Biochemistry Involving Carbon-Fluorine Bonds Filler (ACS 1976)

Biochemistry Involving
Carbon-Fluorine Bonds
In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
Biochemistry Involving
Carbon-Fluorine Bonds
R o b e r t F i l l e r , EDITOR
Illinois Institute of Technology
A symposium sponsored by
the Divisions of Fluorine
and Biological Chemistry
at the 170th Meeting of the
American Chemical Society,
Chicago, Ill., Aug. 26, 1975.
ACS SYMPOSIUM SERIES 28

AMERICAN CHEMICAL SOCIETY
WASHINGTON, D. C. 1976

Library of Congress Data
Biochemistry involving carbon-fluorine fonds.

(ACS symposium series; 28, ISSN 0097-6156)
Includes bibliographical references and index.
1. Organofluorine compounds—Physiological effect—
Congresses. I. Filler, Robert, 1923- . II. American
Chemical Society. Division of Fluorine. III. American
Chemical Society. Division of Biological Chemistry. IV.
Series: American Chemical Society. ACS symposium series;
28.
QP981.F55B56 612'.0157 76-13037
ISBN 0-8412-0335-0 ACSMC8 28 1-215 (1976)
Copyright © 1976
American Chemical Society
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PRINTED IN T H E UNITED STATES O F AMERICA

ACS Symposium Series
R o b e r t F. G o u l d , Series Editor

FOREWORD
The ACS SYMPOSIU
a medium for publishing symposia quickly in book form. The
format of the SERIES parallels that of the continuing ADVANCES
IN CHEMISTRY SERIES except that in order to save time the
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the book. Papers published in the ACS SYMPOSIUM SERIES
are original contributions not published elsewhere in whole or
major part and include reports of research as well as reviews
since symposia may embrace both types of presentation.

PREFACE
Τη recent years there has been increasing interest and research activity
in the biochemistry and medicinal applications of compounds con
taining the carbon-fluorine bond. The medicinal chemistry of fluorinated
organic compounds was last discussed at a symposium held at the national
ACS meeting in Washington, D.C. in September 1971. Simultaneously a
Ciba Foundation symposium on the biochemistry and biological activities
of carbon—fluorine compound
and incisive discussions a symposium, promi
nent contributors to the field participated, were published in 1972 by
Elsevier. A survey of fluorinated compounds of medicinal interest was
presented in the December 1974 issue of Chemical Technology.
The biochemical aspects of C - F chemistry have developed rapidly
since the pioneer studies in the early 1940s by Sir Rudolph Peters, who
elucidated the mechanism of the toxic action offluoroacetateby invoking
the concept of 'lethal synthesis." Special mention should also be made
of the elegant studies since the late 1950s by Charles Heidelberger and
his colleagues on the tumor-inhibitory effects of nucleotides of fluorinted
pyrimidines. Important advances in the biochemistry of organofluorine
compounds continue unabated and a symposium on this subject, co-
sponsored by the Divisions of Fluorine and Biological Chemistry, was
held at the Chicago ACS meeting in August 1975. This volume includes
all ten presentations and discussions at that symposium. The subjects
are of broad interest, ranging from fluorocarboxylic acids as enzymatic
and metabolic probes to the use of perfluorocarbons as "artificial blood."
We hope that these reports will further stimulate those already active
in the field and create a sense of excitement and enlightenment to the
curious. To the newcomer we say "Welcome—don't be afraid of fluorine
compounds—they're lots of fun."
Finally, I wish to express my appreciation to all of the contributors
for their patience and unstinting cooperation in making this book possible.
Illinois Institute of Technology ROBERT FILLER

Chicago, Ill.
January 1976
ix

1
Fluorocarboxylic Acids as Enzymatic and Metabolic
Probes
E R N E S T KUN
University of California, San Francisco, Department of Pharmacology, and the

Cardiovascular Research Institute, San Francisco, Calif. 94143
The development of site specific reagents necessarily

depends on the knowledge of specific biochemical reactions,
which should include the chemical structure of catalysts, sub-
strates, modifiers, and products of enzymatic processes. Very
little is known about catalytic sites of most enzymes, therefore
comparison of substrates with analogues may be one of the experi-
mental approaches to study this problem. It follows also that
extensive information related to analytical enzymology (i.e.,
catalytic properties of isolated enzymes, enzymatic composition
of cells) has to preceed the meaningful application of specific
probes in complex systems. This endeavor is conceptually a
synthetic one and intends to elucidate biological functions. It
is only in relatively rare cases that inhibitors of cellular
systems act in a manner predicted from in vitro enzymology. For
example, a linear multienzymatic process can be indeed regulated
by a rate limiting enzymatic component, susceptible to a specific
inhibitor. In this case the biological significance of the
linear multienzyme system can be studied with success. If acute
inhibition does not cause rapid irreversible changes in cellular
economy, sustained inhibition may trigger a variety of compen-
satory cellular processes, encompassing both intermediary and
macromolecular metabolism. Experimental pathology and toxicology
may benefit from these studies, since pathophysiology of environ-
mental toxic effects is likely to be traced to specific initia-
ting reactions of inhibitors with specific cellular sites. This
experimental subject is at present only in its beginning stages.
In contrast to linear systems-distributive, branching,and
cyclic multienzymatic processes (1) respond in a far more complex
manner to perturbations by a specific inhibitor. Alterations of
steady state concentrations of metabolites, and subcellular changes
of topographic distribution of intermediates of metabolic path-
ways will occur. Most of these consequences are unpredictable
from idealized metabolic charts (2). Experimental results
obtained with relatively uncomplicated fluorocarboxylic acids
indicate that major metabolic pathways are apparently not
1
2 BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS
regulated by properties of enzymatic components alone but more

importantly by a v a i l a b i l i t y of substrates. The role of primary
regulatory enzymes in c e l l u l a r systems, problems of molar ratios
of substrate to enzymes in biological systems have been reviewed>
(3) but the majority of biochemical texts have not incorporated

these advances at present time. The choice of F substitution of
H or OH groups in well known carboxylic acid substrates to
obtain specific inhibitors was based on straight forward chemi-
cal considerations (4). The present paper is restricted to
experimental work related to the mode of action of fluorocarbox-
y l i c acids, developed in our laboratory.
F1uoro-dicarboxylic acids
A series of chemical and enzymological experiments (5,6,7,

8,9,10,11,12,13,14,15,16,17,18,19) with mono and difluoro oxal-
acetates, fluoroglutamates
mal ate, fluorolactate and mal ate dehydrogenases, transaminases,
glutamate and lactate dehydrogenases suggested a reasonably uni-
form pattern of interaction between fluorodicarboxylic acid sub-
strates with respective enzymatic s i t e s . It is of interest that
introduction of one or two F atoms did not change the conforma-
tion of the parent molecule to the extent that i t could not be
recognized by enzymatic s i t e s . In generalênzyme-fluoro-sub-
strate Michaelis-Menten complexes are readily formed, but in
some instances the rates of conversion to products, i . e . , the
process of enzymatic catalysis i t s e l f is d r a s t i c a l l y reduced by
F-substitution. The molecular reasons for this inhibition by F-
substitution are as yet unexplored, and constitute a significant
problem of mechanistically oriented enzymology, worthy of more
detailed investigations. As would be expected F-dicarboxylic
acids behave as r e l a t i v e l y uncomplicated l i n e a r l y competitive
inhibitors with respect to the non-fluorinated substrate mole-
cule. Kinetic analyses of the effect of fluoro-oxalacetate in
bisubstrate systems of mal ate dehydrogenases (19) and lactate
dehydrogenase (14) support this conclusion. A summary of ex-
perimental results is shown in Table I. Whereas monofluoro
oxalacetate is a slowly reacting substrate of MDH, difluoro sub-
s t i t u t i o n converts this substrate homolog to an equally good sub-
strate of MDH to oxalacetate i t s e l f with respect to Vmax,except
difluoro-oxalacetate has a Km of 4.0 mM (about 4.10^ higher than
Km of oxalacetate). Further exploration of this significant
effect of difluoro substitution may shed light on the as yet un-
known molecular mechanism of catalysis of MDH. Since the p r i -
mary purpose of our investigations was to obtain biological
probes, the s t a b i l i t y of F-dicarboxylic acids were also i n v e s t i -
gated in biological systems. Despite the promising in v i t r o
properties of e-monofluoro oxalacetatic acid as an i n ï ï ï b i t o r of
MDH, i n s t a b i l i t y prevented i t s application in complex systems.
As would be expected.monofluoro oxalacetate is susceptible to

SUBSTRATE AND INHIBITORY PROPERTIES OF F-CARBOXYLIC ACIDS
Ma
No. Enzyme F-carboxylic acid 1^ Κ* η
MâXF-S)
1 Kidney MDH(mito) 3-F-oxalacetic 0.5 μΜ 0.5 μΜ 101
2 Kidney MDH(mito) 33'-F2-oxalacetic 4.0 mM 1.0
3 Liver GOT(mito) 33* -F2-oxalacetic 45. μΜ
4 Liver GOT(mito) 3 F-oxalacetic transaminative defluorination
5 Liver GDH 3 F-glutaric 330 μΜ
6 Liver GDH +
a-F-glutamate(NADP ) 1800 μΜ 710 μΜ 13.0
7 Liver GDH +
a-F-glutamate(NAD ) 640 μΜ 330 μΜ 15.0
8 Liver malic enzyme 33'-difluoromalate -- 300 μΜ
(decarboxylating)
9 Kidney MDH (-)-erythrofluoromalate 13 μΜ
10 Muscle LDH L(+) 3-fluorolactate 300 μΜ
LEGEND TO TABLE I: MDH = malate dehydrogenase; GOT = glutamate oxalacetate amino

transferase; GDH = glutamate dehydrogenase; LDH = lactate dehydrogenase; Ma =
molecular a c t i v i t y ; (S) = with physiological substrate; (F-S) = with fluoro-analogue

4 BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
COOH COOH COOH COOH

I I I I
FCH ÇH + Ε
2 CH2 FCH
I I
c=o HCNH ? C=0
I I I HCNH 2
COOH COOH COOH

COOH
COOH COOH COOH

^ I
I HC HCH H0
2 CH2
II I I + NH,
C=NH c=o
C-NH 2
I I
COOH
COOH COOH
Figure 1. Transaminative degradation of monofluoro-oxahcetic
acid

1. KUN Fluorocarboxylic Acids as Probes 5
bivalent metal ion catalyzed decarboxylation to fluoro pyruvate.

The kinetics of this reaction has been studied in detail (20).
Interaction of monofluoro oxalacetate with glutamate-oxalacetate
aminotransferase yields rapid elimination of F~ and Ν Η Λ from a +
system containing both aspartate and the fluoro-acid (b), repre

senting a rare case of elimination reactions characteristic of
pyridoxal-phosphate c a t a l y s i s , as well known from the work of
Snell and his school. This i s shown in Figure 1. From a com
bined use of fluoro-glutarate, a r e l a t i v e l y specific i n h i b i t o r
of GDH (11) and of difluoro-oxalacetate, an i n h i b i t o r of GOT (10)
the regulation of both transaminative and oxidative pathways of
mitochondrial glutamate metabolism was further elucidated (12).
Enzymatic reduction of monofluoro-oxalacetate by MDH on a prepar
ative scale yielded (-)erythrofluoromalic acid (21) which i s an
excellent i n h i b i t o r of malate dehydrogenases in v i t r o . In
isolated hepatocytes this i n h i b i t o r acts only on cytosolic MDH
isoenzyme because i t doe
mitochondrial membrane (cf. 21), therefore i t i s useful as a
c e l l u l a r probe of this MDH isoenzyme. Extensive t r i a l s with
mitochondria and isolated hepatocytes - as models for the study
of complex systems - indicated that only difluoro-oxalacetic and
(-)erythrofluoromalic acidsproved useful. Difluoro-oxalace-
tate, by i n h i b i t i n g GOT,proved to be stable and highly s p e c i f i c
in i t s action. Mono fluoro-malate, besides i n h i b i t i n g cytosolic
MDH is also an effective activator of the c i t r a t e carrier system
f
of the inner mitochondrial membrane, as shown in Figure 2 (cf.

21). When mitochondria are incubated with c i t r a t e and the exit
. Cit F-Mal
I i ι ι ι 1
2 4 6 8 10
Min.
Molecular Pharmacology
Figure 2. Spectrophotometric assay of citrate

entry into mitochondria

of i s o c i t r a t e is monitored by an externally added NADP + Mg + + 2+
isocitrate dehydrogenase i s o c i t r a t e detector system in the dual

wave length spectrophotometer, rapid i s o c i t r a t e efflux is
observed under a wide variety of experimental conditions. This
process is greatly stimulated by (-)erythrofluoromalate (see
Figure 2) as indicated by the large increase of the rate of
extramitochondrial NADPH formation. Fluoromalate appears to act
similar to malate (cf. 22) except i t s effect is not complicated
by penetration and subsequent metabolism in the matrix - as i t
is the case with malate. Consequently (-)erythrofluoromalate is
a more specific and useful activator of the c i t r a t e - i s o c i t r a t e
translocating membrane system than oxidizable dicarboxylic acids
(compare with 22). It is a puzzle why c i t r a t e - i s o c i t r a t e
exchange in energized mitochondria proceeds with the observed
high efficiency (about 80% of added c i t r a t e appears as i s o c i t r a t e
in the extramitochondrial compartment). We présumeras discussed
more extensively l a t e r
through the inner mitochondrial membrane is a more complex
process than a carrier mediated carboxylic acid exchange (22)
and may reflect an energy coupled membrane process of as yet
unknown physiological significance.
In v i t r o chemical and enzymatic studies thus far provided
fluoro malate and difluoro-oxalacetate as candidates useful
as probes in c e l l u l a r systems. The discovery of a valuable tech-
nique, that of isolation of hepatocytes by Berry and Friend (23),
gave significant impetus to further studies. Gluconeogenesis
from lactate was c h a r a c t e r i s t i c a l l y inhibited by difluoro-oxal-
acetate and to a lesser extent by (-)erythrofluoromalate,whereas
glucose formation from pyruvate was more sensitive to fluoro-
malate than to difluoro-oxalacetate (24) as shown in Table II
and Figure 3. Combination of both fluoro-dicarboxylic acids
Effects of fluoro-dicarboxylic acids on rates of gluco-
neogenesis in intact isolated liver cells
Cells (125—225 mg wet weight), obtained from the livers of
animals faeted 18 h, were incubated for 40 min at 37 °C. The
initial substrate concentration was 10 mM and inhibitor
concentration 2.5 m M . The values given are means ± S . E .
with the number of observations in parenthesis. Rates of
glucose formation have been calculated after subtraction
of the average rate observed in the absence of added substrate.
Tabl e II T l l i 8
°·
w a
± - ° ° n m o l x g ^ X m i n " (32 observations)
s 0 8 0 3 1
Substrate . ..... . . . _. . . Apparent

added Inhibitor added Glucose formed i n h l b l t l o n
μπιοί x g" x miu

1 -1
·/·
L-Lactate - 0.45 ± 0.02 (20) -
L-Lactate Fluoromalate 0.34 ± 0.06 (4) 24
L-Lactate Difluorooxaloacetate 0.24 ± 0.04 (4) 56
L-Lactate Fluoromalate,
Difluorooxaloacetate 0.11 ± 0 . 0 5 (3) 76
Pyruvate — 0.51 ± 0.02 (20) —
Pyruvate Fluoromalate 0.21 ± 0.04 (5) 59
Pyruvate Difluorooxaloacetate 0.38 ± 0.08 (4) 25
Pyruvate Fluoromalate, 0.22 ± 0 . 0 7 (4) 57
Difluorooxaloacetate
Fructose - 2.82 ± 0.11 (14) -
Fructose Fluoromalate 2.81 (2) —
Fructose Difluorooxaloacetate 2.Si -- (2) —
European Journal of Biochemistry

J I • . I
0 1 2 3 4 5
Inhibitor concentration (mM)
European Journal of Biochemistry
Figure 3.
on rate of
cells from pyruvate (·) or foctate (O). Each
point is the average of two experiments. Con-
ditions were the same as given in the legend
of Table 1. , with difluorooxaloacetate;
, with fluoromalate.
i n h i b i t gluconeogenesis from both precursors by 76-80% without

s i g n i f i c a n t l y altering c e l l u l a r processes other than those i n v o l -
ved in the transfer of reducing equivalents through the inner mito-
chondrial membrane (25, 26). In preliminary studies we found no
noticable toxic effects of either fluoro carboxylic acids when
injected intravenously (20 mg/100 g body weight) into mice.
Marked changes in glutamate and aspartate concentrations in the
l i v e r indicated that both fluoro carboxylic acids actually pene-
trated l i v e r parenchyma c e l l s . It would appear reasonable to
undertake more extensive in vivo studies with both fluoro carbox-
y l i c aci-ds as a model for a possible experimental chemotherapeu-
t i c approach to metabolic disorders l i k e diabetes, characterized
by abnormally large gluconeogenesis.
Studies with fluorocitrate
a.)Structure of the inhibitory isomer. In contrast to

fluorodicarboxylic acids of apparently low or undetectable acute
toxicity,the most important fluorotricarboxylic acid: monofluoro-
c i t r i c acid, because of i t s remarkable t o x i c i t y , plays a h i s t o r -
ical significance in the f i e l d of biochemical lesions, an area
established by S i r Rudolph Peters (cf. 27). In a series of
classical experiments Peters and his school established that the
site of action of fluorocitrate is localized at an i n i t i a l step of
c i t r a t e metabolism (28). The enzymatic s i t e of action of
fluorocitrate was proposed to be mitochondrial aconitase (27).

This mechanism appears to be incompatible with the results of

Guarrierra-Bobyleva and Buffa (29), who found that after in vivo
administration of toxic doses of fluorocitrate,only the metabo-
lism of c i t r a t e was inhibited in mitochondria whereas c i s - a c o n i -
tate - which is also a substrate of aconitase - is oxidized nor
mally. Because of the apparent uncertainties surrounding both
the chemistry of the toxic isomer of f l u o r o c i t r i c acid and i t s
subcellular mode of action this problem was reinvestigated.
As i l l u s t r a t e d in Figure4 (cf. 30) the four possible isomers
of monofluorocitric acid are formed either from fluoroacetyl-CoA
and oxalacetic acid or from monofluoro-oxalacetic acid and acetyl
CoA. It was shown (31,32) that the toxic isomer i s formed only
by enzymatic condensation of fluoro acetyl CoA with oxalacetate
whereas a l l other isomers had no s i g n i f i c a n t inhibitory effect on
aconitase. Because of the r e l a t i v e l y weak inhibitory effects of
fluorocitrate (Κ · * 50-80 μΜ) the d i s t i n c t i o n between "inhibi
η
tory" and "non-inhibitory

would wish for. Besides i n i t i a l velocity kinetic analyses, we,
as well as others,observed a time dependent, anomalous i n h i b i t i o n
of aconitase a c t i v i t y , but only in the i s o c i t r a t e d hydrogenase
coupled test system, containing either Mg or Mn?+ (see l a t e r for 2+
more details ). Elucidation of the chemical structure of the

toxic fluorocitrate isomer was successful despite the d i f f i c u l t i e s
encountered in the enzymology of aconitase. Synthesis and r e s o l
ution of isomers was accomplished in 1969 (33) and i t was shown
that the electrophoretically separated erythro isomers (Figure 5
and Figure 6) contained the toxic species, which was further re
solved and i d e n t i f i e d as (-)erythrofluorocitric a c i d , correctly
defined as: 1R: 2Rl-fluoro-2-hydroxy-l,2,3-propane tricarboxylic
acid. Crystallographic analysis of rubidium ammonium hydrogen
fluorocitrate dihydrate (34) lent further support to our deduction,
based o r i g i n a l l y on NMR and pk analyses of electrophoretically
resolved diastereoisomers (31,32).
Since the c i t r a t e condensing enzyme plays a key role in the
biosynthesis of (-)erythrofluorocitric acid from fluoroacetic acid
some kinetic characteristics of this enzyme were also determined
with F-acetvl CoA as substrate. Results are shown in Table I I I ,
TABLE 3
Summary of Kinetic Properties of Citrate Synthase from Pig Heart"
Con
stant Substrate or inhibitor Kinetic constant
K m Acetyl-CoA 25 μλί
K m Fluoroacetyl-CoA 23 μλί
K x F l uo roace ty 1-Co A 2.2 μλί
Acetyl-CoA 2.77 (/xmolcs D P N H / m g / m i n )
v Fluoroacetyl-CoA 0.0084.5 (pinoles D P N H / m g / m i n )
' mil
D P N , malate and malate dehydrogenase served as asourceof oxalacetate, and the
β
reaction was followed by appearance of D P N H .
'The Citric Acid Cycle"

KUN Fluorocarboxylic Acids as Probes
F of mulot
(•)-Mixture (•)- Mixture
_ f ~\
\ COOH COOH1 COOH COOH 1
Corbons derived from | | 1 1
oxoloocetote or< H-C-H H-C-F 2 H-C-H F-C-H 2
fluoro-oxaloacetote Λ Λ 1 1 HOOC-i-OH HOOC-fj-OH
|HOOC-C-OH HOOC-C-OH
Carbons derived Γ H-C-F 2 H-C-H F-C-H 2 H-Ç-H
from ocetote or < 1 1
fluoroocetote L C 0 0 H 1 C C O H COOH1 COOH
COO" coo" coo" COO"

Newman projections " O O C ^ L OH H O ^ L COO"
olong C - 2 . C - 3 axis T A J JXj
F ^ C >
(ionized forms) F-V^
Configurational P r e f i x *
fi/S System (2S.3R)- (2R.3S)- (2R.3R)- (2S.3Sh

Racemates (2RS.3SR)- (2RS.3RS)-
E «tensions of carbo-
hydrote-omino acid rules
( C O O H group Ignored) L
« 9"
L
0$Lg- U Og-
t
Racemates ^s^gLg" 0 L .LgC^-

$ t
erythro-L - $ erythro-0 - $ threo-Oj- threo-L,-
Racemates erythro-D L - $ $ fhreo-O L - e f
Provisional Assignments
Weaker acids (Component A) Stronger octds (Component 8 )
H I
From fluoroocetyl-CoA From fluoro-/ Not formed enzymaticolly
oxaloacetate
«- è
enanttomers
"The Citric Acid Cycle"
Figure 4. The isomers of monofluorocitric acid *
* T h e t e r m i n o l o g y o f L. D* a n d erythro D„ originates f r o m Η . B . V i c k c r y [ A M
Suggested N e w N o m e n c l a t u r e f o r the Isomers o f Isocitric A c i d , " / . Biol. Chan.

237, 1739 (1962)1. F o r c l a r i f i c a t i o n o f RS nomenclature, the reader is referred
t o : C a h n , R . S., Ingold, C , a n d P r e l o g , V . : Angew. Client. Intern. Ed. Engl.
3 S 5 ( 5 ) , 511 ( 1 9 6 6 ) , a n d C a h n , R . S., 7. Chem. Educ. 1 1 6 ( 4 1 ) , 508 ( 1 9 6 4 ) .

BIOCHEMISTRY INVOLVING C A R B O N - F L U O R I N E BONDS
0 0 0
C
ο
Y
ο ο ο οο
n
U χ
0 0 0 00 ο 0000 ο ο ν
CL Ο ·— — —· LJL
I 2 3 4 5 6
Journal of Biological Chemistry
Figure 5. Electrophoresis of fluorocitric acid from enzymatic and

chemical syntheses. CS: chemically synthesized fluorocitric acid; P:
picric acid marker; fluorocitric acids I and II (see text) (32).
6£ 5Ό 40 3.0 2.0 ΙΌ 0 ppm ( δ )
CNF CHF C H 2 CH 2 C H ,
I 1 l I 1 I I I t I 1
I I I I i 1 I » 1 1 1 I I t I 1 1 1 1 W i 1 1 1 I I 1 I I 1 t 1 I I I 1 I 1 I 1 1 I t 1 I I I 1 1 1
Journal of Biological Chemistry
Figure 6. Nuclear magnetic resonance spectrum of triethyl

fluorocitrate (32)

(cf. 30). Whereas Km for acetyl CoA and i t s F-analog is the same,
Vmax in the presence of F-acetyl CoA is about l/300th of V mx
measured in the presence of the physiological substrate. Fluoro-

acetyl CoA acts also as an inhibitor of the condensation of acetyl
CoA with oxalacetate.
b.)Mode of action of (-)erythrofluorocitrate. A molecular

mechanism of fluorocitrate t o x i c i t y has to account for the i r r e -
versible cessation of c e l l function in certain s p e c i f i c c e l l s of
the nervous system, known to be the probable anatomical organ s i t e
of this poison (28). Whereas the question of organ s p e c i f i c i t y in
the manifestation of pharmacological responses belongs to the do-
main of physiology, universality of metabolic enzymes in most or-
gans s t i l l provides acceptable in v i t r o models for the study of
biochemical toxicology. Consequently i f i n h i b i t i o n of aconitase
is the mode of action of f l u o r o c i t r a t e , irreversible inhibition
of this enzyme should b
tions of the i n h i b i t o r becaus
most probably present in vivo (compare with slow rates of fluoro-
c i t r a t e biosynthesis cf. 30). Even i f these requirements are met
there is some uncertainty why aconitase i n h i b i t i o n should prove
f a t a l , when inhibitors of other c i t r i c acid cycle enzymes causes
r e l a t i v e l y small t o x i c i t y : eg. malonate is not a powerful poison
(cf. 1), or as stated e a r l i e r , inhibition of malate dehydrogenase
in vivo e l i c i t s no detectable toxic effects. This question is of
particular significance, since the existence of cytoplasmic and
mitochondrial isoenzymes of aconitase (35) make i t uncertain why
the operation of the tricarboxylate cycle could not proceed un-
disturbed i f only one, i . e . , the mitochondrial isoenzyme^were
inhibited (compare with 29). Because of the h i s t o r i c a l l y develop-
ed notion (36) identifying fluorocitrate t o x i c i t y with aconitase
i n h i b i t i o n , the present evidence related to this question w i l l
be examined in d e t a i l . Notorious problems of enzyme i n s t a b i l i t y
at advanced stages of purification are well known to those con-
cerned with the enzymology of aconitase. In a l l but one instance
millimolar concentrations of ascorbate, cysteine, and F e are 2+
required to demonstrate f u l l aconitase a c t i v i t y in v i t r o . More

recent studies concerned with the mechanism of aconitase action
were performed almost entirely in this highly a r t i f i c i a l in v i t r o
environment (37,38). This enzyme had a molecular weight of
68,000. Gawron et a l . (39,40) while unable to reproduce the i s o -
lation procedure of Villafranca and Mildvan (37,38) obtained an
enzyme (mol. weight = 66,000) which had higher s p e c i f i c a c t i v i t y
than reported by e a r l i e r workers. It appears therefore doubtful
that previous in v i t r o studies (37,38) are entirely valid even
within the framework of cuvette enzymology. Non-competitive i n -
hibition of heart aconitase (37) by transaconitate with c i t r a t e
or i s o c i t r a t e as substrates (41) could not be reproduced (42)»
therefore the explanatory mechanism of Villafranca (41) postula-
ting two isomeric forms of aconitase appears to be without firm

experimental foundation. One of the - until recently - unrecog

nized complications of in v i t r o enzymology of aconitase l i e s in
the frequently used NADP^ dependent i s o c i t r a t e dehydrogenase
assay system i t s e l f . As demonstrated in 1974 (43)^various pre
parations of aconitase are susceptible to time dependent i n a c t i -
vation by bivalent cations e g . , Mg or Mn^ , which are necessary
2+ +
constituents of the coupled assay system. Unawareness of this

complication can result in double reciprocal plots of reaction
rates, which can simulate competitive or non-competitive or mixed
types of inhibitions by fluorocitrate with highly variable
apparent Κ·,· values calculated from primary plots. We have also
been misled by these types of experimental observations and pos
tulated e a r l i e r a time dependent inactivation of aconitase by
f l u o r o c i t r a t e , presumably by fluoro-aconitate or i t s defluorina-
ted product (cf. 30). The very low Ki values for fluorocitrate
reported by Brand et a l . (44), assaying aconitase a c t i v i t i e s of
crude mitochondrial extract
are apparently due to the inactivation of aconitase by Mg2 ,
which i s superimposed to the competitive inhibitory effect of
fluorocitrate as reported more recently (43). In view of the
a r t i f i c i a l i t y of the enzymology of aconitase, we pursued this
problem by isolating an electrophoretically homogeneous enzyme
which i s , for a f i n i t e period of time, f u l l y active without any
a r t i f i c i a l activator whatsoever (43). Purification is shown in
Table IV. The molecular weight of this cytoplasmic isoenzyme i s
Table IV
Purification of cytoplasmic aconitase from pig liwr
Purification Total Total acon Specific

slq> protein itase activ activitv
ity at 25° at 25°'
μηιοΐα/ηηη
min/mg
protein
Step 1 (liver
extract) 12,300 1,045 0.134
Step 2 5(i0 730 1.32
Step :> 180 500 2.70
Step I 8 85 10.0
111,000. considerably higher than purified preparations requir

ing Fe^+ cysteine for activation. A much less purified mito
chondrial isoenzyme has also been isolated under conditions
which maintained maximal a c t i v i t y without added activators.
From Figure 7 i t i s evident that fluorocitrate acted as a
l i n e a r l y competitive reversible i n h i b i t o r . From suitable compu
ter models substrate and inhibitory constants were calculated as
%
summarized in Table V. It i s apparent that (-)erythrofluoro-

Ε 40θ|
//·>
Jj I I I L_
— u
COO 2ΌΟΟ OOO 2000
l/S(M)
Figure 7. Competitive inhibition of cy

toplasmic (A) and mitochondrial (B)
aconitase of pig liver by (—)-erythro-
fluorocitrate. Rates of cis-aconitate for
mation
240 nm
at 25°.
plasmic aconitase, and in B, 32 μ% of
mitochondrial aconitase, were used per
test system (5-cm light path; 3-ml vol
ume) at varied concentrations of citrate
(abscissa). Curve 1, 500 μΜ. fluorocitrate;
2,100 μΜ fluorocitrate; 3, no fluorocitrate.
Table V
Siihslrutt: constants fitr citrate ami fl-ixoritralc and
inhihilor constants f<»r (— )-r.rf/thro-Jliiornrilratc
determined at μ/Ι 7.5 (10 w.\t Tris-ΙΚΊ) and 25°
Isoenzyme Km K,
Citrate </-Iso Citrate d Iso-

citrate citrate
Α· Β (Λ; A Β (A)
tnU μΜ μΜ μΜ
Cytoplasmic 220 700 17 18 27 20

Mitochon
drial 420 250 17 00 57 35
" A , determined by the cis-aconitatc assay; B ,

determined by the isocitrate dehydrogenase assay
(Μβ-'" concentration varied between 0.1 and 5.3
nm;.
c i t r a t e behaved as an uncomplicated competitive and reversible

i n h i b i t o r , therefore this kinetic property did not satisfy the
mechanistic requirements set forth by the well known irreversible
and highly potent toxicological effect of f l u o r o c i t r a t e . Although
i t i s apparent that the enzyme requiring no a r i f i c i a l activator i s
probably closer to the enzyme functioning in the c e l l u l a r environ-

mentjits properties are s t i l l different from aconitase located

in isolated mitochondria. It was shown (43) that Mg2 is a +
potent inactivator of the isolated enzyme. In sharp contrast,

lysosome free mitochondria, after accumulating over 200 mM Mg^ +
in the inner membrane and matrix (46) maintain a f u l l y active

intramitochondrial aconitase for several hours at 37°, clearly
indicating that in vitro s e n s i t i v i t y to Mg^ of isolated aconi
+
tase has no relationship to the intramitochondrial c a t a l y t i c

environment of this enzyme. This fact further stresses the need
for extreme caution in projecting cuvette enzymology to b i o l o g i
cal systems.
Defluorination of fluorocitrate by isolated aconitase (37),
has been recently reported by Villafranca and Platus (45) in the
presence of an about 100 fold molar excess of fluorocitrate over
aconitase. This system also contained 10 mM cysteine and 5 mM
Fe^ . It is noteworthy that enzyme preparations requiring no
+
activators f a i l e d to defluorinat
lar results were also obtaine
Gawron (48) did observe in v i t r o defluorination of f l u o r o c i t r a t e ,
again in the presence of 5 mM Fe* , 10 mM cysteine, 30 mM ascor-
+
bate, 0.126 μ moles of aconitase and 1 mM f l u o r o c i t r a t e . Only

3% of 1.0 mM fluorocitrate was defluorinated and this reversible
reaction stopped in 1 to 1.5 minutes. Since inhibition of the
enzyme which occurs during this process is reversible (cf. 45),
whereas, as shown l a t e r , mitochondrial c i t r a t e u t i l i z a t i o n is
inhibited in an irreversible manner by very low concentrations
of f l u o r o c i t r a t e , i t seems unlikely that this phenomenon bears
on the molecular mechanism of fluorocitrate poisoning.
In contrast to kinetic studies with isolated aconitase pre
parations, isolated mitochondria respond to fluorocitrate in a
manner which seems to bear more directly on toxicology. When
the c i t r a t e influx and i s o c i t r a t e efflux (21) of isolated intact
mitochondria are measured following preincubation of mitochondria
with less than micromolar concentrations of (-)erythrofluoro-
c i t r a t e , marked and irreversible inhibition of this process is
obtained. Isocitrate efflux is inhibited when either c i t r a t e or
cis-aconitate are added externally (49). Results are shown in
Figure 8 (a & b) and Table VI. When the mitochondrial membrane
structure was disrupted by the nonionic detergent Triton X-100
f u l l aconitase a c t i v i t y of mitochondria was obtained even in the
presence of low concentrations of fluorocitrate which in the same >
preparation completely inhibited the flux of tricarboxylic acids

into intact mitochondria. It is also apparent (Figure 8b) that
activation of i s o c i t r a t e efflux by fluoromalate is also inhibited
by preincubation with 2 μΜ f l u o r o c i t r a t e . The absence of i n h i b i
tion by 10" to Ι Ο " M fluorocitrate of aconitase of lysed mito
8 6
chondria agreed with in v i t r o enzyme kinetics (43). Fluoroci

trate in intact mitochondria therefore irreversibly inhibited a
membrane associated process essential for energy dependent t r i - '
carboxylic acid translocation. Similar experimental results

Mia
Biochemical and Biophysical Research Communications
Figure 8a. Rates of isocitrate efflux from cis-aconitate

as substrate (1
cis-aconitate
C = cis-aconitate added after 4 min preincubation
without F-citrate; D = mitochondria preincubated
with 5 μΜ F-citrate for 4 min then cis-aconitate addi
y
tion. At arrows, Triton X-100 is added (see Results).

Abbreviations used in Figures 8a and b: C is = cis-
aconitate; F-Cit = fluorocitrate; F-Mai = fluoro-
malate; Cit = citrate; Τ = Triton X-100.
2~ 4 6 8 K) 12
Min
Figure 8b. Rates of isocitrate efflux from citrate (1

mM) as substrate. Upper curve = at 4 min, 1 mM
F-malate added. Lower curve = at 2 min, 2 μΜ F-
citrate; at 4 min, 1 mM citrate; at 7 min, 1 mM F-
malate, being added in succession.
were obtained by Brand et a l . (44). Exchange diffusion of

c i t r a t e in preloaded mitochondria was not inhibited by added
fluorocitrate (44),hence i t was concluded that the anion c a r r i e r
i t s e l f was not the target of inhibition by f l u o r o c i t r a t e . It
must be noted however, that the essential prerequisite for

Table VI
INHIBITION OF ISOCITRATE EFFLUX FROM ADDED CITRATE
AND CIS-ACONITATE BY FLUOROCITRATE
% Inhibition of Isocitrate Efflux
M F-citrate
citrate* cis-aconitate*
1.0 X 1 0 " 8
7 7
1.25 Χ 1 0 " 8
11 13
2.5 Χ 10" 8
31 30
5.0 Χ 10~ 8
61 51
1.0 X 1 0 " 7
5.0 Χ 10" 7
100 78
•Added as s u b s t r a t e s (1 mM) a f t e r 1 minute p r e i n c u b a t i o n w i t h F-citrate
(see Column 1 ) .
inhibition of mitochondrial c i t r a t e transfer i s preincubation of

mitochondria for 2 to 8 minutes with very low concentrations of
fluorocitrate in the absence of c i t r a t e which,if added simul
taneously with fluorocitrate at 5 mM concentration prevents >
i n h i b i t i o n . In preloaded mitochondria c i t r a t e concentration i s

3-4 mM (cf. 44). Itwould be therefore expected that 1(T to 10" 6 8
M fluorocitrate would not be effective under these conditions.

We have reinvestigated this problem in collaboration with Dr.
Eva Kirsten ( v i s i t i n g s c i e n t i s t from the University of Berlin)by
a different experimental technique. Taking advantage of the
extraordinary s t a b i l i t y of lysosome free mitochondria (50) we
have followed the time course of ATP synthetase a c t i v i t y of these
organells for 40-60 minutes. Since ATP synthetase a c t i v i t y under
these conditions i s much faster than substrate permeation into
the matrix, the time course of 32p incorporation into ATP,
induced by external substrate is a sensitive measure of the rate
t
of substrate translocation.
This i s i l l u s t r a t e d in Figure 9. After preincubation of
mitochondria for 10 minutes in the presence of unlabel led Pi +
ADP to deplete endogenous substrates, P + substrates (either 3 2
10 mM c i t r a t e - T r i s , 0.5 mM malate-Tris separately or simul

taneously) were added and the rate of ATP synthesis assayed (51)
by the radiochemical method. The rate of ATP synthesis reached
a plateau in 15 minutes when either substrates were added sep
arately, but proceeded at a high rate for 60 minutes when

MINUTES
Figure 9. Time course of ATP synthesis by lysosome-

free mitochondria (50). After preincubation for 10
min in the presence of 5 mM ADP + 5 m M P<, to
deplete endogenous substrates, 32 Ρ (0.5 to 1.0 X JO 5
dpm) and substrates (see text) were added Τ = 3 0 ° .
c i t r a t e + malate were added simultaneously. This time course i s

characteristic of the kinetics of malate or fluoromalate a c t i v a
ted c i t r a t e translocation (Figure 2). When^during preincubation
of mitochondrials nanomoles of (-)erythrofluorocitrate per mg
mitochondrial protein and 40 mM Mg2+ were present and the reac
tion was started by either glutamate (2.5 mM) plus malate (2.5
mM) or c i t r a t e (10 mM) plus malate (0.5 mM)^the rate of ATP
synthesis was completely inhibited when c i t r a t e was the permeant
substrate but was unaffected when glutamate + malate were the
substrates (FigurelO). It i s interesting that Mg2 accumulation +
(^230 mM into the matrix, cf. 50) caused a 3-fold augmentation

of ATP synthetase a c t i v i t y (compare Figures 9 with 10^ thus Mg 2+
has no inhibitory effect on aconitase in s i t u . The c r i t i c a l

role of the time of preincubation with 50 picomoles fluorocitrate

C
'to
Ο
ί
α
Ό)
Ε
ο
Ε
<
Ο
MINUTES
Figure 10. Conditions were the same as described

for Figure 9, except 40 mM Mg * was also present.
2
C = citrate + malate control; A = glutamate +

malate control; Ό = glutamate + malate + preincu
bation with 50 ρ moles of F-citrate per mg mitochon
drial protein; Β = citrate + malate + preincubation
with 50 ρ moles of F-citrate per mg mitochondrial
protein; Τ = 3 0 ° .
per mg mitochondrial protein is i l l u s t r a t e d by the following

results. Citrate dependent ATP synthetase a c t i v i t y was inhibited
by 52% after 3 minutes preincubation, 63% after 6 minutes pre-
incubationând 90-100% after 12 minutes preincubation. This
time course has direct relationship to the known kinetics of
fluorocitrate poisoning, which has a slow onset but is i r r e v e r s i
ble. Once c i t r a t e metabolism of mitochondria is inhibited by
traces of fluorocitrate no subsequent manipulation can reactivate
this specific process. Penetration of other mitochondrial sub
strates and their subsequent metabolism i s unaffected under con
ditions when c i t r a t e permeation is completely inhibited. No
precise molecular interpretation of these results is as yet

possible, however i t seems clear that predictions based on cu-

vette enzymology of isolated aconitase does not f u l l y account
for these phenomena. Technology of enzyme isolation in a l l but
one case modifies aconitase and makes i t s c a t a l y t i c function de-
pendent on a r t i f i c i a l activators. The preparation which is f u l l y
active without F e + cysteine is inactivated in v i t r o by Mg
2+ 2+
(or Mn ) yet in the intact mitochondria this inactivation by

2+
Mg does not take place, therefore the micro environment of the

2+
mitochondrial enzyme has not yet been reproduced in v i t r o .

Inhibition of the energy dependent c i t r a t e transport by traces
of fluorocitrate in isolated mitochondria exhibits many charac-
t e r i s t i c s which appear to predict the in vivo t o x i c i t y of fluoro-
c i t r a t e . Further analysis of this system and isolation of inner
membrane components of the transport apparatus are necessary for
real progress in this f i e l d . It is significant that Peters (cf.
27) i n t u i t i v e l y predicted that the biochemical lesion in fluoro-
c i t r a t e t o x i c i t y is probabl
chemistry. Our recent
c i a l l y because no biochemically meaningful argument can be pro-
posed for the c r i t i c a l role of c i t r a t e transfer in mitochondrial
and especially in c e l l u l a r metabolism unless an as yet unknown
function of this process in the maintenance of energy transduc-
tion of the inner mitochondrial membrane is postulated.
Acknowledgement
Most of the experimental work was supported by HD-01239,

except the experiments concerned with ATP-synthetase (Figures
9 and 10) was sponsored by GM-20552. E. Kun is a research
Career Awardee of the USPH.
Literature Cited
1. Webb, J.L. "Enzyme and Metabolic Inhibitors," Acad. Press,

New York 1963.
2. Dagley, S. and Nicholson, D.E. "An Introduction to Metabolic
Pathways," John Wiley & Sons, Inc., New York 1970.
3. Sols, A. and Gancedo, C. "Primary Regulatory Enzymes and
Related Proteins," in Biochemical Regulatory Mechanisms in
Eukaryotic C e l l s , (Eds. Kun, E. and G r i s o l i a , S.) Wiley
Interscience, New York 1972.
4. Kun, E. and Dummel, R.J. "Methods in Enzymology, Vol. XIII,"
(Eds. Colowick, Kaplan and Lowenstein) Chapt 79, p.623,
Acad. Press, New York 1969.
5. Kun, E., Grassetti, D.R., Fanshier, D.W. and Featherstone,
R.M. Biochem. Pharmacol. (1958) 158 (207).
6. Kun, E., Fanshier, D.W. and Grassetti, D.R. J . B i o l . Chem.
(1960) 235 (416).
7. Kun, E. and Williams-Ashman, H.G. Nature (London) (1962)

194 (376).
8. Kun, E. and Williams-Ashman, H.G. Biochim. Biophys. Acta
(1962) 59 (719).
9. Kumler, W.D., Kun, E. and Shoolery, J.N. J. Org. Chem. (1962)
27 (1165).
10. Kun, E., Gottwald, L.K., Fanshier, D.W. and Ayling, J.E.
J. Biol. Chem. (1963) 238 (1456).
11. Gottwald, L.K., Ayling, J.E. and Kun, E. J. Biol. Chem.(1964)
239 (435).
12. Kun, E., Ayling, J.E. and Baltimore, B. J. Biol. Chem. (1964)
239 (2896).
13. Kun, E. and Achmatowicz, B. J. Biol. Chem. (1965) 240 (2619).
14. Ayling, J.E. and Kun, E. Mol. Pharmacol. (1965) 1 (255).
15. Gottwald, L.K. and Kun, E. J. Org. Chem. (1965) 30 (877).
16. Cymerman-Craig, J., Dummel, R.J., Kun, E. and Roy, S.K.
Biochem. (1965) 4 (2547).
17. Dupourque, D. and Kun
18. Dupourque, D. and Kun, E. "Methods in Enzymology, Vol. XIII,"
(Eds. Colowick, Kaplan and Lowenstein) Chapt 20, p.116,
Acad. Press, New York 1969.
19. Dupourque, D. and Kun, E. Eur. J. Biochem. (1969) 7 (247).
20. Dummel, R.J., Berry, M.N. and Kun, E. Mol. Pharmacol. (1971)
7 (367).
21. Skilleter, D.N., Dummel, R.J. and Kun, E. Mol. Pharmacol.
(1972) 8 (139).
22. Chappell, J.B. British Med. Bull. (1968) 24 (150).
23. Berry, M.N. and Friend, D.S. J. Cell. Biol. (1969) 43 (506).
24. Berry, M.N. and Kun, E. Eur. J. Biochem. (1972) 27 (395).
25. Berry, M.N., Kun, E. and Werner, H.V. Eur. J. Biochem. (1973)
33 (407).
26. Berry, M.N., Werner, H.V. and Kun, E. Biochem. J. (1974)
140 (355).
27. Peters, R.A. British Med. Bull. (1969) 25 (223).
28. Pattison, F.L.M. and Peters, R.A. "Handbook of Experimental
Pharmacology, Vol. XX," Chapt 8, p.387, Springer Press,
New York 1966.
29. Guarriera-Bobyleva, V. and Buffa, P. Biochem. J. (1969)
113 (853).
30. Kun, E. "The Citric Acid Cycle," (Ed. Lowenstein) Chapt 6,
p.297, Dekker Publications, New York 1969.
31. Fanshier, D.W., Gottwald, L.K. and Kun, E. J. Biol. Chem.
(1962) 237 (3588).
32. Fanshier, D.W., Gottwald, L.K. and Kun, E. J. Biol. Chem.
(1964) 239 (425).
33. Dummel, R.J. and Kun, E. J. Biol. Chem. (1969) 244 (2966).
34. Carrell, H.L. and Glusker, J.P. Acta Crystallogr. (1973)
29 (4364).
35. Eanes, R.Z. and Kun, E. Biochim. Biophys. Acta (1971)
227 (204).
36. Morrison, J.F. and Peters, R.A. Biochem. J. (1954) 58 (473).

37. Villafranca, J.J. and Mildvan, A.S. J. Biol. Chem. (1971)

246 (772).
38. Villafranca, J.J. and Mildvan, A.S. J. Biol. Chem. (1972)
247 (3454).
39. Gawron, O. Sr., Kennedy, M.C. and Bauer, R.A. Biochem. J.
(1974) 143 (717).
40. Gawron, O. Sr., Waheed, Α . , Glaid III, A.J. and Jaklitsch, A.
Biochem. J. (1974) 139 (709).
41. Villafranca, J.J. J. Biol. Chem. (1974) 249 (6149).
42. Gawron, O. Sr., manuscript submitted for publication.
43. Eanes, R.Z. and Kun, E. Mol. Pharmacol. (1974) 10 (130).
44. Brand, M.D., Evans, S.M., Mendes-Mourao, J. and Chappell, J.B.
Biochem. J. (1973) 134 (217).
45. Villafranca, J.J. and Platus, E. Biochem. Biophys. Res. Comm.
(1973) 55 (1197).
46. Kun, E. (unpublished experiments).
47. Peters, R.A. and Shorthouse
48. Gawron, O. Sr. (personal communication).
49. Eanes, R.Z., Skilleter, D.N. and Kun, E. Biochem. Biophys.
Res. Comm. (1972) 46 (1619).
50. Kun, E. manuscript submitted to Biochemistry.
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Discussion
Professor Kun
Q. What sort of methods or approaches are you using for
isolating citrate carrier systems?
A. For the membrane carrier systems we are in the process of
developing techniques of hydrophobic chromatography.
Q. On the affinity binding constants? You permit this substrate
to remain attached to the carrier you isolated?
A. Yes.
Q. Chappell suggested that the tricarboxylate carrier is not
inhibited by F-citrate. Are you suggesting that F-citrate
inhibits energy coupling of the carrier?
A. The exchange diffusion carrier of Chappell was tested for
inhibition by fluorocitrate (ref. 44) in citrate preloaded
mitochondria under conditions (i.e., at 4 mM citrate concen
trations), when also in our hands fluorocitrate inhibition is
prevented by the simultaneous presence of citrate. As dis-

cussed, preincubation with very low concentrations of fluoro-

c i t r a t e , in the absence of c i t r a t e are the required condi-
tions to show inhibition of c i t r a t e translocation. Since
c i t r a t e translocation in our experiments occurs in energized
mitochondria ( i . e . , ATP dependent) i t is possible that F-
c i t r a t e inhibits energy coupling. No precise answer to the
question is as yet available.
Q. Would you comment on the electrophoretic separation of

aconitase.
A. Yes, we have done that. It is electrophoretically homo-

geneous .
Q. Did you use a buffer?
A. Yes, we have describe

Pharmacology and I woul
(see ref. 43).

2
Biochemistry and Pharmacology of Ring-Fluorinated
Imidazoles
KENNETH L. KIRK and LOUIS A. COHEN

Laboratory of Chemistry, National Institute of Arthritis, Metabolism, and
Digestive Diseases, National Institutes of Health, Bethesda, Md. 20014
Medicinal chemists
the value of fluorine in the design of analogues of metabolically
significant molecules (1, 2). The high electronegativity of fluo
rine can effect a marked alteration in electron-density d i s t r i b u
tion, pK, conformation, etc.; simultaneously, this atom, by virtue
of i t s small van der Waals radius (1.35 A), offers minimal steric
interference to binding of the analogue at a specific macromolec
3
ular s i t e . The effective size of fluorine attached to an sp
carbon may be considered to fall between those of hydrogen and the
2
hydroxyl group, while fluorine on an sp carbon is probably some
what smaller — the result of lone pair overlap with the adjacent
pi system (3, 4). Effective size undoubtedly varies, also, with
solvation effects and specific lone pair interactions.
C = C ― <--> C ― C = +
Ring-fluorinated analogues of a variety of aromatic and

heteroaromatic biomolecules have been synthesized and evaluated as
agonists and antagonists of their natural relatives. We realized,
some years ago, that the imidazole ring, one of the most ubiquit
ous and important of natural heteroaromatic systems, could claim
no documented fluoro derivatives, and initiated an effort to fill
this gap. After we had exhausted the classical synthetic routes
(5, 6), abandonment of the effort seemed inevitable. In other
studies, we had found that imidazolediazonium ions, which are un
usually stable to heat, are transformed photochemically to highly
reactive species, possibly carbonium ions or carbenes (with expul
sion of molecular nitrogen) (7). It seemed reasonable that such
reactive species might capture fluoride and other poorly nucleo
p h i l i c anions. Indeed, the f i r s t ring-fluorinated imidazole was
obtained in 1971 by photolysis of ethyl 4-diazoniumimidazole-5-
carboxylate in 50% aqueous fluoroboric acid (8, 9). Subsequently,
a wide variety of both 2- and 4-fluoroimidazoles were prepared for
chemical and biological studies, of which many are still in pro
gress.
23

S y n t h e t i c Methods
The most general procedure f o r the s y n t h e s i s of 4 - f l u o r o -

imidazoles i s i l l u s t r a t e d i n F i g u r e 1 (10, 11), using aminoimid-
azole precursors. Unless they have the c a p a b i l i t y f o r resonance
overlap with an e l e c t r o n s i n k ( n i t r o , cyano, carboxylate, etc.) at
C-5, 4-aminoimidazoles show an i n s t a b i l i t y resembling that of
v i n y l a m i n e s , and cannot normally be i s o l a t e d without p a r t i a l de-
composition. C a t a l y t i c hydrogénation of 4 - n i t r o i m i d a z o l e s proved
u n s a t i s f a c t o r y as a source of n o n s t a b i l i z e d 4-aminoimidazoles;
however, z i n c dust r e d u c t i o n of the n i t r o group i n 50% f l u o r o b o r i c
a c i d , which proceeded r a p i d l y and q u a n t i t a t i v e l y at low tempera-
t u r e , became the method of choice. Immediately upon completion of
r e d u c t i o n , the aminoimidazole i s d i a z o t i z e d w i t h sodium n i t r i t e i n
s i t u , and the r e s u l t i n g diazonium i o n i s subjected to p h o t o l y s i s ,
again i n s i t u . A f t e r n e u t r a l i z a t i o n of the f l u o r o b o r i c a c i d med-
ium, the product i s u s u a l l
o v e r a l l y i e l d s (based o
Since the other products of p h o t o l y s i s are g e n e r a l l y h i g h l y p o l a r ,
hydroxylated imidazoles, they are not removed by the e x t r a c t i o n
s o l v e n t and do not i n t e r f e r e with p u r i f i c a t i o n .
In c o n t r a s t to the 4-amino s e r i e s , 2-aminoimidazoles show
the s t a b i l i t y to be expected of a l k y l a t e d guanidines. These com-
pounds are generated by c a t a l y t i c hydrogénation of 2-arylazoimid-
azoles which, i n t u r n , are obtained by coupling of the imidazole
w i t h aryldiazonium i o n (Figure 2 ) . Although such coupling occurs
predominantly at C-2, smaller q u a n t i t i e s of the 4- and 2,4-bis-
a r y l a z o d e r i v a t i v e s are formed (12). I t i s e s s e n t i a l that the
d e s i r e d isomer be freed of these contaminants ( u s u a l l y by column
chromatography) p r i o r to hydrogénation, s i n c e p u r i f i c a t i o n of the
r e s u l t a n t 2-aminoimidazole has proved extremely l a b o r i o u s . The
pure 2-aminoimidazole i s then d i a z o t i z e d and the diazonium i o n i s
photolyzed i n s i t u (13).
This photochemical method has proved i t s e l f of v a l u e , w i t h
respect to r a p i d i t y , convenience, and y i e l d , i n the synthesis of
r i n g - f l u o r i n a t e d d e r i v a t i v e s of s e n s i t i v e or complex aromatic
systems (£.£., a l k a l o i d s (14) and catecholamines (15)), as w e l l as
other heteroaromatic systems, such as t h i a z o l e (7) and p y r r o l e
(16).
Special Properties
A s i n g l e f l u o r o s u b s t i t u e n t , at C-2 or C-4, reduces the bas-

i c i t y of the imidazole r i n g by 5-6 pK u n i t s and increases i t s
a c i d i t y (NH—*N~) by 4-5 u n i t s (17) . The magnitudes of these
e f f e c t s are s i g n i f i c a n t l y greater than those of the other halogens
and i n d i c a t e that, i n c o n t r a s t to the fluorobenzenes, the i n d u c t -
i v e e f f e c t of f l u o r i n e on the imidazole r i n g overwhelms any
e l e c t r o n - r e l e a s i n g e f f e c t due to resonance. I n c o n s i s t e n c i e s i n
the l ^ F nmr s i g n a l s f o r the f l u o r o i m i d a z o l e s i n d i c a t e f u r t h e r that

2. KIRK AND COHEN Ring-Fluorinated Imidazoles 25
£H CH NHAc
2 2
0 N
2 N ^CH^NHAc
/=( HN0
3
Zn
50%HBF 4
-10
e
CH CH NHAc
7\
CH2CH2NHAC 2 2
=
N^NH 2. h*. -10" N^NH
Figure 1
ArN^ .R ArN ^
2 ^R
ArN +2
pH 8-9 1
N^NH NyNH
NaAr NeAr
Pt.Hî
AcOH Mutt be removed
before hydrogenolysn
+ ArNH 2
N
y N H
NM 2
Figure 2

these compounds cannot be t r e a t e d i n p a r a l l e l w i t h the f l u o r o -

benzenes (17).
The f l u o r i n e atom a t C - 4 , unless a c t i v a t e d by an e l e c t r o n
s i n k a t C-5, has shown no evidence of r e a c t i v i t y toward nucleo-
p h i l e s ; i n c o n t r a s t , 2 - f l u o r o i m i d a z o l e s are moderately r e a c t i v e
toward displacement, p a r t i c u l a r l y i n the ring-protonated form
(13), while the f l u o r o i m i d a z o l e anion i s s i g n i f i c a n t l y more s t a b l e
(Figure 3 ) . Thus, c t - N - t r i f l u o r o a c e t y l - 2 - f l u o r o h i s t i d i n e cannot be
deacylated by a c i d h y d r o l y s i s without l o s s of the f l u o r i n e atom,
but the compound i s r e a d i l y deacylated i n m i l d base. Another
consequence of the r e a c t i v i t y of 2 - f l u o r o i m i d a z o l e s i s the f a c i l e
condensation to c y c l i c t r i m e r s (Figure 4), which appear t o be
l a r g e r i n g , heteroannular aromatic systems.
Since imidazole i t s e l f has been found to undergo f a c i l e
hydrogen-isotope exchange a t C-2 but not a t C-4 (18), we expected
an even more f a c i l e exchang w i t h 4 - f l u o r o i m i d a z o l e s Surprising
l y , the l a t t e r compound
a wide v a r i e t y of c o n d i t i o n s
change r a p i d l y a t C-4 above pH 10, but much more slowly a t n e u t r a l
pH. Thus, a route became a v a i l a b l e f o r the s p e c i f i c t r i t i u m
l a b e l l i n g of 2 - f l u o r o h i s t i d i n e and 2-fluorohistamine, compounds
which subsequently proved t o have extensive biochemical u t i l i t y .
The a b i l i t y o f F-2 t o a c t i v a t e H-4 i s q u i t e unique; t o date, no
other s u b s t i t u e n t a t C-2, whether more o r l e s s e l e c t r o n e g a t i v e
than f l u o r i n e , ^ h a s demonstrated a c a p a b i l i t y of such magnitude.
A route t o 2-[ H ] - 4 - f l u o r o h i s t i d i n e was developed r e c e n t l y , based
on our observation that these exchange processes a r e subject to
strong b u f f e r c a t a l y s i s .
B i o l o g i c a l Properties
Several d e t a i l e d r e p o r t s o f s t u d i e s w i t h f l u o r o i m i d a z o l e s
have already been published or a r e i n press; the m a j o r i t y , how-
ever, a r e s t i l l i n t h e i r e x p l o r a t o r y stages. The f o l l o w i n g d i s -
c u s s i o n does not represent a comprehensive l i s t i n g of these stud-
i e s ; r a t h e r , i t presents a sampling, intended t o demonstrate the
v a r i e t y and scope o f the b i o l o g i c a l a p p l i c a t i o n s of f l u o r o i m i d -
a z o l e s and, h o p e f u l l y , t o suggest d i r e c t i o n s f o r f u r t h e r a p p l i c a -
tion.
2 - F l u o r o h i s t i d i n e . When t r i t i a t e d 2 - f l u o r o - L - h i s t i d i n e i s
administered t o mice subcutaneously, the amino a c i d i s r a p i d l y
d i s t r i b u t e d throughout the animal. Ten minutes a f t e r a d m i n i s t r a -
t i o n , the p r i n c i p a l organs ( i n c l u d i n g b r a i n ) show t r i t i u m l e v e l s
2-7 times that o f the blood. A f t e r 72 h r , two-thirds of the
o r i g i n a l t r i t i u m count i s s t i l l w i t h i n the animal, and h a l f o f
t h i s amount i s found i n i n s o l u b l e p r o t e i n f r a c t i o n s (19). Since
we had shown that replacement of the 2 - f l u o r o group by any other
s u b s t i t u e n t prevents exchange o f isotope a t C - 4 , and s i n c e a l l the
t r i t i u m could be back exchanged from the p r e c i p i t a t e d p r o t e i n

