [78]. The available data on the effects of flumazenil suggest
that augmentation of GABAergic tone induced by natural BZ ligands with agonist
properties contributes substantially to the manifestations of HE in a majority of
patients with liver failure. Specifically, improvements of one to two of the classical
clinical stages of HE have been frequently documented following flumazenil admin-
istration [9, 10, 22, 72, 73]. The efficacy of flumazenil in reversing manifestations
of HE may be related primarily to the degree to which concentrations of BZ agonists
are elevated in the brain. Thus, the apparently greater efficacy of flumazenil in amelio-
rating HE in humans than in animals with FHF may be due largely to the much
higher brain levels of BZs in some patients with FHF than in animals with FHF
[22, 31, 33, 40]. The proportions of patients with HE who respond to flumazenil are
similar (about 60%) for those with both FHF and cirrhosis [65, 74], suggesting that
natural BZs contribute to HE in both of these clinical settings. Thus, HE due to FHF
and HE complicating cirrhosis do not appear to be completely distinct entities.
Whether the response of a patient with HE to flumazenil is an index of prognosis
[74, 91, 95] is currently uncertain.
While an amelioration of HE associated with administration of flumazenil provides
strong evidence that BZ agonists contribute to the manifestations of HE, it does
not follow that a lack of improvement in encephalopathy in a model of liver failure
[8185] or a patient with liver disease following administration of flumazenil
provides evidence against a contribution of BZ agonists to the manifestations of HE.
Such a lack of an overt effect of flumazenil may occur if manifestations of enceph-
alopathy are compounded by the presence of encephalopathogenic factors other
than those attributable to uncomplicated hepatocellular failure alone, for example,
cerebral edema, increased intracranial pressure, hypoxic brain damage, and hypoglyce-
mia. Thus, as FHF or end-stage chronic liver disease progresses to the agonal stages
of liver failure many factors, in addition to those responsible for HE, may contribute
to an encephalopathic state [9, 88]. Furthermore, whenever an encephalopathy consid-
ered to be HE does not respond to flumazenil, it is important not only to determine
if manifestations of HE are being masked by other encephalopathies, but also to

question whether the diagnosis of HE is correct. A confident diagnosis of HE
depends on clinical judgment and experience. There is no simple test to confirm a
diagnosis of HE [9, 88]. Features of patients with liver disease that may be associated
with a lack of response of HE to flumazenil include stage IV encephalopathy, cerebral
edema, and early death [22, 91, 95].
The observations that flumazenil rarely reverses HE completely may be interpreted
as suggesting that pathogenic mechanisms other than BZ agonist-induced augmen-
tation of GABAergic tone also contribute to HE. For example, mechanisms not
involving BZ receptor ligands may mediate increased GABAergic tone in HE [16].
Clearly, flumazenil-induced ameliorations of HE cannot be explained by the weak
agonist intrinsic activity of the drug. The data on the effects of flumazenil on HE in
man may underestimate the magnitude of the contribution of natural BZs to HE for
the following reasons: (1) other complicating metabolic disturbances may have masked
the contribution of natural BZ agonist ligands to encephalopathy [10, 22, 65, 74];
(2) the design of published controlled trials of flumazenil in patients with HE may
not have been optimal; and (3) flumazenil, because of its weak partial agonist intrinsic
activity [64], does not have the properties of a pure BZ antagonist [94].
Whenever the effects of flumazenil on HE are being assessed, it is essential to
exclude recent ingestion of pharmaceutical BZs [28, 96]. In this context, it may not
be easy to ascertain whether a patient with liver disease has taken pharmaceutical
BZs recently, as several natural BZs and pharmaceutical BZs appear to be identical
[27]. Clearly, it is inappropriate in an assessment of the effects of flumazenil on HE
to exclude patients in whom a screening test for BZs in blood is positive [97, 98],
as such positive tests may be attributable to the consequences of liver failure [22].
On the contrary, a positive assay for BZs in a patient with liver failure and HE may
indicate that therapy with flumazenil is likely to be effective.
BZ Receptor Ligands Other than Flumazenil
Exposure to the BZ receptor antagonist Ro 14-7437 markedly increased the sponta-

neous activity of isolated Purkinje neurons from a rabbit model of FHF, but had no
effect on the spontaneous activity of control neurons (Fig. 6.3). The latter observa-
tion is compatible with Ro 14-7437 being classified as a pure BZ receptor antago-
nist with minimal agonist or inverse agonist intrinsic activity (Fig. 6.2). Furthermore,
incubation of Purkinje neurons from the model of HE, but not control neurons, with
subthreshold concentrations of Ro 14-7437 reduced their sensitivity to the neuroin-
hibitory effect of the GABAmimetic, muscimol. In contrast, there was no difference
in sensitivity between neurons from the model of HE and control neurons to the
depressant actions of the alpha-adrenoceptor agonist, phenylephrine [64]. These
observations demonstrate a differential responsiveness of neurons from a model of
HE to ligands that interact with the GABAA-BZ receptor complex and provide
further support for reversible binding of BZ ligands with agonist properties to BZ
receptors on the GABAA receptor complex in HE.
140
Change in Spontaneous Firing

120
Rate, (% of Control)
100
80
60
40
20
0
7 6 5
20
40
Log Drug, [M]
Fig. 6.3 Concentrationresponse curves for the effects of the pure benzodiazepine receptor antagonist,
Ro 14-7437, on the spontaneous activity of Purkinje neurons from control rabbits (closed tri-
angles ) and rabbits with hepatic encephalopathy (open triangles). Data are means SEM.
Ro 14-7437 (0.57.5 mM) had no effect on the activity of neurons from control rabbits, but elicited
a robust increase in the spontaneous activity of neurons from rabbits with hepatic encephalopathy.
The concentration of drug that increased neuronal activity by 50% (EC50) was 1.43 mM. The increased
spontaneous activity of neurons from rabbits with hepatic encephalopathy can be explained by
their disinhibition as a consequence of displacement of ligands with agonist properties from central
BZ receptors
Two other compounds that have been classified as BZ receptor antagonists with
weak partial inverse agonist properties, Ro 15-4513 and Ro 15-3505 (sarmazenil)
[57, 10, 11] (Fig. 6.2), have been administered to animal models of FHF. Both
compounds were shown to induce robust behavioral and electrophysiological
ameliorations of encephalopathy in the models studied [80, 82, 83, 85]. In one study
these BZ receptor ligands were shown not to improve the neurological status of
control animals with uremic encephalopathy [83]. Theoretically, improvements of HE
induced by these agents could arise as a consequence of either or both of their two
well-known properties: (1) their ability to act as antagonists and displace BZ
agonist ligands from central BZ receptors; and (2) their ability to induce an analeptic
effect as a consequence of their weak partial inverse agonist intrinsic activity at
central BZ receptors. The ameliorations of HE induced by these agents did not
appear to be predominantly due to their partial inverse agonist properties for two
reasons: (1) the doses of these drugs that induced ameliorations of HE did not mediate
any unequivocal behavioral or electrophysiological effects in normal animals
[80, 82, 83]; and (2) the administration of subconvulsive doses of the full inverse
agonist, DMCM, to a rat model of HE did not efficaciously reverse HE, but induced
a preconvulsive state [80], whereas the ameliorations of HE induced by Ro 15-4513
and Ro 15-3505 were characterized by normal coordinated motor activity and
exploratory behavior in the absence of any clearly recognizable preconvulsive state
[80, 82, 83]. Furthermore, in one of the studies in which positive data were obtained
using flumazenil, behavioral and electrophysiological ameliorations of HE were
more robust following the administration of Ro 15-4513 than flumazenil [80].
Nevertheless, the possibility that a component of the observed ameliorations of HE

following administration of Ro 15-4513 or Ro 15-3505 was due to the inverse agonist
properties of the administered compound, and was therefore independent of natural
BZ receptor agonist ligands, cannot be excluded.
A third less well-known property of BZ receptor antagonists with weak partial
inverse agonist properties may also be relevant to the potential for these ligands to
ameliorate HE. Increased brain levels of neurosteroids, such as allopregnanolone,
that are positive allosteric modulators of GABAA receptors, are known to occur in
HE and are recognized as a potential mechanism of increased GABAergic tone [16].
Allopregnanolone has been shown to potentiate GABA-induced currents in cultured
hippocampal neurons. This phenomenon was attenuated by Ro 15-4513, but not by
flumazenil [99]. Thus, the beneficial effect of Ro 15-4513 on HE may be mediated,
at least in part, by its ability to reduce the effects of certain neurosteroids on the
function of the GABA neurotransmitter system.
Concluding Perspectives
Both direct and indirect evidence for an association between increased levels of
BZ receptor agonists and HE have been generated and support the hypothesis
that natural BZs with agonist properties contribute to the manifestations of overt and
subclinical HE by potentiating the action of GABA. Mean concentrations of BZ
receptor ligands in the brain in HE, when expressed in units of diazepam (or oxazepam)
equivalents, are probably sufficient to induce subtle derangements of psychomotor
function (subclinical HE) and mild sedation [22, 40, 50], but not all of the manifes-
tations of HE. Furthermore, these mean concentrations appear to be less than those
induced by encephalopathogenic doses of diazepam. However, as the BZ receptor
ligands present in the brain in HE may be functionally heterogenous [22, 31, 33, 40],
it may be inappropriate to make any assumption regarding their functional significance
from concentrations expressed in diazepam (or oxazepam) equivalents. Furthermore,
in the brain of a rabbit model of FHF, BZ receptor ligands are heterogenously
distributed [30]. Thus, their concentrations in certain regions of the brain may
greatly exceed their mean concentration for the whole brain. In addition, the
neuroinhibitory effects of BZ receptor ligands with agonist properties, and, hence,
the contribution of such ligands to HE, may be enhanced if the availability of GABA
at GABAA receptors is increased in liver failure [16, 22, 100, 101]. Indeed, increased
sensitivity of isolated Purkinje neurons from a model of HE [64, 102] and of the
brain of patients with cirrhosis and impaired hepatocellular function [103] to a
pharmaceutical BZ has been documented. Thus, the sensitivity of the GABAA
receptor complex to BZ agonist ligands is increased in HE.
Ideally, an assessment of the contribution of BZ receptor agonist ligands to HE
requires evaluating the responsiveness of the encephalopathy to the intravenous
administration of a pure BZ receptor antagonist, which is devoid of any inverse
agonist intrinsic activity and preferably, unlike flumazenil, is also devoid of any
agonist intrinsic activity. In the management of HE, the contribution of natural BZs
to the encephalopathy could theoretically be completely reversed by administering
a BZ receptor antagonist devoid of any intrinsic activity. If an antagonist with weak
partial agonist properties, such as flumazenil, is administered, the improvement
in encephalopathy induced by the drug may represent an underestimation of the
contribution of natural BZs to the encephalopathy, because potent agonist molecules
would be replaced by weaker agonist molecules on BZ receptors. Conversely, if an
antagonist with weak partial inverse agonist properties is administered, the resultant
improvement in encephalopathy might, theoretically, overestimate the contribution
of natural BZs to the encephalopathy, because any decrease in encephalopathy
attributable to its partial inverse agonist properties would be independent of the
presence of natural BZ receptor ligands. However, this line of reasoning has not
been supported by the documented responses of animal models of FHF to certain
BZ receptor antagonists with weak partial inverse agonist properties, specifically
Ro 15-3505 and Ro 15-4513 [80, 82, 83, 85].
The association of increased levels of natural BZ receptor agonists with HE pro-
vides a strong rationale for the use of a BZ receptor antagonist as a component of a
therapeutic regimen designed to facilitate optimization of mental function in patients
with HE. Such a regimen may include an intravenous infusion of a BZ receptor
antagonist, which, in contrast to intravenous bolus injections, would be likely to
induce a more predictable and sustained response. Two phases in the development
of a new drug for treatment of HE can be recognized: phase Idemonstration that
a drug of a particular class can induce an amelioration of HE; phase IIidentifying
a drug of that class that has optimal therapeutic properties. The effect demonstrated
in phase I may not necessarily be therapeutically significant. With regard to the use
of BZ receptor antagonists in the management of HE, the available data on the
effects of flumazenil on HE in humans indicate that phase I has been completed.
However, it is clear from the above discussion that the properties of flumazenil do
not conform to those of an ideal BZ receptor antagonist for use in the management
of HE [94]. Such a compound has not yet been identified and may not yet have been
synthesized. Consequently, phase II is incomplete. The properties of such an ideal
BZ receptor ligand would include: (1) slow rate of metabolism, so that its ameliorat-
ing effects on HE would be sustained; (2) high affinity and specificity for central BZ
receptors; (3) no toxic effects; and (4) minimal (agonist or inverse agonist) intrinsic
activity, so that no phenomena attributable to intrinsic activity would be apparent
after administration of a wide range of doses of potential therapeutic relevance
(Fig. 6.2).
At this time the only BZ antagonist preparation available for clinical use is
flumazenil for parenteral administration. The impressive case study that demon-
strated a complete and sustained amelioration of intractable chronic portal-systemic
encephalopathy in a middle-aged woman treated with flumazenil 25 mg orally twice
daily [78] should prompt the conduct of an appropriately designed trial to assess
more definitively the efficacy of orally administered flumazenil in the treatment of
patients with chronic portal systemic encephalopathy. A positive result in such a
trial may indicate a place for a preparation of flumazenil for oral administration in
the treatment of chronic HE on an outpatient basis. However, an oral preparation

of flumazenil has yet to be made available for such a study. Furthermore, so far BZ
receptor antagonists with partial inverse agonist properties have not yet been evalu-
ated in humans with HE, in part, because of concern over their assumed convulsive
potential. Ro 15-3505 (sarmazenil) appears to be a weaker partial inverse agonist
than Ro 15-4513 and to have a higher affinity for central BZ receptors than flumazenil
[5, 22]. Factors that contribute to the greater efficacy of both sarmazenil and Ro
15-4513 than flumazenil in ameliorating HE in animal models may include their
higher affinity for central BZ receptors and different pharmacokinetic properties,
rather than their partial inverse agonist properties, which were not shown to be
significant in the context of inducing ameliorations of HE in animal models [22, 80,
82, 83, 85]. Extrapolation from relevant findings in animal models of FHF [80, 82,
83, 85] to man may be used to justify postulating that sarmazenil may act as a BZ
receptor antagonist in patients with HE without any partial inverse agonist effects
becoming clinically apparent following administration of low doses that may well be
efficacious in ameliorating HE. Thus, although high doses of sarmazenil are considered
to have convulsive potential, the safety profile of low, and possibly therapeutically
efficacious, doses may well be sufficiently acceptable to permit carefully conducted
clinical trials of its administration to patients with HE. Furthermore, the possibility
that ameliorations of HE induced by BZ receptor antagonists with weak partial
inverse agonist properties, such as Ro 15-4513, may be mediated, at least in part, by
their ability to attenuate neurosteroid-induced potentiation of GABAergic tone [99],
may imply that the convulsive potential of such agents when used to treat HE is
even less than may have been assumed.
There are exciting prospects for improved treatment of HE with novel BZ recep-
tor antagonist ligands, if the pharmaceutical industry can be motivated to synthe-
size and make available additional BZ receptor ligands with properties that are
superior to those of flumazenil, particularly with respect to their rate of metabolism
and intrinsic activity. If such new ligands are generated it is important that they be
made available for testing experimentally in animal models of HE and, subse-
quently if indicated, in trials in humans with HE complicating both acute and
chronic liver failure.
This is one of the last publications of E. Anthony Jones who died unexpectedly on January 23rd
2012. His memory will live-on in his many proteges (K.D. Mullen).
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Part II
Diagnosis
Chapter 7
Diagnosis of Overt Hepatic Encephalopathy
Karin Weissenborn
Keywords Diagnostic approach Acquired hepatocerebral degeneration Hepatic

myelopathy Magnetic resonance spectroscopy Magnetic resonance imaging
Introduction
Deficits in attention, visual perception, visuo-spatial construction, motor speed and

accuracy are early symptoms of hepatic encephalopathy (HE) [1]. Due to subtle
nature of these cognitive deficits, psychometric testing is required to detect the earli-
est grade of cerebral dysfunction in HEthe so-called minimal HE (mHE). Later on
clinically obvious psychomotor slowing, increasing alterations of consciousness,
pyramidal and extrapyramidal as well as cerebellar symptoms and signs occur.
Patients are classified into grades IIV HE according to the degree of altered con-
sciousness (as determined by the New Haven Scale) [2]. Lethargy and psychomotor
slowing is classified as grade I HE, disorientation is considered to represent grade II
HE, somnolence and stupor grade III HE and coma grade IV HE. The accompanying
motor symptoms indicate a dysfunction of the cerebellar, extrapyramidal and pyra-
midal system, and can be detected by a thorough neurological examination even in
patients who appear clinically unaffected on the first view [3]. But the frequency and
extent of motor symptoms such as asterixis, ataxia, dysarthria, tremor, rigidity, hypo-
mimia and hypokinesia, as well as hyperreflexia, spasticity and extensor plantar
responses increases with increasing grade of HE. In patients with acute liver failure,
HE (Type A HE) is accompanied and even masked by the effects of increasing brain
edema [4]. These patients develop neurological symptoms more rapidly than those
with Type C HE, and in contrast to the latter they also suffer epileptic seizures [5].
K. Weissenborn, MD (*)
Department of Neurology, Hannover Medical School, Carl-Neuberg-Strasse 1,
Hannover 30625, Germany
e-mail: weissenborn.karin@mh-hannover.de
98 K. Weissenborn
In patients with cirrhosis, clinically overt HE usually occurs episodically. It is

often precipitated by dietary protein overload, gastrointestinal bleeding, infection,
electrolyte imbalance or CNS active medication. A few patients, however, present
with chronic progressive symptoms with Parkinsonism, dystonia or choreatic move-
ments as acquired hepatocerebral degeneration (AHD) and/or spastic paraparesis as
hepatic myelopathy (HM).
The symptoms of HE are manifold and of course none of the symptoms of hepatic
encephalopathy (HE) or hepatic myelopathy (HM) are specific. Thus, the diagnosis
of HE or HM can be made only by exclusion of other possible causes of brain or
spinal cord dysfunction, and can be proven only by a positive response to the respec-
tive therapy.
The appropriate diagnostic procedure depends on the quality and time-course of
the symptoms presented, the underlying liver disease and the patients co-morbidities
and medication.
Diagnostic Approach
Biochemical Analysis
Metabolic disturbances, which may affect brain function, besides hyperammonemia

have to be considered. These include hyponatremia [6] in patients with liver cirrho-
sis and hypoglycemia in patients with acute liver failure [4].
Measurement of serum ammonia is not indicated for routine diagnosis of HE in
clinical practice. An increased serum ammonia level may indicate HE as the cause
of a patients altered mental status, but other potential sources of hyperammonemia
have to be excluded, and hyperammonemia per se is no proof that cerebral dysfunc-
tion in the individual patient under study is due to elevated ammonia levels. Mean
ammonia levels increase with increasing grade of HE, but there is a substantial
overlap between values at different stages [7].
While the assessment of plasma ammonia levels is not of diagnostic use in
patients with HE, it appears to be worthwhile for the estimation of patients progno-
sis in case of acute liver failure [8, 9]. Arterial ammonia levels >124 mmol/L indi-
cate a higher risk of cerebral herniation, seizures and death [9].
Brain Imaging
Even in the presence of significant metabolic alterations, brain imaging should be

done in every patient who develops disorientation, somnolence or stupor to exclude
other possible causes such as intracranial bleeding. Chronic subdural hematoma
7 Diagnosis of Overt Hepatic Encephalopathy 99
may present with alterations of cognition and consciousness exclusively, but with
no focal neurological signs, and is more frequent in patients with coagulopathy
specially so in patients with alcoholic liver disease. If available, magnetic resonance
imaging (MRI) should be preferred to cranial computed tomography (CCT) because
MRI offers the opportunity to look also for the characteristic signs of Wernickes
encephalopathythe most important differential diagnosis of hepatic encephalopa-
thy, especially in patients with alcoholic liver disease [10].
MRI has been shown to be pathological in about two-thirds of alcoholics with
clinically proven Wernickes encephalopathy (WE) and in about 100% of published
WE cases in nonalcoholics [11]. Symmetric lesions are usually seen in the thalami,
mamillary bodies, tectal plate and the periaquaeductal area. But in addition, cerebel-
lar and cortical lesions as well as lesions in the splenium and the caudate nucleus
have been observed [11, 12]. Long-TR (repetition time) MR images are considered
the most sensitive technique for the diagnosis of WE and contrast enhancement of
the mamillary bodies may be the only sign of WE [13]. In practice, the radiologist
should be aware of the differential diagnosis of Wernickes encephalopathy to be
able to consider this in patients with symptoms of HE.
Of note, the symmetric pallidal signal alterations in T1-weighted images, which
are frequently observed in patients with cirrhosis, are not diagnostic for hepatic
encephalopathy [14]. They are due to an increased manganese deposition in brain
tissue with a preference to basal ganglia and indicate the presence of significant
porto-systemic shunts [15]. Newer MR imaging techniques such as MR volumetry,
diffusion-weighted imaging and magnetization transfer imaging have been used to
study hepatic encephalopathy, but none of these techniques have been evaluated for
its diagnostic use [14]. Magnetic resonance spectroscopy (MRS) of the brain in
patients with liver cirrhosis has consistently shown a decrease in myo-inositol and
choline signal intensity accompanied with an increase in glutamate/glutamine sig-
nal intensity [14]. These alterations correlate with the degree of hepatic encephal-
opathy [16] and improve with medical treatment [17] or liver transplantation [18].
But, again, the use of MRS for diagnosing HE has still to be established since the
characteristic alterations seen in patients with cirrhosis and HE may be present also
in cirrhotic patients without any signs of HE [19], and they cannot exclude the pres-
ence of another pathology that does not affect MRS such as, for example, drug
effects or thiamine deficiency.
Lumbar Puncture
Sub-acute development of disorientation and alteration of consciousness are also

frequent symptoms of encephalitis, but encephalitis has rarely to be considered in
the differential diagnosis of HE. In case there is any doubt, lumbar puncture should
be performed.
100 K. Weissenborn
Electroencephalogram
The electroencephalogram (EEG) is slowed in patients with hepatic encephalopathy.

Higher grades of HE are typically associated with theta or delta-dominated EEG
and frontal triphasic waves. But, again, this alteration is not specific for HE but can
be seen also with other metabolic disturbances such as hyponatremia or uraemia
[20]. Thus, the EEG cannot be recommended as a diagnostic tool. Instead, it can be
used for follow-up examinations and monitoring of treatment effects after the
diagnosis has been made [20].
Diagnosis of Chronic Progressive HE
Differential diagnosis of AHD is more demanding than differential diagnosis of

episodic HE. Patients with AHD present with Parkinsonism, dystonia, dyskinesia
and choreatic movements. The symptoms may develop more rapidly than in classic
neurodegenerative disorders such as Parkinsons disease, but they may also show a
very slow progress over years, and even remain stable for some time [21]. The most
frequent feature of AHD is Parkinsonism [22]. Differential diagnosis between AHD
and Parkinsons disease (PD) should consider the difference in symptom progres-
sion, the symmetry in motor symptoms in AHD in contrast to PD, the presence of
action tremor in AHD but not PD and the absence of the characteristic shuffling gait
of patients with Parkinsons disease in AHD [22, 23]. Again, brain imaging by CT
or MRI does not help in the differential diagnosis. The analysis of the striatal
dopamine D2 receptor binding capacity (which is not altered in patients with devel-
oping Parkinsons disease) combined with an analysis of the striatal dopamine
transporter binding capacity might add useful information [24, 25]. Both dopamine
D2 receptor and dopamine transporter binding have been shown to be compromised
in a patient with cirrhosis and hepatic encephalopathy by single photon emission
tomography (SPET) [24].
Differential diagnosis between AHD and the so-called Parkinson plus or atypical
Parkinson syndromes is more difficult as both syndromes share the combination of
Parkinsonian symptoms and cerebellar or pyramidal symptoms and the SPET
results. Here, again the time-course of symptom development may help to distin-
guish between these different entities. Unfortunately, the response to treatment
cannot be used for differential diagnostic purposes in this situation. AHD does not
respond to the usual ammonia-lowering therapeutic strategies working for episodic
HE, but some patients respond to dopaminergic drugs [26, 27].
Differential diagnosis of hepatic myelopathy is less demanding. Again, the clinical
symptoms are not specific, but they are quite characteristic: a rapidly progressive
spastic paraparesis without any sensory symptoms that binds the patient to a wheel-
chair within a few months in the absence of spinal cord lesions visible on MRI and
accompanied by normal cerebrospinal fluid analysis results [28, 29]. Again the
7 Diagnosis of Overt Hepatic Encephalopathy 101
diagnosis cannot be tested by the response to ammonia-lowering therapies, as HM

has been shown not to respond to the usual therapy of HE. Of note, however, there
are several reports of the beneficial effects of liver transplantation for both AHD and
HM [28, 30, 31].
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Chapter 8
Diagnosis of Minimal Hepatic Encephalopathy
Jennifer Y. Montgomery and Jasmohan S. Bajaj
Keywords Minimal hepatic encephalopathy Covert hepatic encephalopathy

Cirrhosis Psychometric testing Neurophysiological testing
Introduction
The current classication of hepatic encephalopathy (HE) is the well-known West

Haven criteria, which is based on impairment in consciousness, intellectual func-
tion, and behavior (Table 8.1) [1]. The use of the West Haven scale alone is incon-
sistent when grading patients in stages 0 through 2 because it relies on subjective
assessments by clinicians, which may vary by individual clinician and across multi-
center trials [2]. Nonspecic signs and symptoms are often used in differentiating
between stages 0 and 1; therefore, there is a lack of reproducibility and inconsis-
tency. The approach to HE as a continuum on the spectrum of neurocognitive
impairment in cirrhosis (SONIC) is now promoted [3, 4]. In a round table at the 14th
International Society for Hepatic Encephalopathy and Nitrogen Metabolism
(ISHEN) meeting, it was suggested that patients with minimal HE and grade I HE
should be grouped together, with the conjoined group termed as covert HE [5].
J.Y. Montgomery, MD
Department of Internal Medicine, Virginia Commonwealth University Health System,
Richmond, VA 23298, USA
J.S. Bajaj, MBBS, MD, MS (*)
Department of Gastroenterology, Hepatology and Nutrition,
Virginia Commonwealth University and McGuire VA Medical Center,
1201 Broad Rock Boulevard, Richmond, VA 23249, USA
e-mail: jsbajaj@vcu.edu
104 J.Y. Montgomery and J.S. Bajaj
Table 8.1 West Haven criteria for the classication of hepatic

encephalopathy
Grade Characteristics
0 No detectable abnormalities in personality or behavior
I Trivial lack of awareness
Euphoria or anxiety
Sleep disturbance, altered mood
Shortened attention span
Impaired addition and/or subtraction
Asterixis may be present
II Lethargy, apathy
Disorientation to time, amnesia of recent events
Subtle to obvious personality changes
Inappropriate behavior
Slurred speech
Asterixis is present
III Somnolence, semi-stupor
Confusion, responsive to verbal stimuli
Gross disorientation
Bizarre behavior
Clonus, nystagmus, positive Babinski sign
Asterixis is usually absent
IV Coma
Unresponsive to verbal and/or noxious stimuli
No verbal, eye, or oral response
Table 8.2 Revised classication

Performance on
New classication Old classication Mental status specialized testsa Asterixis
Unimpaired Normal No impairment No impairment Not present
Covert HE Minimal HE No impairment Impairment Not present
Grade I
Overt HE Grade II Disorientation Impairment/ Present (except
Grade III through coma abnormal in coma)
Grade IV
a
Neuropsychometric or neurophysiological tests
Patients with neuropsychological or neurophysiological abnormalities without

disorientation and asterixis would be classied as having covert HE, while those
with West Haven grade II or above would be classied as overt HE. Patients with no
clinical, neuropsychometric, or neurophysiological changes would be classied as
unimpaired (Table 8.2) [5].
Therefore, for the rest of the chapter we will be using the terms covert/minimal HE.
8 Diagnosis of Minimal Hepatic Encephalopathy 105
Diagnostic Methods
Unlike the diagnosis of overt hepatic encephalopathy, in which a physical and mental
status exam shows clear evidence of impairment, the diagnosis of covert/minimal
hepatic encephalopathy is less apparent [6]. Covert/minimal hepatic encephalopathy
shows abnormalities on psychometric testing, particularly in areas of attention
(demonstrated by loss of vigilance, disorientation), executive functions (problem-
solving, planning, judgment), visuo-spatial coordination, and psychomotor speed
(reaction times) [4]. These in turn can lead to learning and memory impairment.
Underlying many of these decits is also an impaired response inhibition [3].
Therefore, testing strategies focus on dening abnormalities related to these domains
using (a) neuropsychological or (b) neurophysiological tests. An overall description
of key tests used is presented in Table 8.3.
Neuropsychological Testing
Experts agree that a battery of tests that measure multiple cognitive domains is more
reliable and reproducible than a single test [1]. An example of a standardized battery
is the portosystemic encephalopathy (PSE) syndrome test (or Psychometric Hepatic
Encephalopathy Score [PHES]) [7]. It includes number connection test A (NCT-A),
NCT-B, digit symbol test (DST), line-tracing test (LTT), and serial-dotting test
(SDT). The ISHEN practice guidelines recommend the PHES because it is rela-
tively cross-cultural, easily applied and relies on nonverbal tasks that require mini-
mal language translation [8]. The PHES is highly specic for the diagnosis of HE,
with poor prognosis implicated by PHES scores 6, indicating severe abnormali-
ties [9]. The major limitations are the lack of normative reference data outside of
Europe, and varying performances noted among ethnic subgroups. In places with-
out PHES normative data, such as the United States, it is recommended that at least
two of the following neuropsychological tests be used: NCT-A, NCT-B, block-
design test (BDT), and DST. The current denition of minimal hepatic encephal-
opathy (MHE) is based on psychometric test results of two standard deviations less
than normal on at least two of these tests [1].
A second standardized battery, the repeatable battery for the assessment of neu-
ropsychological status (RBANS), was originally designed to assess dementia. It
includes a copyrighted set of tests in ve domains: immediate memory, visuo-
spatial/constructional, language, attention, and delayed memory. RBANS scores
predicted disability independently of liver disease severity [8]. The ISHEN practice
guidelines recommend RBANS due to the rigorous population-based standardiza-
tion in the United States; however, it has not been specically validated in HE.
The inhibitory control test (ICT) is a computerized test of sustained attention,
vigilance, working memory, and response inhibition [10]. During this test, the
patient is asked only to respond to targets and not to lures. Covert/minimal HE
106
Table 8.3 Comparison of currently available psychometric tests