Ν Π η + R S H
• H^piH + R S
"
F F

f r a c t i o n s at pH 11, the analogue must have entered i n t o de novo

p r o t e i n synthesis i n place of h i s t i d i n e ; s i n c e the 2-fluorοimid
azole moiety i s i n t a c t , covalent attachment of the f l u o r o h i s t i d i n e
must have occurred at the amino a c i d s i d e chain. Such i n c o r p o r a
t i o n i n t o the mouse i s blocked by cycloheximide and, i n i s o l a t e d
l i v e r ribosomes, by actinomycin, both drugs being i n h i b i t o r s of
protein synthesis. Studies with r a t p i n e a l gland have s u p p l i e d
f u r t h e r evidence f o r the i n c o r p o r a t i o n of 2 - f l u o r o h i s t i d i n e i n t o
newly synthesized p r o t e i n (20). H i s t i d i n e and i t s 2 - f l u o r o analo
gue are u t i l i z e d c o m p e t i t i v e l y , s i n c e a d m i n i s t r a t i o n of an excess
of h i s t i d i n e reduces f l u o r o h i s t i d i n e i n c o r p o r a t i o n . Presumably, the
h i s t i d i n e analogue i s incorporated i n t o a l l newly synthesized pro
t e i n without d i s c r i m i n a t i o n ; y e t , i t i s conceivable that c e r t a i n
f l u o r i n e - c o n t a i n i n g enzymes may r e t a i n a c t i v i t y . S i n g l e doses of
2 - f l u o r o h i s t i d i n e (up to 250 mg per k i l o ) give no evidence of t o x i
c i t y , organ degeneration or r e t a r d a t i o n of growth of the mouse
over a 30-day p e r i o d . ^
Concentrations of 2 - f l u o r o h i s t i d i n
b a c t e r i o s t a t i c ; i n h i b i t i o n of growth of E. c o l i (wild) i s essen
t i a l l y complete i n 3 hr at 37°, but the i n h i b i t i o n i s reversed by
a d d i t i o n of h i s t i d i n e (20 yg/ml) (21). During the course of the i n
h i b i t i o n , the mass of the c u l t u r e increases about t h r e e f o l d , but
the number of v i a b l e c e l l s increases about n i n e f o l d . These data
suggest that the b a c t e r i a , which contain two to three copies of
t h e i r chromosome during the normal growth phase, are unable to
c a r r y out f u r t h e r chromosomal r e p l i c a t i o n i n the presence of the
drug but are able to undergo c e l l u l a r d i v i s i o n . As shown i n Table
I, 2 - f l u o r o h i s t i d i n e i s the most e f f e c t i v e b a c t e r i o s t a t i c agent of
the s e v e r a l f l u o r o h e t e r o c y c l e s t e s t e d to date.
The preceding s t u d i e s suggest that 2 - f l u o r o h i s t i d i n e may be
u s e f u l wherever p r o t e i n synthesis i n a l i e n organisms or c e l l s
occurs more r a p i d l y than i n the host s p e c i e s . Thus, the amino
a c i d shows a n t i v i r a l behavior i n various i n f e c t e d c e l l c u l t u r e s
(Table I I ) , at concentrations s i g n i f i c a n t l y below those needed to
produce any v i s i b l e e f f e c t on the host c e l l s (22). To our know
ledge, t h i s i s the f i r s t amino a c i d analogue to show a n t i v i r a l
p r o p e r t i e s . Although i t s mechanism of a c t i o n has yet to be e l u c i
dated, b i o s y n t h e s i s of ' f a l s e ' phosphorylases or v i r u s - c o a t p r o t
e i n s are l o g i c a l p o s s i b i l i t i e s .
Small peptides c o n t a i n i n g f l u o r i n e are more r e a d i l y obtained
by t o t a l synthesis than by b i o s y n t h e s i s . Thus, t h y r o t r o p i n - r e l e a s
i n g f a c t o r (TRF) and l u t e i n i z i n g hormone-releasing f a c t o r (LHRF)
have been synthesized with 2- and 4 - f l u o r o h i s t i d i n e as r e p l a c e
ments f o r the s i n g l e h i s t i d i n e residues (23). While the 4 - f l u o r o
analogues are i n a c t i v e , those c o n t a i n i n g 2 - f l u o r o h i s t i d i n e show
20-30% r e l e a s i n g a c t i v i t y . In view of the marked l o s s i n b a s i c i t y

Table I . E f f e c t of F l u o r o Compounds on E. c o l l (wild)
O.D. a f t e r 18 h r a t 37°
Compound added (Klett units)
None 375
2-Fluorohistidine 8
4-Fluorohistidine 375
4-Fluoroimidazole 370
2-Fluorourocanic a c i d 370
2-Fluoro-4-hydroxyethylthiazole 63
Same + thiamine (2 yg/ml) 270
In minimal glucos
s u l t s were obtaine
itor.
Table I I . A n t i v i r a l A c t i v i t i e s of Fluorohistidines
a
Minimal i n h i b i t o r y cone. (pg/ml)
Cell 2-Fluoro- 4-Fluoro- ,

C
Virus culture histidine histidine Ribavirin
VSV d
PRK d
30 e
100 _
HSV-1 PRK 30 >100
-
Vaccinia
VSV
PRK
HeLa
30
30
>100
>100
-10
Coxs. B4 HeLa 30 >100 10
Polio I HeLa 30 >100 30
Measles VERO 10 >100 7
Coxs. B4 VERO 30 >100 30
Required to reduce v i r u s - i n d u c e d c y t o p a t h o g e n i c i t y by 50%.
E s s e n t i a l l y s i m i l a r r e s u l t s were obtained f o r p - f l u o r o -
phenylalanine. An e s t a b l i s h e d a n t i v i r a l agent, Ι-β-D-
ribofuranosyl-1,2,4-triazole-3-carboxamide. Abbreviations
e
are i d e n t i f i e d i n r e f . 32. At concentrations of 100 ug/ml,
morphological a l t e r a t i o n of the host c e l l s was apparent only
a f t e r 3 days.

p-Glu-His-Pro-NH 2 (TRF)
p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 (LHRF)
of the imidazole r i n g f o l l o w i n g i n t r o d u c t i o n of f l u o r i n e , the

e x i s t e n c e of any a c t i v i t y i s s u r p r i s i n g , and these r e s u l t s suggest
that r e c o g n i t i o n of the peptide by i t s receptor may depend more on
o v e r a l l conformation than on imidazole b a s i c i t y .
A - F l u o r o h i s t i d i n e . Despite the minimal s t e r i c consequences

of f l u o r i n e s u b s t i t u t i o n and the s i m i l a r i t y i n r i n g b a s i c i t i e s of
the isomers, 2 - f l u o r o - and A - f l u o r o h i s t i d i n e are r e a d i l y d i f f e r
e n t i a t e d by the h i s t i d i n e t-RNA systems. To date, the evidence
i n d i c a t e s that A - f l u o r o h i s t i d i n e does not s u b s t i t u t e f o r h i s t i d i n e
i n p r o t e i n b i o s y n t h e s i s , nor does t h i s analogue show s i g n i f i c a n t
b a c t e r i o s t a t i c or a n t i v i r a l a c t i v i t (Table d II)
s t u d i e s suggest that th
(17), f l u o r i n e at C-A being , perhaps,
u l a r hydrogen bond. Yet, the A - f l u o r o isomer i s a s u b s t r a t e f o r
b a c t e r i a l h i s t i d i n e decarboxylase (but not f o r the mammalian
enzyme), while both isomers are modest substrates f o r h i s t i d i n e
ammonia-lyase (Table I I I ) . The a b i l i t y of A - f l u o r o h i s t i d i n e to
f u n c t i o n both as a weak s u b s t r a t e and a strong competitive i n h i b
i t o r f o r the l a t t e r enzyme prompted a r e i n v e s t i g a t i o n of i t s mech-
Table I I I . E f f e c t s of F l u o r o h i s t i d i n e s on
H i s t i d i n e Ammonia-Lyase (pH 9, 25°)
Κ or K. V
Compound m
(mM) 1
(uM/mg/min)
L-Histidine 2.7 30
A-F-L-Histidine 1.25 0.85
2-F-L-Histidine 170 1-2
anism of a c t i o n (2A). A n a l y s i s of k i n e t i c data, r e v e r s i b i l i t y ,

i s o t o p e i n c o r p o r a t i o n , and isotope e f f e c t s demonstrates that the
r a t e - l i m i t i n g step f o r t h i s enzyme i s not the breakdown of an
intermediate aminoenzyme (as p r e v i o u s l y supposed), but the almost
concerted l o s s of a 3-hydrogen atom and the c o v a l e n t l y bound amino
group. While the isomers l o s e ammonia at comparable r a t e s , A-
f l u o r o h i s t i d i n e i s bound much more e f f e c t i v e l y .
Urocanase, the enzyme f o l l o w i n g the ammonia-lyase i n the c a t a -
b o l i c sequence f o r h i s t i d i n e (Figure 5), promotes the h y d r a t i o n -
rearrangement of urocanic a c i d (25). While n e i t h e r f l u o r o u r o c a n i c
a c i d i s s i g n i f i c a n t as a s u b s t r a t e (Table IV), the 2 - f l u o r o isomer

2. KIRK AND COHEN Ring-F luorinated Imidazoles 31
Table IV. E f f e c t s of F l u o r o u r o c a n i c A c i d s
on Urocanase (pH 7.4, 25°)
10 K7
m or K
± V
Compound (M) (units/ml)
Urocanic a c i d 400 1.2
4-F-Urocanic acid - -
2-F-Urocanic acid 1 0.008
measurable a c t i v i t y as i n h i b i t o r or
substrate.
i s now a very potent i n h i b i t o

enzyme capable of d i r e c t removal of f l u o r i n e from the imidazole
r i n g . The enzymes which degrade the imidazole r i n g of h i s t i d i n e ,
e v e n t u a l l y to glutamic a c i d , can a l s o operate on the f l u o r o h i s t i -
d i n e s , a l b e i t very slowly. Thus f a r , we have found no evidence f o r
metabolites of these analogues i n v i v o .
As already mentioned, the f l u o r o h i s t i d i n e s can be i n c o r p o -
r a t e d i n t o a p o l y p e p t i d e sequence v i a t o t a l s y n t h e s i s . Thus, the
peptide fragment, r i b o n u c l e a s e - S - ( l - 1 5 ) , has been s y n t h e s i z e d with
4- f l u o r o - L - h i s t i d i n e r e p l a c i n g h i s t i d i n e - 1 2 (27). T h i s f l u o r i n e -
c o n t a i n i n g peptide (4-F-His-RNase-(l-15)) a s s o c i a t e s w i t h RNase-S-
(21-124) as w e l l as, and even more s t r o n g l y than, RNase-S-(l-20).
A v a r i e t y of c r i t e r i a suggest that the f l u o r o h i s t i d i n e - c o n t a i n i n g
aggregate has a three-dimensional s t r u c t u r e very s i m i l a r to that
of the complex, RNase-S'. The noncovalent recombination of RNase-
5- (l-15) or -(1-20) w i t h RNase-S-(21-124) r e s t o r e s p r a c t i c a l l y a l l
the enzymatic a c t i v i t y of the o r i g i n a l enzyme, RNase-A; the a n a l -
ogous complex with 4-F-His-RNase-(l-15) i s t o t a l l y devoid of a c t -
i v i t y , although i t i s probably s t i l l capable of b i n d i n g s u b s t r a t e .
The r e s u l t s provide strong support f o r the proposal that His-12
f u n c t i o n s as a general a c i d - g e n e r a l base c a t a l y s t i n the enzymatic
process — a r e d u c t i o n of 5 u n i t s i n the pK of the r i n g being
more than ample to remove t h i s c a t a l y t i c c a p a b i l i t y .
Fluorohistamines. Two types of r e c e p t o r , termed HI and H2,

have been i d e n t i f i e d as mediators of histamine's b i o l o g i c a l
a c t i o n s . C h a r a c t e r i z a t i o n of these r e c e p t o r s , and of histamine's
b i o l o g i c a l r o l e s , have been hampered by l a c k of agents which s e l -
e c t i v e l y b i n d one type. Recently, i t has been found that v a r i o u s
s u b s t i t u t i o n s at C-2 or C-4 of the histamine r i n g s e l e c t i v e l y de-
crease b i n d i n g f o r H2 or HI r e c e p t o r s , r e s p e c t i v e l y . Thus, 2-
phenylhistamine r e t a i n s some 20% of histamine's potency at HI
r e c e p t o r s , but i s l e s s than 0.1% as e f f e c t i v e at H2 r e c e p t o r s .
In a p r e l i m i n a r y study, 2-fluorohistamine was found (28) to have

/CH -CHC00H
2 CH=CHCOOH
NH 2
Lyase
-NH3
Urocanase
+H2Q
CL £H CH C00H
H00CCHCH2CH C00 2
H
NH
CH= NH
Figure 5
-do
HO-CHl
H0-CH2
ON OH
5-FICAR ribavirin 5-AICAR
5-Fluoro-L β-D-ribo- 1 -β-D-ribofuranosyî- 5-amino-l -β-Ό-riho-
furanosylimidazole- l 2,4-triazoh-3-
y furanosuUmidazole-
4-carboxamide carboxamide 4-carboxamide
Figure 6

2. ΚΓΑΚ AND COHEN Ring-Fluonnated Imidazoles 33
equal or greater a f f i n i t y f o r HI r e c e p t o r s ( i n guinea p i g ileum)

than the p r e v i o u s l y most e f f e c t i v e HI a g o n i s t , 2-aminoethylthia-
z o l e . By analogy, 4-fluorohistamine might act as a s e l e c t i v e agon
i s t f o r H2 r e c e p t o r s , a p o s s i b i l i t y now under i n v e s t i g a t i o n .
2-Fluorohistamine was a l s o found to be an e f f e c t i v e s t i m u l a t o r of
c y c l i c AMP formation i n b r a i n s l i c e s , which c o n t a i n both HI and
H2 receptors (29).
Fluoroimidazole Ribosides. R e l a t i v e l y simple imidazole

d e r i v a t i v e s occupy key r o l e s as intermediates i n the b i o s y n t h e s i s
of the purine n u c l e o s i d e s . A major approach to the design of
a n t i v i r a l agents i s based on analogues of Imidazole species which
might i n t e r f e r e with the b i o s y n t h e s i s of n u c l e o s i d e s or of n u c l e i c
a c i d s (Figure 6 ) . Thus, r i b a v i r i n , a t r i a z o l e analogue of 5-AICAR,
commands s e r i o u s a t t e n t i o n as a broad-spectrum a n t i v i r a l agent
(30) . We have synthesized 5-FICAR a f l u o r o analogue of 5-AICAR
(31) ; t h i s compound show
s i m i l a r to that of r i b a v i r i
to b l o c k the b i o s y n t h e s i s of both DNA and RNA. R i b a v i r i n has been
shown to act by i n h i b i t i n g the enzyme, IMP dehydrogenase (33), and
we assume, t e n t a t i v e l y , that 5-FICAR f u n c t i o n s at the same p o i n t
i n the b i o s y n t h e t i c pathway.
Future Plans
The r e s u l t s of these and other s t u d i e s i n f l u o r o i m i d a z o l e

chemistry and biochemistry have r a i s e d i n t e r e s t i n g questions f o r
the f u t u r e . Some aspects of the p h y s i c a l and chemical p r o p e r t i e s
of f l u o r o i m i d a z o l e s do not conform to expectations based on data
f o r other imidazole systems and suggest, » that some sub
s t i t u t e d imidazoles a r e , at b e s t , b o r d e r l i n e aromatic systems.
Further understanding may be provided by a study of d i f l u o r o
imidazoles: 4 , 5 - d i f l u o r o i m i d a z o l e has been synthesized and i s ,
indeed, found to have anomalous p r o p e r t i e s ; d e s p i t e a r a t h e r ex
t e n s i v e e f f o r t , however, the 2 , 4 - d i f l u o r o isomer has not y e t been
prepared.
On the b i o l o g i c a l s i d e , we may ask, , why the isomeric
f l u o r o h i s t i d i n e s show such marked d i f f e r e n c e s i n b i n d i n g and r e
sponse to h i s t i d i n e - s p e c i f i c enzymes; whether the replacement of
h i s t i d i n e by f l u o r o h i s t i d i n e i n an enzyme sequence i n v a r i a b l y
leads to l o s s of a c t i v i t y ; whether the 2 - f l u o r o i m i d a z o l e s can be
made s u f f i c i e n t l y r e a c t i v e to f u n c t i o n as covalent a f f i n i t y l a b e l s
f o r receptor s i t e s . H o p e f u l l y , these and other questions w i l l
have been answered before the next F l u o r i n e Symposium.

Literature Cited
(1) Goldman, Peter, Science (1969), 164, 1123.
(2) Ciba Foundation, "Carbon-Fluorine Compounds," Elsevier,
Amsterdam, 1972.
(3) Reference 2, p. 211.
(4) Belsham, M. G., Muir, A. R., Kinns, Michael, Phillips,
Lawrence, and Twanmoh, Li-Ming, J. Chem. Soc., Perkin Trans.
2 (1974), 119.
(5) Pavlath, A. E. and Leffler, A. J., "Aromatic Fluorine Com
pounds," Reinhold, New York, 1962.
(6) Hudlicky, Miklos, "Organic Fluorine Chemistry," Plenum Press,
New York, 1970.
(7) Kirk, K. L. and Cohen, L. Α., unpublished observations.
(8) Kirk, K. L. and Cohen, L. Α., Symposium on Fluorine in Medic
inal Chemistry, 162nd National Meetin f th America Chem
ical Society, Washington
(9) Kirk, K. L. and Cohen, L. Α., J. Amer. Chem. Soc. (1971),
93, 3060.
(10) Kirk, K. L. and Cohen, L. Α., J. Amer. Chem. Soc. (1973),
95, 4619.
(11) Kirk, K. L. and Cohen, L. Α., J. Org. Chem. (1973), 38, 3647.
(12) Nagai, Wakatu, Kirk, K. L., and Cohen, L. Α., J_. Org. Chem.
(1973),38,1971.
(13) Kirk, K. L., Nagai, Wakatu, and Cohen, L. Α., J. Amer. Chem.
Soc. (1973),95,8389.
(14) Lousberg, R. J. J. Ch. and Weiss, Ulrich, Experientia (1974),
30, 1440.
(15) Kirk, K. L., manuscript in preparation.
(16) Lowenbach, W. A. and King, M. M., unpublished data.
(17) Yeh, H. J. C., Kirk, K. L., Cohen, L. Α., and Cohen, J. S.,
J. Chem. Soc., Perkin Trans. 2 (1975), 928.
(18) Wong, J. L. and Keck, J. H., Jr., J. Org. Chem. (1974), 39
2398, and earlier references cited therein.
(19) Kirk, K. L., McNeal, Elizabeth, Cohen, L. Α., and Creveling,
C. R., manuscript in preparation.
(20) Klein, D. C., Kirk, K. L., Weller, J. L., and Parfitt, A. G.,
Mol. Pharmacol., in press.
(21) Furano, Α. V., Kirk, K. L., and Cohen, L. Α., unpublished
data.
(22) De Clercq, Erik, Kirk, K. L., and Cohen, L. Α., unpublished
data.
(23) Monahan, Μ. Α., Vale, Wylie, Kirk, K. L., and Cohen, L. Α.,
unpublished data.
(24) Klee, C. Β., Kirk, K. L., Cohen, L. Α., and McPhie, Peter,
J. Biol. Chem. (1975), 250, 5033.
(25) Kaeppeli, Franz, and Retey, Janos, Eur. J. Biochem. (1971),
23, 198, and earlier references cited therein.
(26) Klee, C. B., Kirk, K. L., and Cohen, L. Α., unpublished data.

(27) Dunn, Β. Μ., DiBello, Carlo, Kirk, K. L., Cohen, L. Α., and
Chaiken, I. M., J. Biol. Chem. (1974), 249, 6295.
(28) Dismukes, R. Κ., unpublished data.
(29) Dismukes, R. K., Rogers, Michael, and Daly, J. W.,
Neurochemistry, in press.
(30) Khare, G. P., Sidwell, R. W., Witkowski, J. T., Simon, L. N.,
and Robins, R. K., Antimicrob. Ag. Chemother. (1973), 3, 517.
(31) Reepmeyer, J. C., Kirk, K. L., and Cohen, L. Α., Tetrahedron
Letters, in press.
(32) De Clercq, Erik, Luczak, Miroslav, Reepmeyer, J. C., Kirk, K.
L., and Cohen, L. Α., Life Sciences (1975), 17, 187.
(33) Streeter, D. G., Witkowski, J. T., Khare, G. P., Sidwell,
R. W., Bauer, R. J., Robins, R. Κ., and Simon, L. Ν., Proc.
Nat. Acad. Sci. USA (1973), 70, 1174.
Discussion
Q. Has there been a check of monitoring on whether C-F cleavage

occurs?
A. I assume you r e f e r to s t a b i l i t y i n b i o l o g i c a l systems. The
normal pathway f o r h i s t i d i n e degradation i n v o l v e s conversion
to urocanic a c i d , and, u l t i m a t e l y , to glutamic a c i d . As I had
i n d i c a t e d , 2 - f l u o r o h i s t i d i n e i s a very poor s u b s t r a t e f o r the
f i r s t two enzymes i n t h i s pathway; a t the t h i r d s t e p , f l u o
r i d e i o n would be r e l e a s e d , together with innocuous degrada
t i o n products. 4 - F l u o r o h i s t i d i n e can be transformed slowly to
4 - f l u o r o u r o c a n i c a c i d , which i s probably a dead end. Except
f o r t h i s data, we have found no i n d i c a t i o n s f o r enzymatic
removal of f l u o r i n e .
Q. Have you t e s t e d the b i o l o g i c a l a c t i v i t y of f l u o r o a l k y l i m i d a -

z o l e s - r e p l a c i n g F by CF3?
A. We have not made any C F ^ - s u b s t i t u t e d i m i d a z o l e s .
Q. As f a r as you know, are any of them known?

A. Yes. A s e r i e s of 4 - t r i f l u o r o m e t h y l i m i d a z o l e s have been
reported by Baldwin and h i s colleagues a t Merck Sharp & Dohme
[ c f . J . Med. Chem. (1975), l g , 895] and 2 - t r i f l u o r o m e t h y l
imidazoles by Lombardino and Wiseman [ J . Med. Chem. (1974),
17, 1182.]
Q. What do you think would be the long-term s t a b i l i t y of the C-F

bond i n 2-fluoroimidazoles?
A. Simple compounds, such as 2 - f l u o r o i m i d a z o l e and 2 - f l u o r o - 4 -
methylimidazole can be kept i n d e f i n i t e l y i n the s o l i d s t a t e
at -80°, but t r i m e r i z e i n a month or two a t 0 ° . In c o n t r a s t ,
2 - f l u o r o h i s t i d i n e appears to be i n d e f i n i t e l y s t a b l e a t room
temperature. D i l u t e s o l u t i o n s of 2 - f l u o r o h i s t i d i n e i n phos
phate b u f f e r (pH 7) have been kept at 0° f o r months without
evidence of d e t e r i o r a t i o n .

Q. Can you say anything about the a n t i - a r t h r i t i c a c t i v i t y of the

fluorohistidines ?
A. T e s t i n g has not yet been done. We are aware of the p o s s i -
b i l i t i e s i n t h i s d i r e c t i o n and hope to get such a study going
soon.
Q. I'd likÇgto ask whether j g u ever considered the use of r a d i o -

active F as a probe? F has a h a l f - l i f j g O f 110 minutes.
A. Yes, we have. Studies on the s y n t h e s i s of F imidazoles were
i n i t i a t e d some time ago at the Atomic Energy Commission i n
Bucharest.
Q. You're acquaintecjgwith the work of A l Wolf and others a t

Brookhaven with F fluor©phenylalanine and other b i o l o g i c a l -
l y i n t e r e s t i n g molecules? ^g
A. Yes, I am. A l Wol
p o i n t of p a r t i c u l a
the b l o o d - b r a i n b a r r i e r very q u i c k l y , and so there i s c o n s i d -
e r a b l e i n t e r e s t i n t h i s compound f o r b r a i n s c i n t i g r a p h y .

3
2-Fluoro-L-Histidine: A Histidine Analog Which
Inhibits Enzyme Induction
DAVID C. KLEIN
Section on Physiological Controls, Laboratory of Biomedical Sciences, National
Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, Md. 20014
KENNETH L. KIRK
Section on Biochemical Mechanisms f Chemistry Nationa Institut
of Arthritis, Metabolism, and
Institutes of Health, Bethesda
Amino acid analogs have a long and interesting history (1,2)

and have been extremely valuable to biochemists, pharmacologists,
and clinicians as analytical tools and therapeutic agents. Some
of the best established and most useful of these compounds are
para-fluorophenylalanine, para-chlorophenylalanine, and alpha-
-methyldopa. The total number of such compounds which have been
synthesized with the hopes of producing potent and specific tools
probably is in the thousands.
Potential Actions of Amino Acid Analogs
The stratagem usually employed with an amino acid analog is

to have i t enter cells and substitute for the parent compound
(1,2); the structural modification characterizing the analog pre-
cludes the normal function or metabolic fate of the parent com-
pound. Substitution of the analog for the parent compound could
involve substitution in a metabolic pathway resulting in an alter-
ed metabolite. Additionally, a metabolic pathway could be inhib-
ited by an amino acid analog by several mechanisms, including
competitive or noncompetitive inhibition of a specific enzyme or
false feedback. Lastly, substitution for the parent compound in
the synthesis of proteins could result in altered proteins. Such
replacement of an amino acid analog for the parent compound in the
primary structure of a protein could alter a protein through a
number of modes of action, including alteration of the secondary,
tertiary, or quarternary structure, alteration of the active site
of an enzyme, and alteration of the regulatory site of an enzyme.
Role of Histidine in Enzymes
Histidine is one of the most important amino acids involved

in the catalytic action of enzymes (3,40. It functions via the
imidazole group, which can act as a general acid, general base,
37

or n u c l e o p h i l e . This c a t a l y t i c r o l e represents the g e n e r a l l y

recognized f u n c t i o n of h i s t i d i n e i n enzymes (5). Some of the
enzymes i n which h i s t i d i n e i s a t the a c t i v e s i t e i n c l u d e RNAse,
glucose-6-phosphatase, and chymotrypsin.
Recently, evidence has appeared i n d i c a t i n g h i s t i d i n e may
have a f u r t h e r r o l e i n p r o t e i n s (6). I t i s now g e n e r a l l y accepted
that the a c t i v i t i e s of many enzymes are regulated v i a covalent
chemical m o d i f i c a t i o n of enzyme p r o t e i n (5_ ]_>Q) » i n c l u d i n g phos
9
phorylation. S p e c i f i c p r o t e i n kinases c a t a l y z e phosphorylations,

and s p e c i f i c phosphatases c a t a l y z e dephosphorylation. As a r e
s u l t , the a c t i v i t i e s of enzymes can be turned on and turned o f f
q u i c k l y . The phosphorylation of p r o t e i n s occurs v i a covalent
phosphorylation of s p e c i f i c amino a c i d s , with the r e s u l t i n g
formation of both a c i d - s t a b l e and a c i d - l a b i l e phosphate bonds.
The a c i d - s t a b l e bonds appear to be formed with the hydroxy groups
of s e r i n e and threonine, whereas the a c i d - l a b i l e bonds appear to
be formed with the imidazol
The importance o
obvious; i t i s a l s o p o s s i b l e that the imidazole group may be
important i n the r e g u l a t i o n of enzyme a c t i o n . The a v a i l a b i l i t y
of a h i s t i d i n e analog i d e n t i c a l i n s i z e and shape to h i s t i d i n e
but l a c k i n g the f u n c t i o n a l c h a r a c t e r i s t i c s imparted by the
imidazole group would be of general i n t e r e s t .
2-Fluoro-L-Histidine: Stratagem
2 - F l u o r o - L - h i s t i d i n e was synthesized p r i m a r i l y t o provide

an analog of h i s t i d i n e which would have the same s i z e and
shape as h i s t i d i n e , but which would be devoid of the normal
a c t i o n imparted by the imidazole group (9). (For a more d e t a i l e d
d i s c u s s i o n see L. Cohen, t h i s Symposium Series.) Fluorine i s
about the same s i z e as the hydrogen i t r e p l a c e s i n h i s t i d i n e
NH 2
ι
CHsCHCOOH
2-FLUORO-L-HISTIDINE
(SYNTHESIS: Kirk ETAL., J. Am. Chem. Soc.,55,8389 (1973))

3. KLEIN AND KIRK 2-Fluoro-L-Histidine 39
and thus w i l l not introduce major s t e r i c m o d i f i c a t i o n s of h i s t i -

dine. F l u o r i n e - s u b s t i t u t i o n , because of the strong e l e c t r o n
withdrawing e f f e c t of f l u o r i n e , w i l l s u b s t a n t i a l l y a l t e r the pK
of the imidazole group. The r e s u l t i n g compound w i l l be essen-
t i a l l y i n a c t i v e , as compared to the parent compound, as a c a t a -
l y s t i n enzymes.
T h i s e f f e c t of f l u o r i n e s u b s t i t u t i o n i s demonstrated i n ^
Table I. In t h i s experiment, the degree of t r a n s f e r of a [ C ] -
a c e t y l group from [ C ] a c e t y l coenzyme A to the aromaticâmine
tryptamine was determined by measuring the amount of N-[ C]-
acetyltryptamine formed. Imidazole c a t a l y z e d t h i s t r a n s f e r ,
whereas 2 - f l u o r o i m i d a z o l e d i d not. Likewise, on a t h e o r e t i c a l
b a s i s , f l u o r i n e s u b s t i t u t i o n would preclude the phosphorylation
of imidazole. Thus, i f 2 - f l u o r o - L - h i s t i d i n e were incorporated by
c e l l s i n t o p r o t e i n s i n p l a c e of h i s t i d i n e these p r o t e i n s could not
be phosphorylated at a h i s t i d i n residue i f th histidin
were p a r t i c i p a t i n g i n th
2-fluoro-L-histidine-containin
Pineal N-Acetyltransferase: Regulation
We have had a long standing i n t e r e s t i n an enzyme i n the

p i n e a l gland, N - a c e t y l t r a n s f e r a s e (10). T h i s enzyme c a t a l y z e s
the formation of N-acetylated d e r i v a t i v e s of a number of aromatic
amines (10,11,12), such as N-acetyltryptamine and N - a c e t y l s e r o t o -
nin (N-acetyl-5-hydroxytryptamine). I t i s p h y s i o l o g i c a l l y impor-
tant because the d a i l y 50-100 f o l d change i n the a c t i v i t y of
t h i s enzyme r e g u l a t e s l a r g e changes i n the c o n c e n t r a t i o n of sero-
t o n i n i n the p i n e a l gland and l a r g e changes i n the production of
N - a c e t y l s e r o t o n i n and melatonin (5-methoxy-N-acetyltryptamine),
the p u t a t i v e hormone of the p i n e a l gland.
As i s true of the a c e t y l t r a n s f e r r e a c t i o n presented above, i t
i s thought that N - a c e t y l t r a n s f e r a s e molecules i n general act
through the imidazole group of h i s t i d i n e by a t t a c k on the t h i o -
a c e t y l group of a c e t y l CoA with the r e s u l t i n g formation of the
unstable i m i d a z o l e - a c e t y l bond and the subsequent t r a n s f e r of the
a c e t y l group to an acceptor amine (3). A f u r t h e r r o l e of imida-
z o l e i n N - a c e t y l t r a n s f e r a s e molecules might be to accept phosphate
groups, although d i r e c t evidence f o r t h i s does not e x i s t . The i n -
crease i n enzyme a c t i v i t y which occurs on a d a i l y b a s i s appears
to depend not only upon p r o t e i n s y n t h e s i s but a l s o , i n yet an
f f
undefined way, upon the a c t i o n of adenosine 3 ,5 -monophosphate
( c y c l i c AMP). I t i s not c l e a r , however, whether c y c l i c AMP a c t i -
vates new enzyme molecules, as they are being synthesized, stimu-
l a t e s the formation of new enzyme molecules, or has both a c t i o n s .
We f e e l there i s some evidence to suggest that enzyme a c t i v i t y
may be regulated by an a c t i v a t i o n - i n a c t i v a t i o n mechanism. T h i s
i s p r i m a r i l y because N - a c e t y l t r a n s f e r a s e a c t i v i t y can be r a p i d l y
destroyed, probably via enzyme i n a c t i v a t i o n (13,14). I f an
a c t i v a t i o n - i n a c t i v a t i o n mechanism p l a y s a r o l e i n the r e g u l a t i o n

Table I
Comparison of the c a t a l y t i c a c t i v i t y of imidazole and 2 - f l u o r o -

imidazole i n an a c e t y l t r a n s f e r r e a c t i o n .
14
Reaction a d d i t i o n s N-[ C]Acetyltryptamine formed at Λ
( f i n a l concentrations) 37°C (nanomoles/60 min incubation)

14
[ C ] A c e t y l - C o A (0.5 mM),
tryptamine (10 mM) 0.14
14
[ C ] A c e t y l - C o A (0.5 mM) ,
tryptamine (10 mM), Λ
imidazole (100 mM) 13.4
14
[ C ] A c e t y l - C o A (0.5 mM)
tryptamine (10 mM),
2 - f l u o r o i m i d a z o l e (10
X H
Tubes were prepared by adding 25 y l volumes of 2 mM [ C ] -
acetyl-CoA (S.A. = 1 yCi/ymole), 40 mM tryptamine, 400 mM imid
a z o l e , and 400 mM 2 - f l u o r i m i d a z o l e (9), as i n d i c a t e d above. A l l
chemicals were d i s s o l v e d i n .1 M sodium phosphate b u f f e r , pH 6.9.
B u f f e r was added to the r e a c t i o n tubes to b r i n g the f i n a l volume
to 100 y l . Data i s given as the mean of 3 determinations which
were w i t h i n 10% of the mean. -,
*The i d e n t i f i c a t i o n of the N-[ C]-acetyltryptamine e x t r a c t e d
i n t o chloroform was confirmed by two dimensional TLC [chloroform,
methanol, g l a c i a l a c e t i c a c i d (90:10:1) followed by e t h y l ace
t a t e ] ; 88.7% of the r a d i o a c t i v i t y a p p l i e d to a pre-coated s i l i c a
g e l p l a t e , F-254 (Brinkman Instruments Co.) was i s o g r a p h i c with
s y n t h e t i c N-acetyltryptamine.
Chlorof2£m e x t r a c t i o n of a tryptamine-free r e a c t i o n c o n t a i n
ing 0.5 mM [ C]acetyl-CoA and 100 mM imidazole y i e l d e d a small
amount^gf r a d i o a c t i v i t y equivalent to l e s s than 0.02 nanomoles
of N-[ C]acetyltryptamine.
Chloroform e x t r a c t i o n of a tryptamineTfree r e a c t i o n c o n t a i n
ing 100 mM 2 - f l u o r o i m i d a z o l e and 0.5 mM [ C]acetyl-CoA y i e l d e d
no r a d i o a c t i v i t y .

3. KLEIN AND KIRK 2-Fluoro-L-Histicline 41
of N - a c e t y l t r a n s f e r a s e , i t may i n v o l v e c y c l i c AMP. As pointed

out above, i t i s known that the a c t i v i t i e s of other enzymes are
regulated by c y c l i c AMP (7). Furthermore, the a c t i v i t i e s of some
of these enzymes a r e c l o s e l y c o r r e l a t e d with t h e i r phosphorylation
state. In view of our s p e c u l a t i o n that h i s t i d i n e phosphorylation
may be i n v o l v e d i n the r e g u l a t i o n of the a c t i v i t y of N - a c e t y l -
t r a n s f e r a s e and that h i s t i d i n e might be at the a c t i v e s i t e of N-
a c e t y l t r a n s f e r a s e , the study of the e f f e c t s of analogs of h i s t i -
dine on the a c t i v i t y of N - a c e t y l t r a n s f e r a s e became of p a r t i c u l a r
interest.
The E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on the Induction of P i n e a l

N - A c e t y l t r a n s f e r a s e A c t i v i t y in vivo.
A v a l u a b l e t e s t of the p r a c t i c a l use of any amino a c i d ana-

l o g i s to determine i f i t w i l l a c t i n an i n t a c t normal animal
without producing sever
i n i t i a l experimental e f f o r t
a c t i v i t y of N - a c e t y l t r a n s f e r a s e i n the p i n e a l gland can be stimu-
l a t e d by catecholamines a c t i n g d i r e c t l y on the c e l l s c o n t a i n i n g
t h i s enzyme (11). Predict-ably, the a c t i v i t y of t h i s enzyme can
a l s o be stimulated i n the i n t a c t animal by the i n j e c t i o n of these
and s i m i l a r compounds, i n c l u d i n g i s o p r o t e r e n o l (15). We found
that s t i m u l a t i o n of N - a c e t y l t r a n s f e r a s e by i s o p r o t e r e n o l was
blocked by i n j e c t i o n s of 2 - f l u o r o - L - h i s t i d i n e (16).
We d i d not, however, detect any acute adverse s i d e e f f e c t s of
t h i s compound, nor d i d we f i n d that the i n j e c t i o n of a s i m i l a r
dose of h i s t i d i n e could b l o c k the i n d u c t i o n of the enzyme.
Although t h i s experiment showed that 2 - f l u o r o - L - h i s t i d i n e could
act in v i v o , i t d i d not i n d i c a t e whether i t was a c t i n g d i r e c t l y
on the p i n e a l gland, nor d i d i t provide any other i n f o r m a t i o n
regarding the mode of a c t i o n of t h i s compound.
In Vitro Studies on the Induction of P i n e a l N - A c e t y l t r a n s f e r a s e

Activity.
E a r l y i n our s t u d i e s on p i n e a l N - a c e t y l t r a n s f e r a s e we d i s -
covered that we could t r e a t p i n e a l glands i n organ c u l t u r e w i t h
adrenergic agents, i n c l u d i n g a d r e n a l i n and i s o p r o t e r e n o l , and i n
t h i s way i n c r e a s e the a c t i v i t y of N - a c e t y l t r a n s f e r a s e (11). T h i s
provided an e x c e l l e n t model system f o r f u r t h e r s t u d i e s with 2-
fluoro-L-histidine.
E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on the Adrenergic Induction

of N - A c e t y l t r a n s f e r a s e A c t i v i t y . When p i n e a l glands are t r e a t e d
with 2 - f l u o r o - L - h i s t i d i n e the adrenergic s t i m u l a t i o n of N - a c e t y l -
t r a n s f erase a c t i v i t y i s s u b s t a n t i a l l y reduced (Table I I I ) . T h i s
b l o c k i n g e f f e c t of 2 - f l u o r o - L - h i s t i d i n e was seen w i t h glands
which were removed from animals and t r e a t e d immediately with
i s o p r o t e r e n o l and 2 - f l u o r o - L - h i s t i d i n e , or with glands which had

Table I I
In vivo i n h i b i t i o n by 2-F-HIS of the isoproterenol-induced i n

crease i n p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y .
N-Acetyltransferase A c t i v i t y
Exp Group Treatment (nmole/gland/hour)
1 A Isoproterenol 12.6 +1.42
Β Isoproterenol,
2-F-HIS 3.0 + .58
C Control 0.2 + .011
2 A Isoprotereno
D Isoproterenol,
HIS 8.33 + 1.05
Animals (100-gram male Sprague-Dawley Rats) were deprived of

food overnight. Groups Β and D were i n j e c t e d subcutaneously i n
the lower back r e g i o n with a s o l u t i o n of h i s t i d i n e (HIS) or a
suspension o f 2 - f l u o r o - L - h i s t i d i n e (2-F-HIS) (250 mg/kg; 25mg/0.25
ml s a l i n e ) a t 9:00 a.m. Groups A, B, and D were i n j e c t e d sub
cutaneously i n the nape of the neck with i s o p r o t e r e n o l (20 mg/kg,
2.0 mg/0.1 ml s a l i n e ) at 10:00 a.m. Groups Β and D r e c e i v e d
d u p l i c a t e 2-F-HIS or HIS i n j e c t i o n s at 10:30 a.m. A l l animals
were k i l l e d a t noon and t h e i r p i n e a l glands were removed r a p i d l y .
Values are based on 4 glands; data are presented as the mean +
S.E. (From K l e i n e t a l . , 16).

3. KLEIN AND KIRK 2~Fluoro-L-Histidine 43
been c h r o n i c a l l y denervated (16). Chronic denervation removes

a l l n e u r a l elements from the gland; the observation that 2 - f l u o r o -
L - h i s t i d i n e acted i n denervated glands i n d i c a t e d that i t was a c t
i n g on p i n e a l c e l l s d i r e c t l y .
E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on the C y c l i c AMP Induction

of N - A c e t y l t r a n s f e r a s e . Catecholamines s t i m u l a t e the a c t i v i t y of
p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y by f i r s t s t i m u l a t i n g the
production of c y c l i c AMP (10). One p o s s i b l e mechanism of a c t i o n
of 2 - f l u o r o - L - h i s t i d i n e was i n h i b i t i o n of the a c t i o n of c y c l i c
AMP. We evaluated t h i s by determining whether 2 - f l u o r o - L - h i s t i
dine could block the s t i m u l a t i o n of N - a c e t g l t r g n s f e r a s e a c t i v i t y
as caused by a d e r i v a t i v e of c y c l i c AMP, Ν ,2' d i b u t y r y l adeno
f f
s i n e 3 ,5 -monophosphate ( d i b u t y r y l c y c l i c AMP). The s t i m u l a t i o n
of N - a c e t y l t r a n s f e r a s e a c t i v i t y caused by t h i s compound was block
ed by 2 - f l u o r o - L - h i s t i d i n e (Table I I I ) T h i s i n d i c a t e d that 2-
f l u o r o - L - h i s t i d i n e was probabl
sequence of events whic
and f o l l o w s both the i n t e r a c t i o n of catecholamines w i t h a receptor
and the production of c y c l i c AMP.
S p e c i f i c i t y of 2 - F l u o r o - L - H i s t i d i n e I n h i b i t i o n of Enzyme Induction
Our f i n d i n g that the i n d u c t i o n of p i n e a l N - a c e t y l t r a n s f e r a s e

a c t i v i t y was blocked by 2 - f l u o r o - L - h i s t i d i n e r a i s e d the question
of whether the i n h i b i t o r y e f f e c t s of t h i s compound were s p e c i f i c
to t h i s enzyme, or whether the i n d u c t i o n of other enzymes could
be i n h i b i t e d . To examine t h i s important question we studied the
s t e r o i d i n d u c t i o n of t y r o s i n e amino t r a n s f e r a s e i n f e t a l l i v e r ,
the benz[a]anthracene i n d u c t i o n of a r y l hydrocarbon hydroxylase
a c t i v i t y i n l i v e r c e l l s , and the spontaneous i n c r e a s e of o r n i t h i n e
decarboxylase a c t i v i t y i n both mouse mammary t i s s u e and the
p i n e a l gland. In a l l cases the e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e
were examined i n in vitro systems (16).
S t e r o i d Induction of L i v e r Tyrosine Amino Transferase. The

a c t i v i t y of t y r o s i n e amino t r a n s f e r a s e i n l i v e r can be stimulated
by treatment with a low concentration of the s t e r o i d dexametha-
sone (17). We determined that the i n d u c t i o n of t y r o s i n e amino
t r a n s f e r a s e a c t i v i t y i n f e t a l l i v e r expiants by s t e r o i d treatment
was blocked by 2 - f l u o r o - L - h i s t i d i n e (Table IV, 16).
Benz[a]anthracene Induction of L i v e r A r y l Hydrocarbon

Hydroxylase A c t i v i t y . The a c t i v i t y of l i v e r a r y l hydrocarbon
hydroxylase, a membrane bound enzyme, can be induced by low con
c e n t r a t i o n s of a number of compounds (18), i n c l u d i n g benz[a]-
anthracene. Using a c e l l c u l t u r e system, i t was found that the
i n d u c t i o n of the a c t i v i t y of t h i s enzyme was s u b s t a n t i a l l y blocked
by 2 - f l u o r o - L - h i s t i d i n e (Table V, 16).

Table I I I
I n h i b i t i o n by 2-F-HIS of the drug-Induced increase i n p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y i n organ

culture
Surgical N-Acetyltransferase A c t i v i t y
Experiment Pretreatment Treatment i n Organ C u l t u r e (nmole/gland/hour)
1 None HIS (0-11 hours) 0.2 + 0.03
HIS (0-11 hours);

None 10.5 + 1.5.
I s o p r o t e r e n o l (3-11 h r s )
HIS, 2-F-HIS (0-11 hours);

None I s o p r o t e r e n o l (3-11 hours) 4.9 + 0.31 *
-HIS (0-11 hours);

None I s o p r o t e r e n o l (3-11 hours) 16.4 + 2.74
None -HIS, 2-F-HIS (0-11 hours);

I s o p r o t e r e n o l (3-11 hours) 3.2 + 0.56 *
None -HIS (0-11 hours);

I s o p r o t e r e n o l (3-11 hours) 10.4 + 2.11

None -HIS, 2-F-HIS, (0-11 hours);

SCGX -HIS (0-11 hours);
I s o p r o t e r e n o l (3-11 hours) 18.8 + 3.00
SCGX -HIS, 2-F-HIS (0-11 hours);

Surgical N-Acetyltransferase A c t i v i t y
Experiment Pretreatment Treatment i n Organ C u l t u r e (nmole/gland/hour)
3 None -HIS (0-11 hours);

d i b u t y r y l c y c l i c AMP (3-11 h r s ) 22.7 + 3.43
None -HIS, +2-F-HIS (0-11 hours);

d i b u t y r y l c y c l i c AMP (3-11 hrs) 2.6 + 0.64*
Superior c e r v i c a l ganglionectomy (SCGX) was performed 14 days p r i o r t o organ c u l t u r e . P i n e a l

glands were removed between 10:00 and 12:00 a.m. and placed i n t o c u l t u r e . The c o n c e n t r a t i o n o f
2 - f l u o r o - L - h i s t i d i n e (2-F-HIS) was 3 mM; and, when present, the c o n c e n t r a t i o n of h i s t i d i n e (HIS) was
0.1 mM. I s o p r o t e r e n o l was added i n 5 y l of 0.01 M HC1 t o a f i n a l c o n c e n t r a t i o n of 10 yM. D i b u t y r y l
c y c l i c AMP was added i n 5 y l of c u l t u r e medium t o a f i n a l c o n c e n t r a t i o n of 1 mM.
Each value i s based on 4 glands. Data a r e given as the mean + S.E.
* S t a t i s t i c a l l y l e s s than glands t r e a t e d with e i t h e r i s o p r o t e r e n o l or d i b u t y r y l c y c l i c AMP
Ρ < .01. S t a t i s t i c a l a n a l y s i s was performed using Student's " t " t e s t . (From K l e i n et a i . , 16).

Table IV
The e f f e c t of 2-F-HIS on the dexamethasone i n d u c t i o n of t y r o s i n e

amino t r a n s f e r a s e a c t i v i t y i n f e t a l r a t l i v e r expiants i n c u l t u r e
Treatment i n c u l t u r e (hours) Tyrosine amino t r a n s f e r a s e
(0-3) (3-11) a c t i v i t y (nmole/mg protein/min)
Control Control 6.5
Control Dexamethasone 54.2
2-F-HIS 2-F-HIS 6.6
2-F-HIS 2-F-HIS
+ dexamethasone 10.4
A f t e r a 24-hour i n c u b a t i o n period with 1 mM h i s t i d i n e , the

t i s s u e was t r a n s f e r r e d to f r e s h medium c o n t a i n i n g 0.1 mM h i s t i d i n e
f o r the i n d i c a t e d treatments. The concentration of 2 - f l u o r o - L -
h i s t i d i n e (2-F-HIS) was 3 mM. Dexamethasone treatment was i n i -
t i a t e d by adding 5 y l of c u l t u r e medium c o n t a i n i n g dexamethasone
r e s u l t i n g i n a f i n a l medium concentration of 5 yM. Each value i s
the average of the means of d u p l i c a t e determinations performed on
d u p l i c a t e c u l t u r e s . The d u p l i c a t e means d i d not d i f f e r by more
than 3 nanomoles/mg protein/minute. (From K l e i n e t a l . , 16).
Spontaneous Increase of O r n i t h i n e Decarboxylase A c t i v i t y .

O r n i t h i n e decarboxylase i s involved i n the s y n t h e s i s of p o l y -
amines, which appear to play a r o l e i n the process o f c e l l
d i v i s i o n and gene r e g u l a t i o n (19). The a c t i v i t y of t h i s enzyme
i s g e n e r a l l y high under two circumstances: f o l l o w i n g traumatic
treatment o f t i s s u e , such as during regeneration of l i v e r or
immediately a f t e r t i s s u e i s placed i n t o c u l t u r e , and during
normal growth of t i s s u e .
The a c t i v i t y of o r n i t h i n e decarboxylase increases i n both
mouse mammary t i s s u e expiants (20) and i n p i n e a l glands (un-
published r e s u l t s , D. C. K l e i n and T. Oka) soon a f t e r these
t i s s u e s are placed i n t o c u l t u r e . We examined the e f f e c t of 2-
f l u o r o - L - h i s t i d i n e on the spontaneous increase of o r n i t h i n e
decarboxylase a c t i v i t y i n these systems. I t was found that 2-
f l u o r o - L - h i s t i d i n e d i d not block the spontaneous increase of t h i s
enzyme i n the mouse mammary t i s s u e (Table VI) but d i d block i t i n
the p i n e a l gland (Table V I I ) .
The l a c k of an e f f e c t of 2 - f l u o r o - L - h i s t i d i n e on the increase
of o r n i t h i n e decarboxylase i n the mouse mammary t i s s u e may be due

3. KLEIN AND KTRK 2-Fluoro-L-Histidine 47
Table V
The e f f e c t of 2-F-HIS on the benz[a]anthracene i n d u c t i o n of a r y l

hydrocarbon hydroxylase i n l i v e r hepatoma (Hepa-1) c e l l s
Treatment i n C u l t u r e (hours) A r y l hydrocarbon hydroxylase

hydroxylase a c t i v i t y (pmoles/
(0-3) (3-11) ιmg protein/min)
Control Control 19
Control Benz[a]anthracene 159
2-F-HIS 2-F-HIS 6.2
2-F-HIS 2-F-HIS +
benz[a]anthracen
C e l l s were obtained from a stock maintained i n c u l t u r e and

t r a n s f e r r e d to 15 X 60 mm Falcon dishes c o n t a i n i n g 3 ml of
Waymouth MAB medium with 10% f e t a l c a l f serum f o r 72 hours u n t i l
75-80% confluency was achieved. The medium was then replaced
with 2 ml of BGJ c o n t a i n i n g 0.1 mM h i s t i d i n e f o r the treatments
d e t a i l e d above. The c o n c e n t r a t i o n of 2 - f l u o r o - L - h i s t i d i n e (2-F-
HIS) was 3 mM. Benz[a]anthracene was added i n a concentrated
s o l u t i o n r e s u l t i n g i n a f i n a l c o n c e n t r a t i o n of 13 μΜ. Data are
presented as the means of d u p l i c a t e determinations (18) performed
on d u p l i c a t e c u l t u r e s . The i n d i v i d u a l values d i d more than 5%.
(From K l e i n et al., 16).
American Chemical
Society Library
1155 16th St. N. W.
Washington, D. C. 20036
Table VI
E f f e c t o f 2-F-HIS on the spontaneous increase of o r n i t h i n e de-

carboxylase a c t i v i t y i n midpregnant mouse mammary expiants.
Ornithine ^ c a r b o x y l a s e a c t i v i t y
Treatment (picomoles of C 0^ produced/mg t i s s u e / h r )
Not incubated 5.0
Incubated (0-3 hours)
Control 41.2
+ HIS (3 mM) 33.6
+ 2-F-HIS (3 mM) 33.
Midpregnancy mouse (CH~/Hen) mammary expiants were used (20)

( 2 - f l u o r o - L - h i s t i d i n e , 2-F-HIS; h i s t i d i n e , HIS). The c u l t u r e
medium used was M 199 ([HIS] - 0.175 mM). Data a r e presented as
the mean enzyme a c t i v i t y i n two c u l t u r e s . Enzyme a c t i v i t y i n
each c u l t u r e i s based on d u p l i c a t e determinations, which a r e
w i t h i n 1% of the mean. (From K l e i n et a i . , 16).

Table VII
The e f f e c t of 2-F-HIS on the spontaneous i n c r e a s e of o r n i t h i n e decarboxylase a c t i v i t y i n p i n e a l

glands.
N-Acetyltransferase
activity Ornithine^decarboxylase a c t i v i t y
Treatment (nmols/gland/hr) (picomoles of C 0^ produced/gland/hr)
Not incubated 0.15 + .02 6.38 + 0.86
Incubated
C o n t r o l (0-11 hrs) 67.45+6.47
2-F-HIS (0-11 hrs) 34.52 + 9.5*
C o n t r o l (0-3 hrs)
+ I s o p r o t e r e n o l (3-11 hrs) 13.24 + 4.17
2-F-HIS (0-11 hrs)

+ I s o p r o t e r e n o l (3-11 hrs) 4.37 + 0.84
P i n e a l organ c u l t u r e s were prepared j s d e s c r i b e d i n Table I I . Each datum i s presented as the

mean (+ S.E.) of 4 to 6 determinations. For the determination of o r n i t h i n e decarboxylase a c t i v i t y ,

i n d i v i d u a l glands were sonicated i n 100 y l of the assay b u f f e r (20) Immediately a f t e r being removed
from c u l t u r e . The homogenate was stored at -20C f o r 24 hours, thawed, and t r a n s f e r r e d t o a r e a c t i o n

v i a l f o r enzyme assay. The f i n a l volume of the r e a c t i o n was 125 y l and c o n t a i n e d , p y r i d o x a l phos
phate (40 yM), d i t h i o t h r e i t o l (5 mM) , EDTA (4 mM) , T r i s - H C l (50 mM), and DL-[1-C ] o r n i t h i n e (20 yM,
s p e c i f i c a c t i v i t y = 43 yCi/ymole). * S i g n i f l e a n t l y lower than incubated glands not t r e a t e d with 2-
F-HIS, ρ > 0.01. (From K l e i n e t a l . , 16).
to the higher c o n c e n t r a t i o n of h i s t i d i n e i n the c u l t u r e medium.

T h i s would block the e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e , as
discussed below. A l t e r n a t i v e l y , mammary t i s s u e may contain more
h i s t i d i n e or destroy 2 - f l u o r o - L - h i s t i d i n e more q u i c k l y .
General Metabolic E f f e c t s of 2-Fluoro-L-Histidine
The observation that 2 - f l u o r o - L - h i s t i d i n e blocked the induc-

t i o n of s e v e r a l enzymes r a i s e d the p o s s i b i l i t y that t h i s compound,
although not apparently t o x i c to the i n t a c t animal, was i n f a c t
h i g h l y t o x i c to t i s s u e i n general. To i n v e s t i g a t e t h i s p o s s i b i l -
i t y we examined the e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e on s e v e r a l
metabolic parameters (16).
We found that a f t e r 24 hours of treatment w i t h 2 - f l u o r o - L -
h i s t i d i n e , RNA s y n t h e s i s was not decreased (Table V I I I ) . T h i s was
remarkable i n view of th
dependency of i t upon enzym
s y n t h e s i s a small i n h i b i t i o due to 2 - f l u o r o - L - h i s t i d i n e was
apparent immediately. T h i s i n h i b i t o r y e f f e c t d i d not, however,
i n c r e a s e during the next 24 hours of treatment.
E f f e c t s of 2 - F l u o r o - L - H i s t i d i n e on Hydroxyindole-O-Methyl-
transferase A c t i v i t y . Another t o p i c of i n t e r e s t was the e f f e c t
of 2 - f l u o r o - L - h i s t i d i n e on the steady-state l e v e l s of a t h i r d
enzyme i n the p i n e a l gland, hydroxyindole-O-methyl-transferase
(10). I t i s g e n e r a l l y thought that the steady s t a t e l e v e l of an
enzyme depends upon both the ongoing production and d e s t r u c t i o n
of enzyme molecules. Thus, even though a l a r g e increase i n
hydroxyindole-O-methyltransferase a c t i v i t y could not be s t u d i e d ,
as i n the case of the enzymes examined above, i t seemed probable
that the steady-state l e v e l s of t h i s enzyme d i d i n f a c t r e f l e c t
enzyme production at a r a t e equal to that of enzyme degradation.
The e f f e c t of a 24-hour treatment w i t h 2 - f l u o r o - L - h i s t i d i n e
on the a c t i v i t y of t h i s enzyme, which converts N - a c e t y l s e r o t o n i n
to melatonin, was examined. 2 - F l u o r o - L - h i s t i d i n e d i d not a l t e r
the a c t i v i t y of t h i s enzyme (Table IX).
E f f e c t of 2 - F l u o r o - L - H i s t i d i n e on the I n c o r p o r a t i o n of
Histidine into Protein. The l a c k of a n o n s p e c i f i c e f f e c t of 2-
f l u o r o - h i s t i d i n e on s e v e r a l of the metabolic parameters examined
l e d us to suspect that t h i s amino a c i d analog may be a c t i n g on
a s p e c i f i c mechanism, which was i n v o l v e d i n the i n d u c t i o n of a l l
the enzymes we examined. Whereas i t d i d not seem that p r o t e i n
s y n t h e s i s was h a l t e d by 2 - f l u o r o - L - h i s t i d i n e , i t d i d seem p o s s i b l e
that 2 - f l u o r o - L - h i s t i d i n e was a c t i n g by v i r t u e of being i n c o r -
porated i n t o p r o t e i n i n p l a c e of h i s t i d i n e .
We approached t h i s hypothesis by f i r s t determining i f the
i n h i b i t i o n of enzyme i n d u c t i o n by 2 - f l u o r o - L - h i s t i d i n e was
accompanied by an i n h i b i t i o n of the i n c o r p o r a t i o n of h i s t i d i n e .

Table V I I I
The e f f e c t of long term 2-F-HIS treatment i n organ c u l t u r e on

the i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o macromolecules i n p i n e a l
glands incubated w i t h [ C]LEU and [ H ] u r i d i n e f o r a three hour
period.
R a d i o a c t i v e precursor i n TCA
, insoluble material
14 J
[ C]LEU [ H]Uridine
Treatment i n Organ Culture (nanomoles/gland) (picomoles/gland)
C o n t r o l (0-3 hours) 0.34 + 0.041 1.04 + .098
2-F-HIS (0-3 hours) 0.27 + 0.031 0.87 + .167
C o n t r o l (0-24 hours)
2-F-HIS (0-27 hours) 0.28 + 0.018* 1.30 + .122
P i n e a l glands were removed between 10:00 and 12:00 A.M. and

placed i n t o organ c u l t u r e . The c u l t u r e medium contained no HIS.
The concentration-of 2-F-HIS was 3 mM. [ H ] U r i d i n e (3μΜ, S.A.=
21yCi/mole) and [ C]LEU (0.38 mM,S.A.= 8.62 yCi/ymole) were
present f o r only the f i n a l three hours of the i n c u b a t i o n p e r i o d .
Values, which are based on 4 glands, are computed from the S.A.
of the r a d i o a c t i v e p r e c u r s o r s and the r a d i o a c t i v i t y pgr gland
p r e c i p i t a t e . Data are presented as the means + S.E. Statisti
c a l l y l e s s than the value f o r c o n t r o l glands incubated f o r 27
hours (p < 0.01) but not s i g n i f i c a n t l y l e s s than [ C]LEU i n c o r
p o r a t i o n i n t o glands treated f o r 3 hours with 2-F-HIS. (From
K l e i n et a l . , 16).

Table IX
Lack of an e f f e c t of long-term treatment with 2-F-HIS on the

a c t i v i t y of hydroxyindole-O-methyltransferase.
Hydroxyindole-O-methyltransferase
Treatment i n Organ Culture activity(picomoles/gland/hour)
Not incubated 69.2 + 11.1
C o n t r o l (0-24 hours) 109.2 + 19.1
2-F-HIS (0-24 hours) 132.8 + 8.4
C o n t r o l (0-48 hours) 88.8 + 6.0
2-F-HIS (0-48 hours
P i n e a l glands were removed between 10:00 and 12:00 A.M. and

placed i n t o organ c u l t u r e i n medium c o n t a i n i n g 0.1 mM HIS. Each
value i s based on 4 glands and i s presented as the mean (+ S.E.).
The concentration of 2-F-HIS was 3 mM (from K l e i n et a l . , 16).
14
IJ^was found that i n the presence of 7 μΜ [ C ] h i s t i d i n e that
[ C ] h i s t i d i n e could be incorporated i n t o p r o t e i n . More i n t e r e s t
i n g , however, was the f i n d i n g that i n the presence of 2 - f l u o r o -
L - h i s t i d i n e t h i s i n c o r p o r a t i o n was blocked (Table X).
The e f f e c t s of^glevated e x t r a c e l l u l a r concentrations of
h i s t i d i n e on both [ C ] h i s t i d i n e i n c o r p o r a t i o n and i n d u c t i o n
of N - a c e t y l t r a n s f e r a s e by i s o p r o t e r e n o l were examined i n the
presence of 2 - f l u o r o - L - h i s t i d i n e (Figure 1). I t was found that
the i n h i b i t o r y e f f e c t s of 2 - f l u o r o - L - h i s t i d i n e could be overcome
by higher concentrations of h i s t i d i n e . This observation pointed
to the explanation that 2 - f l u o r o - L - h i s t i d i n e was a c t i n g by
d i r e c t l y competing with h i s t i d i n e as a substrate i n p r o t e i n
synthesis.
3
I n c o r p o r a t i o n of [ Η]2-Fluoro-L-Histidine i n t o P r o t e i n
A d i r e c t method of determining whether 2 - f l u o r o - L - h i s t i d i n e

was a c t u a l l y being incorporated i n t o p r o t e i n was to incubate
glands with r a d i o l a b e l e d 2 - f l u o r o - L - h i s t i d i n e and recover the
l a b e l l e d amino a c i d from p r o t e i n . We have synthesized [ H]2-
f l u o r o - L - h i e t i d i n e and have found that i t i s incorporated i n t o
p r o t e i n and that t h i s i n c o r p o r a t i o n can be blocked by c y c l o h e x i -
mide (an i n h i b i t o r of p r o t e i n synthesis) and by high concentra
t i o n s of h i s t i d i n e . In a d d i t i o n we have been able to enzymati-
c a l l y d i g e s t l a b e l l e d p r o t e i n and recover a r a d i o a c t i v e compound
which has the same chromatographic and e l e c t r o p h o r e t i c c h a r a c t e r -

i s t i c s as does a u t h e n t i c ^ 2 - f l u o r o - L - h i s t i d i n e . This r a d i o l a b e l l e d
compound apparently i s [ H ] 2 - f l u o r o - L - h i s t i d i n e (21).
T h i s set of observations provide necessary support f o r the
hypothesis that 2 - f l u o r o - L - h i s t i d i n e i s a c t i v e because i t i s
incorporated i n t o newly synthesized p r o t e i n .
A l t e r n a t i v e Mechanisms of A c t i o n of 2-Fluoro-L-Histidine
Although the above evidence i s necessary to accept the

hypothesis that 2 - f l u o r o - L - h i s t i d i n e i s a c t i v e because i t i s
incorporated i n t o p r o t e i n , t h i s f i n d i n g might be c o i n c i d e n t a l
and not causative. We have thus examined other p o s s i b l e modes o f
a c t i o n of 2 - f l u o r o - L - h i s t i d i n e .
F i r s t l y , i t seemed p o s s i b l e that 2 - f l u o r o - L - h i s t i d i n e was
a c t i v e only a f t e r decarboxylation to 2-fluorohistamine. This,
however, was proven improbabl becaus 2-fluorohistamin itself
was without e f f e c t on th
f e r a s e a c t i v i t y (16). Secondly
f l u o r o - L - h i s t i d i n e to assays of N - a c e t y l t r a n s f e r a s e d i d not i n h i b
i t enzyme a c t i v i t y , e l i m i n a t i n g the p o s s i b i l i t y that 2-fluoro-L-
h i s t i d i n e was a competitive or noncompetitive i n h i b i t o r of enzyme
a c t i v i t y (16). T h i r d l y , we thought that 2 - f l u o r o - L - h i s t i d i n e
might have induced the production o f an i n h i b i t o r of N - a c e t y l -
t r a n s f e r a s e a c t i v i t y . We examined t h i s p o s s i b i l i t y by adding
Figure 1. The effect of histidine (HIS) on N-acetyltransf erase activity and [ C]HIS 14
incorporation in pineal glands treated with isoproterenol and 2-fluoro-L-histidine (2-F-

HIS). Data was collected from 4 experiments; a different concentration of HIS was
used in each. Percent inhibition is calculated from the following: 100 [isoproterenol
value—(isoproterenol + 2-F-HIS value)] isoproterenol value. In each experiment a
set of 4 glands was treated with 10 Μ isoproterenol and a second set of 4 glands was
μ
treated with 10 Μ isoproterenol and 3 mM 2-F-HIS (16).

μ

Table Χ
3
E f f e c t of 2-F-HIS on r a d i o a c t i v i t y incorporated i n t o p r o t e i n of glands incubated with [ H]LEU and
r\:]HIS.
R a d i o a c t i v i t y i n TCA-insoluble m a t e r i a l
(nmoles of r a d i o a c t i v e amino acid/gland)
~ -, N-Acetyltransferase A c t i v i t y
Treatment i n Organ Culture [ H]LEU [ C]HIS (nmole/gland/hour)
Control (0-11 hours) 1.25 + .071 .30 + .019 0.34 + .068
2-F-HIS (0-11 hours) 1.17 + .158 .117 + .018* 0.18 + .034
Control (0-3);
Isoproterenol (3-11 hours) 1.65 + .076 .41 + .022 14.3 + .034
2-F-HIS (0-11 hours);

Isoproterenol (3-11 hours) 1.43 + .075 .126 + .005' 3.68 + .548
P i n e a l glands were removed between 10:00 and 12:00 and were placed into,organ c u l t u r e . The
c u l t u r e medium contained 0.38 mM [ H]LEU (S.A. = 51.2 yCi/umole) and 1 mM [ C]HIS (S.A. = 24
yCi/ymole). The c o n c e n t r a t i o n of 2-F-HIS was 3 mM. I s o p r o t e r e n o l was added i n 5 y l of 0.01 mM HCl
to a f i n a l c o n c e n t r a t i o n of 10 yM. At the end of the experiment glands were sonicated i n 100 y l of
2 mM p e n i c i l l a m i n e i n 0.01 M sodium phosphate b u f f e r , pH 6.9, at 4°C. T h i s treatment preserves

enzyme a c t i v i t y d u r i n g handling. A 50 y l sample of each s o n i c a t e was used f o r enzyme assay; a 25
y l sample was used f o r p r e c i p i t a i o n of TCA i n s o l u b l e m a t e r i a l . Values, which are based on 4-5
glands, are computed from the S. A. of the r a d i o a c t i v g amino a c i d and the r a d i o a c t i v i t y per gland

p r e c i p i t a t e . ^ Data are presented as the mean + S.E. S i g n i f i c a n t l y l e s s than c o n t r o l value
(P < .01). S i g n i f i c a n t l y l e s s than group t r e a t e d with i s o p r o t e r e n o l alone (P < .01) (From K l e i n
et a l . , 16).
3. KLEIN A N D KIRK 2-Fluoro-L-Histidine 55
homogenates of glands t r e a t e d with i s o p r o t e r e n o l and 2 - f l u o r o - L -

h i s t i d i n e to homogenates of glands t r e a t e d only with i s o p r o t e r e n
o l (16). The r e s u l t i n g enzyme a c t i v i t y was the sum of the a c t i
v i t i e s o f each homogenate, i n d i c a t i n g that an i n h i b i t o r probably
was not present i n the i s o p r o t e r e n o l + 2 - f l u o r o - L - h i s t i d i n e homo
genate. F i n a l l y , we added 2 - f l u o r o - L - h i s t i d i n e to glands which
had already been treated with i s o p r o t e r e n o l f o r 3 hours. I f 2-
f l u o r o - L - h i s t i d i n e were i n a c t i v a t i n g newly formed enzyme mole
c u l e s , i t would have caused a r a p i d i n a c t i v a t i o n of enzyme a c t i v
ity. T h i s , however, was not observed (16).
Conclusions
The r e s u l t s o f our s t u d i e s i n d i c a t e that 2 - f l u o r o - L - h i s t i d i n e

c e r t a i n l y i s not an a c u t e l y t o x i c compound but that i t can i n h i b
i t the i n d u c t i o n of s e v e r a l without b l o c k i n RNA synthe
s i s , without i n h i b i t i n
out a l t e r i n g the a c t i v i t
One explanation of these r e s u l t s i s that 2 - f l u o r o - L - h i s t i d i n e
i s incorporated i n t o enzyme p r o t e i n , and that i n c o r p o r a t i o n r e
s u l t s i n the s u b s t i t u t i o n of 2 - f l u o r o - L - h i s t i d i n e f o r h i s t i d i n e
i n the primary s t r u c t u r e o f p r o t e i n s . The s u b s t i t u t i o n could
block the i n d u c t i o n of enzyme a c t i v i t y by causing changes i n the
s t r u c t u r e of the enzyme, by producing n o n - f u n c t i o n a l a c t i v e s i t e s
i n the enzymes, or by b l o c k i n g phosphorylation o f h i s t i d i n e at a
r e g u l a t o r y s i t e on the enzyme.
We have not, however, shown whether i n c o r p o r a t i o n of 2-
f l u o r o - L - h i s t i d i n e i n t o complete enzyme p r o t e i n a c t u a l l y occurs.
T h i s i s one of our f u t u r e goals. We may f i n d , a l t e r n a t i v e l y ,
that 2 - f l u o r o - L - h i s t i d i n e blocks the production o f complete
molecules of any p r o t e i n i n which i t i s incorporated. Net
i n c o r p o r t a t i o n of r a d i o l a b e l l e d l e u c i n e might appear n e a r l y
normal because an increased number of p r o t e i n fragments were
synthesized. I t a l s o has not been shown whether the phosphoryl
a t i o n of enzyme p r o t e i n i s blocked by the s u b s t i t u t i o n of h i s t i
dine with 2 - f l u o r o - L - h i s t i d i n e . T h i s and other f a s c i n a t i n g
problems regarding the a c t i o n of 2 - f l u o r o - L - h i s t i d i n e are present
l y being studied. The s o l u t i o n s of t h i s problem w i l l not only
provide us with information regarding the a c t i o n of 2 - f l u o r o - L -
h i s t i d i n e , but w i l l a l s o provide b a s i c information about b i o l o g
i c a l processes i n general.
LITERATURE CITED
1. Shive, W. and Skinner, C. G. "Metabolic Inhibitors, A
Comprehensive Treastise" pp. 1-60, Academic Press, New York
(1963).
2. Fowden, L., Lewis, D., and Tristram, Η., Adv. Enz. (1968)
29, 89-163.
3. Jencks, W. P. "Catalysis in Chemistry and Enzymology,"

pp 163-168, McGraw Hill, New York (1969).

4. Kirsch, J., Ann. Rev. Biochem. (1973) 42, 205-234.
5. Lehninger, "Biochemistry" pp 217-248, Worth Publishers, Inc.,
New York (1975).
6. Chen, C. C., Smith, D. L., Bruegger, B. B., Halpern, R. Μ.,
and Smith, R. Α., Biochemistry (1974) 13, 3785-3790.
7. Segal, H. L., Science (1973) 180, 25-32.
8. Langan, Τ. Α., Proc. Nat. Acad. Sci. USA (1968) 64, 1267-
1271.
9. Kirk, K. L., Nagai, W. and Cohen, L. Α., J. Am. Chem. Soc.,
(1973) 95, 8389-8392.
10. Klein, D. C. "The Neurosciences: Third Study Program" pp.
509-519, MIT Press, Cambridge, Mass. (1974).
11. Klein, D. C. and Weller, J. L., J. Pharmacol. Exp. Ther. 186,
516-527.
12. Deguchi, T., J. Neuroche
13. Klein, D. C. and Weller
14. Binkley, S., Klein, D. C. and Weller, J. L., J. Neurochem.
(1976) (in press).
15. Axelrod, J., Science (1974) 184, 1341-1348.
16. Klein, D. C., Kirk, K. L., Weller, J. L., Oka, T., Parfitt,
Α., and Owens, I. S., Molec. Pharmacol. (1976) (in press).
17. Kenny, F. T., "Mammalian Protein Metabolism" pp 131-145,
Academic Press, New York (1970).
18. Gielen, J. E., and Nebert, D. W. J. Biol. Chem. (1972) 247,
7591-7801.
19. Russel, D. Η., "Polyamines in Normal and Neoplastic Growth"
Raven Press, New York (1973).
20. Oka, T. and Perry, J., J. Biol. Chem. (1976) (in press).
21. Klein, D. C., Kirk, K. L. and Weller, J. L. (manuscript in
preparation).

4
Thymidylate Synthetase: Interaction with 5-Fluoro and
5-Trifluoromethyl-2'-Deoxyuridylic A c i d
DANIEL V. SANTI, ALFONSO L. POGOLOTTI, THOMAS L. JAMES,

YUSUKE WATAYA, KATHRYN M. IVANETICH, and STELLA S. M. LAM
Department of Biochemistry and Biophysics, and Department of Pharmaceutical
Chemistry, University of California, San Francisco, Calif. 94143
Thymidylate synthetas
of 2'-deoxyuridylate (dUMP) to thymidylate (TMP) with the
concomitant conversion of 5, 10-methylene tetrahydrofolate
(CH FAH ) to 7, 8-dihydrofolate (FAH ) as depicted in F i
2 4 2
gure 1 (for a recent review, see reference 1). In this process

the hydrogen at C-6 of FAH is directly transferred to the methyl
4
group of TMP (2).

One of our objectives over the past few years has been to
establish the mechanism of catalysis of thymidylate synthetase.
Extensive investigations of chemical counterparts (3-6) have in
dicated that the reaction is initiated by attack of a nucleophile at
the 6-position of dUMP and that many, if not all, reactions along
the pathway are facilitated by analogous nucleophilic catalysis.
The proposed mechanism of this enzyme, as derived from
investigations of chemical models is illustrated in Figure 2.
It is proposed that the reaction is initiated by attack of a
nucleophilic group of the enzyme to the 6-position of dUMP.
In this manner, the 5-position of dUMP could be made sufficient
ly nucleophilic (viz I, Figure 2) to react with CH FAH or an 2 4
equivalent reactive species of formaldehyde. Thus, the initial

condensation product between dUMP and CH F A H is now 2 4
generally accepted (1, 7) to be one which is covalently bound to

the enzyme and saturated across the 5, 6-double bond of dUMP
(II). Proton abstraction from II would give the intermediate
enolate III. As with the chemical models, III should readily
undergo a β-elimination to produce the highly reactive exocyclic
methylene intermediate IV and FAH , bound to the enzyme in
4
close proximity. Intermolecular hydride transfer from FAH to 4
IV would yield dTMP, FAH , and the native enzyme. It should

2
be emphasized that all of the aforementioned reactions and inter
mediates have direct chemical counterparts, and are in complete
accord with all available biochemical data.
With the availability of a stable enzyme from an amethop
terin resistant strain of Lactobacillus casei (8, 9) and facile
methods for its purification (9-11), we undertook studies which
57

Figure 2. Suggested sequence for the thymidylate

synthetase reaction. All pyrimidine structures have a
l-(5-phospho-2'-deoxyribosyl) substituent and R =
CH NHC H COGlu.
t 6 k

4. SANTi E T A L . Thymidylate Synthetase 59
might p r o v i d e d i r e c t s u p p o r t f o r p r o p o s a l s w h i c h w e r e b a s e d on
the a f o r e m e n t i o n e d n o n e n z y m i c m o d e l s . A l t h o u g h a n u m b e r of
d i r e c t i o n s h a v e b e e n p u r s u e d t o w a r d s t h i s o b j e c t i v e , the f o l
l o w i n g s u m m a r i z e s o u r i n v e s t i g a t i o n s of the i n t e r a c t i o n of t h y
m i d y l a t e synthetase w i t h 5 - f l u o r o - 2 ' - d e o x y u r i d y l a t e ( F d U M P )
and 5 - t r i f l u o r o m e t h y l - 2 - d e o x y u r i d y l a t e ( C R d U M P ) . Studies of
f
these two f l u o r i n a t e d n u c l e o t i d e s have p r o v i d e d c o n v i n c i n g e v i

d e n c e for the m e c h a n i s m of this e n z y m e ; i n a d d i t i o n , they h a v e
p r o v i d e d i n s i g h t into how these d r u g s act, and how the r e a c t i
v i t y of f l u o r i n a t e d m o l e c u l e s m i g h t be u t i l i z e d i n the d e s i g n of
e n z y m e i n h i b i t o r s . We e m p h a s i z e that a n u m b e r of other l a b o
r a t o r i e s have been engaged i n s i m i l a r i n v e s t i g a t i o n s , and r e
g r e t that s p a c e does not p e r m i t c o m p l e t e c i t a t i o n of a l l the
e x c e l l e n t s t u d i e s p e r f o r m e d i n this a r e a .
5 - F l u o r o - 2 ' -deoxyuridylat
s o m e t i m e ( 12, 13) tha
of t h y m i d y l a t e s y n t h e t a s e , but the n a t u r e of i n h i b i t i o n has b e e n
the topic of c o n s i d e r a b l e c o n t r o v e r s y (_1_4). S i n c e the 6 - p o s i t i o n
of 1 - s u b s t i t u t e d 5 - f l u o r o u r a c i l s i s quite s u s c e p t i b l e t o w a r d n u c
l e o p h i l i c attack ( 15- 17), we s u s p e c t e d that F d U M P m i g h t e x e r t
its i n h i b i t o r y effect by r e a c t i o n w i t h the p r o p o s e d n u c l e o p h i l i c
c a t a l y s t of t h y m i d y l a t e s y n t h e t a s e . Studies f r o m this (18, 19)
and o t h e r (20, 21 ) l a b o r a t o r i e s h a v e s i n c e d e m o n s t r a t e d this
to be the c a s e .
A s i m p l i f i e d d e p i c t i o n of the i n t e r a c t i o n s of F d U M P and
C H ^ F A H ^ is g i v e n below i n F i g u r e 3.
E-FdUMP
E-FdUMP-CH FAH
ι—ι 1
Ε '4 - Ê.FdUMP.CH FAH
2
2 4
Figure 3

U s i n g the i s o t o p e t r a p p i n g m e t h o d (2_2), we have found that the

two l i g a n d s , F d U M P and C H ^ F A H ^ , i n t e r a c t w i t h the e n z y m e
i n a r a n d o m f a s h i o n , a n d that f o r m a t i o n of the i n i t i a l t e r n a r y
c o m p l e x ( E ^ F d U M P ^ C H ^ F A H ^ ) i s at l e a s t p a r t i a l l y r a t e d e t e r -
mining. F r o m e q u i l i b r i u m b i n d i n g techniques we h a v e a s c e r -
tained that the d i s s o c i a t i o n constantes of both b i n a r y c o m p l e x e s
(K^ a n d K ^ ) a r e a p p r o x i m a t e l y 10" M . T h e f i r s t t e r n a r y c o m -
plex which is f o r m e d with F d U M P , C H ^ F A H . and enzyme is
d e p i c t e d as E - F d U M P ' C H ^ F A H ^ a n d does not i n v o l v e c o v a l e n t
bonds. Interestingly, analogous r e v e r s i b l e t e r n a r y complexes
m a y be f o r m e d u s i n g a n a l o g s of the c o f a c t o r . T h r o u g h s t u d i e s
of the i n t e r a c t i o n of F d U M P and a n a l o g s of C H ^ F A H ^ we h a v e
a s c e r t a i n e d that t h e r e i s a s t r i k i n g s y n e r g i s m i n b i n d i n g of
l i g a n d s to this p r o t e i n . T h a t i s , the a f f i n i t y of e i t h e r l i g a n d
f o r the cognate b i n a r y c o m p l e x i s c a . two o r d e r s of m a g n i t u d e
g r e a t e r than the a f f i n i t y f o r the f r e e e n z y m e With many a n a -
l o g s of C H ^ F A H ^ , thi
that i t m a y be p h y s i c a l l
T h e s e t e r n a r y c o m p l e x e s show i n t e r e s t i n g u l t r a v i o l e t d i f -
f e r e n c e s p e c t r a w h i c h m a y be u s e d f o r t h e i r q u a n t i t a t i o n a n d
characterization. Shown i n F i g u r e 4 a r e d i f f e r e n c e s p e c t r a o b -
tained w i t h C H 2 F A H ^ a n d 5, 8 - d e a z a f o l i c a c i d ; s i m i l a r d i f -
f e r e n c e s p e c t r a h a v e a l s o b e e n o b t a i n e d w i t h a n u m b e r of other
a n a l o g s of f o l i c a c i d . C h a r a c t e r i s t i c of these d i f f e r e n c e s p e c t r a
i s a peak at c a . 330 n m a n d , u s u a l l y , a t r o u g h at c a . 290 n m .
A l t h o u g h the e x a c t r e a s o n f o r the s p e c t r a l changes w h i c h o c c u r
upon f o r m a t i o n of the r e v e r s i b l e t e r n a r y c o m p l e x e s i s y e t u n -
k n o w n , we s u g g e s t that they r e s u l t f r o m p e r t u r b a t i o n s of the p -
a m i n o b e n z o y l g l u t a m a t e m o i e t y of the c o f a c t o r a n a l o g s w h i c h r e -
sult f r o m t h e i r e n v i r o n m e n t w i t h i n the t e r n a r y c o m p l e x . T h i s
p e r t u r b a t i o n i s b e l i e v e d to be a m a n i f e s t a t i o n of a c o n f o r m a t i o n -
a l change w h i c h i s r e l a t e d to the a f o r e m e n t i o n e d s y n e r g i s m i n
b i n d i n g of the two l i g a n d s . T h e d i f f e r e n c e s p e c t r u m of the
E » F d U M P C H F A H c o m p l e x ( F i g u r e 4) i s s i m i l a r to those o b -
e
2 4
s e r v e d f o r the c o f a c t o r a n a l o g s , s u g g e s t i n g that s i m i l a r c h a n -
ges i n e n v i r o n m e n t o c c u r w i t h the n a t u r a l c o f a c t o r , C H ^ F A H . .
T h e r e i s one s t r i k i n g d i f f e r e n c e i n that t h e r e i s a l o s s of d i f f e -
r e n t i a l a b s o r b a n c e at 269 n m i n the c o m p l e x f o r m e d w i t h
C H ^ F A H ^ (18, 1 9 ) w h i c h w e h a v e not o b s e r v e d i n c o m p l e x e s
f o r m e d w i t h c o f a c t o r a n a l o g s ; the r e a s o n f o r this w i l l b e c o m e
a p p a r e n t i n the e n s u i n g d i s c u s s i o n .
The complex f o r m e d with thymidylate synthetase, F d U M P ,
and the n a t u r a l c o f a c t o r C H J A H . , h a s b e e n e x t e n s i v e l y i n v e s -
t i g a t e d . T h i s c o m p l e x i s e x t r e m e l y tight a n d m a y r e a d i l y be
i s o l a t e d b y a v a r i e t y of t e c h n i q u e s (18, 1 9 , 21, 23, 24). Using
r a d i o a c t i v e F d U M P and C H , F A H ^ , the e n z y m e h a s b e e n
t i t r a t e d a n d shown to p o s s e s s two F d U M P a n d two c o f a c t o r
b i n d i n g s i t e s p e r m o l e ( 1 9 ) . T h e s t o i c h i o m e t r y of b i n d i n g h a s
b e e n v e r i f i e d i n a n u m b e r of l a b o r a t o r i e s b y a v a r i e t y of m e -
thods ( 11, 25, 26). T h i s i s i n a c c o r d w i t h the e a r l i e r f i n d i n g

that t h y m i d y l a t e s y n t h e t a s e f r o m L . c a s e i has two a p p a r e n t l y

i d e n t i c a l subunits of M W 35, 000 e a c h (9, 27).
Studies of the r a t e of a s s o c i a t i o n of F d U M P w i t h the
E - C H - , F A H . c o m p l e x (k ) and its d i s s o c i a t i o n (k ) have
r r
2 4 ^ v
on 7
off '
x
E-CHÂH^+f îl]FdUMP Ε-Cr^FArJ· [hi]FdUMP

k
off
a l l o w e d us t o ^ c ^ l c u l a t e the d i s s o c i a t i o n c o n s t a n t of the c o m p l e x
to be c a . 10 M . T h i s p r o v i d e s a n e x p l a n a t i o n f o r the d i s
c r e p a n c i e s i n R e v a l u e s r e p o r t e d f o r F d U M P in the l i t e r a t u r e ;
n a m e l y , p r e v i o u s e x p e r i m e n t s w e r e u s i n g c o n c e n t r a t i o n s of e n
z y m e h i g h e r than the and w e r e i n c o n c e n t r a t i o n r a n g e s
w h e r e F d U M P was b e h a v i n g as a s t o i c h i o m e t r i c i n h i b i t o r . The
kinetically determined i s a p p r o x i m a t e l y 10 - f o l d l o w e r
than that f o r the b i n a r y c o m p l e x ; i n e f f e c t the p r e s e n c e of the
c o f a c t o r i n c r e a s e s the
10 k c a l / m o l i n b i n d i n g
t u r e of the i n t e r a c t i o n of F d U M P and t h y m i d y l a t e s y n t h e t a s e i n
v o l v e s changes w h i c h o c c u r w i t h i n the bound t e r n a r y c o m p l e x .
S e v e r a l l i n e s of e v i d e n c e d e m o n s t r a t e r a t h e r c o n c l u s i v e l y
that a c o v a l e n t bond i s f o r m e d b e t w e e n F d U M P and t h y m i d y l a t e
s y n t h e t a s e w i t h i n the c o m p l e x , (a) T h e E - ^ l F d U M P ^ C r L F A H ^
c o m p l e x m a y be t r e a t e d w i t h a n u m b e r of p r o t e i n d é n a t u r a n t s
( u r e a , g u a n i d i n e h y d r o c h l o r i d e , e t c . ) without a p p a r e n t
l o s s of p r o t e i n - b o u n d r a d i o a c t i v i t y . W i t h few e x c e p t i o n s ,
s u c h t r e a t m e n t i s s u f f i c i e n t to d i s r u p t n o n c o v a l e n t i n t e r a c -
tions b e t w e e n low m o l e c u l a r w e i g h t l i g a n d s and t h e i r p r o t e i n
receptors, (b) U p o n f o r m a t i o n of the c o m p l e x , t h e r e i s a d e -
c r e a s e of a b s o r b a n c e at 269 n m w h i c h c o r r e s p o n d s to s t o i c h i o -
m e t r i c l o s s of the p y r i m i d i n e c h r o m o p h o r e of F d U M P . This re-
s u l t s t r o n g l y s u g g e s t s that the 5, 6 - d o u b l e bond of the p y r i m i -
dine is s a t u r a t e d i n the bound c o m p l e x , (c) T h e r a t e of d i s -
s o c i a t i o n of [6- H ] F d U M P f r o m the c o m p l e x shows a s e c o n d a r y
t r i t i u m i s o t o p e effect ( k ^ / k ^ ) of 1.23. T h i s would c o r r e s p o n d
to k £ j / k j 3 1. 15 and c l e a r l y α errions traite s that the 6 - c a r b o n
=
of the h e t e r o c y c l e u n d e r g o e s sp to sp r e h y b r i d i z a t i o n d u
r i n g the p r o c e s s as r e q u i r e d i f the 5, 6 - d o u b l e bond of F d U M P
i s s a t u r a t e d i n the c o m p l e x , (d) P r o t e o l y t i c d i g e s t i o n of
the c o m p l e x y i e l d s a p e p t i d e w h i c h i s c o v a l e n t l y bound to both
F d U M P and C H ^ F A H ^ . T h e u l t r a v i o l e t and f l u o r e s c e n c e s p e c
t r a of this p e p t i d e a r e c h a r a c t e r i s t i c of 5 - a l k y l t e t r a h y d r o f o l a t e s
a n d , as w i t h the n a t i v e c o m p l e x , t h e r e i s no e v i d e n c e of u l t r a
v i o l e t a b s o r p t i o n of the F d U M P c h r o m o p h o r e .
F r o m t h e s e l i n e s of e v i d e n c e , together w i t h i n f o r m a t i o n g a
t h e r e d f r o m m o d e l c h e m i c a l c o u n t e r p a r t s , the s t r u c t u r e of the
e n z y m e ' F d U M P ' C H ^ F A H ^ c o m p l e x i s c u r r e n t l y b e l i e v e d to be as
d e p i c t e d i n F i g u r e 5. H e r e , a n u c l e o p h i l e of the e n z y m e has a d
d e d to the 6 - p o s i t i o n of F d U M P , and the 5 - p o s i t i o n of the p y r i
m i d i n e i s c o u p l e d to the 5 - p o s i t i o n of F A H . v i a the m e t h y l e n e

Figure 4. Dashed line: Ultraviolet difference spectra

of FdUMP, CH FAHt and thymidylate synthetase vs.
h
CHgFAHi and thymidylate synthetase. Solid line:

FdUMP, 5,8-deazofohte and thymidylate synthetase
vs. enzyme and 5,8-deazafolate.
Figure 5. Structure of the FdUMP · CH · F A t f j thymidylate synthetase ternary com-

t
plex where X represents a nucleophile of one of the enzyme amino acids

g r o u p of the c o f a c t o r . A s i m i l a r structure was proposed f r o m

e v i d e n c e o b t a i n e d i n d e p e n d e n t l y i n another l a b o r a t o r y (20).
R e f e r r i n g to F i g u r e 5, it i s noted that the a s s i g n e d s t r u c
ture f o r the E F d U M P * C H > F A r J c o m p l e x i s a n a l o g o u s to one of
e
the p r o p o s e d s t e a d y state i n t e r m e d i a t e s of the n o r m a l e n z y m i c

r e a c t i o n ( v i z II, F i g u r e 2). T h e y d i f f e r i n that II p o s s e s s e s
a p r o t o n at the 5 - p o s i t i o n of the n u c l e o t i d e w h i c h i s a b s t r a c
ted i n a s u b s e q u e n t s t e p , w h e r e a s the E ' F d U M P - C H ^ F A H ^ c o m
p l e x ( F i g u r e 5) p o s s e s s e s a s t a b l e f l u o r i n e at the c o r r e s p o n d i n g
position. T h u s , i t a p p e a r s that F d U M P behaves as a ' q u a s i - 1
substrate 11
f o r this r e a c t i o n . T h a t i s , it e n t e r s into the c a t a
l y t i c r e a c t i o n as d e p i c t e d f o r the s u b s t r a t e d U M P i n F i g u r e 2
up to the p o i n t w h e r e a n i n t e r m e d i a t e i s f o r m e d w h i c h c a n p r o
c e e d no f u r t h e r ; i n effect, a c o m p l e x i s t r a p p e d w h i c h r e s e m
b l e s a steady state i n t e r m e d i a t e ( v i z II, F i g u r e 2) of the n o r m a l
catalytic reaction.
We have r e c e n t l y obtaine
a n FdUMP* C r L F A H . * peptid
t i o n of the F O U M P M C H ^ A H ^ • t h y m i c j v l a t e s y n t h e t a s e c o m p l e x .
A s shown i n F i g u r e 6, the 94 M H z F s p e c t r u m c o n s i s t s o f a
doublet of t r i p l e t s l o c a t e d 87. 2 p p m u p f i e l d of the e x t e r n a l r e -
f e r e n c e / t r i f l u o r o a c e t i c a c i d . O u r c u r r e n t i n t e r p r e t a t i o n of this
s p e c t r u m i s as f o l l o w s : T h e d o u b l e t i s c a u s e d b y s p l i t t i n g of the
F r e s o n a n c e b y the p r o t o n at the 6 - p o s i t i o n of the u r a c i l r i n g
(H . ) w i t h a c o u p l i n g constant ^ of 32. 5 H z . E a c h c o m p o n e n t
of m e d o u b l e t i s s p l i t f u r t h e r into a t r i p l e t ( i n t e n s i t y r a t i o (1:2:1)
c a u s e d b y c o u p l i n g of the f l u o r i n e w i t h the a d j a c e n t m e t h y l e n e
p r o t o n s ( H ^ ) of the c o f a c t o r w i t h the m a g n i t u d e of the c o u p l i n g
constant b e i n g Λ 9 . 2 H z . T h e i n n e r m o s t l i n e s of the t r i p
l e t s o v e r l a p s o the F r e s o n a n c e a p p e a r s to be a quintet w i t h
i n t e n s i t y r a t i o 1:2:2:2:1.
It h a s b e e n w e l l e s t a b l i s h e d that the t r i g o n a l g e o m e t r y of
the c a r b o n y l a t o m s i n u r a c i l d e r i v a t i v e s s a t u r a t e d a c r o s s the
5, 6 - d o u b l e bond r e s u l t s i n a h a l f - c h a i r c o n f o r m a t i o n w i t h s u b -
stituents o n c a r b o n a t o m s 5 a n d 6 s t a g g e r e d (28-30) as c o m
m o n l y fowjid i n c y c l o h e x a n e .
The F spectrum, in conjunction with p r e v i o u s l y reported
u l t r a v i o l e t and f l u o r e s c e n c e s p e c t r a l data (3J.), of the F d U M P »
C H ^ F A H . * peptide y i e l d s d e f i n i t i v e e v i d e n c e f o r i t s s t r u c t u r e .
T h e d o u b l e t of t r i p l e t s i m p l i e s that the f l u o r i n e - b o n d e d c a r b o n i s
f l a n k e d b y C H a n d C H g r o u p s ( i . e. C H C F C P ^ ) .
2 T h e C H , of
c o u r s e , o c c u r s at the 6 - p o s i t i o n of the n u c l e o t i d e w h i c h i s a t
t a c h e d to the n u c l e o p h i l e of the e n z y m e . T h e m o s t l o g i c a l a s
s i g n m e n t f o r the C H - , i s the b r i d g i n g g r o u p between the n u c l e o t i d e
and c o f a c t o r a s d e p i c t e d f o r the n a t i v e c o m p l e x i n F i g u r e 5.
B a s e d o n the s t a b i l i t y of the F d U M P ' C r ^ F A H . » peptide i n the
a b s e n c e o f a n t i o x i d a n t s , we h a v e s u r m i s e d that the C F ^ g r o u p
b r i d g e s the n u c l e o t i d e to the 5 - a n d n o t to the 1 0 - n i t r o g e n o f the
cofactor.
It i s p o s s i b l e to a s s i g n the s t e r e o c h e m i s t r y of the

19
F NMR
Figure 6. 94 MHz Fluorine-19 nmr spectrum of the FdUMP · CH FAH

t k · peptide
substituents w h i c h have a d d e d a c r o s s the 5, 6 - d o u b l e bond of

F d U M P to g i v e the t e r n a r y c o m p l e x by c o m p a r i n g the o b s e r v e d
c o u p l i n g constants w i t h those f r o m e x t e n s i v e l y s t u d i e d m o d e l s .
A s shown i n F i g u r e 7, the 5 - f l u o r o and 6 - h y d r o g e n of the
F d U M P * C H ^ F A H ^ * peptide a r e p r o p o s e d to be i n a t r a n s p s e u d o -
axial conformation. T h e e n z y m e n u c l e o p h i l e and c o f a c t o r a r e
t h e r e f o r e trans_ p s e u d ο e q u a t o r i a l .
T h e a d d i t i o n of a n u c l e o p h i l e of t h y m i d y l a t e s y n t h e t a s e to
the 6 - p o s i t i o n of d U M P is a p r i m a r y event i n the e n z y m e - c a t a
l y z e d r e a c t i o n ( F i g u r e 8). T h e r e s u l t a n t c a r b a n i o n (1) r e a c t s
w i t h C H , F A H ^ to p r o d u c e a n i n t e r m e d i a t e w i t h a s t r u c t u r e
analogous to tnat of the t e r n a r y c o m p l e x f o r m e d w i t h F d U M P
and the c o f a c t o r (2, 3). A b s t r a c t i o n of the 5 - h y d r o g e n f o l l o w e d
by a s e r i e s of steps i n v o l v i n g r e d u c t i o n of the one c a r b o n u n i t
a n d e l i m i n a t i o n of the n u c l e o p h i l e r e s u l t s i n the o b s e r v e d p r o
ducts of the r e a c t i o n . T h e s e steps h a v e b e e n p r e v i o u s l y d e p i c
ted i n d e t a i l i n F i g u r e 2. W i t h the l o g i c a l a s s u m p t i o n that
the n o r m a l e n z y m e - c a t a l y z e d r e a c t i o n o c c u r s i n a m a n n e r s i m i
l a r to the f o r m a t i o n of the t h y m i d y l a t e s y n t h e t a s e « F d U M P ·
C r | F A H ^ c o m p l e x , s e v e r a l d e t a i l s c o n c e r n i n g the m e c h a n i s m of
the n o r m a l e n z y m i c r e a c t i o n m a y be i n f e r r e d .
F i r s t , the o v e r a l l s t e r e o c h e m i c a l pathway of the e n z y m e -
c a t a l y z e d r e a c t i o n m a y be d e d u c e d . T h e a d d i t i o n of the n u c l e o
p h i l e and c o f a c t o r a c r o s s the 5, 6 - d o u b l e bond m u s t o c c u r i n
a t r a n s f a s h i o n . C o n s e q u e n t l y , the s u b s e q u e n t e l i m i n a t i o n of
the 5 - h y d r o g e n and the e n z y m i c n u c l e o p h i l e m u s t o c c u r as a c i s -
elimination .

SANTi E T A L . Thymidyhte Synthetase
Figure 7. Stereochemical projection of the

FdUMP · CH FAH
t h· peptide as determined hy
its F nmr spectrum; R = 5-phospho-2'deoxy-
19
ribosyl
Figure 8

S e c o n d , the s t e r e o c h e m i s t r y c o n c e r n i n g the t r a n s i e n t i n
t e r m e d i a t e s shown i n F i g u r e 8 m a y be i n f e r r e d . T h e m e c h a n i s
tic d e t a i l s d i s c u s s e d below f o l l o w f r o m the p r i n c i p l e that a
g r o u p r e a c t i n g w i t h the π - s y s t e m of the u r a c i l h e t e r o c y c l e ap
p r o a c h e s a p p r o x i m a t e l y p e r p e n d i c u l a r to the plane of the r i n g ;
by m i c r o s c o p i c r e v e r s i b i l i t y , a s i m i l a r o r i e n t a t i o n i s r e q u i r e d
w h e n a g r o u p d e p a r t s to r e f o r m the π - s y s t e m . T h u s , the i n i
t i a l attack of the n u c l e o p h i l e of the e n z y m e at the e l e c t r o p h i l i c
6 - c a r b o n of d U M P s h o u l d be p e r p e n d i c u l a r to the plane of the
heterocycle. T h e r e s u l t a n t c a r b a n i o n (1) w i l l be d e l ^ c a l i z e d
throughout the c a r b o n y l g r o u p s and w i l l be h i g h i n sp c h a r a c
ter. T h e a p p r o a c h of C H ^ F A H ^ to the 5 - p o s i t i o n w o u l d ^
p e r p e n d i c u l a r to the plane of the r i n g a n d , b a s e d on the F
data p r e s e n t e d a b o v e , t r a n s to the n u c l e o p h i l e attached to the
6 - p o s i t i o n . A s a r e s u l t , as shown i n s t r u c t u r e 2, the c o f a c t o r
w o u l d e x i s t i n a p s e u d o a x i a l p o s i t i o n , and the 5 - h y d r o g e n w o u l d
be p s e u d o e q u a t o r i a l . F o
the p r o t o n f r o m the 5 - p o s i t i o
s i t i o n (3) p r i o r to its a b s t r a c t i o n to f o r m the c a r b a n i o n (4).
This mechanistic interpretation necessitates a previously u n r e
c o g n i z e d c o n f o r m a t i o n a l c h a n g e , w h i c h o c c u r s a f t e r a d d i t i o n of
the c o f a c t o r but b e f o r e a b s t r a c t i o n of the 5 - p r o t o n ; and r e s u l t s
i n r i n g i n v e r s i o n about the 5 - a n d 6 - p o s i t i o n s of the n u c l e o t i d e
19
i n t e r m e d i a t e s {i.e. 2-*\3). T h e r e s u l t s of F n m r studies
d e s c r i b e d h e r e a r e p r e l i m i n a r y ; a d e t a i l e d n m r study of the
i n t e r a c t i o n of F d U M P w i t h t h y m i d y l a t e synthetase w i l l be p u b
lished elsewhere.
5 - T r i f l u o r o m e t h y l - 2 ' - d e o x y u r i d y l a t e (C ξ d U M P ) . CEdUMP
i s a potent i n h i b i t o r of t h y m i d y l a t e s y n t h e t a s e . R e y e s aria
H e i d e l b e r g e r have r e p o r t e d that u p o n p r e i n c u b a t i o n C E ^ d U M P
c a u s e s i r r e v e r s i b l e i n h i b i t i o n of t h y m i d y l a t e synthetase
f r o m E h r l i c h A c s i t e s c e l l s (32). B a s e d on the o b s e r v a t i o n that
t r i f l u o r o m e t h y l u r a c i l ( C F ~ U ) a c y l a t e s a m i n e s i n aqueous media to
give u r a c i l - 5 - c a r b o x a m i d e s (33), it was s u g g e s t e d that the i r
r e v e r s i b l e i n a c t i v a t i o n of t h y m i d y l a t e synthetase m i g h t r e s u l t
f r o m a s i m i l a r a c y l a t i o n of a n a m i n o g r o u p at o r n e a r the
a c t i v e site of the e n z y m e as shown i n F i g u r e 9 (32).
A q u e s t i o n that a r o s e i s why the tr i f l u o r o m e t h y l g r o u p at
the 5 - p o s i t i o n of u r a c i l d e r i v a t i v e s s h o u l d be at a l l s u s c e p t i b l e
to these r e a c t i o n s . T h e c a r b o n - f l u o r i n e bond i s quite s t r o n g
(34) and a n outstanding c h a r a c t e r i s t i c of m o s t t r i f l u o r o m e t h y l
groups is their unusual r e s i s t a n c e toward c h e m i c a l d e g r a d a
t i o n . A s r e l e v a n t e x a m p l e s , it is noted that b e n z o t r i f l u o r i d e s ,
d e r i v a t i v e s of 6 - t r i f l u o r o m e t h y l u r a c i l s (35), d e r i v a t i v e s of 2-
t r i f l u o r o m e t h y l - 4 - o x o p y r i m i d i n e s (36) and 5 - t r i f l u o r o m e thy Ι
ό - a z a u r a c i l (37) a r e quite stable t o w a r d h y d r o l y t i c r e a c t i o n s .
In c o n t r a s t , U is r a p i d l y c o n v e r t e d into 5 - c a r b o x y u r a c i l
( C U ) (33 ) i n b a s i c m e d i a a n d , although s o m e w h a t s l o w e r , n u
c l e o s i d e s of C F ^ U a r e c o n v e r t e d into the c o r r e s p o n d i n g

4. SANTi E T A L . Thymidylate Synthetase
Figure 9
n u c l e o s i d e s of C U (38-40)
and C F g d U R p r o v i d e s C
f r o m p y r i m i d i n e s (41).
A n u m b e r of other c o m p o u n d s have been r e p o r t e d to h a v e r e
a c t i v e C - F g r o u p s (see r e f e r e n c e 42). T h e r e a c t i v i t y of C - F
bonds i n m o s t c a s e s has b e e n a t t r i b u t e d to h y p e r c o n j u g a t i v e e f
f e c t s (43, 44), h y d r o g e n bonding effects (44, 45), and d i r e c t d i s
p l a c e m e n t (S.^2) r e a c t i o n s (46). M o d e l r e a c t i o n s i n v o l v i n g the
h y d r o l y s i s o f c o m p o u n d s p o s s e s s i n g C - F bonds w e r e e x a m i n e d
in this l a b o t a t o r y i n an a t t e m p t to u n d e r s t a n d the u n d e r l y i n g f e a
t a r e s w h i c h r e s u l t e d i n the r e a c t i v i t y of s o m e of these m o l e
c u l e s (42). F r o m s u c h s t u d i e s we p r o p o s e d that C - F bond l a b i -
l i z a t i o n u s u a l l y i n v o l v e s one of s e v e r a l g e n e r a l m e c h a n i s m s ,
(a) A s d e p i c t e d i n F i g u r e 10a, p r o t o n r e m o v a l i s at an a t o m
a to the c a r b o n b e a r i n g the f l u o r i n e a t o m w i t h the r e s u l t a n t
negative c h a r g e , e i t h e r i n a s t e p w i s e o r c o n c e r t e d m a n n e r ,
a i d i n g i n the f o r m a t i o n of an i n t e r m e d i a t e (fluoro) a l k e n e .
D e p e n d i n g on the s t a b i l i t y of the a l k e n e , it m a y o r m a y not
react with solvent, (b) T h e p r o t o n m a y be s i t u a t e d on a n a t o m
s u c h that the n e g a t i v e c h a r g e r e s u l t i n g f r o m the i o n i z a t i o n of
the p r o t o n can e x e r t its i n f l u e n c e t h r o u g h an extended π - s y s t e m
( F i g u r e 10b). (c) W h e n the c o m p o u n d i s an a l l y l i c f l u o r i d e i n c a
pable of u n d e r g o i n g either of the m e c h a n i s m s d e s c r i b e d a b o v e ,
it m a y u n d e r g o n u c l e o p h i l i c ( M i c h a e l - t y p e ) attack at the β - c a r -
bon w i t h a s s i s t a n c e by the d e v e l o p i n g c a r b a n i o n to give an
i n t e r m e d i a t e s i m i l a r to those p r e v i o u s l y d e s c r i b e d ( F i g u r e
10c). In any of the a b o v e , t r i f l u o r o m e t h y l g r o u p s give
c a r b o x y l i c a c i d s o r d e r i v a t i v e s , d i f l u o r o m e t h y l g r o u p s give
a l d e h y d e s or k e t o n e s , and f l u o r o m e t h y l g r o u p s give a l c o h o l s
or alkenes.
M o s t of the C - F bond c l e a v a g e s thus f a r r e p o r t e d c a n be
e x p l a i n e d then i n t e r m s of the a f o r e m e n t i o n e d m e c h a n i s m s ; the
a b i l i t y of the c o m p o u n d s to f o r m o l e f i n i c i n t e r m e d i a t e s of the
type d e s c r i b e d a p p e a r s n e c e s s a r y f o r s u c h r e a c t i o n s to o c c u r .
T h e m e c h a n i s m ( s ) b y w h i c h the o l e f i n i c i n t e r m e d i a t e s a r e t r a n s
f o r m e d to p r o d u c t s i s b e l i e v e d to i n v o l v e a l t e r n a t e a d d i t i o n of

H
I I 5=t -X^-C-^F
— X-C—F
—X=C —product (a)
H
ι I Α Γ \ Ι Α
— X-(C=C) - C - F - X - * ( C = C ) i-C-L-F — •
— X=C—(C=C) —C=C n l > - » product (b)
N u :
—c=Q-c—F -*• —c—cic-ί —»
> ι ι /
—C—C=C —»—^-product (c)
I \
Nu
Figure 10
ι /
—C—CF. — C = C
I J
\
H F HO
II I II
—C—COH -*• — C — C — F v H
II yl , \ I
_ ! = / O H
^ - U o
\
c
Figure 11
n u c l e o p h i l e (or solvent) to the i n t e r m e d i a t e , and e l i m i n a t i o n of

f l u o r i d e i o n . A p o s s i b l e m e c h a n i s m f o r h y d r o l y s i s of the C F ^
g r o u p is d e p i c t e d i n F i g u r e 11 a n d , as shown, m a y i n v o l v e the
i n t e r m e d i a c y of a c y l f l u o r i d e s and k e t e n e s i n the t r a n s f o r m a
tion of a t r i f l u o r o m e t h y l g r o u p to a c a r b o x y l a t e f u n c t i o n , a l
though these i n t e r m e d i a t e s h a v e , as yet, not been d e t e c t e d .