Time needed
U.S. norms Copy- Expertise for adminis-
Test Domains tested available? righted? needed? tration (s/min) Limitations
Paper and pencil Number connection test Psychomotor speed Y N N 30120 s Poor specicity
psychometric A (NCT-A)a
tests NCT-Ba Psychomotor speed N Y Y 13 min Poor specicity
Set shifting (improved over
Divided attention NCT-A)
Digit symbol test Psychomotor speed Y Y Y 2 min Very sensitive; can be
(DST)a Attention used as an early
indicator
Line tracing test (LTT)a Psychomotor speed N Y N 10 min
Visuo-spatial
reasoning
Serial dotting test Psychomotor speed N Y N 14 min Only tests
(SDT)a psychomotor speed
Block design test Psychomotor speed Y Y Y 1020 min
(BDT) Praxis
Visuo-spatial
reasoning
Repeatable battery for Psychomotor speed Y Y Y 35 min Limited studies
the assessment of Visuo-spatial involving HE
neuropsychological reasoning
status (RBANS) Language
Verbal, visual,
J.Y. Montgomery and J.S. Bajaj
working memory
Time needed
U.S. norms Copy- Expertise for adminis-
Test Domains tested available? righted? needed? tration (s/min) Limitations
8
Computerized Inhibitory control test Attention Y (limited) N N 15 min Requires high

psychometric (ICT) Response inhibition functioning patients
tests Vigilance with knowledge
Working memory of computers
Cognitive drug research Attention N Y N 1520 min Requires high
(CDR) Working memory functioning patients
Episodic memory with knowledge
of computers
Neurophysiological Electroencephalography Generalized brain Y (local) N Y Varies
tests (EEG), mean activity
dominant frequency
Visual evoked Interval between Y (local) N Y Varies Highly variable results
potentials (VEPs) activity and
visual stimulus
Brainstem auditory Cortical response Y (local) N Y Varies Shown to have
evoked potentials after auditory inconsistent results
Diagnosis of Minimal Hepatic Encephalopathy
stimulus in HE patients
P300 cognitive evoked Infrequent stimulus Y (local) N Y Varies Requires patient
potentials embedded in cooperation;
irrelevant stimuli potential for good
diagnostic results
Critical icker Visual discrimination N N N 10 min Requires high
frequency (CFF) General arousal functioning patients
a
These ve tests are part of the psychometric hepatic encephalopathy score (PHES)
107
patients had longer reaction times, lower rate of target response, higher rate of lure
response than unimpaired patients, with a sensitivity of 87% and specicity of 77%
[11]. Impairment demonstrated on ICT is signicantly associated with motor vehi-
cle accidents and trafc violations [12, 13]. Other studies showed that ICT targets
are a better differentiator than lures alone, and that ICT outcome lures are more
valuable if adjusted for target accuracy [14]. ICT is a free and easily administered
test; however, even though the equipment has been standardized, there still remain
signicant variations in the threshold levels used and the test requires intense patient
concentration.
The Cognitive Drug Research (CDR) battery of tests was developed by Cognitive
Drug Research Ltd. With over 50 parallel forms of each task, it tests ve domains:
attention, continuity of attention, speed of memory, quality of episodic and working
memories. Covert/minimal HE patients were found to have impairment in all
domains, worsened after a nitrogen challenge and improved with liver transplanta-
tion [15]. The CDR has been validated in the United Kingdom and is available for
approximately 50 USD.
These neuropsychological tests are well-documented and extensively tested;
however, they have many limitations. Results are often greatly inuenced by the
patients age, educational status, and cultural/ethnic background; therefore local,
population-based normative values are necessary. The choice of which battery/
test to select should be driven by availability of local normative data as well as
expertise [5].
Neurophysiological Testing
Neurophysiological tests involve specialized, computer-assisted techniques and are

offered under the supervision of a neurologist. They are recommended to be used
in conjunction with neuropsychological tests [1]. The advantages of neurophysio-
logical testing are the absence of learned effects, objective data, and the high
specicity of the response [16]. These tools provide objective data on brain electri-
cal activity and do not require patient cooperation, which allows comparisons
between multiple centers.
Electroencephalography (EEG) is the electrophysiological technique most fre-
quently used to assess neuropsychiatric status in cirrhotic patients [17]. An EEG
reects cortical neuronal activity and shows generalized slowing of the background
activity with characteristic triphasic waves in overt HE [18]. In covert/minimal HE,
the mean dominant frequency is slowed, and has been shown to correlate with PHES
abnormalities [16]. EEG abnormalities were detected in 840% of MHE patients,
but this wide range may reect variations in technique (quality of the recording and
the analysis performed).
Evoked potentials measure the latency between an applied stimulus and the
brains ability to sense it [19]. They are small phasic potentials elicited in response
to sensory, motor, and cognitive events. Visual evoked potentials (VEPs) can be
ash, pattern-reversal, or motion-elicited. Abnormalities are observed in pure

optic nerve disorders, demyelinating processes, metabolic abnormalities and psy-
chotropic medications, but to date, results in cirrhotics have been difcult to inter-
pret [19]. Somatosensory evoked potentials (SEPs) are measured following brief
electric shocks administered via skin electrodes to large, mixed-type peripheral
nerves (i.e., median/ulnar nerves at the wrist, peroneal nerve at the knee, tibial
nerve at the ankle). A prolongation of the peak and interpeak latencies N20N65
in one or more cortical SEPs was observed in up to 50% of covert/minimal HE
patients. These ndings suggest that SEPs may be a promising tool in the diagno-
sis of HE [16, 20].
Brain electrical responses to an auditory stimulus can likewise be measured.
The P300 event-related potential (P300ERP) uses an infrequent stimulus embedded
in a series of otherwise irrelevant frequent stimuli (called oddball paradigm) [21].
Patients are asked to identify and keep count of the rare stimuli (oddballs). The
potential is evoked independent of the delivery modality used (visual, auditory,
olfactory). A typical response peaks within 250500 ms after the stimulus; a delay
greater than 2.5 SD of the age-controlled mean indicates a dysfunctional response.
Increases in the latency of responses are recorded in more than half of encephalo-
pathic patients, but there were no signicant differences between latencies of covert/
minimal HE patients and unimpaired controls. Therefore, the P300ERP alone has
limited diagnostic potential in this situation [16].
The critical icker frequency (CFF) measures the maximum frequency at which
a ickering light can still be perceived to icker [22]. As the frequency of light
pulses is decreased, the frequency at which the ickering light no longer appears
fused is called the CFF threshold. When using a threshold of 39 Hz, CFF strongly
correlates with PHES and was shown to accurately diagnose 7383% of covert/
minimal HE patients, with higher sensitivity and specicity than use of P300ERP
alone [23]. Covert/minimal HE patients were distinguished from unimpaired
patients with a sensitivity of 55% and specicity of 91100% [24]. CFF decreases
with aging and can be affected by medications (sedatives, psychotropic drugs, caf-
feine), and equipment (luminance and the color of the transmitted light) [5]. These
variables should be taken into considerationage-adjusted values should be used
and should be compared with normative reference data. Though it requires patient
cooperation and binocular vision, CFF is a simple, reproducible test that is not lim-
ited by educational status, which allows for its widespread use in the diagnosis of
covert HE [5].
Smooth pursuit eye movements (SPEM) are the conjugate eye movements used
to track smooth predictable trajectories of targets, such as small dots. Impairment
of SPEM can be observed when patients can no longer track the dot trajectory
accurately, leading to anticipatory and corrective saccadic movements which
appear jerky or with a cogwheel pattern. This can occur in many clinical situations,
including cirrhotics with hepatic encephalopathy. The degree of SPEM impairment
reects neuropsychiatric status. In patients with covert HE, there were clear dis-
ruptions of smooth pursuit with interspersed anticipatory and corrective catch-up
saccades [25].
Other Markers of Cognitive Dysfunction
In addition to cognitive dysfunction, patients with covert HE are known to have a

higher degree of extrapyramidal signs (EPS) and impairment in motor abilities,
most likely due to manganese deposits in the basal ganglia which affect dopaminer-
gic transmission [26]. Motor signs can include speech abnormalities, facial expres-
sion, resting tremor, intention tremor, nger dexterity, rigidity, gait disturbance, and
postural stability [27]. Patients with covert HE were more prone to the development
of EPS, and there is a strong correlation between the severity of EPS and neuropsy-
chological impairment [27]. However, these are nonspecic and their analysis may
be subjective. Metabolomics offers a quantitative examination of underlying meta-
bolic derangements which occur with liver dysfunction. It is applicable to a broad
spectrum of metabolites and has previously been used in the diagnosis of cancer,
coronary artery disease and diseases of the CNS. Jimenez et al. studied cirrhotic
patients with covert/minimal and found increased serum glucose and lactate levels
along with decreased serum choline and lipids [28]. A detailed approach to metabo-
nomics is needed with respect to covert/minimal HE before this can be used
routinely.
Hepatic encephalopathy is not readily identied by structural abnormalities in
the brain, although there is evidence that T1-pallidal hyperintensity may be seen on
MR images [29]. However for the most part, neuroimaging serves to exclude other
causes of brain disease in suspected HE when clinically indicated. Magnetic reso-
nance spectroscopy is a study of metabolites in various brain regions, which shows
an increase in glutamate/glutamine ratio with a compensatory decrease in myoinosi-
tol and choline in patients with hepatic encephalopathy [30]. The limited availabil-
ity and expense of an MRI scanner capable of two-dimensional MRS techniques
limits the use of this modality.
Barriers Against Testing for Covert/Minimal HE

and Consensus on Future Trials
Although the majority of clinicians agree that covert/minimal HE is a signicant

problem and requires outpatient testing, many barriers exist that prevent routine
testing. The inability to have insurance companies reimburse for testing, the extra
time added to outpatient visits, the lack of standardized norms, the reliance on psy-
chological expertise to administer and interpret test results, and the expensive and
copyrighted testing procedures are all contributors [31].
In the round table discussion at the 14th ISHEN meeting, it was concluded
that trials involving minimal/covert HE patients should be randomized and pla-
cebo-controlled. Patient populations in such trials should exclude patients receiv-
ing treatment for overt HE, and those with prior episodes of overt HE, as this was
a confounder in the classication of performance on neuropsychological tests.
In single-center or proof-of-concept studies, test operators should be experienced in

the use, administration, and interpretation of that particular test(s) and appropriate
normative reference data should be available. The test(s) should also be validated
for use in the selected patient population. In multi-center trials, there needs to be
more information on the interchangeability and standardization of tests; in the
interim, the use of two or more validated tests are recommended [5].
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Chapter 9
The Electroencephalogram in Hepatic
Encephalopathy
Piero Amodio
Keywords Electroencephalogram Triphasic waves EEG quantitative analysis

EEG spectral analysis Prognosis Event-related response Cognitive potential
P300 Neurophysiology
Principles of EEG Functioning
The electroencephalogram (EEG) represents the time-course of the difference of

electric potentials that are recorded on the scalp by electrodes placed over specific
sites, which are called derivations, with respect to a reference derivation. The EEG
tracing ultimately depends on the electric current generated by the synchronized
postsynaptic potentials of thousands of pyramidal cells of the fourth layer of the
brain cortex that are placed under the recording derivations. Unlike the ECG, it is
not possible for the EEG to have a reference derivation without any electric activity,
either on the scalp (cephalic derivation) or outside the scalp (extra-cephalic deriva-
tion). The electric activity of the reference derivation can only be minimized after
EEG recording (post-processing) by mathematical work-up [1]. Alternatively to
display the EEG as it is has been recorded, the EEG activity can be displayed in a
bipolar way (i.e. the time-course of the difference of electric potential between two
cephalic derivations), or as the voltage difference between the considered derivation
and the average of all the other derivations.
The EEG has low spatial resolution; in contrast, it has great time resolution. This
property allows the use of the EEG to detect the time-course of the electric poten-
tials that are evoked by sensory or cognitive stimuli, once the background EEG
activity is removed by mathematical handling of the signals.
P. Amodio, MD (*)
Department of Medicine, University Hospital of Padova,
Via Giustiniani, 2, Padova 35128, Italy
e-mail: piero.amodio@unipd.it
114 P. Amodio
The cortical neuronal electric activity that produces the EEG is modulated by
both physiological and pathological diencephalic and brain-stem influences. In
addition, the electric activity is extremely sensitive to metabolic and toxic influences;
therefore, the EEG is a reliable tool to detect metabolic brain dysfunction, even if in
clinical practice the EEG is manly used to detect the abrupt, abnormal electric dis-
charges characterizing epilepsy.
Clinical Scenario
The EEG is a tool that provides a functional assessment of the nervous system;
therefore, its domain is similar to that of clinical examination and complementary to
that of neuroimaging. When compared to clinical examination, the EEG provides
more quantitative assessment, which is potentially amenable for follow-up and
remains interpretable in non-cooperative patients.
In a few circumstances, the EEG is highly useful to diagnose the disease causing
confusion or comatose patterns. Examples are non-convulsive seizures, viral
encephalitis and spongiform encephalitis. Highly indicative patterns are also pro-
vided by stroke, subdural hematoma and malingering (Fig. 9.1).
In other cases, the EEG, similar to other functional evaluations (and similar to
clinical evaluation), is generally non-specific and analogous patterns can be found
Fig. 9.1 In this patient, hyperammonaemia and coma occurred after surgical porto-systemic
shunt. The diagnosis of HE was ruled out by the electroencephalogram (EEG) that disclosed
a massive suffering on the right hemisphere: a massive right stroke had occurred during the
surgical procedure
9 The Electroencephalogram in Hepatic Encephalopathy 115
in a wide variety of pathophysiological events, from transient, primary, subcortically

or metabolically induced cortical dysfunction to irreversible cortical problems.
Nonetheless, in these conditions, the pattern of EEG alterations reflects the severity
of underlying brain dysfunction. Therefore, the objection that EEG is useless for the
diagnosis of HE, because it is relatively unspecific, is meaningless and depends on
the confusion between the concept of differential diagnosis (diagnosis of a disease
vs. another disease) and of disease severity.
EEG Patterns in HE
Therefore, apart from the possibility that the EEG discloses another cause of delirium/
coma in a patient with cirrhosis, the EEG provides useful information to quantify
brain dysfunction in hepatic encephalopathy (HE). In fact, HE is associated with the
occurrence of EEG patterns that present a relationship, albeit rough, with the behav-
ioural features of the syndrome, as was proven by Parsons-Smith et al. [2] more than
50 years ago. This rough correlation with the behavioural feature does not imply that
EEG reflects brain dysfunction at a lower level than behaviour: simply they reflect two
different, albeit correlated, domain of brain function. In addition, since the behav-
ioural expression of neurologic disorder is highly influenced by the premorbid condi-
tions of the patient, by his/her will to cooperate and by other confounding factors, the
EEG can reflect biological alterations more than behaviour ability [3].
The first EEG sign of HE is a low-frequency alpha rhythm disturbed by random
waves in the theta range over both hemispheres. A certain degree of frontalization
of alpha activity was proven by quantified analysis of EEG performed by short
epoch, dominant activity, cluster analysis (SEDACA) [4]. Waves in the theta band
are generally observed in the temporal areas, but they may also be observed in the
frontal areas or diffusely on the scalp.
The increase in HE severity causes a progressive increase in theta band activity
that diffuses over both hemispheres along with high voltage arrhythmic delta band
activity. At this stage, triphasic waves are usually discernable. These are synchronous
waves with anterior dominance that appear in groups or runs, have a fronto-occipital
lag time, and are superimposed on the basic slow thetadelta rhythm. Although
triphasic waves are frequent in HE, they are not specific and can also be observed in
other types of metabolic encephalopathies (uremic, hyponatremia) or in drug intoxi-
cations (lithium, valproate, baclofen) [57].
Burst of high voltage frontal intermittent delta activity (FIRDA) or occipital
intermittent delta activity (OIRDA) can occur. In addition, the EEG reactivity to eye
opening (block of alpha rhythm) progressively decreases in parallel with the increase
in HE severity. A further increase in HE severity, in comatose patients, is character-
ized by an EEG tracing formed only by high voltage arrhythmic delta waves; finally
in severe coma, arrhythmic delta activity decreases both in frequency and amplitude
until reaching the shape of a flat EEG [8]. Once a flat EEG is reached, the information
obtainable by the EEG is saturated and further useful information on brain activity
can be obtained by somatosensory evoked potentials (SSEP) [9].
116 P. Amodio
The Objective Quantification of EEG in HE
The patterns of EEG in HE are clear for an expert; however, the inter-observer
repeatability of a classification based on pattern recognition is poor [10]. A simple
way to improve the repeatability of EEG evaluation of HE is provided by the visual
measuring of basic background frequency on posterior derivations [10]. This
approach is limited by the fact that the first stages of HE are characterized by the
mixing of rare activities in the theta range to alpha rhythm, therefore producing high
subjectivity in the estimation of basic frequency. Similarly, in more severe cases of
HE, when theta and delta activities are mixed together, the estimation of basic
rhythm is unreliable. These limitations can be overcome by objective quantification
of digitalized EEG. The simplest way to obtain proper quantification is provided by
the spectral analysis of about 6090 s of bipolar EEG signals from posterior deriva-
tions [11]. In addition, temporal-occipital (T3-O1 and T4-O2) as well as central-
occipital (Cz-O1, Cz-O2) and biparietal (P3-P4) or parieto-occipital derivations are
useful [11, 12]. Spectral analysis can be performed either by non-parametric (fast
Fourier transform) or parametric (autoregressive) procedures [13]. Using spectral
analysis, a simple quantification of EEG alteration in HE is possible: at the begin-
ning theta activity increases, later a decrease of the barycentre of the frequencies
(expressed by the mean dominant frequency (MDF)1) occurs and, lastly, an increase
in delta activity is detectable (Fig. 9.2; Table 9.1).
A stage of low power, very low delta frequency occurs in severe coma, before the
stage of flat EEG: spectral analysis can be misleading in these circumstances and no
study using quantified EEG on this stage of HE has been published.
A possible limitation of the current criteria for quantification of EEG in HE is
given by the fact that they consider only background activity from few derivations,
missing the information coming from triphasic waves or the relationship across
rhythms and their spatial distribution. Other modes/methods of quantification can
be obtained by more sophisticated techniques, such as principal component analy-
sis, SEDACA, neural network procedures and coherency esteem, which can par-
tially overcome these limitations [4, 10, 14].
Clinical Information from the EEG in HE
The EEG quantification provides good assessment both of the risk for development
of overt HE and mortality at 1-year follow up (Table 9.2) in patients who do not
display symptoms of overt HE at the time of examination. These observations provide
1
The MDF is given by the ratio of the sum of each frequency band multiplied by its electric power
over the total electric power of the interval of examined frequencies.
Fig. 9.2 The classification of EEG alterations based on spectral analysis: grade 1 is characterized
by an increase in theta activities (35%), grade 2 by a significant reduction of the mean dominant
frequency (MDF) (<6.8 Hz) without a massive increase in delta activity, grade 3 by a reduction of
the MDF with a massive increase in delta activity (49%). In the circle, a triphasic wave pattern is
shown. In severe coma, the spectral measures lose their meaning: the activity tends to disappear, in
the figure the apparent activity is due to gasping (personal observations, classification according to
Amodio et al. [12])
Table 9.1 EEG classification of HE based on spectral measures from bipolar biparietal deriva-
tions (P3-P4)
Grade MDF Theta relative power (%) Delta relative power (%)
Normal >6.8 <35 <49
Grade 1 >6.8 35
Grade 2 6.8 35 <49
Grade 3 6.8 Irrelevant 49
Reprinted from Amodio et al. [12] 1999. With permission from Elsevier
evidence that EEG is a valuable tool to investigate HE in clinical practice, even in

routine examination of outpatients with cirrhosis. In fact, EEG has the advantage to
provide data that are independent of patients cooperation and education level: as
against psychometric evaluation and is complementary to it for evaluation of patients
with covert HE.
EEG has proven to have higher predictive value on survival in minimal HE subjects
compared to psychometric testing and cognitive evoked potentials (P300 latency) [15].
118
Table 9.2 One year risk of dying and risk of developing bouts of overt HE according to EEG features (comparison of visual reading based on pattern recogni-
tion, visual reading based on the esteem of posterior basic frequency and spectral analysis)
Visual reading based on pattern recognition Visual estimate of posterior basic frequency Spectral analysis
Events per 100 Odds ratio Events per 100 Odds ratio Events per 100 Odds ratio
Grade patients-year (CI 95%) Grade patients-year (CI 95%) Grade patients-year (CI 95%)
Risk of 0 9 1 0 14 1 0 13 1
dying 1 19 1.9 (0.57.4) 1 37 2.8 (1.17.2) 1 41 4.3 (1.810.1)
2 21 2.6 (0.710.0) 23 86 8.8 (4.019.4) 23 57 6.3 (2.615.1)
34 103 12.6 (3.743.2)
Risk of 0 29 1 1 35 1 0 33 1
bouts of 1 35 1.1 (0.52.6) 2 95 2.3 (1.24.3) 1 72 2.0 (1.13.6)
overt HE 2 78 2.2 (1.04.7) 34 166 3.9 (2.07.5) 23 139 3.5 (1.96.6)
34 163 5.3 (3.411.6)
Modified from Amodio et al. [10] 2006. With permission from Elsevier
P. Amodio
Fig. 9.3 The massive improvement of EEG features after a session of MARS in a patient with HE
in acute on chronic liver failure. On the left, before treatment, a very slow EEG tracing with a high
number of triphasic waves (circles), after treatment the mental state improves (from grade 23 to
grade 1 HE) and the EEG tracing shows the disappearance of triphasic waves (personal observation)
In addition, even minor changes in EEG dynamics correlate with changes on psy-
chometric test findings as demonstrated by our group [12].
The EEG is particularly appropriate to show objectively the changes in brain
function following treatment of HE (Fig. 9.3). The improvement related to liver
transplantation is also extremely remarkable [16] and EEG may have a role in the
evaluation of post-transplant neurological complications [17].
Obviously, being a functional measure and similar to psychometric investigation,
subtle EEG changes in patients with a low pre-test probability of HE (well pre-
served liver function and absence of porto-systemic shunt) should be proven to
depend on HE and not to other causes of brain dysfunction. It should be noted,
however, that this is true also for psychometric dysfunction in patients with a low a
priori probability of HE: other causes of mild cognitive impairment different from
HE should be ruled out.
EEG Responses Evoked by Cognitive Tasks
An alternative method to use the EEG for the study of HE is the extraction of
cognitive potentials related to execution of a cognitive task. Of these, the P300 elic-
ited by the oddball paradigm was the most widely used for the study of the mildest
120 P. Amodio
manifestations of HE. This technique has limited clinical applicability [9], and its
clinical utility in comparison with the EEG for the detection and monitoring of HE
is doubtful [15, 18]. Other evoked responses, such as lateralized readiness potential
allow distinction between selective and executive phase of a response [19].
Conclusions
In conclusion, the EEG, especially with quantitative techniques, provide useful

information, independent of educational bias, for the assessment, follow up and
monitoring treatment of individuals with cirrhosis with covert, mild or severe HE.
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Chapter 10
Brain Imaging in Hepatic Encephalopathy
Rita Garca-Martnez and Juan Crdoba
Keywords Magnetic resonance Ammonia Manganese Brain edema Brain

atrophy
Abbreviations
ADC Apparent diffusion coefficient

Cho Choline containing compounds
DWI Diffusion-weighted imaging
FLAIR Fast fluid-attenuated inversion recovery
fMRI Functional magnetic resonance imaging
Glx Glutamine/glutamate
HE Hepatic encephalopathy
LT Liver transplantation
MR Magnetic resonance
MT Magnetization transfer
PET Positron emission tomography
SPECT Single-photon emission computed tomography
WML White matter focal T2-weighted lesions
R. Garca-Martnez, PhD J. Crdoba, PhD (*)

Liver Unit, Department of Internal Medicine, Vall dHebron Hospital,
Barcelona 08035, Spain
e-mail: humrita77@yahoo.es; jcordoba@vhebron.net
124 R. Garca-Martnez and J. Crdoba
Introduction
Neuroimaging techniques provide the possibility to assess the central nervous

system of patients that experience hepatic encephalopathy (HE). There are many
available methods that can be applied, which are classified according to the physical
principles of the technique (e.g., computed tomography, magnetic resonance (MR),
positron emission tomography [PET]). Some techniques (conventional T1 or T2
MR-imaging) are considered standard diagnostic methods that are available in most
centers and are very useful in the evaluation of acute confusional syndrome, because
they allow exclusion of other neurologic disease and may reveal typical signs of
HE [1]. Other techniques have been relevant to understand the pathogenesis of HE
and may become of clinical interest in the future [2]. The most common presenta-
tion of HE is in cirrhotic patients (HE type-C). This population is also the most
frequently included in neuroimaging studies. One of the limitations of the studies
that have been performed is the difficulty to separate the disturbances caused by
liver failure (without impact on brain function) from those that are directly involved
in HE. In addition, these patients exhibit frequent co-morbidities that may partici-
pate in the neurological manifestations, such as cerebrovascular disorders or alco-
hol-induced injury. This chapter aims to review the contribution of neuroimaging
techniques to understanding the pathogenesis, diagnosis and monitoring of HE,
emphasizing the role of magnetic resonance.
Brain Features That Are Relevant for Neuroimaging
Manganese Accumulation
Liver failure may lead to the accumulation in the brain of manganese, a metal that
is excreted in the bile. Increased blood levels are seen in the presence of large portal-
systemic shunts. Manganese shows a preference to accumulate in some areas of the
brain, such as the basal ganglia, due to the presence of specific carriers that relate to
the participation of manganese in certain metabolic pathways. Manganese has a
key role in the normal functioning of several enzymes including mitochondrial
superoxide dismutase [3], glutamine synthetase, and phosphoenolpyruvate car-
boxykinase [4]. The metal was first considered to be neurotoxic more than 150
years ago, when workers employed in grinding black oxide of manganese devel-
oped an unsteady gait and muscle weakness [5]. Since that time, many cases of
manganese neurotoxicity (manganism), a neurological disease characterized by
psychological and neurological abnormalities has been reported [6]. It has some
similarities to Parkinsons disease and has been reported particularly in miners,
smelters, welders, and workers involved in the alloy industry. Typically, patients
exhibit extrapyramidal changes that include hypokinesia, rigidity, and tremor.
10 Brain Imaging in Hepatic Encephalopathy 125
Ammonia
Patients with liver failure or portal-systemic shunting have elevated levels of

circulating ammonia, which enters the brain through the bloodbrain barrier,
increasing the brainblood ammonia concentration ratio. PET studies using 13NH3
provide evidence of the increased bloodbrain ammonia transfer and brain ammo-
nia utilization rates in patients with chronic liver failure [7]. This hyperammone-
mia results in profound astrocyte changes, including Alzheimer type II changes
seen commonly in longstanding type C HE and astrocyte swelling in acute HE.
One of the effects of ammonia is inducing a rise in brain glutamine in astrocytes,
the only cell in the brain that has glutamine synthetase. The rise in glutamine may
cause an increase in the intracellular osmolality and induce compensatory meta-
bolic changes to counteract the osmotic imbalance induced by intra-astrocytic glu-
tamine accumulation. In chronic liver failure, there is enough time for activation of
effective compensatory mechanisms of cellular adaptation to the osmotic changes:
glial accumulation of one organic osmolyte, glutamine, should lead to the loss of
other organic osmolytes, such as myo-inositol, taurine, and choline. This osmo-
regulatory mechanism may account for the protection against massive edema in
chronic liver failure [8].
Brain Edema
Brain water secondary to severe liver failure [9] and to hyperammonemia (urea
cycle disorders) has been clearly documented. A large increase in brain water which
results in intracranial hypertension is seen more often but not exlusively in patients
with fulminant hepatic failure [10]. The relevance of more subtle cerebral edema is
a matter of much debate, primarily because this is a common finding in patients with
HE in chronic liver disease. Experimental studies indicate that brain edema is mostly
located in the astrocytes, but may also be located in the interstitial compartment and
be secondary to disturbances in the bloodbrain barrier [11].
Brain Atrophy
Neuropathological studies have documented loss of brain tissue in chronic liver

failure, especially in patients with long-lasting HE manifestations and large porto-
systemic shunts [12]. This is usually referred to as acquired hepatocerebral degen-
eration [13], and it may have milder forms that are more common than previously
recognized.
Magnetic Resonance: T1 High Signal Intensity
Since the introduction of MR imaging in clinical practice, it has been well described
that the majority of patients with cirrhosis or portal-systemic shunts exhibit a bilat-
eral, symmetrical high signal intensity at the globus pallidus and substantia nigra
(Fig. 10.1) [14]. The signal may increase after performing a portal-systemic
shunting with transjugular intrahepatic portal-systemic stent placement [15] and
reverses after normalization of liver function [16] or after occlusion of congenital
portal-systemic shunts [17]. The most plausible explanation for the increased T1
signal is a rise in manganese concentration (a paramagnetic substance) in the CNS,
with preferential deposition in the globus pallidus [18]. The arguments favoring the
manganese hypothesis include the dramatic blood and CSF manganese increase
in patients with cirrhosis and pallidal hyperintensities [19], normalization of MR
signal abnormalities and manganese levels after liver transplantation [16], and the
several-fold increase in manganese concentrations from pallidal samples obtained
at autopsy in cirrhotic patients [20].
This manganese-related MR signal abnormality has also been described in non-
cirrhotic patients, such as those receiving total parenteral nutrition [21], patients
with occupational exposure to manganese from welding [22], and patients with
noncirrhotic portal vein thrombosis or congenital portal-systemic bypass and no
intrinsic hepatocellular disease [23]. In all these situations, the MR signal changes
resolve after discontinuation of manganese intake [21]. Similar findings were
observed in a patient with Alagilles syndrome [24], an autosomal dominant disorder
characterized by cholestasis, intrahepatic bile duct paucity, end-stage liver disease,
and elevated blood manganese.
Bilateral basal ganglia T1 signal changes have also been observed in several
conditions unrelated to increased brain manganese levels (e.g., nonketotic hyperg-
lycemic episodes, hypoxic-ischemic encephalopathy, basal ganglia calcification,
neurofibromatosis type I, and Japanese encephalitis), although the high signal inten-
sity occurring in these conditions does not usually show symmetrical, predomi-
nantly pallidal involvement [25].
Although pallidal hyperintensities are found in about 90% of cirrhotic patients,
these signal alterations are not closely linked to the presence of HE. It has been
shown that cirrhotic patients with no clinical, neuropsychological, or neurophysi-
ological signs of HE can also show severe signal alterations, while others with
manifest HE may present only slight signal alterations [2628]. Moreover, longitu-
dinal studies have shown quick regression of HE after liver transplantation, while
T1 signal abnormalities need up to 1 year to resolve (Fig. 10.1) [28, 29]. The clinical-
MR discrepancy may, therefore, be explained by the fact that T1 high signal inten-
sity cannot be used as a quantitative measure of tissue manganese, as it represents
only semiquantitative measurement of abnormal manganese deposition. Thus, it is
possible that manganese accumulation participates in the pathogenesis of HE only
after reaching a certain threshold, which may not be clearly identified by MR. An
interesting study supports the concept that the presence of parkinsonism is related
Fig. 10.1 Transverse T1-weighted MR images of the brain in chronic liver failure. Observe the
bilateral and symmetric high T1 signal change involving the globus pallidus (white arrow)
to the extension of the high signal intensity to midbrain structures (particularly

substantia nigra), as this MR feature is unique to patients with cirrhosis-related
parkinsonism. These data provide a good explanation for the apparent clinical
radiological discrepancies. The current understanding is that T1 high signal inten-
sity identifies the deposition of paramagnetic substances that participate in the
pathogenesis of chronic rigidity-akinesia. From a clinical perspective, the value of
T1 high signal intensity in the diagnosis of chronic neurological manifestations has
not been assessed. However, the clinical experience indicates that the absence of T1
high signal intensity is a strong argument against interpreting the neurological
manifestations as secondary to liver failure [30].
1
H-MR Spectroscopy: Metabolites
MR detects the relaxation properties of some atoms (most usual isotopes are 1H,
31
P, 23Na, 13C) in strong magnetic fields and according to how the data are processed
can generate high-resolution images or spectrum of several metabolites that
contain the atoms that are studied. The spectrum contains a series of metabolites
that are displayed as peaks at different frequencies (Fig. 10.2). 1H magnetic reso-
nance spectroscopy shows relative to creatine an increase in glutamine/glutamate
(Glx) signal and a decrease of choline containing compounds (Cho) and myo-
inositol. Abnormalities in the Glx signal have been interpreted as an increase
Fig. 10.2 MR of the brain in a patient that exhibits grade II HE that was repeated 6 weeks later
when the patient exhibited minimal HE. MR-spectroscopy shows an increase in the peaks that
contain glutamine (Glx) and a decrease of the peaks containing myo-inositol (mIns) and choline
(Cho). After improving HE, the peak of Glx decreased and the peak of mIns increased. Other peaks
correspond to n-acetyl-aspartate (NAA), and creatine (Cr)
in brain glutamine secondary to the metabolism of ammonia in astrocytes.