T h e above p r o v i d e d i n s i g h t into the p o s s i b l e m e c h a n i s m s

by w h i c h C F ^ d U M P m i g h t a c t as an a c y l a t i n g agent. T o o b -
tain d i r e c t s u p p o r t i n g e v i d e n c e , the m e c h a n i s m s of h y d r o l y t i c
r e a c t i o n s of 5 - t r i f l u o r o m e t h y l u r a c i l a n d its N - a l k y l a t e d d e r i -
v a t i v e s w e r e e x a m i n e d i n d e t a i l (47). T h e r e s u l t s of these
studies a r e s u m m a r i z e d b e l o w , and d e p i c t e d i n F i g u r e s 12-14.
A l l r e a c t i o n s a p p e a r to p r o c e e d by f o r m a t i o n of a h i g h l y
reactive intermediate having an exocyclic difluoromethylene
g r o u p at the 5 - p o s i t i o n w h i c h s u b s e q u e n t l y r e a c t s w i t h w a t e r
o r h y d r o x i d e i o n i n a s e r i e s of r a p i d steps to give c o r r e s p o n -
ding 5 - c a r b o x y u r a c i l s . F o r those d e r i v a t i v e s w h i c h p o s s e s s
an i o n i z a b l e p r o t o n at the 1 - p o s i t i o n , the p r e d o m i n a n t m e c h a -
n i s m i n v o l v e s i o n i z a t i o n to the conjugate b a s e and a s s i s t a n c e
by the 1 - a n i o n i n the e x p u l s i o n of f l u o r i d e i o n ( F i g u r e 12).
W h e n i o n i z a t i o n at the 1 - p o s i t i o n i s p r e c l u d e d by the p r e s e n c e
of a n a l k y l substituent ( F i g u r e 13), a c y l a t i o n r e a c t i o n s p r o c e e d
by r a t e d e t e r m i n i n g attac
the n e u t r a l o r n e g a t i v e l
v i d e the r e a c t i v e i n t e r m e d i a t e . In o r d e r to o b t a i n s u i t a b l e i n -
t r a m o l e c u l a r m o d e l s , and to v e r i f y the p r i m a r y s i t e of r e a c t i o n
of 1 - s u b s t i t u t e d d e r i v a t i v e s , a s e r i e s of 1-(a>-aminoalkyl)tri-
f l u o r o m e t h y l u r a c i l s w e r e p r e p a r e d and t h e i r h y d r o l y s e s e x a -
m i n e d ( F i g u r e 14). N e i g h b o r i n g g r o u p p a r t i c i p a t i o n was a p p a -
r e n t w h e r e attack of the a m i n o g r o u p on the 6 - p o s i t i o n of the
h e t e r o c y c l e r e s u l t s i n the f o r m a t i o n of f i v e - , s i x - , and
s e v e n - m e m b e r e d r i n g s ; i n the c a s e of 1 - ( 3 - a m i n o p r o p y l ) - 5 - t r i -
f l u o r o m e t h y l u r a c i l , apparent f i r s t - o r d e r constants w e r e m o r e
than 10 t i m e s g r e a t e r than s i m p l e 1 - a l k y l d e r i v a t i v e s not
possessing a neighboring nucleophile.
W i t h r e g a r d to the e n z y m i c r e a c t i o n , the s a l i e n t f e a t u r e of
these studies i s that the t r i f l u o r o m e t h y l g r o u p of C F ^ d U M P
d e r i v a t i v e s w o u l d o n l y b e h a v e as an a c y l a t i n g agent when a s e -
c o n d a r y d r i v i n g f o r c e i s f u r n i s h e d by r e a c t i o n s w h i c h o c c u r at
other p a r t s of the h e t e r o c y c l e . T h a t i s , it i s n e c e s s a r y that a
n u c l e o p h i l e i s a d d e d to the 6 - p o s i t i o n of the h e t e r o c y c l e ; i n
this m a n n e r , the n o r m a l l y i n e r t t r i f l u o r o m e t h y l g r o u p w o u l d be
c o n v e r t e d into a h i g h l y r e a c t i v e e x o c y c l i c d i f l u o r o m e t h y l e n e
i n t e r m e d i a t e w h i c h m i g h t then a c y l a t e a n u c l e o p h i l i c g r o u p of
the e n z y m e .
In a c c o r d w i t h p r o p o s a l s f o r the i n v o l v e m e n t of n u c l e o p h i -
l i c c a t a l y s i s i n the e n z y m i c r e a c t i o n (viz I, F i g u r e 2), these
studies l e d us to p r o p o s e a r e l a t e d m i n i m a l m e c h a n i s m f o r the
r e p o r t e d i r r e v e r s i b l e i n a c t i v a t i o n of t h y m i d y l a t e synthetase b y
CFjdUMP. In the pathway d e p i c t e d i n F i g u r e 15, it was s u g -
g e s t e d that j u x t a p o s e d w i t h i n the a c t i v e s i t e , a n u c l e o p h i l i c
g r o u p of the e n z y m e (:Z) adds to the 6 - p o s i t i o n of C F ~ d U M P ,
p r o m o t i n g the e x p u l s i o n of f l u o r i d e i o n and the f o r m a t i o n of a
r e a c t i v e e x o c y c l i c d i f l u o r o m e t h y l e n e i n t e r m e d i a t e s i m i l a r to
those e n c o u n t e r e d i n o u r m o d e l s t u d i e s . The reactive inter-
m e d i a t e w o u l d then be t r a p p e d by a n u c l e o p h i l i c g r o u p of

Figure 13

4. SANTI E T A L . Thymidylate Synthetase 71
Figure 15

the e n z y m e to g i v e , a f t e r a n u m b e r of s t e p s , the a c y l a t e d
enzyme.
Subsequent to c o m p l e t i o n of these m o d e l s t u d i e s , the i n t e r -
a c t i o n of C F ^ d U M P w i t h t h y m i d y l a t e synthetase was r e i n v e s -
tigated i n another l a b o r a t o r y u s i n g the e n z y m e f r o m L . c a s e i
(21). T h e s e w o r k e r s o b s e r v e d that C F ^ U M P , C H . F A F L a n d
t h y m i d y l a t e synthetase f o r m e d a tight t e r n a r y c o m p l e x w n i c h
was i s o l a t a b l e by d i s c g e l e l e c t r o p h o r e s i s u n d e r n o n - d e n a t u r i n g
c o n d i t i o n s . H o w e v e r , u n l i k e the F d U M P * C H ^ F A H ^ e n z y m e
c o m p l e x , no change i n the d i f f e r e n c e s p e c t r a w a s o b s e r v e d
w h e n C F g d U M P was u s e d . F u r t h e r m o r e , g e l e l e c t r o p h o r e -
s i s i n the p r e s e n c e of a p r o t e i n d é n a t u r a n t r e s u l t e d i n a p -
p a r e n t d e s t r u c t i o n of the c o m p l e x . A f t e r d e n a t u r a t i o n of
the c o m p l e x , the n u c l e o t i d e p r o d u c t was o b s e r v e d to m i -
g r a t e i d e n t i c a l l y w i t h authentic C F ^ d U M P on D E A E - c e l l u -
l o s e p a p e r . F r o m these r e s u l t s i t was c o n c l u d e d that C - F
bonds of the n u c l e o t i d
n u c l e o p h i l e of the e n z y m
C F dUMP.
R e c e n t r e s u l t s o b t a i n e d i n this l a b o r a t o r y a r e not i n
a c c o r d w i t h t h e s e f i n d i n g s . A l t h o u g h the m e c h a n i s m of r e -
a c t i o n of C F ^ d U M P w i t h t h y m i d y l a t e synthetase has not b e e n a s -
c e r t a i n e d at this t i m e , i t a p p e a r s c e r t a i n that C - F bond c l e a -
v a g e i s c a t a l y z e d by the e n z y m e , p r o b a b l y v i a n u c l e o p h i l i c c a -
t a l y s i s . O u r p r e l i m i n a r y r e s u l t s w h i c h l e a d to this c o n c l u s i o n
are s u m m a r i z e d below.
C o n t r a r y to the p r e v i o u s r e p o r t (2 1), we o b s e r v e that s u b -
t r a c t i o n of the u l t r a v i o l e t s p e c t r u m of e n z y m e a n d C F L F A F L
f r o m that of the e n z y m e , C H ^ F A H ^ a n d C F ^ d U M P p r o d u c e s a
d i f f e r e n c e s p e c t r a ( F i g u r e 16a) w h i c h i s v e r y s i m i l a r to that
o b s e r v e d w i t h the F d U M Ρ · C H ^ F A H ^ · e n z y m e t e r n a r y c o m p l e x
( F i g u r e 4). A s w i t h the t h y m i a y l a t e s y n t h e t a s e ^ F d U M P -
C H ^ F A H ^ c o m p l e x , t h e r e i s the c h a r a c t e r i s t i c i n c r e a s e of a b -
s o r b a n c e at 330 n m and a d e c r e a s e at 261 n m ; the l a t t e r is i n
a c c o r d w i t h s a t u r a t i o n of the 5, 6 - d o u b l e bond of the n u c l e o t i d e .
U p o n a d d i t i o n of s o d i u m d o d e c y l s u l f a t e , the o n l y d i f f e r e n t i a l
a b s o r b a n c e i s that c h a r a c t e r i s t i c of a n u c l e o t i d e w i t h λ »
267 n m ; although this i s u p f i e l d f r o m the m a x i m u m of
C F ^ d U M P , we have not y e t a s c e r t a i n e d w h e t h e r a n a l t e r a t i o n
of s t r u c t u r e i s i n v o l v e d o r whether the s h i f t i s a r t i f a c t u a l .
In the a b s e n c e of C H ^ F A H the i n c u b a t i o n of t h y m i d y l a t e
s y n t h e t a s e and C F ^ d U M F ^ ( r a t i o 1:6) f o r 20 m i n u t e s at 2 2 °
r e s u l t s i n 89% i n a c t i v a t i o n of the e n z y m e . T h i s i s i n a c c o r d
w i t h p r e v i o u s r e p o r t s on the effect of this n u c l e o t i d e on the e n
z y m e f r o m E h r l i c h a s c i t e s c e l l s (32). The difference spec
t r a of C F g d U M P and e n z y m e vs e n z y m e i s shown i n F i g u r e 16b.
T h e m a x i m u m of C F ^ d U M P at 261 n m d e c r e a s e s , and a t r a n
sient b r o a d peak a p p e a r s w h i c h has a b s o r b t i o n u p to c a .
340 n m . A f t e r 1 h o u r , the f i n a l s p e c t r u m e x h i b i t s a m a x i m a
at 276 n m , r e s e m b l i n g 5 - a c y l d e r i v a t i v e s of d U M P . Paper

ι ι ι ι ι
260 300 340
W A V E L E N G T H
Figure 16. Ultraviolet difference spectra, (a) Dashed line: CF dUM? s CH FAH
y t and
k
thymidylate synthetase vs. CH FAHt k and thymidylate synthetase. Solid line: after treat-
ment with sodium dodecyl sulfate, (b) Dashed line: CF dUMP s and thymidylate syn
thetase vs. thymidylate synthetase after 20 seconds. Solid line: after 1 hr. Broken line:
ultraviolet spectrum of CF dUMP.s

c h r o m a t o g r a p h y of this r e a c t i o n m i x t u r e shows a s i n g l e spot

w h i c h m o v e s s l i g h t l y s l o w e r than the s t a r t i n g m a t e r i a l
(C^dUMP). A l t h o u g h this p r o d u c t h a s not yet b e e n i d e n t i f i e d ,
i t i s not 5 - c a r b o x y - d U M P . W h e n C R d U M P was t r e a t e d w i t h
a l i m i t i n g a m o u n t of t h y m i d y l a t e synthetase (50:1) f o r 23 h o u r s
at 2 2 ° , we w e r e a b l e to d e t e c t that at l e a s t 0.3 e q u i v a l e n t s of
F ~ w e r e r e l e a s e d , d e m o n s t r a t i n g that C - F bonds of the n u c l e o
tide w e r e i n d e e d l a b i l i z e d .
A l t h o u g h these r e s u l t s a r e too p r e l i m i n a r y to p e r m i t d e f i n i
tive i n t e r p r e t a t i o n , c e r t a i n c o n c l u s i o n s m a y be r e a c h e d a n d s p e
c u l a t i o n s m a y be f o r w a r d e d . It i s c l e a r that the i n t e r a c t i o n of
C I ^ d U M P and t h y m i d y l a t e synthetase i n the a b s e n c e of c o f a c
tor m a y r e s u l t i n c l e a v a g e of C - F bonds of the n u c l e o t i d e as
w e l l as i n a c t i v a t i o n of the e n z y m e . F r o m the a f o r e m e n t i o n e d
m o d e l s t u d i e s , it i s m o s t r e a s o n a b l e to p r o p o s e that a c t i v a t i o n
of the C - F bond r e q u i r e s a d d i t i o n of a n u c l e o p h i l e of the e n z y m e
tu the 6 - p o s i t i o n of th
i n a c t i v a t i o n i s not k n o w n
than the r e a c t i o n l e a d i n g to C - F bond c l e a v a g e . It i s a l s o a p
p a r e n t that the p r e s e n c e of C H ^ F A H ^ m o d u l a t e s this r e a c t i o n
i n s o m e y e t unknown m a n n e r ; tne e n z y m e * C F ^ d U M P C H 2 F A H ^#
c o m p l e x h a s u l t r a v i o l e t s p e c t r a l q u a l i t i e s quite s i m i l a r to
those of the c o m p l e x f o r m e d w i t h F d U M P , i n d i c a t i n g
that the 5, 6 - d o u b l e bond of d U M P i s s a t u r a t e d i n the
ternary complex. Further experiments are i n progress which
a i m to e l u c i d a t e the m e c h a n i s m of i n t e r a c t i o n of d U M P with
thymidylate synthetase.
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5
The Effect of Aliphatic Fluorine on Amine Drugs
RAYW.FULLERandBRYANB.MOLLOY
The Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Ind. 46206
Fluorine is th
(Table I), and its presence in a molecule can greatly
affect the ionization of acids and bases (2). The
withdrawal of electrons toward fluorine on a carbon
atom adjacent to a carboxyl carbon or an amino-bearing
carbon increases the carboxyl group's ability to re
lease a proton and decreases the amine's ability to
accept a proton. The carboxylic acid becomes a
stronger acid, and the amine becomes a weaker base.
For example, the pK of ethylamine is >10 compared to
a
5.7 forβ,β,β-trifluoroethylamine(3).
We have studied some sympathomimetic amine drugs
having reduced basicity due to fluorine substituted on
the β carbon. The influence of ionization on the ac
tivity of sympathomimetic amines has previously been
considered (4), but with the exception of compounds
having substituents directly on the nitrogen, no such
amines having pK values below physiological pH appear
a
to have been available (5). An advantage of the β,β-
difluoro compounds is that the substitution is in a
position of the molecule that is not a major site of
metabolic attack nor a site known to be involved in
binding to physiological receptors, and the small size
of the fluorine atoms (Table I) results in minimal
steric alteration in the molecule. Thus the changes
in the pharmacological properties of the amines as a
result of β,β-difluoro substitution probably are due
to the change in ionization.
In solution an amine exists in the following
equilibrium,
-H+
RNH3+ V ^ RNH2
+H+
77

the p o s i t i o n o f t h e e q u i l i b r i u m depending on t h e p K a
o f t h e amine and t h e pH o f t h e s o l u t i o n . When t h e pH

i s e q u a l t o t h e p K , 50% o f t h e drug m o l e c u l e s a r e p r o -
a
t o n a t e d ( c a t i o n i c ) and 50% a r e n o n p r o t o n a t e d ( n e u t r a l ) .
When t h e pH i s one u n i t below t h e p K , j u s t o v e r 90% a
o f t h e m o l e c u l e s e x i s t i n t h e c a t i o n i c form, and when

the pH i s two u n i t s below t h e p K more t h a n 99% o f t h e
a
m o l e c u l e s a r e charged. I n t h i s paper we compare some

p r i m a r y amines w i t h p K v a l u e s w e l l above p h y s i o l o g i c a l
a
pH t o t h e i r 3 , 3 - d i f l u o r o d e r i v a t i v e s h a v i n g p K v a l u e s
a
below p h y s i o l o g i c a l pH. The p a r e n t amines a r e n e a r l y

c o m p l e t e l y c a t i o n i c whereas t h e 3 , 3 - d i f l u o r o deriva-
t i v e s e x i s t p r e d o m i n a n t l y as n e u t r a l m o l e c u l e s a t
p h y s i o l o g i c a l pH. Presumably as a r e s u l t o f t h i s
charge d i f f e r e n c e , t h e p h a r m a c o l o g i c p r o p e r t i e s o f t h e
d r u g s — b o t h t h e b i o l o g i c a l f a t e and t h e b i o l o g i c a l
actions of the d r u g s — a r
TABLE I
Fluorine in Relation to Other Halogen Atoms 1
Atom Van der Waals Electronegativity

radius, value
0
A
F 1. 35 4. 0
Cl 1. 80 3. 0
Br 1.95 2.8
I 2. 15 2.5
H 1.2 2. 1
1
Data from (1).
Amphetamine
The e f f e c t on i o n i z a t i o n o f amphetamine produced

by t h e p r e s e n c e o f one o r two f l u o r i n e atoms on t h e
3 carbon i s shown i n T a b l e I I . A s i n g l e f l u o r i n e atom
reduces t h e p K v a l u e by more than one pH u n i t . The
a
p K o f t h e monofluoro d e r i v a t i v e o f amphetamine i s
a
s t i l l above p h y s i o l o g i c a l pH, however, so t h a t most o f

5. FULLER AND MOLLOY Aliphatic Fluorine 79
the monofluoro compound i s i o n i z e d a t p h y s i o l o g i c a l pH.

More t h a n 99% o f t h e m o l e c u l e s o f amphetamine e x i s t as
c a t i o n s a t p h y s i o l o g i c a l pH. In c o n t r a s t , t h e d i f l u o r o
compound has a p K below p h y s i o l o g i c a l pH and thus
a
would e x i s t m a i n l y as a n e u t r a l m o l e c u l e r a t h e r t h a n as
a c a t i o n i n t h e body.
TABLE II
Effect of ^ Fluorine Substution on the

Ioni zation οf Amphetamine
Compound Distribution of Ion ic Forms

at pH 7. 4
RNH 2
RNH z+
\ y-CH CHNH 2 2
\=y 1
CH 3
(/ y-CHCHNH 8. 35 10 90
\=/ 1 1
F CH 2
X y-CF CHNH
2 2 6.97 73 27
\=y 1
CH 3
The i n t e r a c t i o n o f amphetamine w i t h b i o l o g i c a l
macromolecules ought t o be p r o f o u n d l y a f f e c t e d by t h e
f l u o r i n e s u b s t i t u t i o n and a t t e n d a n t i o n i c changes,
s i n c e t h e t r a n s p o r t and enzymic systems t h a t determine
d i s t r i b u t i o n and m e t a b o l i s m o f t h e drug s h o u l d a c t
d i f f e r e n t l y on a n e u t r a l u n p r o t o n a t e d amine compared
to a c a t i o n . A number o f s t u d i e s on t h e i n t e r a c t i o n
o f amphetamine and i t s f l u o r i n a t e d d e r i v a t i v e s w i t h
enzymes and o t h e r p r o t e i n s i n v i t r o and o f t h e pharma
c o l o g i c c h a r a c t e r i s t i c s o f t h e s e drugs i n v i v o have
borne o u t t h e e x p e c t e d a l t e r a t i o n s i n p r o p e r t i e s o f
amphetamine r e s u l t i n g from f l u o r i n e s u b s t i t u t i o n .
In V i t r o I n t e r a c t i o n s . β,β-Difluoro substitution
i n c r e a s e s t h e a c t i v i t y o f amphetamine as an i n v i t r o
s u b s t r a t e f o r l u n g N - m e t h y l t r a n s f e r a s e (6) o r l i v e r
microsomal deaminase (Ί). On t h e o t h e r H a n d , β , β -

d i f l u o r o s u b s t i t u t i o n d e c r e a s e s t h e a c t i v i t y o f amphet
amine as an i n h i b i t o r o f m i t o c h o n d r i a l monoamine o x i
dase (Table I I I ) . L i k e w i s e , β , β - d i f l u o r o s u b s t i t u t i o n
d i m i n i s h e s t h e b i n d i n g o f amphetamine t o b o v i n e serum
albumin (Table I I I ) . Thus r e d u c i n g t h e p K o f amphet a
amine by β , β - d i f l u o r o s u b s t i t u t i o n can e i t h e r enhance

or i n h i b i t i t s i n t e r a c t i o n s with v a r i o u s b i o l o g i c a l
macromolecules i n v i t r o .
TABLE III
Effect of &jβ-Difluoro Substitution on Two Properties

of Amphetamine : Monoamine Oxidase Inhibition
and Binding to Bovine Serum Albumin
Drug
Amphetamine 3. 23 82
ft, β-Difluoroamphetamine 2. 56 45
MAO inhibition was measured with rat liver mito

chondrial enzyme and ^C'-tryptamine as substrate. The
pl50 value is the negative log of the molar concentra
tion of inhibitor producing 50% inhibition. Binding to
4% bovine serum albumin in pH 7.4 sodium phosphate buf
fer was measured with 10 micromolar drug concentration;
after f i l t r a t i o n through Aminco Centriflo u l t r a f i l t r a
tion cones drug levels were assayed colorimetrically
with methyl orange.
In V i v o P r o p e r t i e s . The e f f e c t o f β - f l u o r i n e sub
s t i t u t i o n on t h e d i s t r i b u t i o n o f amphetamine among
v a r i o u s body t i s s u e s a f t e r d r u g a d m i n i s t r a t i o n t o ex
p e r i m e n t a l a n i m a l s was s t r i k i n g . F i g u r e 1 shows t h e
t i s s u e d i s t r i b u t i o n o f amphetamine, β-fluoroamphetamine,
and β , β - d i f l u o r o a m p h e t a m i n e i n r a t s one hour a f t e r t h e
drugs were i n j e c t e d i n t r a p e r i t o n e a l l y a t e q u i m o l a r
doses. H i g h e s t t i s s u e l e v e l s o f amphetamine were i n
l u n g , and l o w e s t l e v e l s were i n t h e f a t , a l l t i s s u e s
h a v i n g h i g h e r l e v e l s than b l o o d . The r e l a t i v e d i s t r i
b u t i o n o f t h e monofluoro d e r i v a t i v e was s i m i l a r , l e v e l s
i n a l l t i s s u e s e x c e p t l u n g b e i n g about t h e same as
t h o s e o f amphetamine. T h i s s i m i l a r i t y i s e x p e c t e d
s i n c e t h e monofluoro compound, l i k e amphetamine, i s
m o s t l y p r o t o n a t e d a t p h y s i o l o g i c a l pH. I n marked con
t r a s t was t h e d i s t r i b u t i o n o f t h e d i f l u o r o d e r i v a t i v e .

5. FULLER A N D MOLLOY Aliphatic Fluorine 81
H i g h e s t l e v e l s o f t h i s d r u g were i n f a t , t h e t i s s u e
t h a t c o n t a i n e d l o w e s t l e v e l s o f t h e o t h e r two amines.
L e v e l s o f t h e d i f l u o r o compound i n o t h e r t i s s u e s were
c o n s e q u e n t l y reduced; f o r example, t h e l e v e l s i n b r a i n
were o n l y about o n e - f o u r t h t h o s e o f amphetamine. This
d i s t r i b u t i o n a t one hour i s s i m i l a r t o t h a t seen a t
o t h e r times ( 7 ) , i . e . t h e r e i s l i t t l e r e d i s t r i b u t i o n
o f drug and t K e r a t e o f d i s a p p e a r a n c e o f t h e drugs from
a l l t i s s u e s i s about t h e same.
S i n c e t h e predominant p h a r m a c o l o g i c e f f e c t s o f
amphetamine r e s u l t from i t s a c t i o n on t h e b r a i n , we
compared r e g i o n a l d i s t r i b u t i o n o f amphetamine and 3 , 3 -
d i f l u o r o a m p h e t a m i n e i n b r a i n (Table I V ) . The drugs
were g i v e n a t doses chosen t o produce comparable whole
brain levels. Some d i f f e r e n c e s i n d i s t r i b u t i o n among
the v a r i o u s anatomi region noted but thes
not as g r e a t as t h
v a r i o u s o r g a n s . Th
amphetamine i n b r a i n was n o t s i g n i f i c a n t l y a l t e r e d
by d i f l u o r o s u b s t i t u t i o n (1) .
TABLE IV
Regional Distribution of Amphetamine and

3, β-Dif'luoroamphetamine in Rat Brain
Brain Region Drug Level, nanomoles/g

(% of Total Brain Weight)
Amphetamine 3> $-Difluoro
amphetamine
Cerebral hemispheres 46 ± 4 57 ± 2
(62-67%)
Cerebellum (14-17%) 18 ± 6 33 ± 3
Midbrain (8-10%) 54 ± 4 92 ± 6
Brain stem (6-10%) 65 ± 20 71 ± 7
Hypothalamus (3-4%) 86 ± 11 84 ± 6
Drugs were injected i . p . (amphetamine at 10 mg/kg,

3,3-difluoroamphetamine at 40 mg/kg) 1 hour before the
rats were killed. Mean values and standard errors for
5 rats per group are shown.

J u s t as t h e t i s s u e d i s t r i b u t i o n o f amphetamine i s
changed by β, β - d i f l u o r o s u b s t i t u t i o n , so a l s o i s t h e
metabolism o f t h e drug changed. A l t h o u g h t h e h a l f -
l i v e s o f amphetamine and β , β - d i f l u o r o a m p h e t a m i n e a r e
about t h e same i n r a t s , t h e pathways o f m e t a b o l i s m f o r
the two drugs a r e d i f f e r e n t (Table V ) . Amphetamine i s
m e t a b o l i z e d i n t h e r a t p r e d o m i n a n t l y by h y d r o x y l a t i o n
on t h e p a r a p o s i t i o n o f t h e a r o m a t i c r i n g , whereas
β , β - d i f l u o r o a m p h e t a m i n e appears n o t t o be m e t a b o l i z e d
by p a r a - h y d r o x y l a t i o n a t a l l (7). Instead the d i
f l u o r o compound i s m e t a b o l i z e d r a p i d l y by o x i d a t i v e
d e a m i n a t i o n (7). T i s s u e l e v e l s o f amphetamine and i t s
h a l f - l i f e a r e i n c r e a s e d by agents l i k e d e s m e t h y l i m i p r a -
mine t h a t i n h i b i t a r o m a t i c h y d r o x y l a t i o n , whereas t i s
sue l e v e l s o f β , β - d i f l u o r o a m p h e t a m i n e a r e i n c r e a s e d by
t y p i c a l i n h i b i t o r s o f microsomal enzymes l i k e SKF 525A
and DPEA {7)· Inductio
by c h r o n i c phénobarbita
the r a t e o f removal o f β , β - d i f l u o r o a m p h e t a m i n e from
t i s s u e s a f t e r i t i s i n j e c t e d i n t o r a t s b u t has no
e f f e c t on t h e r a t e o f removal o f amphetamine (0).
β , β - D i f l u o r o s u b s t i t u t i o n increases the a c t i v i t y of
amphetamine as a s u b s t r a t e f o r microsomal deaminases,
an e f f e c t t h a t can be shown i n v i t r o . Apparently the
d i f l u o r o s u b s t i t u t i o n abolisKes the a c t i v i t y of
amphetamine as a s u b s t r a t e f o r t h e a r o m a t i c hydroxy-
l a t i n g system, though t h i s system i s d i f f i c u l t t o
study i n v i t r o . The f a i l u r e o f t h e d i f l u o r o compound
t o be H y d r o x y l a t e d i l l v i v o may be due t o one o r b o t h
of the f o l l o w i n g reasons: (a) i t i s more r e a d i l y
deaminated t h a n i s amphetamine, (b) i t may be l e s s
r e a d i l y h y d r o x y l a t e d t h a n amphetamine.
TABLE V
Effect of &,$-Difluoro Substitution on

Amphetamine Metabolism in the Rat
Half-life
Drug
in Brain Major Metabolic Route
Amphetamine 1.δ hrs Ring hydroxylation
β, $-Difluoro 1.5 hrs Oxidative deamination

amphetamine

3,3-Difluoroamphetamine has been shown t o i n c r e a s e

locomotor a c t i v i t y i n mice t o the same e x t e n t as
amphetamine (9), though a p p r o x i m a t e l y f o u r t i m e s h i g h e r
doses o f the 3 i f l u o r o compound have t o be g i v e n .
L i k e w i s e about f o u r times h i g h e r doses o f t h e d i f l u o r o
compound a r e r e q u i r e d t o produce e q u i v a l e n t drug l e v e l s
i n b r a i n compared t o amphetamine, so t h e i n t r i n s i c
a b i l i t y o f t h e d i f l u o r o compound t o cause CNS s t i m u l a -
t i o n seems about e q u a l t o t h a t o f amphetamine.
In mice, t h e h a l f - l i f e o f t h e d i f l u o r o compound
(0.3 hours) i s much s h o r t e r than i s t h a t o f amphetamine
(0.9 h o u r s ) . A p p a r e n t l y t h i s d i f f e r e n c e i s r e l a t e d t o
the f a c t t h a t mice have t h e enzymic machinery t o metab-
o l i z e amphetamine by o x i d a t i v e d e a m i n a t i o n (10), and
s i n c e t h e d i f l u o r o d e r i v a t i v e i s a much b e t t e r
s u b s t r a t e f o r d e a m i n a t i o n i t i s m e t a b o l i z e d much more
r e a d i l y i n mice t h a
of amphetamine and 3 , 3
s i m i l a r r e s u l t s i n mice and i n r a t s .
One use t h a t has been made o f 3 , 3 - d i f l u o r o a m p h e t -
amine i s as a t o o l t o e l u c i d a t e i n t e r r e l a t i o n s h i p s
among the v a r i o u s a c t i o n s o f amphetamine. Gessa, C l a y
and B r o d i e (11) had s u g g e s t e d t h a t the h y p e r t h e r m i a
f o l l o w i n g ampEetamine i n j e c t i o n i n t o r a t s was a d i r e c t
consequence o f t h e e l e v a t i o n o f plasma f r e e f a t t y
acids. We found t h a t 3 , 3 - d i f l u o r o a m p h e t a m i n e injected
at an e q u i m o l a r dose e l e v a t e d f r e e f a t t y a c i d s t o t h e
same e x t e n t as amphetamine but produced no h y p e r t h e r m i a
(12), showing t h a t t h e s e two e f f e c t s o f amphetamine
were c o m p l e t e l y d i s s o c i a b l e by v i r t u e o f d i f l u o r o
substitution. Presumably the h y p e r t h e r m i c response t o
amphetamine i s due t o an a c t i o n i n b r a i n , hence 3*3-
d i f l u o r o a m p h e t a m i n e produces h y p e r t h e r m i a o n l y when i t
i s i n j e c t e d a t higher doses.
Another d i f f e r e n c e between amphetamine and 3,3-
d i f l u o r o a m p h e t a m i n e i s t h e i n a b i l i t y o f t h e l a t t e r drug
to cause d e p l e t i o n o f n o r e p i n e p h r i n e l e v e l s i n b r a i n
and h e a r t (Table V I ) . H i g h doses o f amphetamine have
l o n g been known t o d e p l e t e n o r e p i n e p h r i n e i n t h e s e
t i s s u e s (13). We gave t h e d i f l u o r o compound a t f o u r
times the~cTose o f amphetamine t o produce e q u i v a l e n t
drug l e v e l s i n b r a i n and h e a r t but s t i l l found no
reduction of norepinephrine l e v e l s . The p r e c i s e
mechanism(s) by which amphetamine lowers n o r e p i n e p h r i n e
l e v e l s i s s t i l l u n c e r t a i n , but h y d r o x y l a t e d m e t a b o l i t e s
may p l a y a r o l e i n t h i s e f f e c t . The f a i l u r e o f 3/3-
d i f l u o r o a m p h e t a m i n e t o lower n o r e p i n e p h r i n e c o u l d t h e n
be e x p l a i n e d because i t i s not m e t a b o l i z e d by hydroxy-
lation.

TABLE VI
Inability of β,ë-Difluoroamphetamine to Cause

Amphetamine-Like Depletion of Tissue Norepinephrine
Norepinephrine Levels, vg/g

Treatment Group Heart Brain
Control 0.89±.05 0.40±.01
dl-Amphetamine 0.58±.01 (P<.001) 0.351.01 (P<.05)

0.1 mmole/kg
dl-&,&-Difluoro- 0.91±.06 (ns) 0.41±.03 (ne)

amphetamine
0.4 mmole/kg
Drugs were injected i.p. 6 hours before the rats were

killed. Mean values and standard errors for 5 rats per
group are shown.
Phenethylamine
The i o n i z a t i o n o f phenethylamine i s a f f e c t e d by
β - f l u o r i n e s u b s t i t u t i o n i n much t h e same way as t h a t o f
amphetamine (Table V I I ) . A s i n g l e f l u o r i n e reduces t h e
p K , and a second f l u o r i n e f u r t h e r reduces t h e p K t o
a a
below p h y s i o l o g i c a l pH.
TABLE VII
Effect of Fluorine Substitution on the

Ionization of PhenethyI amine
Distribution of Ionic Forms

Compound pK a at pH 7.4
RNH2 RNH3+
CH2CH2NH2 9.55 99
Ο CHCH 2NH 2
I
F
8. 20 13 87
^~^-CF2CH2NH2 6. 75 82 18

Figure 1. Tissue distribution of amphetamine in rats as affected by

fluorine substitution on the β carbon.
Drugs were injected i.p. at 0.1 mmole/kg 1 hr before groups of 5
rats were killed. Mean values and standard errors are shown (7).
Figure 2. Effect of β fluorine atoms on the

activity of phenethylamine as an enzyme
substrate.
The phenethulamines were compared at 4
mM with the N-methyltransjerase and at
1 mM with monoamine oxidase.

In V i t r o I n t e r a c t i o n s . The e f f e c t o f β - f l u o r i n e
s u b s t i t u t i o n on the a c t i v i t y o f phenethylamine as a
s u b s t r a t e f o r two enzyme systems i n v i t r o i s shown i n
F i g u r e 2. A d d i t i o n o f one and two f l u o r i n e atoms
p r o g r e s s i v e l y r e d u c e d t h e a c t i v i t y o f phenethylamine
as a s u b s t r a t e f o r m i t o c h o n d r i a l monoamine o x i d a s e b u t
p r o g r e s s i v e l y i n c r e a s e d a c t i v i t y as a s u b s t r a t e f o r
l u n g N - m e t h y 1 t r a n s f e r a s e . The l a t t e r enzyme p r o b a b l y
i s n o t i m p o r t a n t i n phenethylamine metabolism i n v i v o
but i l l u s t r a t e s t h a t l o w e r i n g t h e p K o f t h e amine can
a
i n c r e a s e as w e l l as d e c r e a s e i t s a f f i n i t y f o r enzymes.
The a b i l i t y o f monofluoro and d i f l u o r o phenethylamines
t o i n h i b i t t h e o x i d a t i o n o f 14c-phenethylamine by t h r e e
p r e p a r a t i o n s o f monoamine o x i d a s e i s shown i n F i g u r e 3.
There was a s u b s t a n t i a l d i f f e r e n c e i n t h e i n h i b i t o r y
a c t i v i t y o f t h e two compounds and t h i s d i f f e r e n c e
appeared t o r e s u l t e n t i r e l
values. P l o t t i n g percen
c o n c e n t r a t i o n o f p r o t o n a t e d form o f t h e two i n h i b i t o r s
r e v e a l e d t h a t t h e p o i n t s f e l l a l o n g t h e same l i n e ; t h i s
f i n d i n g s u g g e s t s t h a t t h e RNH3+ form i s t h e a c t i v e
i n h i b i t o r o f monoamine o x i d a s e and t h a t t h i s form o f
b o t h compounds has e q u a l a f f i n i t y f o r t h e enzyme.
In V i v o P r o p e r t i e s . Phenethylamine i t s e l f was
v e r y r a p i d l y degraded by monoamine o x i d a s e when i t was
i n j e c t e d i n t o a n i m a l s , b u t t h e d i f l u o r o d e r i v a t i v e was
l e s s r e a d i l y d e s t r o y e d . I t s l e v e l s were h i g h e r t h a n
those o f phenethylamine i n a l l t i s s u e s , and t h e t i s s u e
d i s t r i b u t i o n o f t h e d i f l u o r o compound resembled v e r y
much t h a t o f β , β - d i f l u o r o a m p h e t a m i n e ( 1 4 ) .
Thus d i f l u o r o s u b s t i t u t i o n on phenethylamine l e d
t o g r e a t e r b i o l o g i c a l s t a b i l i t y whereas d i f l u o r o
s u b s t i t u t i o n on amphetamine d e c r e a s e d b i o l o g i c a l
s t a b i l i t y a t l e a s t i n some s p e c i e s . Figure 4
i l l u s t r a t e s t h i s d i f f e r e n c e i n the e f f e c t o f d i f l u o r o
s u b s t i t u t i o n on i n v i t r o metabolism o f phenethylamine
and amphetamine E y l i v e r homogenates. The metabolism
b e i n g measured i s d e a m i n a t i o n i n b o t h c a s e s ; microsomal
enzymes a r e r e s p o n s i b l e f o r t h e d e a m i n a t i o n o f
amphetamine, whereas m i t o c h o n d r i a l monoamine o x i d a s e
i s r e s p o n s i b l e f o r the d e a m i n a t i o n o f p h e n e t h y l a m i n e .
β , β - D i f l u o r o a m p h e t a m i n e was more r a p i d l y d e s t r o y e d than
amphetamine, b u t β , β - d i f l u o r o p h e n e t h y l a m i n e was l e s s
r a p i d l y destroyed than phenethylamine. In r a t s , t h e
a d d i t i o n a l metabolic route of para-hydroxylation
o p e r a t e s on amphetamine i n v i v o , so i n f a c t amphetamine
i s d e s t r o y e d a t about t h e same r a t e as d i f l u o r o a m p h e t
amine i n r a t s b u t more s l o w l y i n mice. Phenethylamine

(aj Soluble MAO (rat] (b) Mitochononal MAO (rat) (c) Mitochondrial MAO (human)
10 100 1000 10 100 1000 10 100 1000

Micromolar concentration of inhibitor
/ / /
10 100 1000 10 100 1000 10 100 1000
Micromolar concentration of RNH 3
+
form of inhibitor
Figure 3 Inhibition of three monoamine oxidase preparations by β-monofluoro-

phenethylamine (dots) and bu β,β-difluoro-phenethylamine (half circles). The substrate
was phenethylamine (.2 mM). Total inhibitor concentration is shown at the top, and
the concentration of the protonated form of the inhibitor (pH 7.4) is shown at the
bottom. Monoamine oxidase was solubilized from rat liver mitochondria (a), or in
tact liver mitochondria were used as enzyme source (b) and (c).
(a) lb)
minutes
Figure 4. Effect of β,β-difluoro substitution on the in vitro degra

dation of (a) amphetamine and (b) phenethylamine by rat liver
homogenates.
The amines were added at .125 mM concentrations to liver homoge
nates.

i s d e s t r o y e d more r a p i d l y t h a n d i f l u o r o p h e n e t h y l a m i n e
b o t h i n mice and i n r a t s .
In mice, t h e c e n t r a l s t i m u l a n t a c t i v i t y o f 3 , 3 -
d i f l u o r o p h e n e t h y l a m i n e becomes a p p a r e n t a t lower doses
than w i t h phenethylamine i t s e l f , b u t i n mice p r e t r e a t e d
w i t h an i n h i b i t o r o f monoamine o x i d a s e t h e c o n v e r s e i s
t r u e (14). Two f a c t o r s a r e i n v o l v e d : the greater
b i o l o g i c a l s t a b i l i t y o f t h e d i f l u o r o compound and t h e
more f a v o r a b l e ( f o r b r a i n ) t i s s u e d i s t r i b u t i o n o f
phenethylamine i t s e l f . I n normal mice t h e m e t a b o l i c
d i f f e r e n c e s a r e more i m p o r t a n t and so t h e d i f l u o r o com-
pound i s more a c t i v e as a s t i m u l a n t than i s p h e n e t h y l -
amine. When t h e m e t a b o l i c d i f f e r e n c e s a r e e l i m i n a t e d
by i n h i b i t i o n o f t h e enzyme r e s p o n s i b l e (monoamine
o x i d a s e ) , then phenethylamine has more s t i m u l a n t
a c t i v i t y t h a n t h e d i f l u o r o compound because o f t h e
1
l a t t e r s tendency t
b r a i n and o t h e r o r g a n s
p-Chloroamphetamine
p-Chloroamphetamine i s a drug o f s p e c i a l i n t e r e s t
because o f i t s s e l e c t i v e e f f e c t s on s e r o t o n i n neurons
in brain. p-Chloroamphetamine l e a d s t o a r a p i d
d e c r e a s e i n t h e l e v e l s o f s e r o t o n i n and i t s major
metabolite, 5-hydroxyindoleacetic a c i d , i n b r a i n . In
the r a t and some o t h e r s p e c i e s , p-chloroamphetamine
leads further to c y t o t o x i c d e s t r u c t i o n o f serotonin
neurons as i n d i c a t e d by r e m a r k a b l y l o n g - l a s t i n g
d e c r e a s e s i n parameters s p e c i f i c a l l y a s s o c i a t e d w i t h
s e r o t o n i n neurons i n b r a i n ( t r y p t o p h a n h y d r o x y l a s e ,
s e r o t o n i n and 5 - h y d r o x y i n d o l e a c e t i c a c i d l e v e l s ; h i g h -
a f f i n i t y s e r o t o n i n uptake) (15,16) and by d i r e c t
h i s t o l o g i c e v i d e n c e ( 1 7 ) . TEïïs i t was o f i n t e r e s t t o
study t h e d i f l u o r o d e r i v a t i v e o f p-chloroamphetamine.
The r e d u c t i o n o f t h e p K o f p-chloroamphetamine
a
by 3 , 3 - d i f l u o r o s u b s t i t u t i o n i s shown i n T a b l e V I I I .
The t i s s u e d i s t r i b u t i o n o f p-chloroamphetamine, which
resembles t h a t o f amphetamine i t s e l f , was a l t e r e d by
the d i f l u o r o s u b s t i t u t i o n . 3,3-Difluoro-p-chloro-
amphetamine l o c a l i z e d t o an even g r e a t e r e x t e n t i n f a t
than d i d 3 , 3 - d i f l u o r o a m p h e t a m i n e , a p p a r e n t l y t h r o u g h
a c o n t r i b u t i o n o f the c h l o r i n e t o the o v e r a l l l i p o p h i l -
i c i t y o f t h e m o l e c u l e i n a d d i t i o n t o t h e reduced i o n i -
z a t i o n due t o t h e d i f l u o r o s u b s t i t u t i o n (18). The
h a l f - l i f e o f t h e d i f l u o r o d e r i v a t i v e was l e s s t h a n t h a t
of p-chloroamphetamine i n r a t b r a i n (IS) , presumably
because o f more f a c i l e d e a m i n a t i o n o f t h e d i f l u o r o
compound. When h i g h doses o f t h e d i f l u o r o compound
were i n j e c t e d t o produce drug l e v e l s i n b r a i n

TABLE VIII
Effect of β ^-Difluoro Substitution on the

Ionization of p-Chloroamphetamine
Distribution of Ionic Forms

Compound pK a at pH 7.4
RNH2 RNH3+
p-Chloroamphetamine SK3 1 99
ë>,ë>-Difluoro-p- 6.8 80 20
Chloroamphetamine
e q u i v a l e n t t o those
t o n i n and 5 - h y d r o x y i n d o l e a c e t i
were d e p l e t e d t o about t h e same e x t e n t as by p - c h l o r o -
amphetamine a t 6 hours (Table I X ) . However, t h e r e was
TABLE IX
Brain Serotonin and 5-Hydroxyindoleaoetic Acid

Levels after Injection of p-Chloroamphetamine
or its β,β-Di fluoro Derivative
Drug Treatment Serotonin 5HIAA
% of Control
p-Chloroamphetamine
(0.1 mmole/kg)
6 hrs 46 ± 3 * 54 + 2*
24 hrs 43 ± 3 * 50 t 2*
β, ë-Difluoro-
p-chloroamphetamine
(0.4 mmole/kg)
6 hrs 44 ± 1* 56 ± 3*
24 hrs 97 t 6 95 t 2
^Significant drug effect, P<.01 (5 rats/group).

a d i f f e r e n c e i n d u r a t i o n o f the a c t i o n of the two

compounds. With the d i f l u o r o d e r i v a t i v e , b r a i n
5 - h y d r o x y i n d o l e l e v e l s had r e t u r n e d t o normal w i t h i n
24 hours (Table I X ) . On the o t h e r hand, p-chloroam-
phetamine lowers b r a i n 5 - h y d r o x y i n d o l e s not o n l y 24
hours (Table IX) but f o r s e v e r a l months (15,.16) a f t e r
a single injection. T h i s l o n g - l a s t i n g efïect appears
t o be due t o a t o x i c a c t i o n o f p-chloroamphetamine on
s e r o t o n i n neurons (17). Thus the d i f l u o r o compound has
the same s h o r t - t e r m e f f e c t as p-chloroamphetamine on
b r a i n s e r o t o n i n but l a c k s the n e u r o t o x i c e f f e c t of the
p a r e n t d r u g . Whether t h i s d i f f e r e n c e i s due t o the
i n a b i l i t y o f the d i f l u o r o compound t o be c o n v e r t e d t o a
n e u r o t o x i c m e t a b o l i t e as happens w i t h p-chloroamphet-
amine o r to some o t h e r f a c t o r s cannot be known a t
present.
The i n i t i a l l o w e r i n
p-chloroamphetamine
amine depends on the a c t i v e t r a n s p o r t o f t h o s e drugs
i n t o s e r o t o n i n neurons, s i n c e t h e i r e f f e c t s are b l o c k e d
by i n h i b i t i o n o f t h a t a c t i v e uptake (19). Continual
r e u p t a k e o f p-chloroamphetamine i s n e c e s s a r y f o r the
d e p l e t i o n o f s e r o t o n i n t o be m a i n t a i n e d , s i n c e t r e a t
ment w i t h an uptake i n h i b i t o r a t e a r l y t i m e s a f t e r
p-chloroamphetamine i n j e c t i o n can r e v e r s e the depletion
of s e r o t o n i n (20). Thus one p o s s i b i l i t y i s t h a t the
d i f l u o r o compound i s not so e f f i c i e n t l y r e - t a k e n up by
the membrane pump on the s e r o t o n i n neuron. Consistent
w i t h t h i s p o s s i b i l i t y . i s the r e c e n t f i n d i n g (D. T. Wong
and F. P. Bymaster, u n p u b l i s h e d data) t h a t the d i f l u o r o
compound has o n l y about o n e - t e n t h of the a f f i n i t y f o r
the s e r o t o n i n uptake pump as does p-chloroamphetamine
(determined by t h e i r r e l a t i v e a b i l i t y t o i n h i b i t h i g h -
a f f i n i t y s e r o t o n i n uptake i n t o synaptosomes i l l v i t r o ) .
O t h e r Amines
We have been i n t e r e s t e d i n a p p l y i n g the p K a lower

i n g t h r o u g h β , β - d i f l u o r o s u b s t i t u t i o n t o amine drugs
whose l o c a l i z a t i o n i n a d i p o s e t i s s u e would be e x p e c t e d
t o enhance t h e i r p h a r m a c o l o g i c a l a c t i o n s , i . e . whose
t a r g e t t i s s u e would be f a t .
One l i n e o f i n v e s t i g a t i o n has d e a l t w i t h agents
that i n h i b i t l i p o l y s i s . N i c o t i n i c a c i d (I) lowers
serum f r e e f a t t y a c i d l e v e l s and i s used i n the t r e a t
ment o f h y p e r l i p o p r o t e i n e m i a (21). One of the s i d e
e f f e c t s t h a t l i m i t s the u s e f u l n e s s o f n i c o t i n i c a c i d i s
f l u s h i n g due t o v a s o d i l a t a t i o n (21) . We knew t h a t
3 - a m i n o m e t h y l - p y r i d i n e (II) had a n t i l i p o l y t i c a c t i v i t y
l i k e n i c o t i n i c a c i d b o t h i n v i t r o and i n v i v o ; the

•CH2CH2NH2 CF2CH2NH2
III IV
amine i s m e t a b o l i z e
t i n i c a c i d i n v i v o , so i t s i n v i v o a c t i v i t y i s due
almostly e n t i r e l y to n i c o t i n i c a c i d . T h i s amine t h e n
o f f e r s no advantages o v e r n i c o t i n i c a c i d as an a n t i -
l i p o l y t i c drug.
We thought t h a t s u b s t i t u t i o n o f f l u o r i n e s t o
reduce t h e pKa o f an amine o f t h i s s o r t might a c h i e v e
two o b j e c t i v e s : ( 1 ) r e t a r d the o x i d a t i v e deamination
o f t h e amine and ( 2 ) cause t h e amine t o l o c a l i z e p r e
f e r e n t i a l l y i n adipose t i s s u e . The u l t i m a t e purpose
would be t o reduce s i d e e f f e c t s w h i l e r e t a i n i n g t h e
a n t i l i p o l y t i c a c t i v i t y of n i c o t i n i c acid. Though t h e
p r e c i s e mechanisms o f f l u s h i n g caused by n i c o t i n i c a c i d
are n o t known, i t seemed l i k e l y t h a t an amine r a t h e r
t h a n an a c i d — a n d i n p a r t i c u l a r an amine t h a t was con
c e n t r a t e d m o s t l y i n f a t — m i g h t be f r e e o f t h i s s i d e
effect.
To approach t h i s o b j e c t i v e , i t was f i r s t n e c e s s a r y
to lengthen the side chain o f 3-aminomethylpyridine t o
provide a s i t e f o r f l u o r i n e s u b s t i t u t i o n . 3-Amino-
e t h y l - p y r i d i n e ( I I I ) was s y n t h e s i z e d and found t o have
a c t i v i t y as an a n t i l i p o l y t i c agent i n v i v o and i n
v i t r o ; t h e a c t i v i t y i n v i t r o i n d i c a t e s t h a t t h e amine
i t s e l f i s a c t i v e and does n o t r e q u i r e c o n v e r s i o n t o t h e
acid. F i n a l l y t h e β , β - d i f l u o r o d e r i v a t i v e o f 3-amino-
e t h y l - p y r i d i n e (IV) was s y n t h e s i z e d and s t u d i e d as an
a n t i l i p o l y t i c drug. Some méthodologie d i f f i c u l t i e s
were e n c o u n t e r e d i n measuring drug l e v e l s , b u t semi-
q u a n t i t a t i v e d a t a i n d i c a t e t h e drug was l o c a l i z e d t o
some e x t e n t (though perhaps n o t as much as expected)
i n adipose t i s s u e . However, a c t i v i t y as an a n t i l i p o -
l y t i c agent was n o t enhanced. A p p a r e n t l y t h e d i f l u o r o

s u b s t i t u t i o n d i d not a d e q u a t e l y r e t a r d the o x i d a t i v e
d e a m i n a t i o n o f t h i s compound. Furthermore the s u b c e l l -
u l a r l o c a l i z a t i o n o f the drug may not have been p r o p e r
f o r optimum a n t i l i p o l y t i c a c t i o n . N i c o t i n i c a c i d may
a c t on the a d e n y l c y c l a s e c o n t a i n e d i n the c e l l
membrane (22), whereas the d i f l u o r o s u b s t i t u t e d amine
may be p r e 3 o m i n a n t l y l o c a l i z e d i n s i d e the a d i p o c y t e
such t h a t the c o n c e n t r a t i o n a t the p r e c i s e s u b c e l l u l a r
s i t e o f a c t i o n i s not h i g h e r t h a n t h a t o f n i c o t i n i c
acid. Whatever the e x p l a n a t i o n , the a p p l i c a t i o n o f pK
a
r e d u c t i o n t h r o u g h d i f l u o r o s u b s t i t u t i o n was not s u c -
c e s s f u l i n t h i s instance.
We have a l s o s t u d i e d 3 , 3 - d i f l u o r o s u b s t i t u t e d
N - c y c l o p r o p y 1 - p h e n e t h y l a m i n e s , compounds which are
i r r e v e r s i b l e i n h i b i t o r s o f monoamine o x i d a s e . Mono-
amine o x i d a s e i n h i b i t o r s have been used c l i n i c a l l y t o
t r e a t mental d e p r e s s i o
pharmacologic a c t i o
t h a t has p o t e n t i a l c l i n i c a l a p p l i c a t i o n i n v o l v e s
a d i p o s e t i s s u e as a t a r g e t o r g a n .
Stock and Westermann (2_3) have shown t h a t mono-
amine o x i d a s e i n h i b i t o r s e l e v a t e n o r e p i n e p h r i n e l e v e l s
i n adipose t i s s u e . The n o r e p i n e p h r i n e i s presumably
c o n t a i n e d i n a d r e n e r g i c nerve endings t h a t c o n t r o l the
c y c l i c A M P - a c t i v a t e d l i p a s e i n the f a t c e l l s and c o n s e -
q u e n t l y the r a t e o f l i p o l y s i s . Exposure t o c o l d l e a d s
t o i n c r e a s e d m o b i l i z a t i o n o f f a t depots and e l e v a t i o n
o f plasma f r e e f a t t y a c i d l e v e l s . In r a t s whose a d i -
pose t i s s u e n o r e p i n e p h r i n e l e v e l s had been i n c r e a s e d by
monoamine o x i d a s e i n h i b i t i o n , exposure t o c o l d produced
a g r e a t e r i n c r e a s e i n plasma f r e e f a t t y a c i d s than
o c c u r r e d i n c o n t r o l r a t s (23) .
Of c o u r s e the use of monoamine o x i d a s e i n h i b i t o r s
would have e f f e c t s i n the b r a i n and o t h e r organs i n n e r -
v a t e d by the a d r e n e r g i c system as w e l l , so one could
not s e l e c t i v e l y a f f e c t f a t m o b i l i z a t i o n i n t h i s way.
We wondered i f the r e d u c t i o n o f p K a v a l u e s by 3/3-
d i f l u o r o s u b s t i t u t i o n might enhance the l o c a l i z a t i o n o f
the monoamine o x i d a s e i n h i b i t o r i n f a t and thus make
i t s e f f e c t s somewhat s e l e c t i v e , m a x i m i z i n g enhanced
l i p i d m o b i l i z a t i o n w h i l e m i n i m i z i n g CNS e f f e c t s and
e f f e c t s on the c a r d i o v a s c u l a r system.
The p o s s i b l e use o f l i p o l y t i c agents i n t r e a t i n g
o b e s i t y by m o b i l i z i n g f a t depots has l o n g been
considered. The i d e a of u s i n g a drug t o enhance
endogenous l i p o l y t i c s t i m u l i as opposed t o a drug t h a t
d i r e c t l y caused l i p o l y s i s seemed e s p e c i a l l y i n t e r e s t i n g .
Thus we have p r e p a r e d 3 , 3 - d i f l u o r o - N - c y c l o p r o p y l - p -
c h l o r o p h e n e t h y l a m i n e and s t u d i e d i t s p r o p e r t i e s as an
i n h i b i t o r of monoamine o x i d a s e . N - C y c l o p r o p y l amines

o f t h i s s o r t have l o n g been known t o i n h i b i t monoamine

o x i d a s e i r r e v e r s i b l y (24). The 3 , β - d i f l u o r o compounds,
l i k e t h e p a r e n t N - c y c l o p r o p y l a m i n e s , were found t o
i n h i b i t t h e enzyme i n a manner t h a t was n o t r e v e r s i b l e
by d i a l y s i s .
The 3 / 3 - d i f l u o r o d e r i v a t i v e had t h e e x p e c t e d lower
p K v a l u e (Table X ) . However when we i n j e c t e d t h e
a
drugs i n t o r a t s and measured monoamine o x i d a s e i n h i b i

t i o n i n v a r i o u s t i s s u e s , we d i d n o t observe any s e l e c
t i v i t y i n t h e i n h i b i t i o n o f t h e enzyme i n f a t (Table
XI). A g a i n méthodologie d i f f i c u l t i e s have kept us from
o b t a i n i n g adequate d a t a on d i s t r i b u t i o n o f t h e d r u g s ,
but we can assume from d a t a on s i m i l a r drugs t h a t t h e
d i f l u o r o compound would l o c a l i z e i n f a t . Why then was
i t n o t a more e f f e c t i v e i n h i b i t o r o f monoamine o x i d a s e
in this tissue? P o s s i b l y t h e monoamine o x i d a s e i n t h e
fat tissue i s locate
much g r e a t e r e x t e n t
drug i s l o c a l i z e d c h i e f l y i n t h e a d i p o c y t e s . F o r what-
ever reason, s e l e c t i v e i n h i b i t i o n o f adipose t i s s u e
monoamine o x i d a s e was n o t a c h i e v e d .
The 3 / 3 - d i f l u o r o s u b s t i t u t e d monoamine o x i d a s e
i n h i b i t o r s may n o n e t h e l e s s have i n t e r e s t i n g uses as
pharmacological t o o l s . The f l u o r o s u b s t i t u e n t s might
p e r m i t NMR s t u d i e s t h a t would r e v e a l something o f t h e
n a t u r e o f t h e a c t i v e s i t e o f monoamine o x i d a s e t o which
the i n h i b i t o r i s i r r e v e r s i b l y bound.
TABLE X
Ionization of N-Cy alopropyl-4-ohlorophenethylamine

and its & &-Difluoro
3 Derivative
At pH 7. 4
y % as
Compound PaK
RNH2 RNH 3+
8. 3 20 80

TABLE XI
Inhibition of Monoamine Oxidase in Rat Tissues In_ Vivo
Cl^~^-CR2CH2NH<^
Experiment 1, R = H Experiment 2. R = F
Tissue Group MAO Activity Inhibition MAO Activity Inhibition
Liver Control 513 ± 21 450 ± 23

Drug 213 ± 15 59 137 + 6 69
Brain ControI 107 ± 2 114 ± 1

Drug 52 ± 1 51 42 ± 1 63
Adrenals ControI 106 ± 5 109 ± 5

Drug 40 ± 1 62 42 ± 2 62
Kidneys Control 57 ± 1 51 ± 1
Drug 22 ± 1 61 20 ± 1 60
Heart ControI 25 ± 1 25 ± 2

Drug 10 ± 1 60 13 ± 1 46

Fat ControI 17 ± 1 24 ± 1
Drug 7 + 1 59 11 ± 1 55
Drugs were injected i.p. at 50 mg/kg 1 hour before the rats were killed. Monoamine
oxidase activity in tissue homogenates was assayed with ^-^C-tryptamine as substrate.
Summary
S u b s t i t u t i o n o f one o r two f l u o r i n e atoms on t h e

8 carbon o f a r y l a l k y l a m i n e drugs markedly reduced the
pK a o f the amines. Monofluoro s u b s t i t u t i o n reduced the
p K more than 1 pH u n i t , but the p K was s t i l l above
a a
p h y s i o l o g i c a l pH. In g e n e r a l , monofluoro s u b s t i t u t i o n
l e d t o s m a l l changes o r no change i n p h a r m a c o l o g i c
p r o p e r t i e s o f the d r u g s . Difluoro substitution further
reduced the p K a t o below p h y s i o l o g i c a l pH. Thus a t
p h y s i o l o g i c a l pH the d i f l u o r o compounds a r e m o s t l y
n e u t r a l whereas p a r e n t amines a r e n e a r l y c o m p l e t e l y
cationic. Presumably as a r e s u l t o f t h i s e f f e c t on
pK , d i f l u o r o s u b s t i t u t i o n markedly a l t e r e d the
a
p r o p e r t i e s o f amphetamine and p h e n e t h y l a m i n e s .
As a r e s u l t o f d i f l u o r o s u b s t i t u t i o n amphetamine
became a b e t t e r s u b s t r a t
whereas phenethylamin
m i t o c h o n d r i a l monoamine o x i d a s e i r i v i t r o ; amphetamine
was bound l e s s r e a d i l y t o albumin; amphetamine and
phenethylamine became b e t t e r s u b s t r a t e s f o r l u n g
N - m e t h y l t r a n s f e r a s e ; amphetamine, phenethylamine and
p-chloroamphetamine were d i s t r i b u t e d d i f f e r e n t l y i n
mouse and r a t t i s s u e s , l o c a l i z i n g l e a s t i n f a t w i t h o u t
d i f l u o r o s u b s t i t u t i o n b u t most i n f a t w i t h t h a t
s u b s t i t u t i o n ; amphetamine metabolism i n the r a t was
s h i f t e d from p a r a - h y d r o x y l a t i o n t o o x i d a t i v e deamina-
t i o n ; the metabolism o f d i f l u o r o a m p h e t a m i n e but not
amphetamine i n r a t s was a c c e l e r a t e d by phénobarbital
p r e t r e a t m e n t t o i n d u c e microsomal enzymes i n l i v e r ;
amphetamine h y p e r t h e r m i a i n r a t s and l o c o m o t o r s t i m u -
l a t i o n i n mice d e c r e a s e d p r o p o r t i o n a l t o drug l e v e l s
i n b r a i n ; amphetamine l o s t the a b i l i t y t o d e p l e t e
h e a r t and b r a i n n o r e p i n e p h r i n e even a t h i g h doses;
1
p-chloroamphetamine s a b i l i t y t o lower b r a i n s e r o t o n i n
l e v e l s i n i t i a l l y was n o t a l t e r e d e x c e p t t o the e x t e n t
t h a t drug l e v e l s i n b r a i n were lower, but the neuro-
t o x i c e f f e c t o f p-chloroamphetamine on s e r o t o n i n
neurons was l o s t .
These r e s u l t s i l l u s t r a t e the importance o f i o n i z a -
t i o n i n the i n t e r a c t i o n o f amine drugs w i t h b i o l o g i c a l
macromolecules b o t h i n v i t r o and i r i v i v o . Fluorine
s u b s t i t u t e d near the n i t r o g e n , because o f i t s s t r o n g
e l e c t r o n e g a t i v i t y , can reduce the b a s i c i t y o f t h e amino
group t o the e x t e n t t h a t i t s i o n i z a t i o n a t p h y s i o l o g i -
c a l pH i s s h a r p l y r e d u c e d . F l u o r i n e s u b s t i t u t i o n thus
a l t e r s many o f the p h a r m a c o l o g i c p r o p e r t i e s o f such
amine drugs.

Acknowledgments
We thank D r s . W. S. M a r s h a l l , W. N. Shaw, and

R. E . Toomey f o r t h e i r c o l l a b o r a t i o n i n t h e s t u d i e s
on t h e p y r i d i n e compounds. We a l s o a r e g r a t e f u l f o r
the t e c h n i c a l a s s i s t a n c e o f Kenneth L. Hauser i n t h e
s y n t h e s i s o f f l u o r i n a t e d d e r i v a t i v e s and o f H a r o l d D.
Snoddy, B e t t y W. Roush, and John C. Baker i n t h e
biological studies.
Literature Cited
1. Pauling, L. "The Nature of the Chemical Bond"
p. 93 & 260, 3rd ed., Cornell University Press,
Ithaca, 1960.
2. Loncrini, D. F. and Filler, R., Advances in
Fluorine Chemistr
3. Henne, A. L. an
(1955) 77, 1901-1902.
4. Lewis, G. P., Brit. J. Pharmacol. (1954) 9, 488-
493.
5. Vree, Τ. Β., Muskens, A. Th. J. Μ., and van
Rossum, J. Μ., J. Pharm. Pharmacol. (1969) 21,
774-775.
6. Fuller, R. W. and Roush, B. W., Res. Comm. Chem.
Pathol. Pharmacol. (1975) 10, 735-738.
7. Fuller, R. W., Molloy, Β. B., and Parli, C. J. In
"Psychopharmacology, Sexual Disorders, and Drug
Abuse" pp. 615-624, Avicenum Press, Prague, 1973.
8. Fuller, R. W., Parli, C. J., and Molloy, Β. B.
Biochem. Pharmacol. (1973) 22, 2059-2061.
9. Fuller, R. W., Molloy, Β. B., Roush, B. W. and
Hauser, K. L., Biochem. Pharmacol. (1972) 21,
1299-1307.
10. Dring, L. G., Smith, R. L., and Williams, R. T.,
Biochem. J. (1970) 116, 425-435.
11. Gessa, G. L., Clay, G. Α., and Brodie, Β. B.,
Life Sci. (1969) 8, 135-141.
12. Fuller, R. W., Shaw, W. Ν., and Molloy, Β. B.,
Arch. Int. Pharmacodyn. (1972) 199, 266-271.
13. Moore, Κ. E., J. Pharmacol. Exptl. Therap., (1963)
142, 6-12.
14. Fuller, R. W., Snoddy, H. D., Molloy, Β. B., and
Hauser, K. L., Psychopharmacologia (1973) 28,
205-212.
15. Sanders-Bush, Ε., Bushing, J. Α., and Sulser, F.,
Eur. J. Pharmacol., (1972) 20, 385-388.
16. Fuller, R. W. and Snoddy, H. D., Neuropharmacol.,
(1974) 13, 85-90.

17. Harvey, J. A., McMaster, S. E., and Yunger, L. Μ.,

Science (1975) 187, 841-843.
18. Fuller, R. W., Snoddy, H. D., and Molloy, Β. B.
J. Pharmacol. Exptl. Therap. (1973) 184, 278-284.
19. Fuller, R. W. and Molloy, Β. B., Adv. Biochem.
Psychopharmacol. (1974) 10, 195-205.
20. Fuller, R. W., Perry, K. W., and Molloy, Β. B.,
Eur. J. Pharmacol. (1975) 33, 119-124.
21. Levy, R. I. and Frederickson, D. S., Postgrad.
Med. (1970) 47, 130-136.
22. Peterson, M. J., Hillman, C. C. and Ashmore, J . ,
Mol. Pharmacol. (1968) 4, 1-9.
23. Stock, K. and Westermann, E. O., J. Lipid Res.,
(1963) 4, 297-304.
24. Mills, J . , Kattau, R., Slater, I. H. and Fuller,
R. W., J. Med Chem (1968) 11 95-97
Question and Answer Period

Q. Have you prepared any ring-hydroxylated compounds?
A. No. We particularly wanted Ç> Ç>-difluoro-p- y
hydroxy amphetamine but have not been able to
synthesize it. There may be a problem of stabil
ity with compounds having both a hydroxy I and
-CF2R group on the ring.
Q. Is fluoride ion a metabolite of these difluoro
compounds?
A. We do not know whether any of the fluorine is
removed metabolically.
Q. About the possibility of changing the basicity of
the nitrogen in the difluoro compounds—have you
looked at the effect of the 3,3-difluoro substi-
tuent on transport across biological membranes?
A. Mostly in indirect ways. I think the large change
in tissue distribution produced by the 3,3-
difluoro substitution i s a manifestation of
altered transport across various biological mem-
branes in the complex system of the whole animal.
Dr. David Wong has looked at the ability of 3*3-
difluor ο-p-chlorο amphetamine to inhibit the trans
port of monoamines across synaptosomal membranes
in vitro; the difluoro compound is less effective
as an inhibitor than is p-chloroamphetamine
itself.

Q. Do you know what t h e p r o d u c t s a r e from o x i d a t i v e

d e a m i n a t i o n o f t h e β , β - d i f l u o r o compounds?
A. Dr. John Parti has isolated the oxime> the ketone,

and the alcohol as in vitro metabolites of β-
difluoroamphetamine. He has found that the oxime
of difluorophenylacetone i s formed as a metabolite
in vivo in rats and is excreted in urine in free
and conjugated form.

6
Metabolic and Transport Studies with
Deoxyfluoro-monosaccharides
N.F.TAYLOR,A.ROMASCHIN,andD.SMITH
Department of Chemistry, University of Windsor, Ontario N9B 3P4 Canada
The rational
fluorocarbohydrates and related compounds as probes
for the study of enzyme specifity, carbohydrate metab-
olism and transport in biological systems has been
elaborated in a previous symposium (1). Such com-
pounds have now been used to study carbohydrate metab-
olism in yeast cells (2), Ps. fluorescens (3) and
E. coli (4); enzyme specificity of glycerol kinase (5),
yeast hexotinase (6), phosphoglucomutase and UDPG
pyrophosphorylase (7) and glycerol-3-phosphate dehydro-
genase (8); carbohydrate transport in hamster intes-
tine (9) and the human erythrocyte (10), (11). The
wide range of synthetic fluorinated carbohydrates and
related compounds now available has been extensively
reviewed (12). As will be evident from our recent
studies, however, many detailed biochemical studies l 4
will be limited until the introduction of C and/or
into fluorocarbohydrates has been accomplished.
The Transport of D-Glucose Across the Human Erythrocyte
Membrane.
The question of the exact nature of the carrier
protein(s) and translocation mechanism for the trans-
port of D-glucose across the erythrocyte membrane is
still debated (13). However, the saturation kinetics
obtained for D-glucose and glucose analogues are in
accordance with a facilitated transport mechanism
which allows binding of the sugar molecule to one or
more sites of a receptor protein in the membrane. A
comprehensive study of the specificity of this binding
has been undertaken by a number of workers (14), (15),
(16), (17), In particular the comparative inhibition
and comparative transport studies of D-glucose with a
number of monodeoxy-D-glucoses and monodeoxymonofluoro-
99

D - g l u c o s e s p r o v i d e k i n e t i c parameters which p e r m i t
assignment o f hydrogen bonds between s p e c i f i c oxygen
atoms i n D-glucose and r e c e p t o r s i t e s i n t h e c a r r i e r
protein. Thus u s i n g the o p t i c a l method o f Sen and
Widdas (1_5) and t h e s i m p l i f i e d r a t e e q u a t i o n (18) f o r
the e x i t o f g l u c o s e from p r e - l o a d e d e r y t h r o c y t e s , we
have shown (11_) t h a t r e p l a c i n g the oxygen f u n c t i o n a t
C 3 o f D-glucose by f l u o r i n e t o g i v e 3 - d e o x y - 3 - f l u o r o -
D-glucose does n o t s i g n i f i c a n t l y change t h e h a l f - s a t
u r a t i o n c o n s t a n t (K ) f o r t h e c a r r i e r p r o t e i n (Table 1).
x
In c o n t r a s t 3-deoxy-D-glucose has l o s t t h i s a b i l i t y t o
hydrogen bond a t C 3 and c o n s e q u e n t l y has a lower a f f i n
i t y f o r the c a r r i e r p r o t e i n ( h i g h e r Κ v a l u e ) .
χ In
a d d i t i o n the K v a l u e f o r 5 - t h i o - D - g l u c o s e ( T a y l o r , N.F.
x
& Gagneja, G.L. u n p u b l i s h e d r e s u l t ) s u g g e s t s t h a t t h e

r i n g oxygen a t C 5 o f D-glucose i s a l s o i n v o l v e d w i t h
hydrogen bonding (Tabl
T a b l e 1. Kx and V m a x v a l u e s o f D-glucose
and d e r i v a t i v e s a t 37°
V
max
Sugar Κ (mM)
χ (mmol. L i t r e ^ min ^)
D-Glucose 3.9 640

3-Deoxy-3-fluoro- 2.3 600
D-glucose
3-Deoxy-D-glucose 15.3 340
5-Thio-D-glucose 15.0 500
K i n e t i c parameters were d e t e r m i n e d as p r e v i o u s l y
d e s c r i b e d (11).
These r e s u l t s a r e i n agreement w i t h B a r n e t t e t a l . (10}

who showed by i n h i b i t i o n s t u d i e s t h a t u n l i k e 3-deoxy-
D - g l u c o s e , 3 - d e o x y - 3 - f l u o r o - D - g l u c o s e b i n d s t o the
c a r r i e r p r o t e i n as w e l l as D - g l u c o s e . Furthermore,
t h e importance o f the β - o r i e n t a t i o n o f -OH a t in
D-glucose was s u g g e s t e d by the h i g h K i v a l u e s f o r
1-deoxy-D-glucose and α - D - g l u c o s y l f l u o r i d e and t h e low
Ki value f o r 3-D-glucosylfluoride. These t r a n s p o r t
and i n h i b i t i o n s t u d i e s p r o v i d e e v i d e n c e f o r t h e
p r o p o s a l by Kahlenburg and Dolansky (19) t h a t t h e
oxygen f u n c t i o n s l o c a t e d a t C i , C 3 and t h e r i n g oxygen
a t C 5 o f the C l - c o n f o r m a t i o n o f 3-D-glucopyranose
( F i g u r e 1 ) , a r e c o n s i d e r e d t o be n e c e s s a r y f o r e f f e c t
i v e hydrogen bonding o f D-glucose t o t h e t r a n s p o r t
protein. T h i s model a g r e e s w i t h the d e t e c t i o n o f three
d i f f e r e n t r e c e p t o r groups (- N H 2 , - SH and i m i d a z o l e )
on t h e p r o t e i n a s s o c i a t e d w i t h g l u c o s e t r a n s p o r t (20)
and i s a l s o c o n s i s t e n t w i t h a r e c e n t l y proposed model

6. TAYLOR E T A L . Deoxyfluow-monosaccharides 101
(21) f o r t h e mode o f a c t i o n o f c y t o c h a l a s i n Β (22)

i n h i b i t i o n o f g l u c o s e t r a n s p o r t i n t h e human e r y t h r o
cyte. Thus a D r i e d i n g m o l e c u l a r model ( F i g u r e 2) o f
c y t o c h a l a s i n Β (I) r e v e a l s an almost i d e n t i c a l s p a t i a l
d i s t r i b u t i o n o f t h e f o u r oxygen atoms l o c a t e d a t C I ,
C19, C18 and C4 t o t h o s e l o c a t e d a t C5, C l , C2 and C3
o f t h e C l - c o n f o r m a t i o n o f 3-D-glycopyranose ( F i g u r e 1 ) .
At l e a s t t h r e e o f t h e s e s i t e s a t R, R l and R3 ( F i g u r e
2) a r e i m p l i c a t e d i n hydrogen bonding t o t h e c a r r i e r
p r o t e i n f o r D-glucose and p a r t i a l l y e x p l a i n why
c y t o c h a l a s i n Β i s a c o m p e t i t i v e i n h i b i t o r o f D-glucose
t r a n s p o r t i n t h e human e r y t h r o c y t e w i t h K i , 1.2 χ 10"'
M (21) .
A f u r t h e r point o f i n t e r e s t r e s i d e s i n the f a c t
t h a t when whole r e d b l o o d c e l l s a r e i n c u b a t e d w i t h 3-
d e o x y - 3 - f l u o r o - D - g l u c o s e f o r 24 h r s a t 37°C a s m a l l
but s i g n i f i c a n t r e l e a s
( H a l t o n , D. & T a y l o r
200mM 3 - d e o x y - 3 - f l u o r o g l u c o s e C-F c l e a v a g e r e a c h e s a
maximum (^ 1%) a f t e r 24 hours i n c u b a t i o n ( F i g u r e 3 ) .
U n l i k e t h e c o n t r o l s such c e l l s c o m p l e t e l y l o s e t h e i r
a b i l i t y to transport glucose. The s i g m o i d a l c u r v e
i n d i c a t e s m u l t i p l e k i n e t i c s f o r t h e mechanism o f
f l u o r i d e r e l e a s e and one p o s s i b l e e x p l a n a t i o n f o r t h e œ
r e s u l t s would be t h e concommittant c o - v a l e n t attachment
o f t h e g l u c o s e r e s i d u e t o one o f t h e r e c e p t o r s i t e s o f
the c a r r i e r p r o t e i n a s s o c i a t e d w i t h g l u c o s e t r a n s p o r t .
Such a mechanism i s shown ( F i g u r e 4) i n which 3-deoxy-
3-fluoroglucose (II) hydrogen bonds t o t h e r e c e p t o r
p r o t e i n , e l i m i n a t e s HF t o produce t h e e p o x i d e ( I I I )
which i s a t t a c k e d by t h e n u c l e o p h i l i c p r o t e i n t o p r o -
duce ( I V ) . We have r e c e n t l y s y n t h e s i s e d 3H-C-(3)-3-
d e o x y - 3 - f l u o r o - D - g l u c o s e (Lopes, D. & T a y l o r , N.F.
u n p u b l i s h e d r e s u l t s ) i n o r d e r t o e s t a b l i s h whether
g l u c o s y l a t i o n o f a membrane p r o t e i n has i n f a c t o c c u r r -
ed. Such a r e a c t i o n may p e r m i t i s o l a t i o n o f t h e
c a r r i e r p r o t e i n f o r D-glucose.
M i c r o b i a l Metabolism o f Deoxyfluoro-D-glucoses
Our p r e v i o u s s t u d i e s (3) demonstrated t h a t 3-deaxy-
fluoro-D-glucose (II) i s m e t a b o l i s e d by whole r e s t i n g
c e l l s o f P £ . f l u o r e s c e n s , w i t h r e t e n t i o n o f t h e C-F
bond, t o produce 3 - d e o x y - 3 - f l u o r o - D - g l u c o n i c a c i d ( V ) .
C e l l - f r e e e x t r a c t s o f t h i s organism o x i d i s e d 3FG
f u r t h e r t o 3 - d e o x y - 3 - f l u o r o - 2 - k e t o - D - g l u c o n i c a c i d (VI).
I t has a l s o been shown t h a t t h e same enzymes t h a t
o x i d i s e D-glucose ( g l u c o s e o x i d a s e and g l u c o n a t e de-
hydrogenase) o x i d i s e (II) and t h a t (II) and (V) a r e
c o m p e t i t i v e i n h i b i t o r s o f g l u c o n o k i n a s e (23) . In
o r d e r t o study f u r t h e r t h e s p e c i f i c i t y o f t h e s e enzymes

102 BIOCHEMISTRY INVOLVING CARBON
Figure 1. Stereospecific binding sites of

β-D-glucopyranose in the Cl-conforma-
tion to a transport protein . . . Hydrogen
bonds. R, Rl and R3 represent receptor
sites on the transport protein.

6. T A Y L O R E T A L . Deoxyfluoro-monosaccharides 103
Ί9Ν
Figure 2. Model of cytochalasin B C

R1
gen bonds. X = -CH ?h.t R, RI, R2 and

R3 represent receptor sites on the transport
R2 R3 protein.
Figure 3. Fluoride ion released after incubation of human

erythrocytes at 37° with different concentrations of 3-deoxy-3-
fluoro-D-glucose: A 50 mM, · 100 mM, Ο ^50 mM, • 200
mM. Fluoride ion determinations were by the fluoride elec
trode (26) and the preparation of the erythrocytes as previ
ously described (11). The initial control fluoride ion concent
ration was 1.0 X 10' M.
4

towards o t h e r d e o x y f l u o r o - D - g l u c o s e s we have examined

the b i o c h e m i c a l e f f e c t s o f t h e i s o m e r i c 4-deoxy-4-
fluoro-D-glucose (VII) {24) on whole r e s t i n g c e l l s and
c e l l - f r e e e x t r a c t s o f Ps. f l u o r e s c e n s ( H i l l , L. &
T a y l o r , N.F. u n p u b l i s h e d r e s u l t s ) .
CH OH2
COOH COOH
-H
H—I—OH
H-f-OH
CH OH2
(ID (V) (VI) (VII)
Warburg r e s p i r o m e t r y i n d i c a t e d t h a t u n l i k e (II) (VII)

i s n o t o x i d i s e d by whole c e l l s o f P£. f l u o r e s c e n s b u t
t h e r e i s an immediate r e l e a s e o f f l u o r i d e a n i o n f F i g u r e
5). U s i n g 2.5mM o f 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e t h e
r a t e o f C-F c l e a v a g e i s l i n e a r o v e r t h e f i r s t f o u r
hours and a f t e r 24 hours 94% o f t h e c o - v a l e n t f l u o r i n e
i s r e l e a s e d as f l u o r i d e a n i o n (no F " was d e t e c t e d i n
the absence o f c e l l s ) . The p o s s i b l e d e f l u o r i n a t e d
products o f t h i s r e a c t i o n are D-glucose, D-galactose,
4-deoxy-D-glucose and 3,4-anhydro-D-galactose. Only
the l a t t e r two compounds a r e l i k e l y s i n c e D - g a l a c t o s e
and D - g l u c o s e , even i n t h e p r e s e n c e o f f l u o r i d e a n i o n ,
are o x i d i s e d by t h e organism. T . l . c . a n a l y s i s o f t h e
c e l l s u p e r a t a n t s and i n t r a c e l l u l a r c o n t e n t s , however,
has f a i l e d t o r e v e a l any new c a r b o h y d r a t e components.
When c e l l - f r e e e x t r a c t s o f Ps. f l u o r e s c e n s a r e c h a l -
l e n g e d w i t h 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e (VII) we f i n d
t h a t no s i g n i f i c a n t de-flùorination o c c u r s . Thus i n
the c o n c e n t r a t i o n range 2 - 2 0 ymoles t h e i n i t i a l r a t e
and e x t e n t o f o x i d a t i o n o f (VII) by c e l l - f r e e e x t r a c t s
is shown (Table 2 ) . The o x i d a t i o n o f (VII) i s com-
p l e t e w i t h i n 2 hours and 2 g atoms o f oxygen/mole
o f s u b s t r a t e a r e consumed.

6. TAYLOR ET A L . Deoxyfluoro-monosaccharides 105
(ID (III)
H" (IV)
Figure 4. Possible mechanism
of fluoride release from 3-
deoxy-3-fluoro-D-glucose.
protein X = Ν or S.
·
60
Hours
Figure 5. Release of fluoride anion from 2.5 mM 4-deoxy-4-fluoro-D-

glucose by whole cells of Ps. fluorescens. • Fluoride anion. A Oxygen
uptake. Six Warburg flasks were used. Each flask contained 5 fimoles
4-deoxy-4-fluoro-D-glucose, 20 mg dry wt cells and 0.67M phosphate
buffer to 2.0 ml in main well. Flask contents were incubated at 30°C
with shaking. At time intervals the contents of each flask were centri
fugea at 4 040 g for 20 min and fluoride ion determinations made with
a fluoride electrode (26) on the supernatants. Warburg conditions for
oxygen uptake: 30°C, reaction vol 2.0 ml. Fhsk contained 1.0 ml 0.67M
phosphate buffer and 0.5 ml 5 ^moles of 4-deoxy-4-fluoro-D-glucose in
main well and 0.2 ml 20% KO H aq. in center well. Reaction was initiated
by tipping 0.5-ml cell suspension (24 mg dry wt) in 0.67M phosphate
buffer from side arm. Endogenous respiration subtracted (1434 μΐ in
480 min).

T a b l e 2.
O x i d a t i o n o f 4-deoxy-4-fluoro-D-glucose by
c e l l - f r e e e x t r a c t s o f Ps. f l u o r e s c e n s .
Rate o f Moles 0 / 2
Amount o f oxidation Net mole o f

4-fluoroglucose ymoles O 2 / y l oxygen substrate
ymoles hr/mg p r o t e i n consumed oxidised
2.0 0.07 50 1.12
5.0 0.18 110 0.99
10.0 0.26 225 1.00
20.0 0.34 440 0.96
5.0 (glucose) 0.38 115 1.03
Warburg c o n d i t i o n s : 30°C, gas phase, a i r . R e a c t i o n
volume, 2.0 m l . Each f l a s k c o n t a i n e d 37 mg c e l l - f r e e
e x t r a c t p r o t e i n , 1 ymole NAD, 0.67 M phosphate b u f f e r ,
pH, 7.0 made up t o 1.5 l i th mai compartment
S i d e arm c o n t a i n e d 0.
w e l l 0.2 ml 20% KOHag pape
was i n i t i a t e d by t y p i n g c o n t e n t s from t h e s i d e arm.
Endogenous r e s p i r a t i o n (317 y l oxygen i n 150 min)
subtracted.
A f t e r o x i d a t i o n o f (VII) was complete, s i l i c a g e l
t . l . c . a n a l y s i s (EtOAC:ACOH:H 0 3:3:1) o f t h e c e l l - f r e e
2
e x t r a c t r e v e a l e d t h e absence o f ( V I I ) ( R , 0.6) and t h e

F
presence o f a new component (RF 0.45). By analogy

w i t h t h e e s t a b l i s h e d m e t a b o l i c pathway o f g l u c o s e (27)
and 3 - d e o x y - 3 - f l u o r o - D - g l u c o s e {23) i n t h i s organism,
t h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h e two s t e p o x i d a -
t i o n o f (VII) by g l u c o s e o x i d a s e and g l u c o n a t e dehydro-
genase t o 4 - d e o x y - 4 - f l u o r o - D - g l u c o n i c a c i d (VIII) and
4-deoxy-4-fluoro-2-keto-D-gluconic a c i d (IX) r e s p e c -
tively.
ÇOOH COOH
-OH h o
HCH-H H
(VII)
H- H-
H H
CH °H
2 &H2OH
(VIII) (IX)

6. TAYLOR ET AL. Deoxyfluoro-monosaccharides 107
I s o l a t i o n and c h a r a c t e r i s a t i o n o f (IX) i s c u r r e n t l y
being i n v e s t i g a t e d . The e x t e n s i v e d e f l u o r i n a t i o n o f
(VII) by whole c e l l s o f Ps. f l u o r e s c e n s and the r e t e n -
t i o n o f the C-F bond on t r e a t m e n t o f fVTI) w i t h c e l l -
f r e e e x t r a c t s suggest t h a t C-F c l e a v a g e o c c u r s a t the
cell-wall/membrane l e v e l o f the organism. The mode o f
uptake o f D-glucose by Ps. f l u o r e s c e n s i s not known.
However, i n a c l o s e l y r e l a t e d s p e c i e s Ps. a e r u g i n o s a ,
i t has been shown (28) t h a t a l t h o u g h the phosphoenol-
p y r u v a t e p h o s p h o t r a n s f e r a s e system (29) i s not i n v o l v e d ,
the t r a n s p o r t o f D-glucose i s energy dependant and,
t h e r e f o r e , l i k e l y t o have a c a r r i e r p r o t e i n system.
A s i m i l a r g l u c o s e t r a n s p o r t system may be p r e s e n t i n
Ps. f l u o r e s c e n s and t h e f a i l u r e o f t h e whole c e l l t o
o x i d i s e 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e (VII) may be due t o
a s t e r e o s p e c i f i c r e a c t i o n of (VII)with a c a r r i e r
p r o t e i n system i n whic
i s r e l e a s e d as f l u o r i d
p e r m i t the sugar r e s i d u e (at C 4 ) t o become a t t a c h e d t o
p r o t e i n i n the membrane and account f o r the f a c t t h a t
we a r e unable t o d e t e c t any m e t a b o l i t e e i t h e r o u t s i d e
o r i n s i d e the c e l l d u r i n g the i n c u b a t i o n p e r i o d . Some
s u p p o r t f o r t h i s p o s s i b i l i t y i s p r o v i d e d by (a) the
f a c t t h a t when whole c e l l s are p r e - i n c u b a t e d w i t h
2.5mM 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e f o r 12 hours and
c h a l l e n g e d w i t h 2 - 8mM g l u c o s e s i g n i f i c a n t i n h i b i t i o n
o f the r a t e o f r e s p i r a t i o n o c c u r s ( F i g u r e 6 ) . T h i s
would be c o n s i s t e n t w i t h a b l o c k e d g l u c o s e t r a n s p o r t
s i t e ( s ) a l t h o u g h as t h e g l u c o s e c o n c e n t r a t i o n i s
i n c r e a s e d t o 20 ymoles some r e c o v e r y o f r e s p i r a t i o n i s
apparent. The p o s s i b i l i t y t h a t a s m a l l u n d e t e c t a b l e
amount o f 4 - d e o x y - 4 - f l u o r o - D - g l u c o s e o r a n o n - o x i d i s -
able f l u o r i n a t e d metabolite i s i n h i b i t i n g glucose
metabolism and/or t r a n s p o r t i s o f c o u r s e not e x c l u d e d
by t h e s e r e s u l t s . (b) Ps. f l u o r e s c e n s i s unable t o
grow on a m i n e r a l s a l t s medium w i t h 4 - d e o x y - 4 - f l u o r o -
g l u c o s e (VII) as a s o l e carbon source a l t h o u g h f l u o r i n e
i o n i s r e l e a s e d i n t o the medium. S e v e r a l examples a r e
known where the r e l e a s e o f f l u o r i d e from a C-F com-
pound by a b a c t e r i u m a l l o w s the f r e e n o n - f l u o r i n a t e d
fragment t o s e r v e as a source o f carbon and energy f o r
growth. Thus a Pseudomonad has been i s o l a t e d which
grows on f l u o r o a c e t a t e as a r e s u l t o f C-F c l e a v a g e (30).
S i m i l a r l y , a Pseudomonad has been i s o l a t e d which w i l l
grow on m o n o f l u o r o c i t r a t e a f t e r f l u o r i d e r e l e a s e (31).
The i n a b i l i t y o f 4 - d e o x y - 4 - f l u o r o g l u c o s e ( V I I ) , t o a c t
as a carbon source f o r Ps. f l u o r e s c e n s , d e s p i t e C-F
c l e a v a g e , may be due t h e r e f o r e , t o the attachment o f
the sugar r e s i d u e t o some c e l l u l a r component.
It i s expected t h a t the s y n t h e s i s o f ^-^C-(l)-4-

Figure 6. Oxidation of D-glucose by rest

ing whole celh of Ps.fluorescensafter pre
incubation with 2.5 mM 4-deoxy-4-fluoro-
D-glucose. Pre-incubation conditions: 30°C,
reaction volume 2.0 ml, time 12 hr. Each
Warburg flask contained 1.0 ml 0.67M phos
phate buffer, 0.5 ml 5 μmoles of 4-deoxy-4-
fluoro-D-glucose or 5 ^moles D-glucose in
main well and 0.5-ml cell suspension (28
mg dry wt) in 0.67M phosphate buffer in
side arm. Pre-incubation was initiated by
tipping contents from side arm. Oxidation
of added D-glucose, Warburg conditions:
30°C, reaction volume 2.5 ml, gas phase,
air. After preincubation period, 0.5 ml of
μmole quantities of D-glucose were added
from the side arm, 0.3 ml 20% KOHag
added to the center well. Endogenous res
piration subtracted (879 μΐ in 240 min).
I Average oxidation of 5, 10,
μτηοΙβ8 glucose after pre-incubation
5 μmole glucose.
• 10 μmole, Δ 15 μmole, A 20 μmole glu
cose added after pre-incubation of celh
with 2.5 mM 4-deoxy-4-fluoro-D-glucose.
Temp. ^
Figure 7. (a) Temperature programmed gas chromato-

gram of locust haemolymph and (b) Locust fat body
utilizing OV-17 (7.5%) phase at 170°C followed by a
4°C/min heating rate. Peaks: A = a-D-glucose, Β =
β-D-glucose, C = trehalose.

f l u o r o - D - g l u c o s e , based on a K i l i a n i e x t e n s i o n o f
3- d e o x y - 3 - f l u o r o - D - a r a b i n o s e {22) 14
with Na CN, w i l l
a l l o w us t o a s c e r t a i n the a c t u a l m e t a b o l i c f a t e o f
4- d e o x y - 4 - f l u o r o - D - g l u c o s e (VII) and i t s d e f l u o r i n a t e d
product. In o r d e r t o examine the b a c t e r i a l s p e c i f i c i t y
o f t h i s C-F c l e a v a g e we have r e c e n t l y examined the
b i o c h e m i c a l e f f e c t s o f (VII) on E. c o l i (ATCC 11775)
and shown t h a t no s i g n i f i c a n t C-F c l e a v a g e by whole
c e l l s or c e l l - f r e e e x t r a c t s occurs. A s m a l l but
s i g n i f i c a n t uptake o f (VII) i s o b s e r v e d (0.06 mg/mg
d r y weight o f b a c t e r i a ) . U s i n g a n o t h e r s t r a i n o f E.
c o l i (ATCC 1494 8) we have a l s o demonstrated ( L o u i e ,
L i - Y u & T a y l o r , N.F. u n p u b l i s h e d r e s u l t s ) t h a t (VII)
p r e v e n t s u t i l i z a t i o n o f l a c t o s e i n t h i s o r g a n i s m by
u n c o m p e t i t i v e i n h i b i t i o n o f the i n d u c t i o n o f β -
galactosidase. Our r e s u l t s are s i m i l a r t o t h o s e r e
p o r t e d f o r the e f f e c t
on E. c o l i ( 4 ) .
T o x i c i t y of 3-deoxy-4-fluoro-D-glucose i n Locusta
înigratoria and S c h i s t o c e r c a g r e g a r i a
A l t h o u g h 3 - d e o x y - 3 - f l u o r o - D - g l u c o s e (II) d i s p l a y s
s e v e r a l p h y s i o l o g i c a l and b i o c h e m i c a l e f f e c t s i n r a t s
(33), i t i s not t o x i c . Coupled w i t h the f a c t t h a t
r a t s e x c r e t e l a r g e q u a n t i t i e s o f unchanged (II) v i a
u r i n e and f a e c e s and a l s o the r e l a t i v e l y l a r g e q u a n t i -
t i e s o f (II) (5g/Kg body weight) n e c e s s a r y t o evoke
a b i o c h e m i c a l response i t was c o n s i d e r e d t o be o f some
i n t e r e s t t o s t u d y an a n i m a l o r g a n i s m which r e q u i r e d a
s m a l l e r dosage o f (II) and r e t a i n e d water t o a g r e a t e r
extent. Two c l o s e l y r e l a t e d A f r i c a n l o c u s t s p e c i e s ,
S c h i s t o c e r c a g r e g a r i a and L o c u s t a m i g r a t o r i a were
chosen f o r t h i s p u r p o s e . In b o t h 10-14 day a d u l t
i n s e c t s (II) was t o x i c (LDCQ 5mg/g l o c u s t t i s s u e ) when
i n j e c t e d i n t o the haemocoel. T h i s compound was a l s o
found t o be t o x i c when o r a l l y i n g e s t e d . T o x i c i t y was
e v i d e n c e d by p r o g r e s s i v e l o s s o f m o t o r a c t i v i t y w i t h
d e a t h o c c u r r i n g between 30 hours t o 4 d a y s . These
symptoms s u g g e s t e d t h a t a slow m e t a b o l i c p o i s o n i n g was
occurring.
U s i n g the gas c h r o m a t o g r a p h i c p r o c e d u r e o u t l i n e d
by F o r d and Candy (34) f o r o b t a i n i n g a c a r b o h y d r a t e
scan, l e v e l s of v a r i o u s steady s t a t e n e u t r a l carbo-
h y d r a t e m e t a b o l i t e s were assayed from v a r i o u s l o c u s t
t i s s u e s (Figure 7). I t was found t h a t l e v e l s o f (II)
d i s a p p e a r e d r a p i d l y from hemolymph, f a t body and f l i g h t
muscle w i t h i n two hours o f i n j e c t i o n . A n a l y s i s of
e x c r e t a i n d i c a t e d t h a t a s i g n i f i c a n t p o r t i o n o f the
i n j e c t e d dose was l o s t . Gas c h r o m a t o g r a p h i c r e s u l t s

Figure 8. (a) Temperature programmed gas

chromatogram of Locust haemolymph after 3-
deoxy-3-fiuoro-D-glucose injections. M = me
tabolite, A = a-D-glucose, Β = β-D-glucose,
C = trehalose, (b) Locust fat body after similar
treatment.
T i me
Figure 9. (a) Gas chromatogram (isothermal

conditions) of locust hemolymph after 3-deoxy-d-
fluoro-D-glucose and glucose administration
using OV-17 stationary support. M = 3-deoxy-
3-fluoro-glucitol G = glucitol, A = a-D-glucose
y y
Β = β-D-glucose. (b) Using E301 stationary

support.

i n d i c a t e d t h a t a new m e t a b o l i t e appeared i n the n e u t r a l

carbohydrate f r a c t i o n f o l l o w i n g a d m i n i s t r a t i o n of ( I I ) .
T h i s c a r b o h y d r a t e was shown t o be the a l d i t o l o f ( I I ) ,
namely, 3 - d e o x y - 3 - f l u o r o - D - g l u c i t o l (X) by gas chrom-
a t o g r a p h i c and TLC comparison o f the s y n t h e t i c a l l y
made a l d i t o l (Lopes, D. and T a y l o r , N.F. u n p u b l i s h e d
r e s u l t s ) . t o the one o b t a i n e d from l o c u s t t i s s u e
e x t r a c t s ( F i g u r e s 8 and 9 ) . Furthermore, a f t e r i n s e c t s
were p o i s o n e d w i t h a 10.8 mg dose o f (II) and 12 hours
l a t e r hemolymph g l u c o s e l e v e l s a r t i f i c a l l y r a i s e d by
a 10.8 mg dose o f g l u c o s e the presence o f 0.4 - 0.5
mg/100mg t i s s u e o f (X) and 0.3 - 0.6 mg/100mg t i s s u e
D - g l u c i t o l was d e t e c t e d . T h i s i s the f i r s t example o f
s o r b i t o l metabolism t o be d e t e c t e d i n e i t h e r o f t h e s e
species. Under normal c o n d i t i o n s s o r b i t o l was not
d e t e c t a b l e i n the n e u t r a l carbohydrate f r a c t i o n . This
phenomenon may hav
(a) the r a t e o f norma
g r e a t e r than i t s r a t e o f f o r m a t i o n , (b) T h i s was not
an important m e t a b o l i c pathway i n the l o c u s t and was
o n l y d e t e c t a b l e due t o i n h i b i t i o n .
S o r b i t o l m e t a b o l i s m has been r e p o r t e d i n the s i l k
worm (35) and mosquito (36). An e x t e n s i v e r e v i e w o f
p o l y o l s and t h e i r metabolism has been o f f e r e d by
T o u s t e r and Shaw (37) . Our r e s u l t s suggested t h a t the
pathway from g l u c o s e t o f r u c t o s e v i a s o r b i t o l was
a c t i v e i n t h e s e l o c u s t s p e c i e s and t h a t (II) b l o c k e d
the subsequent c o n v e r s i o n o f s o r b i t o l t o f r u c t o s e
t h r o u g h the i n h i b i t o r y a c t i o n o f (X) on t h e enzyme
s o r b i t o l dehydrogenase ( F i g u r e 10). A precursor
product r e l a t i o n s h i p i s e v i d e n t between (II) and (X)
i n b o t h hemolymph and f a t body ( F i g u r e 11). Initially
i t was thought t h a t the enzyme s o r b i t o l dehydrogenase
was found i n t h e hemolymph o f the l o c u s t . T h i s i s the
case i n silkworm (35). No a c t i v i t y , however, was
a s s a y a b l e i n l o c u s t hemolymph u s i n g 0.1M s o r b i t o l and
+
lOmM n i c o t i n a m i d e adenine d i n u c e l t i d e (NAD ) as sub-
strates. Subsequent study r e v e a l e d t h a t the enzyme
a c t i v i t y was c o n f i n e d t o the f a t body. U s i n g about
10 f o l d p u r i f i e d enzyme p r e l i m i n a r y r e s u l t s have
suggested t h a t t h i s enzyme i s not v e r y a c t i v e i n l o -
custs having a K M f o r s o r b i t o l i n the o r d e r o f 0.05M.
P r e l i m i n a r y r e s u l t s suggest t h a t (X) i s a c o m p e t i t i v e
i n h i b i t o r o f the enzyme w i t h a KiÔ.lM. These r e s u l t s
a l s o suggest t h a t the pathway o f s o r b i t o l m e t a b o l i s m
from g l u c o s e t o f r u c t o s e i s not v e r y important t o the
t o t a l m e t a b o l i s m o f the l o c u s t . Therefore, i t i s
d i f f i c u l t t o r a t i o n a l i z e how the i n h i b i t i o n o f such
a minor pathway can m a n i f e s t t o x i c a c t i o n . It i s
q u i t e p o s s i b l e t h a t some h i t h e r t o u n d e t e c t e d

Dehydrogenase
Figure 10. Sorbitol conversion pathway.
Time in m i n u t e s
Figure 11. Levels of 3-deoxy-3-fluoro-D-glucose (II)

and 3-deoxy-3-fluoro-D-glucitol (X) in locust hemolymph
and fat body after a 3.6-mg injection of (II). (II)
in hemolymph (X) in hemolymph (II) in fat
body.

metabolites are responsible for t o x i c i t y .

C h e f u r k a (38), (39.) has suggested a means o f
14
e v a l u a t i n g glucose metabolism i n i n s e c t s using 1 - C
14
and 6 - C l a b e l l e d D-glucose by examining t h e r e l a t i v e
4
r a t e s o f ^ C02 e v o l u t i o n . T h i s t e c h n i q u e may be a
u s e f u l t o o l f o r m o n i t o r i n g any o t h e r changes i n t h e
pathways o f g l u c o s e m e t a b o l i s m induced by (II) o r i t s
metabolites. Our s t u d i e s on t h e mode o f t o x i c i t y w i l l
3
be f u r t h e r e d when and/or H - l a b e l l e d f l u o r o g l u c o s e s
become a v a i l a b l e b u t t h e s e p r e l i m i n a r y r e s u l t s i n d i c a t e
t h a t some f l u o r i n a t e d c a r b o h y d r a t e s a r e n o t o n l y t o x i c
t o i n s e c t s b u t may a l s o a c t as probes f o r t h e d e t e c t i o n
o f unsuspected m e t a b o l i c pathways.
Acknowledgment
T h i s work i s s u p p o r t e
C o u n c i l o f Canada.
Abstract
(a) The use of deoxyfluoro-D-glucose as probes
for the study of glucose transport across the human
erythrocyte membrane is discussed. These studies are
also related to the mode of action of cytochalasin Β
inhibition of D-glucose across this membrane,
(b) Evidence is presented to show that 4-deoxy-4-
fluoro-D-glucose is not oxidised by whole resting cells
of Ps. fluorescens (ATCC 12633) but an extensive
release of fluoride anion occurs. With cell-free
extracts of Ps. fluorescens however, 4-deoxy-4-fluoro-
D-glucose is oxidised to the extent of 2 g atoms of
oxygen/mole of substrate. Possible reasons for this
C-F cleavage are discussed. (c) 3-Deoxy-3-fluoro-D-
glucose is toxic to Locusta migratoria (L.D.50 5mg/g
and is metabolised by an NAD-linked sorbitol dehydro
genase, which is located in the fat body of the insect,
to 3-deoxy-3-fluoro-D-glucitol. The activity of the
partially purified enzyme is low with Km, 0.05M for
sorbitol. 3-Deoxy-3-fluoro-glucitol is a competitive
inhibitor with K i , 0.1M.

Literature Cited.
1. Ciba Fdn. Symposium: "Carbon-Fluorine Compounds:
Chemistry, Biochemistry and Biological Activities."
pp 1-417. Associated Scientific Publishers, New
York, 1972.
2. Woodward, B., Taylor, N.F. & Brunt, R.V., Biochem.
Pharmacol. (1971), 20 1071-1077.
3. Taylor, N.F., White, F.H. & Eisenthal, R.E.,
Biochem. Pharmacol. (1972), 21 347-353.
4. Miles, R.J. & S.J. J. Gen. Microbiol. (1973)
76 305-318.
5. Eisenthal, R.E., Harrison, R., Lloyd, W.J. &
Taylor, N.F., Biochem. J. (1972), 130 199-205.
6. Bessel, E.M. & Thomas, P., Biochem. J. (1973) 131
843-850.
7. Wright, J.A., Taylor N.F. Brunt R.V & Brownse
R.W., Chem. Commun
8. Silverman, J.B., Barbiatz, P.S., Mahajan, K.P.,
Buschek, J. & Foudy, T.P., Biochemistry (1975) 14
2252-2258.
9. Barnett, J.E.G., Ralph, A. & Monday, K.A.,
Biochem. J. (1970) 118 843-850.
10. Barnett, J.E.G., Holman, J.D. & Monday, K.A.,
Biochem. J. (1973)131211-221.
11. Riley, G.J. & Taylor, N.F., Biochem. J. (1973) 135
773-777.
12. Kent, P.W., Ciba Fdn. Symposium: "Carbon-Fluorine
Compounds". pp 169-208.
13. Lieb, W.R. & Stein, W.D., Biochim. Biophys. Acta
(1972), 265 187-207.
14. LeFevre, P.G., Pharmacol. Rev. (1961) 13 39-70.
15. Sen, A.K. & Widdas, W.F., J. Physiol (London)
(1962) 160 392-403.
16. Lacko, L. & Burger, M. Biochem. J. (1962) 83
622-625.
17. Baker, G.F. & Widdas, W.F., J. Physiol. (London)
(1972) 271 10p.
18. Miller, D.M. in "Red Cell Structure and Function".
(Jamieson, G.A. & Greinwalt, Τ.A. Eds.) pp 240-292,
J.B. Lippincott & Co. Philadelphia & Toronto, 1969.
19. Kahlenberg, A. & Dolansky, D., Canad. J. Biochem.
(1972) 50 638-643.
20. Bloch, R.J. Biol. Chem. (1974) 249 1814-1822.
21. Taylor, N.F. & Gagneja, G.L., Proc. Canad. Fed.
Biol. Soc. (1975) 18 p 4.
22. Aldridge, D.C., Armstrong, J.J. & Speake, R.N.,
J. Chem. Soc. (1967) 1667-1676.
23. Taylor, N.F., Hill, L. & Eisenthal, R.E.,
Canad. J. Biochem. (1975) 53 57-64.

24. Barford, A.D., Foster, A.B., Westwood, J.H., Hall,

L., & Johnson, R.N., Carbohydr. Res. (1971), 19
49-61.
25. Umbreit, W.W., Burris, R.H. & Stauffer, J.F.,
"Manometric Techniques" pp 1-61. Burgess Publish
ing Co., Minneapolis, Minn. 1964.
26. Woodward, B., Taylor, N.F., & Brunt, R.V., Analyt.
Biochem. (1971) 36 303-309.
27. Wood, W.A. & Schwerdt, R.F., J. Biol. Chem. (1953)
201 501-511.
28. Midgley, M. & Dawes, E.A., Biochem. J. (1973) 132
141 -
29. Kundig, W., Ghosh, S. & Roseman, S., Proc. Nat.
Acad. Sci. (1964) 52 1067-1074.
30. Goldman, P. J. Biol. Chem. (1965) 240 3434-3438.
31. Kirk, Κ., Goldman P. Biochem J (1970) 117
409-410.
32. Wright, J.A., an
(1967) 3 333-339.
33. Riley, G.J., Ph.D. Thesis University of Bath U.K.
(1973).
34. Ford, W.C.L., Candy, D.J., Biochem. J. (1972)
130, 1101.
35. Faulkner, P., Biochem. J. (1958) 68 375.
36. Handel, E. Van, Comp. Biochem. Physiol. (1969) 29
1023.
37. Touster, Ο., Shaw, D.R.D., Physiol. Rev., (1962)
42 181.
38. Chefurka, W., in "The Physiology of Insecta".,
Volume 2, pages 641-656, Academic Press Inc.,
New York, 1965.
39. Chefurka, W., Ela, R., and Robinson, J.R.,
J. Insect Physiol. (1970) 16 2137-2156.
40. Hall, L.D., Johnson, R.N., Foster, A.B. and
Westwood, J.H., Canad. J. Chem. (1971) 49 236-240.
41. Lin, S. and Spudich, J.A., J. Biol. Chem. (1974)
249 5778-5783.

Q. Is the conformation o f glucose, 3-deoxy-3-fluoro

g l u c o s e and 3-deoxy-glucose t h e same?
A. Although there is no direct experimental evidence,
t h e s e t h r e e hexoses p r o b a b l y have t h e same p r e
dominate C l - c o n f o r m a t i o n i n aqueous s o l u t i o n w i t h
a l l t h e s u b s t i t u e n t s equitorial. There is some
e v i d e n c e , however, t h a t t h e i n t r o d u c t i o n o f f l u o r i n e
can d i c t a t e which c o n f o r m a t i o n t h e sugar r i n g w i l l
adopt. Thus Hall et al (40) have shown t h a t i n
the case o f t h e a c e t y l a t e d D - x y l o s y l f l u o r i d e
d e r i v a t i v e s t h e ß-anomer adopts t h e unexpected
c o n f o r m a t i o n i n which all t h e s u b s t i t u e n t s a r e
axially disposed.
Q. Does g l u c o s e b i n d w i t h c y t o c h a l a s i n B?
A. No. The b i n d i n
and our own kinetic results (21) in which c y t o
c h a l a s i n Β is shown t o be a c o m p e t i t i v e inhibitor
( K i , 10- M) o f g l u c o s e t r a n s p o r t in t h e human
7
e r y t h r o c y t e suggest t h a t c y t o c h a l a s i n Β b i n d s t o a
c a r r i e r p r o t e i n and n o t t o g l u c o s e .

7
Organic Fluorocompounds in Human Plasma:
Prevalence and Characterization
W. S. GUY
Department of Basic Dental Sciences, University of Florida, Box J424,
Gainsville, Fla. 32610
D. R. TAVES
Department of Pharmacology and Toxicology, University of Rochester,
Rochester, N.Y. 14642
W. S. BREY, JR.
Department of Chemistry, Universit
Taves discovered tha
tained two distinct forms of fluoride (1-4). Only one of these
was exchangeable with radioactive fluoride. The other, non
-exchangeable form was detectable as fluoride only when sample
preparation included ashing. This paper is concerned with three
aspects of this newly discovered, non-exchangeable form: 1) its
prevalence in human plasma, 2) how its presence in human plasma
affects the validity of certain earlier conclusions about the
metabolic handling of the exchangeable form of fluoride, and
3) its chemical nature.
Preliminary work in this laboratory suggested that the non
-exchangeable form was widespread in human plasma but did not
exist in the plasma of other animals. Ashing increased the amount
of fluoride an average of 1.6 ± 0.25 SDμM(range 0.4-3.0) in
samples of plasma from 35 blood donors in Rochester, N.Y. (5).
No such fluoride was detectable (above 0.3 μM) in blood serum
from eleven different species of animal including horse, cow,
guinea pig, chicken, rabbit, sheep, pig, turkey, mule and two
types of monkey (6).
Standard methods for analysis of exchangeable fluoride in
serum have in the past included ashing as a step in sample
preparation (7). Taves showed that the amount of fluoride in
serum that would mix with radioactive fluoride was only about
one-tenth the amount generally thought to be present based on
analyses using these older methods (4). When plasma samples from
individuals living in cities having between 0.15 and 2.5 ppm
fluoride in their water supply were analysed by these older
methods, no differences were found between the averages for the
different cities. This led to the conclusion that "homeostasis
of body fluid fluoride content results with intake of fluoride
up to and including that obtained through the use of water with a
fluoride content of 2.5 ppm" (8). If the non-exchangeable form
of fluoride predominated in these samples, differences in the
exchangeable fluoride concentration would probably not have been
apparent, and it would be unnecessary to postulate such rigorous
117

homeostatic c o n t r o l mechanisms f o r f l u o r i d e .
In t h i s study plasma samples were c o l l e c t e d from a t o t a l of
106 i n d i v i d u a l s l i v i n g i n f i v e d i f f e r e n t c i t i e s with between 0.1
and 5.6 ppm f l u o r i d e i n t h e i r p u b l i c water supply. These were
analyzed f o r both forms of f l u o r i d e . I n t h i s way the r e l a t i o n
ship between exchangeable f l u o r i d e c o n c e n t r a t i o n i n the plasma
and the consumption of f l u o r i d e through d r i n k i n g water was r e
evaluated, and the prevalence of the non-exchangeable form was
further studied.
With respect to the chemical nature of the non-exchangeable
form of f l u o r i d e s e v e r a l l i n e s of evidence suggested that i t was
some s o r t of organic fluorocompound of intermediate p o l a r i t y ,
t i g h t l y bound to plasma albumin i n the blood. I t migrated with
albumin during e l e c t r o p h o r e s i s of serum a t pH nine (3) and was
not u l t r a f i l t e r a b l e from serum ( 2 ) . Attempts at d i r e c t e x t r a c
t i o n from plasma with s o l v e n t
petroleum ether and e t h y
Treatment of albumin s o l u t i o n (prepared by e l e c t r o p h o r e s i s of
plasma) with c h a r c o a l a t pH three d i d remove the bound f l u o r i n e
f r a c t i o n . And f i n a l l y , when plasma p r o t e i n s were p r e c i p i t a t e d
with methanol a t low pH the f l u o r i n e f r a c t i o n o r i g i n a l l y bound to
albumin appeared i n the methanol-water supernatant i n a form
which s t i l l r e q u i r e d ashing to r e l e a s e f l u o r i n e as i n o r g a n i c
f l u o r i d e (5) . Based on these c o n s i d e r a t i o n s the non-exchangeable
form of f l u o r i d e i n human plasma i s r e f e r r e d to as "organic
f l u o r i n e " throughout the r e s t of t h i s paper.
In order to f u r t h e r c h a r a c t e r i z e the organic f l u o r i n e
f r a c t i o n , i t was p u r i f i e d from 20 l i t e r s of pooled human plasma
and c h a r a c t e r i z e d by f l u o r i n e nmr.
M a t e r i a l s and Methods
A n a l y t i c a l Methods. Values f o r organic f l u o r i n e were c a l c u

l a t e d by taking the d i f f e r e n c e between the amount of i n o r g a n i c
f l u o r i d e i n ashed and unashed p o r t i o n s of the same m a t e r i a l .
The f o l l o w i n g procedure was used to prepare ashed samples:
1) samples (sample s i z e f o r plasma was 3 ml) were placed i n
platinum c r u c i b l e s and mixed with 0.6 mmoles of low
f l u o r i d e MgCl and 0.1 mmoles of NaOH, 2) these were d r i e d on a
2
h o t p l a t e and then ashed (platinum l i d s i n place) f o r 2-4 h r a t

600° C i n a muffle furnace which had been modified so that the
chamber received a flow of a i r from outside the b u i l d i n g (room
a i r increased the blank and made i t more v a r i a b l e ) , and 3) ashed
samples were d i s s o l v e d i n 2 ml of 2.5 Ν H S0^ and t r a n s f e r r e d t o
2
polystyrene d i f f u s i o n dishes using 2 r i n s e s with 1.5 ml of water.

The f o l l o w i n g procedure was used f o r s e p a r a t i o n of f l u o r i d e
from both ashed and unashed samples: 1) samples (sample s i z e f o r
unashed plasma was 2 ml) were placed i n d i f f u s i o n dishes (Organ
C u l t u r e Dishes, Falcon P l a s t i c s , Oxnard, C a l i f . , absorbent

7. G U YE T A L . Fluorocompounds in Human Pfosma 119
removed, r i n s e d with water), a c i d i f i e d with 2 ml of 2.5 Ν H SO^, 2
and a g i t a t e d with a g e n t l e s w i r l i n g a c t i o n on a l a b o r a t o r y shaker

f o r 30 min to remove C0 ; 2) f o r each sample the trapping s o l u
2
t i o n (0.5 ml, 0.01 Ν NaOH + p h e n o l t h a l e i n - p - n i t r o p h e n o l i n d i c a t o r )

was placed i n a small polystyrene cup i n the c e n t e r - w e l l of the
d i f f u s i o n d i s h , 1 drop of 10% T r i t o n - X 100 was added to the
sample to decrease surface t e n s i o n and make the d i f f u s i o n r a t e
more uniform between samples c o n t a i n i n g plasma and those not, the
l i d with a small hole made near i t s l a t e r a l margin was sealed
i n t o p l a c e with petroleum j e l l y , 0.02 ml of 4% hexamethyldisilox-
ane (Dow Corning, F l u i d 200, 0.65 c s , Midland, Mich.) i n ethanol
was i n j e c t e d through the hole i n the l i d i n t o the sample, and the
hole was sealed Immediately with petroleum j e l l y and a s t r i p of
p a r a f f i n f i l m ; and 3) samples were d i f f u s e d with g e n t l e s w i r l i n g
f o r a t l e a s t 6 hr, d i f f u s i o n was terminated by breaking the s e a l
and trapping s o l u t i o n
checked a t t h i s p o i n t t
and d r i e d i n a vacuum oven (60° C, 26 in-Hg vacuum, i n the
presence of a NaOH d e s i c c a n t ) .
F l u o r i d e was determined by potentiometry with the f l u o r i d e
e l e c t r o d e . The system used c o n s i s t e d of a f l u o r i d e e l e c t r o d e
o r i e n t e d i n an i n v e r t e d p o s i t i o n (model 9409A, Orion Research Inc.
Cambridge, Mass.), a calomel reference e l e c t r o d e ( f i b e r t y p e ) , a
p l a s t i c vapor s h i e l d which j u s t f i t t e d over the bodies of both
e l e c t r o d e s forming an enclosed sample chamber i n which water-
saturated t i s s u e paper was placed above the sample to prevent
e v a p o r i z a t i o n of the sample, and a high impedence voltmeter
(model 401, O r i o n ) .
Samples were prepared and read i n the f o l l o w i n g way: 10 μΐ
of 1 M HAc was drawn i n t o a polyethylene m i c r o p i p e t t e (Beckman
Micro Sampling K i t , Spinco Div., Beckman I n s t . Co., Palo A l t o ,
C a l i f . ) and deposited i n t o the cup c o n t a i n i n g the r e s i d u e from
the trapping s o l u t i o n a f t e r d r y i n g ; the f l e x i b l e t i p of the
m i c r o p i p e t t e was used to wash down the w a l l s of the cup; and the
s o l u t i o n was then t r a n s f e r r e d to the surface of the f l u o r i d e
e l e c t r o d e and the reference e l e c t r o d e brought i n t o p o s i t i o n .
Surfaces of the two e l e c t r o d e s were b l o t t e d dry between samples.
Samples were read i n order of i n c r e a s i n g expected concentra
t i o n and sets of samples were read between bracketing c a l i b r a t i o n
standards. These standards were used i n two d i f f e r e n t ways
during a run. F i r s t , they were flooded onto the e l e c t r o d e
surfaces to e q u i l i b r a t e them to concentrations expected f o r
samples and to make them uniform. This procedure permitted the
a n a l y s t to take reasonably s t a b l e readings f o r samples w i t h i n
one minute. Secondly, they were used i n 10 y l volumes f o r
readings used i n preparing the standard curve.
Values f o r i d e n t i c a l samples ( u s u a l l y t r i p l i c a t e s ) were
averaged and the average blank was subtracted from sample means.
These were then d i v i d e d by the average f r a c t i o n a l recovery of

f l u o r i d e (usually 90 to 95%) i n standards treated the same way as

the sample s e t .
P l a s t i c w a r e (Falcon P l a s t i c s ) was used f o r a l l a n a l y t i c a l
procedures to avoid contamination by f l u o r i d e from g l a s s . Liquid
volume measurements were made with 1, 5 and 10 ml p o l y s t y r e n e
p i p e t t e s and a polycarbonate volumetric f l a s k (100 ml).
Reagents were p u r i f i e d to i n s u r e uniformly low blanks.
Water was r e d i s t i l l e d and d e i o n i z e d . A c e t i c a c i d and ammonia
were r e d i s t i l l e d . F l u o r i d e contamination i n MgCl2 ( a n a l y t i c a l
grade) was reduced by preparing a 1 M s o l u t i o n c o n t a i n i n g HC1 to
pH 1 and scrubbing with hexamethyldisiloxane vapor i n a column
through which the s o l u t i o n was continuously r e c y c l e d . Following
scrubbing the s o l u t i o n was b o i l e d to one t h i r d volume to remove
any r e s i d u a l v o l a t i l e s i l i c o n e s and then made j u s t b a s i c with
NHi+OH. F l u o r i d e contamination i n I^SO^ was reduced by repeated
e x t r a c t i o n s of a 6.7 Ν
then b o i l i n g to one t h i r
Buffered c a l i b r a t i o n standards were made from the same NaOH
and HAc stock s o l u t i o n s as f o r samples.
The blanks f o r ashed samples ranged between 0.2 and 1.5
nmoles f l u o r i d e and were t y p i c a l l y about 0.5 nmoles. The blanks
were smaller f o r unashed samples; these ranged between 0.05 and
0.2 nmoles f l u o r i d e and were t y p i c a l l y about 0.1 nmoles.
Factors a f f e c t i n g recovery of f l u o r i d e during d i f f u s i o n
were i n v e s t i g a t e d w i t h F~ t r a c e r . Recovery during d i f f u s i o n
was 97% a f t e r 80 min from 5 ml containing 2 ml of plasma.
Increasing the a c i d i t y of the sample up to 5 N, the volume of the
sample up to 7.5 ml, the amount of c o l d F~ up to 1 pmole, the
amount of f l u o r i d e complexors up to 1 ymole of ThiNC^)^ had no
m a t e r i a l e f f e c t on the r a t e of f l u o r i d e d i f f u s i o n . The absence
of both plasma and detergent i n the sample compartment markedly
slowed the r a t e of d i f f u s i o n . Not shaking the sample a l s o slowed
the r a t e of d i f f u s i o n . Increasing the a l k a l i n i t y of the trapping
s o l u t i o n to 0.1 Ν increased the r a t e of d i f f u s i o n but the lower
concentration, 0.01 N, was r e q u i r e d here to permit a lower i o n i c
strength i n the sample reading s o l u t i o n .
O v e r a l l recovery of added cold f l u o r i d e was measured. In
samples containing n e i t h e r plasma nor detergent the
recovery a f t e r 6 hr d i f f u s i o n averaged 93% and 95% f o r ashed and
unashed samples, r e s p e c t i v e l y . In samples containing plasma the
recovery was 95% a f t e r 3 hr d i f f u s i o n .
The degree to which f l u o r i n e from organic fluorocompounds
could be f i x e d as i n o r g a n i c f l u o r i d e by ashing v a r i e d from l e s s
than 1% f o r v o l a t i l e compounds l i k e p - a m i n o b e n z o t r i f l u o r i d e ,
m-hydroxybenzotrifluoride, b e n z y l f l u o r i d e and benzotrifluoride
to over 80% f o r l e s s v o l a t i l e compounds l i k e 5 - f l u o r o u r a c i l ,
f l u o r o a c e t a t e and p - f l u o r o p h e n y l a l a n i n e .
Methods used here f o r s e p a r a t i o n of f l u o r i d e ( d i f f u s i o n at
rm. temp.) (9) and i t s q u a n t i t a t i o n ( f l u o r i d e e l e c t r o d e ) (10) are
considered to be q u i t e s p e c i f i c f o r f l u o r i d e . One p o t e n t i a l l y

7. GUY E T A L . Fluorocompounds in Human Plasma 121
important i n t e r f e r e n c e , however, was c o d i f f u s a b l e organic a c i d s

which might p a r t i a l l y n e u t r a l i z e the trapping s o l u t i o n and thus
lower the pH of the b u f f e r e d reading s o l u t i o n . Indeed, i t was
found that samples c o n t a i n i n g r e l a t i v e l y l a r g e concentrations of
a c e t i c acid (e.g., f r a c t i o n s 2, 3 and 4 from step 4 i n the
p u r i f i c a t i o n system) completely n e u t r a l i z e d the trap w i t h i n a
few hours. The s i g n i f i c a n c e of t h i s problem i n the a n a l y s i s of
f l u o r i d e i n blood plasma was i n v e s t i g a t e d i n two ways. F i r s t ,
four samples of human plasma were allowed to d i f f u s e f o r three
weeks, and no change i n the c o l o r of the phenolphthalein i n d i c a
tor i n the trapping s o l u t i o n was observed. Secondly, samples
c o n t a i n i n g the same plasma were d i f f u s e d f o r d i f f e r e n t periods
up to 158 hr and the apparent f l u o r i d e was determined. No changes
were observed between samples which c o r r e l a t e d with d i f f u s i o n
time.
The s e n s i t i v i t y o
the blank r a t h e r than th
R e p r o d u c i b i l i t y v a r i e d with the amount of f l u o r i d e being
measured. The c o e f f i c i e n t of v a r i a t i o n averaged 55% i n the low
range (samples c o n t a i n i n g 0.25 to 0.75 nmoles F ) and 6.6% i n
the high range (10-12 nmoles F " ) .
Blood Plasma. Human plasma was obtained from blood banks

i n f i v e c i t i e s . According to p u b l i c records these c i t i e s had
not changed the f l u o r i d e c o n c e n t r a t i o n of t h e i r p u b l i c water
supply f o r a t l e a s t s i x years p r i o r to o b t a i n i n g the samples.
Samples were r e c e i v e d i n i n d i v i d u a l polyethylene bags which were
p a r t of the Fenwall ACD blood c o l l e c t i o n system. In blood
c o l l e c t i o n using t h i s system 450 ml of blood i s drawn i n t o a bag
containing 67.5 ml of anticoagulant a c i d c i t r a t e dextrose (ACD)
s o l u t i o n . When the c e l l s are removed the ACD s o l u t i o n remains
i n the plasma. Because of t h i s d i l u t i o n of plasma a c o r r e c t i o n
f a c t o r of 1.3 was a p p l i e d to values obtained here f o r the
c o n c e n t r a t i o n of f l u o r i d e . The p o t e n t i a l e r r o r i n t h i s f a c t o r
was ± 0 . 1 because of v a r i a t i o n between standard l i m i t s f o r
hematocrit and minimum volume of the blood donation. Bovine
blood was obtained a t slaughter and mixed immediately with ACD
s o l u t i o n i n 1 l i t e r polyethylene b o t t l e s .
E l e c t r o p h o r e s i s . A continuous flow e l e c t r o p h o r e t i c
separator (model FF-3, Brinkman I n s t . , Inc., Westbury, N.Y.) was
employed. Sample flow r a t e was 2.3 ml/hr, b u f f e r flow r a t e was
72 ml/hr, v o l t a g e was 0.67 kv, and current was 140 mamp. Separa
t i o n took 19 hr. P l a t e s e p a r a t i o n was 1 mm and operating tempera
ture was between 2 and 4° C. The b u f f e r was 0.12% (NHi ) C0 , + 2 3
made by bubbling C 0 from dry i c e i n t o r e d i s t i l l e d ΝΗίψΟΗ u n t i l

2
the pH reached 9.0.