Disturbances of Cho and myo-inositol have been interpreted as a compensatory
response to the increase in intracellular osmolality caused by the accumulation of
glutamine in astrocytes. Disturbances in MR-spectroscopy have been proposed as
a signature of HE because the severity of these changes has been associated with
HE [31]. The origin of these disturbances appears to be ammonia. In patients with
cirrhosis subjected to ammonia load, 1H-MRS consistently show increases in the
Glx signal accompanied by myo-inositol depletion, and decreases in the choline
signal [3234].
Longitudinal studies have assessed the evolution of 1H-MR spectroscopy
abnormalities after liver transplantation (LT) and demonstrated their reversibility
[26, 27, 34]. However, time to normalization for each metabolite is different. The
Glx peak normalizes within the first 12 months (except in cases of high peaks)
after liver transplant, whereas myo-inositol normalizes slower, and may take 37
months. This reversibility precedes the disappearance of pallidal hyperintensity
after LT and correlates with improvements in neurologic manifestations [35].
The data provided by MR-spectroscopy may be refined using a two-dimensional
analysis. In a study performed in patients with minimal HE, a decrease in myo-
inositol was the most accurate predictor for minimal HE compared with MR
imaging or neuropsychological tests [36]. The decrease in the peak of myo-inositol
is exacerbated by hyponatremia, as can be expected from the functional role of

myo-inositol. In a prospective study of prognostic factors for development of overt
HE among 61 cirrhotic patients, low levels of myo-inositol were associated to
hyponatremia, which was the major risk factor of overt HE [37]. However, these
metabolic changes have also been observed in patients with cirrhosis and neither
clinical nor psychometric or neurophysiologic signs of cerebral dysfunction. The
current understanding is that the increase in brain Glx and the decrease in myo-
inositol reflect the neurometabolic changes associated with HE, but they are not
perfect indicators of neuronal function and clinical manifestations.
Magnetic Resonance: Brain Water
Standard MR may reveal changes in the volume of brain parenchyma, such as

decrease in the size of ventricles, cortical sulcal effacement, and attenuation of the
signal intensity of brain parenchyma. However, mild to moderate accumulation of
water may not be apparent. Studies comparing the brain of patients before and after
liver transplantation show an increase in the size of the ventricles and a decrease in
white matter lesions that are compatible with resolution of mild edema after liver
transplantation [38]. The increase in brain water has been confirmed with water
quantification maps, a sophisticated method of MR that measures directly the
amount of water in the tissue [39]. Conventional MR imaging techniques, such as
T2-weighted signal-intensity reveal no abnormalities. However, other MR imaging
sequences have been applied in the recent years for the assessment of brain in liver
diseases. These include magnetization transfer imaging, fast fluid-attenuated inver-
sion recovery (FLAIR) imaging, and diffusion-weighted imaging (DWI), which are
more sensitive to detect changes in brain water. These studies support the hypothe-
sis of low-grade diffuse brain edema in patients with chronic liver disease.
Magnetization Transfer
Magnetization transfer (MT) imaging is based on the interaction between protons in

a relatively free environment and those in which movement is restricted [40].
Exchange of this saturated magnetization with free water reduces the signal intensity
observed in the subsequent MR imaging. The degree of signal-intensity loss depends
on the attenuation of the macromolecules in the interrogated tissue. Different
studies assessed MT ratios in the brain of patients with cirrhosis [27, 41, 42] showing
all of them had low values in different regions of the brain. Compared to other
diseases, the decrease in MT ratio is mild (10%) and without significant abnor-
malities on conventional T1- and T2-weighted images. In addition, this decrease
returns to almost normal values after liver transplant in parallel with improvement
in neuropsychological alterations [27], supporting the hypothesis of changes in
brain water rather than reduction in brain tissue.
FLAIR
This MR imaging sequence is used to nullify signal from fluids, such as cerebrospinal
fluid. In the subsequent image, abnormalities that are normally covered by bright
fluid signal can be revealed. Several studies have assessed the brain of cirrhotic
patients with this technique [4346]. Two findings emerge from these studies:
(a) High signal intensity along the hemispheric white matter or around the corti-
cospinal tract was observed in cirrhotic patients with an improvement after LT
[43] or after resolution of the episode of HE (Fig. 10.3) [46]. Although similar
findings have been observed in diseases which pathologic bases are axonal loss
or demyelination, several factors lead to reject this interpretation and to support
the presence of mild brain edema in cirrhotic patients. The progressive normal-
ization in prospective follow-up in parallel with improvement of cognitive
function, normal values of neuronal marker (N-acetyl-aspartate/creatine) in 1H-
MR spectroscopy, and lack of other signs concordant with loss of brain tissue
in other MR imaging sequences are more consistent with the concept of low-
grade brain edema.
(b) White matter focal T2-weighted lesions (white matter lesions [WMLs]) are
attributed to degenerative small-vessel cerebrovascular disease (Fig. 10.4).
They are often seen in MR images of general population over 60 years old, with
a progressive increase in their volume and associated to cognitive decline [47].
In patients with cirrhosis, these lesions showed a decrease in their volume after
improvement of HE [45] and after liver transplant [38, 44]. The shrinkage of
Fig. 10.3 Serial transverse T2-weighted fast-FLAIR images obtained in a patient with liver cir-
rhosis during an episode of hepatic encephalopathy. Observe the symmetric areas of increased
signal along the corticospinal tract in both cerebral hemispheres (upper panel). This signal abnor-
mality almost completely reversed after liver transplant (lower panel)
Fig. 10.4 (a) Baseline MR study (transverse fast-FLAIR T2-weighted imaging) of a 56-year-old
patient with hepatitis C cirrhosis without overt hepatic encephalopathy. Multiple focal white mat-
ter lesions in both cerebral hemispheres were attributed to small vessel disease. (b) A new scan
obtained 2 years later during an episode of hepatic encephalopathy shows marked increase in size
of these focal white matter lesions. (c) A new follow-up scan after complete resolution of neuro-
logical symptoms shows decrease in size of the white matter lesions. This last scan was almost
identical to the first study
these lesions is closely associated with the improvement of neuropsychological

function and this is the opposite behavior observed in WMLs attributable to
small-vessel cerebrovascular disease. The most plausible explanation for this
feature is the existence of brain edema.
Diffusion-Weighted Imaging
While MT or FLAIR sequences are sensitive to detect an increase in the content of

brain water, these techniques cannot discriminate whether this water is intracellular
or extracellular. DWI allows calculating an apparent diffusion coefficient (ADC),
which represents a tool to understand the interaction between water and the cellular
barriers in its movement. Several studies revealed a significant increase in brain

water diffusivity, more pronounced with the severity of HE [4850]. A study per-
formed in 13 cirrhotic patients showed that induced hyperammonemia causes
osmotic changes (increase in glutamine and decrease in myo-inositol) and a
significant increase in ADC demonstrating that ammonia can lead to changes in
brain water distribution [51]. A prospective study among patients with overt HE
showed that after the resolution of the episode, there is a decrease in the ADC in the
parietal gray matter suggesting a flux between extracellular and intracellular com-
partments [52]. Furthermore, in a study performed among 40 cirrhotic patients,
ADC showed a good correlation with neuropsychological test and its ability to pre-
dict the development of overt HE [53]. According to the basis of diffusivity [54],
increase in ADC indicates augmentation of water in the extracellular compartment
and consequently, does not support the astrocytic swelling as the cause of diffuse
low-grade brain edema in cirrhosis.
Brain Size
Neuroradiological studies have documented brain atrophy in patients with cirrhosis,

which is aggravated in those with history of alcohol abuse [55]. Volumetric MR
techniques [56] have been applied to cirrhotic patients after liver transplant to assess
changes in brain size after the resolution of low-grade brain edema [57]. This tech-
nique has shown that the normalized brain volume (according to a standard cranial
size) was smaller in patients with prior HE after adjustment for age. This finding,
together with the persistence of cognitive deficits in those patients with prior HE
[57, 58], supports the hypothesis of structural damage secondary to HE [59]. Voxel-
based morphometry has also observed a decrease in brain density in patients with
cirrhosis compared with healthy controls in several areas of the brain, which was
more pronounced in those patients with alcohol etiology and was related to the
severity of liver failure. The decrease in brain density was associated with neurop-
sychological performances and persisted after liver transplant [60].
Functional Studies
In addition to changes in the structure and metabolism of brain tissue, patients with
HE experience disturbances in neuronal function. These disturbances are heteroge-
neously distributed in the first stages of HE and are more conspicuous in some
regions. The reasons for the higher vulnerability of some regions, such as the basal
ganglia, the hippocampus, the corticospinal tract or the anterior cingulate cortex, is
not known, but probably relates to specific neurochemical properties of these
regions.
Magnetic Resonance
Functional magnetic resonance imaging (fMRI) measure the hemodynamic response

(change in blood flow) associated to different brain areas during predetermined
tasks. The selected tasks are related to specific brain areas and allow examining the
function of this region. This technique requires patients collaboration so that is
suitable for patients with low-grade HE. Few studies have evaluated cirrhotic
patients with fMRI. These methods are capable of demonstrating the functional
impairment, such as disturbances in attention [61] or the need to recruit more brain
areas to carry out simple tasks [62]. These disturbances are indicative of the cogni-
tive disturbance, but are not specific to HE.
Nuclear Imaging Techniques
These techniques consist of assessing with PET or single-photon emission computed

tomography (SPECT); the signal generated by radioisotopes that are administered
intravenously. The radioisotopes are linked to molecules, such as deoxyglucose,
which provides functional and metabolic information of the brain. The most relevant
limitations are the spatial resolution, and the lack of information about the anatomi-
cal structures and the detected metabolic data.
PET provides quantitative data of isotope distribution (nCi/mL); the main iso-
topes used to study brain function are 15O, 13N, 11C, and 18F. In contrast, SPECT
provides only relative measurements of the radioactivity (counts/mL) and uses
uncommon biological elements such as 99mTc. However, SPECT is more easily
available and less expensive than PET. Most recent application is the bimodal imag-
ing that combines the anatomical images of CT or MR with functional images.
PET has been applied to investigate cerebral ammonia metabolism (13N-ammonia
PET) in parallel with cerebral glucose utilization (18F-fluorodesoxyglucose PET)
[63]. In this study, plasma ammonia levels correlated with ammonia metabolism of
the brain and with MR-spectroscopy in white matter. MR spectroscopy showed also
a correlation with cerebral glucose utilization. However, ammonia metabolism and
glucose utilization were not associated. The study suggests that cerebral ammonia
metabolism is important in the development of HE but is not the only factor.
Energy impairment has been proposed to play a role in the pathogenesis of HE.
Oxygen consumption (15O-oxygen PET) and cerebral blood flow (15O-water PET)
have been investigated in cirrhotic patients with and without HE, and compared to
healthy controls [64]. HE induced a decrease in oxygen consumption and cerebral
blood flow. The analysis of flow-metabolism coupling indicated that the decrease in
blood flow was not the cause, but the consequence of reduced brain energy metabo-
lism. It has been proposed that the inability to use the delivered oxygen of patients
with HE relates to a specific inhibition associated with oxidative metabolism in
mitochondria [65].
One of the possible clinical applications of PET/SPECT is the differential diagnosis

with other neurological disorders, such as Alzheimers or Parkinsons disease, for
which there are specific radioligands that are of clinical help [66]. Future studies
should confirm its utility for difficult to diagnose cases.
Conclusions
Neuroimaging has had a rapid development in the last years and the data obtained
from the brain of patients with different stages of liver disease provided us with a
better understanding of the pathogenesis of hepatic encephalopathy. These tech-
niques have been useful in the comprehension of the role of manganese in neuro-
logic complications of liver disease, the development of diffuse low-grade brain
edema, and the possible permanent damage associated with this metabolic condi-
tion. The wide information obtained with these tools support their use in monitoring
hepatic encephalopathy and evaluating the effect of new therapeutic measures.
Financial Support CIBEREHD is supported by Instituto de Salud Carlos III, Madrid, Spain.
Rita Garca-Martnez has been supported by grant CM07/00109.
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Part III
Treatment
Chapter 11
Disaccharides in the Treatment
Praveen Sharma and Shiv Kumar Sarin
Keywords Hepatic encephalopathy Disaccharides Lactulose
Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome, which may

complicate acute or chronic liver failure. It is characterized by changes in mental
state including a wide range of neuropsychiatric symptoms ranging from minor
signs of altered brain function to deep coma. Traditionally, HE is graded according
to the West Haven criteria, which dene HE grades IIV based on the presence of
specic clinical signs and symptoms and their severity [1, 2]. However, patients
with cirrhosis present with a continuous severity spectrum of neuropsychological
symptoms ranging from entire normality (HE 0) up to obvious decits [3]. Even in
minimal HE (MHE) without obvious clinical symptoms, neuropsychological and
neurophysiological testing uncovers decits which impact on the quality of life and
the tness to drive a motor vehicle [47].
There is consensus that ammonia is a key toxin in HE, which may sensitize the
brain to the different precipitating factors (Fig. 11.1) [8, 9]. Astrocytes play an
important role in the pathogenesis of HE with consequences on neuronal function.
At the neurophysiological level, the motor decits seen in patients with HE are
characterized by a pathologically altered oscillatory coupling within the central
motor system and the cognitive decits are assigned to pathologically alter oscilla-
tory activity in higher cognitive brain areas [10, 11]. In chronic liver disease, urea
synthesis is impaired and the brain acts as an alternative major ammonia detoxication
pathway. Astrocytes have the ability to eliminate ammonia by the synthesis of
P. Sharma, MD, DM S.K. Sarin, MD, DM, FNA, FNASc (*)

Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi 110070, India
e-mail: shivsarin@gmail.com
142 P. Sharma and S.K. Sarin
Fig. 11.1 Pathogenesis of brain swellingammonia
glutamine through amidation of glutamate by the enzyme glutamine synthetase

[1214]. Hyperammonemia leads to the accumulation of glutamine within astro-
cytes, which exerts an osmotic stress that causes astrocytes to take in water and
swell. Further support for the ammoniaglutaminebrain water hypothesis has been
provided by inducing hyperammonemia in patients with cirrhosis through the oral
administration of an amino acid solution. An increase in brain glutamine, reduction
in magnetization transfer ratio, and signicant deterioration in neuropsychological
function were suggestive of an increase in brain water [15].
Nonabsorbable Disaccharides and Mechanism of Action
For over 25 years, nonabsorbable disaccharides have been the rst-line drug treat-
ment for lowering the production and absorption of ammonia in HE. Current therapies
for HE are based on ammonia lowering, with the hypothesis that the colon is the
11 Disaccharides in the Treatment of Hepatic Encephalopathy 143
Fig. 11.2 Therapies for hepatic encephalopathy based on site of action
primary organ that generates ammonia (Fig. 11.2) [2, 16]. Lactulose is the most
commonly utilized nonabsorbable disaccharide for HE. Lactulose, a synthetic disac-
charide, is comprised of the monosaccharides lactose and galactose, and is available
as a syrup. Doses are generally titrated to achieve two to four semisoft stools daily,
with typical doses of 20 g/30 mL orally 34 times per day. A second nonabsorbable
disaccharide, lactitol, has also been used in the treatment of HE, but it is not currently
commercially available in the United States [17, 18]. Lactitol (p-galactosido-sorbitol)
is a disaccharide analog of lactulose which is neither absorbed nor broken down in
the small intestine, but is extensively metabolized by colonic bacteria. It is produced
in a highly soluble crystalline powder form which is reported to be less sweet in
taste than lactulose. Clinical trials have reported lactitol dosages of 10 g every 6 h,
0.5 g/kg/day twice daily and lactitol powder 66.7 g/100 mL titrated to two bowel
movements daily (mean equivalent of approximately lactitol 30 g/day) [17, 19].
Both lactulose and lactitol get metabolized by the bacteria in the colon to acetic and
lactic acid. This acidication of the colon not only creates a hostile environment for
the survival of intestinal bacteria with urease activity involved in the production of
ammonia in the gut, but also facilitates the conversion of NH3 to nonabsorbable
NH4+. Both effects result in reduced levels of ammonia in the colon and portal blood.
Nonabsorbable disaccharides also cause a fourfold increase in fecal nitrogen excre-
tion due to their cathartic effect [2022].
Clinical Efficacy of Nonabsorbable Disaccharides
Lactulose or Lactitol Versus Placebo or No Intervention
Overt Hepatic Encephalopathy
The nonabsorbable disaccharides have been a mainstay of therapy for HE for

decades, and have been extensively studied in several small clinical trials since the
late 1960s for this indication. In most of these trials, patients had cirrhosis with
acute, chronic or minimal hepatic encephalopathy [2330]. Oral lactulose was used
in majority of these studies though some had also used lactitol and lactulose enemas
[26]. Uribe et al. [26] performed a double-blind, controlled trial to study the efcacy
of acidifying enemas of lactitol and lactose vs. nonacidifying tap-water enemas in 45
episodes of acute portosystemic encephalopathy. A favorable response to treatment
was obtained in 19 (86%) of the patients receiving lactitol enemas and in 14 (78%)
of those receiving lactose enemas. They concluded that acidifying agents like lac-
tose and lactitol are effective and superior to tap-water enemas for the treatment of
acute nitrogenous portosystemic encephalopathy. In most of the studies, the daily
mean doses of lactulose ranged from 30 to 80 g (median 50 g) to obtain two to three
semisoft stools per day. The median duration of treatment was 15 days (range 5360
days). None of the trials followed up patients after the end of treatment. A recent
meta-analysis evaluated 22 clinical trials in order to better assess the utilization of
nonabsorbable disaccharides in the management of HE when compared with pla-
cebo, no intervention, or antimicrobials. Compared with placebo or no intervention,
lactulose and lactitol seemed to reduce the risk of no improvement of hepatic
encephalopathy (relative risk 0.62, 95% condence interval 0.460.84). However,
high quality trials found no signicant effect of lactulose or lactitol on the risk of no
improvement (0.92, 0.422.04), whereas low quality trials found a signicant
benecial effect of lactulose or lactitol (0.57, 0.400.83) [31, 32]. At the present
time, however, there is a lack of sufcient evidence to thoroughly refute the use of
nonabsorbable disaccharides for the treatment of HE.
Minimal Hepatic Encephalopathy
Trials in patients with minimal hepatic encephalopathy (MHE) found that lactulose or
lactitol signicantly reduced the risk of no improvement assessed by various psycho-
metric tests (0.61, 0.470.79). Compared with placebo or no intervention, lactulose
and lactitol had no signicant effect on mortality but tended to lower blood ammonia.
Reported adverse events were not serious, and all originated from the gastrointestinal
tract (diarrhea, atulence, abdominal pain, or nausea) [31]. A summary analysis of
some clinical trials of nonabsorbable disaccharides vs. placebo/no treatment is shown
in Table 11.1. In a recent study, Prasad et al. [30] found that treatment with lactulose
improved both cognitive function and health-related quality of life, as measured by
the Sickness Impact Prole, when compared against a no treatment patient group.
Table 11.1 Comparison of nonabsorbable disaccharides and placebo or no treatment for hepatic encephalopathy
Trial Study design Patients No. Treatment Assessment Efcacy
Simmons et al. [23] Parallel AHE + CHE 26 Lactulose/glucose Clinical grading, Lactulose = glucose
ammonia, stool
production
Rodgers et al. [24] Crossover CHE 6 Lactulose/sorbitol Clinical grading, Lactulose = sorbitol
EEG, ammonia
Corazza et al. [25] Parallel CHE 32 Lactulose/placebo Encephalopathy Lactulose better than
intensity score, placebo
ammonia
Uribe et al. [26] Parallel AHE 15 Lactulose enema Mortality, clinical Lactulose > placebo
grading
Horsmans et al. [27] Parallel MHE 14 Lactulose/placebo Psychometric tests, Lactulose > placebo
ammonia levels
Watanabe et al. [28] Parallel MHE 36 Lactulose/no treatment Three psychometric Lactulose > placebo
11 Disaccharides in the Treatment of Hepatic Encephalopathy
tests, ammonia
Dhiman et al. [29] Parallel MHE 26 Lactulose/no treatment Psychometric tests Lactulose > placebo
Prasad et al. [30] Parallel MHE 61 Lactulose/no treatment Psychometric tests Lactulose > placebo
and HRQOL
AHE acute hepatic encephalopathy; CHE chronic hepatic encephalopathy; MHE minimal hepatic encephalopathy; EEG electroencephalography
145
Lactulose Versus Lactitol for the Treatment

Lactulose and lactitol both have been used for the treatment of HE and lactulose has
been compared with lactitol in various studies (Table 11.2) [18, 19, 3340]. In a
meta-analysis by Blanc et al. [39], evaluated parameters were portosystemic enceph-
alopathy index of Conn after treatment, the percentage of improved patients, and the
percentage of patients who had ill effects related to the treatment (atulence, diar-
rhea). The duration of the treatment ranged from 3 to 6 months. All studies found a
similar efciency with both drugs. However, they exhibited some discrepancies in
the relative frequency of adverse reactions (atulence). Meta-analysis showed no
statistical differences in the portosystemic encephalopathy index after lactitol or
lactulose treatment. The percentage of improved patients after lactitol or lactulose
was similar [39]. In contrast, the analysis revealed a higher frequency (p less than
0.01) of atulence in patients treated with lactulose compared with those treated
with lactitol. In conclusion, this meta-analysis shows no statistical difference
between therapeutic effects of lactitol and lactulose, but it does show a higher fre-
quency of atulence with lactulose [39]. However, an another meta-analysis by
Camm et al. [40] showed that lactitol was as effective as other disaccharides in the
treatment of encephalopathy: pooled odds ratio was 0.83, 95% condence interval
was 0.381.82. Patients experienced fewer side effects during treatment with lacti-
tol, but the pooled odds ratio was not statistically signicant. In all studies, lactitol
was considered more palatable [33, 38]. Clinical effectiveness of lactitol, in long-
term treatment of chronic encephalopathy, is similar to those of lactulose. It seems
that lactitol has lower side effects than lactulose.
Comparison of Lactulose and Antimicrobial

Agents for Hepatic Encephalopathy
Antimicrobial agents have long been utilized as an alternative treatment option for
patients intolerant or unresponsive to nonabsorbable disaccharides. Neomycin and
other antimicrobials are utilized as a treatment modality in HE due to their ability
to inhibit ammonia production by intestinal bacteria [41]. Other antimicrobials,
including metronidazole and vancomycin, have been studied to a more limited
extent than neomycin (Table 11.3) [4244]. Orlandi et al. [43] conducted a random-
ized study in order to compare the course of HE in patients treated with neomycin
plus magnesium sulfate or with lactulose. The treatment groups were similar in
terms of clinical characteristics, fatalities, recovery rate from grade 1 encephalopa-
thy, and disappearance rate of neuropsychiatric signs. Transitions from severe to
grade 1 or 0 encephalopathy showed a 0.17 (NS) difference in favor of neomycin.
Early therapy and evidence of precipitating factors showed a favorable prognostic
signicance.
Table 11.2 Comparison of lactulose and lactitol for hepatic encephalopathy
Treatment
Trial Study design Patients No. duration Assessment Efcacy
Lanthier et al. [33] Crossover CHE 5 6 months Clinical examination, Lactulose = lactitol
psychometric tests,
ammonia levels, EEG,
cerebral blood ow
Morgan and Hawley [18] Parallel, double AHE 25 5 days Psychometric tests, EEG, Lactulose = lactitol
blind PSE index
Heredia et al. [19] Parallel AHE 40 5 days Mortality, clinical grading, Lactulose = lactitol
PSE grade, adverse events
Heredia et al. [34] Randomized, CHE 25 6 months Psychometric tests, ammonia Lactulose = lactitol
crossover levels, EEG, PSE index
Morgan et al. [35] Double-blind, MHE 9 3 months Psychometric tests, ammonia Lactulose = lactitol
random- levels, EEG
ized,
crossover
Riggio et al. [36] Parallel CHE + MHE 31 6 months PSE index, new episodes Lactulose = lactitol
of HE, adverse events
Grandi et al. [37] Cross-over CHE 40 PSE index, adverse events Lactulose = lactitol
Pai et al. [38] Parallel AHE 45 5 days PSE index, adverse events Lactitol > lactulose
Blanc et al. [39] Meta-analysis CHE 77 36 months PSE index Lactulose = lactitol
Camm et al. [40] Meta-analysis CHE PSE index Lactulose = lactitol
AHE acute hepatic encephalopathy; CHE chronic hepatic encephalopathy; MHE minimal hepatic encephalopathy; PSE portosystemic encephalopathy
147
Table 11.3 Comparison of lactulose and neomycin, metronidazole for hepatic encephalopathy
No. of Duration of
Trial Study design patients treatment Assessment Efcacy
Conn Neomycin vs. 29 10 days each Mental status, Neomycin = lactulose
et al. lactulose arm before asterixis score,
[41] (double- crossover EEG, ammonia
blind, levels,
randomized, PSE index
crossover)
Atterbury Parallel 47 7 days Mental status, Neomycin = lactulose
et al. asterixis score,
[42] EEG, ammonia
levels,
PSE index
Orlandi Single blind 173 14 days Mental status, Neomycin = lactulose
et al. asterixis score,
[43] EEG, ammonia
levels,
HE change
EEG electroencephalography
Comparison of Lactulose and Rifaximin for Hepatic

Encephalopathy
Rifaximin is a poorly absorbed synthetic antimicrobial with a broad spectrum of

antibacterial activity, including both aerobic and anaerobic Gram-positive and
Gram-negative organisms. Due to its low rate of systemic absorption, rifaximin
appears to be relatively safe [45]. Many studies have demonstrated the efcacy of
rifaximin in the treatment of overt HE (grade 1) (Table 11.4) [4656]. In addition,
a randomized, double-blind, dose-ranging study demonstrated that rifaximin at
doses of 1,200 and 2,400 mg/day for 7 days signicantly improved HE [46]. In a
meta-analysis by Als-Nielsen et al. [31, 32] compared with antibiotics, patients tak-
ing lactulose or lactitol had a signicantly higher risk of no improvement of hepatic
encephalopathy (1.24, 1.021.50). They also found no signicant difference in
response to treatment between aminoglycosides and rifaximin (p = 0.2 by test of
interaction) or when trials were stratied by quality or type of hepatic encephalopa-
thy. It was also found that there was no signicantly different effect on mortality
between nonabsorbable disaccharides and antibiotics (0.90, 0.481.67) or on
adverse events (1.62, 0.574.58). None of the reported adverse events were serious,
and all originated from the gastrointestinal tract (diarrhea, atulence, abdominal
pain, or nausea). In a meta-analysis by Jiang et al. [57], ve trials involving 264
patients met all the inclusion criteria. There was no signicant difference between
rifaximin and nonabsorbable disaccharides on improvement in patients with hepatic
encephalopathy (relative risk [RR] 1.08; 95% condence interval [CI], 0.851.38;
p = 0.53). RR was 0.98 (95% CI: 0.851.13; p = 0.74) for acute hepatic encephalopathy
Table 11.4 Comparison of lactulose and rifaximin for hepatic encephalopathy
No. of
Trial Study design patients Duration of treatment Assessment Efcacy
Festi et al. [46] Lactulose (open-label) 21 21 Neurological signs of HE, Rifaximin = lactulose
asterixis score, HRNB,
EEG, ammonia levels
Bucci and Palmieri [50] Lactulose (double-blind, 58 15 Neurological status, asterixis Rifaximin > lactulose
double-dummy) score, HRNB, cancelation
tasks, EEG, ammonia levels
Massa et al. [52] Lactulose (double-blind, 40 15 HE index severity, mental Rifaximin > lactulose
double-dummy) status, cancelation tasks,
HRNB, EEG
Fera et al. [54] Lactulose (double-blind, 40 First 2 weeks of each Mental status, asterixis score, Rifaximin > lactulose
double-dummy) month for 3 months cancelation tasks, HRNB,
EEG, ammonia levels, PSE
index
Mas et al. [53] Lactitol (double-blind, 103 510 days Mental status, asterixis score, Rifaximin = lactitol
double-dummy) EEG, ammonia levels, PSE

index, psychometric tests
Leevy et al. [55] Lactulose (crossover) 145 >6 months lactulose > HE grade, asterixis score Rifaximin > lactulose
6 months rifaximin
Paik et al. [56] Lactulose (open-label) 54 7 days Ammonia levels, apping Rifaximin = lactitol
tremor, mental status, HE
index, psychometric tests
Jiang et al. [57] Meta-analysis 264 Rifaximin = lactitol
149
in 157 patients and 0.87 (95% CI: 0.401.88; p = 0.72) for chronic hepatic enceph-
alopathy in 96 patients, respectively. There was no signicant difference between
rifaximin and nonabsorbable disaccharides on diarrhea (RR = 0.90; 95% CI: 0.17
4.70; p = 0.90). However, a signicant difference in favor of rifaximin on abdominal
pain (RR = 0.28; 95% CI: 0.080.95; p = 0.04) was identied. Rifaximin is not supe-
rior to nonabsorbable disaccharides for acute or chronic hepatic encephalopathy in
the long-term or short-term treatment except that it may be better tolerated. Further
studies on larger populations are required to provide more sufcient evidence for
assessment of the use of rifaximin.
Disaccharides Versus Other Therapy

for Hepatic Encephalopathy
Loguercio et al. [58] studied 40 patients with cirrhosis on a dietary protein regimen
of 1 g/kg b.w., determined the effect on chronic hepatic encephalopathy of long-
term administration of Enterococcus faecium (SF68) vs. lactulose. The patients
received one of the two treatments for three periods of 4 weeks, each separated by
drug-free 2-week intervals. The efcacy of treatment was assessed by arterial blood
ammonia concentration, mental status, number connection (Reitans part A) test,
and ash-evoked visual potentials. At the end of the third period, the reduction in
both blood ammonia concentrations and Reitans test times was more enhanced in
patients on SF68 than in patients on lactulose. In conclusion, SF68 is at least as use-
ful as lactulose for the chronic treatment of chronic hepatic encephalopathy; it has
no adverse effects, and treatment can be interrupted for 2 weeks without losing the
benecial effects. Sushma et al. [59] conducted a prospective randomized double-
blind study to evaluate the efcacy of sodium benzoate in the treatment of acute
portosystemic encephalopathy. Seventy-four consecutive patients with cirrhosis or
surgical portosystemic anastomosis and hepatic encephalopathy of less than 7 days
duration were randomized to receive lactulose (dose adjusted for 2 or 3 semiformed
stools/day) or sodium benzoate (5 g twice daily). Assessment of response included
mental status, asterixis, arterial ammonia level, electroencephalogram and number-
connection test. The incidence of side effects was similar in the two treatment
groups. The cost of lactulose for one course of therapy was 30 times that of sodium
benzoate. They concluded that sodium benzoate is a safe and effective alternative to
lactulose in the treatment of acute portosystemic encephalopathy. However, sodium
benzoate is not routinely used due to fear of high sodium load and no change in
ammonia level after its use.
Rossi-Fanelli et al. [61] conducted a controlled study in two groups of 20 cir-
rhotic patients with deep coma in order to compare the efcacy of intravenous
branched-chain amino acid solutions in 20% glucose (group A) vs. lactulose plus
glucose in isocaloric amount (group B). Complete mental recovery was obtained in
70% of patients in group A and in 47% in group B. They concluded that, branched-
chain amino acids are at least as effective as lactulose in deep hepatic coma.
However, in a meta-analysis of 11 randomized trials (556 patients) assessing BCAA

vs. carbohydrates, neomycin/lactulose, or isonitrogenous control they found no evi-
dence of an effect of BCAA on improvement of hepatic encephalopathy in trials
with adequate generation of the allocation sequence (RR 1.01, 95% CI 0.841.23,
three trials), adequate allocation concealment (RR 1.09, 95% CI 0.891.33, ve tri-
als), or adequate double-blinding (RR 1.20, 95% CI 0.831.73, three trials). They
did not nd convincing evidence that BCAA had a signicant benecial effect on
patients with hepatic encephalopathy (Table 11.5) [62].
Disaccharides for Primary Prophylaxis

Certain patients are at risk of development of overt HE, such as patients with minimal
hepatic encephalopathy and those with advanced liver disease [6365]. Recently, our
group has shown in a randomized trial [66] involving 120 (48%) patients, receiving
either lactulose (n = 60) or no lactulose (n = 60). Twenty (19%) of 105 patients, fol-
lowed up for 12 months, developed an episode of overt HE. Six (11%) of 55 in the
lactulose group and 14 (28%) of 50 in the no lactulose group (p = 0.02) developed HE.
Ten (20%) of 50 patients in the no lactulose group and 5 (9%) of 55 patients in the
lactulose group died (p = 0.16). On multivariate analysis, Childs score and presence
of MHE at baseline were signicantly associated with development of HE. Lactulose
is effective in the primary prevention of HE.
Variceal bleed is an important precipitating factor for HE in patients with cir-
rhosis. In a randomized trial [67], we enrolled 70 patients with acute variceal bleed
into group 1 (lactulose, n = 35) and group 2 (no lactulose, n = 35). Nineteen (27%)
patients developed HE, 5 patients (14%) in the lactulose group and 14 patients
(40%) in no lactulose group (p = 0.03). On multivariate analysis, only baseline arte-
rial ammonia, blood requirement during hospital stay, and lactulose therapy were
predictors for the development of HE. Hence, lactulose was effective in preventing
HE in these patients. We, therefore, recommend lactulose (3060 mL/day) so that
patients pass two to three semiformed stools in a day.
Disaccharides for Secondary Prophylaxis

The emergence of HE after transjugular intrahepatic portosystemic shunt (TIPS) is

of major concern for patients undergoing this procedure for refractory ascites or for
prevention of variceal rebleeding. This clinical complication tends to occur within
the rst few days post-procedure.
Although the majority of post-TIPS HE episodes are mild and responsive to
pharmacological therapy, there are some cases where intractable HE develops and
152
Table 11.5 Disaccharides vs. other therapy for hepatic encephalopathy

Type of
Trial Study design No. of patients patients Treatment Assessment Efcacy
Loguercio et al. [58] Parallel 40 CHE Lactobacillus SF68/lactulose PSE parameters, SF68 = lactulose
adverse events
Sushma et al. [59] Parallel 74 AHE Sodium benzoate/lactulose Mortality, PSE Sodium
parameters benzoate = lactulose
Fiaccadori [60] Parallel 23 AHE + CHE BCAA/BCAA + lactulose/lactulose Clinical grading BCAA + lactu-
lose > BCAA/lactulose
Rossi-Fanelli [61] Parallel 40 AHE BCAA/lactulose Clinical grading BCAA = lactulose
P. Sharma and S.K. Sarin
hospitalization is required. There are limited data on the use of drug therapy for the
prophylaxis of HE after a TIPS procedure or in patients who have recovered from
an episode of HE who may benet from pharmacological prophylaxis to prevent
future recurrences. Until recently, there has not been any conclusive evidence to
support routine use of pharmacological prophylaxis for this purpose. Riggio et al.
[68] conducted the rst randomized controlled trial utilizing lactitol or rifaximin as
pharmacological prophylaxis for post-shunt HE. Seventy-ve consecutive patients
with cirrhosis undergoing a TIPS procedure were randomized to receive lactitol
60 mL/day, rifaximin 1,200 mg/day or no treatment. Patients in the rifaximin or no-
treatment groups were allowed administration of a sorbitol enema (120 mL) in cases
of minimal bowel movement (<1 bowel movement/day). Treatments were contin-
ued for 1 month post-TIPS or until the occurrence of an episode of HE. There was
no signicant difference in the rate of HE occurrence among the three patient groups
(p = 0.97).
Two recent clinical trials have been conducted to evaluate the efcacy of lactu-
lose or rifaximin used concomitantly with lactulose, as secondary prophylaxis of
overt HE compared with placebo [69, 70]. Sharma et al. [69] conducted a single-
center, open-label, randomized controlled trial in 125 cirrhotic patients who had
recovered from at least one previous episode of HE. Patients were randomized to
receive either lactulose 3060 mL/day or placebo. Development of overt HE was
the primary study endpoint. At the end of a median follow-up time of 14 months,
signicantly more patients in the placebo group (30 of 64 patients [46.8%]) than in
the lactulose group (12 of 61 patients [19.6%]) developed HE (p = 0.001).
In a recent multicenter double-blind randomized clinical trial to assess the sec-
ondary prevention of HE, Bass et al. [70] enrolled 299 cirrhotic patients with a his-
tory of at least two episodes of overt HE to receive either rifaximin (550 mg twice
daily; n = 140) or placebo (n = 159) for a period of 6 months. All enrolled patients
had Model for End-Stage Liver Disease (MELD) scores of <25. More than 90% of
patients in both groups were also maintained on concomitant lactulose therapy.
A signicantly lower percentage of patients in the rifaximin group (22.1%) experi-
enced a breakthrough HE episode during the study period than in the placebo group
(45.9%), with a hazard ratio (HR) of 0.42 (95% CI 0.28, 0.64; p < 0.001). In addi-
tion, there was a signicantly reduced risk of hospitalization in the rifaximin patient
group when compared with placebo; 19 patients in the rifaximin group (13.6%) vs.
36 patients in the placebo group (22.6%), with a corresponding HR of 0.50 (95% CI
0.29, 0.87; p = 0.01). No signicant difference in the incidence of adverse events
was found between the two groups. Overall, this pivotal study has demonstrated a
clinically relevant benet of rifaximin as pharmacological prophylaxis of HE in cir-
rhotic patients with a recent history of overt HE. The addition of rifaximin to a
standard lactulose regimen may offer advantages in terms of decreasing risk of both
breakthrough HE episodes as well as hospitalizations when compared with lactu-
lose alone. Further, in a sub-analysis of patients from the United States and Canada,
patients who received rifaximin 550 mg b.i.d. had signicantly higher time-weighted
average scores for overall QoL on the Chronic Liver Disease Questionnaire than
those who received placebo (p = 0.0093) [71]. The mean time-weighted average
Table 11.6 Disaccharides for secondary prophylaxis of hepatic encephalopathy

No. Duration
of of
Trial Study design patients treatment Assessment Efcacy
Sharma Lactulose 140 14 months Psychometry Lactulose > no
et al. [69] (open-label) and CFF treatment
Bass Rifaximin + lactulose 299 6 HE clinical Rifaximin > placebo
et al. [70] (randomized,
double-blind,
placebo-
controlled)
QoL scores across all six subdomains of the Chronic Liver Disease Questionnaire
were also signicantly improved with rifaximin 550 mg b.i.d. compared with pla-
cebo (p < 0.05 for each) (Table 11.6). Hence, we recommend lactulose and rifaximin
for the secondary prophylaxis of HE.
Conclusion
Current pharmacotherapy for the management of HE is fairly limited, mainly

because of the complex and relatively limited understanding of the pathophysiology
of the disorder. Although the evidence base supporting a pivotal role of ammonia is
robust, in everyday clinical practice a consistent correlation between the concentra-
tion of ammonia in the blood and the manifest symptoms of HE is not observed.
More recently, the synergistic role of inammation and infection in modulating the
cerebral effects of ammonia has been shown to be important. The most commonly
utilized pharmacological agents include the nonabsorbable disaccharides lactulose
and lactitol, and the antimicrobial agent rifaximin. Recent literature has questioned
the clinical efcacy of disaccharides in improving morbidity and mortality in
patients with HE and, although antimicrobial agents such as rifaximin have had an
established role in the treatment of encephalopathy, its use in high-grade HE needs
more data. Until we have more denitive agents nonabsorbable disaccharide lactu-
lose still continues to be the rst-line therapy for the prevention, treatment, and
secondary prophylaxis of hepatic encephalopathy.
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52. Massa P, Vallerino E, Dodero M. Treatment of hepatic encephalopathy with rifaximin: double
blind, double dummy study versus lactulose. Eur J Clin Res. 1993;4:718.
53. Mas A, Rodes J, Sunyer L, Rodrigo L, Planas R, Vargas V. Comparison of rifaximin and lactitol
in the treatment of acute hepatic encephalopathy: results of a randomized, double-blind, double-
dummy, controlled clinical trial. J Hepatol. 2003;38:518.
54. Fera G, Agostinacchio F, Nigro M, et al. Rifaximin in the treatment of hepatic encephalopathy.
Eur J Clin Res. 1993;4:5766.
55. Leevy CB, Phillips JA. Hospitalizations during the use of rifaximin versus lactulose for the
treatment of hepatic encephalopathy. Dig Dis Sci. 2007;52:73741.
56. Paik YH, Lee KS, Han KH, et al. Comparison of rifaximin and lactulose for the treatment of
hepatic encephalopathy: a prospective randomized study. Yonsei Med J. 2005;46(3):399407.
57. Jiang Q, Jiang XH, Zheng MH, Jiang LM, Chen YP, Wang L. Rifaximin versus nonab-
sorbable disaccharides in the management of hepatic encephalopathy: a meta-analysis.
Eur J Gastroenterol Hepatol. 2008;20(11):106470.
58. Loguercio C, Del Vecchio Blanco C, Coltorti M. Enterococcus lactic acid bacteria strain SF68
and lactulose in hepatic encephalopathy: a controlled study. J Int Med Res. 1987;15(6):33543.
59. Sushma S, Dasarathy S, Tandon RK, Jain S, Gupta S, Bhist MS. Sodium benzoate in the treat-
ment of acute hepatic encephalopathy: a double-blind randomized trial. Hepatology. 1992;16(1):
13844.
60. Fiaccadori F, Ghinelli F, Pelosi G, et al Selective amono acid solutions in hepatic encephalopa-
thy treatment (a preliminary report). Ric Clin Lab. 1980;10(2):41122.
61. Rossi-Fanelli F, Riggio O, Cangiano C, Cascino A, De Conciliis D, Merli M, et al. Branched-
chain amino acids vs lactulose in the treatment of hepatic coma: a controlled study. Dig Dis
Sci. 1982;27(10):92935.
62. Als-Nielsen B, Koretz RL, Kjaergard LL, Gluud C. Branched-chain amino acids for hepatic
encephalopathy. Cochrane Database Syst Rev. 2003;(2):CD001939.
63. Romero-Gomez M, Boza F, Garcia-Valdecasas MS, Garca E, Aguilar-Reina J. Subclinical
hepatic encephalopathy predicts the development of overt hepatic encephalopathy. Am
J Gastroenterol. 2001;96:271823.
64. Das A, Dhiman RK, Saraswat VA, Verma M, Naik SR. Prevalence and natural history of sub-
clinical hepatic encephalopathy in cirrhosis. J Gastroenterol Hepatol. 2001;16:5315.
65. Hartmann IJ, Groeneweg M, Quero JC, Beijeman SJ, de Man RA, Hop WC, et al. The prognostic
signicance of subclinical hepatic encephalopathy. Am J Gastroenterol. 2000;95:202934.
66. Sharma P, Agrawal A, Sharma BC, Sarin SK. Primary prophylaxis of hepatic encephalopathy
in patients with cirrhosis: an open labeled randomized controlled trial of lactulose versus no
lactulose. Indian J Gastroenterol. 2010;29 Suppl 1:A8.
67. Sharma P, Agrawal A, Sharma BC, Sarin SK. Prophylaxis of hepatic encephalopathy in acute
variceal bleed: a randomized controlled trial of lactulose versus no lactulose. J Gastroenterol
Hepatol. 2011;26(6):9961003.
68. Riggio O, Masini A, Efrati C, et al. Pharmacological prophylaxis of hepatic encephalopathy
after transjugular intrahepatic portosystemic shunt: a randomized controlled study. J Hepatol.
2005;42:6749.
69. Sharma BC, Sharma P, Agrawal A, et al. Secondary prophylaxis of hepatic encephalopathy: an
open-label randomized controlled trial of lactulose versus placebo. Gastroenterology.
2009;137:88591.
70. Bass NM, Mullen KD, Sanyal A, et al. Rifaximin treatment in hepatic encephalopathy. N Engl
J Med. 2010;362(12):107181.
71. Sanyal A, Bass N, Mullen K, et al. Rifaximin treatment improved quality of life in patients
with hepatic encephalopathy: results of a large, randomized, placebo-controlled trial [abstract
15]. J Hepatol. 2010;52 Suppl 1:S7.
Chapter 12
Antibiotic Treatment for Hepatic
Encephalopathy
Keywords Lactulose Rifaximin Hepatic encephalopathy Neomycin Small

intestinal bacterial overgrowth
Introduction
Long before lactulose was introduced as a therapy for hepatic encephalopathy (HE),
an assortment of antibiotics were used to treat HE. Chlortetracycline was used in the
1950s [1, 2] and soon after neomycin became a commonly used treatment [3, 4].
Neomycin efficacy was not originally tested in randomized controlled trials (RCT)
and indeed no therapy was subjected to rigorous test in that era. Neomycin dosing
was in the range of 13 g orally every 6 h for 5 days and the general impression was
it had some efficacy in the treatment of HE. Toxicity, particularly, in the form of
hearing loss and renal failure was a major concern, especially if longer courses of
therapy were employed. Lower doses of neomycin became popular for a time, but
the publication of Strauss et al. seriously questioned the efficacy of neomycin when
correction of precipitating factors alone was found to be effective as neomycin plus
the correction of precipitating factors [5]. Neomycin is still used to manage intractable
recurrent HE, but less toxic alternative antibiotics are now available to reduce episodes
of HE in patients who still have these bouts of HE despite lactulose therapy.
One very interesting facet of neomycin therapy that came to light in the 1950s
was its relationship to intestinal glutaminase activity. Neomycin was definitely
shown to inhibit this enzyme. It was proposed that this action rather than its antibiotic
properties was responsible for its effect on HE [6]. In general, the observation was
that antibiotics with activity against anaerobic bacteria were more effective than those
with antiaerobic bacteria activity. In the 1970s, based on very limited data, lactulose
became the preferred therapy for the treatment of HE [7]. Nonetheless, metronidazole
and vancomycin were shown to have efficacy in the treatment of HE even though
neither of these was compared to placebo in the RCT setting. Paromomycin and
vancomycin also were featured as a treatment option for HE and other antibiotics
were also proposed as possible therapies to employ.
Mechanism of Action of Antibiotics
The basic premise for the improvement of HE with antibiotics was simply that this
therapy reduced ammonia generation in the gut from enteric bacteria. Whether erad-
ication or reduction of bacterial flora acted primarily in the small or large bowel was
a topic of considerable interest. Sherlock and coworkers and more recently Dhiman
and coworkers have demonstrated that small bowel bacterial overgrowth (SBO) was
common in cirrhotic patients [8, 9]. Could SBO be a major contributor to HE? Is
some of the efficacy of antibiotics due to suppression or clearance of small bowel
bacteria? We still do not have an answer. However, it should be noted that less than
10% of oral metronidazole reaches the colon and yet this antibiotic is felt to have
efficacy in treating HE [10]. It can be further speculated that the main bacterial
culprits for HE were anaerobes since vancomycin was also noted to be effective in
treating lactulose-resistant HE [11]. This drug is no longer used because of the fear
of induction of vancomycin-resistant enterococci (VRE). Nonetheless, its use was
needed to support the concept that a nonabsorbable antibiotic with activity against
anaerobic bacteria was a potentially effective therapy for HE. This association of
antibiotics with anaerobic activity being possible effective therapy for HE still con-
tinues to be observed (e.g.: nitosoxanide). However, there still is no clear evidence
supporting eradication of small bowel bacteria alone being the main mechanism of
action of these types of antibiotics. Most are in association with major effects on
colonic flora which may provide the setting for clostridium difficile overgrowth.
Returning to neomycin therapy as mentioned previously, its main mechanism of
action appears to have been (at least at the very high doses used) inhibitor of intes-
tinal glutaminase. As demonstrated in rat experiments, the majority of portal vein
ammonia comes from glutaminase activity and not from intestinal bacteria.
Neomycin was also associated with reports of villous atrophy which potentially could
have eradicated the glutaminase activity [12, 13]. However, the direct enzyme
inhibiting action seems a more likely cause of reduction in ammonia coming from
the gut. Certainly, the antianaerobic bacterial action of neomycin and possibly also
paromomycin seems less important in the reports of improvement of the HE with
oral amino glycosides.
Despite the data in germ-free rats, it seems possible that suppression of intestinal flora
does reduce production of ammonia by preventing breakdown of nitrogen-containing
compounds. These would arise to some extent from partially hydrolyzed proteins
12 Antibiotic Treatment for Hepatic Encephalopathy 161
Table 12.1 Lists the antibiotics studied for the treatment of hepatic encephalopathy
FDA approved
Rifaximin 550 mg PO twice daily (Mainly recommended for prophylaxis of recurrent overt
HE) [14]
Off label agents
Metronidazole 250 mg four times daily [10]
Neomycin 24.5 g daily in divided doses [3, 7]
Vancomycin 12 g daily in divided doses [15]
Paromomycin 1 g four times a day [16]
Nitazoxanide 500 mg twice daily [14]
from the diet and exudates from the intestinal tract. Fecal incubation studies
have shown significant production of ammonia when hydrolyzed proteins are
added to fecal incubation containing anaerobic flora. If the small bowel motility is
reduced in cirrhotic patients, as demonstrated in at least two studies, then the risk
of SBO is increased [8, 9]. More studies are needed to establish if SBO is prevalent
in cirrhotics.
A peculiarity of antibiotic rifaximin is its differential bioavailability in the small
as opposed to large bowel. Rifaximin is largely insoluble unless exposed to bile salts.
Hence, the drug is an active antibacterial (aerobic and anaerobic) agent in the small
bowel. When bile salts are reabsorbed, its antibacterial activity is significantly
reduced. This is evident in the generally mild effect on colonic flora induced by the
antibiotic. However, it may be important to consider that bile salt delivery to the gut
may be markedly reduced, especially in cholestatic liver disease. Potentially this
may reduce the efficacy of rifaximin in this type of situation.
Published Data on Antibiotic Therapy for Hepatic

Encephalopathy
Most, if not all, of the literature on the efficacy of antibiotics in the treatment of HE
is not placebo controlled. Majority of the studies compare antibiotic therapy to
nonabsorbed disaccharides or to other antibiotics. When antibiotics have been
compared to nonabsorbable disaccharides, there is a trend in favor of greater
efficacy of antibiotics. However, the toxicity of many antibiotics used in the past
was felt to outweigh the possible superiority of this form of treatment. Table 12.1 lists the
antibiotics with recommended doses from various studies (see Table 12.2).
The systematic analysis published by Als-Nielsen et al. is a useful resource [21].
Some criticism of this study of this study has been voiced in that some published
studies were arbitrarily excluded from the system analysis. Nonetheless, this review
had a major impact on this perspective of the efficacy of agents to treat HE. Primarily
what was noted was the extreme paucity of data fulfilling RCT criteria with pla-
cebo control. This was very important because of the already entrenched view that
lactulose was a well-proven therapy. This perspective was so strongly held that
Table 12.2 Summarizes important trials involving antibiotics for treatment of hepatic
encephalopathy
Intervention (number
of study subjects
Investigators Study details in each arm) Conclusion
Bass et al. [17] Double-blind Rifaximin 550 mg bid Rifaximin significantly
placebo-controlled (140) vs. placebo reduced the risk of an
multicentric study. (159) (>90% of episode of hepatic
Duration6 subjects in both encephalopathy and
months groups were on reduced the risk of
lactulose) hospitalization because
of HE
Mas et al. [18] Randomized Rifaximin 1,200 mg/ Rifaximin is a safe
double-blind day (50) vs. alternative therapy to
double dummy lactitol 60 g/day lactitol in the treatment
study. Duration (53) of acute hepatic
510 days encephalopathy
Strauss et al. [5] Randomized Neomycin 1.5 g q6 Compared to placebo (with
double-blind (20) vs. placebo correction of precipitat-
study. Duration (19) ing factors) neomycin
5 days shortened the duration
of hepatic encephalopa-
thy but this difference
was not statistically
significant
Parini et al. [19] Randomized study Paromomycin Rifaximin proved to be as
in acute episode 1,500 mg/day (15) effective as paromomy-
of hepatic vs. rifaximin cin in treating acute
encephalopathy. 1,200 mg/day (15) episode of hepatic
Duration10 encephalopathy
days
Pedretti et al. [20] Randomized study. Rifaximin 400 mg q 8 Rifaximin is at least as
Duration21 (15) vs. neomycin effective as neomycin
days 1 g q 8 (15) in achieving clinical
improvement in hepatic
encephalopathy and
reducing ammonia
levels
Tarao et al. [15] Randomized Vancomycin 2 g q Vancomycin seems to be
double-blind 12 h (12) vs. effective in chronic
crossover study. lactulose (12) portal systemic
Duration8 encephalopathy in
weeks patients who are not
helped by lactulose
alone
Morgan et al. [10] Randomized Neomycin 1 g Q6 (9) Metronidazole is as
double-blind vs. metronidazole effective as neomycin
study. 200 mg Q 6 (9) in the treatment of
Duration7 days hepatic encephalopathy
Conn et al. [7] Randomized Neomycin 1 g q 8 Neomycin is as effective in
double-blind (33) vs. lactulose the treatment of hepatic
double dummy (33) encephalopathy
12 Antibiotic Treatment for Hepatic Encephalopathy 163
there was a virtual ban on placebo-controlled trials [22]. This factored strongly in
the design of a recent trial of rifaximin treatment of patients at risk for recurrent
bouts of HE [20]. Over 90% of patients continued to stay on lactulose while rifaximin
or placebo was added to their therapeutic regimen. The 58% reduction in further
episodes of overt HE clearly indicated that it had a significant therapeutic action.
References
1. Mc Jr DW. Metabolism and toxicity of ammonia. N Engl J Med. 1957;257(22):107681.

2. Martini GA, Strohmeyer G, Doelle W. [The treatment of hepatic coma with antibiotics
(chlortetracycline, neomycin)]. Medizinische. 1959;52:254953.
3. Dawson AM, Mc LJ, Sherlock S. Neomycin in the treatment of hepatic coma. Lancet.
1957;273(7008):12628.
4. Summerskill WH. Hepatic coma in liver failure and gastro-intestinal haemorrhage treated with
neomycin. Br Med J. 1958;2(5108):13225.
5. Strauss E, Tramote R, Silva EP, Caly WR, Honain NZ, Maffei RA, et al. Double-blind randomized
clinical trial comparing neomycin and placebo in the treatment of exogenous hepatic enceph-
alopathy. Hepatogastroenterology. 1992;39(6):5425.
6. Hawkins RA, Jessy J, Mans AM, Chedid A, DeJoseph MR. Neomycin reduces the intestinal
production of ammonia from glutamine. Adv Exp Med Biol. 1994;368:12534.
7. Conn HO, Leevy CM, Vlahcevic ZR, Rodgers JB, Maddrey WC, Seeff L, et al. Comparison of
lactulose and neomycin in the treatment of chronic portal-systemic encephalopathy. A double
blind controlled trial. Gastroenterology. 1977;72(4 Pt 1):57383.
8. Martini GA, Phear EA, Ruebner B, Sherlock S. The bacterial content of the small intestine in nor-
mal and cirrhotic subjects: relation to methionine toxicity. Clin Sci (Lond). 1957;16(1):3551.
9. Gupta A, Dhiman RK, Kumari S, Rana S, Agarwal R, Duseja A, et al. Role of small intestinal
bacterial overgrowth and delayed gastrointestinal transit time in cirrhotic patients with minimal
hepatic encephalopathy. J Hepatol. 2010;53(5):84955.
10. Morgan MH, Read AE, Speller DC. Treatment of hepatic encephalopathy with metronidazole.
Gut. 1982;23(1):17.
11. Tarao K, Ikeda T, Hayashi K, Sakurai A. Successful use of vancomycin hydrochloride in the
treatment of lactulose-resistant chronic hepatic encephalopathy. J Gastroenterol Hepatol.
1989;4 Suppl 1:2846.
12. Faloon WW, Jacobson ED. Malabsorption during neomycin administration. Gastroenterology.
1961;40:4478.
13. Jacobson ED, Faloon WW. Malasorptive effects of neomycin in commonly used doses. JAMA.
1961;175:18790.
14. Basu AP, Rayapudi K, Estevez J, Brown RS. A pilot study utilizing nitazoxanide for hepatic
encephalopathy in chronic liver disease program and abstracts of the 59th annual meeting of the
American Association for the study of liver diseases (abstract), 31 Oct4 Nov; 2008. p. 1742.
15. Tarao K, Ikeda T, Hayashi K, Sakurai A, Okada T, Ito T, et al. Successful use of vancomycin
hydrochloride in the treatment of lactulose resistant chronic hepatic encephalopathy. Gut.
1990;31(6):7026.
16. Tromm A, Griga T, Greving I, Hilden H, Huppe D, Schwegler U, et al. Orthograde whole gut
irrigation with mannite versus paromomycine+lactulose as prophylaxis of hepatic encephal-
opathy in patients with cirrhosis and upper gastrointestinal bleeding: results of a controlled
randomized trial. Hepatogastroenterology. 2000;47(32):4737.
18. Mas A, Rodes J, Sunyer L, Rodrigo L, Planas R, Vargas V, et al. Comparison of rifaximin and
lactitol in the treatment of acute hepatic encephalopathy: results of a randomized, double-blind,
double-dummy, controlled clinical trial. J Hepatol. 2003;38(1):518.
19. Parini P, Cipolla A, Ronchi M, Roda A. Effect of rifaximin and paromomycin in the treatment
of portal-systemic encephalopathy. Curr Ther Res. 1992;52(1):349.
20. Pedretti G, Calzetti C, Missale G, Fiaccadori F. Rifaximin versus neomycin on hyperammoniemia
in chronic portal systemic encephalopathy of cirrhotics. A double-blind, randomized trial. Ital
J Gastroenterol. 1991;23(4):1758.
systematic review of randomised trials. BMJ. 2004;328(7447):1046.
22. Mullen KD, Amodio P, Morgan MY. Therapeutic studies in hepatic encephalopathy. Metab
Brain Dis. 2007;22(34):40723.
Chapter 13
Ornithine Phenylacetate: A Novel Strategy
for the Treatment of Hepatic Encephalopathy
Maria Jover-Cobos, Nathan A. Davies, Yalda Sharifi, and Rajiv Jalan
Keywords Hepatic encephalopathy Ornithine phenylacetate Acute liver failure

Brain edema Ammonia Glutaminase Glutamine synthetase ADMA Nitric
oxide NFkB
Introduction
Although the exact pathophysiological mechanisms of hepatic encephalopathy

(HE) remain the subject of investigation, studies over the past 100 years have dem-
onstrated a central role of ammonia. The mechanisms include ammonia-induced
changes in neurotransmitter synthesis and release, neuronal oxidative stress, impaired
mitochondrial function, and osmotic disturbances resulting from astrocytic metabolism
of ammonia to glutamine. Systemic hyperammonemia has been largely found in
patients with HE with underlying cirrhosis and acute liver failure (ALF).
Neuropathological examination of the brains obtained from patients who died
with HE shows that astrocytes are the main cells to show physical alterations [1].
Patients with ALF develop raised intracranial pressure (ICP), which uncorrected
may result in cerebral herniation, culminating in the death of about 30% of patients
[2]. In patients with ALF, the astrocytes are swollen and in cirrhosis these cells show
changes in their morphology to Alzheimer type II astrocytosis [3]. Similar changes
can be induced in cultured astrocytes following incubation with ammonia [1, 4].
Furthermore, infusion of ammonia into rats with portacaval shunt results in brain
swelling, and ammonia is thought to produce astrocytic edema through the ammonia
glutamine brain swelling hypothesis [5].
M. Jover-Cobos, PhD N.A. Davies, PhD, BSc Y. Shari, MD, BAO, BCh, LRCP,
SI&MRCP (UK) R. Jalan, MBBS, MD, PhD, FRCPE, FRCP (*)
UCL Institute of Hepatology, Royal Free Hospital, University College of London,
Upper Third UCL Medical School, Pond Street, London NW3 2PF, UK
e-mail: r.jalan@ucl.ac.uk
166 M. Jover-Cobos et al.
Minimal hepatic encephalopathy (MHE) is a clinical condition that occurs in

patients with cirrhosis and is dened by the existence of a series of neurophysiological
changes that go unnoticed in routine examination and has a strong impact on quality
of life, altering memory, concentration, and attention. The increase in brain water
correlates with the severity of MHE suggesting that this is important in its patho-
genesis [6]. Direct evidence for the ammonia hypothesis was provided in patients
with cirrhosis. In this study, hyperammonemia induced by the administration of
amino acid solution mimicking the hemoglobin (emulating gastrointestinal bleeding)
alters neuropsychology, brain magnetic resonance spectroscopy (MRS), and magneti-
zation transfer ratio (MTR) in cirrhotic patients [7]. Other studies conrmed that a
high arterial ammonia level predicts brain herniation, clinical manifestations of
cerebral edema, increased ICP, and mortality in ALF patients [811].
Studies in patients and animal models have also indicated a role for inammation
in the pathogenesis of HE. However, it has been shown that induced hyperammonemia
on the background of inammation produces HE indicating that the effects of
ammonia are synergistic with inammation [12, 13]. Further evidence for the synergy
between ammonia and inammation has more recently been provided in animal studies
suggesting that hyperammonemia may prime the brain to the effects of inammation
and alter the NO-cGMP pathway [14, 15]. HE treatment remains an unmet clinical
need [1619]. Hence, ammonia reduction remains an important therapeutic target
for the treatment of HE in liver disease. Recent studies using a novel therapeutic
approach, ornithine phenylacetate (OP), may provide a useful treatment for patients
with hyperammonemia.
Interorgan Ammonia Metabolism
The main mechanism for ammonia removal is urea production by hepatocytes. In liver
disease, this function is compromised resulting in elevated ammonia levels, and
other ammonia-regulating pathways in multiple organs assume important
signicance; see Fig. 13.1 [20].
Studies focusing on interorgan ammonia metabolism in patients with cirrhosis
indicate that the liver, muscles, kidney, and the small bowel are important in regulating
the circulating levels of ammonia. Contrary to popular belief, it has been shown that
at least 5060% of total gut ammonia is derived from uptake of glutamine, which is
metabolized to glutamate and ammonia by the enzyme glutaminase (GA) [21, 22].
Ammonia that would normally be converted to urea by the liver increases to toxic
levels. In this situation, the enzyme glutamine synthetase (GS) plays a pivotal role
in ammonia detoxication, effectively removing ammonia during the conversion of
glutamate to glutamine [23]. Studies of the administration of l-ornithine l-aspartate
(LOLA) and OP are based on using GS as a major alternative ammonia detoxication
pathway. Antibiotics, probiotics, and symbiotic have been used as modulators of
intestinal ammoniagenesis as well as in the prevention of systemic inammation.
These studies are based on the hypothesis that luminal bacteria produce the majority
13 Ornithine Phenylacetate: A Novel Strategy for the Treatment 167
Fig. 13.1 Interorgan metabolism in health (a) and cirrhosis (b)
of ammonia (gut sterilization). Lactulose has been the most popular treatment, but
there is little evidence to support routine use [24]; it offers no clear benet in ALF
[25], where HE remains the major determinant of death [26]. Current approaches
for treatment of HE are interventions targeting inammation such as the use of
hypothermia, and antibiotics such as Rifaximin. These treatments have shown some
promise but the Rifaximin approach thus far has not been shown to successfully
reduce ammonia levels [27]. Hence, GS and GA are current and future targets for
therapy.
Ornithine Phenylacetate as a New Treatment

for Hepatic Encephalopathy
Leading the Hypothesis
Currently, there is no specic treatment of proven value for Type A HE and only
liver transplantation remains a denitive treatment for long-term benet. Studies in
animal models of liver failure suggested that the administration of a mixture of the
amino-acids, LOLA, is associated with a lowering of plasma ammonia [23]. It is
thought that the mechanism represents the conversion of l-ornithine to glutamate in
the muscle, suggesting that the muscle could be targeted as an alternative site of
ammonia detoxication [17, 23]. A large, placebo-controlled trial in ALF failed to
show any benet on ammonia level, encephalopathy grade, or survival [28]. In addi-
tion, the role of aspartate remains unclear. Aspartate infusions in animals were not
shown to result in a reduction in ammonia levels indicating that aspartate was
unlikely to be the precursor of glutamate/glutamine and that ornithine was likely to
be the active component of LOLA.
According to the above stated reasoning, the administration of LOLA would
generate glutamine, which would only temporarily reduce ammonia, as this glutamine
would be recycled in the small bowel to produce more ammonia [29]. Phenylacetate
and its prodrug phenylbutyrate (converted to phenylacetate in vivo) have been used
for the hyperammonemia which occurs due to urea cycle enzyme deciencies [30].
Phenylacetate combines covalently with the glutamine derived from glutamate to
produce phenylacetylglutamine which is excreted by the kidneys. However, this
therapy has not been attempted previously in cirrhosis as these patients do not have
increased glutamine levels. The studies of interorgan ammonia trafcking, the lessons
from LOLA observations, and the current use of phenylacetate to treat urea cycle
disorders have led to the hypothesis. The concomitant administration of ornithine
and phenylacetate act synergistically to produce a sustained reduction in ammonia
concentration [29].
The Mechanism of Action of Ornithine Phenylacetate
Decreasing Plasma Levels of Ammonia: Direct Effect on Ammonia

Metabolism Enzymes in Liver Failure
In preliminary studies, it has been shown that the combination of ornithine with
phenylacetate to treat hyperammonemia in cirrhosis is effective in animal models.
Administration of OP results in increased conversion of glutamate to glutamine by
stimulation of GS activity in the muscle with the subsequent excretion of pheny-
lacetylglutamine in the urine, a reaction in which one molecule of ammonia is
removed. GA has been found to contribute to hyperammonemia in cirrhosis and
in MHE animal models [21, 31]. It has also been discovered that variations in the
promoter region of the GA gene is associated with the development of HE in a cohort
of patients with cirrhosis [32]. These ndings suggest developing approaches to
target GA to prevent ammonia release and HE as a valid therapeutic strategy. Recent
data show that OP treatment for 5 days intraperitoneally resulted in normalization
of GA activity in the gut, indicating that OP effectively restricts the production
of in vivo ammonia in a cirrhotic model [33]. Mechanism of action of OP on the
metabolism of ammonia is shown in Fig. 13.2.
Fig. 13.2 Mechanism of action of OP in GS and GA enzymes. GS is stimulated in muscle by

glutamate increased levels. At the same time PAGN is formed and excreted in the urine. In addition,
GA is restored to normal levels in the gut
Decreasing Brain Edema and Motor-Evoked Potentials
Cytotoxic brain edema and intracranial hypertension occurring in encephalopathy

ALF patients account for a large number of deaths owing to cerebral herniation.
It has been shown that in chronic liver failure there is a low-grade brain edema [34]
that is resolved after transplantation. In this novel approach to targeting the altered
interorgan ammonia metabolism in liver failure, OP utilizes the activity of GS
to trap ammonia as glutamine and phenylacetate facilitates its excretion as pheny-
lacetylglutamine [1113]. Effectiveness of this approach with OP has been conrmed
in animal models of cirrhosis and ALF. The reduction (50%) of plasma ammonia
was associated with (a) an improvement in grade of HE in cirrhotic patients and
(b) a reduction in ICP in ALF. OP treatment reduced ammonia concentrations
signicantly which was associated with a reduction in brain water and the brain
myo-inositol levels were signicant increased, showing an improvement in brain
metabolism [29, 35]. In a devascularized pig model of ALF the rise in arterial
ammonia was attenuated with OP which was accompanied by a signicant decrease
in extracellular brain ammonia and prevention of intracranial hypertension in pigs
with ALF [36].
Physical symptoms of MHE have been detected by motor-evoked potentials
(MEP) which examines the function of signal transmission along the nerve, which
is perturbed by low-grade brain edema. Similar disturbances have been found in
patients with cirrhosis using magnetic resonance (MR), with signs compatible with
low-grade edema along the corticospinal tract. These abnormalities were related to
functional impairment detected by transcranial magnetic stimulation and were
found to be reversed after liver transplantation. Recently, the assessment of MEP in
awake rats has been validated to monitor HE in animal models of liver failure
(portacaval anastomosisPCA) and precipitated HE (simulated gastrointestinal
bleedGiB). These models have been utilized to test the efcacy of OP [37], dem-
onstrating that OP treatment prevents the neurophysiological abnormalities induced
by the GiB insult in the PCA animals. Administration of OP over differing time
periods (3 h and 3 days) as a pretreatment prevents the decrease in the amplitude
and increase in MEP latency at 6 h post GiB [38].
Indirect Effect of Ammonia Metabolism: Cytokines, Nitric

Oxide/ADMA Inflammation Pathway in the Brain
In cirrhotic patients it has been shown that the effects of hyperammonemia are
synergistic with inammation [13]. The effects on cell swelling by cytokines in
ammonia-sensitized cultured astrocytes have also been shown [12]. However, the
mechanisms by which ammonia produces brain swelling are still subject of much
investigation. Although the effects on inammatory processes have been found to
contribute to the formation of cerebral edema, it is not clear whether ammonia
promotes inammation or both are independent factors. Inammatory pathways
identied as contributing to the edema include cyclo-oxygenase, nitric oxide (NO)/
cyclic guanosine monophosphate (cGMP) signaling, and cytokine release [34, 39, 40].
Hyperammonemia could increase bloodbrain-barrier permeability to systemic
cytokines. It is also possible that several factors associated with the systemic
inammatory response syndrome could modulate brain dysfunction induced by
hyperammonemia. These processes may help to explain the differences that some-
times exist between lower ammonia levels and observed brain impairment in
some patients. It has been shown that the presence of HE grade 3/4 correlates better
with inammation than with ammonia plasma levels [41], though extracellular brain
ammonia levels may be signicantly higher. One recent study showed that in a cir-
rhosis animal model in which plasma and brain cytokines were markedly elevated
following administration of lipopolysaccharide (LPS), pretreatment with OP pre-
vented increased levels of TNFa and IL-6 (trend) in plasma and in brain induced by
LPS. Moreover, OP reduced LPS-induced development of precoma/coma and wors-
ening of brain edema. It is well known that the transcription of NFkB directly
increases proinammatory cytokines and leads to induction of nitric oxide syn-
thase [42]. OP reduced iNOS and NFkB expressions in cortical brain of cirrhotic
animals indicating that ammonia reduction may modulate neuroinammation [43].
In cirrhosis, a paradox exists between reduced intrahepatic NO generation and
excess NO in the splanchnic circulation. Splanchnic vasodilatation leads to vasocon-
striction of numerous vascular beds, including the liver, kidneys, and has signicant
effects on the brain. Asymmetric dimethylarginine (ADMA) is an endogenous inhibi-
tor of eNOS (endothelial nitric oxide synthase), the levels of which are increased in
liver failure [44, 45]. It has been shown that treatment of cirrhotic rats with OP resulted
in restoration of the NOS pathways (reduction in ADMA levels, increased eNOS
activity, reduced caveolin-1) [43]. The reduction in arterial ammonia concentration
Fig. 13.3 Sites of action of OP on the neuroinammatory cascade
with OP may prevent LPS-induced worsening of HE and brain edema. It was therefore
not surprising to note that treatment of animals with OP resulted also in restoring
nitric oxide signaling (see Fig. 13.3).
Conclusions
In summary, the mechanism by which OP directly reduces ammonia levels in cirrhosis

is by increasing muscle glutamine synthesis activity, subsequently trapping and
increasing ammonia excretion as phenylacetylglutamine, with the concomitant
normalization of gut glutaminase activity. The reduction on ammonia (by OP) leads
to a reduction in ICP in ALF and is associated with an improvement in inammation
and NO pathways in the context of chronic liver disease. Moreover, OP modulates
iNOS and NFkB mechanisms and prevents LPS-induced brain edema in cirrhotic
rats. Studies to date have indicated that OP is safe and patient studies in MHE
and HE is needed to establish OP as a treatment for this signicant complication of
liver disease.
Conflict of Interest UCL has licensed its invention ornithine phenylacetate in hepatic encephal-
opathy to Ocera and Prof. Jalan is the named inventor on the patents.
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Endotoxemia produces coma and brain swelling in bile duct ligated rats. Hepatology.
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16. Ortiz M, Jacas C, Cordoba J. Minimal hepatic encephalopathy: diagnosis, clinical signicance
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17. Shawcross D, Jalan R. The pathophysiologic basis of hepatic encephalopathy: central role for
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18. Gerber T, Schomerus H. Hepatic encephalopathy in liver cirrhosis: pathogenesis, diagnosis
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19. Blei AT. Diagnosis and treatment of hepatic encephalopathy. Baillieres Best Pract Res Clin
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Part IV
Special Topics
Chapter 14
Sleep Disorders and Hepatic Encephalopathy
Sara Montagnese
Keywords Sleep quality Sleep timing Sleepiness Diurnal preference Melatonin

Circadian rhythms
This chapter will focus on sleep disturbances and their pathophysiology in patients
with cirrhosis. Sleep disturbances will be divided into night sleep disturbance, abnor-
mal sleep timing and daytime sleepiness. The relationship between each of these key
features of the sleepwake prole and hepatic encephalopathy, if any, will be dis-
cussed. A separate section will cover the available information on the pathophysiology
of sleep alterations in this patient population. Finally, treatment will be discussed.
Night Sleep Disturbance
Up to 5065% of patients with cirrhosis complain of unsatisfactory sleep [14].