P u r i f i c a t i o n System. Steps i n the p u r i f i c a t i o n system are

summarized i n t a b l e I. In the f i r s t step one l i t e r of plasma
(pooled from 5-6 i n d i v i d u a l s ) was d i a l y s e d i n seamless c e l l u l o s e
tubing (1 i n . diameter) against 20 l i t e r s of water at 4° C. The
d i a l y s a t e was changed twice at 24 hr i n t e r v a l s . In the second
step d i a l y s e d plasma was f r e e z e d r i e d .
In the t h i r d step the d r i e d powder from e l e c t r o p h o r e s i s
was extracted with methanol i n a soxhelet e x t r a c t i o n apparatus
(model 6810 G, Ace Glass, Inc., Vineland, N.J.). C e l l u l o s e
e x t r a c t i o n thimbles (model 6812 G, Ace Glass) were soaked
overnight i n methanol. Operating c o n d i t i o n s were 25° to 30° C
under a vacuum of 24 in-Hg. Coolant f o r the condenser was 80%
ethanol; i n l e t temperature was -10° to -20° C and o u t l e t tempera
ture was -10° to 0°. Two l i t e r s of methanol were r e f l u x e d
through the apparatus f o r a period of 4 hr and approximately
400 ml were l o s t to evaporatio
were placed i n the f l a s
In the f o u r t h step the r e s i d u e from the methanol e x t r a c t
was f r a c t i o n a t e d according to the method described by Siakotos
and Rouser (11) f o r separating l i p i d and n o n - l i p i d components.
The method i s based on l i q u i d - l i q u i d p a r t i t i o n i n a column
containing a dextran g e l (Sephadex G-25, coarse, beaded,
Pharmacia F i n e Chemicals, Inc., N.Y.). Four eluents are used:
1) 500 ml chloroform/methanol, 19/1, saturated with water,
2) 1000 ml of a mixture of 5 p a r t s of chloroform/methanol,
19/1, and 1 part of g l a c i a l a c e t i c a c i d , saturated with water,
3) 500 ml of a mixture of 5 p a r t s chloroform/methanol, 19/1, and
1 part g l a c i a l a c e t i c a c i d , saturated with water, and 4) 1000 ml
of methanol/water, 1/1. T h e i r method was modified f o r use here
by i n c r e a s i n g the column length to that a t t a i n e d by using a f u l l
100 grams of dextran beads. Sample s i z e corresponded to that
from 2.5 l i t e r s of the o r i g i n a l plasma.
In the f i f t h step the residues from eluents 2 and 3 from two
runs of step four were combined, a p p l i e d to a s i l i c i c a c i d
column, and e l u t e d by reverse flow with an exponential gradient
of i n c r e a s i n g amounts of methanol i n chloroform. The column
(model SR 25/45, 2.5 cm i . d . χ 45 cm, Pharmacia) was f i l l e d to
a height of 30 cm with s i l i c i c a c i d ( U n i s i l , 100-200 mesh,
Clarkson Chem. Co., Inc., W i l l i a m s p o r t , Pa., heat a c t i v a t e d at
110° C f o r 2 days) and was washed with a complete set of e l u t i o n
s o l v e n t s before use. The gradient maker (model 5858, set 4,
Ace Glass Co.) was f i l l e d with 1 l i t e r of methanol i n the upper
chamber and 2 l i t e r s of chloroform i n the lower. The flow r a t e
was adjusted by the height of the s o l v e n t r e s e r v o i r s to an
average of 3 ml/min f o r the f i r s t l i t e r of eluent. The sample
had to be t r a n s f e r r e d to the column by repeated washings with
chloroform because of i t s low s o l u b i l i t y i n t h i s s o l v e n t . This
u s u a l l y r e q u i r e d about 30 ml of chloroform t o t a l . Dead volume
for the system as 90 ml. F r a c t i o n s of 15 ml volume were
c o l l e c t e d i n c a r e f u l l y cleaned g l a s s tubes.

7. GUY E T A L . Fluorocompounds in Human Plasma 123
Table I
PROCEDURE FOR PURIFICATION

OF FLUOROCOMPOUNDS FROM BLOOD PLASMA
Fraction Fraction
Treated Treatment Removed
blood plasma step 1: exhaustive smaller, water-

d i a l y s i s against s o l u b l e components
d i s t i l l e d water
plasma p r o t e i n s & step 2: lyophili- water

protein-bound zation
substances i n
water s o l u t i o n
plasma p r o t e i n s &
protein-bound extraction—sox-
substances h e l e t , 25°C, 24
in-Hg vacuum
plasma l i p i d s step 4: column l i p i d s of low

chromatography— p o l a r i t y and
liquid-liquid residual polar
p a r t i t i o n on contaminants
Sephadex
polar l i p i d s step 5: column unknown: severa1

chromatography— yellow f r a c t i o n s
adsorption on
s i l i c i c acid
Figure 1. Relationship between the concentration of

fluoride in human plasma and the concentration of fluo-
ride in the drinking water

Figure 2. Relationship between the concentration of organic

fluorine in human pfosma and the concentration of fluoride in
the drinking water
TUBE NO.
Figure 3. Separation of fluoride and organic fluorine in

human plasma by electrophoresis. A sample (about 45
ml) of human phsma was electrophoresed in pH 9 buffer
and fractions between the sampling port (near tube 72)
and the positive pole (near tube 1) were analyzed for the
fluoride content of both ashed and unashed aliquots.
Relative concentrations of proteins were estimated by
absorbance at 280 nm.

7. GUY ET AL. Fluorocompounds in Human Plasma 125
Tubing and f i t t i n g s to the columns were p o l y t e t r a f l u o r o e t h y -

lene (supplied l a r g e l y by Chromatronix, Inc., Berkeley, C a l i f . ) .
A l l solvents were r e d i s t i l l e d . Methanol and chloroform were
ACS c e r t i f i e d ( F i s h e r S c i e n t i f i c Co.) and a c e t i c a c i d was
a n a l y t i c a l reagent grade, U.S.P. ( M a l l i n c k r o d t Chemical Works,
St. L o u i s ) . Solvents were removed from samples i n a f l a s h
evaporator.
NMR. The nmr spectrum was obtained on a V a r i a n XL-100

spectrometer with N i c o l e t Technology F o u r i e r Transform accessory.
The sample was d i s s o l v e d i n an approximately 1/1 mixture of
CH OH and CDCI3 and s p e c t r a were run i n a 5 mm tube.
3 External
r e f e r e n c i n g to CFCI3 was used f o r the chemical s h i f t s , and these
are expressed with p o s i t i v e numbers to lower f i e l d ( i . e . , higher
frequency). E x t e r n a l l o c k was used. T y p i c a l c o n d i t i o n s were a
pulse length of 15 microseconds
c y c l e s of 2.5 sec, and a
processing.
Results
Values f o r i n o r g a n i c f l u o r i d e (F~) and organic f l u o r i n e

(R-F) i n 106 plasma samples from humans l i v i n g i n f i v e c i t i e s
are shown i n t a b l e I I . These data show that the average f l u o r i d e
c o n c e n t r a t i o n i n plasma i s d i r e c t l y r e l a t e d to the f l u o r i d e
c o n c e n t r a t i o n i n the water supply, and that the average organic
f l u o r i n e concentration i n plasma i s not. No r e l a t i o n s h i p between
f l u o r i d e i n plasma and organic f l u o r i n e i n plasma was apparent
by i n s p e c t i o n of values f o r i n d i v i d u a l samples. The d i s t r i b u t i o n s
of the values w i t h i n c i t i e s are shown i n f i g u r e s 1 and 2. In
both cases the d i s t r i b u t i o n s appear to be l o g normally d i s t r i b u t e d
with only 3 or 4 i n d i v i d u a l s s u r p r i s i n g l y deviant. In the cases
of the two i n d i v i d u a l s with l i t t l e or no apparent organic f l u o r i n e
( f i g u r e 2, Andrews group, l e f t margin), the i n o r g a n i c f l u o r i d e
l e v e l s were both i n excess of 7 μΜ, making the d i f f e r e n c e measure
ment f o r organic f l u o r i n e d i f f i c u l t . The o v e r a l l mean value f o r
organic f l u o r i n e was 1.35 ± 0.85 SD μΜ.
Plasma was electrophoresed i n an attempt to reproduce the
f i n d i n g s of Taves (3) using plasma from another i n d i v i d u a l .
Results shown i n f i g u r e 3 c l o s e l y match those found e a r l i e r i n
that a predominant form of organic f l u o r i n e appeared to migrate
with albumin at pH 9, and i n that organic and i n o r g a n i c forms
were c l e a r l y separated.
The recovery, mass balance and p u r i f i c a t i o n f a c t o r s f o r steps
i n the p u r i f i c a t i o n system l i s t e d i n t a b l e I are recorded i n
t a b l e I I I . These data show that about o n e - t h i r d of the o r i g i n a l
amount of organic f l u o r i n e i n plasma i s recovered i n the major
peak from s i l i c i c a c i d chromatography. Another t h i r d i s accounted
for i n other f r a c t i o n s and the r e s t i s not accounted f o r ,
presumably because of adsorption to surfaces of containers i n

Table I I
CONCENTRATION OF FLUORIDE (F~) AND ORGANIC FLUORINE

(R-F) IN BLOOD PLASMA SAMPLES FROM FIVE CITIES HAVING
DIFFERENT FLUORIDE CONCENTRATIONS IN THEIR WATER SUPPLY
[F"] i n Plasma , uMa

TR-Fl i n Plasma ***, yM
C , d
C i t y ([F~] i n Mean± Diff.° Mean! Diff.
Water, ppm) SD(n) Range P<.05 SD(n) Range P<.05
Albany, N.Y. 0.38± 0.14- 1.2± 0.3-

(<.D 0.21 1.1 0.6 2.6
(30) (30)
Rochester, 0.89±
N.Y.(1.0) 0.75 4.2 1.2 6.8
(30) (30)
n.s. n. s.
Corpus C h r i s t i , 1.0± 0.60- 1.3± 0.4-
Tex.(0.9) 0.35 1.7 0.9 3.9
(12) (12)
sig. n. s.
Hillsboro, 1.9± 0.60- 2.3± 1.5-
Tex.(2.1) 0.9 2.6 0.6 2.8
(4) (4)
sig. sig.
Andrews, Tex. 4.3± 1.4- 1.1± 0.1-
(5.6) 1.8 8.7 0.5 2.3
(30) (30)
^Sach value used i n the computation was the average of a t l e a s t

three r e p l i c a t e analyses and was corrected f o r d i l u t i o n by
b ACD s o l u t i o n by m u l t i p l y i n g i t by 1.3.
taken to be the d i f f e r e n c e between the amount of inorganic
f l u o r i d e measured i n ashed and unashed a l i q u o t s of the same
c sample
^by t - t e s t assuming equal v a r i a n c e i n each group
The d i f f e r e n c e between Rochester and Andrews i s s t a t i s t i c a l l y
significant.

7. GUYE TAL. Fluorocompounds in Human Plasma 127
Table I I I
MASS BALANCE, RECOVERY AND PURIFICATION

FACTOR FOR STEPS IN THE PURIFICATION SYSTEM
a b
Dry Wt. Amt. R-F Recovery Purifi-
Fraction grams nmoles % cation
human plasma 200 1725

(ACD, 2.5 l i t e r ±273(6)
batch)
Methanol E x t r a c t i o n
extract 10.
residue — 105 6.1
±37(4) ±1.0
Sephadex Column
Fraction I — 125 7.3

±18(4) ±1.6
Fractions II + I I I 1.29 1195 69.3 108 X
±129(6) ±13.3
F r a c t i o n IV — 118 6.8
±29(4) ±1.2
S i l i c i c A c i d Column
d
major peak .03° 630 36.5 2,440 X
d
other peaks — 240 13.9
combined
fmean ± SD(n)
percent of the amount of R-F i n the o r i g i n a l plasma sample,
mean ± SD
estimate based on weighing the contents of two tubes i n the center
^ of the major peak
estimate based on area under peaks from graph

I * I 1
U 1
< 1
" l " » H » I M ι Γ -
0 10 20 30 40 SO 60 70 80 90 100
TUBE NO.
Figure 4. Distribution of organic fluorine from human and bovine plasma in
fractions from silicic acid chromatography

7. GUY E T AL. Fluorocompounds in Human Pfosma 129
which samples were placed.

The blank f o r the p u r i f i c a t i o n process was obtained by
using bovine r a t h e r than human plasma. No organic f l u o r i n e was
d e t e c t a b l e i n the o r i g i n a l bovine sample but as a f u r t h e r check
the sample was d i a l y s e d to remove i n o r g a n i c f l u o r i d e to f a c i l i
t a t e making the measurement f o r organic f l u o r i n e by d i f f e r e n c e .
Some organic f l u o r i n e was apparent i n d i a l y s e d bovine plasma:
0.13 ± 0.11 SD μΜ (n=6), a s t a t i s t i c a l l y s i g n i f i c a n t though
small d i f f e r e n c e . This t r a c e amount of organic f l u o r i n e c l e a r l y
was not found i n the same s i l i c i c f r a c t i o n s as the dominant peak
from human plasma as shown i n f i g u r e 4.
Human plasma had been stored i n polyethylene bags w i t h ACD
s o l u t i o n . A n a l y s i s of ACD s o l u t i o n from unused blood bags and
a n a l y s i s of blood plasma before and a f t e r p l a c i n g i t i n the bags
showed that not more than 5% of the organic f l u o r i n e i n human
plasma could have come fro t h i
The d i s t r i b u t i o n o
s i l i c i c a c i d chromatography figur
batches corresponding to 5 l i t e r s of the o r i g i n a l plasma each.
There i s c l e a r l y one dominant peak l y i n g i n approximately the
same e l u t i o n p o s i t i o n f o r each batch (the exact p o s i t i o n v a r i e d
with column use and the degree of h y d r a t i o n of the s i l i c i c a c i d
adsorbant). There were always some s m a l l e r secondary peaks, but
they v a r i e d i n s i z e and p o s i t i o n r e l a t i v e to the major peak.
The sample used f o r c h a r a c t e r i z a t i o n by nmr was obtained by
combining the f r a c t i o n s c o n t a i n i n g the major peaks i n each of
the four batches. Much of the m a t e r i a l from batches one and two
had been used f o r other purposes p r i o r to t h i s combination. The
combined sample was rechromatographed on s i l i c i c a c i d and a
s i n g l e sharp peak obtained. The f i n a l sample was taken from the
c e n t r a l p o r t i o n of that peak and contained 3.3 μπιοΐββ of organic
fluorine.
Four sample runs were made on the nmr spectrometer w i t h
15,000 to 17,000 scans each and with a sweep width of 15,151 Hz
i n a l l but one run, where i t was 7,576. The r e s u l t s of a l l runs
were c o n s i s t e n t w i t h the spectrum shown i n f i g u r e 5 and the
chemical s h i f t s shown i n t a b l e IV. A blank run on the solvent
mixture showed no i n s t r u m e n t a l a r t i f a c t s which might have
contributed to the spectrum. Chemical s h i f t s determined f o r
p e r f l u o r o - o c t a n o i c a c i d are a l s o included i n t a b l e IV. Compari
son of the s h i f t s i n the unknown with that of p e r f l u o r o - o c t a n o i c
a c i d show that there i s a constant d i f f e r e n c e i n s h i f t s of about
2 ppm except f o r the -CF - peak next to the f u n c t i o n a l group (peak
2
E) where the s h i f t i s about 6 ppm. Only the l a t t e r i s enough to

be considered a s i g n i f i c a n t d e v i a t i o n s i n c e e x t e r n a l r e f e r e n c i n g
was used f o r each. The d i f f e r e n c e i n s h i f t f o r peak Ε i s
c o n s i s t e n t with the presence of amide or e s t e r d e r i v a t i v e s , or
p o s s i b l y w i t h the presence of a s u l f o n i c a c i d d e r i v a t i v e as the
f u n c t i o n a l group. One e x p l a n a t i o n f o r the a d d i t i o n a l peaks i n
the spectrum i s the presence of branched isomers, peaks A and Β

Table IV
RESULTS OF NMR SPECTROSCOPIC ANALYSIS
Chemical S h i f t , ppm
Perfluoro-
Peak octanoic Suggested
Designation Sample Acid Assignments
A -70.7 -CF groups a t

3
branch p o i n t s
Β -71.9
terminal -CF i n 3
C -80.0 branched isomers
D -81.
Ε -114.3 -120.2 -CF - next to X b

2
F -120.3 -123.1
-CF - i n 2
G -121.5 -124.2 -CF -CF -CF -

2 2 2
- C F - next to
2
H -122.3
branch p o i n t s
I -126.0 -127.6 -CF - next to
2
terminal -CFo
E x t e r n a l r e f e r e n c i n g t o CFC1 was used f o r the chemical

3
s h i f t s , and these a r e expressed with p o s i t i v e numbers

, to lower f i e l d ( i . e . , higher frequency),
where X i s l i k e l y to be -CO-Y
wXjlLt
ι ι I ι ι ι ι I ι ι ι ι I ι ι ι ι I ι ι ι I ι ι ι ι I ι i ι ι I i ι ι ι I i ι ι ι I ι 1
AB CD Ε FGH I
Figure 5. NMR spectrum of organic fluorocompound(s) isolated from human
pfosma

7. GUY ET AL. Fluorocompounds in Human Plasma 131
representing - C F 3 groups at branch p o i n t s , peak C the -CF 3
groups two carbons removed from the branch p o i n t s , and peak H

representing -CF - next to the branch p o i n t s .
2
The sample was reanalyzed f o r organic f l u o r i n e f o l l o w i n g

c h a r a c t e r i z a t i o n by nmr to check f o r contamination; no a d d i t i o n a l
f l u o r i n e was apparent. The degree to which f l u o r i n e from
p e r f l u o r o - o c t a n o i c a c i d i s f i x e d as i n o r g a n i c f l u o r i d e during
ashing was found to be 21 ± 3 SD % (n=3).
Discussion
These f i n d i n g s suggest that there i s widespread contamination

of human t i s s u e s with t r a c e amounts of organic fluorocompounds
derived from commercial products. A l l a v a i l a b l e i n f o r m a t i o n on
t h i s subject i s i n accordance with t h i s i n t e r p r e t a t i o n . A
s e r i e s of compounds havin
here f o r the predominan
i s widely used commercially poten proper
ties. For example, they are used as water and o i l r e p e l l e n t s i n
the treatment of f a b r i c s and l e a t h e r . Other uses i n c l u d e the
production of waxed paper and the formulation of f l o o r waxes
(12). The f i n d i n g s presented here that the c o n c e n t r a t i o n of
organic f l u o r i n e was not r e l a t e d to the c o n c e n t r a t i o n of inorgan-
i c f l u o r i d e e i t h e r i n blood or i n the p u b l i c water supply, and
the e a r l i e r f i n d i n g that there was l i t t l e or no organic f l u o r i n e
i n the blood of animals other than human (6) are a l l i n keeping
with environmental sources such as these.
The prevalence of organic f l u o r i n e i n human plasma i s
probably q u i t e high s i n c e 104 of the 106 plasma samples tested
here and a l l 35 i n an e a r l i e r study (5) had measurable q u a n t i t i e s .
The prevalence of the p a r t i c u l a r compounds i s o l a t e d and charac-
t e r i z e d here, i . e . , p e r f l u o r o f a t t y a c i d ( C - C ) d e r i v a t i v e s , i s
6 8
not known s i n c e the s t a r t i n g m a t e r i a l f o r each batch shown i n

f i g u r e 4 was pooled from between 25 and 30 i n d i v i d u a l s and
s i n c e only about one t h i r d of the o r i g i n a l organic f l u o r i n e
content was accounted f o r i n the f r a c t i o n s c o n t a i n i n g these
compounds (see t a b l e I I I ) .
Peaks other than the one c h a r a c t e r i z e d by nmr appear i n the
chromatograms shown i n f i g u r e 4 suggesting that human plasma
contains other forms of organic fluorocompounds. They are
probably not v o l a t i l e compounds l i k e freons s i n c e i t i s d o u b t f u l
that these would be detected by the a n a l y t i c a l methods used i n
t h i s study. They correspond i n s o l u b i l i t y to very p o l a r l i p i d s
s i n c e they appear i n f r a c t i o n s two and three i n the f o u r t h p u r i -
f i c a t i o n step. According to the authors of the method used i n
that step the f i r s t eluent contains most f a t s , the second and
t h i r d eluents c o n t a i n very p o l a r f a t s l i k e g a n g l i o s i d e s and
c e r t a i n b i l e a c i d s i n a d d i t i o n to compounds l i k e urea, phenyla-
l a n i n e and t y r o s i n e . The l a s t f r a c t i o n contains water s o l u b l e
n o n - l i p i d compounds (11). Components of these other peaks are

l e s s p o l a r than the compounds i n the predominant peaks i n

accordance with the methanol-in-chloroform gradient used to
e l u t e them i n the f i f t h p u r i f i c a t i o n step. Other forms not seen
i n s i l i c i c a c i d f r a c t i o n s may a l s o e x i s t s i n c e only about h a l f
the o r i g i n a l organic f l u o r i n e was recovered i n these f r a c t i o n s .
The a c t u a l amounts of the p e r f l u o r i n a t e d f a t t y a c i d
d e r i v a t i v e s i n human plasma i s not known both because
i n d i v i d u a l plasma samples were not assayed f o r these p a r t i c u l a r
compounds and because the degree to which organic f l u o r i n e from
these compounds i s converted to i n o r g a n i c f l u o r i d e during ashing
i e not known. Metal s a l t s of p e r f l u o r i n a t e d f a t t y a c i d s have been
reported to decompose at 175 to 250° C forming 00 , v o l a t i l e 2
p e r f l u o r i n a t e d o l e f i n s one carbon s h o r t e r , and one atom of

f l u o r i d e per molecule (13). About 3 f l u o r i n e atoms per molecule
of p e r f l u o r o - o c t a n o i c a c i d were f i x e d as i n o r g a n i c f l u o r i d e by
ashing methods used here
f l u o r i d e a f t e r ashing f r a c t i o n
4 probably represent somewhere between o n e - t h i r d and one times
the molar amount.
L i t t l e has been published about the metabolic handling and
t o x i c o l o g y of p e r f l u o r i n a t e d f a t t y a c i d d e r i v a t i v e s . Computer
a s s i s t e d l i t e r a t u r e searches using Medline, T o x l i n e and Chemcon
developed no i n f o r m a t i o n on these s u b j e c t s . This was s u r p r i s i n g
with respect to the widespread commercial use of such compounds.
I t would appear from information presented here that r a p i d
e x c r e t i o n of such compounds i n t o u r i n e i s u n l i k e l y s i n c e they
are bound to albumin i n the blood. On t h i s t o p i c i t can a l s o be
s t a t e d that other chemicals are u s u a l l y not t o x i c i n blood con
c e n t r a t i o n s s i m i l a r to those found here f o r organic f l u o r i n e .
The c o n c e n t r a t i o n of organic f l u o r i n e i n human plasma may
be changing with time. In 1960 Singer and Armstrong reported
that the plasma of 70 i n d i v i d u a l s r e s i d i n g i n communities with
1 ppm or l e s s f l u o r i d e i n t h e i r p u b l i c water supply had an
average c o n c e n t r a t i o n of f l u o r i d e of 8.8 μΜ (8). They prepared
t h e i r samples by ashing them and then d i s t i l l i n g f l u o r i d e from
the ash a c i d i f i e d with p e r c h l o r i c a c i d (7). Thus, i t seems
l i k e l y that t h e i r values f o r " f l u o r i d e " would have included
organic f l u o r i n e had i t been present. Assuming that i n o r g a n i c
f l u o r i d e concentrations at that time were s i m i l a r to those found
i n t h i s study (see t a b l e I I ) , the organic f l u o r i n e component
would exceed 7 μΜ. In 1969 the same i n v e s t i g a t o r s using the
same method reported an average f l u o r i d e c o n c e n t r a t i o n of 4.5 μΜ
f o r 6 plasma samples each pooled from at l e a s t 3 i n d i v i d u a l s
supposedly l i v i n g i n f l u o r i d a t e d communities (14). This
corresponds to an organic f l u o r i n e component of only about 4 μΜ.
Organic f l u o r i n e c o n c e n t r a t i o n presented here averages only
1.35 μΜ. Therefore, there may have been a decrease i n the
c o n c e n t r a t i o n of organic f l u o r i n e i n human plasma s i n c e the l a t e
f
1950 s % An a l t e r n a t e explanation might be that d i f f e r e n c e s i n

7. G U Y E T A L . Fluorocompounds in Human Plasma 133
the a n a l y t i c a l methods or d i f f e r e n c e s i n the sample populations

caused these values to vary.
Organic f l u o r i n e i s the predominant form of f l u o r i n e i n
human blood except where the c o n c e n t r a t i o n of f l u o r i d e i n
d r i n k i n g water i s high ( i n which case f l u o r i d e predominates, see
t a b l e I I ) . T h i s together with the f i n d i n g reported here that
there i s no apparent r e l a t i o n s h i p between the concentrations of
organic f l u o r i n e and i n o r g a n i c f l u o r i d e i n plasma helps e x p l a i n
why i n e a r l i e r s t u d i e s (8) no r e l a t i o n s h i p was found between
plasma f l u o r i d e determined i n ashed samples and the f l u o r i d e
content of the p u b l i c water supply. The data i n t a b l e I I show
that when methods s p e c i f i c f o r i n o r g a n i c f l u o r i d e are a p p l i e d , a
c l e a r r e l a t i o n s h i p between f l u o r i d e i n plasma and f l u o r i d e i n
the p u b l i c water supply (between 0.1 and 5.6 ppm) can be
demonstrated. Thus, there i s no need to p o s t u l a t e the existence
of such r i g o r o u s homeostati
f l u o r i d e as suggested e a r l i e
concentrations f o r i n d i v i d u a l s l i v i n g i n the same c i t y as reported
here r e f l e c t the balance e s t a b l i s h e d between f l u o r i d e i n blood
and that i n bone mineral over periods of years. These f i n d i n g s do
not c o n t r a d i c t a passive homeostatic c o n t r o l mechanism i n
which bone m i n e r a l damps swings i n blood f l u o r i d e c o n c e n t r a t i o n
over r e l a t i v e l y shorter periods of time.
The values presented here f o r the average i n o r g a n i c f l u o r i d e
c o n c e n t r a t i o n of plasma from i n d i v i d u a l s l i v i n g i n a community
having about 1 ppm f l u o r i d e i n the water supply are c o n s i s t e n t
with recent f i n d i n g s of others using s i m i l a r methods (14, 15).
Literature Cited
1. Taves, D.R., Nature (1966), 211, 192.
2. Taves, D.R., Nature (1968), 217, 1050.
3. Taves, D.R., Nature (1968), 200, 582.
4. Taves, D.R., Talanta (1968), 15, 1015.
5. Guy, W.S., "Fluorocompounds of Human Plasma: Analysis,
Prevalence, Purification and Characterization", doctoral
thesis, University of Rochester, Rochester, N.Y., 1972.
6. Taves, D.R., J. Dental Res. (1971), 50, 783.
7. Singer, L. and Armstrong, W.D., Anal. Chem. (1959), 31, 105.
8. Singer, L. and Armstrong, W.D., J. Appl. Physiol. (1960), 15,
508.
9. Taves, D.R., Talanta (1968), 15, 969.
10. Frant, M.S. and Ross, Jr., J.W., Science (1966), 154, 1553.
11. Siakotos, A.N. and Rouser, G., J. Amer. Oil Chem. Soc. (1965)
42, 913.
12. Bryce, H.G., "Industrial and Utilitarian Aspects of Fluorine
Chemistry" in "Fluorine Chemistry", Vol. V, J.H. Simon, ed.,
Academic Press, N.Y., 1964.
13. Hals, L.J., Reid, T.S. and Smith, G.H., J. Amer. Chem. Soc.
(1951), 73, 4054.
14. Singer, L. and Armstrong, W.D., Arch. Oral Bio.(1969), 14, 1343.
15. Singer, L. and Armstrong, W.D., Biochem. Med. (1973), 8, 415.

Q. I wonder if you t r i e d to c o r r e l a t e w i t h i n i n d i v i d u a l s the

l e v e l of organic f l u o r i n e with age.
A. I t would c e r t a i n l y be i n t e r e s t i n g to have t h i s information
but unfortunately we cannot supply i t at t h i s time. An expedi-
tious approach might be to analyze cord blood from i n f a n t s of
mothers who had not received f l u o r i n e - c o n t a i n i n g anesthetics at
childbirth. I t would a l s o be of i n t e r e s t to know whether
i n d i v i d u a l s l i v i n g i n i s o l a t e d regions have organic f l u o r i n e i n
t h e i r blood plasma.
Q. Did you say the sample analyzed by nmr contained methyl
alcohol?
A. Yes, I d i d .
Q. Methyl a l c o h o l w i l l r e a c t very r a p i d l y with f l u o r i n a t e d
acids. The nmr spectrum may, t h e r e f o r e , represent that of
methyl ester d e r i v a t i v e s .
A. Methanol was a l s o use
p u r i f i c a t i o n system. Th
presence of methyl ester d e r i v a t i v e s of p e r f l u o r i n a t e d f a t t y
acids (C6-C8) and t h e i r branched isomers.

8
Intravenous Infusion of Cis-Trans Perfluorodecalin
Emulsions in the Rhesus Monkey
LELAND C. CLARK, JR., EUGENE P. WESSELER, SAMUEL KAPLAN,
and CAROLYN EMORY
Children's Hospital Research Foundation, Elland and Bethesda Aves.,
Cincinnati, Ohio 45229
ROBERT MOORE
Sun Ventures, Inc., Marcus-Hook, Pa. 19061
DONALD DENSON
Stanford Research Institute, Menl
Introduction
Interest in the possibility of making fluorocarbon-based
artificial blood began following the discovery by Clark (1) that
animals survived the breathing of oxygen-saturated FC75 (3M Com-
pany). Since that time over 150 papers have been published, a
FASEB Symposium (2) and several industrial symposia have been held
on the subject. Reviews by Sloviter (3) and Geyer (4) have been
published. A large number of perfluorochemicals (PFC) have been
screened for this purpose. Most PFC carry oxygen in biologically
significant quantities. But most are ruled out for practical use
because, after performing their oxygen-carrying function, they re-
main in the body, largely in the liver, after being taken up main-
ly by the mononuclear phagocytes as are all PFC. So far it seems
that only those containing fluorine and carbon or fluorine, carbon
and bromine in their structure leave the body. Although we have
not completed our testing of the PFC on hand and expect to test
new ones in the future, nonetheless, of the PFC tested so far,
perfluorodecalin (PFD) emerges as the best because it leaves the
liver in a reasonable time and has a vapor pressure compatible
with intravenous use. As an emulsion, its acute intravenous toxi-
city in the mouse is very low. It is available in commercial
quantities and can be partially purified by distillation. For
these, and other reasons, it was selected for testing as artifi-
cial blood in a non-human primate, the rhesus monkey. We previ-
ously reported (5) on the infusion of perfluorodecalin in one
awake rhesus monkey. This paper describes the first results of
further tests in 19 monkeys and 10 dogs and outlines the beginning
of a protocol for its possible evaluation as a blood substitute in
man. Of the 19 monkeys, 10 were infused with 10% PFD, 4 with 20%
PFD emulsions, and 4 with only a "hypotensive test dose".
The data included here is only a part of that on hand; it has
been selected to illustrate the salient problems and findings. It
is to be interpreted in terms of our previous publications (6-18)
on this subject.
135

Data on oxygen s o l u b i l i t y i n PFC presented at t h i s meeting

i s being expanded and w i l l be the subject of a separate report
(19).
Methods and m a t e r i a l s
Nineteen monkeys were s e l e c t e d from the I n s t i t u t e of Develop-

mental Research's r e s i d e n t p o p u l a t i o n . Many of these monkeys had
been p r e v i o u s l y used f o r t e s t i n g drugs i n t e r a t o l o g i c a l research.
Three were males and had not been given drugs. The monkeys were
housed i n s t a i n l e s s s t e e l cages i n a i r c o n d i t i o n e d rooms and were
fed a d i e t of Purina monkey chow (Code 5038) supplemented with
bananas, oranges, and raw peanuts. They were given p e r i o d i c s k i n
t e s t s f o r t u b e r c u l o s i s and were given 11 mg/kg i s o n i a z i d e d a i l y
impregnated as a s o l u t i o n on a sugar cube. The monkeys, a l l of
which presumably have lun mites chest d arrival
and weighed weekly. P u b l i
used f o r monkeys obtaine
lowed i n the l a b o r a t o r y . The dogs were beagles obtained from a
commercial source.
The f i r s t monkey tested and reported (_5) , was a young male.
Two of the monkeys i n the present s e r i e s were a l s o males. The
v e t e r i n a r i a n s were unable to a s s i g n an approximate age to any mon-
key but, judging by t h e i r teeth and t h e i r behavior, many were very
old.
The p e r f l u o r o d e c a l i n used i n these experiments was purchased
from ISC Chemicals, L t d . , Avonmouth, B r i s t o l , England and p u r i f i e d
by s p i n n i n g band d i s t i l l a t i o n using a Perkin-Elmer NFA-200 Auto-
annular s t i l l . The f r a c t i o n b o i l i n g between 91°C and 93°C at
180 mm pressure was used f o r these s t u d i e s .
The P l u r o n i c (PF68) s o l u t i o n was made by d i s s o l v i n g 200 gm i n
s t e r i l e water (Abbott), brought up to 1 l i t e r , and f i l t e r i n g suc-
c e s s i v e l y through 5.0, 0.8, and 0.22 uM M i l l i p o r e f i l t e r s at 4°C.
This stock was d i l u t e d j u s t before use, the i o n i c composition was
adjusted so the emulsion contained h a l f the s a l t s required f o r
Ringer's and the pH was adjusted with Tham (28).
Emulsions were made i n a G a u l i n Model 15M l a b homogenizer.
Parts which were exposed to the emulsion were autoclaved. The
shear pressure was preset to 6000 l b s / i n ^ and the gauge was r e -
moved. Homogenization was continued u n t i l the o p t i c a l d e n s i t y
plateaued. The emulsion was placed i n s t e r i l e Pyrex v i a l s or one
l i t e r b o t t l e s , frozen and preserved at -70°C.
S t e r i l i t y t e s t s , performed by adding 2 ml i n t o 20 ml of a
supplemented peptone broth prepared by Becton-Dickinson and Co.,
were conducted on random samples of the emulsion. A l l t e s t s show-
ed the emulsion to have no b a c t e r i a l growth.
Chemical methods o f s t e r i l i z a t i o n i n c l u d i n g the use of pro-
p i o l a c t o n e were avoided, except with one monkey, Melvin 05), who
r e c e i v e d emulsion s t e r i l i z e d with t h i s compound.

8. CLARK ET AL. Cis-Trans Perfluorodecalin Emulsions 137
Two batches of 10% by volume PFD i n 5% PF68 and two batches

of 20% by volume PFD i n 10% PF68 were prepared. A l l of the blood
pressure t e s t doses were from the same batch (1 l i t e r ) of 10%
PFDE and were taken from small Pyrex v i a l s which were thawed j u s t
before use. A l l of the monkeys i n f u s e d with 10% PFDE r e c e i v e d
emulsion (4 l i t e r s ) from the second batch where i n d i v i d u a l bot-
t l e s were thawed j u s t before use. Two of the monkeys, C2 and C3,
r e c e i v e d 20% PFDE from a t h i r d batch (1.5 l i t e r ) . Two, 74 and
113, r e c e i v e d 20% PFDE from a f o u r t h batch (4 l i t e r ) . The f i r s t
three batches were prepared from pooled PFD (ISC a n a l y t i c a l r e f -
erence S173/A) while the f o u r t h batch was prepared from PFD (ISC
a n a l y t i c a l references S172/Aand S148/B). A l l the 10% PFDE and the
f i r s t two 20% PFDE emulsions were s t o r e d i n i c e water u n t i l used
but the 20% PFDE f o r 74 was warmed before a d m i n i s t r a t i o n and that
used f o r 113 was bubbled with oxygen at room temperature f o r three
hours before i n f u s i o n
Blood samples wer
through i n d w e l l i n g catheter
syringes having the dead space f i l l e d with heparin (1000 USP
u n i t s / m l ) . The samples from awake r e s t r a i n e d monkeys were ob-
tained by venipuncture. Precautions were taken to be sure that
the blood samples were not d i l u t e d with the i s o t o n i c s o l u t i o n s
used to r i n s e the sampling l i n e s . Samples f o r blood gases, pH,
and packed c e l l volume were analyzed immediately. The remaining
blood was c e n t r i f u g e d and the plasma analyzed by Autotechnicon
SMA 12 procedures (21)• Venous blood samples were taken at ran-
dom from L. C l a r k , to determine the v a r i a b i l i t y of the same blood
f a c t o r s being measured i n the monkey. Most of the samples from
L. C l a r k were taken during the f a s t i n g s t a t e , a l l those reported
here were taken while L. C l a r k was i n a s t a t e of w e l l - b e i n g , and
a l l were processed l i k e monkey blood. Samples were taken p e r i o d i -
c a l l y from the monkey p r e v i o u s l y published (5). A l l samples not
analyzed immediately were c h i l l e d i n an ice-water bath. Approxi-
mately 4800 determinations were performed as part of the research
reported here.
Test f o r hypotensive effect
The monkeys were f a s t e d overnight and a n e s t h e t i z e d with i n -

travenous sodium p e n t o b a r b i t a l i n the morning. They breathed hu-
m i d i f i e d oxygen. The a r t e r i a l pressure was measured by means of
a p e d i a t r i c c u f f and a Model 802 Doppler (Parks E l e c t r o n i c s Lab-
o r a t o r y , Oregon) using the r a d i a l a r t e r y . 0.05 ml/kg of emulsion
was i n j e c t e d i n t r a v e n o u s l y and r i n s e d i n with 5 ml of Ringer's
s o l u t i o n . A second t e s t dose was given w i t h i n f i v e minutes a f t e r
the f i r s t i n the monkey but a f t e r the blood pressure returned to
normal, 10 or 20 minutes, i n the dog. A s i m i l a r procedure was
used f o r each v i a l of emulsion using the beagle w i t h i n one day of
the t e s t i n the monkey. The blood pressure i n the dog was measur-
ed with a mercury manometer and d i r e c t cannulation of the femoral

artery.
Twelve monkeys were subjected to biopsy before PFDE i n f u s i o n
and nine a f t e r . The o v e r n i g h t - f a s t e d monkeys were anesthetized
with sodium p e n t o b a r b i t a l and a 2 or 3 gram piece of l i v e r was ex-
c i s e d under d i r e c t v i s i o n , f i x e d , and examined by l i g h t and e l e c -
tron microscopy using methods p r e v i o u s l y published (18). Parts of
these post i n f u s i o n samples were analyzed f o r PFD by sodium b i -
phenyl combustion and by GLC. C o n t r o l blood samples were taken
p r i o r to making the biopsy i n c i s i o n . Post biopsy blood samples
were taken one hour a f t e r the i n c i s i o n was c l o s e d and again on the
f o l l o w i n g day from the r e s t r a i n e d animal.
For the i n f u s i o n , the animal i s anesthetized with pentobarbi-
t a l and kept asleep with pentothal. An endotracheal tube i s i n -
serted and the animal's head covered with a transparent bag being
flushed with humidified oxygen. The femoral v e i n s and the r a d i a l
a r t e r y are cannulated with s t e r i l e p l a s t i c tubes and the r i g h t
heart i s c a t h e t e r i z e d wit
On the day of i n f u s i o
and ECG of the monkey have been recorded f o r s e v e r a l minutes and
look s t a b l e , the f i r s t samples of blood are taken f o r blood gas
analyses. A 2 ml sample of blood i s drawn into a h e p a r i n i z e d 2 ml
syringe from the r i g h t heart and the r a d i a l a r t e r y . I f the p02
and pC0 readings obtained on these samples are w i t h i n normal l i m -
2
i t s , a l a r g e (12.5 ml/kg) sample of blood i s removed. This blood

is c e n t r i f u g e d and analyzed as described elsewhere. At t h i s p o i n t
21 ml/kg of 57o human albumin i s infused with a Sage Instrument
syringe pump at 6 ml/min.
Another 12.5 ml/kg of blood i s removed c a r e f u l l y so that the
blood pressure does not f a l l below 80 mm Hg. The hematocrit of
t h i s blood i s determined and i f i t i s not one h a l f the o r i g i n a l
hematocrit, another 4 ml/kg i s infused and another hematocrit run.
To date, t h i s procedure has always reduced the hematocrit approxi-
mately 507o. In f i v e of the f i r s t s i x monkeys infused 50 ml/kg of
Ringer's s o l u t i o n was given i n place of the albumin.
The PFDE i s infused i n four batches and a r t e r i a l and mixed
venous blood gases and pH are measured a f t e r each of the four
batches are i n .
A f t e r the i n f u s i o n , the a r t e r i a l cannula and a l l but one of
the venous cannulae are removed and the i n c i s i o n s c l o s e d . The an-
imal i s kept i n the operating room u n t i l i t begins to waken. Then
i t i s t r a n s f e r r e d to a recovery room and c l o s e l y watched f o r the
next 12 hours. F l u i d s may be given intravenously i f r e q u i r e d .
U s u a l l y the monkeys are awake and d r i n k i n g l i q u i d s or even e a t i n g
by l a t e afternoon.
The autopsies are conducted under the auspices of the Medical
School's V e t e r i n a r y Department. The organs are inspected, photo-
graphed, weighed and h a l f of each organ i s placed i n n e u t r a l 10%
phosphate-buffered f o r m a l i n s o l u t i o n and h a l f i s placed i n 957o
ethanol a f t e r portions of each organ are removed f o r f i x i n g , s t a i n -
ing and examining under l i g h t microscopy.

Results and d i s c u s s i o n
I n t e r l a b o r a t o r y v a r i a t i o n i n the a n a l y s i s of three p e r f l u o r i -
nated l i q u i d s i s given i n Table 1. I t i s apparent that the d i f -
f i c u l t y l i e s i n g e t t i n g q u a n t i t a t i v e recovery of f l u o r i n e and not
of carbon. Some a n a l y t i c a l l a b o r a t o r i e s obtained such inaccurate
r e s u l t s that they abandoned attempts to analyze the samples. An-
other l a b o r a t o r y i s continuing i t s e f f o r t s to develop a method.
No d i f f i c u l t y was encountered, i n preparing the PFD emul-
s i o n s . Those preserved at -70°C remained f o r months with no d i s -
c e r n i b l e change. The seven day LD50 i n mice of the i n f u s e d 10%
PFDE was 140 ml/kg; the LD50 of the 20% PFDE was 69 ml/kg.
Measurement of p a r t i c l e s i z e d i s t r i b u t i o n i n three emulsions
was performed at Sun Ventures by a technique (26) i n v o l v i n g e l e c -
tron microscopy and the r e s u l t s are shown i n F i g u r e 1.
Tables 2 and 3 represent the changes i n blood chemistry found
upon biopsy of the l i v e r
l i t t l e meaning because
glycolysis. The increased l a c t i c dehydrogenase a c t i v i t y may have
come from the damaged l i v e r . There i s no doubt that c r e a t i n e
kinase increased as a r e s u l t of the procedure but i t i s known to
increase a f t e r any kind of surgery (25).
C o n t r o l values f o r blood samples from a human primate are
shown i n Table 7 and those from awake monkeys i n Table 5.
One reason the monkey was s e l e c t e d f o r these experiments i s
that i t very o f t e n behaves l i k e the human i n i t s response to drugs.
The dog sometimes shows b i z a r r e responses to emulsions and s o l u -
t i o n s c o n t a i n i n g s u r f a c t a n t s . In Table 4 i t can be seen that no
monkey t e s t e d , but a l l dogs, s u f f e r e d a drop i n blood pressure.
It sometimes takes h a l f an hour f o r the dog to recover but once i t
has recovered a second dose has very l i t t l e or no e f f e c t .
In Tables 10 and 11 i t can be seen that n e i t h e r the anesthe-
s i a nor the s u r g i c a l manipulations i n v o l v e d i n g i v i n g a t e s t dose
had a s i g n i f i c a n t e f f e c t upon the blood components analyzed ex-
cept f o r c r e a t i n e kinase where a small but s i g n i f i c a n t increase
occurred. The abnormally high values f o r twenty-four hours on
monkey 80 are due to the f a c t that she was moribund and died two
hours l a t e r a f t e r s e v e r a l attempts at r e s u s c i t a t i o n .
The d e t a i l s concerning the replacement of withdrawn blood by
Ringer's s o l u t i o n and/or 5% human albumin are given i n Table 16.
The amounts of PFDE i n f u s e d are shown. For our purpose here we
have r e f e r r e d to Dextran 40 and albumin as osmotic or o n c o t i c l i q -
quids. Albumin, however, has a longer h a l f l i f e i n the body and
of course the PF68 i n PFDE has o n c o t i c a c t i v i t y . 5% human a l b u -
min was given p r e v i o u s l y to one monkey with no d i s c e r n i b l e e f f e c t .
That we had considerable d i f f i c u l t y i n maintaining blood b a l -
ance a f t e r phlebotomy and PFDE i n f u s i o n i s apparent from the t a b l e .
Most of the d i f f i c u l t i e s happened during the evening of the day of
the i n f u s i o n and were thought to be due to the f a c t that most of
the PF68 and water administered had been excreted l e a v i n g the

Figure 1. Particle size distribution as determined by electron micros-

copy

animals hypovolemic and although awake they were not a l e r t and

were weak. When Dextran 40 was given on two occasions (C2, C3)
four or f i v e hours post i n f u s i o n the animals responded to the
point where they were d i f f i c u l t to r e s t r a i n . They a l s o drank
Gatorade and ate. One which r e c e i v e d Dextran twenty-four hours
l a t e r , f o l l o w i n g an a l l night slow b l e e d i n g from a p o o r l y t i e d i n
cannula, r e v i v e d only b r i e f l y . Most of the 10% PFD monkeys that
r e c e i v e d albumin during the phlebotomy r e q u i r e d l e s s f l u i d l a t e r .
Two (74, 113) that r e c e i v e d 20% PFDE d i e d , p o s s i b l y because they
were overloaded, but more l i k e l y because the PFDE they were given
had been a t room temperature too long and coalescence of p a r t i c l e s
had begun. Our experience i n maintaining blood and f l u i d balance
i n thousands of dogs and human p a t i e n t s has not served us w e l l
with the rhesus. I t seems to us that the rhesus monkey, p a r t i c
u l a r l y the o l d monkey, i s a very d e l i c a t e and f r a g i l e animal.
The main f i n d i n g i Tabl 6 i tha th mixed
tension increased i n a l
none of those r e c e i v i n g 2p0
beyond 100 mm, the approximate p o i n t where a l l the hemoglobin i s
saturated. In three of the monkeys r e c e i v i n g 20% PFDE the mixed
venous p 0 went above 100 mm.
2 In monkey C3 the a r t e r i a l p 0 2 was
on the low s i d e and t h i s could not be increased by chest and/or
heart massage. At autopsy t h i s monkey was found to have severe
emphysema. T h i s low a r t e r i a l p 0 may account, at l e a s t i n p a r t ,
2
f o r the low venous p0 . 2 At autopsy C3 was found to have lungs

h e a v i l y i n f e s t e d with mites.
The heart r a t e decreased and the r e s p i r a t i o n increased as a
r e s u l t of PFDE i n f u s i o n as shown i n Table 8. The Τ t e s t s shown
i n t h i s t a b l e i n d i c a t e there i s no d i f f e r e n c e i n these responses
to the 10% and 20% PFDE.
There was no change i n a r t e r i a l pressure as a r e s u l t of PFDE
i n f u s i o n as shown i n Table 9. From these and other s t u d i e s we
have concluded that there i s an increase i n pulmonary a r t e r i a l
pressure followed by a r e t u r n to normal i n about an hour. Because
of u n c e r t a i n t y about the l o c a t i o n (RV or PA) of the catheter t i p
(x-ray equipment was not a v a i l a b l e to us during these s t u d i e s ) the
data i n Table 8 cannot be analyzed s t a t i s t i c a l l y . However i t can
be seen that i n a l l of the cases where the pressure was low before
i n f u s i o n , probably i n d i c a t i n g that pulmonary a r t e r i a l pressure was
being monitored, there was a d i s t i n c t i n c r e a s e .
Tables 12 and 13 give the r e s u l t s of a n a l y z i n g the blood of
monkeys r e c e i v i n g 10% PFDE and Table 13 gives the average values
and standard e r r o r s f o r t h i s data. Some of the apparent decreases
i n c o n c e n t r a t i o n of blood components are due to the f a c t that
about h a l f the blood was removed and d i l u t e d by Ringer's, albumin,
and PFDE. The extent to which d i l u t i o n per se a f f e c t e d the r e
s u l t s can be best judged by the extent to which the hematocrit de
creased. While the t a b l e shows a decrease i n a l l components ex
cept t o t a l b i l i r u b i n , i f these are " c o r r e c t e d " f o r d i l u t i o n by

Figure 2. Light microscopic view of the liver of a mon

key before infusion. H, hepatocyte; N, nucleus; S, sinus
oid; Toluidine blue 0. χ 850.
Figure 3. Liver specimen taken at sacrifice two weeks

after infusion with a 20% emulsion shows hepatocytes
essentially unchanged morphologically. Cytoplasm of
is completely filled with particles of PP5, and bulges into

the lumen of sinusoids. H, hepatocyte; N, nucleus; M,
mononuclear phagocyte. Toluidine blue 0. X 850.
Figure 4. Eleven months after infusion, the liver shows

normal morphology and no structures remained which
could be identified unequivocally as fluorocarbon. Other
features were within normal limits. H, hepatocyte; N,
nucleus; S, sinusoid. Toluidine blue 0. X 850.

d i v i d i n g by 0.32, the hematocrit f a c t o r , the r e s u l t s i n d i c a t e an

increase i n everything except c h o l e s t e r o l . The percentage change
i n the components before and a f t e r c o r r e c t i o n are as f o l l o w s :
TP 41,128; AB 71,222; CA 71,222; IP 90,281; GL 74,231; BN 88,275;
VA 44,138; CT 80,250; TB 140,438; AP 51,159; LD 75,234; GO 45,141;
CK 142,444; CH 18,56; PV 32,100.
The blood chemistry changes found before and a f t e r i n f u s i o n
of 20% PFDE are shown i n Table 14.
Twenty-eight samples were taken from the monkey i n f u s e d with
10% PFDE about a year ago and the mean values are shown i n Table
15. The mean a l k a l i n e phosphatase i s higher than that shown i n
Table 3 and Table 5 but t h i s would be expected because Melvin i s
growing r a p i d l y . A l l the other data are normal according to the
information we have accumulated here.
Over a three month p e r i o d the average weight gain of the
eight s u r v i v i n g monkeys i n f u s e d with PFDE t significantl
d i f f e r e n t from s i x monkey
Morphology. Three of nineteen monkeys are p i c t u r e d here

which represent the p r e i n f u s i o n , post i n f u s i o n and long term r e
covery of the l i v e r of a l l monkeys examined to date.
The normal, or p r e i n f u s i o n morphology of the l i v e r d i f f e r e d
s l i g h t l y from that of other commonly used l a b o r a t o r y mammals ( f i g .
2). V a r i a t i o n s included a marked e l e v a t i o n i n hemosiderin depos
i t s i n Kupffer c e l l s , g e n e r a l l y a s s o c i a t e d with age, numerous
large autophagic vacuoles (10 μ) and the presence of long tubular
s t r u c t u r e s i n the mitochondria. These p e c u l i a r i t i e s have a l s o
been observed by others (27).
Within twenty four hours a f t e r i n f u s i o n of emulsions of PP5,
p a r t i c l e s begin to appear w i t h i n the phagocytic c e l l s of the
s i n u s o i d s of the l i v e r , other organs, and the c i r c u l a t i o n . Just
a f t e r i n f u s i o n , while p a r t i c l e s are i n the bloodstream, the poly-'
morphonuclear c e l l s show a few cytoplasmic p a r t i c l e s . Most a f
fected m o r p h o l o g i c a l l y by the i n f u s i o n of emulsions are the c e l l s
of the mononuclear phagocytic system. These c e l l s engulf i n d i v i
dual p a r t i c l e s or clumps of p a r t i c l e s u n t i l they reach very l a r g e
proportions ( f i g . 3) as they r e s t i n the l i v e r s i n u s o i d s , s p l e n i c
pulp or s i m i l a r area. A f t e r a few days they may aggregate i n t o
small nodules of e p i t h e l i a l - t y p e c e l l s s i m i l a r to those seen i n
other f o r e i g n body responses ( f i g . 3).
The hepatocytes are not changed d r a m a t i c a l l y by the i n f u s i o n
of emulsions ( f i g . 4) but a small number of p a r t i c l e s do enter the
cytoplasm. These d i m i n i s h , however, and u l t i m a t e l y disappear by
unknown means. Residual e f f e c t s of the occupation of the l i v e r
(and of course other comparable c e l l systems i n the body) by PFC
i n f a c t have not been observed m o r p h o l o g i c a l l y , s i n c e mito
chondria, smooth and rough endoplasmic r e t i c u l u m , lysosomes,micro-
bodies, n u c l e i , e t c . , appear i n d i s t i n g u i s h a b l e from c o n t r o l s
( f i g s . 5,6). A l l the specimens shown were obtained at biopsy.

Figure 5. Electron microscopic view of liver from the same monkey as figure 3. A
very small percent of the cytoplasm is occupied by particles of fluorocarbon (small
arrow). Organelles appear unchanged. H, hepatocyte cytoplasm; N, nucleus; M, mono-
nuclear phagocyte with cytoplasm full of fluorocarbon particles (large arrows). Uranyl
acetate, lead citrate. X 8,500.
Figure 6. Liver from the same monkey as figure 4 shows organelles which appear
normal. Fluorocarbon particles, no longer identified in the cytoplasm of hepatocytes or
mononuclear phagocytes, have apparently left the liver unchanged. M, mitochondria;
N, nucleus; H, hepatocyte cytoplasm. Uranyl acetate, lead citrate. X 4,000.

Post mortem f i n d i n g s . Of ten monkeys which r e c e i v e d 10% PFDE;

eight are a l i v e . One, M e l v i n , has s u r v i v e d over a year. One died
the day a f t e r i n f u s i o n from slow b l e e d i n g around a venous cannula.
One (117) died eleven weeks a f t e r the i n f u s i o n from anesthesia
preparatory to surgery f o r l i v e r biopsy. Of the four monkeys
which r e c e i v e d 20% PFDE two died w i t h i n twenty four hours f o l l o w -
ing the i n f u s i o n and were thought to have died because the emul-
s i o n had begun to d e t e r i o r a t e . P o s s i b l y t h e i r c i r c u l a t i o n s were
overloaded. Two s u r v i v e d i n apparent good h e a l t h but were s a c r i -
f i c e d at one and two weeks post i n f u s i o n because t h e i r l e g s be-
came ischemic and p o s s i b l y gangrenous, f o l l o w i n g l i g a t i o n of t h e i r
femoral a r t e r i e s as a part of the process of r e c o r d i n g t h e i r a r t e -
r i a l pressure. Femoral a r t e r i e s can be l i g a t e d i n cats and dogs
with no v i s i b l e e f f e c t but primates are s u s c e p t i b l e to n e c r o s i s i f
the femoral a r t e r i a l c i r c u l a t i o n i s compromised. One monkey's
femoral a r t e r y was s u c c e s s f u l l y r e p a i r e d ; t h i s animal (117) l i v e d
eleven weeks with norma
femoral a r t e r y , d i r e c t pressure
tery. One monkey s u f f e r e d p a r t i a l l o s s of the f i n g e r s of the l e f t
hand f o l l o w i n g l i g a t i o n of the r a d i a l . One i n j u r e d monkey, 6, was
s a c r i f i c e d s h o r t l y a f t e r i t was purchased, and before i t was used
as a c o n t r o l f o r organ weights. Monkey 80 b l e d to death overnight
because a femoral a r t e r y was punctured during an attempt at cathe-
terization.
The organ weights obtained at autopsy are shown i n Table 17.
Because of the d e a r t h of normal data i n the rhesus very l i t t l e can
be s a i d about the e f f e c t s of i n f u s i o n . The l i v e r of C2 and the
spleen of C3 are q u i t e p o s s i b l y enlarged.
A l l the monkeys were found to have lung mites, and some were
considered h e a v i l y i n f e s t e d . Many of the lungs were s t i f f and d i d
not c o l l a p s e .
More d e t a i l e d r e p o r t s of the f i n d i n g s with l i g h t microscopy
w i l l be the s u b j e c t of a separate r e p o r t .
Sixteen p i e c e s from v a r i o u s p a r t s of the l i v e r of one monkey
were analyzed and were found to range from 0.61% to 2.05% with a
mean of 1.45%.
Information obtained by sodium biphenyl combustion of the
l i v e r and GLC are shown i n Table 18. There was of course c i r c u l a -
t i n g PFDE i n monkeys 62, 74, and 113. Depending on the dose, i t
r e q u i r e s s e v e r a l days f o r i t to completely disappear from the
blood. The PFD disappears from the blood both by evaporation
through the lungs and s k i n and by being engulfed by the scavenger
c e l l s of the body.
I n t e r e s t i n g l y , a t r a c e could be detected i n Melvin's l i v e r
almost a year a f t e r the i n f u s i o n .
There i s c o n s i d e r a b l e v a r i a b i l i t y between monkeys i n the way
i n which they sequester the PFD i n t h e i r l i v e r s and spleen, and
the r a t e at which i t leaves. The h a l f l i f e of PFD i n the l i v e r i s
probably about two weeks. In the mouse, most PFD i s gone i n two
weeks.