More specically, they complain of increased sleep latency (difculties falling
asleep) and excessive sleep fragmentation (numerous night awakenings) [1, 3, 4].
Questionnaire-based sleep complaints are substantiated by quantitative sleep quality
parameters, such as wrist actigraphy (i.e. the recording of movement over days/
weeks by means of an accelerometer worn as a wrist watch), which documents
activity over the whole 24 h and numerous sleep interruptions [1, 5, 6].
Night sleep disturbance seems to be more common in patients with cirrhosis than
in patients with other chronic illnesses, for instance, renal failure [1], and is detectable
also in well-compensated patients with cirrhosis [1, 4], with no obvious reasons for
S. Montagnese, MD, PhD (*)

Department of Medicine, University of Padova,
Via Giustiniani, 2, Padova 35128, Italy
e-mail: sara.montagnese@unipd.it
178 S. Montagnese
disturbed sleep such as severe itching, tense ascites or the need to empty their
bladder repeatedly overnight because of treatment with diuretics.
While night sleep disturbance has been traditionally associated with hepatic
encephalopathy, there are limited experimental animal data [7, 8] and virtually no
human data to support this contention. Crdoba et al. found no difference in the
prevalence of sleep disturbance in relation to psychometric performance in 44
patients with cirrhosis, 24 (55%) of whom had minimal hepatic encephalopathy [1].
In a study by Montagnese et al., which was designed to assess the relationship
between sleep behaviour and neuropsychiatric performance, no association was
observed between the presence of night sleep disturbance and either the presence or
the severity of hepatic encephalopathy [4]. Finally, Spahr et al. showed that the
histamine H1 blocker hydroxyzine improves sleep quality in patients with cirrhosis
and minimal hepatic encephalopathy but not their cognitive performance [5], thus
dissociating the two sets of symptoms.
Delayed Sleep Timing
The rst study to assess sleep timing in patients with cirrhosis was that of Crdoba
et al. [1]. In this study, sleep timing was assessed, in relation to sleep quality and diur-
nal preference (eveningness/morningness), in a group of healthy volunteers, a group
of patients with cirrhosis and a control diseased group of patients with renal failure
[1]. An association was observed between delayed sleep habits/evening preference
and impaired sleep quality in patients with cirrhosis, while no such association existed
in the healthy and disease control groups. These ndings were conrmed by
Montagnese et al., who described signicant correlations between diurnal preference
and sleep quality scores, with evening patients taking longer to fall asleep and sleep-
ing worse [4]. The observed delays in sleep habits in a subgroup of patients with cir-
rhosis (approximately 60 min compared to the healthy population) were shown to be
independent of employment status in a smaller, subsequent study [6].
The interest in sleep timing amongst chronobiologists and sleep scientists has
grown considerably over the recent years. It has been shown that even in the healthy
population, individuals who are more alert in the evening and have late/delayed
sleep habits (owls) can experience difculties in complying with the living and
working constraints of the Western world, which requires them to be operative in
the early part of the day [9]. These difculties, which can translate into morning
trafc accidents and poor school/work performances, become particularly prominent
when evening subjects are forced to a sudden 60-min advance of their sleepwake
schedule, on the spring switch to light saving time. There is even some indication
that the transition to light saving time might be associated with an increase in the
incidence of myocardial infarction [10]. The 60-min delay relating to light saving
time is comparable to the delay in sleepwake habits exhibited by patients with
cirrhosis compared to the healthy population, although the latter is chronic rather
than suddenly and externally imposed. Nevertheless, the prognostic relevance of
abnormal sleep timing in this patient population is worthy of further research.
14 Sleep Disorders and Hepatic Encephalopathy 179
Daytime Sleepiness
So-called inversion of the sleepwake pattern, manifest as an inability to rest at

night and profound daytime somnolence, was rst recognised as a sign of overt hepatic
encephalopathy by Sherlock et al. in a case series of 17 patients with varying degree
of hepatic dysfunction, accompanied by severe neurological abnormalities [11]. This
paper is often quoted as indicating that sleepwake inversion and disturbed nocturnal
sleep are both features of hepatic encephalopathy. However, the patient population
was extremely heterogeneous, with several individuals having noncirrhotic acute
hepatic failure, and the accompanying neurological alterations were very severe.
Nevertheless, excessive daytime sleepiness has been subsequently described in
individuals with cirrhosis and milder neuropsychiatric impairment [14]. In at least
in one study, an association was observed between excessive sleepiness and the
presence/degree of hepatic encephalopathy. In the same paper, a correlation was
described between a sleepiness scale and the degree of electroencephalographic
slowing [4]. These observations t the hypothesis that hepatic encephalopathy can
be interpreted, at least to some extent, as a syndrome of decreased vigilance [12].
Indeed, some of the electroencephalographic features of hepatic encephalopathy are
reminiscent of those observed during the wakesleep transition [13].
In summary, night sleep disturbance, especially in the way of increased sleep
latency and interrupted night sleep, is common in patients with cirrhosis, regardless
of the presence of hepatic encephalopathy. In addition, a subset of these patients
tend to have delayed sleep habits (bed and wake-up times delayed by approximately
an hour compared to the healthy population), independently of their daytime
commitments [1, 6]. These individuals also exhibit more pronounced night sleep
disturbance. Excessive daytime sleepiness and daytime napping are also common
in patients with cirrhosis and they are associated with the presence of hepatic
encephalopathy [4]. There is little evidence, in the studies performed to date, of a
relationship between night sleep disturbance and daytime sleepiness (patients who
are sleepy in the daytime are not necessarily those who sleep badly at night), sug-
gesting that their occurrence may reect different disease processes. Finally, sleep
wake alterations have been shown to severely impinge on quality of life in this
patient population [4, 14]. Nonetheless, they are not routinely screened for [15] and
they tend to be managed in a nonspecic, potentially inappropriate fashion.
Physiological Sleep Regulation
The currently accepted two-process model of human sleep regulation postulates the
interaction between a circadian and a homeostatic mechanism [16].
Circadian sleep regulation is responsible for the alternation of periods of high/
low sleep propensity, in relation to dark/light cues, and irrespective of preceding
sleepwake behaviour. The suprachiasmatic nuclei of the hypothalamus are the site
of the master circadian clock, which generates circadian rhythms. In humans,
180 S. Montagnese
a 60
Plasma melatonin (pg/ml) 50
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Fig. 14.1 The black lines illustrate the normal, 24-h rhythm of plasma melatonin, which is virtually
absent in the daytime, starts rising in the evening, peaks in the middle of the night, and then gradually
declines. Part of the changes observed in the 24-h melatonin prole of patients with cirrhosis, such as
prolonged melatonin peaks and high daytime levels (green line (a)), can be ascribed to impaired
hepatic melatonin metabolism, while others, such as an overall rhythm delay involving both the onset
and the offset of the peak (green line (b)) suggest central circadian dysfunction
the average, endogenous circadian period is approximately 24.18 h and must be

constantly synchronised, or entrained, to the 24-h day by external inuences [17].
Light, which is the major external time cue, reaches the suprachiasmatic nuclei by
afferent projections from the retina, primarily via the retino-hypothalamic tract. In
turn, the suprachiasmatic nuclei project to the pineal gland, regulating the produc-
tion of melatonin, which can be thought of as a neuroendocrine transducer of the
light/dark cycle [18]. Thus, in healthy individuals, melatonin synthesis increases
soon after the onset of darkness, peaks in the middle of the night and then gradually
declines (Fig. 14.1). The nocturnal rise in melatonin synthesis is associated with an
increased propensity to sleep and is acutely suppressed by light exposure, as a result
of a rapid decrease in pineal serotonin N-acetyltransferase activity [19]. Melatonin
is hydroxylated and sulphated to 6-sulphatoxymelatonin (aMT6s), primarily in the
liver, and aMT6s is subsequently excreted in the urine. Minor amounts of melatonin
are excreted unchanged, conjugated with glucuronic acid or react with active oxy-
gen species, leading to the formation of the pharmacologically active compounds of
the kynurenines family [20].
Homeostatic sleep regulation is responsible for the increase in sleep propensity
when sleep is curtailed or absent and its dissipation during sleep. The term homeo-
static refers to the fact that the system counteracts deviations from an average
reference level of sleep. The pioneering studies of Blake and Gerard showed that
both the arousal threshold and the dominance of slow electroencephalographic
waves are high in the initial part of sleep and progressively decrease [21]. The initial
dominance of slow-wave activity has been conrmed in subsequent studies [22] and
it has also been shown that sleep deprivation produces an increase in slow-wave
activity in the recovery night [23]. In contrast, a daytime nap attenuates slow-wave
activity in the subsequent sleep episode [24]. Taken together, these ndings indicate
that slow-wave activity reliably reects prior history of sleep and wake. The exact
neurochemical correlates of human sleep homeostasis remain unknown, but there is
evidence that adenosinergic neurotransmission might play an important role [25].
The separation of the circadian and homeostatic processes is useful for descrip-
tive purposes. However, it is the ne-tuned interaction between these two processes
that enables sleep consolidation, optimal waking performance and relatively brief
sleepwake and wakesleep transitions [26]. It is common experience that, no matter
how long the preceding wake period, it is still easier to sleep during the night, when
it is dark, than during the day.
Sleep Regulation in Patients with Cirrhosis
It has been assumed, on rst principles, that, as melatonin is metabolised in the

liver, its disposition would be delayed in patients with cirrhosis (Fig. 14.1a).
Abnormalities have been observed, including high daytime plasma melatonin con-
centrations [27], low urinary aMT6s concentrations [28] and a reduction in the
clearance of exogenously administered melatonin [29], which point to impaired
hepatic metabolism. In a study where plasma melatonin and urinary aMT6s were
assessed simultaneously over a 36-h period (two nights plus one day), 24-h mela-
tonin clearance was shown to be comparable to that of healthy controls, while
overnight melatonin clearance (thus clearance at a time when the hormone levels
are highest) was reduced [30]. In the same and in other studies, correlations were
observed between the delay in plasma melatonin/urinary aMT6s peaks and
the degree of hepatic failure [6, 31, 32].
182 S. Montagnese
However, other circadian abnormalities have also been described in patients with
cirrhosis, namely, delays in the nocturnal rise of plasma melatonin and in its time to
peak [27, 30, 33], suggesting dysfunction of the central circadian clock rather than
impaired melatonin disposition (Fig. 14.1b). The function of the retinal-hypothalamic
axis, and thus of the circadian clock, can be assessed by measuring melatonin sup-
pression (i.e. the rapid decrease in melatonin plasma levels in response to the expo-
sure of the retina to light at night [34]) and/or by measuring the 24-h prole of at least
two variables out of melatonin, cortisol and core body temperature, the rhythm of
each of which is strongly connected to the phase of the circadian clock. Montagnese
et al. demonstrated parallel delays in the onset of plasma melatonin/plasma cortisol
rhythms and attenuated melatonin sensitivity to light in a group of 20 patients with
cirrhosis, thus suggesting that some degree of central circadian dysfunction exists in
this patient population [30, 35]. Bernardi et al. [36] and Velissaris et al. [33] reported
normal cortisol rhythms in patients with cirrhosis but in both studies the number of
samples was smaller than required for accurate estimates of cortisol rhythm timing
and controls were not exercised for light exposure, which might have biased the
results. Interestingly, in the study by Montagnese et al., melatonin sensitivity to night
light (melatonin suppression) was inversely correlated with the timing of the mela-
tonin peak [30], supporting Steindls original hypothesis that the observed delays in
the 24-h melatonin prole depend on a dysfunctional retinal-hypothalamic axis [27].
Similar circadian abnormalities have been reported in blind individuals; however,
these show considerably more variation in their melatonin and cortisol proles, with
advanced, delayed and free-running rhythms all being described [37].
Some attempt has been made to correlate the changes in the melatonin rhythm
with the sleep disturbances observed in patients with cirrhosis, but the ndings have
been inconclusive [27, 30, 38]. Montagnese et al. have suggested that circadian
rhythm delays in this patient population are associated with delayed sleep habits,
although not necessarily with impaired sleep quality. The combination of evening
preference, delayed sleep habits, impaired sleep quality and delayed circadian
rhythms is reminiscent of delayed sleep phase syndrome [39], a circadian disorder
characterised by considerable delays in sleep onset and wake times. The goal of
treatment is to resynchronise the circadian clock with the 24-h light/dark cycle:
structured sleepwake schedules and avoidance of exposure to bright light in the
evening are advised. In addition, exposure to bright light shortly after awakening in
the morning [40] and/or administration of melatonin 56 h before habitual sleep
time [41] have been shown to advance the timing of sleep. In patients with cirrhosis,
naturally occurring delayed sleep phase syndrome might be exacerbated by delayed
hepatic melatonin metabolism, increasing its prevalence and modulating its clinical
features [6, 42].
Virtually no information is available on the effect of hepatic transplantation on
circadian abnormalities in patients with cirrhosis, but one encouraging case report
suggests that transplantation can revert melatonin arrhythmia [43].
Limited information is available on homeostatic sleep regulation in patients with
cirrhosis. Polysomnography has been performed in a limited number of studies but
with the aim of evaluating indices of hepatic encephalopathy rather than homeostatic
Fig. 14.2 Diagram summarising the potential pathophysiological mechanisms of sleepwake

alterations in patients with cirrhosis. Continuous arrows mark associations or causal relationships
that are supported by the studies performed to date; dashed arrows mark hypothetical associations/
causal relationships. HE hepatic encephalopathy; aMT6s 6-sulphatoxymelatonin
indices per se. Correlations were established between the clinical severity of
encephalopathy and ammonia levels on one hand and the degree of disruption of
sleep architecture on the other [44]. No matter how profound, the disturbances in
sleep architecture remained reversible, in parallel with lowered ammonia levels
and improved neuropsychiatric performance [44].
Decreased density of the adenosine receptor A1AR has also been described in
both cortical and subcortical regions of the brain of patients with cirrhosis in one
positron emission tomography/magnetic resonance imaging study [45], thus poten-
tially implicating the homeostatic system in sleep deregulation in these patients;
however, sleepwake proles were not obtained in this study.
In summary, the pathophysiology of sleepwake disturbance in patients with
cirrhosis remains poorly understood. Circadian regulation has been studied in some
depth, while less is known about homeostatic regulation. In addition, virtually no
information is available on: (a) genetic predisposition, (b) sympathetic/parasympa-
thetic transmission of the circadian clock signal to the periphery, and (c) function/
dysfunction of the hepatic clocks, which may all play a role (Fig. 14.2).
Treatment of SleepWake Abnormalities

in Patients with Cirrhosis
Limited therapeutic options are available to treat sleepwake disturbances in patients

with cirrhosis. This is for a number of reasons: (a) sleepwake alterations are not
formally assessed in routine hepatological practice, (b) their pathogenesis has not
184 S. Montagnese
been fully elucidated, (c) patients with cirrhosis are extremely sensitive to psychoac-
tive drugs [46], and (d) hepatic impaired disposition of common hypnotics can result
in accumulation and oversedation [47]. Even when an aetiological treatment was
attempted by Spahr et al., who administered the histamine H1 blocker hydroxyzine
to a group of patients with minimal encephalopathy and sleep alterations, some risk
of precipitating severe hepatic encephalopathy was observed [5]. All these issues
result in underdiagnosis and cautious, aspecic and potentially inappropriate man-
agement strategies. Further elucidation of the pathophysiological mechanisms may,
in time, lead to the development of specic therapies. Meanwhile:
1. Routine assessment of night sleep quality, sleepwake timing habits and diurnal
somnolence should be performed.
2. Sleep and light hygiene practices, to include regular sleepwake schedules,
exposure to bright, natural light in the early hours of the morning and avoidance
of exposure to bright light in the evening should be encouraged.
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Chapter15
Hepatic Encephalopathy and Driving
Matthew R. Kappus and Jasmohan S. Bajaj
Keywords Driving Minimal hepatic encephalopathy Simulation Legal Motor

vehicle Crashes Traffic violations
Introduction
Hepatic encephalopathy is characterized as a spectrum of neuropsychiatric symp-

toms in the absence of other known brain disease [1]. Hepatic encephalopathy
ranges from overt and severe disturbances to minimal hepatic encephalopathy
(MHE), once described as low-grade or subclinical encephalopathy. These
patients lack the neurologic exam findings and historical symptoms for diagnosis,
and instead manifest with subtle neuro-cognitive deficits and psychomotor abnor-
malities, primarily affecting immediate memory, attention, visual spatial abilities,
and fine motor skills [2]. As MHE is estimated to have a prevalence of 2280%
[37], this becomes particularly important when pertaining to the responsibility of
driving an automobile or commercial vehicle.
M.R. Kappus, MD
Department of Internal Medicine, Virginia Commonwealth University Health Systems and
Physicians, 1250 East Marshall Street, PO Box 980509, Richmond, VA 23298-0509, USA
J.S. Bajaj, MBBS, MD, MS (*)
Department of Gastroenterology, Hepatology and Nutrition, Virginia Commonwealth
University and McGuire VA Medical Center, 1201 Broad Rock Boulevard,
Richmond, VA 23249, USA
e-mail: jsbajaj@vcu.edu
188 M.R. Kappus and J.S. Bajaj
Driving and Society
Driving is a dangerous activity, and has a significant impact on our society with over
34,000 fatalities and 2,120,000 injuries in the United States in 2008 alone [8].
Individual characteristics of the driver predominate in the causation of most motor
vehicle crashes, and certain underlying conditions predispose drivers to their occur-
rence [8]. It is expected for states, through the department of motor vehicles or
transportation safety, to detect, examine, and regulate problem drivers. These drivers
include those inexperienced, the elderly, the intoxicated, or those with episodic loss
of consciousness (i.e., epilepsy). Restrictions are imposed upon the new driver to
protect both society and the driver. More frequent written and performance exami-
nations are required to detect impairment in the elderly. Awareness of this link
between medical conditions like diabetes, dementia, cardiac disease, stroke, and
epilepsy and car accidents [9] brings forward the importance of how to assess medical
fitness to drive [10]. As the population ages, the prevalence of medical conditions
known to impair driving, like stroke, obstructive sleep apnea, dementia, polyphar-
macy, will increase. Frighteningly, most medical conditions are not even considered
by the state licensing agencies, and legislation to restrict the impaired driver is slow.
Among those conditions which leave patients with a questionable ability to drive is
MHE. More evidence is beginning to emerge on the adverse effects of MHE on
daily functioning and guidelines do not currently exist for evaluating capacity to
operate a motor vehicle.
Skills Needed to Drive
Skills expected for safely driving a vehicle includes physical mobility and psychomo-
tor coordination, visual and audio perception, and higher cognitive ability and attention
span. Decisions are made on a tactical, strategic, and operational level [11]. The act of
driving requires a person to incorporate different senses, coordinating different actions
such as speeding up, slowing down, passing, and turning. Visual perception allows the
person to see obstacles, and audio perception helps drivers detect warning signals such
as car horns and the sound of other vehicles on the road. Drivers are required to main-
tain attention to the road, and minimize other distractions such as other passengers or
the surrounding environment. Cognitive ability allows the driver to make decisions and
determine reaction time, navigate an environment, and executive decision making in
order to determine traveling speeds, and follow local driving laws.
Why Should MHE Affect Driving
MHE encompasses neuro-cognitive impairment [10]. Studies done with Alzheimer

patients have shown that impairment of attention and speed of mental processing
both exhibited in patients with MHEaffect an individuals ability to react to
15 Hepatic Encephalopathy and Driving 189
unexpected traffic conditions [12]. MHE is characterized by defects in visuo-spatial

assessment, attention span, working memory, and speed of information processing
and motor abilities [13]. Diminished driving ability in these patients has been
demonstrated [14]. This pattern of disease suggests involvement of the subcortical
brain centers [15]. It is present in up to 80% of cirrhotics, and psychometric testing
has indicated that between 15 and 75% of patients with MHE were unable to safely
drive a motor vehicle [16, 17].
The presence of MHE alone does not necessarily predict inability to drive a car
[17]. Cognitive examinations have demonstrated depressed cognition, however, not
necessarily inability to drive a car [17]. Cognitive exams such as the FEV 5 (German
guidelines for driver qualification and evaluation) and the BGL (German guidelines
for expertise on driver aptitude) have been used to evaluate the neuropsychological
fitness for patients with MHE to drive. The results of both of these computerized
tests and a real driving test with a driving instructor assessment showed progres-
sively poor test results paralleled increased severity of hepatic encephalopathy.
Interestingly, however, according to the judgment of the driving instructor, 39 and
48% of overt and minimal HE patients, respectively, could still drive a car. The driv-
ing instructors only went so far as to deem overt and MHE doubtful and unfit to
drive in 61 and 52%, respectively, of the time [17]. This demonstrates that while
these patients are cognitively impaired, there may be still some debate as to who
still can safely drive.
Evidence of Driving Impairment
On-Road Driving Studies
Two early studies performed in the 1980s by Schomerus et al. [16] and Watanabe
et al. [18] categorized a large fraction of patients with cirrhosis unfit to drive as
judged by on-road testing (4460%). Around the same time, Srivastava et al. [19] in
a pilot study evaluated driving on a live road test in 15 cirrhotic patients, nine of
which had MHE, and they failed to detect impaired performance while driving a car.
These conflicting studies renewed interest in the field and indicated that larger stud-
ies would lead to further investigation of driving fitness in cirrhotic patients
(Table 15.1).
A 2004 study conducted by Wein et al. [20] evaluated 48 cirrhotics, 14 with
MHE, using a standardized 90 min real-life road test. The evaluation by a profes-
sional driving instructor, unaware of the diagnoses or reason for the test, showed
that driving competence was scored lower in patients with MHE. Ratings in patients
with cirrhosis without MHE were scored as being similar to the control group. This
study suggested that MHE should be considered a medical condition that increases
the risk of automobile accidents. The conflicts of the Wein [20] study with earlier
Table 15.1 Available studies and results of minimal hepatic encephalopathy and driving
Total no./% Results in MHE
Study and location with MHE MHE diagnosis Testing patients
Schomerus et al. 40/25% EEG Driving test 100% Unsafe
[16]; Germany
Watanabe et al. 16/100% Reaction time Driving test 44% Unsafe
[18]; Japan
Srivastava et al. 15/60% Psychometric Driving test Similar to controls
[19]; Chicago tests
Wein et al. [20]; 48/29% Psychometric Driving test X10 intervention
Germany tests Behavior Worse rating
rating Worse driving
Kircheis et al. 51/53% Psychometric Driving test Simulated or
[56]; Germany tests Driving instructor driving
Self-evaluation test required
Biochemical analysis
studies [19] may be attributed to the larger sample size used in the Wein study, a
study group with a more advanced stage of disease, or a more demanding on-road
driving test. There were several variables unaccounted for in all studies, and further
testing was needed.
Simulation Studies
While on-road tests are largely standardized, they do not ensure similar conditions
and also have medico-legal implications. In contrast, driving simulation can be used
to test the detailed cognitive response in MHE. A study of navigation and driving in
cirrhotic patients with MHE was performed on a driving simulator [21]. This study
showed that while impairment of attention, response inhibition, and visuo-motor
coordination exists in MHE [2226], working memory problems for navigational
purposes also exists [27]. Working memory is a key component of completing an
executive task by allowing an individual to rapidly adapt to new situations by recall-
ing previous experience [21]. Patients underwent testing with three psychometric
tests, and driving skills were assessed by using a driving simulation program. There
was a significantly higher rate of collisions in the MHE group compared to all other
groups. All patients incurred accidents when asked to overtake a slow moving vehi-
cle by crossing into the lane of oncoming traffic, or when a simulation dog darted
out into the driving field. This shows a miscalculation of time needed to overtake the
other vehicle, and a failure to react to a stimulus, respectively. In this study, patients
with MHE also had more difficulty following mapped directions, and incurred a
higher number of incorrect turns compared to the other drivers. The study went on
to further correlate a significant relationship between the number of positive
psychometric tests with number of incorrect turns, though this same correlation was
not statistically significant when it came to number of collisions [21]. By computer
simulation there are demonstrated difficulties with attention and response inhibi-
tion, skills required for safe navigation in patients with MHE.
Real-Life Driving Outcomes
The self-reported traffic violations and motor vehicle accidents in a cohort of cir-
rhotic patients have been performed [28]. The results indicated that patients with
cirrhosis reported more events than controls, and that patients with concomitant
diagnoses of MHE had the highest rate of events compared to cirrhotics without
MHE, or even those patients on psychoactive drugs. This was an important finding,
as this is one of the first studies to document objectively higher rates of motor
vehicle accidents and traffic violations in patients with MHE. However, the self-
reported nature as well as the retrospective design introduced bias. Another
confounding feature of this study is the potential of the effect of etiology of liver
disease, such as hepatitis C on the neurophysiologic and neuropsychological fea-
tures used to establish a diagnosis of MHE [29]. Likewise, the effects of prolonged
alcohol abuse leading to cirrhosis may have prolonged and subtle neurophysiologic
abnormalities, therefore independently impacting driving outcome.
Therefore, the authors undertook a prospective validation in which they found
that patients with MHE diagnosed by the ICT were at significantly higher risk for
future driving offenses [14]. They also reported that MHE patients had a higher risk
of traffic violations and motor vehicle crashes over the past year with self-report as
well as through official driving records. Self-report of driving offenses was compa-
rable to the official driving records [14].
The current evaluation shows that MHE patients are likely to have driving
difficulties on the road and on a simulator. These findings actually translate into
worse driving outcomes.
Additional Challenges Faced by MHE Patients While Driving
In addition to the neuropsychological deficits patients with MHE contend with,

they also have a chronic debilitating illness which puts them at risk for increased
fatigue. Of the factors that contribute to human error leading to motor vehicle
accidents, a major one is fatigue [30]. Fatigue is associated with driving difficulties
in healthy individuals and in patients with attention deficits. This is demonstrated
both in a simulator and during live driving scenarios [3133]. An age-matched

control study was conducted observing the affect of fatigue on driving in patients
with recent overt hepatic encephalopathy (OHE), MHE, and a control group [34].
The study compared driving skills in the first half and second half of an extended
driving period, and it was felt that patients with MHE had a significant worsening of
simulated driving skill with time related to fatigue. Interestingly, patients with MHE
were the only group to show a significant increase in the number of collisions in the
second half compared with the first when compared to controls and OHE. Patients
with OHE did not show significant difference as they were already impaired at
baseline. In the second half of the driving simulation, MHE patients had worsen-
ing in their rate of collisions, off road excursions, and their speeding. A survey at
the end of the simulation period asking drivers after driving I feel tired, MHE
and OHE patients showed no difference in how many answered yes; however,
there was a significantly increased number when compared to the control group.
Patients with MHE have several dimensions of impairment in their driving abili-
ties, and this study highlighted that fatigue [34] is one, in addition to reduced
reaction time and navigational ability [20, 21]. Decreased driving ability due to
fatigue from processing multiple sensory inputs during driving is important to con-
sider in patients with MHE, especially because they lack the chronic feeling of
fatigue that patients with OHE experience. Fatigue is one way that patients with
MHE may be able to self-realize their inability to operate a motor vehicle; however,
this is not present at the beginning of the driving task, but rather manifests later in
the driving experience.
Besides fatigue, patients with MHE also lack insight into their own impair-
ment, and insight into a problematic process is the first step towards seeking inter-
vention. In 2008, Bajaj et al. [35] used a 26-item scale named the driving behavioral
survey (DBS), validated by Barkley et al. [36] in patients with attention deficit
hyperactivity disorder (ADHD), to test the hypothesis of whether patients with
MHE did in fact have insight into their driving disability. This scale was used
because patients with MHE and ADHD struggle with attention impairment [21].
The study outcomes demonstrated that patients with MHE rated themselves
equivalent to controls and cirrhotic patients without MHE despite having
significantly worse driving performance on simulation driving. Also, the study
enlisted observers who rated patients driving abilities, and observers rated MHE
patients as poorer drivers compared with those patients without MHE or controls.
MHE is difficult to evaluate in the clinical setting, and evaluation of self-aware-
ness in this group is challenging [3740]. If patients lack insight into their clinical
disease, it will be up to healthcare providers to inquire about driving records to
perhaps make patients more aware of their problem. This is important not only
because motor vehicle accidents are a leading cause of death in the United States
[41], but also because of the negative impact of cirrhosis on survival after trauma
and traffic accidents [4144].
Determining Fitness to Drive in Minimal Hepatic

Encephalopathy
Treatment of MHE Pertaining to Driving
Patients with MHE may not be safe to drive in certain cases; however, therapy,
either medically or behaviorally, can improve driver ability. Therapy with gut-
specific agents like lactulose and rifaximin has been relied upon to clear cognition
in patients with OHE, and may be useful in patients with MHE [17, 4548]. Bajaj
et al. [49] randomly assigned in a double-blinded manner patients with known MHE
to placebo or rifaximin and demonstrated improved ability by driving simulation.
They also measured cognition with a set of cognitive battery tests, the NCT-A, NCT-
B, the DST, and the ICT. A greater proportion of those given the rifaximin interven-
tion improved driving outcomes (decreased collisions, illegal turns, and speeding
tickets), as well as improvement towards normal in the NCT-B, DST, BDT, and the
ICT cognitive tests. Interestingly, there was no significant difference between the
two groups with respect to the number of collisions. This may reflect that those
patients randomized to rifaximin may have been able to grasp insight into their poor
driving based upon collisions, and perhaps this reflected improved working mem-
ory, response inhibition, and cognitive flexibility. This would suggest that therapy
improved the cortical network feedback between frontal, insular, and the parietal
regions [49].
Still unknown is whether cognitive rehabilitation could have a benefit in patients
with MHE as it has been shown to have in stroke and brain injury patients. Limited
research has been done with brain injury patients with respect to specific cognitive
and behavioral aspects of motor vehicle operation. The design of these studies has
been to test whether training exercises involving visuo-motor tracking, divided
attention, performance feedback, and social reinforcement can improve the safety
of driving in this patient population. Results have indicated that training resulted
in improved performance during live driving simulation, and there may be a
significant therapeutic effect in using specific training exercises in patients with
stroke and brain injury [50]. These same training techniques have not yet been
understood in patients with MHE, and this will be an area of significant research
in the future.
Legal Ramifications
Currently, only 76% of states in the United States have a medical advisory board
overseeing driver regulation [51]. None of the 50 states even mentions oversight of
patients with altered mental status as a result of liver-related disease, which would
include both overt and MHE [51]. Only a subset, 12%, of states has a mandated
system in which physicians are required to report medically impaired patients, and
in those states reporting was suboptimal due to burden to the physician [51]. Only
32 states provide legal immunity to physicians for reporting these unsafe drivers,
and transcending the legal ramifications, physicians face the ethical decision of pro-
tecting an individuals right to privacy vs. the right of safety for society. For violat-
ing either of these, a physician may be legally liable.
As there is no written law definitive to this topic, and the present laws are subject
to interpretation of the legal system. The American Medical Association (AMA)
has released a guide to physicians assessing and counseling elderly drivers [52].
While this document does not address patients with MHE or OHE specifically, it
does provide a set of tools which may be useful for physicians. The overwhelming
recommendation is for the physician and patient, with family, to have a candid dis-
cussion, and that if there is a strong threat to public safety, it is both desirable and
ethical to notify the authorities. It is in this way that the burden still rests with the
states in making the final determination of driving safety.
Because physicians are not specifically trained in fitness to drive evaluations,
they must act in the best interest of the patient and society while following the local
laws [53]. Physicians are advised to follow the applicable local laws on mandatory
reporting; inform the patient and their family of the potential impairment; and if
possible, recommend a fitness to drive evaluation by a driving instructor trained in
detecting impairment. Physicians can also educate the public and legislatures to
advocate for changes in driving-related legislation.
As of now, this is still a burdensome task as the present tools used to diagnose
MHE are not easily used in a clinical setting. It is hopeful that in the future, a more
simple and direct way of being able to identify these patients will be available. Until
then, it will be the duty of the physician to report patients at risk to the proper gov-
ernmental agency for further driving evaluation.
Summary
OHE is usually clinically evident and obviates driving, but the challenge arises once
acute episodes have resolved, or patients present with MHE. It is evident that HE
consists of spectrum of neuro-cognitive deficits. The most mild of these deficits
consists of cognitive and attention deficits and are compounded by impaired response
inhibition, working memory, and visuo-motor coordination [54, 55], all of which
are important skills for driving a vehicle. These patients lack insight into their prob-
lem, and more easily develop fatigue, which contributes to the danger [21, 49, 56].
Diagnosis of MHE requires specialized testing and is often difficult to detect, and
physician reporting to state driver regulatory organizations is riddled with ethical
and legal dilemmas. The effect of hepatic encephalopathy on driving is complex and
affects patients and the general population alike. These effects and exciting new
treatment strategies are being actively investigated.
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subclinical hepatic encephalopathy. Dig Dis Sci. 2000;45:154952.
47. Prasad S, Dhiman RK, Duseja A, et al. Lactulose improves cognitive functions and health-
related quality of life in patients with cirrhosis who have minimal hepatic encephalopathy.
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48. Watanabe A, Sakai T, Sato S, et al. Clinical efficacy of lactulose in cirrhotic patients with and
without subclinical hepatic encephalopathy. Hepatology. 1997;26:14104.
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Gastroenterology. 2009;137(170615):e19.
Chapter 16
Nutrition and Hepatic Encephalopathy
Manuela Merli, Michela Giusto, and Oliviero Riggio
Keywords Hepatic encephalopathy Liver cirrhosis Nutrition Protein energy

malnutrition Protein Ammonia
Introduction
Hepatic encephalopathy (HE) is a neurologic syndrome characterized by a wide

spectrum of neuropsychiatric changes and alterations in neuromuscular function
which are seen in patients with severe liver insufciency. HE may be present in
patients with acute liver failure and is included among the symptoms for the diag-
nosis of fulminant hepatic failure: in these patients survival is poor due to the severe
prognosis of the underlying liver disease. Most frequently HE is seen in patients
with chronic liver disease. It has been estimated that 3040% of patients with liver
cirrhosis will experience overt HE during the natural history of the disease.
Symptoms of HE in chronic liver disease may appear as acute reversible episodes
frequently associated with a precipitating cause. However, episodes of HE may
also be recurrent with intermittent neurological symptoms negatively affecting the
patients self-sufciency [1]. Frequent HE precipitating events are constipation,
hypokalemia, alkalosis, hyponatremia, hypovolemia, gastrointestinal bleeding,
dehydration, infections, surgery, renal failure, diuretics, and psychoactive medica-
tions. Patients with more severe liver insufciency and those with spontaneous or
articially created porto-systemic shunts are at higher risk of HE.
Some cirrhotic patients, even if recognizable clinical symptoms of brain
dysfunction are lacking, may show an abnormal performance when submitted to
M. Merli, MD (*) M. Giusto, MD O. Riggio, MD

Department of Clinical Medicine, University Sapienza Roma,
Viale DellUniversita 37, Rome 00185, Italy
e-mail: manuela.merli@uniroma1.it
200 M. Merli et al.
psychometric or computerized tests or electrophysiological techniques. The term

minimal HE (MHE) has been proposed to dene this condition [2]. This cognitive
dysfunction may be present in 6070% of cirrhotic patients; it may affect quality of
life and even impair the execution of simple and complex tasks such as car driving
[3]. The patients with MHE are considered to be at risk for the development of
overt HE.
HE represents the second-most frequent cause of decompensation in cirrhotic
patients after ascites and before variceal bleeding. Prognostic signicance of HE
in liver cirrhosis has been recognized in many reports [4].
The mechanisms involved in the pathogenesis of HE are still a matter of debate
and multiple factors are probably involved in the genesis of this neurologic syndrome.
Gut-derived nitrogenous compounds are usually released from the intestine. These
metabolites are normally detoxied by the liver but in cirrhosis their hepatic clear-
ance is impaired. Portosystemic shunts causes blood to by-pass the liver and this
also reduces metabolites detoxication. When ammonia or other gut-derived toxins
accumulate in the blood they may also reach the brain through the bloodbrain
barrier, and exert a toxic effect on the brain function. Several other compounds,
such as mercaptans, short-chain fatty acids amines, g-aminobutyric acid (GABA),
endorphins, glutamate, endogenous benzodiazepine agonists, tryptophan and its
metabolites have also been proposed [5]. As far as the mechanism involved in the
central nervous system is concerned, in the last few years, astrocytes swelling has
been identied as an important process negatively inuencing the neuronal neurotrans-
mission as well as the brain energy production rate. The astrocytes swelling
hypothesis is able to explain one of the key features of HE, namely, that the syn-
drome is precipitated by heterogeneous factors [6]. Infection, for example, induces
the astrocytes swelling by endotoxins and proinammatory cytokines [6].
Protein-Calorie Malnutrition in Cirrhosis
Alterations in nutritional status are a frequent nding in patients with cirrhosis [79]
either of alcoholic [7] or nonalcoholic origin [9]. The prevalence of malnutrition in
cirrhosis has been reported to be as high as 6590%. Patients with more advanced
liver disease are more frequently malnourished [8]. On the other hand, more sophis-
ticated methods to evaluate body composition have shown that alteration in cell mass
or muscle function may be found also in compensated cirrhosis [10, 11]. The best
denition of malnutrition in cirrhotic patients is protein-calorie malnutrition (PCM),
in fact both lean and fat tissue may be depleted. Depletion of adipose tissue is more
frequent in women while muscle tissue is more often compromised in men [8, 9].
Multiple factors are involved in the etiology of PCM in chronic liver disease
(Table 16.1), dietary intake is inadequate to meet energy expenditure, absorption is
compromised, and substrate utilization is impaired due to liver disease. A variety of
events decrease the ability of the cirrhotic patient to control their dietary intake.
It is commonly described that when these patients present new clinical symptoms,
16 Nutrition and Hepatic Encephalopathy 201
Table 16.1 Main causes Anorexia

of malnutrition in cirrhotic Dietary restriction
patients
Unpalatable diet
Dysgeusia
Other gastrointestinal symptoms
Ascites
Hepatic encephalopathy
Frequent hospitalization
Diuretic therapy
Lactulose treatment
Pancreatic insufciency
Bacterial overgrowth
Rapid transition to a starvation prole
Decreased protein synthesis
Disturbances in substrates utilization
they get worried that consumption of food would worsen their conditions. Patients
with liver cirrhosis show an increased severity of gastrointestinal symptoms which
has been associated with impaired nutritional status and health-related quality of
life [12]. In cirrhotic patients intestinal transit is reduced [13] and ascites reduces
the postprandial gastric volumes and accommodation [14], further compromising
nutritional intake. Paradoxically, nutrition is further neglected when these patients
are hospitalized to treat the complications of the disease: they are starved to be sub-
mitted to endoscopy, ultrasonography, contrast imaging, or other invasive procedures.
The hospital staff in charge of the patient often considers nutrition of lower rele-
vance with respect to the other complications that need to be treated.
Cirrhotic patients are further penalized due to more rapid transition to a starvation
prole. The liver, in fact, plays a central role in many metabolic pathways and the
metabolic disturbances consequent to liver derangement induce profound
modications in substrate utilization [1517]. Insulin resistance, manifested as high
levels of circulating insulin and impaired glucose utilization [15, 18] affects the
ability of glucose storage through glycogen synthesis. As a consequence the cir-
rhotic liver is unable to adequately derive glucose from glycogen through glycog-
enolysis and after a short-term fasting, gluconeogenesis is enhanced in these patients
to produce glucose. To provide substrates for gluconeogenesis, alanine and glycerol
are mobilized from muscle and adipose tissue deposits, respectively, thus causing
skeletal muscle and adipose tissue catabolism. Previous studies have shown that a
different eating pattern with 47 small meals, including a late-evening snack, by
avoiding prolonged starvation periods, may improve the nitrogen economy in these
patients and reverse this abnormal substrate oxidation [19, 20]. In a recent study, Plank
et al. [21] provided in a group of cirrhotic patients a late-evening nutritional supple-
ment over a 12-months period to test the hypothesis that, by shortening the night
fast, the total protein stores would improve. They observed that total body protein,
measured by neutron activation analysis, increased signicantly in these patients
throughout the observation period compared to baseline.
202 M. Merli et al.
Interestingly, the presence of nutritional alterations should not be considered

only as a consequence of chronic liver disease, but it may even accelerate the natu-
ral history of the disease and adversely affect the patients outcome. Prospective
studies on large series of cirrhotic patients have in fact shown that severe malnutrition,
as well as the presence of depletion in lean body mass, represents an independent
prognostic factor in the survival of patients with liver cirrhosis [22, 23].
Nutrition, Diet, and HE
There are several reasons why nutrition and diet should be carefully managed in
cirrhotic patients to prevent or treat HE
1. PCM can be involved in the pathogenesis of HE.
2. The patients diet (mainly protein intake) has been invoked both as precipitating
factor and as treatment of HE.
3. Episodes of HE further inuence the dietary consumption due to the patients
attitude about feeding and physician prescriptions.
Role of Malnutrition in HE
Recent available information on interorgan ammonia exchange in liver cirrhosis

have suggested that, in cirrhotic patients, due to the inability of urea synthesis in the
failing liver, the muscle may have a crucial role in ammonia detoxication [24].
The muscle can remove ammonia from the circulation and release it as glutamine.
Although this metabolic pathway does not result in a denitive ammonia disposal
(as glutamine reaches the small intestine and the colon mucosa, where it is converted
once again into glutamic acid and ammonia), it has been proposed that muscle
depletion may have relevant implications in favoring HE [6] (Fig. 16.1). An alternative
mechanism to explain the possible relationship between muscle mass and HE takes
into account that an increased glutamine release from muscle may also derive from
an increased muscle protein catabolism. In this case the excess glutamine is drained
to the small intestine and kidney and its conversion to glutamic acid and ammonia
contributes to increase the whole body ammonia disposal. Patients with cirrhosis
and malnutrition often have reduced muscle mass [25]. Despite the potential rele-
vance of the correlation between malnutrition and HE, few studies have dealt with
this topic and denite conclusions are still lacking. Campillo et al. [26] have previ-
ously reported that HE is independently related to low caloric intake in hospitalized
patients with liver cirrhosis, but a potential link between nutritional status and
cognition was not investigated.
Two studies have examined more recently the potential role of malnutrition in
the development of HE with conicting results. Srs et al. [27] examined 223
Fig. 16.1 Interorgan ammonia metabolism in health and in liver cirrhosis. GS glutamine-
synthetase; HE hepatic encephalopathy. Adapted from Wright et al. [6], with permission from
John Wiley & Sons, Inc
patients with nonalcoholic cirrhosis. These patients had a complete nutritional

assessment (BMI, anthropometric measurements and bioelectric impedance analy-
sis, and indirect calorimetry) and were evaluated for the presence of clinically overt
HE (West Haven criteria). Fifty-ve percent of these patients had grade 1 HE and
7% grade 2 or 3; 38% had no evidence of neurologic impairment. Nutritional status
and metabolic variables were similar in patients with and without HE and multivari-
ate analysis failed to show that these parameters were independently related to HE.
The authors conclude that malnutrition and catabolism did not seem to be indepen-
dent risk factors for the presence of HE in patients with liver cirrhosis.
Kalaitzakis et al. [28] performed a prospective study in 128 patients with cirrhosis
of various etiology evaluating HE, malnutrition, and diabetes. In this study, patients
with MHE were also included. Forty percent of the patients were malnourished,
26% had diabetes, and 34% had HE. Patients with malnutrition suffered more
frequently from HE when compared to those with good nutritional status (p = 0.03).
Plasma ammonia levels showed a correlation with muscle mass (r = 0.28, p = 0.003)
and insulin resistance (r = 0.42, p < 0.001). Malnutrition and diabetes were indepen-
dently correlated with the time needed to perform the number connection test A.
In conclusion, due to methodological differences, these studies reached different
conclusions and more studies are probably needed to better clarify the relation
between nutritional status and HE. Despite the lack of clear evidence, muscle wasting
and protein catabolism in cirrhotic patients should always be avoided and an
adequate nutritional intake to maintain their muscle mass should be implemented.
In fact, protein malnutrition may favor HE through indirect mechanism. We have
recently shown that protein malnutrition in cirrhotic patients is an independent risk
factor for severe infections [29]. Bacterial infections, due to increased endotoxins or
through a cytokine release, are a well-known trigger for HE.
204 M. Merli et al.
Diet as a Treatment of HE
The restriction of protein intake has traditionally been considered as a rule for the
treatment of HE [30]. This was originated from old experimental studies showing
that in porto-caval shunted dogs meat feeding caused neurological symptoms. Later
on it was reported that the symptoms of HE were controlled by a low-protein diet,
containing about 20 g proteins a day [31]. Based on these observations the restriction
of protein intake in cirrhotic patients with HE became a common practice. It should
also be considered that the therapeutic armamentarium for HE was extremely lim-
ited at that time and protein restriction was one of the few treatment options.
In the last decade, the increased knowledge on the progressive deterioration of
nutritional status in liver cirrhosis and improved comprehension of metabolic alter-
ations in chronic liver disease has questioned the opportunity to adopt a severe and
prolonged protein restriction in the treatment of HE [32].
It has been recognized that protein restriction may increase muscle catabolism
and the release of the amino acids, with a consequent elevation in serum ammonia
concentrations and worsening of HE. In fact, while the limitation in protein intake
tries to reduce the dietary nitrogen load to the liver, it increases, on the other hand,
the nitrogen derived from muscle catabolism. The main goal of an adequate protein
feeding in cirrhotic patients should be to avoid muscle protein breakdown.
Morgan et al. [33] have examined the relationship between protein intake and
changes in HE in a large number of patients with alcoholic hepatitis, 63% with HE,
during the rst weeks of hospitalization. All the patients were encouraged to eat a
prescribed adequate diet and their 24-h dietary intake was assessed weekly. These
authors reported that lower the protein intake in the previous week, the higher was
the deterioration in their mental status suggesting that a diet lacking adequate
protein content could favor HE. On the other hand, those patients who improved
their mental status were those in whom a higher protein intake was recorded during
the previous week.
Protein requirement and protein utilization were investigated in malnourished
cirrhotic patients showing that long-term oral refeeding with increased amounts
of proteins and energy intake was associated with high nitrogen retention and was
able to induce signicant protein synthesis [34].
In 1997, the European Society of Parenteral and Enteral Nutrition published
specic guidelines for nutrition in liver disease and transplantation [35]. These
guidelines stated for the rst time the higher protein requirements in cirrhotic
patients and recommended a diet including at least 1.2 g/kg of proteins every day.
Even the presence of HE was not considered a reason to decrease protein content in
diet of at least 1.01.5 g/kg/day. If any protein restriction was needed, this was
recommended to be only transient [35]. More recently, the same recommendations
were also conrmed in the guidelines for the use of enteral nutrition in patients with
liver diseases [36].
A randomized study was performed to better clarify if a normal or high protein
diet could be recommended in patients with overt HE [37]. All patients included
were hospitalized for episodic HE; none had alcoholic hepatitis, recent alcohol
intake, gastrointestinal bleeding, benzodiazepine intake, and neurologic, respiratory,
or cardiovascular comorbidities. Patients were randomized to receive two different
diets: either a normal protein diet (1.2 g/kg/day) or a strict low-protein diet (0 g
proteins for days 03, 12 g proteins for days 46, 24 g proteins for days 710) for
14 days through a nasogastric tube. A single lactulose enema was administered
followed by Neomycin therapy in both groups. Ten patients in each group
completed the study. Patients following a normal protein diet showed a similar
improvement in HE, while the low-protein diet caused an increase in protein
breakdown during the rst days. Apparently, therefore, protein restriction did not
cause any major benet on HE while, on the other hand, the low-protein diet
exacerbated protein breakdown. These results were a further support to the safety
of a normal protein intake during HE and demonstrated the harmful effect of a
low-protein diet. Further reports have given evidence to the fact that a high-calorie
high-protein diet might be well tolerated in patients with overt HE [38]. More
recently a randomized study, performed in patients with compensated cirrhosis and
MHE, suggested that eating a regular breakfast meal (500 kcal and 21 g of proteins)
may improve their cognitive performance with regard to attention and executive
function [39].
HE Influences the Patients Attitude About Feeding

and Physician Prescriptions
In spite of the advice of experts in the eld [35, 40, 41], medical practitioners and
dietitians often feel that protein restriction is necessary in patients with HE. This has
been demonstrated by a number of surveys in different countries showing that
moderate (3050 g/day) or severe (<30 g/day) restriction of dietary protein intake
was frequently prescribed for patients with HE. This opinion was reported either
among medical practitioners [42] or in gastroenterologists [30] or in dietitians [43].
This perspective may induce the patients to believe that, after an episode of HE,
protein restriction is advisable even as a long-term strategy.
Current Guidelines About Nutrition in Hepatic Encephalopathy
Patients with overt HE usually have advanced liver disease. As recommended in

current guidelines their diet should provide at least 30 kcal/kg of body weight,
3035% of calories as fat, and the remaining 5055% as carbohydrates. The protein
intake is expected to reach at least 11.2 g/kg of protein per day to maintain nitrogen
homeostasis but requirements may be increased to 1.5 g/kg/day in malnourished
patients to avoid endogenous protein breakdown (Table 16.2). Oral intake should be
206 M. Merli et al.
Table 16.2 Recommended energy and protein intake in liver cirrhosis

Nonprotein energy
Clinical condition (kcal/kg/day) Protein or amino acids (g/kg/day)
Compensated cirrhosis 2535 1.01.2
Cirrhosis with malnutrition and/or 3540 1.5
inadequate nutrient intake
Cirrhosis and low-grade 2535 Transient 1.01.5 if protein
encephalopathy intolerance: vegetable protein
or BCAA supplement
Cirrhosis and high-grade 2535 0.51 BCAA-enriched amino-
encephalopathy acid solution
Reprinted from Plauth et al. [35] 1997, with permission from Elsevier
Frequent meals (47 per day with a late-evening meal). Provide micronutrients and vitamins if
correction is needed. In patients with clinical signs of malnutrition, oral intake should be encour-
aged and nutritional supplements may be added to provide the needed requirements. When nutritional
supplementation is insufcient to obtain the desired intake, enteral nutrition should be considered.
Parenteral nutrition is used only when enteral feeding is not possible or impracticable
encouraged and diet needs to be cared for in these patients for adequate nutritional
support. The consumption of 46 small meals a day is advisable and the positive
role of breakfast and a late-evening meal has been already discussed. Whenever a
patient is unable to maintain an adequate oral intake, oral nutrition supplements or
tube feeding should be considered [36]. Enteral nutrition (EN) improves nutritional
status, reduces complications, and prolongs survival in hospitalized cirrhotic patients
with malnutrition; the possibility that the use of nasogastric tube may induce gastro-
intestinal bleeding is not supported by the literature. For patients who cannot be
adequately fed by EN or in whom EN is contraindicated, parenteral nutrition (PN)
may be helpful. PN is indicated when the patient is considered unlikely to resume
normal oral nutrition within the next 57 days irrespective of current nutritional
status. When a patient comes to the hospital in hepatic coma, he/she may need to
be supported with a complete parenteral nutrition [44].
Patients with recurrent HE may present specic problems with regard to the
maintenance of an adequate protein intake due to protein intolerance or a general-
ized decreased in food intake associated with the alteration in mental status.
The utilization of alternative sources of proteins might be of help either as a more
tolerable substitute of animal proteins or as a nutritional supplement.
Vegetable proteins have been claimed to be better tolerated than animal proteins
in cirrhotic patients with HE [45]. The benecial effects of a vegetable protein diet
(green vegetables, fruits, cereals, and pulses) include a higher intestinal clearance of
nitrogen-waste products due to the high ber content able to induce a greater
bacterial mass, a shortened transit time, and a reduced colonic pH entrapping ammo-
nia in the intestinal lumen. The clinical advantage of vegetable protein diets in HE
has been reported in small series of patients with chronic HE but not conrmed by
all authors [45]. Bloating, atulence, and early satiety are frequent in those consum-
ing vegetarian diets and may cause poor tolerance in the long term. To obtain a more
palatable and a more varied dietary regimen, vegetables may be supplemented with
cheese and other milk-derived dietary products. A diet including vegetables, fruits,
cereals, and milk-derived products planned to ensure at least 30 kcal/kg/day and

1.2 g of proteins/kg/day was well tolerated in 153 cirrhotic patients hospitalized
with overt HE [38].
Branched-chain amino acids (BCAA: isoleucine, leucine, and valine) are essential
amino acids with a peculiar role in whole-body nitrogen metabolism. They have a
stimulatory effect on protein synthesis, secretion of hepatocytes growth factor, glu-
tamine production, and inhibitory effect on proteolysis. BCAA supplementation may
ameliorate HE by promoting ammonia detoxication and through a competitive action
on amino-acid transport across the bloodbrain barrier [46]. Based on this possible
pharmacologic effects in cirrhotic patients with encephalopathy, BCAA were ini-
tially used as intravenous solutions in patients with severe hepatic coma as far as 25
years ago. Results of meta-analyses on the benecial effects of BCAA in the treatment
of HE and on patients survival were controversial. When BCAA were administered
as long-term oral supplementation it appeared, however, that BCAA allowed a higher
protein intake without inducing encephalopathy in cirrhotic patients with chronic
recurrent HE or either improving the neurologic symptoms when they were present
[47]. The nutritional and anticatabolic effect of BCAA may ameliorate the symptoms
of HE also through the improvement of muscle protein catabolism. A long-term oral
supplementation with BCAA in patients with advanced liver disease has been shown
to increase serum albumin concentration [48] and even to have benecial effects on
the progression-free survival [49, 50]. In a double-blind randomized trial comparing
BCAA supplementation with an equicaloric (maltodextrine) or an equinitrogen
(lactoalbumin) supplementation for 12 months, a signicant reduction in the number
of hospital admissions and the length of hospital stay was seen in patients treated with
BCAA as compared with controls [49]. The main problem in the study was a higher
noncompliance and low palatability of the BCAA formulation. The second study [50]
enrolled 622 patients with cirrhosis treated (long term) with BCAA supplementation
vs. diet alone; in the treated group, a signicant improvement in a composite end-
point [(progression to liver failure or death) (hazard ratio: 0.67, condence interval:
0.490.93)] and a tendency to a reduced HE incidence was reported.
In conclusion, although the use of BCAA supplementation may be limited by the
patients compliance and its availability (due to its high costs), there is a growing
evidence that these amino acids may help intolerant patients to reach the amount of
protein intake needed and prevent endogenous protein breakdown, thus represent-
ing a valid tool in the management of those cases at an advanced stage of the disease
and HE.
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Chapter 17
Hepatic Encephalopathy in Patients
with Transjugular Intrahepatic
Portosystemic Shunt (TIPS)
Martin Rssle and Wulf Euringer
Keywords Transjugular shunt TIPS Hepatic encephalopathy Prediction of HE

Prevention of shunt-induced HE Portosystemic pressure gradient
Introduction
Hepatic encephalopathy (HE) is a complex phenomenon which depends on both

liver dysfunction and portosystemic shunting. Thus, patients with acute hepatic fail-
ure may develop HE without any portosystemic shunting while patients with huge
shunts may develop HE without significant liver dysfunction [1]. In patients with
cirrhosis, HE (Type C HE) is a result of both components with variable contribu-
tion. In addition, most of these patients may suffer from non-hepatic, secondary
factors such as hypovolemia, hypotension, anaemia, electrolyte imbalance, renal
failure, malnutrition, and wasting which may contribute to HE.
The Eck fistula [2] was the first animal model demonstrating that diversion of the
portal blood flow results in a cerebral disease which can be provoked by meat inges-
tion [3]. Since then, numerous models have been investigated using portosystemic
shunts or experimental liver cirrhosis [4]. However, there is no animal model avail-
able which closely resembles the human situation with a combination of shunting
and cirrhosis. The transjugular intrahepatic portosystemic shunt (TIPS) is an ideal
model to investigate HE because the technique allows repeated measurements in
M. Rssle, MD (*)
Department of Gastroenterology and Radiology, University Hospital Freiburg,
Hugstetter Strasse 55, Freiburg 79106, Germany
e-mail: martin-roessle@t-online.de
W. Euringer, MD
Department of Radiology, University Hospital Freiburg,
Hugstetter Strasse 55, Freiburg 79106, Germany
212 M. Rssle and W. Euringer
humans of portal hemodynamics, shunt flow, and parameters of liver function as

well. In the last 2 decades, it has been increasingly applied for the treatment of
symptomatic portal hypertension and this has markedly stimulated the investigation
of HE [5]. However, most studies available are of questionable quality due to a number
of problems such as inappropriate design, inappropriate methods, and unblinded
investigators.
The following article tries to critically summarize the present knowledge obtained
in TIPS patients and discuss the difficulties of its assessment, prediction, prevention,
and treatment on the basis of the present literature.
Assessment and Incidence of HE
Several aspects suggest that assessment of HE was inappropriate in most of the

previous studies. In particular, previously applied tests for the assessment of HE
severity are now questioned. These include the West-Haven criteria which is subjec-
tive in nature with high inter-observer variability regarding the staging of lower
grades of HE [6, 7]. The diagnosis of minimal HE (mHE) is an even greater problem
which in part may be due to the limitations of the most often applied paper pencil
tests [611]. To overcome these problems, the critical flicker frequency test (CFF)
was introduced and investigated for the assessment of mHE and overt HE [12, 13].
This test seems to be an objective and reproducible diagnostic tool with little bias in
training effects, education, and time of testing or inter-observer variability. A recent
study by Kircheis et al. [14] compared the results obtained with CFF to a battery
of computerized psychometric tests and clinical assessment by the West-Haven
criteria which served as gold standards for mHE and overt HE. Sixty-three patients
were investigated before and after TIPS and compared to a control group of 34
age-matched cirrhotic patients without TIPS. A clear correlation was found between
HE severity assessed with the West-Haven criteria, CFF, and the psychometric tests
at baseline. However, the TIPS-induced changes assessed by the West-Haven criteria
were only reflected by the CFF. Neither the worsening nor the improvement of the
HE severity after TIPS implantation was reflected in respective changes in the different
computerized psychometric test results. The lack of correlation of most of the psycho-
metric tests with HE severity (West-Haven criteria) and the CFF in the longitudinal
assessment after TIPS was interpreted as a lack of reliability and reproducibility of
the psychometric tests. Overall, these results question the usefulness of psychometric
tests for follow-up investigations in clinical trials.
In addition to the methodological problems, the design of most studies may not
be appropriate to investigate HE. First, longitudinal cohort studies may be biased by
comparing a retrospective evaluation before the TIPS (past history of HE together
with the assessment at index hospitalization) with a prospective evaluation after the
TIPS. Except in the study by Kircheis et al. [14], exact data on the observation
period before the procedure are not given [1522]. Second, in the numerous random-
ized trials summarized and analysed in respective meta-analyses comparing TIPS
17 Hepatic Encephalopathy in Patients with Transjugular Intrahepatic 213
with medical treatment of variceal bleeding [23, 24] or refractory ascites [25], the
evaluation of HE may be biased because investigators were not blinded. It can be
assumed that investigators expected a higher risk of HE in the TIPS groups, a fact
which may have influenced the result. Third, HE often presents as an episodic and
self-limiting disease which may not be evaluated by regular outpatient visits.
Assessment using a diary has not been performed so far.
The incidence of post-TIPS encephalopathy varies from 15 to 48% [1422].
In controlled trials comparing TIPS with alternative forms of therapy, the inci-
dence of encephalopathy was always greater in patients who received TIPS. Thus,
a meta-analysis including studies in patients treated for variceal bleeding showed
1-year probabilities of overt HE of 19 and 35% for medical and TIPS treatment, respec-
tively [23]. In another analysis comparing medical treatment with TIPS, one serious
HE episode occurred for every eight TIPS procedures [24]. In patients with ascites,
a recent meta-analysis of individual patient data [25] showed that the cumulative
probability of developing the first episode of HE during follow-up was not different
between TIPS and paracentesis groups (P = 0.36 by log-rank) and a similar result
was found for the development of severe HE (P = 0.46 by log-rank). However, when
the average number of episodes-per-patient was considered, patients allocated to
TIPS had significantly more episodes of HE with regard to both, total number of episodes
(1.13 1.93 vs. 0.63 1.18, P = 0.006) and number of severe episodes (0.68 1.0 vs.
0.24 0.50, P = 0.008).
The recent study by Kircheis et al. [14] was designed to assess HE in a sufficient
number of patients receiving TIPS as well as in controls not receiving the intervention.
Their data showed a stable HE severity in the control group during an observation
period of 442 428 days with only minimal changes in CFF. In the TIPS group,
however, HE severity did not change in 44% of the patients, deteriorated in 35%,
and improved in 21% of the patients. Thus, while controls remained stable, TIPS
had the potential to deteriorate as well as to improve HE.
Prediction of HE After TIPS
Since HE depends on both liver function and portosystemic shunting, parameters of

liver function as well as of shunting may predict HE in patients with cirrhosis with
or without TIPS. In addition, biometrical variables may be important such as age
and gender. Furthermore, in most patients with advanced cirrhosis secondary factors
such as low blood pressure, renal insufficiency, electrolyte abnormalities, malnutrition,
etc. may contribute to HE (Table 17.1). It is the nature of portosystemic shunting to
worsen the hepatogenic factors of HE. However, the shunt may improve some of the
secondary factors making the prediction of HE more complex and unclear. Accordingly,
a variety of patterns of predictors including primary, biometrical, and secondary
prognostic factors have been suggested. Most often, increased age, advanced liver
failure (expressed by elevated bilirubin), a history of encephalopathy before TIPS
insertion, and serum sodium concentration [1618, 20] have been found to predict
Table 17.1 Secondary factors contributing to HE and effect of the TIPS treatment
Prominent feature Direct effect of TIPS
Cerebral Older age, pre-existing vascular or alcoholic damage No direct effect
Cardiac Systolic and diastolic dysfunction due No direct effect
to cirrhosis or toxins
Hemodynamic Hypovolemia due to vasodilatation and ascites Improved by TIPS
production
Renal Hepatorenal syndrome Improved by TIPS
Hematologic Anaemia due to GI-bleeding and hypersplenism Improved by TIPS
causing fatigue
Nutritional Muscle wasting may affect ammonia metabolism Improved by TIPS
Metabolic Hyponatremia, nocturnal hypoglycaemia, hypo- Improved by TIPS
phosphatemia, vitamin B deficiency, thiamine
deficiency
HE. With respect to pre-TIPS HE as a predictor of post-TIPS HE, the study by