General d i s c u s s i o n
Although most of our r e s u l t s have been discussed above we

think i t may be d e s i r a b l e to add some general comments about our
concepts and our f i n d i n g s .
Our work on a r t i f i c i a l blood i s prompted by our experience
with thousands of open heart surgery p a t i e n t s and a f a m i l a r i t y
with the problems surrounding plasma, plasma s u b s t i t u t e s , par-
e n t e r a l s o l u t i o n s and the use of donor blood.
Various substances have been used over the past s e v e r a l dec-
ades as plasma s u b s t i t u t e s . C e r t a i n of these substances, such as
Dextran and plasma albumin, have earned a d e f i n i t e place i n c l i n -
i c a l medicine f o r maintaining c i r c u l a t o r y volume. G e n e r a l l y , such
s o l u t i o n s d i s s o l v e only 2 or 3 ml of oxygen per 100 ml, while
whole blood d i s s o l v e s about 20 ml per 100 ml.
In order to meet the oxygen demands of the body the c a r d i a c
output must be increase
words, the way i n which
c a r r y i n g c a p a c i t y of blood i s to work harder and pump more blood.
Because fluorocarbons c a r r y l a r g e q u a n t i t i e s of oxygen they can be
used to increase the oxygen c a p a c i t y of the c i r c u l a t i n g blood and
t h e r e f o r e decrease the work of the heart. Of course, a weak,
f a i l i n g , or i n j u r e d heart may not be able to increase i t s output
to meet oxygen needs and shock and death may f o l l o w . Therefore, a
primary f u n c t i o n of a r t i f i c i a l blood i s to decrease the work of
the heart. Oxygen c a r r y i n g l i q u i d s w i l l be most u s e f u l i n c o n d i -
tions where the c a r d i a c output i s low or the blood volume i s be-
low normal.
The only other way to increase the oxygen c a p a c i t y of blood
i s to add stroma-free hemoglobin but t h i s has not proven to be
p r a c t i c a b l e yet. Hemoglobin i s e a s i l y s t o r e d and i t may someday
be p o s s i b l e to use hemoglobin from other animals, such as the cow
and the p i g . One of i t s f a u l t s i s i t s high rate of e x c r e t i o n .
Synthetic oxygen chelates seem to be f a r i n the f u t u r e . PFC a r t i -
f i c i a l blood, l i k e stroma-free hemoglobin, has no blood types.
Our research on l i q u i d breathing of p e r f l u o r o c h e m i c a l l i q u i d s
formed the e a r l y b a s i s of the work reported here. It indicated,
f o r example, that c e r t a i n h i g h l y f l u o r i n a t e d l i q u i d s were probably
biologically inert. I t has l e d to the use by Dr. David M. Long,
as we hear today, of brominated p e r f l u o r i n a t e d l i q u i d as X-ray
contrast agents i n the d i a g n o s i s of lung disease. Perfluorocarbon
l i q u i d s may a l s o be u s e f u l some day i n the treatment of lung d i s -
eases, such as by washing out o b s t r u c t i v e m a t e r i a l s .
I t would be h i g h l y d e s i r a b l e to have a s u i t a b l e water s o l u b l e
s y n t h e t i c oxygen solvent to increase the oxygen c a p a c i t y of plasma
or plasma s u b s t i t u t e s . PFC i n general are good oxygen and carbon
d i o x i d e s o l v e n t s but they are completely i n s o l u b l e i n water.
Therefore they can only be used as emulsions. This introduces at
l e a s t three problems. (a) B i o l o g i c a l l y s u i t a b l e e m u l s i f i e r s must
be found and used. (b) C e r t a i n PFC cannot be e m u l s i f i e d and

c e r t a i n others form only unstable emulsions. (c) Because emul-

sions c o n s i s t of suspended p a r t i c l e s they are g r a d u a l l y removed
from the c i r c u l a t i o n and deposited i n part i n the l i v e r and
spleen.
We have e l e c t e d to concentrate our e f f o r t s on a study of the
use of p e r f l u o r o d e c a l i n p r i m a r i l y because i t g r a d u a l l y leaves the
body (16), but a l s o because i t has an acceptable vapor pressure,
and i s a good oxygen and carbon dioxide s o l v e n t . I t r e a d i l y forms
an emulsion having a low o p t i c a l d e n s i t y but must, at present, be
preserved at -70°C. For comparison, we and others have found that
FC47 forms a f i n e p a r t i c l e emulsion, as judged by o p t i c a l d e n s i t y ,
which i s f a r more s t a b l e than that produced by PFD but once en-
trapped i n the l i v e r i t remains f o r years. PFD i n a d d i t i o n , un-
l i k e , f o r example, p e r f l u o r o m e t h y l d e c a l i n with ten isomers, has
only two.
A fluorocarbon having but a s i n g l e molecular c o n f i g u r a t i o n
such as adamantane woul
mers such as PFD. PFD a f t e
l a t i o n as described i n t h i s paper, was analyzed at Stanford Re-
search I n s t i t u t e by GLC and f i e l d i o n i z a t i o n mass spectrometry and
reported to be 96.4% pure with p o s s i b l y eight d i s t i n c t compounds
present having molecular weights ranging from 68 to 484.
A few m i l l i t e r s of the two isomers of PFD, having the pub-
l i s h e d p r o p e r t i e s , were l a b o r i o u s l y prepared by gas chromatography.
It may be that the very property of being i n e r t , which makes some
of these fluorocarbons p o t e n t i a l l y u s e f u l i n medicine, makes them
d i f f i c u l t to p u r i f y , c h a r a c t e r i z e , and i d e n t i f y . Of course, high
p u r i t y and u n i f o r m i t y are r e q u i r e d f o r medical use.
The l i s t of u s e f u l e m u l s i f i e r s i s very short indeed because
they must be non-toxic, non-hemolytic and cause no undesirable
p h y s i o l o g i c a l r e a c t i o n s . Only two such substances e x i s t at the
present time. S p e c i a l egg p h o s p h o l i p i d (Vitrum, Sweden) has been
used e x t e n s i v e l y o u t s i d e of the United States as an e m u l s i f i e r f o r
intravenous f a t emulsions f o r c l i n i c a l use. This e m u l s i f i e r can
only be used to formulate emulsions under very c a r e f u l l y c o n t r o l l -
ed c o n d i t i o n s , as to p r e p a r a t i o n , temperature, exposure to a i r and
other f a c t o r s . I t i s capable of e m u l s i f y i n g PFD. The other emul-
s i f i e r , P l u r o n i c F68 (Wyandotte, USA), at the present time i s not
used c l i n i c a l l y i n the USA as a component of intravenous emul-
sions of any kind. I t has found extensive use as an e m u l s i f i e r
for PFC emulsions i n research with animals. I t was s e l e c t e d f o r
the PFDE used here i n the rhesus because i t forms emulsions with
PFD and i s r e l a t i v e l y non-toxic and s t a b l e .
Most of previous work with PFDE was done using the mouse, the
cat, and the dog. We decided to perform the t e s t s reported here
i n the rhesus (macaca raulatta) monkey to determine i f the primate
responded d i f f e r e n t l y .
We found that the hypotensive e f f e c t of small doses of emul-
s i o n d i d not occur i n the primate. Measurements of the LD50 of
plasma from the dog at the depth of the blood pressure drop

i n d i c a t e d that no t o x i c substance was present and we tend t h e r e -

fore to agree with W r e t l i n d (23, 24) that t h i s i s some k i n d of a
cardiovascular reflex.
Emulsions prepared from PFD and PF68 are d i f f i c u l t to charac-
t e r i z e as to p a r t i c l e s i z e and c o n c e n t r a t i o n of PF68 i n the aque-
ous phase a f t e r e m u l s i f i c a t i o n . There i s no one method f o r meas-
u r i n g p a r t i c l e s i z e i n the ranges i n v o l v e d here which i s g e n e r a l -
l y accepted. O p t i c a l d e n s i t y measurement continues to be the best
means to monitor the making of emulsions from any given PFC. I t
probably cannot be used to compare p a r t i c l e s i z e when d i f f e r e n t
PFC s t r u c t u r e s are i n v o l v e d .
Space w i l l not permit d i s c u s s i o n of the complex f a c t o r s i n -
volved i n maintaining f l u i d balance by means of osmotic, o n c o t i c ,
and h y d r o s t a t i c f o r c e s . S u f f i c e to say that we attempt to main-
t a i n o n c o t i c a c t i v i t y by the s h o r t - a c t i n g P l u r o n i c and Dextran and
by the long a c t i n g plasma albumin We found that Dextran given
s e v e r a l hours a f t e r the
f i c i a l e f f e c t ; i t has brough
s t a t e to one of near normal.
The d i f f e r e n c e i n the f a t e of the two monkeys given a warmed
20% emulsion and a cooled 20% emulsion would seem to suggest that
warming an emulsion i n v i t r o i s apparently completely d i f f e r e n t
than warming i t i n v i v o . Not only does the emulsion appear to be
much more s t a b l e i n v i v o than i n v i t r o , even though f a r above room
temperature, but judging from the b l u i s h haze i n the plasma of
some of these monkeys, the p a r t i c l e s i z e may even have decreased.
This may be due not only to the e m u l s i f y i n g p r o p e r t i e s of
blood, but to the mechanical mixing e f f e c t s i n the c a r d i o v a s c u l a r
system and the unique c h a r a c t e r i s t i c s of the l i n i n g of the blood
vessels.
I t should be borne i n mind that most of the chemical analyses
of blood reported here are done on plasma d i a l y z e d through a c e l -
lophane membrane and should t h e r e f o r e not be a f f e c t e d by the pres-
ence of PFD. T o t a l p r o t e i n , albumin, t o t a l b i l i r u b i n , and l a c t i c
dehydrogenase were analyzed without passing through a d i a l y s i s
membrane and the measurement could have been a f f e c t e d .
Summary
Of nineteen monkeys s e l e c t e d f o r t h i s study, fourteen were

used f o r the i n f u s i o n of p u r i f i e d p e r f l u o r o d e c a l i n i n the form of
10 and 20% by volume emulsions. Four received only t e s t doses and
one was s a c r i f i c e d before use as a c o n t r o l . P l u r o n i c F68 was used
as the e m u l s i f y i n g agent. Previous to the i n f u s i o n of PFDE, blood
was removed to decrease the hematocrit by h a l f and t h i s blood was
replaced by e i t h e r Ringer's s o l u t i o n or 5% human albumin. Nine
out of ten monkeys infused with 10% PFDE survived. Two of four
monkeys infused with 20% PFDE survived. Mixed venous p 0 i n c r e a s - 2
ed i n a l l monkeys and exceeded 100 t o r r i n three monkeys, which

r e c e i v e d 20% of PFDE. The s u r v i v i n g monkeys appear to be i n good

8. CLARK ETAL. Cis-Trans Perfluorodecalin Emulsions 149
h e a l t h . Three monkeys were s a c r i f i c e d , one as a c o n t r o l , two be

cause o f compromised c i r c u l a t i o n t o the l e g . One succumbed during
anesthesia preparatory to biopsy and one from a s u r g i c a l accident
during cannulation. Aside from a drop i n c h o l e s t e r o l there were
only questionable changes i n about 20 blood components analyzed.
Small (0.05 ml/kg) doses o f PFDE given t o dogs i n v a r i a b l y caused a
pronounced drop i n blood pressure but none occurred i n the mon
keys. The morphologic changes i n the l i v e r of the monkeys were
r e v e r s i b l e with no s i g n of damage. The major problems encountered
were d i f f i c u l t y i n o b t a i n i n g pure PFD, making s t a b l e emulsions,
o b t a i n i n g a n a l y s i s of compounds and t i s s u e s f o r PFD, working with
the f r a g i l e c a r d i o v a s c u l a r and r e s p i r a t o r y systems of the rhesus
and judging optimum f l u i d balance post phlebotomy and post i n f u -
sion.
Abbreviations
NO Number
SP Samples
TP T o t a l P r o t e i n (gm%)
AB Albumin (gm%)
CA Calcium (mg%)
IP Inorganic Phosphrous (mg%)
GL Glucose (mg%)
BN Blood Urea Nitrogen (mg%)
UA U r i c A c i d (rag%)
CT C r e a t i n i n e (mg%)
TB T o t a l B i l i r u b i n (mg%)
AP A l k a l i n e Phosphatase (mU/ml) (EC 3.1.3.1)
LD L a c t i c Dehydrogenase (mU/ml) (EC 1.1.1.27)
GO Glutamic-oxaloacetic Transaminase (mU/ml) (EC 2.6.1.1)
CK Creatine Kinase (mU/ml) (EC 2.7.3.2)
CH C h o l e s t e r o l (mg%)
PV Packed C e l l Volume (%), hematocrit
PF Packed PFD Volume (%), f l u o r o c r i t
PFC Perfluorochemical
PFD D i s t i l l e d Perfluorodecalin
PFDE P e r f l u o r o d e c a l i n Emulsion
C Control
GLC Gas-Liquid Chromatography
MK Monkey
PTD Post Test Dose
M Mean
SE
Τ
RI Ringer's s o l u t i o n
BL Blood
LD50 Mean l e t h a l dose, i . v . i n j e c t i o n
ME The monkey ( r e f . 5), named Melvin
PP5 In the s e c t i o n on morphology t h i s i s used to designate d i s
t i l l e d perfluorodecalin.

Acknowled gemen t s
The authors wish to thank Dr. George M i l l e r f o r guidance i n

s u r g i c a l techniques used f o r t i s s u e biopsy. We consulted C h r i s t
Taraborski concerning chemical problems. The a s s i s t a n c e of the
f o l l o w i n g persons i s g r a t e f u l l y acknowledged: Dr. Marian L.
M i l l e r , Dr. Steele F. M a t t i n g l y , Dr. Jag L a i , Frank Knapke, Pat
Turner, L i l a m Stanley, Margaret Kelm, Steven Jones, David
DeForest, Barbara Cincush, Stanley Gaines, Eleanor C l a r k , Eleanor
Brinkmoeller, Sandra Hoffman, and Dotty O ' R e i l l y of Sun Ventures.
The P l u r o n i c F68 was a g i f t from Dr. I r v i n g Schmolka of Wyandotte.
This research i s supported i n part by grants HL17586, HL17353,
GM21475, HD05221, from the N a t i o n a l I n s t i t u t e s of Health, grant
74 619 from the American Heart A s s o c i a t i o n and a grant from the
Southwestern Ohio Chapter of the American Heart A s s o c i a t i o n .
Shortly a f t e r mailin
Technicon I n d u s t r i a l Systems, Tarrytown, New York 10591, v i a
Richard Carr, 1000 Crest C i r c l e , C i n c i n n a t i , Ohio 45208, a d d i t i o n -
a l data f o r normal values f o r c l i n i c a l blood chemistry i n the
rhesus. One report (29) gives the normal values f o r blood from
f o r t y - s i x female and t h i r t y - t h r e e male rhesus. Another report (30)
gives the mean and range f o r eleven blood component analyses f o r
f i f t y rhesus.

Trade Name PP5 Fomblin E4
h F F 2 CF,
Structure £o-CF-CF ]- 2 n n=6 CF CHF-(OCF2CF> F

3 4
CFo
h • Fa
%F %c %F %C %F %C
Calculated 74.01 25.99 67.70 21.40 70.28 21.43
Lab A 74.19 26.10 72.65 21.14 72.93 21.90

73.1b 26.35 72.73 21.32 73.39 22.01
73.53 71.73
73.94 72.32
71.85
Lab Β 78.24 84.05

78.32 83.90
Lab C 66.35 65.76

77.02 73.59
76.72 73.79

Lab D 74.22 25.93 68.60 21.81 70.14 21.31
74.09 25.78 68.78 21.59 70.35 21.30

Table 1 Comparison of a n a l y t i c a l results.
NO SP TP AB CA IP GL BN UA CT TB AP LD GO CK PV
gm% gm% mg% mg% mg% mg% mg% mg% rag% mU/ml mU/ml mU/ml mU/ml %
62 C 7.3 4.8 9.3 4.7 198 14 0.07 0.8 0.2 187 242 40 71 40
1H — — >778 38
48H 8.0 4.6 10.0 4.1 93 16 0.15 0.9 0.2 227 330 48 183 38
86A C 7.7 3.9 9.0 2.6 116 11 0.29 1.0 0.1 152 226 18 <27 43
1H 7.6 3.9 9.2 2.4 97 14 0.31 1.0 0.2 167 480 42 192 43
24H 8.2 3.9 10.1 5.9 149 16 0.29 1.3 0.3 167 336 36 250 38
71 C 8.2 3.5 9.8 3.0 105 20 0.20 0.9 0.2 580 175 20 40 41
1H 8.3 3.3 10.1 3.5 108 21 0.15 0.9 0.2 610 439 38 830 42
24H 8.4 3.6 10.0 4.6 122 12 0.19 0.9 0.2 > 350 294 48 380 37
117 C 8.1 4.0 9.3 5.4 81 14 0.30 0.8 0.2 110 660 48 58 40
1H 7.4 3.8 9.2 5.0 78 15 0.19 0.8 0.1 107 493 47 500 40
24H 7.6 3.7 9.7 5.7 185 15 0.47 1.1 0.2 135 381 51 220 37
33 C 7.2 3.7 8.8 2.4 119 15 0.28 1.1 0.2 128 317 50 67 39
1H 7.4 3.8 9.4 4.0 80 15 0.19 0.8 0.2 156 569 66 640 42
24H 7.8 3.9 10.0 4.2 109 21 0.25 1.1 0.2 170 373 57 245 39
58 C 8.8 5.7 10.5 4.6 77 16 0.19 1.1 0.2 194 583 86 > 778 46
1H 7.5 5.2 10.1 4.3 72 16 0.09 0.8 0.3 195 820 113 > 778 43
24H 7.5 4.5 9.5 4.6 225 27 0.28 1.5 0.6 232 > 600 > 300 > 788 31
74 C 7.2 4.9 9.5 4.7 75 18 0.12 1.1 0.2 138 195 38 39 39
1H 6.8 4.5 9.0 4.5 36 18 0.10 0.9 0.3 140 252 44 210 35
48H 7.6 4.2 9.6 6.0 122 19 0.19 1.1 0.1 186 224 40 435 37
113 C 7.3 3.9 8.9 3.8 72 22 0.17 0.8 0.2 223 192 53 52 40
1H 7.2 3.7 8.9 3.4 70 24 0.12 0.7 0.2 234

109 60 144 42
24H 7.6 4.1 9.6 2.9 111 16 0.21 0.8 0.3 236 207 83 86 38

Table 2 Blood chemistry before and a f t e r l i v e r biopsy and before t e s t dose o r i n f u s i o n with PFDE i n
the monkey.
continued on Table 3
NO SP TP AB CA IP GL BN UA CT TB AP LD GO CK PV
gm% gm% mg% mg% mg% mg% mg% mg% mg% mU/ml mU/ml mU/ml mU/ml %
80* C 7.4 4.5 9.9 4.4 87 19 0.1 1.0 0.2 122 185 38 <27 45
24H 7.6 4.0 10.0 12.0 176 24 0.5 1.3 0.2 177 380 78 835 43
95 C 6.9 4.4 9.0 1.3 111 16 0.3 1.0 0.2 65 191 25 <25 42
1H 6.9 4.3 9.0 2.9 97 17 0.2 0.9 0.2 68 388 51 210 45
24H 7.2 4.3 9.3 2.1 122 15 0.2 1.1 0.2 83 321 96 300 39
98 C 7.9 4.5 9.0 4.0 92 14 0.1 1.0 0.1 97 410 38 27 42
1H 7.5 4.4 8.6 5.0 62 15 0.2 0.8 0.2 108 509 55 280 38
24H 8.0 4.6 9.5 3.3 110 15 0.1 1.1 0.2 99 282 38 220 40
97A C 8.1 4.9 9.3 2.9 104 18 0.2 1.5 0.2 75 204 33 < 25 44
1H 7.8 4.7 9.1 3.2 66 19 0.2 1.2 0.4 80 570 76 280 46
24H 8.4 5.1 10.7 3.3 119 17 0.1 1.2 0.1 105 364 51 210 44
Mean C 7.7 4.4 9.4 3.6 103 16 0.20 1.0 0.2 171 298 41 51 42
1H 7.4 4.2 9.3 3.8 77 17 0.17 0.9 0.2 186 463 59 365 41
24H 7.8 4.2 9.8 4.9 143 18 0.27 1.1 0.2 156 326 60 305 39
S.Ε. C 0.17 0.19 0.15 0.36 10 0. 9 0.02 0.06 0.01 45 50 5.5 6.5 0,
1H 0.14 0.19 0.16 0.29 7 1.1 0.02 0.05 0.03 52 64 7.4 84 1,
24H 0.14 0.15 0.14 0.93 13 1.6 0.04 0.07 0.04 20 20 7.4 75 1,
Table 3 Blood chemistry before and a f t e r l i v e r biopsy and before t e s t dose or i n f u s i o n with PFDE i n

the monkey.
β
C sample taken from the a n e s t h e t i z e d monkey before biopsy. IH = sample one hour a f t e r i n c i s i o n c l o s

ed. 24 and 48H = 24 and 48 hours a f t e r i n c i s i o n c l o s e d .
*bone marrow b i o p s i e d .
BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
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98 7.9 4.4 7.9 3.1 84 18 0.16 0.9 0.2 112 395 39 21 138 6.9 103 14 157 42
7.7 4.2 9.2 3.3 77 18 0.11 0.9 0.2 106 370 38 38 138 5.6 104 18 145 42
113 8.3 4.9 9.2 4.0 90 18 0.19 0.9 0.2 157 148 42 104 151 4.3 108 25 133 43
8.2 4.6 9.1 4.4 96 20 0.38 0.9 0.2 135 297 58 450 145 4.9 106 14 162 42
74 7.5 4.3 8.7 3.6 111 17 0.29 1.0 0.2 192 280 53 91 148 4.8 104 13 133 42
7.5 4.1 7.8 3.3 99 18 0.29 0.9 0.2 156 312 54 86 141 6.3 105 16 129 40
62 8.1 4.5 8.1 3.9 82 15 0.20 0.9 0.3 169 404 47 87 143 6.5 107 13 143 45
7.9 4.5 8.6 4.0 88 16 0.28 1.0 0.3 150 476 50 188 139 5.4 105 15 134 44
95 7.5 4.1 8.6 3.6 69 15 0.12 0.9 0.2 84 351 44 50 141 4.6 104 18 167 41
7.3 3.8 8.4 3.3 74 14 0.20 0.9 0.2 77 383 49 30 138 4.8 103 20 148 42
71 7.7 4.2 9.0 3.8 62 15 0.15 0.9 0.2 130 310 52 83 144 5.3 107 13 210 45
7.6 4.1 9.1 3.4 85 15 0.20 1.0 0.2 128 324 60 124 142 4.5 106 18 207 42
58 7.5 4.3 9.4 2.5 58 18 0.29 0.8 0.2 110 347 42 42 148 5.0 108 20 175 43
7.9 4.3 10.0 3.2 67 14 0.19 1.0 0.2 188 250 47 70 150 3.6 110 20 156 44
33 7.8 4.1 9.1 2.8 74 17 0.30 0.9 0.3 119 306 39 18 148 5.4 109 14 170 42
8.0 4.3 9.6 3.2 94 14 0.21 1.1 0.2 133 166 30 40 150 3.8 115 12 145 43
97A 8.0 4.4 10.2 3.6 89 19 0.29 0.9 0.2 78 427 81 188 151 5.5 110 10 170 41
8.3 4.5 9.8 4.1 82 14 0.17 1.0 0.1 80 254 37 40 150 3.7 108 13 144 42
86A 8.0 4.3 9.6 2.0 77 14 0.20 0.7 0.2 119 252 30 15 150 5.4 107 18 190 43
8.2 4.5 9.8 2.8 85 12 0.15 0.9 0.1 124 155 25 32 152 4.0 110 19 159 43
117 7.6 3.9 7.9 4.3 104 15 0.31 0.6 0.2 73 323 27 16 150 5.1 111 9 460 39
7.9 4.1 9.6 4.4 90 16 0.20 0.8 0.1 73 240 28 34 152 4.3 113 13 159 42

Mean 7.8 4.3 9.0 3.5 84 16 0.22 0.9 0.2 122 308 44 84 146 5.0 107 16 173 42
S.Ε. 0.06 0.05 0.15 0.14 2.9 0.45 0.02 0.02 0.01 7.9 19 2.8 21 1.1 0.19 0.70 0.84 14.8 Ο.ι

Table 5 A n a l y s i s of blood and plasma from awake monkeys a f t e r biopsy and blood pressure t e s t dose
but before PFDE i n f u s i o n
pOo pC0 2
PH
AORTIC VENOUS AORTIC VENOUS AORTIC VENOUS
B D A B D A B D A B D A B D A B D A
•
Cl 378 455 369 43 68 56 36 39 33 42 39 36
C4 400 415 478 40 53 58 36 38 41 44 47 47 7.34 7.34 7.34 7.30 7.27 7.28
C5 360 484 524 39 62 81 41 39 39 44 47 48 7.36 7.32 7.32 7.33 7.23 7.26
62 311 436 452 46 50 61 35 35 33 45 30 44 7.40 7.34 7.41 7.37 7.31 7.30
86A 378 416 489 33 43 59 30 26 26 37 33 35 7.52 7.48 7.54 7.48 7.42 7.43
71 369 420 318 34 57 52 43 38 39 45 50 45 7.44 7.46 7.43 7.43 7.37 7.38
117 416 461 494 31 45 63 25 28 30 34 40 40 7.52 7.49 7.44 7.45 7.42 7.37
33 227 407 306 45 57 71 29 34 34 35 43 37 7.39 7.38 7.41 7.35 7.31 7.37
58 341 430 448 33 38 52 28 32 34 36 42 42 7.43 7.54 7.54 7.36 7.46 7.46
C2 332 414 440 36 64 135 42 36 30 46 46 41
C3 295 352 354 38 69 87 40 39 36 43 45 42
74 379 466 494 42 122 233 23 23 35 28 26 42 7.42 7.41 7.50 7.43 7.36 7.44
113 48 150 233 22 23 22 7.40 7.40 7.43
Mean 349 430 430 39 68 95 34 34 34 39 39 40 7.42 7.42 7.44 7.39 7.36 7.37
S.E. 15.7 10.5 22.5 1.6 9.3 19 2. 1 1.7 1.,3 2. 1 2.5 1.9 0.22 0.28 0.28 0.19 0.25 0.24
Table Blood gases and pH before, during, and a f t e r i n f u s i o n o f PFDE i n the monkey.

6
" A o r t i c " r e f e r s to samples removed from the cannula i n s e r t e d i n t o the a o r t a v i a the femoral a r t e r y .

"Venous" r e f e r s to mixed venous samples removed from a catheter e i t h e r i n the r i g h t v e n t r i c l e or the
pulmonary a r t e r y . Blood gas tensions are i n mm. o f mercury. Β = before, D = during i n f u s i o n ,
A = a f t e r . The average uncorrected barometric pressure i n C i n c i n n a t i i s 744ramHg.
DATE TP AB CA IP GL BN UA CT TB AP LP GO CK CH
gm% gm% mg% mg% mg% mg% mg% mg% mg% mU/ml mU/ml mU/ml mU/ml mg%
11-08-73 7.4 4.8 9.6 3.6 14 7.5 0.4 71 205 64 235

12-07-73 7.9 5.0 9.5 2.8 81 14 7.8 0.4 71 215 88 213
01-09-74 8.1 4.8 9.6 3.6 94 18 6.8 0.4 57 221 74 252
05-08-74 7.6 5.1 9.9 3.6 111 11 8.7 1.0 0.4 52 222 67 225
06-27-74 8.1 5.3 10.0 3.4 133 11 8.5 1.0 0.3 55 220 70 248
07-24-74 7.8 5.2 10.1 3.5 111 9 9.2 1.1 0.4 61 201 69 247
09-03-74 7.8 5.0 9.8 3.3 100 10 7.7 1.1 0.5 58 217 86 244
10-10-74 7.1 5.0 9.3 3.3 77 15 8.7 1.0 0.4 64 166 68 206
11-09-74 7.7 5.1 10.1 4.1 97 11 7.7 1.0 0.5 56 210 68 254
12-09-74 7.6 5.5 9.6 3.6 130 10 7.1 1.1 0.5 72 219 82 82
03-19-75 7.3 4.4 9.5 3.1 119 12 6.9 0.9 0.5 53 225 92 104 220
07-10-75 8.0 4.9 9.9 3.3 125 12 8.1 1.0 0.6 57 260 138 40 250
08-14-75 7.6 4.6 9.6 2.8 118 11 8.1 1.0 0.5 60 190 72 36 221
Mean 7.60 4.97 9.80 3.4 108 12.2 7.9 1.0 0.4 60.5 206 79.8 65.5 235
S.E. 0.30 0.28 0.22 0.35 18.3 2.5 0.73 0.06 0.07 06.9 3.6 01.9 33.0 16.8
Normal 1 6.0 3.5 8.5 2.5 50 10 2.0 0.7 0.2 25 100 7 15 150

Range ) 8.0 5.0 10.5 4.5 120 20 7.0 1.5 1.0 125 225 40 110 300

Table 7 Random blood samples from L.C. used as l a b o r a t o r y c o n t r o l f o r the monkeys.
HEART RATE RESPIRATION RATE

(BEATS/MINUTE) (BREATHS/MINUTE)
BEFORE DURING AFTER BEFORE DURING AFTER

MK NO INFUSION INFUSION INFUSION INFUSION INFUSION INFUSION
CI 207 200 210 42 66 87

CA 216 203 212 37 42 52
C5 213 200 198 32 47 42
62 193 148 140 36 36 51
86A 187 189 170 25 31 36
71 209 190 171 25 58 84
117 238 205 179 30 40 48
33 212 189 186 41 43 42
58 213 186 179 31 36 42
Mean 210 190

S.E. 5.10 6.0
C2 214 201 180 34 39 51

C3 174 156 152 70 77 72
74 170 174 162 23 32 34
113 206 176 164 47 65 70
Mean 191 177 165 44 53 57

S.E. 12.8 10.7 6.7 11.7 12.3 10.3
Mean 204 186 177 36 47 55

T-Test 1.85 1.25 1.52 -1.45 -1.01 -0.27
Table 8 Pulse and r e s p i r a t i o n before during and a f t e r infusion

of 10% PFDE (Cl-58) and 20% PFDE (C2-113)

8. CLARK E T AL. Cis-Trans Perfluorodecalin Emulsions 159
ARTERIAL PRESSURE RV OR PA PRESSURE

mm H g mm H g
BEFORE DURING AFTER BEFORE DURING AFTER
MK NO INFUSION INFUSION INFUSION INFUSION INFUSION INFUSION
CI 134 126 107 10 14 16

C4 111 122 121 8 16 12
C5 115 120 121 2 9 18
62 88 79 75 3 15 20
86A 111 111 110 8 17 12
71 112 105 101 11 19 19
117 100 108 100 7 19 20
33 88 95 102 12 19 18
58 102 102 96 6 11 9
Mean 107 108

S.E. 5.07 5.1
C2 101 111 120 8 14 19

C3 89 93 97 16 18 18
74 88 71 82 8 18 21
113 107 124 124 5 19 19
Mean 96 100 106

S.E. 5.36 13.28 11.44
Mean 104 105 104

T-Test 1.33 0.75 -0.22
Table 9 Blood pressures i n the rhesus monkey before during and

a f t e r i n f u s i o n of 10% PFDE (Cl-58) and 20% PFDE (C2-13)

NO SP TP AB CA IP GL BN UA CT TB AP LP GO CK CH PV
62 C 7.1 4.0 8.7 1.8 67 19 0.35 0.8 0.1 168 364 98 65 138 41.0
1u 44.0
±n
24H 8.1 4.7 9.1 2.5 77 15 0.15 1.0 0.1 236 333 105 95 133 43.5
86A C 7.0 3.7 9.1 3.8 94 14 0.32 0.9 0.1 158 162 32 46 178 40.0
1H 6.7 3.4 8.7 2.6 91 14 0.21 0.8 0.1 164 212 34 250 175 43.0
24H QNS 90 172 38.5
71 C 7.1 3.8 9.2 3.5 126 22 0.18 1.1 0.1 153 292 38 110 189 39.0
1H 7.0 3.8 9.1 2.9 89 21 0.20 1.0 0.2 154 420 52 420 196 38.5
24H 7.8 3.9 9.8 3.4 105 12 0.20 1.1 0.2 152 394 117 550 191 40.0
117 C 7.1 4.0 8.9 4.7 75 15 0.10 0.7 0.1 75 225 23 170 163 42.0
1H QNS 39.0
24H 7.2 3.7 9.0 5.3 132 14 0.21 1.0 0.2 156 444 82 196 126 37.0
33 C 6.6 3.6 8.7 3.8 132 16 0.21 1.0 0.2 86 162 30 39 149 39.5
1H 6.7 3.6 8.8 1.4 68 15 0.15 0.8 0.2 108 249 34 112 159 41.0
24H 7.8 4.4 10.1 2.6 103 17 0.28 1.2 0.2 102 256 40 82 146 40.0
58 1H QNS 110 34.0
24H 7.1 4.0 9.8 2.9 96 14 0.28 1.3 0.4 163 302 47 132 165 39.0
74 C 6.7 3.5 8.0 3.6 74 16 0.12 0.6 0.2 207 125 26 54 107 39.5
1H 5.8 2.9 7.5 2.2 71 16 0.08 0.5 0.2 193 138 26 104 104 44.0
24H 7.0 4.1 8.5 5.5 128 16 0.22 1.0 0.2 197 352 36 130 102 40.0
113 C 7.1 4.3 8.4 3.9 81 19 0.12 0.7 0.2 116 184 38 56 142 38.0
1H 6.9 4.2 8:4 4.2 71 19 0.09 0.6 0.2 128 143 38 102 138 42.0
24H 8.2 4.5 9.1 5.6 125 14 0.30 1.2 0.1 145 174 34 130 141 43.0
80 C 6.3 3.2 8.9 4.0 168 27 0.20 0.8 0.3 122 367 63 854 84 34.0

1H 5.0 2.6 14.9 6.6 102 24 0.23 0.7 0.2 104 362 67 854 72 28.0
24H 6.8 3.4 7.6 10.8 218 70 0.47 2.4 0.4 137 3950 480 847 111 32.0

95 C 7.1 3.3 8.4 3.4 92 16 0.25 0.9 0.1 76 207 40 48 116 41.0
1H 6.6 3.0 8.4 3.0 78 17 0.13 0.8 0.2 79 286 44 720 133 49.5
24H 7.0 3.9 9.6 2.3 97 16 0.14 1.2 0.1 88 256 53 112 128 40.0
Table 10 Blood chemistry before and a f t e r t e s t dose of PFPE continued on Table 11

NO SP TP AB CA IP GL BN UA CT TB AP LD GO CK CH PV
98 C 7.0 4.0 8.4 3.0 82 17 0.18 0.8 0.1 86 187 23 30 131 39.0
1H 6.7 3.8 8.4 1.9 76 17 0.06 0.7 0.2 98 204 28 60 141 44.0
24H 7.7 4.3 9.0 3.2 89 13 0.09 1.0 0.2 101 275 30 44 119 37.0
97A C 7.1 3.9 9.3 1.3 76 18 0.21 0.8 0.2 72 189 24 72 143 41.5
1H 7.1 4.0 8.9 2.4 56 17 0.12 0.9 0.2 64 156 18 38 151 42.0
Mean C 6.9 3.8 8.7 3.3 97 18 0.20 0.8 0.2 120 224 40 140 140 39.5
1H 6.5 3.5 9.2 3.0 78 18 0.14 0.8 0.2 121 241 38 277 141 40.8
24H 7.5 4.1 9.2 4.5 117 15 0.23 1.2 0.2 148 309 60 219 139 39.1
S.E. C 0.09 0.10 .13 0.31 10 1. 2 0.02 0.04 0.02 14 26 7.1 76 9.7 0.65
1H 0.24 0.19 .77 0.55 5 1. 1 0.02 0.05 0.01 15 35 5.2 98 13.0 1.66
24H 0.17 1.13 .24 0.84 13 0. 6 0.04 0.14 0.04 15 29 11.5 79 8.6 0.9Ç
Table 11 Blood chemistry before and a f t e r t e s t dose of PFDE
NO TP AB CA IP GL BN UA CT TB AP LD Gp Na Κ Ç1 CO? HCT
Ν 28 28 28 28 28 28 27 28 28 28 28 28 26 26 26 26 28
M 8.1 4.5 10.2 5.0 112 19.3 .29 1.17 0.2 390 387 58 160 5.1 112 15 37
SE .29 .15 .27 .37 4.6 1.21 .02 0.37 .02 25.8 52 11 1.60 0.33 0.91 1.7 1.6

Table 15 Blood chemistry f o r one year's sampling a f t e r i n f u s i o n of PFDE i n M e l v i n .

NO SP TP AB CA IP GL BN UA CT TB AP LD GO CK CH PV PF
CI 1DPP 7.8 —
3.8 9.1 5.7 140 11 0.50 0.8 0.2 "500 340 30 194 42 0
IPP 7.2 4.0 8.7 7.4 83 12 0.19 0.6 0.2 "500 340 30 148 131 39 0
PI 4.9 2.6 7.3 7.3 55 10 0.21 0.7 0.2 348 292 24 142 28 12
C4 8DPP 8.7 4.1 9.7 5.2 134 12 0.18 0.8 0.1 202 406 33 44 0
1DPP 9.2 4.7 11.3 4.5 92 21 0.36 0.9 0.2 182 377 52 66 149 38 0
IPP 6.0 3.1 8.9 4.4 63 20 0.10 0.6 0.1 158 194 28 88 92 38 0
PI 5.8 3.0 8.2 6.2 68 18 0.10 0.6 0.2 166 519 51 190 12 20 12
C5 8DPP 7.9 4.2 9.9 4.8 112 18 0.28 0.8 0.2 208 258 31 112 200 44 0
1DPP 7.8 4.3 10.3 5.5 76 18 0.33 0.9 0.2 200 252 31 114 183 43 0
IPP 6.8 3.9 9.2 4.6 99 19 0.19 0.6 0.3 207 299 36 210 181 36 0
PI 2.2 1.2 6.6 4.2 81 14 0.09 0.5 0.1 82 155 17 80 44 14 12
62 1DPP 7.4 4.4 10.0 1.8 72 19 0.20 1.0 0.2 145 313 42 50 156 44 0
IPP 6.5 3.6 8.4 2.6 85 16 0.20 0.8 0.2 162 286 43 102 122 38 0
PI 1.7 0.9 5.9 2.9 64 14 0.12 0.7 0.1 47 92 17 38 24 11 13
2HPI 2.1 1.1 6.0 2.5 123 15 0.18 0.9 0.1 69 148 26 80 48 13 13
86A 1DPP 7.8 4.5 9.4 3.4 82 15 0.28 1.0 0.1 135 162 38 68 220 46 0
IPP 6.5 3.9 8.4 4.0 97 11 0.09 0.7 0.2 129 183 27 52 152 39 0
MP 6.0 4.4 8.5 3.6 95 11 0.02 0.3 0.3 69 133 17 38 98 25 0
PI 3.1 2.2 6.8 3.5 65 10 0.02 0.7 0.3 43 102 10 38 36 13 12
1DPI 6.2 3.8 9.2 5.8 125 18 0.34 1.1 0.1 88 1520 325 857 46 13 6
71 8DPP 7.2 4.3 9.5 3.0 93 17 0.29 1.0 0.1 104 190 43 26 220 45 0
IPP 6.0 3.6 8.5 3.2 102 18 0.12 0.6 0.2 114 233 32 62 190 40 0
MP 6.0 4.6 8.7 3.0 92 16 0.10 0.7 0.3 58 147 17 26 124 24 0
PI 3.1 2.4 7.2 2.8 69

14 0.08 0.7 0.3 44 117 13 90 55 9 13
3DPI 6.1 3.4 8.7 4.8 97 24 0.30 0.9 0.2 230 " 1160 183 1502 190 15 4
117 1DPP 7.4 4.0 10.0 5.0 102 18 0.01 0.8 0.1 79 170 16 8 182 40 0

IPP 6.0 3.2 8.2 3.8 93 18 0.04 0.5 0.2 83 200 23 54 145 32 0
MP 5.9 4.1 8.5 3.7 79 18 0.06 0.6 0.2 52 125 12 36 82 20 0
PI 3.3 2.2 7.0 3.4 70 15 0.02 0.6 0.2 68 94 8 40 34 8 13
2DPI 6.3 3.4 9.1 3.8 122 16 0.21 0.8 0.2 214 > 600 265^ 123 17 4
Table 12 Blood chemistry before and a f t e i: 10% PFDE. Continued on Tab
NO SP TP AB CA IP GL BN UA CT TB AP LD GO CK CH PV PF
33 7DPP 8.0 4.5 9.7 3.2 83 16 0.11 0.9 0.1 116 137 28 8 166 44 0
IPP 5.9 3.1 8.2 4.0 76 16 0.10 0.9 0.1 137 273 28 232 130 36 0
MP 5.7 3.5 8.4 3.9 66 15 0.11 0.9 0.1 73 174 18 134 78 22 0
PI 3.3 2.2 7.1 3.5 42 14 0.15 0.9 0.2 54 213 15 144 34 12 11
Ο TV Ό 11
Α.Λ. 1
jDïr 1 Τ JL
58 6.7 2.4 7.7 4.0 53 14 0.23 0.9 0.1 258 256 105 90 220 48
2DPP 0
IPP 5.7 3.1 8.9 1.5 82 17 0.19 0.9 0.3 199 408 94 374 153 35 0
MP 5.5 3.9 9.2 1.2 82 15 0.21 0.9 0.5 122 258 57 240 87 23 0
PI 2.9 2.2 7.2 0.9 58 13 0.15 0.8 0.4 62 207 42 204 23 9 10
2DPI 5.9 3.3 10.0 4.4 89 19 0.40 1.1 0.4 237 1740 412 3640 70 8 4
Mean DPP 7.8 4.1 9.7 4.2 94 16 0.25 0.89 0.15 194 260 41 74 188 44 0
IPP 6.3 3.5 8.6 3.9 87 16 0.14 0.69 0.20 188 268 38 147 144 37 0
MP 5.8 4.1 8.7 3.1 83 15 0.10 0.68 0.28 75 167 24 95 94 23 0
PI 3.2 2.9 6.9 3.8 70 14 0.11 0.71 0.21 98 194 22 105 34 14 12.1
DPI 6.1 4.1 9.2 4.7 108 19 0.28 0.98 0.23 192 1473 296 6601 107 13 3.8
S.E. DPP 0.22 0.20 0.28 0.39 8. 3 1. 0 0.04 0.03 0.02 36 29 7 19 10 0.9 0
IPP 0.17 0.13 0.12 0.57 4. 4 1. 1 0.02 0.05 0.02 44 26 8 38 11 0. 9 0
MP 0.11 0.22 0.16 0.55 5. 8 1. 3 0.04 0.12 0.07 14 27 9 46 9 1. 0 0
PI 0.42 0.75 0.22 0.11 7. 1 0. 8 0.02 0.04 0.03 32 44 5 21 5 2. 0 0.3
DPI 0.10 0.82 0.31 0.49 10.4 2. 0 0.05 0.09 0.07 41 207 56 4993 37 1. 8 0.9

Table 13 Blood chemistry before and a f t e r i n f u s i o n of 10% PFDE i n 9 monkeys.

(DPP = days pre-phlebotomy, IPP = immediately pre-phlebotomy, MP = mid-phlebotomy, PI = immediately
p o s t - i n f u s i o n , HPI = hours p o s t - i n f u s i o n , DPI = day (post-infusion). *This value i s considered
erroneous and has been e l i m i n a t e d from the mean and standard e r r o r .
ο ο rH ο ο <r ο Ο ο CM ο ο Ο CO Ο Ο ο ο Ο O Ο Ο
PM
PM rH m CO CM CM CO rH
vO rH rH -d"
> ON CM CM m <r m *d" rH ο m <f co rH vO CO rH CM 00 rH CO CM m Λ *
PH m CO CO CM rH CO CM rH <r CO CM rH rH <f CO CM rH £ ω
Ό Ι
r-* 00 CM m vO <r <* m ON Ο vO ON vO 00 Ο CO ON CM ON
CJ rH CM ON ο <r CO CM ON
CO 00 m ο ON m rH ON m CM rH rH CO π
rH rH rH rH rH rH rH rH rH rH
fi "
Ο ο 00 vO CM vO 00 <r vO vO 00 Ο Ο Ο Ο vO 00 ο ^d- CO *d" ON Φ
θ0 k—|
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rH ON ON
rH m CO ^d- CO <f <f m ΟΟ vO CM co co Ο m
e
vO -d" rH CM CO CM CM CM CM rH rH rH rH rH CO ο
<f Φ 4-1
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43
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ο CO CO rH CM CM rH co CO CM rH m vO co rH CO co <f rH CO
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43
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PM PM PM PM Μ CO D
PM PM PM PM PM PM PM PM PM PM PM PM PM Τ 3 MM
Q P ^ M Q P M M Q P M P J M Q P M P H M Q PM PM PM M PM ΡΜ ΡΜ Μ rH G
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4 HR. POST OVER 4 HR.
PHLEBOTOMY INFUSION INFUSION POST INFUSION TOTALS IN AND OUT
OUT IN OUT IN OUT IN IN OUT IN
NO WT BL RI AB TO BL PFDE RI TO BL RI OS TO RI OS TO BL RI OS PFDE TO BAL
Cl 3.2 10 4 0 4 6 60 9 69 18 4 0 4 0 0 0 34 17 0 60 77 43
OA 3.2 28 54 0 54 4 50 5 55 3 5 0 5 0 0 0 35 64 0 50 114 79
C5 4.4 26 55 0 55 3 50 3 53 6 3 0 3 0 0 0 35 61 0 50 111 76
62 6.6 26 53 0 53 2 50 2 52 7 12 19D 31 0 11D 11 35 68 30D 50 148 113
86A 6.1 30 3 29 32 2 50 3 53 4 2 3A 5 0 0 0 36 8 31A 50 89 53
71 8.0 26 4 25 29 2 50 2 52 3 3 0 3 0 0 0 31 9 25A 50 84 53
117 6.8 26 3 21 24 2 50 3 53 4 1 3A 4 7 9A 16 32 14 33A 50 97 65
33 7.4 16 2 33 35 2 50 2 52 3 1 0 1 0 0 0 21 5 33A 50 88 67
58 7.1 26 5 25 30 2 50 3 53 3 1 0 1 0 0 0 31 9 25A 50 84 53
C2 3.0 26 49 0 49 4 50 6 56 1 5 0 5 0 23D 23 41 60 23D 50 133 92
C3 3.8 26 53 0 53 3 50 3 53 8 9 16D 25 0 0 0 37 65 16D 50 131 94
74 6.2 25 5 30 35 2 50 3 53 8 27 16A 43 16 8A 24 35 51 54A 50 155 120
113 5.6 26 7 25 32 1 50 3 53 4 0 0 0 0 0 0 31 10 25A 50 85 54
Table 16 Blood out and f l u i d s i n b e f o r e , during and a f t e r i n f u s i o n of PFDE.
Monkey weight i s i n kilograms. Blood out and f l u i d s i n are expressed as ml/kg. OS = osmotic or o n c o t i c ,
D = Dextran 40, A = albumin, BL = blood, RI = Ringer's s o l u t i o n , TO = t o t a l , B a l = balance or t o t a l i n
minus t o t a l out.

EMULSION BODY
NO. RECEIVED WEIGHT LIVER SPLEEN LUNG KIDNEYS
7.PFDE kg
62 10 7.2 26..3 1.1 11.8 3.7
117 10 6.4 22..9 1.9 7.2 4.4
C2 20 3.0 47,.5 2.3 12.3 5.7
C3 20 4.2 37..7 6.3 13.6 5.1
74 20 6.4 32..3 1.2 14.9 5.0
113 20 5.7 29..0 1.5 9.9 4.6
M
- 5.5 32..6 2.4 11.6 4.8
S.E.
- 0.7 4,.0 0.9 1.2 0.3
6 0 7.2 17..0 0.7 5.1 3.4

80
C7
0
0
4.0
3.9
36..6
26 8
0.9
1.0
8.8
13.3
-
5.8
M
-
S.E.
-0
FAS 3.6 19..2
- 19.0
-
Table 17 Organ weights obtained at autopsy. FAS = normal organ
weights f o r the rhesus monkey reported by FASEB (20).
A l l organ weights expressed as gms/kg of body weight.
DAYS POST PFD PFD

NO. INFUSION INFUSED FOUND GLC
gm gm %
62 1 64 7.7 12 8,000
74 1 120 24 20 12,000
113 1 109 14 13 1,400
C3 8 74 17 23 7,000
C2 14 58 13 22 10,000
CI 42 37 2.1 5.6 1,500
C4 47 31 1.3 4.2 1,700
C5 56 43 2.1 4.8 1,800
86A 64 59 0.3 0.5 4
71 70 78 3.4 4.4 1,000
117 77 66 0.96 1.0 800
58 84 69 0.2 0.3 40
ME 332 25 0.1 0.4 1
Table 18 PFD content of l i v e r obtained a t biopsy or autopsy as

determined by sodium biphenyl combustion or vapor phase
GLC.
Data on Monkeys C l , C4, C5, 86A, 71, 58 and ME were

obtained from samples obtained a t biopsy; the l i v e r was
assumed to be 37« of the body weight. R e l a t i v e peak
area was obtained from a pen w r i t i n g i n t e g r a t o r on the
s t r i p chart.

Literature cited
1. Clark, Jr., Leland C. and Gollan, Frank. Science (1966)
152, 1755-1756.
2. Clark, Jr., Leland C., Editor. Federation Proceedings (1970)
29, 1696-1820.
3. Sloviter, Henry A. Medical Clinics of North America (1970)
54, 787-795.
4. Geyer, Robert P. The New England Journal of Medicine (1973)
289, 1077-1082.
5. Clark, Jr., Leland C.; Kaplan, Samuel; Emory, Carolyn and
Wesseler, Eugene P. "Progress in Clinical and Biological Research,
Vol. I. Erythrocyte Structure and Function", edited by George J.
Brewer, 589-600, Alan R. Liss, Inc., New York (1975).
6. Gollan, Frank and Clark, Jr., Leland C. The Alabama Journal
of Medical Sciences (1967) 4 336-337
7. Gollan, Frank and
(1966) 9, 191.
8. Gollan, Frank and Clark, Jr., Leland C. Transaction of the
Association of American Physicians (1967) 80, 102-110.
9. Clark, Jr., Leland C.; Kaplan, Samuel; Becattini, Fernando
and Benzing III, George. Federation Proceedings (1970) 29,
1764-1770.
10. Spitzer, Hugh L.; Sachs, George and Clark, Jr., Leland C.
Federation Proceedings (1970) 29, 1746-1750.
11. Clark, Jr., Leland C.; Kaplan, Samuel and Becattini, Fernando.
Presented at the American Association for Thoracic Surgery 50th
Annual Meeting, Washington, D.C. (Abstract No. 34), April 8, 1970.
Pediatric Research (1970) 4, 464 (Abstract 113).
The Journal of Thoracic and Cardiovascular Surgery (1970) 60,
757-773.
14. Clark, Jr., Leland C.; Bacattini, Fernando and Kaplan, Samuel.
Triangle (1972) 11, 115-122.
15. Clark, Jr., Leland C.; Becattini, Fernando and Kaplan, Samuel.
The Alabama Journal of Medical Sciences (1972) 9, 16-29.
16. Clark, Jr., Leland C.; Becattini, Fernando; Kaplan, Samuel;
Obrock, Virginia; Cohen, David and Becker, Charles. Science
(1973) 181, 680-682.
17. Clark, Jr., Leland C.; Wesseler, Eugene P.; Kaplan, Samuel;
Miller, Marian L.; Becker, Charles; Emory, Carolyn; Stanley,
Lilam; Becattini, Fernando and Obrock, Virginia. Federation
Proceedings (1975) 34, 1468-1477.
18. Miller, Marian L.; Clark, Jr., Leland C.; Wesseler, Eugene P.;
Stanley, Lilam; Emory, Carolyn and Kaplan, Samuel. The Alabama
Journal of Medical Sciences (1975) 12, 84-113.
19. Wesseler, Eugene P.; Iltis, Ron and Clark, Jr., Leland C.
The solubility of oxygen in highly perfluorinated liquids. In
preparation.

20. Altman, P h i l i p L. and Dittmer, Dorothy S., E d i t o r s .

" B i o l o g i c a l Handbooks: Growth i n c l u d i n g r e p r o d u c t i o n and morpho-
l o g i c a l development". Federation of American S o c i e t i e s f o r Ex-
perimental Biology, Washington, D.C. (1962).
21. Technicon SMA12 procedures were used f o r most of these a n a l -
yses. The exact methods used are on f i l e here.
22. Calderwood, H. W.; Modell, J . H.; Rogow, L.; Tham, M. K. and
Hood, C. I. Anesthesiology (1973) 39, 488-495.
23. W r e t l i n d , A r v i d . J o u r n a l o f N u t r i t i o n , Metabolic Diseases and
D i e t e t i c s (1972) 14, 1-57.
24. W r e t l i n d , A r v i d . The pharmacological basis f o r the use of f a t
emulsions i n intravenous n u t r i t i o n . Department of N u t r i t i o n and
Food Hygiene. The N a t i o n a l of I n s t i t u t e of P u b l i c Health Stock-
holm 60, Sweden.
25. Galen, R. S. and Gambino, S. R. C l i n i c a l Chemistry (1975)
21, 272.
26. Pooley, F. D.; Kanellopoulos
(1972) 3, 486-496.
27. S t e i n , R. J . ; R i c h t e r , W. R.; Rdzok, E. J.; Moize, S. M. and
B r y n j o l f s s o n , G. "Use of nonhuman primates i n drug e v a l u a t i o n " ,
e d i t e d by Harold Vogtborg, 187-199, U n i v e r s i t y of Texas Press,
A u s t i n (1968).
28. Here we have given a summary of the procedure used f o r making
emulsion. A p r e c i s e d e t a i l e d account f o r making a v a r i e t y of PFC
emulsions i s on f i l e and can be provided on request.
29. Busey, W i l l i a m and W i l l n e r , Howard. Normal biochemical p a r a -
meters o f rhesus monkeys and beagle dogs. Presented at the
Technicon Symposium, "Automation i n A n a l y t i c a l C h e m i s t r y , " New
York, N . Y . , October 4, 1967. Published by Technicon C o r p o r a t i o n .
30. Technicon I n d u s t r i a l System brochure e n t i t l e d , "The Technicon
SMA 6/60 micro multichannel biochemical a n a l y z e r , " August 23, 1974.
Technicon number 4229-9-4-2.

D i s c u s s i o n of the paper of Dr. Leland C. Clark, J r .
Q. How long does i t take f o r the pulmonary a r t e r i a l pressure to

r e t u r n to normal?
A. About an hour.
Q. So that i t was back to normal before the m a t e r i a l was cleared

from the blood?
A. Yes, i t takes over a day before i t i s cleared from the blood.
Q. Do you have any studies on the perfluoromethyladamatane

s t r u c t u r e you showed?
A. We have made p r e l i m i n a r y studies on a small batch which was

somewhat impure bu
and a l s o leaves th
Q. In the a b s t r a c t you mentioned brominated and i o d i n a t e d per-

fluorocarbons as X-ray c o n t r a s t agents. Would you l i k e to
comment on that?
A. Dr. Long w i l l soon t e l l us the s t o r y of the X-ray c o n t r a s t

agents. We have found that the i o d i n a t e d compounds are very
l i t t l e , i f any, more X-ray opaque than the brominated com-
pounds, at 55KV. The i o d i n a t e d compounds are unstable i n the
presence of l i g h t and even though the l i b e r a t e d iodine could
be removed by m e t a l l i c s i l v e r there would be other decomposi-
t i o n products there. We have j u s t about given up on i o d i n e .
Q. How do the iodinated compounds hold up i n the animal?
A. I f they decompose i n the presence of l i g h t you could almost

count on t h e i r breakdown i n the body. We've had some animals
survive small doses. Some of t h i s information has been pub-
l i s h e d . (Clark, Leland C , J r . , Eugene P. Wesseler, Samuel
Kaplan, Marian L. M i l l e r , Charles Becker, Carolyn Emory,
Lilam Stanley, Fernando B e c a t t i n i and V i r g i n i a Obrock. Emul-
sions of p e r f l u o r i n a t e d solvents f o r i n t r a v a s c u l a r gas trans-
port. F e d e r a t i o n Proceedings 34, 1468-1477 (1975); C l a r k ,
Leland C., J r . , Eugene P. Wesseler, Marian L. M i l l e r and
Samuel Kaplan. Ring versus s t r a i g h t chain p e r f l u o r o c a r b o n
emulsions as a r t i f i c i a l blood. J o u r n a l of M i c r o v a s c u l a r
Research 8, 320-340 (1974)).
Q. What are your l i m i t a t i o n s on v o l a t i l i t y of the p e r f l u o r o -

carbons?

A. When used as a r t i f i c i a l blood the vapor pressure must be

below 50 torr at 38°C. Higher vapor oressures, perhaps much
higher vapor pressures can be w e l l t o l e r a t e d f o r l i q u i d
breathing i f 1007 oxygen i s used as the gas phase.
o
Q. Do you think these compounds w i l l ever be u s e f u l i n man?
A. I f I didn't I wouldn't be working so hard. This has been a

major e f f o r t of ours f o r over eight years.
Q. How about toxicity?
A. When p e r f l u o r o d e c a l i n i s p u r i f i e d by c a r e f u l d i s t i l l a t i o n i n
a spinning band column the t o x i c i t y decreases to the point
where the LD50 of a 10% by volume emulsion i s over 200 ml/kg.
200 ml/kg i s about three times the blood volume of the mouse
The lower b o i l i n g f r a c t i o n
r i a l as r e c e i v e d .
Assessment of the t o x i c i t y of PFC emulsions i s complicated by

the f a c t that i t i s s t i l l d i f f i c u l t to c h a r a c t e r i z e the f i n a l
product with a high degree of c e r t a i n t y . Hence one i s often
unsure whether d i f f e r e n c e s are due to the p h y s i c a l p r o p e r t i e s
of the emulsions themselves or to the v a r i o u s impurities i n
d i f f e r e n t PFC.
Generation of f l u o r i d e ion by s o n i c a t i o n , a former complica-

t i o n of emulsion t o x i c i t y t e s t i n g , has been p r a c t i c a l l y elim-
inated by the discovery by Geyer that by s a t u r a t i n g the l i q -
uids being sonicated with carbon dioxide, f l u o r i d e i o n i s not
generated. Nitrogen, helium, or hydrogen do not work.