Kircheis et al. [14] did not confirm previous findings using a different methodology.
Predictors, other than biometrical ones, have limited value in patients with an acute
bleed since bleeding limits the interpretation of laboratory and psycho-neurological
parameters.
In the meta-analysis of individual patient data [25], independent predictors of
post-TIPS HE in patients treated for refractory ascites were baseline mean arterial
pressure (MAP) (HR 0.93, CI 0.890.98; P = 0.004), MELD score at baseline (HR
1.068, CI 1.0061.13; P = 0.032), and post-TIPS porto-systemic pressure gradient
(HR 0.93, CI 0.870.99; P = 0.048). An explanation for the correlation of baseline
MAP with HE may be that a low MAP reflects poor liver and brain perfusion
together with advanced disease. Unfortunately, the study by Salerno et al. [25] does
not provide thresholds with lower or higher probabilities of HE after TIPS. However,
with a mean MAP of 87 mmHg and a mean MELD score of 12.5, values of <80 mmHg
or above 15, respectively, may be regarded as risk indicators for HE.
With respect to the hepatic arterial or portal hemodynamics, no correlation
could be seen between the increase in hepatic arterial blood flow after TIPS
(so-called buffer response) and the incidence of post procedural HE [26, 27].
However, patients with a loss of portal blood flow before TIPS are protected against
HE after TIPS [28, 29].
Most of the variables predicting HE are also predictors for survival after TIPS.
As a reliable marker of liver function bilirubin plays a dominant role in predicting
both HE and mortality in patients not having cholestatic disease. A value above
3 mg/dL is critical and above 5 mg/mL a relative contraindication for a TIPS in
bleeders as well as in ascites patients [3034]. As shown recently [14], the extent of
pre-TIPS HE is a major predictor for long-term survival in patients undergoing
TIPS implantation. Patients with pre-existing manifest HE have a significantly
reduced survival rate after TIPS implantation. This prognostic information was
obtained by a pre-TIPS CFF measurement. Those patients with a CFF below 37 Hz
prior to TIPS implantation have a significantly reduced long-term survival after
TIPS implantation than those with a CFF above this threshold [14].
Since TIPS is expected to worsen HE, it is not surprising that attention has been
focused exclusively on the factors predicting HE. However, as shown recently, TIPS
may also improve HE [14]. If those patients could be selected with sufficient accu-
racy, TIPS may rather be an indication than a contraindication. This requires the
knowledge of predictive markers for a positive effect of the TIPS on pre-existing
HE which have not been investigated so far. Candidate predictors for an improve-
ment of HE after TIPS may in particular be some secondary factors of HE
(Table 17.1) which have a high potential of improvement by the TIPS treatment.
One of these factors is the systemic circulation. It has been demonstrated that
within a short time after the TIPS, the central venous pressure and the arterial blood
pressure increase with a concomitant decrease of the heart rate [32]. In addition, the
hyperdynamic circulation improves almost reaching normal values for the periph-
eral resistance and cardiac output within 1 year of follow-up [32]. This is accompa-
nied by normalization of the plasma renin activity, aldosterone, and norepinephrine
concentrations [32]. As a consequence of the improvement in the systemic hemody-
namics after TIPS, renal function also normalizes during a 1 year follow-up which
is accompanied by an improvement of the dilutional hyponatremia [32].
Malnutrition and muscle wasting are seen in most patients with cirrhosis and
refractory ascites and have a negative effect on survival and HE [35, 36]. Wasting
may not only affect the ammonia metabolism in muscle, but may also lead to
decreased synthetic function of the liver. This affects many biochemical and even
hemodynamic variables such as decrease in serum albumin concentration present
in patients with cirrhosis. In contrast to serial paracentesis, which leads to a protein
loss of about 200 g/10 L of ascites removed (including the albumin substitution of
8 g/L), TIPS increases the plasma albumin concentration and increases body weight
despite resolution of ascites [31]. In addition, three studies show a significant
improvement in dry weight, total body nitrogen, total body fat, and total body pro-
tein [3739].
Prevention of HE
Attempts have been made to limit shunting with its negative effects on liver
function and HE by reducing the diameter of the shunt. The degree of shunting
can be visualized semi-quantitatively by portography. As demonstrated in Fig. 17.1,
a reduction of the pressure gradient by 50% or more of the pre-TIPS gradient
commonly results in a loss of portal liver blood flow equivalent to 100% shunting.
Fortunately, the loss or the reduction of the portal perfusion induces an immediate
rise in the arterial blood flow which is known as the arterial buffer response [27].
As demonstrated by endoluminal flow measurements during the TIPS procedure,
the calculated average arterial liver perfusion per minute increased from 599 100 mL/
min before to 749 161 mL/min after TIPS creation [40]. The effect occurred within
seconds after opening of the shunt and disappeared also within seconds after its
balloon occlusion.
Fig. 17.1 Portal hepatic blood flow in a cirrhotic patient before and after the TIPS implantation.
Before TIPS, anterograde portal blood flow is seen. The implantation of an 8-mm stent resulted in
a reduction of the pressure gradient by 50% from 24 to 12 mmHg leading to a complete deviation
of the portal blood flow through the shunt
It is assumed that the arterial buffer response does not fully compensate for the
loss of the portal hepatic perfusion. Therefore, the use of smaller shunts (<10 mm)
has been proposed to prevent complete diversion of the portal blood flow and also
efflux of arterial flow retrograde through the portal vein. This is supported by the
fact that larger shunts with a lower portal-systemic pressure gradient were identified
as an independent risk factor for post-TIPS HE and survival [25, 4143]. In the
study by Casado et al. [42], 23 out of 25 patients who developed HE after TIPS had
a pressure gradient <12 mmHg. The close correlation between the reduction of the
pressure gradient and post-shunt HE has also been demonstrated in previous studies
with surgical shunts [44]. Wider shunts with a greater pressure reduction resulted in
significantly increased incidence of HE. Thus, a recent uncontrolled study in patients
with ascites suggested that an incomplete stent expansion during TIPS construction
may reduce the occurrence of HE [45]. However, this study used self-expandable
nitinol stents which do not hold a given smaller diameter but expand to their nomi-
nal diameter due to the memory character of the nitinol. This is not the case with the
Viatorr stent which retains the adjusted diameter.
To further prove the advantage of smaller shunts with respect to HE, a recent
randomized study compared 8 mm with 10 mm TIPS using a covered stent graft
[46]. The study was stopped prematurely because of a less efficient control of com-
plications of portal hypertension, which could have been predicted. Thus, among
the 12 patients with the smaller stent, one patient re-bled and 5 continued to have
high-risk varices which needed further treatment for reducing the risk of rebleeding
(i.e. re-TIPS, endoscopic ligation, variceal embolization, or beta-blockers).
When patients treated for refractory ascites were considered, the majority of patients
(6 out of 10 patients) in the 8-mm stent group continued to have ascites and needed
to be submitted to repeated paracentesis after the procedure. Because of the reduced
efficiency, the authors did not recommend the use of 8 mm stents without having
the possibility to correct shunt efficiency. This possibility, however, is provided by
using stents with a nominal diameter of 10 mm that can be expanded to 8 mm only

and enlarged to 10 mm if necessary. Such an approach can be achieved using the
Viatorr stent (Gore) which allows stepwise enlargement due to its specific design.
Not only the shunt diameter, but also the design of the stent has been found to
influence the occurrence of HE. It has recently been shown that the covered stent
(Viatorr) has a reduced risk of HE and may, therefore, be preferred in patients with
a higher risk of HE [47]. However, this finding is invalid because the diameters of
the covered stents were smaller as compared to those of the bare stents and, there-
fore, the groups were not comparable [48]. In contrast, covered stents can be
expected to have even a higher risk of HE than bare stents. As shown with bare
stents, HE usually becomes clinically apparent 23 weeks after TIPS insertion and
then begins to decline (as measured by the portosystemic encephalopathy index)
at 6 months [15, 18]. This may be due to shunt dysfunction over time which is a
common phenomenon after placement of bare stents. With the use of covered stent
grafts, post-TIPS HE is no more confined to the postoperative period but remains a
long-term problem because of the improved patency of these stents [20]. Therefore,
in a patient with a higher risk of shunt-induced HE, uncovered stents may be preferred
because they may narrow with time preventing worsening of liver function or HE,
whereas patients with a low risk of these complications may have a covered stent a
priori to avoid unnecessary revisions.
Treatment of Shunt-Induced HE
Treatment of HE is medical in most of the patients and consists of lactulose and

non-absorbable antibiotics (neomycin or rifaximin) [15, 16]. In most patients, the
encephalopathy rapidly improves with supportive and standard therapy. In case of
medical therapy failure, the TIPS diameter can be reduced or the shunt occluded
[4952]. In this situation, one should be assured that the patients quality of life
benefits more from improving HE than it worsens by the reaccumulation of ascites.
Fortunately, in a series of 1,000 patients, the need for shunt reduction for debilitating
HE was only 3% [52]. According to one randomized study, pharmacological prophy-
laxis of HE is not effective and cannot be recommended for routine use [53].
Conclusions
The present knowledge of the incidence of HE after TIPS implantation and its
prediction is limited. Studies assessing HE may be biased by inappropriate methods
and design and may, therefore, provide rather qualitative but not quantitative infor-
mation. Without doubt, TIPS certainly increases the incidence of HE but the degree
of the effect is not sufficiently quantified. In addition, the factors predicting improvement
of HE by TIPS need to be established.
Prevention of HE may be achieved by creation of smaller shunts with the potential

of further dilatation in case of insufficient efficacy with respect to control of ascites
or variceal bleeding. Even in patients with refractory ascites, hepatorenal syndrome
or hydrothorax that are likely to require larger shunts, smaller shunts may be implanted
and dilated further after a stable course within the following 3 months.
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2006;17:160510.
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45. Thalheimer U, Leandro G, Samonakis DN, et al. TIPS for refractory ascites: a single centre
experience. J Gastroenterol. 2009;44:108995.
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shunt created with covered stents with different diameters: results of a randomized controlled
trial. J Hepatol. 2010;53:26772.
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in patients treated by transjugular intrahepatic portosystemic shunts: long-term results of a
randomized multicenter study. Liver Int. 2007;27:7427.
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may not always be desirable! Hepatology. 2004;40:4957.
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2005;42:6749.
Chapter 18
Quality of Life in Hepatic Encephalopathy
Jillian Kallman Price and Zobair M. Younossi
Keywords Quality of life Health-related quality of life Health utilities
Defining Quality of Life and Health-Related Quality of Life
Quality of life (QoL) is a general term for the assessment of the impact of health,
social, economic, and environmental factors on an individuals well-being. Although
the denition for QoL has not yet been standardized, the World Health Organization
(WHO) has dened QoL as individuals perceptions of their position in life in the
context of the culture and value systems in which they live, and in relation to their
goals, expectations, standards, and concerns [1, 2]. Other denitions of QoL have
included a patients perception of his/her ability to perform functions such as work,
their cognitive capabilities, the physical effects of the illness and concomitant psycho-
logical conditions (e.g., anxiety, depression, and aggressiveness), sexual problems,
and the patients relationship with their family and healthcare providers [1, 36].
Health-related quality of life (HRQL) deals primarily with the health-related
aspect of QoL [7]. In the context of chronic liver disease (CLD) and its complica-
tions such as hepatic encephalopathy (HE), assessing the impact of HE and its
J.K. Price, MS
Outcomes Research Program, Betty and Guy Beatty Center
for Integrated Research, Inova Fairfax Hospital, Falls Church, VA, USA
Z.M. Younossi, MD, MPH (*)
Beatty Liver and Obesity Research Center, Inova Fairfax Hospital,
Claude Moore Education and Research Building, 3rd Floor,
3300 Gallows Road, Falls Church, VA 22042, USA
e-mail: zobair.younossi@inova.org
222 J.K. Price and Z.M. Younossi
treatment on patients HRQL are important [6, 7]. In addition, patients with HE
have profound impairment of both their physical and mental aspects of HRQL,
affecting HE patients ability for self-care and other daily activities [8]. Currently,
many of the HRQL measures of impairment available in this patient population
depend upon the subjective perspective and self-reporting of the patient. It is neces-
sary to determine the areas of functionality and HRQL that are valued most by each
patient and to address perceived symptoms or limitations impacting their QoL.
HRQL can be measured both quantitatively with objective, validated, reliable
measures and qualitatively. Both forms of assessment and their utility will be
elaborated upon in this chapter. HRQL measures can also feed into health utility
calculations, which will also be discussed.
Generic Versus Disease-Specific Health-Related Quality

of Life Measures and Combined/Composite Measures
Two types of HRQL measures are currently usedgeneric and disease specic [5].
There is a growing belief that these two types of instruments measure different
aspects of patients HRQL and are therefore considered to be complementary [9,
10]. In some recent studies, both types of HRQL measures are used to provide the
full spectrum of HRQL issues.
Generic Health-Related Quality of Life Measures
Generic HRQL measures allow for the global assessment of HRQL across a wide
variety of diseases. Assessment results for a generic HRQL instrument can be com-
pared across diseases (e.g., in general, patients with CLD have better HRQL than
patients with congestive heart failure) and are also compared with general or so-
called healthy population norms. The limitation of generic measures is that it may
not assess all issues important for a specic disease (e.g., generic measures may not
have a question regarding apprehension related to potential for liver failure in the
future) and may not be responsive to subtle yet clinically signicant changes for a
specic disease state over time [5, 11]. Examples of generic HRQL measures which
have been used in hepatic encephalopathy studies are the Medical Outcomes Survey
Short Form 36 (MOS SF-36 or SF-36), the Sickness Impact Prole (SIP) and the
Nottingham Health Prole which will be further discussed later in this chapter
(Tables 18.1 and 18.2).
Many generic measures of HRQL will have a series of subscales or domains
measuring different global contributors to HRQL. Some have been designed to
measure specic symptoms tied to HRQL in great detail. Often these symptoms
may be included in a generic HRQL measure, but not in the detail desired for a
specic disease. The addition of a symptom-specic generic assessment to a test
18 Quality of Life in Hepatic Encephalopathy 223
Table 18.1 Quality of life (QoL) measures used in hepatic encephalopathy assessment
QoL measure Type of measure Total number of items
Medical Outcomes Survey Short Form 36 Generic 36
(SF-36)
Sickness Impact Prole (SIP) Generic 136
Nottingham Health Prole Generic 38
Chronic Liver Disease Questionnaire (CLDQ) Liver disease-specic 29
National Institute of Diabetes and Digestive Liver disease-specic 47
and Kidney Disease-QoL Assessment
Questionnaire (NIDDK-QA)
Hepatitis Quality-of-Life Questionnaire (HQLQ) Modular/Combination SF-36 + 15
Liver Disease Quality of Life Questionnaire Combination SF-36 + 75
(LDQOL)
battery may be desired (e.g., self-care, confusion, emotional behavior), especially if

a disease or treatment-related side effect is a common complaint or is believed to
strongly impact HRQL in the population to be studied.
Disease-Specific Health-Related Quality of Life Measures
Disease-specic measures of HRQL are tailored to a specic illness or category of

illness (e.g., Chronic Liver Disease Questionnaire (CLDQ), and Liver Disease
Quality of Life Questionnaire (LDQOL 1.0), National Institute of Diabetes and
Digestive and Kidney Disease-QoL Assessment Questionnaire (NIDDK-QA)
[1016] (for more detailed information on these instruments, please refer to
Tables 18.1 and 18.2). By focusing on the unique concerns and challenges of a
specic disease, disease-specic measures tend to be more responsive than generic
instruments.
Combination Instruments
Occasionally a test will be designed to capture both generic HRQL information and
disease-specic information. These combination instruments such as modular
instruments may have some ease in administration, and reduce the overlap of ques-
tions that can occur when both generic and disease-specic HRQL assessments are
used. However, unless portions of the questionnaire overlap signicantly or com-
pletely with an existing generic measure, it is difcult to compare the results to
other generic measure study ndings. The LDQOL is a combined instrument com-
prising the SF-36 plus 75 liver disease-specic questions. The Hepatitis Quality of
Life Questionnaire (HQLQ) is another example of disease-specic questions com-
bined with the SF-36 (Tables 18.1 and 18.2).
Table 18.2 QoL measures: areas of measurement for domains and scales
Health-related quality of life (HRQL) Total number of domains/
measure scales Domain/scale scores
Medical Outcomes Survey Short Form 8 Physical functioning (PF)
36 (SF-36) Role limitation-physical (RP)
Bodily pain (BP)
General health (GH)
Vitality (VT)
Social functioning (SF)
Role limitation-emotional (RE)
Mental health (MH)
Sickness Impact Prole (SIP) 12 Sleep and rest
Eating
Work
Home management
Recreation and pastimes
Ambulation
Mobility
Body care and movement
Social interaction
Alertness
Emotional behavior
Communication
Nottingham Health Prole Part I: 6 Energy (Part I)
Pain (Part I)
Emotional reactions (Part I)
Sleep (Part I)
Social isolation (Part I)
Physical immobility (Part I)
Part II: 1 Daily living (Part II)
Chronic Liver Disease Questionnaire 6 Abdominal symptoms
(CLDQ) Activity
Emotional function
Fatigue
Systemic symptoms
Worry
National Institute of Diabetes 4 Liver disease symptoms
and Digestive and Kidney Physical function
Disease-QoL Assessment Health satisfaction
(NIDDK-QA) Overall well-being
Liver Disease Quality-of-Life 12 Symptoms of liver disease
Questionnaire (LDQOL) Effects of liver disease
Concentration
Memory
Quality of social interaction
Health distress
Sleep problems
Loneliness
Hopelessness
Stigma of liver disease
Sexual functioning
Sexual problems
Health Utilities and Quality of Life
Another important type of measurement related to QoL is the measurement of health

utilities, and are measured on an interval scale of 01.0, with 0 reecting states of
health equivalent to death and 1 reecting perfect health. In health economics,
health utilities are often combined with survival estimates and aggregated across
individuals to generate quality-adjusted life years (QALYs) for use in costutility
analyses and comparison of different healthcare interventions [1720]. The main
indirect methods of utility measurement are: the use of generic preference instru-
ments (EQ-5D, SF-6D, and HUI); the use of disease-specic preference measures;
and mapping from a disease-specic HRQL instrument to a generic instrument. The
EQ-5Ds Visual Analog Scale (VAS) has been used in an HE treatment study [21].
The Health Utilities Index (HUI) has correlated strongly with global measures of
QoL such as the SF-36 and has also been applied to CLD [22]. A scoring method
for the SF-36, the SF-6D, has also been used to derive health utility scores, and has
correlated well in a CLD population [23]. Table 18.3 contains additional informa-
tion on the healthy utilities measures listed above.
Quantitative Versus Qualitative Measurement of Quality of Life
Quantitative assessment of HRQL allows us to quantify the relative impacts of

hepatic encephalopathy on patients daily activities to evaluate the impact of specic
treatment strategies on a patients HRQL. A qualitative approach can provide deeper
insights into the issues affecting HRQL in patients with hepatic encephalopathy,
Table 18.3 Health utilities measures

European Quality of Medical Outcomes
Life-Visual Analog Health Utilities Index Survey Short Form-6D
Scale (EQ-5D VAS) (HUI) (MOS SF-6D or SF-6D)
Number of items N/A 17 36
Number of scales/ Selection of a HUI2 attributes: Physical functioning,
domains/ number between sensation, mobility, social functioning,
classications/ 000 and 100 emotion, cognition, mental health,
attributes self-care, pain, vitality, pain, and
fertility role limitations
HUI3 attributes: vision,
hearing, speech,
ambulation,
dexterity, emotion,
cognition, pain
Selected citations Poo et al. [21] Younossi et al. [40] Dan et al. [41]
and explain potentially ambiguous or contradictory ndings of the quantitative

models. Both methods can become a rst step in the development of reliable and
valid HRQL measures tailored to specic clinical populations [13].
Chronic Liver Disease and Health-Related Quality of Life
In the recent years, QoL research has become increasingly essential to assess the
impact of CLD and its treatment, not only on important clinical outcomes but also
on patients well-being. HRQL monitoring is a vital part of the management of
potential transplant patients as a tool to assist in minimizing both human and
economic impacts of cirrhosis [2427]. Impairment of HRQL correlates strongly
with severity of liver disease [28, 29], and other complications of cirrhosis, as
well as repeated hospitalization [25, 2831]. In fact, patients with CLD have been
shown to exhibit impaired HRQL, on par with chronic obstructive pulmonary
disease and congestive heart failure [28, 32, 33]. In addition, cirrhotic patients
tend to present with more severe gastrointestinal symptoms than the general pop-
ulation, which is associated with decreased HRQL [16, 34]. Also, patients with
early cirrhosis showed better QoL as measured by the SF-36, as against those
with advanced cirrhosis [8, 16, 29]. Therefore, disease severity is associated with
worsening HRQL scores.
Health-Related Quality of Life in Hepatic Encephalopathy
Patients with hepatic encephalopathy have been shown to have lower HRQL than
the general population [8, 35]. Using SF-36, patients with cirrhosis undergoing ini-
tial screening for liver transplant candidacy, in a combined group of overt hepatic
encephalopathy (excluding grade III HE and above) and minimal (as dened by the
Reitan trial test) hepatic encephalopathy, were found to have lower mental and
physical component scores (MCS and PCS) on SF-36 than those without hepatic
encephalopathy, irrespective of the Child-Pugh score. In addition, those with mini-
mal hepatic encephalopathy (MHE) had worse MCS scores than patients without
hepatic encephalopathy [8]. SF-36 domains most affected were role limitation-emo-
tional, mental health, and social functioning [8]. An abbreviated SIP was also used
in a study assessing the uctuating course of MHE [36]. Several other studies have
also used the SIP to measure HRQL in patients with hepatic encephalopathy [37] as
well as assessing HRQL in patients pre-liver transplantation with varying severities
of hepatic encephalopathy [38]. In one such study, severity of hepatic encephalopa-
thy prior to liver transplantation was shown to correlate with QoL post liver trans-
plantation [38]. Finally, a lower HRQL score, depicting severe impairment, has
been shown to predict HE recurrence [39].
Health-Related Quality of Life

and Minimal Hepatic Encephalopathy
As noted previously, a number of researchers have found signicant QoL impair-

ment in patients with MHE. MHE has been shown to independently impair HRQL,
socio-economic status, and daily functioning in multiple studies [16, 29, 35, 37, 40,
41]. In fact, MHE can also impact the patients adherence to treatment which can
lead to increased dependence on providers [7, 42]. Additional studies have reported
that 4450% of patients with MHE are unt to work or were not regularly employed,
compared with 15% of patients without MHE [37, 43]. Furthermore, MHE may
have a negative impact on tness to drive an automobile [19, 20, 40, 4447]. Patients
with MHE showed signicant impairment on 11 scales of the SIP, the psychosocial
and physical subscores, and in total SIP score, particularly in social interaction,
alertness, emotional behavior, sleep, work, home management, and recreation and
pastimes [35]. More in-depth studies of MHE using disease-specic HRQL instru-
ments are currently underway.
Impact of Treatment of Hepatic Encephalopathy and Minimal

Hepatic Encephalopathy on Health-Related Quality of Life
Treatment of hepatic encephalopathy can positively impact HRQL [21, 32, 43, 48,
49]. Treatment options for hepatic encephalopathy include nonabsorbable disac-
charides (e.g., lactulose) [48] and nonabsorbable antibiotics (e.g., rifaximin) [50].
Although not available in the United States, l-ornithine-l-aspartate has also been
used to treat HE [32, 43]. HRQL data using VAS of the EQ-5D survey for
l-ornithine-l-aspartate used for treatment of HE showed signicantly greater
improvements than lactulose [21]. In another study using CLDQ-D, l-ornithine-
l-aspartate for 8 weeks improved HRQL domain scores [32].
The effect of nonabsorbable disaccharide, lactulose, on HRQL of cirrhotic
patients with minimal HE has also been assessed [21, 32, 35]. Patients with MHE
after 3 months of treatment with lactulose improved cognitive performance and
showed increased HRQL as measured by the SIP [35]. Nonabsorbable disaccharide,
lactulose, however, may be associated with gastrointestinal symptoms which might
reduce HRQL [51].
Finally, patients with MHE who were treated with rifaximin, a non-absorbable
antibiotic, for 8 weeks have also been shown to have improvements in cognitive
function and HRQL, as measured by the SIP [52]. Improvements on neuropsycho-
logical tests correlated with improved HRQL. Specically, mean total SIP score
showed signicant improvement in the rifaximin group. A recent study found that
rifaximin signicantly improved HRQL in patients with cirrhosis and recurrent HE
[39]. In fact, the study suggested that HRQL of patients with HE using CLDQ was
profoundly impaired. After 8 weeks of treatment with Rifaximin, all domain scores
of CLDQ showed signicant improvement as compared to baseline or compared to

lactulose [39]. This study suggests that effective treatment of HE with this antibi-
otic will not only result in reduction of recurrence of HE but also in improvement
of HRQL.
Summary
It has been long suspected that symptoms of hepatic encephalopathy impair func-
tioning and well-being of patients with cirrhosis in a variety of ways. Recent studies
have shown that impairment of HRQL does indeed correlate strongly with both
overt HE [8, 24, 29, 49, 53] and minimal HE [3335, 49, 51, 5456]. Additionally,
treatment of overt and minimal HE can lead to improvement of the patients HRQL.
Therefore, measurement of HRQL remains an important bellwether of a patients
condition in overt hepatic encephalopathy, MHE, and CLD. The specic impact on
HRQL of HE and MHE versus other symptoms of liver disease and treatment side
effects needs to be studied in greater detail.
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37. Groeneweg M, Quero JC, De Bruijn I, Hartmann IJ, Essink-bot ML, Hop WC, et al. Subclinical
hepatic encephalopathy impairs daily functioning. Hepatology. 1998;28:459.
38. Tarter RE, Switala J, Plial J, Havrilla J, Van Thiel DH. Severity of hepatic encephalopathy
before liver transplantation is associated with quality of life after transplantation. Arch Intern
Med. 1992;152:2097101.
39. Sanyal A, Younossi ZM, Bass NM, Mullen KD, Poordad F, Brown RS, Vemuru RP, Jamal M,
Huang S, Merchant K, Bortey E, Forbes WP. Rifaximin treatment improved quality of life in
patients with hepatic encephalopathy: results of a large, randomized, placebo-controlled trial.
J Hepatol. 2011;52(S7):15.
40. Schomerus H, Hamster W. Quality of life in cirrhotics with minimal hepatic encephalopathy.
41. Quero Guillen JC, Groeneweg M, Jimenez Saenz M, et al. Is it medical error if we do not
screen cirrhotic patients for minimal hepatic encephalopathy? Rev Esp Enferm Dig. 2002;94:
54457.
42. Miyazaki ET, Dos Jr SR, Miyazaki MC, Domingos NM, Felicio HC, Rocha MF, et al. Patients
on the waiting list for liver transplantation: caregiver burden and stress. Liver Transpl.
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43. Kircheis G, Wettstein M, vom Dahl S, Hussinger D. Clinical efcacy of L-ornithine-L-aspartate
in the management of hepatic encephalopathy. Metab Brain Dis. 2002;17:453.
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patients with liver cirrhosis with special reference to driving ability. Metab Brain Dis. 1995;10:
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to drive. Hepatology. 2004;39:73945.
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for accidents and trafc violations. Am J Gastroenterol. 2007;103:19039.
47. Bajaj JS, Hafeezullah M, Hoffman RG, et al. Navigation skill impairment: another dimension
of driving difculties in minimal hepatic encephalopathy. Hepatology. 2008;47(2):596604.
48. Als-Nielsen B, Gluud LL, Gluud C. Nonabsorbable disaccharides for hepatic encephalopathy
(review). Cochrane Database Syst Rev. 2004;(2):CD003044. doi:10.1002/14651858.
CD003044.pub2.
49. Les I, Doval E, Flavia M, Jacas C, Cardenas G, Esteban R, et al. Quality of life in cirrhosis is
related to potentially treatable factors. Eur J Gastroenterol Hepatol. 2010;22(2):2217.
50. Festi D, Vestito A, Mazella G, Roda E, Colecchia A. Management of hepatic encephalopathy:
focus on antibiotic therapy. Digestion. 2006;73 Suppl 1:94101.
51. Kalaitzakis E, Bjrnsson E. Lactulose treatment for hepatic encephalopathy, gastrointestinal
symptoms, and health-related quality of life. Hepatology. 2007;466:94950 (Letter).
52. Sidhu SS, Goyal O, Mishra BP, Sood A, Chhina RS, Soni RK. Rifaximin improves psychomet-
ric performance and health-related quality of life in patients with minimal hepatic encephal-
opathy (the RIME trial). Am J Gastroenterol. 2011;106(2):30716.
53. Afendy A, Kallman JB, Stepanova M, Younoszai Z, Aquino RD, Bianchi G, et al. Predictors
of health-related quality of life in patients with chronic liver disease. Aliment Pharmacol Ther.
2009;30(5):46976.
54. Kalaitzakis E, Josefsson A, Bjornsson E. Type and etiology of liver cirrhosis are not related to
the presence of hepatic encephalopathy or health-related quality of life: a cross-sectional study.
BMC Gastroenterol. 2008;8:46.
55. Bao ZJ, Qiu DK, Ma X, Fan Z-P, Zhang G-S, Huang Y-Q, et al. Assessment of health-related
quality of life in Chinese patients with minimal hepatic encephalopathy. World J Gastroenterol.
2007;13(21):30038.
56. Company L, Zapater P, Perez-Mateo M, Jover R. Extrapyramidal signs predict the develop-
ment of overt hepatic encephalopathy in patients with liver cirrhosis. Eur J Gastroenterol
Hepatol. 2010;22(5):51925.
Chapter 19
Liver Transplantation and Hepatic
Encephalopathy
Dileep K. Atluri and Kevin D. Mullen
Keywords Hepatic encephalopathy Liver transplantation Graft allocation
Introduction
Liver transplantation (LT) is the definitive treatment for end-stage liver disease and
its associated complications. LT improves and in most cases reverses hepatic
encephalopathy (HE). With the advent of model for end-stage liver disease (MELD),
the importance of HE as a major indication for LT has decreased. As the evidence
for residual effect of HE on posttransplant cognition continues to mount, we may
need to devise a system which includes HE in the decision-making process for LT.
Effect of Liver Transplantation on HE
Most clinicians believe that overt HE resolves after LT. A study by Hockerstedt
et al. [1] showed marked improvement in encephalopathic state of patients with
overt HE after LT. Along with extending life expectancy, LT was noted to improve
D.K. Atluri, MD, MRCP (UK) (*)

Department of Gastroenterology, Metrohealth Medical Center, 2500 Metrohealth Drive,
Cleveland, OH 44109, USA
e-mail: datluri@metrohealth.org
K.D. Mullen, MD, FRCPI
Division of Gastroenterology, Department of Internal Medicine,
Metrohealth Medical Center, Cleveland, OH, USA
234 D.K. Atluri and K.D. Mullen
Fig. 19.1 Chronology of improvement in cognitive function after liver transplantation as observed
in Mattarozzi study
quality of life (QOL) and cognitive functioning of the patients. In a prospective

study by Moore et al., 32 post-LT patients were followed for 9 months after LT.
Their QOL and cognitive functioning improved on successive measurements at 3,
6, and 9 months [2]. Tarter et al. noted that severity of HE is associated with post-LT
improvement in QOL [3].
Some patients with advanced neurodegeneration such as acquired hepatocere-
bral degeneration aka non-Wilsonian hepatolenticular degeneration were also
noted to benefit from LT. Case series by Stracciari, Powell, and Pinarbasi et al. have
demonstrated the reversibility of this condition after LT both clinically and radio-
graphically [47].
Several studies were conducted to evaluate the reversibility of minimal hepatic
encephalopathy (MHE) after LT. In a study done by Tarter et al., performance of
liver disease patients with a battery of neuropsychological tests improved after LT.
19 Liver Transplantation and Hepatic Encephalopathy 235
But, the impairments on 4 of 27 measures remained after transplantation [8]. The

improvement in neuropsychiatric test performance does not appear to be uniform
among the LT patients. In a study by Mechtcheriakov et al., only half of 14 patients
saw improvements in their visuo-constructive performance score. They concluded
that the visuo-motor deficits in liver disease patients resolve only in some of the
patients after LT, whereas a significant number of patients show no improvement
of the visuo-motor and visuo-constructive function [9]. In a larger study by
Mattarozzi et al., cognitive performance was assessed in LT patients for longer
intervals of up to 18 months posttransplantation [10]. In this longitudinal study, it
was noted that cognitive functions such as verbal short-term memory were late to
improve compared to visuo-spatial function and psychomotor speed. Their findings
are shown in Fig. 19.1.
Effect of Liver Transplantation on Findings

of Investigative Modalities for HE
A variety of changes are noted in brain in patients with HE. These changes include
manganese deposition in basal ganglia, brain edema, and increase in intracellular
concentration of glutamine and decrease in choline. These changes can be detected
by magnetic resonance (MR) imaging. These changes are noted to be reversible after
restoration of liver function through LT [1114]. They are detailed in Table 19.1.
Glucose metabolism of brain is altered in HE patients. Decreased glucose
metabolism is particularly evident in cingulate gyrus, frontal lobe, and hippocam-
pus. The glucose metabolism is noted to improve significantly in these areas after
LT [15].
The abnormalities in spectral electroencephalogram (S-EEG) associated with
HE are noted to improve after LT [16].
Residual Cognitive Deficits After Liver Transplantation
Many studies have noted persistent deficits in cognition of liver transplant patients.
This may be attributable to various causes as detailed below.
1. Preexisting central nervous system (CNS) damage:
CNS insults such as trauma, stroke, and Wernickes encephalopathy (WE) can
lead to persistent neurological deficits post-LT. Prolonged alcohol use, in con-
junction with thiamine deficiency, can lead to WE. Pathologically, WE manifests
with mammillary body and thalamic lesions. In a study by Kril and Butterworth,
brains of patients with autopsy proven cirrhosis were examined. WE findings
were discovered in 9 out of 30 alcoholic patients. But, clinical diagnosis of WE
was considered only in two patients. Cerebellar degeneration was found in 17 of
Table 19.1 Changes in findings of diagnostic modalities of HE with liver transplantation

Diagnostic After
modality Pathogenetic mechanism Before transplantation transplantation
PET scan Altered glucose Decreased glucose uptake in Improvement in
metabolism of brain cortical and subcortical glucose uptake
areas
MR imaging Deposition of manganese Bilateral high signal intensity Decrease in
in basal ganglia of globus pallidus and hyperintensity
substantia nigra on on T1-weighted
T1-weighted images images
Accumulation of Increase in glutamine/ Reversal of osmolar
glutamine in astrocyte glutamate signal, adaptation
and osmolar decrease in choline and changes of
adaptation of astrocyte myo-inositol signal astrocyte
Diffuse brain edema Focal high-signal T2 lesions Decrease in white
in subcortical hemispheric matter lesions
white matter on T2/
FLAIR images
Interstitial brain edema Increase in mean diffusivity Reversal of this
in hemispheric white change
matter
S-EEG Cerebral bioelectric Low MDF (mean dominant Significant increase
alterations frequency) and low LogR in MDF and
(LogR _ log10 occipital LogR
alpha-theta ratio) (<0.13)
30 patients [17]. This demonstrates the possibility of clinically unidentified

WE and cerebellar degeneration from alcohol abuse accounting for part of the
post-LT cognitive deficits.
2. Intraoperative injury to brain:
Given the complexity and prolonged nature of the LT procedure, many CNS
insults may occur. Changes in cerebral perfusion pressure and hypotension may
lead to ischemic event. Air embolism, embolic stroke, and cerebral hemorrhage
can also lead to lasting injury to brain [18].
3. Persistence of porto-systemic collaterals:
Porto-systemic shunts may persist after LT. Although HE is uncommon due to
collaterals in a patient with normally functioning graft, exceptions do exist. In a
case report by Herrero et al., recurrent bouts of HE were observed after a suc-
cessful LT with normally functioning graft. This was attributed to collaterals in
the retroperitoneum based on abdominal angiography. After successful embo-
lization of these collaterals, HE did not recur [19].
4. CNS toxicity of immunosuppressant drugs:
Immunosuppressive agents such as tacrolimus and cyclosporine are used after
LT as antirejection drugs. They act by inhibiting T lymphocytes. They also have
neuropsychiatric complications such as diffuse encephalopathy, cerebellar disor-
ders, and posterior leukoencephalopathy [20].
5. Residual effects of prior bouts of overt HE:

The effect of overt HE prior to LT on post-LT neurological outcomes was studied
by Sotil et al. Although this study was limited by small cohort and possible selec-
tion bias, authors suggested the possibility of overt HE having lasting effects on
the neurological outcome of the LT [20].
Importance of HE on Transplant Outcomes
HE is known to affect posttransplant outcomes such as neurological recovery,

survival, and QOL. In a study by Bajaj et al., effect of recurrent bouts of HE on
cognition was assessed. They concluded that episodes of overt HE are associated
with persistent and cumulative deficits in working memory, response inhibition,
and learning. It was also noted that the number of OHE hospitalizations correlated
with severity of residual impairment [21].
Patients with HE at time of listing have decreased 3- and 12-month posttrans-
plant survival compared to their counterparts without HE [22].
Apart from having possible residual neurological effect post-LT, HE is also noted
to be an independent risk factor for the early calcineurin inhibitor-induced neuro-
toxicity (ECIIN) after LT [23]. Acute graft rejection and infections were more
frequent in the ECIIN patients.
Should Hepatic Encephalopathy Be Given Greater

Consideration in Transplant Allocation?
LT is recognized as an effective treatment for advanced liver disease and the demand
for organs has been steadily going up. But the supply of the organs could not keep
pace with the demand. With this demandsupply mismatch, it becomes imperative
to allocate the organs to the recipients most in need. MELD was introduced as an
objective and reliable predictor of short-term mortality in liver disease patients.
This scale is also used currently to assign priority to liver disease patients for organ
allocation. It is based on objective data such as serum bilirubin, creatinine, and
international normalized ratio (INR). MELD has not assigned HE any extra points.
MELD exception study group and conference 2006 (MESSAGE) did not recom-
mend any automatic increase in priority due to HE. They suggested case-by-case
assessments and priority to patients in coma needing endotracheal intubation and/or
increased intracranial pressures [24, 25].
As new evidence for effect of HE on transplant outcomes continues to build,
it becomes necessary to explore ways in which HE can be given higher priority
in transplant allocation.
Conclusion
LT has evolved from experimental procedure to accepted treatment modality for

advanced chronic or fulminant liver failure. LT treats a variety of complications of
liver failure including HE. Overt HE is known to improve after LT, whereas the
improvement in MHE appears to be nonhomogenous, dynamic, and at times
incomplete. There are a range of possible reasons for persistent cognitive deficits
after LT. Possible lasting effect of overt HE on brain is one of them. Overt HE is
also known to affect the post-LT survival, QOL, and modulate ECIIN. As detri-
mental effects of overt HE on transplant outcomes become more apparent, more
emphasis might need to be given for early transplantation in these patients.
References
1. Hockerstedt K, Kajaste S, Isoniemi H, Muuronen A, Raininko R, Seppalainen AM, et al. Tests

for encephalopathy before and after liver transplantation. Transplant Proc. 1990;22(4):15768.
2. Moore KA, McL Jones R, Burrows GD. Quality of life and cognitive function of liver trans-
plant patients: a prospective study. Liver Transpl. 2000;6(5):63342.
3. Tarter RE, Switala J, Plail J, Havrilla J, Van Thiel DH. Severity of hepatic encephalopathy
before liver transplantation is associated with quality of life after transplantation. Arch Intern
Med. 1992;152(10):2097101.
4. Stracciari A, Guarino M, Pazzaglia P, Marchesini G, Pisi P. Acquired hepatocerebral degenera-
tion: full recovery after liver transplantation. J Neurol Neurosurg Psychiatry. 2001;70(1):1367.
5. Stracciari A, Baldin E, Cretella L, Delaj L, DAlessandro R, Guarino M. Chronic acquired
hepatocerebral degeneration: effects of liver transplantation on neurological manifestations.
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6. Powell EE, Pender MP, Chalk JB, Parkin PJ, Strong R, Lynch S, et al. Improvement in chronic
hepatocerebral degeneration following liver transplantation. Gastroenterology. 1990;
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7. Pinarbasi B, Kaymakoglu S, Matur Z, Akyuz F, Demir K, Besisik F, et al. Are acquired hepa-
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8. Tarter RE, Switala JA, Arria A, Plail J, Van Thiel DH. Subclinical hepatic encephalopathy.
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9. Mechtcheriakov S, Graziadei IW, Mattedi M, Bodner T, Kugener A, Hinterhuber HH, et al.
Incomplete improvement of visuo-motor deficits in patients with minimal hepatic encephal-
opathy after liver transplantation. Liver Transpl. 2004;10(1):7783.
10. Mattarozzi K, Stracciari A, Vignatelli L, DAlessandro R, Morelli MC, Guarino M. Minimal
hepatic encephalopathy: longitudinal effects of liver transplantation. Arch Neurol.
2004;61(2):2427.
11. Thomas MA, Huda A, Guze B, Curran J, Bugbee M, Fairbanks L, et al. Cerebral 1H MR
spectroscopy and neuropsychologic status of patients with hepatic encephalopathy. AJR Am
J Roentgenol. 1998;171(4):112330.
12. Rovira A, Minguez B, Aymerich FX, Jacas C, Huerga E, Cordoba J, et al. Decreased white
matter lesion volume and improved cognitive function after liver transplantation. Hepatology.
2007;46(5):148590.
13. Pujol J, Kulisevsky J, Moreno A, Deus J, Alonso J, Balanzo J, et al. Neurospectroscopic altera-
tions and globus pallidus hyperintensity as related magnetic resonance markers of reversible
hepatic encephalopathy. Neurology. 1996;47(6):152630.
14. Chavarria L, Alonso J, Garcia-Martinez R, Aymerich FX, Huerga E, Jacas C, et al. Biexponential
analysis of diffusion-tensor imaging of the brain in patients with cirrhosis before and after liver
transplantation. AJNR Am J Neuroradiol. 2011;32(8):15107.
15. Senzolo M, Pizzolato G, Ferronato C, Chierichetti F, Boccagni P, Dam M, et al. Long-term
evaluation of cognitive function and cerebral metabolism in liver transplanted patients.
Transplant Proc. 2009;41(4):12956.
16. Ciancio A, Marchet A, Saracco G, Carucci P, Lavezzo B, Leotta D, et al. Spectral electroen-
cephalogram analysis in hepatic encephalopathy and liver transplantation. Liver Transpl.
2002;8(7):6305.
patients with end-stage liver disease. Hepatology. 1997;26(4):83741.
18. Stracciari A, Guarino M. Neuropsychiatric complications of liver transplantation. Metab Brain
Dis. 2001;16(12):311.
19. Herrero JI, Bilbao JI, Diaz ML, Alegre F, Inarrairaegui M, Pardo F, et al. Hepatic encephalopa-
thy after liver transplantation in a patient with a normally functioning graft: treatment with
embolization of portosystemic collaterals. Liver Transpl. 2009;15(1):1114.
20. Sotil EU, Gottstein J, Ayala E, Randolph C, Blei AT. Impact of preoperative overt hepatic
encephalopathy on neurocognitive function after liver transplantation. Liver Transpl.
2009;15(2):18492.
21. Bajaj JS, Schubert CM, Heuman DM, Wade JB, Gibson DP, Topaz A, et al. Persistence of
cognitive impairment after resolution of overt hepatic encephalopathy. Gastroenterology.
2010;138(7):233240.
22. Bajaj JS, Saeian K. MELD score does not discriminate against patients with hepatic encephal-
opathy. Dig Dis Sci. 2005;50(4):7536.
23. Balderramo D, Prieto J, Cardenas A, Navasa M. Hepatic encephalopathy and post-transplant
hyponatremia predict early calcineurin inhibitor-induced neurotoxicity after liver transplanta-
tion. Transpl Int. 2011;24(8):8129.
24. Ham J, Gish RG, Mullen K. Model for end-stage liver disease (MELD) exception for hepatic
encephalopathy. Liver Transpl. 2006;12(12 Suppl 3):S1024.
25. Freeman Jr RB, Gish RG, Harper A, Davis GL, Vierling J, Lieblein L, et al. Model for end-
stage liver disease (MELD) exception guidelines: results and recommendations from the
MELD Exception Study Group and Conference (MESSAGE) for the approval of patients who
need liver transplantation with diseases not considered by the standard MELD formula. Liver
Transpl. 2006;12(12 Suppl 3):S12836.
Chapter 20
Future of Hepatic Encephalopathy
Keywords Covert hepatic encephalopathy Rifaximin Psychometric tests Liver

transplantation
One of the major trends we predict for the future is a totally different focus for hepatic
encephalopathy (HE) treatment. Formerly, all trials of new therapeutic agents for
the treatment of HE were performed in patients with overt HE. Since most of these
patients had advanced liver disease and at times multiple precipitating events for
episodes of HE, it was not possible to perform randomized controlled clinical trials
in these patients. This may explain why no new drug was approved for the treatment
of HE in 30 years [1]. These days minimal or covert HE can be reliably detected
and quantified [2, 3]. It is generally seen earlier in the course of liver disease when
precipitating factors are relatively rare. Accordingly, new therapeutic agents will be
tested for efficacy in this population of HE patients.
The total emphasis on overt HE has shifted towards covert HE based on the
growing knowledge of the impact of covert HE. Patients meeting the criteria for
covert HE (generally by psychometric test scores) have been shown to have
(1) decreased quality of life; (2) reduced driving skills; (3) less chance of being
employed; and (4) high probability (60%) of developing overt HE without 18
months [49]. Treatment of covert HE has already been shown to improve/normal-
ize psychometric test scores, driving capacity, and quality of life [1012]. Future
studies will determine if overt HE can be postponed or, even more tantalizingly,
prevented!!!
In future, another trend in HE will be genetic markers for susceptibility to developing
HE. Not all patients with cirrhosis develop overt HE. Why does one patient get
HE at the same level of liver dysfunction as another patient who is free of HE?
The recent discovery of the importance of the profile of glutaminase gene inheri-
tance in modulating the expression of overt HE has provoked great interest [13].
The enzymes were known in the early to mid-1950s as a potential mediator of the
therapeutic action of neomycin [14]. Inhibition of intestinal glutaminase enzyme
activity was proposed to reduce portal vein ammonia levels [15]. Now we have its
potential role in permitting the appearance of overt HE being identified. In the near
future more genetic markers of susceptibility to express or protect from HE will
be identified. This will considerably modify how we manage cirrhotic patients in the
decades to come.
Another new development still requiring more verification is the concept that
overt HE episodes may be associated with less than full recovery to normal brain
function [16]. If this is verified, liver transplantation may be moved up in cirrhotics
to precede the development of overt HE especially in patients with genetic predis-
position to develop overt HE. Short-term data after bouts of overt HE have shown
delayed recovery from bouts of overt HE. These have been identified with either
specific psychometric tests or the inhibitory control test. The question still remains
whether more effective or aggressive therapy over time will reverse these deficits
more fully. Even after successful liver transplantation there are concerns that certain
domains of neurological function will never return to complete normality [17].
Whether brain atrophy so commonly seen in cirrhotics reverses after liver transplan-
tation should be resolved in the next few years.
Future treatments of HE will include more effective agents for lowering ammonia
in the blood. New glutaminase inhibitors are already being developed and tested.
Probiotic agents plus laxatives seem likely to be piloted as a therapy for covert HE
in the coming years [18]. Perhaps the most promising agents are antibiotics with
prominent anaerobic activity. Ever since metronidazole was noted to potentially
have efficacy in treating HE, antibiotics with anaerobic coverage have been tested
for efficacy in treating HE. Nitazoxanide and Rifaximin may be the first in a line of
agents to treat HE [10, 19]. Compounds that provide alternatives to urea for the
excretion of ammonia will continue to be tested and developed [20]. The agents
being tested will do so in patients with covert rather than overt HE because placebo-
controlled trials can be utilized in this patient population.
In summary, the future of HE therapeutic interventions will be greatly enhanced
by enrollment of covert HE patients. We have the ability to detect and quantify this
earlier form of HE. Randomized placebo-controlled trials will be the norm for
initial testing of agents for the efficacy in controlling or preventing HE. Truly,
these are exciting times for the development of new therapeutic agents for the treat-
ment of HE. A key to this new testing paradigm is agreement on computerized
psychometric testing systems that are widely available and validated as measures
of covert HE.
20 Future of Hepatic Encephalopathy 243
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Index
A cerebral edema, 9
Acquired hepatocerebral degeneration cognitive and motor functions, 10
(AHD), 100 cytokines, nitric oxide, 170171
Acute liver failure (ALF) free radical production, 51
Alzheimer type II astrocytosis, 20, 21 inflammation, 3839
bloodbrain barrier, 40 interorgan metabolism, 166167, 203
brain edema, 9, 169 metabolism of, 54
CBF, 9 ornithine phenylacetate, 168169
death of patients, 9 vs. proinflammatory mechanisms, 2425
electron micrograph of cerebral cortex, 21 Antibiotic treatment
ICP, 12 data, 161163
infection vs. inflammation, 3637 mechanism of action, 160, 161
microglial activation, 22, 23 treatment, 162
OX-42 immunohistochemistry, 22, 23 Antioxidants, 6061
proinflammatory cytokine, 38 Aquaporin-4 (AQP4), 59
systemic immune dysfunction, 39 Asymmetric dimethylarginine
ADMA. See Asymmetric (ADMA), 170171
dimethylarginine (ADMA)
AHD. See Acquired hepatocerebral
degeneration (AHD) B
ALF. See Acute liver failure (ALF) BBB. See Bloodbrain barrier (BBB)
American Medical Association (AMA), 194 BCAA. See Branched-chain amino acids
Ammonia (BCAA)
ADMA inflammation pathway, 170171 Benzodiazepine (BZ) receptor ligands
amino acid neurotransmitter systems, 12 agonists and antagonist, 74, 85
blood ammonia levels, 8 in animals and humans, 7677
blood ammonia-reducing therapies, 10 concentrationresponse curves, 85
brain function flumazenil
astrocytic swelling, 1112 controlled studies of, 8182
neurotransmitter imbalance, 1214 efficacy of, 79, 83
brain imaging, 125 FHF, 82, 83
brain swelling, 142 1,4-imidazobenzodiazepine, 79
CBF, 9 observations, 84
DOI 10.1007/978-1-61779-836-8, Springer Science+Business Media, LLC 2012
246 Index
Benzodiazepine (BZ) receptor ligands (cont.) Central nervous system (CNS), 19

plasma clearance, 79 damage, 235, 236
positron emission tomography, 79 toxicity of, 236
traditional approaches, 82 Cerebral blood flow (CBF), 9
uncontrolled studies of, 8081 CFF. See Critical flicker frequency (CFF)
GABA-mediated inhibitory cGMP. See Cyclic guanosine monophosphate
neurotransmission (cGMP)
GABAA/benzodiazepine receptor Chronic liver failure
complex, 7172 bloodbrain barrier, 40
intrinsic activities of, 7274 infection vs. inflammation, 3637
measurements of, 7576 MR images, 127
nomenclature of, 7778 systemic immune dysfunction, 39
Ro 14-7437, 84, 85 Chronic subdural hematoma, 9899
Ro 15-4513 and Ro 15-3505, 74, 85 Cirrhosis
role for neuroinflammation, 2324
antagonism, 7375 protein-calorie malnutrition, 200202
GABAergic tone, 73 sleep disorders
sources of, 78 sleep regulation, 179, 181, 182
Bloodbrain barrier (BBB), 8, 9, 11 sleepwake abnormalities, 183184
acute and chronic liver failure, 40 CNS. See Central nervous system (CNS)
infection, 37 Cognitive drug research (CDR), 107, 108
Brain edema Covert hepatic encephalopathy, 13
astrocytic swelling, 1112 cognitive dysfunction, 110
brain imaging, 125 diagnosis of, 105
development of, 9 neurophysiological testing, 108109
and motor-evoked potentials, 169170 neuropsychological testing, 105, 108
role of glutamine, 12 trials, 110111
Brain imaging Critical flicker frequency (CFF), 107, 109,
ammonia, 125 212, 214
brain atrophy, 125 Cyclic guanosine monophosphate (cGMP)
brain edema, 125 excess of nitric oxide, 11
brain size, 132 impairment of cognitive functions, 10
FLAIR reduction of, 13
diffusion-weighted imaging, 131132
T2-weighted lesions, 130, 131
functional studies D
magnetic resonance, 133 Diet, 202, 204205
nuclear imaging techniques, 133134 Disaccharides
1
H-MR spectroscopy, antimicrobial agents, 146, 148
metabolites, 127129 lactulose and lactitol
magnetic resonance minimal hepatic
brain water, 129 encephalopathy, 144145
T1 high signal intensity, 126127 overt hepatic encephalopathy, 144
manganese accumulation, 124 treatment, 146, 147
Branched-chain amino acids (BCAA), 207 mechanism of action, 142143
BZ receptor ligands. See Benzodiazepine vs. other therapy, 150152, 154
(BZ) receptor ligands primary prophylaxis, 151
rifaximin, 148, 150
secondary prophylaxis, 151
C Driving
CBF. See Cerebral blood flow (CBF) crashes, 188
CDR. See Cognitive drug research (CDR) driving impairment
Index 247
on-road driving studies, 189190 Fulminant hepatic failure (FHF), 77, 8083
simulation studies, 190191 Functional magnetic resonance imaging
minimal hepatic encephalopathy (fMRI), 133
challenges, 191192
cognitive examinations, 189
fitness, 193194 G
legal ramifications, 193194 Gamma-aminobutyric acid (GABA)
motor vehicles, 191 BZ receptor ligands
real-life driving outcomes, 191 GABAA/benzodiazepine receptor
skills, 188 complex, 7172
and society, 188 intrinsic activities of, 7273
traffic violations, 191 hyperammonemia, 13
Glutaminase (GA), 166, 169
Glutamine/glutamate (Glx), 127128
E Glutamine synthetase (GS), 166169
ECs. See Endothelial cells (ECs) Glutathione (GSH), 51
Electroencephalogram (EEG), 100, 108
classification of, 117
clinical information, 116119 H
clinical scenario, 114115 HE. See Hepatic encephalopathy (HE)
cognitive tasks, 119120 Health-related quality of life (HRQL)
EEG patterns, 115 chronic liver disease, 226
objective quantification, 116, 117 definition, 221222
P300, 119 disease-specific measures, 223
principles of, 113114 domains and scales, 224
quantitative assessment, 114 generic, 222224
spectral analysis, 116118 hepatic encephalopathy, 226
triphasic waves, 115117, 119 impact of treatment, 227228
Electron transport chain (ETC), 53 minimal hepatic
Endothelial cells (ECs), 5758 encephalopathy, 227
Enteral nutrition (EN), 206 Health utilities index (HUI), 225
ETC. See Electron transport chain (ETC) Hemeoxygenase-1(HO-1), 49
Extrapyramidal signs (EPS), 110 Hepatic encephalopathy (HE)
cirrhotic patients, 10
covert, 13, 241, 242
F ISHEN, 12
Fast fluid-attenuated inversion recovery liver transplantation, 242
(FLAIR) nomenclature and classification, 13
diffusion-weighted imaging, 131132 overt, 3
T2-weighted lesions, 130, 131 psychometric test, 241, 242
Flumazenil rifaximin, 242
controlled studies of symptoms of, 97, 98
animal models of, 8182 terminology, 13
patients with HE, 82 West Haven criteria, 103, 104
efficacy of, 79 Hepatic myelopathy (HM), 98, 101
FHF, 82, 83 HO-1. See Hemeoxygenase-1(HO-1)
1,4-imidazobenzodiazepine, 79 HRQL. See Health-related quality
observations, 84 of life (HRQL)
plasma clearance, 79 HUI. See Health utilities index (HUI)
positron emission tomography, 79 Hyperammonemia
traditional approaches, 82 accumulation of glutamine, 142
uncontrolled studies of CBF, 9
doses of, 80 glutamate exocytosis, 13
patients with HE, 8081 in vivo model, 11
248 Index
I Malnutrition, 202203
Inducible nitric oxide synthase Matrix metalloproteinase 9 (MMP-9), 9
(iNOS), 50, 51, 53 MELD. See Model for end-stage liver disease
Infection (MELD)
ammonia, 3839 Metabotropic glutamate receptor
bloodbrain barrier, 37 (mGluR) activity, 10
hepatic encephalopathy, 38 Minimal hepatic encephalopathy
vs. inflammation, 3637 (MHE), 166
Inflammation cognitive dysfunction, 110
ammonia, 3839 diagnostic methods, 105107
bloodbrain barrier, 37 driving
and hepatic encephalopathy challenges, 191192
immune dysfunction and cognitive examinations, 189
oxidative stress, 3940 fitness, 193194
role of, 38 legal ramifications, 193194
treatment of, 4042 studies and results of, 190
infection vs., 3637 health-related quality of life, 227
pathogenesis of ALF, 57 neurophysiological testing, 108109
Inhibitory control test (ICT), 105, 107, 108 neuropsychological testing, 105, 108
International Society for Hepatic Encephalopathy nonabsorbable disaccharides, 144145
and Nitrogen (ISHEN), 12, 105, 110 physical symptoms of, 169
Intracellular calcium, 52 presence and severity of, 24
Intracranial pressure (ICP), 9, 12, 165 psychometric tests, 106107
trials, 110111
Mitochondrial permeability transition, 5253
L Model for end-stage liver disease
Lactulose, 159, 162 (MELD), 237
antimicrobial agents, 146, 148 Motor-evoked potentials (MEP), 169170
vs. lactitol MRI. See Magnetic resonance
minimal hepatic encephalopathy, imaging (MRI)
144145 MRS. See Magnetic resonance
overt hepatic encephalopathy, 144 spectroscopy (MRS)
treatment, 146, 147
vs. neomycin, 148
rifaximin, 148, 150 N
Lipopolysaccharide (LPS), 25 Natriuretic peptide clearance receptor
Liver transplantation (NPR-C), 11
CNS, 235, 236 Natriuretic peptides (NPs), 11
cognitive function, 234 Neomycin, 148, 159, 160, 162
effect of, 233235 Neuroinflammation
immunosuppressant drugs, 236 ALF, 2123
intraoperative injury, 236 ammonia vs. proinflammatory
investigative modalities, 235, 236 mechanisms, 2425
porto-systemic collaterals, 236 cirrhosis, 2324
residual cognitive deficits, 235237 CNS complications
transplant allocation, 237 allopregnanolone, 27, 28
transplant outcomes, 237 diagnostic and therapeutic
L-ornithine L-aspartate (LOLA), 166168 implications, 2831
LPS. See Lipopolysaccharide (LPS) glial pathology in liver failure, 2021
liverbrain proinflammatory
signaling, 2526
M Neurotransmitter imbalance
Magnetic resonance imaging (MRI), 99, 100 GABAergic transmission, 13
Magnetic resonance spectroscopy (MRS), 99 glutamatergic transmission, 13
Magnetization transfer (MT) imaging, 129 serotoninergic transmission, 14
Index 249
Neutrophils, 36, 39, 40 Alzheimer typeII astrocytes, 48

Nitrative stress nitric oxide production, 50
astrocyte cultures, 5051 protein tyrosine nitration, 50
experimental animals, 4950 thioacetamide-treated rats, 49, 50
Nitric oxide (NO), 11 in humans, 5152
N-methyl D-aspartate (NMDA) mechanisms of
receptors, 13, 5354 electron transport chain, 53
NPR-C. See Natriuretic peptide clearance glutamine, 5455
receptor (NPR-C) inflammation, 57
NPs. See Natriuretic peptides (NPs) intracellular calcium, 52
Nuclear factor-kappa B (NF-KB), 53 manganese, 56
Nutrition mitochondrial permeability transition,
diet, 202, 204205 5253
energy and protein intake, 206 NMDA receptors, 5354
feeding and physician prescriptions, 205 nuclear factor-kappa B, 53
guidelines, 205207 peripheral benzodiazepine receptor,
liver cirrhosis, 203, 206 5556
malnutrition, 202203 Oxidative stress
protein-calorie malnutrition, 200202 astrocyte cultures, 5051
experimental animals, 4849
humans, 5152
O
OHE. See Overt hepatic encephalopathy (OHE)
ONS. See Oxidative/nitrative stress (ONS) P
Ornithine phenylacetate (OP) Parenteral nutrition (PN), 206
blood ammonia, 10 Parkinsons disease (PD), 100
LOLA, 167, 168 Peripheral benzodiazepine receptor
mechanism of action (PBR), 5556
ADMA inflammation pathway, 170171 PET. See Positron emission tomography (PET)
ammonia metabolism enzymes, 168169 P300 event-related potential (P300ERP), 109
brain edema and motor-evoked PN. See Parenteral nutrition (PN)
potentials, 169170 Portacaval anastomosis (PCA), 170
cytokines, nitric oxide, 170171 Positron emission tomography (PET)
NFKB, 170 ammonia, 125
Overt hepatic encephalopathy (OHE), 3 cerebral ammonia metabolism, 133
chronic progressive HE, 100101 oxygen consumption, 133
diagnostic approach quantitative data, 133
biochemical analysis, 98 Protein-calorie malnutrition (PCM), 200202
brain imaging, 9899 Psychometric hepatic encephalopathy score
electroencephalogram, 100 (PHES), 105
lumbar puncture, 99
nonabsorbable disaccharides, 144
Oxidative/nitrative stress (ONS) Q
antioxidants, 6061 Quality of life (QoL)
in astrocyte cultures chronic liver disease, 226
ammonia, 51 combination instruments, 223224
GSH level, 51 definition, 221222
time-dependent changes, 51 disease-specific HRQL, 223
cellular sources of free radicals domains and scales, 224
endothelial cells, 5758 generic HRQL, 222224
microglia, 57 health utilities, 225
consequences of impact of treatment, 227228
astrocyte swelling/brain edema, 5859 minimal hepatic encephalopathy, 227
neurobehavioral defects, 60 quantitative versus qualitative
in experimental animals measurement, 225, 226
250 Index
R SPECT. See Single-photon emission computed

Reactive oxygen and nitrogen species tomography (SPECT)
(RONS), 11, 12, 52, 59 Spectral electroencephalogram
Reactive oxygen species (ROS), 51 (S-EEG), 235, 236
Repeatable battery for the assessment of Systemic inflammatory response syndrome
neuropsychological status (SIRS), 23, 36, 37
(RBANS), 105, 106
Rifaximin, 148, 150, 161, 162
T
Toll-like receptors (TLRs), 39, 42
S Transjugular intrahepatic portosystemic
Serotoninergic transmission, 14 shunt (TIPS), 151, 153
Single-photon emission computed tomography assessment and incidence, 212213
(SPECT), 133, 134 portal hepatic blood flow, 216
Sleep disorders prediction of HE, 213215
circadian rhythm, 182 secondary factors, 214
cirrhosis shunt-induced HE, 217
sleep regulation, 179, 181, 182 18-kDa Translocator protein (TSPO), 55, 56
sleepwake abnormalities, 183184
daytime sleepiness, 179
delayed sleep timing, 178 V
diurnal preference, 178 Visual analog scale (VAS), 225
melatonin, 180182 Visual evoked potentials (VEPs), 107109
night sleep disturbance, 177178
physiological sleep regulation, 179181
sleep quality, 178 W
Small bowel bacterial overgrowth Wernickes encephalopathy (WE), 99, 235
(SBO), 160, 161 Western blot analysis
Smooth pursuit eye movements oxidized proteins, 49
(SPEM), 109 protein tyrosine nitration, 50
Somatosensory evoked potentials (SEPs), 109 World Gastroenterology Congress, 1, 2

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CLINICAL GASTROENTEROLOGY

For further volumes:

ISBN 978-1-61779-835-1 e-ISBN 978-1-61779-836-8

Library of Congress Control Number: 2012936298

Springer Science+Business Media, LLC 2012

Printed on acid-free paper

Humana Press is part of Springer Science+Business Media (www.springer.com)

Cleveland, OH, USA Kevin D. Mullen

1 Introduction, Nomenclature, and Classification of Hepatic

2 Role of Ammonia in the Pathogenesis of Hepatic

7 Diagnosis of Overt Hepatic Encephalopathy ....................................... 97

9 The Electroencephalogram in Hepatic Encephalopathy .................... 113

Part III Treatment

11 Disaccharides in the Treatment of Hepatic Encephalopathy ............. 141

Part IV Special Topics

14 Sleep Disorders and Hepatic Encephalopathy .................................... 177

Index ................................................................................................................ 245

Jan Albrecht, PhD Department of Neurotoxicology, M. Mossakowski

Arumugam R. Jayakumar, PhD Department of Neuropathology,

Praveen Sharma, MD, DM Department of Hepatology, Institute of Liver

Kevin D. Mullen and Ravi K. Prakash

Keywords Hepatic encephalopathy Terminology Overt hepatic encephalopathy

Fig. 1.1 World Congress of gastroenterology classification of hepatic encephalopathy

(EASL) and International (IASL)] to provide periodic updates on terminology and

1. Ferenci P, Lockwood A, Mullen K, Tarter R, Weissenborn K, Blei AT. Hepatic encephalopathy

Keywords Ammonia Cerebral blood ow Brain edema Glutamine Amino

J. Albrecht, PhD (*)

damage or asymptomatic animals with experimentally induced chronic liver failure

Correlation Between Ammonia Levels in Blood and/or Its Rate

The Role of Ammonia in Alterations of Cerebral

Ammonia and Impairment of Cognitive and Motor Functions

HE is associated with impairment of learning and memory. The complexity of the

Effectiveness of Blood Ammonia-Reducing Therapies

As discussed elsewhere in this book, nonabsorbable disaccharides (lactulose) and

Cellular and Molecular Mechanisms Underlying

Ammonia that enters the brain is metabolized in astrocytes to glutamine in an

Role of Ammonia in Astrocytic Swelling and Brain Edema

critical for the translation of RONS accumulation to astrocytic swelling [34].

Ammonia and the Neurotransmitter Imbalance in HE

Progression of HE through its different stages from normality excitation to coma is

in vitro treatment of astrocytes or neurons strongly suggest that ammonia is largely

The Glutamatergic Transmission

Administration of ammonia to rats results in increased activation of NMDA type of

The GABAergic Transmission

Hyperammonemia is associated with an increased GABAergic tone. The underlying

The Serotoninergic Transmission

Serotonin, the tryptophan-derived inhibitory monoamine, is involved in regulation

Concluding Comments: Gaps in the Knowledge

1. Zieve L. Pathogenesis of hepatic encephalopathy. Metab Brain Dis. 1987;2:14765.

Keywords Neuroinammation Hepatic encephalopathy Liver failure Cirrhosis

Central nervous system (CNS) complications of liver failure include hepatic

R.F. Butterworth, PhD, DSc (*)

circulating ammonia remain the mainstay of clinical management and therapy.

Glial Pathology in Liver Failure

Neuroinflammation in Acute Liver Failure

A signicant correlation exists between the presence of the systemic inammatory

Synergy Between Ammonia and Proinflammatory Mechanisms

There is increasing evidence to support the notion that ammonia-related mechanisms

LiverBrain Proinflammatory Signaling

intriguing possibility, however, is not currently available. More recently, using an

Neuroinflammation and the CNS Complications

Evidence of a link between microglial activation, TLP induction, and neurosteroid

Diagnostic and Therapeutic Implications

Whatever the ultimate mechanism responsible, the consistent ndings of induction