9
Radiopaque Applications of Brominated Fluorocarbon
Compounds in Experimental Animals and Human
Subjects
D. M. LONG
Department of Radiology, School of Medicine, University of California, San Diego, Calif.
M. S. LIU, G. D. DOBBEN, and P. S. SZANTO
Hektoen Institute for Medical Research, Cook County Hospital, Chicago,Ill.60612
A. S. ARAMBULO
College of Pharmacy, Universit
Interest in the biological application of perfluorocarbon
compounds was begun with the imaginative fluid ventilation stu-
1
dies of Clark and Gollan in 1965. In the first decade since the
publication of those experiments, there has been a slowly growing
expansion of background information on the effects of perfluoro-
23
,
carbons on the biological systems. The industrial use of per-
fluorocarbon compounds has helped by making more molecules
available in pure states at reasonable costs. Imaginative bio-
medical workers have asked where these new compounds could help
solve existing medical problems. Our aggressive chemical industry
extended itself in seeking new markets in medicine for the pro-
ducts of their laboratories and plants.
Fluid ventilation as a tool for delivering oxygen to damaged
lungs was our initial goal in experiments with perfluorocarbon
compounds. This application and the dreams of Jacques Cousteau of
a fluid breathing diver in the depths of the ocean seem frustra-
ted for the time being by the unsolved problems of the work of
fluid ventilation and by the mechanical injury to the lungs resul-
ting from long periods of fluid ventilation.
We became interested in the possibility of developing a rad-
iopaque fluorocarbon molecule that would be less irritating and
safer than currently available radiopaque media which can be quite
4
irritating to the lungs. The inert synthetic fluids, either sil-
icone oils or perfluorocarbons, that were available in 1966 did
not possess radiopaque properties. Brominated fluorocarbon liquid
was found to be radiodense. Iodinated fluorocarbon compounds were
found to be chemically unstable and, therefore, unsuitable for
biomedical application. Brominated organic compounds were used
in the past and were found to be inferior to iodinated compounds
when studied with the usual high kilovoltage used in x-rays of
humans. It was argued that brominated compounds, although satis-
factory for small animal studies, would be unsatisfactory in the
larger human subjects. As can be seen in Figure 1, brominated
perfluorocarbon is most satisfactory for use in humans. Lower
kilovoltage or energies are used to get maximum absorption from
171

Figure 1. X-ray of the abdomen

in a healthy subject ten min
oral administartion of 500
neat radiopaque fluorocarbon. Note
the rapid visualization of the je-
junal portion of the small intestine.
Figure 2. Bronchogram after ad-

ministration of 6:1 emulsion of
radiopaque fluorocarbon in a pa-
tient with hemoptysis. A calcified
nodule or broncholith can be seen
in continuity with an eighth gen-
eration bronchus in the lower right
hand corner.

9. LONG ET A L . Brominated Fluorocarbon Compounds 173
t h e b r o m i n e , b u t t h i s a l t e r a t i o n i n t e c h n i q u e h a s p r e s e n t e d no
problems w i t h c u r r e n t l y a v a i l a b l e x - r a y equipment. Emulsions of
r a d i o p a q u e f l u o r o c a r b o n (RFC) were p r e p a r e d t o o b t a i n a m a t e r i a l
w i t h a h i g h e r v i s c o s i t y t o produce a t h i c k e r c o a t i n g o f the t r a -
cheobronchial tree (Figure 2). The e m u l s i o n s w e r e p r e p a r e d i n
high concentrations of fluorocarbon i n physiologic s a l t solution
w i t h P l u r o n i c F-68 as t h e e m u l s i f y i n g a g e n t and were a l s o n o n -
irritating.
A l t h o u g h we h a v e e x a m i n e d a number o f b r o m i n a t e d f l u o r o c a r b o n
4
m o l e c u l e s , most o f o u r s t u d i e s h a v e b e e n p e r f o r m e d w i t h p e r f l u o r -
octylbromide. T h i s compound i s b i o l o g i c a l l y i n e r t and p o s s e s s e s a
v e r y low t o x i c i t y . The LD50 ( T a b l e I) o f C g F B 1 7 i s g r e a t e r than
R
64 m l . / k g . when a d m i n i s t e r e d i n t o t h e g a s t r o i n t e s t i n a l t r a c t . We
h a v e u s e d h i g h e r d o s a g e s o f as much as 128 m l . / k g . w i t h o u t a d v e r s e
e f f e c t s , but these experiments are f a c e t i o u s s i n c e the g a s t r o i n -
t e s t i n a l t r a c t was l o a d e d and o v e r f l o w i n g a t one o r b o t h e n d s b e -
f o r e a l l t h e d o s e was a d m i n i s t e r e d
C F B w n e n
8 17 R injected int
a n i m a l s h a v e b e e n c o m p l e t e l y submerged i n C Q F 7 B and b r e a t h e d
1 r
t h i s f l u i d f o r s h o r t p e r i o d s o f time w i t h s u r v i v a l . The L D ^ Q o f
t h e 10:1 e m u l s i o n o f C g F ^ B j ^ i n t h e l u n g s was g r e a t e r t h a n 4 m l . /
kg. R e p e t i t i v e dosage programs have been p e r f o r m e d i n a n i m a l ex-
p e r i m e n t s w i t h no a d v e r s e e f f e c t s . The e f f i c a c i o u s d o s e s o f t h e
RFC i n human d i a g n o s t i c x - r a y s t u d i e s a r e g i v e n i n T a b l e I . There
i s o b v i o u s l y a w i d e m a r g i n o f t h e r a p e u t i c s a f e t y when RFC i s u s e d
i n these areas of a p p l i c a t i o n .
Gastroenterography
Our i n i t i a l t o x i c o l o g i c a l and d i a g n o s t i c s t u d i e s w e r e d i r e c -
t e d t o w a r d e x a m i n a t i o n o f t h e e f f e c t s o f RFC i n t h e GI t r a c t . It
s h o u l d be remembered t h a t p e r f l u o r o c a r b o n compounds a r e new i n
b i o m e d i c a l f i e l d s , and none o f t h i s f a m i l y o f compounds h a d e v e r
b e e n a d m i n i s t e r e d p u r p o s e f u l l y i n l a r g e d o s e s t o humans. The l a -
b o r a t o r y s t u d i e s i n d i c a t e d RFC w o u l d be among t h e s a f e s t o f d r u g s
or d i a g n o s t i c agents. When g i v e n t o e x p e r i m e n t a l a n i m a l s and h u -
man s u b j e c t s b y t h e GI t r a c t , t h e r e h a v e b e e n no a d v e r s e e f f e c t s .
S p e c i f i c a l l y , t h e r e was no c h a n g e i n t h e b l o o d c o u n t s , s e r u m e n -
zymes, and u r i n a l y s i s . The a n i m a l s showed no c h a n g e i n g r o w t h
p a t t e r n s e v e n when g i v e n r e p e t i t i v e h i g h d o s e s o v e r s h o r t p e r i o d s
of time o r over prolonged i n t e r v a l s . RFC h a s b e e n g i v e n r e p e t i -
t i v e l y i n newborn a n i m a l s u n t i l t h e y a c h i e v e y o u n g a d u l t h o o d and
to adult animals.
The RFC i s o d o r l e s s and t a s t e l e s s , and h a s a low v i s c o s i t y s o
t h a t i t i s easy t o d r i n k . Because o f the e x c e l l e n t w e t t a b i l i t y ,
t h e mouth and o r o p h a r y n x a r e c o a t e d i m m e d i a t e l y a f t e r t a k i n g t h e
material. Some s u b j e c t s h a v e c o m p l a i n e d o f an u n p l e a s a n t o i l y
f e e l i n g i n t h e mouth, b u t m o s t h a v e h a d no s u c h r e s p o n s e . RFC
t r a v e r s e s t h e GI t r a c t more r a p i d l y t h a n f o o d o r o t h e r c o n t r a s t
agents. When RFC i s m i x e d w i t h f o o d and f e d t o d o g s , t h e RFC

Table I
LD50 (Animals) Efficacious Dose
Gastroenterology 64 m l . / k g . Dogs 1-6 m l . / k g .

Rats
Alveolography > 35 m l . / k g . H a m s t e r s 1-2 m l . / k g .

^> 12 m l . / k g . C a t s
Bronchography > 4 m l . / k g . Dogs 0.3-0.6 m l . / k g .
Figure 3. Gastrointestinal series in an "asymptomatic" volun-

teer forty minutes after oral administration of 225 ml of neat
RFC. This subject had a small channel ulcer with pyloro-
spasm and delayed gastric emptying. Note that RFC is still
present in the stomach while the left colon is beginning to
show filling with RFC.

9. LONG ET AL. Brominated Fluorocarbon Compounds 175
l e a v e s t h e f o o d and t r a v e r s e s t h e GI t r a c t a h e a d o f t h e f o o d .
T h i s b e h a v i o r o f RFC h a s b e e n a t t r i b u t e d t o t h e " c r e e p i n g " p r o p -
e r t y o f s u b s t a n c e s w i t h low s u r f a c e t e n s i o n .
I n i t i a l l y , we t h o u g h t t h a t r a p i d g a s t r i c e m p t y i n g m i g h t be a
d i s a d v a n t a g e i n c e r t a i n d i s e a s e s t a t e s ; h o w e v e r , we f o u n d t h i s
p r o p e r t y t o be an a d v a n t a g e i n c l i n i c a l p r a c t i c e . I n F i g u r e 3,
we s e e t h e x - r a y o f a s u b j e c t w i t h a s m a l l , c h a n n e l u l c e r c r a t e r .
G a s t r i c e m p t y i n g o f RFC was d e l a y e d b e y o n d f o r t y m i n u t e s due t o
p y l o r o s p a s m o f a v e r y modest degree. T h i s s u b j e c t was a h e a l t h y
v o l u n t e e r m e d i c a l s t u d e n t who s t a t e d a f t e r t h e s t u d y t h a t he r e -
g u l a r l y had m i l d e p i g a s t r i c d i s c o m f o r t e s p e c i a l l y b e f o r e s l e e p
and d u r i n g t i m e s o f s t r e s s . R e p e a t u p p e r GI s e r i e s w i t h b a r i u m
s u l f a t e d i d n o t r e v e a l any u l c e r c r a t e r o r d e l a y e d g a s t r i c empty-
ing.
One d i s a d v a n t a g e o f RFC i n t h e GI t r a c t o f humans i s t h a t
t h e r e i s i n a d e q u a t e r a d i o p a c i f i c a t i o n o f t h e e s o p h a g u s and f u n d u s
o f t h e stomach. T h i s inadequac
animals.
I n d i c a t i o n s f o r Use o f RFC i n t h e GI T r a c t . O b v i o u s l y , RFC

c a n n o t compete i n c o s t w i t h b a r i u m s u l f a t e f o r GI s t u d i e s . There
a r e s p e c i f i c c i r c u m s t a n c e s i n w h i c h RFC c a n and s h o u l d be u s e d ,
and t h e s e i n d i c a t i o n s r e p r e s e n t an e s t i m a t e d one p e r c e n t o f t h e
t o t a l m a r k e t o f GI x - r a y s t u d i e s . I n any s u b j e c t w h e r e p u l m o n a r y
a s p i r a t i o n o f c o n t r a s t m e d i a i s l i k e l y , RFC w o u l d be u s e f u l due
to i t s lack of t o x i c i t y t o the lungs. These c i r c u m s t a n c e s i n -
c l u d e i n f a n t s , e l d e r l y p a t i e n t s , and weak c a c h e c t i c p a t i e n t s a s
w e l l as a l l s u b j e c t s w i t h symptoms o f t r a c h e o e s o p h a g e a l f i s t u l a .
We h a v e a l s o f o u n d RFC u s e f u l i n p a t i e n t s w i t h i n t e s t i n a l f i s t u -
l a s , perforations, or i n t e s t i n a l obstruction, or patients with
suspected postoperative i l e u s or obstruction. The x - r a y p i c t u r e s
o f t h e s m a l l i n t e s t i n e s h a v e b e e n j u d g e d s u p e r i o r t o t h o s e ob-
t a i n e d w i t h b a r i u m s u l f a t e and c a n be o b t a i n e d i n l e s s t h a n an
hour.
P e r i t o n e a l Contamination w i t h Radiopaque Agents
Leakage o f c o n t r a s t m a t e r i a l such as b a r i u m s u l f a t e i n t o t h e
p e r i t o n e a l c a v i t y c a r r i e s p o t e n t i a l l y s e v e r e c o m p l i c a t i o n s due t o
t h e a c u t e and c h r o n i c i n f l a m m a t o r y r e a c t i o n i n c i t e d b y b a r i u m
sulfate. W a t e r - s o l u b l e o r g a n i c i o d i d e compounds may be u s e d when
p e r f o r a t i o n i s s u s p e c t e d , b u t t h e s e compounds a l s o p r o d u c e a c u t e
inflammation o f the peritoneum. Water-soluble organic iodide
compounds p r o d u c e d i a r r h e a and do n o t g i v e s a t i s f a c t o r y r a d i o p a -
c i f i c a t i o n o f t h e GI t r a c t . RFC i s n o n - i o n i c and d o e s n o t r e s u l t
in diarrhea. RFC h a s b e e n i n j e c t e d i n t o t h e p e r i t o n e a l c a v i t y o f
e x p e r i m e n t a l a n i m a l s i n d o s a g e s o f 16 m l . / k g . , many t i m e s t h e l e -
R
t h a l dosage o f barium s u l f a t e o r o r a l Hypaque. T h e r e was no
a c u t e o r c h r o n i c i n f l a m m a t i o n e v e n i n a n i m a l s o b s e r v e d f o r more
than four years. No e v i d e n c e o f c a r c i n o g e n i c i t y was o b s e r v e d on

histological studies. The C g F ^ B l e a v e s t h e p e r i t o n e a l c a v i t y

7 R
v e r y s l o w l y by v a p o r i z a t i o n and by p h a g o c y t o s i s . Some o f t h e
c
8 17 R
f b w
f a s o u n
d i n s u b c u t a n e o u s lymph n o d e s o f t h e a n t e r i o r a b -
dominal w a l l . P h a g o c y t o s i s o f RFC o c c u r s b y m o n o c y t e s i n t h e p e r -
itoneal cavity. A c c u m u l a t i o n s o f v a c u o l e - l a d e n p h a g o c y t e s c a n be
s e e n i n t h o s e a r e a s w i t h r a d i o d e n s i t y on x - r a y s ( F i g u r e 4). Large
c y s t - l i k e a c c u m u l a t i o n s o f RFC a r e s e e n w i t h i n t h e p e r i t o n e a l c a -
vity. T h i s r e a c t i o n i s comparable t o t h e f o r e i g n body r e a c t i o n
R 1
seen w i t h T e f l o n o r S i l a s t i c * , t h e most i n e r t m a t e r i a l s u s e d i n
medical implants. Such f o r e i g n body r e a c t i o n s a r e f o u n d w i t h any
i n e r t m a t e r i a l with long residence time.
Fluorocarbons o f High Vapor Pressure
The r a d i o p a q u e f l u o r o c a r b o n C ^ F ^ B R was a l s o s t u d i e d i n t h e
peritoneal cavity. T h i s compound h a s a b o i l i n g p o i n t o f 9 8 ° a n d a
v a p o r p r e s s u r e o f 90 T o r r
vity, C Fi3B 6 vaporizeR
tended w i t h f l u o r o c a r b o n gas. C^F-^Bj^ l e a v e s t h e b o d y more

r a p i d l y than C g F ^ B j ^ and i s c o m p l e t e l y e l i m i n a t e d from t h e p e r i -
t o n e a l c a v i t y i n weeks e v e n w i t h l a r g e d o s e s o f 16 m l . / k g . This
p r o p e r t y o f r a p i d v a p o r i z a t i o n and i n t r a c a v i t a r y gas f o r m a t i o n
d o e s n o t p r o d u c e a n y a d v e r s e e f f e c t s i n t h e GI t r a c t o r l u n g s ;
h o w e v e r , when i n j e c t e d i n t o t h e s u b a r a c h n o i d s p a c e , g a s f o r m a t i o n
r e s u l t s i n n e u r o l o g i c a l i n j u r y i n a s i g n i f i c a n t number o f a n i m a l s .
F o r t h i s r e a s o n , we s e l e c t e d C s F i B f o r o u r i n i t i a l i n v e s t i g a -
7 R
t i o n s o f RFC s i n c e e c o n o m i c c o n s i d e r a t i o n s d i d n o t p e r m i t s i m u l -
t a n e o u s a n d p a r a l l e l s t u d i e s w i t h more t h a n o n e RFC compound. T h e
F B
C-6 17 R compound was a l s o n o n - t o x i c a n d may p r o v e t o b e u s e f u l f o r
GI a n d p u l m o n a r y s t u d i e s . C £ F i B v a p o r i z e s w i t h i n t h e GI t r a c t
7 R
thus p r o v i d i n g u s e f u l d i a g n o s t i c p r o p e r t i e s o f gas c o n t r a s t . In
some m a n u f a c t u r i n g p r o c e s s e s , C F B i s the principle
6 1 7 R contaminant
o r i m p u r i t y . Up u n t i l now, we h a v e h e l d s t a n d a r d s o f b e t t e r t h a n
99.9 p e r c e n t p u r i t y f o r C F B . Q T h i s h i g h p u r i t y s t a n d a r d may
1 7 R
n o t b e n e c e s s a r y , a n d i f n o t , c o s t s o f raw m a t e r i a l w o u l d b e r e -
duced s i g n i f i c a n t l y .
Submersion Experiments
S u b m e r s i o n o f h a m s t e r s i n RFC h a s b e e n p e r f o r m e d f o r p e r i o d s
up t o t e n m i n u t e s . When t h e h a m s t e r s w e r e removed f r o m t h e l i -
q u i d , x - r a y s showed t h e l u n g s t o b e f i l l e d w i t h RFC a n d RFC was
a l s o p r e s e n t i n t h e GI t r a c t . L a t e r x - r a y s showed g r a d u a l c l e a r -
ing o f t h e lungs. S i n c e RFC c l e a r s p r i m a r i l y b y v a p o r i z a t i o n , t h e
a r e a s o f t h e l u n g s t h a t a r e b e t t e r v e n t i l a t e d show more r a p i d
clearing. T h u s , t h e a l v e o l o g r a m s w i t h RFC p r o v i d e a p h y s i o l o g i c
picture of different ventilatory patterns i n different parts of
the lungs.
Microscopic examination o f t h e l u n g s one d a y a f t e r submersion
in RFC r e v e a l e d t h e p r e s e n c e o f h y p e r i n f l a t i o n o f t h e a l v e o l i due

9. LONG E T A L . Brominated Fluorocarbon Compounds 177
Figure 4. Photomicrograph of
phagocytic cells one month

after intraperitoneal injection
of 16 ml of neat RFC into the
peritoneal cavity of a rat. Note
the large vacuoles in the mono
cytic cells at the top of the
Hematoxylin and
eosin stain. 17 5χ.
Figure 5. Lung macrophage
of radiopaque fluorocarbon in
cupying the alveoli in a rabbit

two days after a bronchogram
with 10:1 emulsion of RFC.
Hematoxylin and eosin stain.
290X.

to the m e c h a n i c a l e f f e c t s o f the dense f l u o r o c a r b o n ( d e n s i t y 1.93

gm./ml.). M a c r o p h a g e s w i t h v a c u o l e s o f RFC were s e e n i n t h e a l v e -
o l a r spaces ( F i g u r e 5). No e v i d e n c e o f p n e u m o n i t i s due t o RFC was
o b s e r v e d when compared w i t h c o n t r o l a n i m a l s . A t one month, t h e r e
were few m a c r o p h a g e s w i t h v a c u o l e s o f RFC. The m a c r o p h a g e s w i t h
v a c u o l e s o f f l u o r o c a r b o n showed no e v i d e n c e o f i n t r a c e l l u l a r dam-
age, and t h e a l v e o l a r a r c h i t e c t u r e h a d r e t u r n e d t o n o r m a l .
Alveolographic Studies with RFC
X - r a y v i s u a l i z a t i o n o f t h e a l v e o l a r compartment o f t h e l u n g s
c a n n o t be o b t a i n e d w i t h c u r r e n t l y a v a i l a b l e m a t e r i a l . Theoreti-
c a l l y , i t s h o u l d be h i g h l y d e s i r a b l e t o o b t a i n a l v e o l a r s t u d i e s
in l i v i n g subjects without r e s o r t i n g to lung b i o p s i e s . Such i n -
f o r m a t i o n s h o u l d be u s e f u l i n d i f f e r e n t i a t i n g v a r i o u s t y p e s o f
lung diseases. In other areas of medicine precision i n diagnosis
has been e s s e n t i a l i n p r e s c r i b i n
understanding e t i o l o g i
disease. A t f i r s t , we w e r e d i s a p p o i n t e d t o o b s e r v e t h a t RFC
f i l l e d t h e a l v e o l a r compartment and d i d n o t p r o v i d e r a d i o p a c i f i c a -
t i o n o f t h e t r a c h e a and b r o n c h i . I t t h e n became n e c e s s a r y t o d e -
velop unique emulsions f o r tracheobronchography. In t i m e , the
a l v e o l o g r a p h i c s t u d i e s may p r o v e more u s e f u l .
A l v e o l o g r a p h y c a n be a c c o m p l i s h e d w i t h d o s e s t h a t a r e a f r a c -
t i o n o f t h e LD50 d o s e . We h a v e b e e n u n s u c c e s s f u l i n o u r a t t e m p t s
t o n e b u l i z e n e a t RFC i n t o t h e l u n g s . A catheter i s inserted into
t h e t r a c h e a w i t h t o p i c a l a n e s t h e s i a o f t h e l a r y n x and t r a c h e a .
The n e a t RFC d o e s n o t i n d u c e c o u g h i n g o r o t h e r phenomena o f i r r i -
tation. The t o p i c a l a n e s t h e s i a d o e s i n d u c e b r o n c h o s p a s m and hy-
p o x e m i a , b u t t o p i c a l a n e s t h e s i a has b e e n n e e d e d f o r c a t h e t e r
p l a c e m e n t i n s u b j e c t s s t u d i e d t h u s f a r . The s u b j e c t s h a v e com-
p l a i n e d o f p h a r y n g i t i s and l a r y n g i t i s f r o m t h e t o p i c a l a n e s t h e s i a
and t h e c a t h e t e r . We h a v e a l s o o b s e r v e d m i l d e l e v a t i o n s i n o r a l
t e m p e r a t u r e s , w h i t e b l o o d c e l l c o u n t , and o c c a s i o n a l m i l d e l e v a -
t i o n s i n t h e s e r u m enzymes SGOT o r LDH. These e f f e c t s have d i s -
a p p e a r e d i n t w e n t y - f o u r t o f o r t y - e i g h t h o u r s , and i t i s d i f f i c u l t
t o d e t e r m i n e what p a r t o f t h e s e a d v e r s e e f f e c t s a r e due t o t h e
c a t h e t e r p l a c e m e n t , t h e t o p i c a l a n e s t h e s i a and t h e f l u o r o c a r b o n .
I n p a t i e n t s w i t h b u l l o u s emphysema, t h e a l v e o l o g r a m s h a v e
a c c u r a t e l y o u t l i n e d the areas o f normal lung. The emphysematous
b u l l a e h a v e n o t f i l l e d w i t h RFC ( F i g u r e 6 ) . Areas of c e n t r i l o b -
u l a r emphysema h a v e a l s o showed a v o i d on x - r a y s ( F i g u r e 7 ) .
A r e a s o f n o r m a l a l v e o l a r s t r u c t u r e were c l e a r l y d e m o n s t r a t e d i n
p a t i e n t s and h e a l t h y v o l u n t e e r s . Those areas o f l u n g compressed
by emphysematous b u l l a e f i l l e d s l o w l y on a l v e o l o g r a p h y . Similar-
l y , t h e a r e a s o f p o o r l y v e n t i l a t e d o r c o m p r e s s e d l u n g c l e a r e d RFC
more s l o w l y t h a n d i d a r e a s o f w e l l v e n t i l a t e d l u n g . Radiographic
e v i d e n c e f o r t h e RFC h a d c l e a r e d by f o r t y - e i g h t h o u r s .

Figure 6. Alveologram with neat RFC
hemithorax. Note that the fluorocarbon

does not fill the emphysematous bullae.
Figure 7. Close up view of the lower

portions of the left hemithorax in the
same patient as seen in Figure 6. The
compressed areas of the lung can be seen
to contain radiodense material which is
RFC. The punched out areas in the
alveologram represent areas of centri-
lobuhr emphysema verified by histologi-
cal examination of the tissue.

Emulsions of RFC
D i l u t e e m u l s i o n s o f RFC (2:1 v o l u m e t o v o l u m e ) a r e e a s i l y
p r e p a r e d by m i x i n g RFC i n a 6% s o l u t i o n o f P l u r o n i c F-68 i n p h y -
siologic salt solutions. C o n c e n t r a t e d e m u l s i o n s (up t o 15:1) can
be p r e p a r e d b y g r a d u a l a d d i t i o n o f n e a t RFC. The c o n c e n t r a t e d
emulsions are u s e f u l i n bronchography. We u s e e m u l s i o n s w i t h
c o n c e n t r a t i o n s v a r y i n g f r o m 6:1 t o 10:1. Emulsions w i t h concen-
t r a t i o n s l e s s t h a n 6:1 c a u s e c o u g h i n g i n e x p e r i m e n t a l a n i m a l s
s i m i l a r t o t h a t produced by p h y s i o l o g i c s a l t s o l u t i o n . Emulsions
o f c o n c e n t r a t i o n s 6:1 o r g r e a t e r b e h a v e more l i k e n e a t RFC and d o
not produce coughing.
The e m u l s i o n s h a v e a w i d e r a n g e o f p a r t i c l e s i z e i n c l u d i n g
some p a r t i c l e s a s l a r g e a s 1 mm. T h e r e i s some c h a n g e i n p a r t i -
c l e s i z e d i s t r i b u t i o n as w e l l as v i s c o s i t y w i t h t i m e , b u t t h e
s h e l f l i f e o f the emulsions i s v e r y s a t i s f a c t o r y f o r our purpo
ses. Only s l i g h t creamin
r e - e m u l s i f i e d by s h a k i n
5
s i o n s i s n o n - N e w t o n i a n and t h i x o t r o p i c .
Bronchography w i t h RFC Emulsions
B r o n c h o g r a p h y was p e r f o r m e d w i t h t o p i c a l a n e s t h e s i a o f t h e
p h a r y n x and t r a c h e a and i n s e r t i o n o f a t r a c h e a l c a t h e t e r . The
t r a c h e a l c a t h e t e r was p o s i t i o n e d i n t o t h e b r o n c h u s o f c h o i c e , and
10 t o 20 m l . o f 6:1 o r 10:1 e m u l s i o n was i n j e c t e d i n t o t h e m a i n
bronchus w i t h the p a t i e n t apneic i n e x p i r a t i o n . The p a t i e n t was
then asked t o t a k e a deep b r e a t h . Additional injections into l o -
b a r b r o n c h i were p e r f o r m e d as i n d i c a t e d . A p p r o p r i a t e x - r a y s were
t a k e n t o o b t a i n c o m p l e t e i n f o r m a t i o n on t h e s t r u c t u r e o f t h e t r a -
cheobronchial tree. B i l a t e r a l bronchograms are u s u a l l y d e s i r e d
and h a v e b e e n p e r f o r m e d s i m u l t a n e o u s l y w i t h o u t i n c i d e n t . The
x - r a y s o f t h e c h e s t w e r e o b t a i n e d w i t h k i l o v o l t a g e o f 70 t o 75,
somewhat l e s s t h a n t h a t o b t a i n e d w i t h c o n v e n t i o n a l x - r a y s o f t h e
chest.
S a t i s f a c t o r y bronchograms have been o b t a i n e d i n a l l h e a l t h y
v o l u n t e e r s and p a t i e n t s s t u d i e d . E v e n p a t i e n t s w i t h s e v e r e and
f a r a d v a n c e d p u l m o n a r y d i s e a s e h a v e t o l e r a t e d t h e RFC e m u l s i o n s .
Three p a t i e n t s had e x p e r i e n c e d s e v e r e r e s p i r a t o r y d i s t r e s s p r e -
v i o u s l y when t h e y r e c e i v e d b r o n c h o g r a m s w i t h c u r r e n t l y a v a i l a b l e
o r g a n i c i o d i d e b r o n c h o g r a p h i c media. These t h r e e p a t i e n t s t o l e r -
a t e d t h e RFC b r o n c h o g r a m s w e l l .
D e c r e a s e s i n a r t e r i a l p02 were o b s e r v e d i n s u b j e c t s f o l l o w -
i n g t o p i c a l a n e s t h e s i a and h y p o x e m i a and b r o n c h o s p a s m h a v e b e e n
r e p o r t e d by o t h e r s f o l l o w i n g t o p i c a l a n e s t h e s i a o f t h e t r a c h e o -
6 7
bronchial t r e e . ' A f t e r t h e RFC was i n j e c t e d , t h e a r t e r i a l p 0 2
e i t h e r d e c r e a s e d o r i n c r e a s e d o r s t a y e d t h e same. A r t e r i a l hyp-
o x e m i a was c o r r e c t e d by t h e u s e o f s u p p l e m e n t a l n a s a l o x y g e n
breathing.

Biological Disposition
The b i o l o g i c a l d i s p o s i t i o n o f RFC h a s b e e n e x a m i n e d i n e x -
p e r i m e n t a l a n i m a l s r e c e i v i n g RFC b y t h e s e v e r a l r o u t e s s t u d i e d .
The a n i m a l s were s a c r i f i c e d a t d i f f e r e n t i n t e r v a l s a f t e r r e c e i v -
i n g RFC and t h e t i s s u e s were e x t r a c t e d i n h e x a n e . The e x t r a c t s
were a n a l y z e d f o r f l u o r o c a r b o n u s i n g g a s l i q u i d c h r o m a t o g r a p h y .
The b i o l o g i c d i s p o s i t i o n o f RFC g i v e n b y t h e g a s t r o i n t e s t i n a l
r o u t e i s shown i n T a b l e I I . O n l y t h o s e t i s s u e s w i t h t h e h i g h e s t
f l u o r o c a r b o n c o n c e n t r a t i o n s were l i s t e d a l t h o u g h o t h e r t i s s u e s
were a l s o a n a l y z e d . T r a c e amounts o f RFC c o u l d b e s e e n i n x - r a y s
o f t h e a n i m a l s t w e n t y - f o u r h o u r s a f t e r a d m i n i s t r a t i o n , b u t chem-
i c a l a n a l y s i s revealed s i g n i f i c a n t q u a n t i t i e s i n the g a s t r o i n t e s -
tinal tract. T r i v i a l amounts o f RFC were a b s o r b e d f r o m t h e GI
t r a c t as e v i d e n c e d b y t h e s m a l l q u a n t i t i e s o f RFC p r e s e n t i n
o t h e r t i s s u e s . A p r o g r e s s i v e d e c l i n e i n t i s s u e RFC c o n c e n t r a t i o n
o c c u r r e d , and a t t h r e e
r e s i d u a l RFC.
T a b l e s I I I and I V p r e s e n t t h e b i o l o g i c d i s p o s i t i o n d a t a on
RFC a d m i n i s t e r e d i n t o t h e l u n g s f o r a l v e o l o g r a p h y and f o r b r o n -
c h o g r a p h y , r e s p e c t i v e l y . Twenty-one d a y s a f t e r a d m i n i s t r a t i o n o f
a l a r g e d o s e o f 4 m l . A g . o f n e a t RFC, t h e r e w e r e o n l y t r a c e
amounts f o u n d i n t h e t i s s u e s . When RFC e m u l s i o n was g i v e n i n a
d o s a g e o f 2 m l . / k g . , e l i m i n a t i o n was v i r t u a l l y c o m p l e t e b e f o r e
t w e l v e weeks a f t e r a d m i n i s t r a t i o n .
Lymphography
R a d i o p a c i f i c a t i o n o f l y m p h a t i c s t r u c t u r e s was o b t a i n e d b y
i n f u s i o n o f RFC i n t o l y m p h a t i c c h a n n e l s o r i n t o lymph n o d e s .
N e a t RFC was p r e f e r r e d f o r l y m p h o g r a p h y . I n f u s i o n of emulsions
r e s u l t e d i n r a d i o p a c i f i c a t i o n o f the lymphatics t o the f i r s t
lymph node. The e m u l s i o n was r a p i d l y p h a g o c y t o s e d b y t h e c e l l s
i n t h e lymph n o d e , t h e lymph node became e n g o r g e d and t e n s e , and
t h e f l o w o u t o f t h e lymph node was b l o c k e d s o t h a t d i s t a l lympha-
t i c s and lymph n o d e s c o u l d n o t b e v i s u a l i z e d . When n e a t RFC was
i n f u s e d , t h e u p t a k e by t h e lymph n o d e s was l e s s v o r a c i o u s . The
lymph n o d e s became r a d i o p a q u e b u t n o t t e n s e , and t h e d i s t a l lymph
n o d e s and c h a n n e l s were v i s u a l i z e d ( F i g u r e 8 ) . When e x c e s s i v e
q u a n t i t i e s were i n j e c t e d , t h e n e a t RFC s p i l l e d o v e r i n t o t h e p u l -
monary v a s c u l a t u r e .
The L D Q d o s e o f RFC i n c a t s was b e t w e e n 1 . 0 and 1 . 2 5 m l . / k g «
5
The e f f i c a c i o u s d o s e was 0 . 2 m l . / k g . , w h i c h was t h a t q u a n t i t y r e -

q u i r e d t o f i l l t h e l y m p h a t i c s b e f o r e s p i l l over i n t o t h e venous
system. The r e s i d e n c e t i m e o f RFC i n lymph n o d e s was g r e a t e r
than t h r e e years i n dogs. T h i s l o n g r e s i d e n c e t i m e may be an a d -
v a n t a g e , p a r t i c u l a r l y when one i s s t u d y i n g t h e i n v o l v e m e n t o f
lymph n o d e s b y m a l i g n a n t t u m o r s . I o d i z e d o i l , the agent c u r r e n t -
l y used f o r lymphography a l s o has a l o n g r e s i d e n c e t i m e , b u t t h i s
a g e n t i n c i t e d i n f l a m m a t i o n and f i b r o s i s i n lymph n o d e s .

Table II
Biologic Disposition after Gastroenterography
1 Day 3 Days
5
Large Intestines 1 X ΙΟ" 2
7 X ΙΟ"
Stomach & S m a l l Intestines 1.4 X Ι Ο " 3

5 X ίο" 6
Liver 1.6 X Ι Ο " 4

6 X ΙΟ" 5
Lungs 4 X ΙΟ" 5
6 X ίο" 5
Lymph Nodes 5 X ΙΟ" 5

2 X ίο" 5
Fat
T i s s u e l e v e l s i n m l . 8 1 7 ] p e r gram o f t i s s u e a t d i
C F B R
time i n t e r v a l s a f t e r g a s t r o e n t e r o g r a p h y w i t h 16 m l . / k g . o f
C F B
8 17 R* E x p e r i m e n t a l a n i m a l was t h e a d u l t r a t .
Figure 8. Lymphangiography in a dog with

Etniodal injected into the right leg and neat
R
radiopaque fluorocarbon injected into the left

leg. The iodinated oil, Ethiodal , is more R
radiodense than RFC but the difference in

radiodensity does not detract from diagnostic
accuracy.

9. LONG E T AL. Brominated Fluorocarbon Compounds 183
Table I I I
Biologie Disposition after Alveolography
3 Days 1 Week 3 Weeks
4 5 5
Lungs 8 x 1CT 8 χ ΙΟ" 5 χ ΙΟ"
Lymph
Nodes 7 χ ΙΟ" 5
4 χ 1(Γ 4
9 χ ΙΟ"* 6
5 5
Fat 7 χ ΙΟ" 4 χ 10-5 3 χ ΙΟ"
T i s s u e l e v e l s i n m l . C Q F I 7 B p e r gram o f t i s s u e a t d i f f e r e n t
r
time i n t e r v a l s a f t e r a l v e o l o g r a p h y w i t h 4 ml./kg. Experimental

a n i m a l was t h e a d u l t r a t
Table IV
Biologic Disposition a f t e r Bronchography
1 Day 3 Days 1 Week 4 Weeks 12 Weeks
4 4 4 5 6
Lungs 6 χ 10" 2 χ 10~ 1 χ 10~ 1 χ 10" < 1 χ 10"
Lymph
5 4 5 6
Nodes 2.4 χ 1 0 " 3.8 χ 1 0 " 3 χ 10" 4 χ 10~
Fat 7 χ 10" 5
9 χ 10-5 χ x IQ-5 4 X IQ-5 <2 χ 10* 6
T i s s u e l e v e l s i n m l . C Q F ^ B R p e r gram o f t i s s u e a t d i f f e r e n t
t i m e i n t e r v a l s a f t e r b r o n c h o g r a p h y w i t h 2 m l . / k g . o f a 10:1 e m u l
sion. E x p e r i m e n t a l a n i m a l was t h e d o g .

BIOCHEMISTRY INVOLVING CARBON-FLUORINE BONDS
Figure 9. Ventriculomyelogram in a rabbit with neat RFC. The ventri-

culomyelogram was well tolerated and resulted in excellent visualization of
central nervous system structures.
Figure 10. Hepatography and splenography in a rat given 15 ml/kg of

a 67% emulsion of radiopaque fluorocarbon intravenously. The particle
size of the emulsion was 1-1.5 microns. The x-ray on the left was made
prior to injection, and the x-ray on the right was made four days after
injection of the emulsion. The structures of the thin spleen are more
accurately outlined than those of the more dense liver.

Ventriculomyelography
Radiopaque p e r f l u o r o c a r b o n has been used t o enhance r a d i o l o g -

i c a l v i s u a l i z a t i o n o f c e n t r a l nervous system s t r u c t u r e s i n e x p e r i -
8 , 9
mental a n i m a l s . These s t u d i e s have been performed w i t h C Q F ^ B R ,
C7F15BR and C ^ F ^ B R . N e a t RFC h a s b e e n u s e d s i n c e e m u l s i o n s w e r e
too v i s c o u s f o r t h i s purpose. The RFC compounds p r o v e d t o be e f -
f i c a c i o u s f o r o u t l i n i n g appropriate s t r u c t u r e s of the c e n t r a l ner-
v o u s s y s t e m ( F i g u r e 9 ) . The r e s i d e n c e t i m e o f C Q F B 1 7i n the sub-
r
a r a c h n o i d s p a c e was g r e a t e r t h a n t h r e e y e a r s . T h e r e was no a c u t e
i n f l a m m a t o r y r e a c t i o n e l i c i t e d b y CgF-^BR a s e v i d e n c e d by t h e l a c k
o f s i g n i f i c a n t c e l l u l a r and c h e m i c a l c h a n g e s i n t h e c e r e b r o s p i n a l
fluid. No d e m o n s t r a b l e n e u r o l o g i c a l i n j u r y was p r o d u c e d i n e x p e r -
imental animals, e i t h e r i n the acute or c h r o n i c phases. A mild
a r a c h n o i d i t i s was s e e n i n c h r o n i c e x p e r i m e n t s and was m a n i f e s t e d
by t h e a c c u m u l a t i o n o f p h a g o c y t i c m o n o c y t e s i n t h e a r e a s o f f l u o -
rocarbon a c c u m u l a t i o n .
w i l l be o f c l i n i c a l s i g n i f i c a n c
s u p e r i o r t o PantopaqueÇ the c u r r e n t l y a v a i l a b l e c o n t r a s t agent f o r
myelography.
P e r f l u o r o h e x y l b r o m i d e was s t u d i e d b e c a u s e o f t h e r a p i d v a p o r -
i z a t i o n o f t h i s compound a t b o d y t e m p e r a t u r e . The v a p o r p r e s s u r e
o f C ^ F Q ^ B R was a p p r o x i m a t e l y 9 0 T o r r compared t o 1 4 T o r r f o r
C8F17BR and 5 5 T o r r f o r C 7 F B . 1 5 R Previous s t u d i e s demonstrated
t h a t C5F13BR v a p o r i z e d r a p i d l y and d i s a p p e a r e d r a d i o l o g i c a l l y
w i t h i n weeks when i n j e c t e d i n t o t h e p e r i t o n e a l c a v i t y o r s u b c u t a -
neous s p a c e . When i n j e c t e d i n t o t h e s u b a r a c h n o i d s p a c e , C ^ F ^ B R
f o r m e d v a p o r p o c k e t s , and t h e g a s p h a s e was n o t r e a b s o r b e d r a p i d l y
e n o u g h , s o t h a t n e u r o l o g i c a l i n j u r y and e v e n m o r t a l i t y o c c u r r e d i n
animals. 9
The r a t e o f d i s a p p e a r a n c e o f C 7 F 5 B 1 R f r o m t h e b o d y and
the s u b a r a c h n o i d space i s under i n v e s t i g a t i o n i n our l a b o r a t o r y .
Hepatography and Splenography
I n t r a v e n o u s i n f u s i o n s o f e m u l s i o n s o f RFC r e s u l t i n r a d i o p a -
c i f i c a t i o n o f t h e s p l e e n o r t h e s p l e e n and l i v e r . When we t e s t e d
t h e b r o n c h o g r a p h i c e m u l s i o n s f o r i n t r a v e n o u s t o x i c i t y , we o b s e r v e d
r a d i o p a c i f i c a t i o n of the spleen. These emulsions have a l a r g e
particle size. The r a d i o p a c i f i c a t i o n was a p p a r e n t i n t h i r t y m i n -
u t e s and i n c r e a s e d b y f o u r h o u r s . The r a d i o p a c i f i c a t i o n d i m i n i s h -
ed g r a d u a l l y and d i s a p p e a r e d b y a b o u t f o u r weeks. When s m a l l
p a r t i c l e s i z e e m u l s i o n s o f 1 t o 1.5 m i c r o n were i n j e c t e d i n t r a v e n -
o u s l y , r a d i o p a c i f i c a t i o n o f t h e s p l e e n and l i v e r o c c u r r e d ( F i g u r e
10). E m u l s i o n s o f s m a l l e r p a r t i c l e s i z e have been t e s t e d w i t h
equivocable r e s u l t s . T h e s e s t u d i e s o f h e p a t o g r a p h y and s p l e n o g r a -
phy a r e i n an e a r l y s t a g e o f d e v e l o p m e n t s o t h a t a c o n s i d e r a b l e
amount o f r e s e a r c h i s r e q u i r e d t o d e f i n e t h e optimum p a r t i c l e s i z e
and d o s a g e . The RFC i s p h a g o c y t o s e d b y t h e r e t i c u l o e n d o t h e l i a l
c e l l s o f t h e l i v e r and s p l e e n ( F i g u r e 1 1 ) . As s e e n i n F i g u r e 1 1 ,
the r a d i o d e n s i t y achieved i s s u f f i c i e n t f o r d i a g n o s t i c purposes.

Figure 11. Photomicrograph of the

spleen of a rabbit one week after receiv-
ing intravenous injection of large particle
size emuhion of RFC. The spleen con-
tained foci of mononuclear cells with
foamy cytoplasm due to the presence
intracellular fluorocarbon.
Other Areas of Application
B o t h n e a t RFC and e m u l s i o n s o f RFC h a v e b e e n t e s t e d f o r a r -

t h r o g r a p h y a n d h a v e b e e n f o u n d t o be i n a d e q u a t e . RFC h a s a l s o
b e e n e v a l u a t e d i n r e t r o g r a d e u r o g r a p h y , c h o l e c y s t o g r a p h y , and
pancreatography i n experimental animals. The r e s u l t s o b t a i n e d
were comparable t o t h o s e o b t a i n e d w i t h c u r r e n t l y a v a i l a b l e c o n -
t r a s t agents. D e t a i l e d e f f i c a c y and t o x i c i t y s t u d i e s h a v e n o t
been performed t o date i n t h e s e areas o f l i m i t e d frequency o f
use. RFC may b e a f e a s i b l e a l t e r n a t i v e f o r u s e i n p a t i e n t s w i t h
h y p e r s e n s i t i v i t y t o organic iodide contrast agents. I t may a l s o
p r o v e t o be s a f e r i n v i s u a l i z a t i o n o f i n f l a m e d o r f r a g i l e d u c t a l
s y s t e m s where e x t r a v a s a t i o n o f c o n t r a s t m e d i a s u c h a s o r g a n i c
i o d i d e compounds w o u l d e x a c e r b a t e t h e i n f l a m m a t o r y p r o c e s s i n an
organ such as t h e p a n c r e a s .
Summary
M o n o b r o m i n a t e d p e r f l u o r o a k y l compounds h a v e b e e n t e s t e d a s
x-ray c o n t r a s t agents. T h e compound p e r f l u o r o c t y l b r o m i d e i s t h e
agent o f c h o i c e a t t h i s time because o f i t s b i o l o g i c a l i n e r t n e s s ,
low s u r f a c e t e n s i o n , e a s e o f e m u l s i f i c a t i o n and f a v o r a b l e r a t e
o f e l i m i n a t i o n from t h e body. Gastroenterography, bronchography,
and a l v e o l o g r a p h y h a v e b e e n p e r f o r m e d w i t h r a d i o p a q u e f l u o r o c a r -
bon i n e x p e r i m e n t a l a n i m a l s and humans. O t h e r a r e a s o f a p p l i c a -
t i o n are being investigated i n experimental animals.

Acknowledgements: The a u t h o r s w i s h t o e x p r e s s t h e i r g r a t i t u d e t o
M i s s P a t L e e , M r s , F r a n c e s M u l t e r , and M i s s Margo N i e l s o n f o r e x
c e l l e n c e i n l a b o r a t o r y e x p e r i m e n t s , a n d t o D o c t o r s Hugh G. B r y c e ,
Raymond J . S e f f l , J . Dana McGowen, a n d D o n a l d L a Z e r t e o f t h e 3M
Company.
Supported by g r a n t s from t h e C h e m i c a l D i v i s i o n , M i n n e s o t a Mining

and M a n u f a c t u r i n g Company, S t . P a u l , M i n n e s o t a a n d t h e U.S.P.H.S.
Grant GM20998.
Literature Cited
1. Clark, L.C., Jr., an
56: "Survival of mammals breathing organic liquids equilibra
ted with oxygen at atmospheric pressure."
2. Clark, L.C., Jr., Kaplan, S., Becattini, F. J. Thoracic Car
diovascular Surg. (1970) 60, p. 757-773: "The physiology of
synthetic blood."
3. Geyer, R.P. Fed. Proc. (1975) 34, p. 1499-1505: "'Bloodless'
rats through the use of artificial blood substitutes."
4. Long, D.M., Liu, M., Szanto, P.S., Alrenga, D.P., Patel, M.M.,
Rios, M.V., and Nyhus, L.M. Radiology (1972) 105, p. 323-332:
"Efficacy and toxicity studies with radiopaque perfluorocar
bon."
5. Arambulo, A.S., Liu, Μ., Rosen, A.L., Dobben, G., and Long,
D.M. Drug Devel. Commun. (1975) 1, p. 73-87: "Perfluoroctyl
bromide emulsions as radiopaque media."
6. Salisbury, B.G., Metzgir, L.F., Altrose, M.D., Stanley, N.N.,
and Cherniak, N.S. Am. Rev. Respir. Dis. (1974) 109, p. 691:
"Effect of fiberoptic bronchoscopy on respiratory performance
in patients with chronic obstructive pulmonary disease."
7. Miller, W.C. and Awe, R. Am. Rev. Respir. Dis. (1975) 111,
p. 739-741: "Effect of nebulized lidocaine on reactive air
ways . "
8. Dobben, G.D., Long, D.M., Szanto, P.S., Mategrano, V.C., and
Liu, M. Neuroradiology (1973) (6, p. 17-19: "Experimental
studies with radiopaque fluorocarbon in the subarachnoid
space."
9. Brahme, F., Sovak, Μ., Powel, Η., and Long, D.M. Acta Radiol.
Scan. In Press: "Perfluorocarbon bromides as contrast agents
in radiography of the central nervous system."

Discussion
Q. How c a n t h e b r o m o p e r f l u o r o c a r b o n s compete w i t h b a r i u m s u l f a t e
i n s t u d i e s o f t h e GI t r a c t ?
Dr. Long
A. P e r f l u o r o c t y l b r o m i d e and t h e o t h e r s c a n n o t compete w i t h
BaSO/4 on a c o s t b a s i s . However, f o r s e l e c t e d c a s e s , b a r i u m
c a n be d a n g e r o u s . F o r a b o u t 1% o f t h e GI s t u d i e s d o n e , w a t e r
s o l u b l e c o n t r a s t agents are used at the present time. These
c o n t r a s t a g e n t s a r e n o t w e l l t o l e r a t e d , c a u s i n g d i a r r h e a and
i r r i t a t i o n , and d o n ' t g i v e v e r y g o o d x - r a y c o n t r a s t . The
fluorocarbon i s vastly superior. The f l u o r o c a r b o n i s s u p e r -
i o r t o barium i n the s m a l l i n t e s t i n e s a l s o . There are other
ways w h e r e t h e b a r i u m i s n ' t as a d e q u a t e as t h e fluorocarbon.
F o r r e a s o n s t h a t a r e n o t c l e a r , we g e t g o o d c o a t i n g o f t h e
e s o p h a g u s and o t h e part f th stomach i dog with fluoro
carbons , but not i
a b l e t o g e t good c o a t i n
the stomach, so t h a t ' s a l i m i t a t i o n . R a d i o l o g i s t s and s u r -
geons a r e v e r y e n t h u s i a s t i c about t h e s e f l u o r o c a r b o n s i n the
GI t r a c t , a l t h o u g h t h e r e w i l l be an i n c r e a s e d c o s t .
Q. Would y o u comment on t h e C7 compound?

A. We h a v e t r i e d t h e C 7 compound and i t i s v e r y i n t e r e s t i n g . I t
i s n o t e l i m i n a t e d r a p i d l y enough f r o m t h e b o d y t o be u s e f u l
i n myelography. F o r a l v e o l o g r a p h y , y o u c a n u s e t h e Cg, C7 o r
C3 compounds. C5 may be b e t t e r t h a n t h e o t h e r s . For c l i n i c a l
s t u d i e s , we h a v e u s e d t h e Ce compound.
Q. What a b o u t i r r i t a t i o n ?
A. T h e r e i s l i t t l e o r no i r r i t a t i o n from t h i s material.
Q. You m e n t i o n e d i n t h e c a s e o f m y e l o g r a p h y how t h e f l u o r o c a r -
bons are r e t a i n e d f o r a l o n g p e r i o d o f t i m e . Have y o u t r i e d
t o r e l a t e t h a t t o Pantopaque r e t e n t i o n ?
A. Pantopaque i s r e t a i n e d permanently. You d o n ' t remove a l l t h a t
agent. The f l u o r o c a r b o n i s more a c c e p t a b l e t h a n P a n t o p a q u e
s i n c e i t does not cause a r a c h n o i d i t i s l i k e Pantopaque does.
There are water s o l u b l e c o n t r a s t agents t h a t are being used
now i n m y e l o g r a p h y , and we a r e n o t s u r e how t h e s e a r e g o i n g
t o work o u t . I n some o f t h e e a r l i e r s t u d i e s , t h e y d i d n o t ap-
p e a r t o be t o x i c when f i r s t i n j e c t e d . E v e n t h o u g h t h e y com-
p l e t e l y d i s a p p e a r e d , an a r a c h n o i d i t i s o r c h r o n i c i n f l a m m a t o r y
p r o c e s s was i n c i t e d b y t h e i n i t i a l i n j e c t i o n o f t h e w a t e r s o -
l u b l e c o n t r a s t agents. The n o n - i o n i c w a t e r s o l u b l e c o n t r a s t
a g e n t s a r e now b e i n g e v a l u a t e d c l i n i c a l l y t o s e e w h e t h e r t h e y
w i l l be more a c c e p t a b l e , and t h e r e s u l t s h a v e b e e n e n c o u r a g -
ing. The f l u o r o c a r b o n i s v a s t l y s u p e r i o r t o P a n t o p a q u e , t h e
m a t e r i a l c u r r e n t l y used i n almost a l l myelography.

9. LONG E T AL. Brominated Fluorocarbon Compounds 189
Q. I am s u r p r i s e d t h a t y o u do n o t f e e l more f a v o r a b l e a b o u t t h e
u s e o f f l u o r o c a ibons i n t h e s p e c i f i c a p p l i c a t i o n o f m y e l o g r a -
phy.
A. I was n o t c o m p a r i n g f l u o r o c a r b o n s w i t h P a n t o p a q u e , b u t w i t h
the n o n - i o n i c water s o l u b l e c o n t r a s t agent under study. I
t h i n k t h i s c o n t r a s t agent i s p r e f e r r e d , b u t i t i s n o t appro-
v e d b y t h e F.D.A. I t i s b e i n g used i n o t h e r c o u n t r i e s , and
i t i s u n d e r c l i n i c a l i n v e s t i g a t i o n i n t h e U.S. I t looks l i k e
t h e n o n - i o n i c c o n t r a s t a g e n t i s g o i n g t o be a v e r y g o o d a g e n t
b u t f l u o r o c a r b o n s w i l l b e a b l e t o compete w i t h i t u n d e r some
c i r c u m s t a n c e s , such as i n p a t i e n t s w i t h h y p e r s e n s i t i v i t y t o
i o d i n a t e d compounds.

10
Preparation and Physiological Evaluation of Some New
Fluorinated Volatile Anesthetics
DONALD D. DENSON, EDWARD T. UYENO, ROBERT L. SIMON, JR., and

HOWARD M. PETERS
Stanford Research Institute, 333 Ravenswood Ave., Menlo Park, Calif. 94025
Although several adequate fluorinated anesthetics are in

clinical use today, all have disadvantages and possible hazards.
We are synthesizing and evaluating fluorinated ethers for use as
volatile anesthetics. Since these compounds are "inert" gases,
they exert their biologic effects without undergoing any chemical
transformation during administration, residence in the body, and
elimination from the body. It is hoped they will provide the
advantages of currently available fluorinated anesthetics but
preclude their disadvantages (1).
In discussing volatile anesthetics, it is important to un-
derstand the difference between analgesia, narcosis, and anes-
thesia. Analgesia (Stage I) is the loss of pain or numbing of
sensory nerves without loss of consciousness. Narcosis (Stage
II) is a reversible state of analgesia accompanied by stupor or
unconsciousness. Surgical anesthesia (Stage III) is the revers-
ible loss of all modalities of sensation and loss of conscious-
ness. The planes of surgical anesthesia in humans (2) are:
Plane 1: Swallowing reflex lost; respiration regular;
muscle relaxation minimal
Plane 2: Muscle relaxation increased
Plane 3: Muscle relaxation further increased and
suitable for intraabdominal surgery
Plane 4: Skeletal muscle relaxation complete;
possible cyanosis; blood and pulse pressure
falls; pulse rate increases
The level below Plane 4 of surgical anesthesia is respiratory
arrest (Stage IV). While planes 1-3 of surgical anesthesia are
the most important, some consideration must be given to the
190

10. DENSON E T AL. Fluorinated Volatile Anesthetics 191
dangerous a s p e c t s o f Stage I I I , Plane 4, and S t a g e IV. These

l e v e l s must be c a r e f u l l y avoided i f the patient i s to survive
without adverse affects.
Thus the major o b j e c t i v e s of successful a n e s t h e s i a (_2) a r e
to:
(1) Alleviate p a i n by b l o c k i n g s e n s o r y or afferent nerves.

(2) Block mentation to alleviate the mental a n g u i s h and
anxiety resulting from the fear of pain.
(3) Relax t h e m u s c l e s by b l o c k i n g e f f e r e n t o r motor nerves.
(4) Preclude adverse effects of surgery or anesthesia.
Examination of the f l u o r i n a t e d a n e s t h e t i c s i n use today

suggests that fluorinated ethers (as a c l a s s ) offer more advan-
tages than fluorinated hydrocarbons While these ethers are
more u n p r e d i c t a b l e i
three important advantages
b e t w e e n a n a l g e s i a and a n e s t h e s i a , (2) t h e y p r o v i d e more effec-
tive muscle r e l a x a t i o n , and (3) t h e y demonstrate less sensitiza-
tion of myocardial tissue to epinephrine.
H a z a r d s o f F l u o r i n a t e d A n e s t h e t i c s Now i n Use
Halogenated compounds have been u s e d as a n e s t h e t i c agents

since 1847 w i t h t h e d i s c o v e r y o f c h l o r o f o r m . During t h e 1940s,
Robbins r e p o r t e d the f i r s t comprehensive study of fluorocarbons
as potential anesthetics(3).
While any number o f f l u o r i n a t e d compounds have potential
anesthetic properties, wide s c a l e clinical use has been limited
to about eight compounds. T h e f o u r compounds u s e d most o f t e n
are :
Halothane CF CHBrCl
3
Methoxyflurane CH 0CF CHC1

3 2 2
Fluroxene CF CH OCH=CH
3 2 2
Enflurane CHF 0CF CHC1F

2 2
All these compounds a r e a s s o c i a t e d w i t h a c u t e and c h r o n i c toxic-

ities that must be c a r e f u l l y considered (1, 4 ) .
Halothane i s oxidatively metabolized to t r i f l u o r o a c e t i c
acid (5).
NADPH/0 2
CF CHBrCl
3
Hepatocellular damage
pathways

This transformation i s probably harmless with production o f few,

if any t o x i c intermediates. I t i s q u i t e p o s s i b l e however t h a t
aberrant metabolic pathways produce r e a c t i v e i n t e r m e d i a t e s capa-
ble of i n f l i c t i n g h e p a t o c e l l u l a r damage ( 1 ) . Halothane i s
implicated i n producing an u n p r e d i c t a b l e postanesthetic hepatitis
(1), a n d i n some c a s e s s e n s i t i z e s myocardial tissue to epineph-
rine (6) .
Methoxyflurane produces f r e e f l u o r i d e i o n on metabolism ( 5 ) .
NADPH
CH 0CF CHC1
3 2 2
CH OCF CH OH + C I "
3 2 2
\
Urinary
CH 0 + H0CF CC1 H
2 2 2
H0CH CC1 H2 2 + 2F'
In f a c t s i n c e 60-80% o f a l l absorbed m e t h o x y f l u r a n e i s metabo-

lized, relatively high serum f l u o r i d e levels c a n be expected.
These l e v e l s a r e o f t e n h i g h enough t o cause r e n a l t u b u l a r cell
damage, g i v i n g r i s e t o the high output renal failure syndrome ( 4 ) .
This nephrotoxicity i s further complicated by t h e o b s e r v a t i o n
that p h é n o b a r b i t a l may sensitize a t h r e e f o l d o r greater extent of
methoxyflurane metabolism t o f l u o r i d e ion (4).
Fluroxene i s metabolized i n animals t o t r i f l u o r o e t h a n o l and
in humans t o t r i f l u o r a c e t i c acid (5).
CF CH 0CH
3 2 = CH 2 ·* CF C0 H + Urinary
3 2 Metabolites
H i g h serum f l u o r i d e s a n d h e p t o c e l l u l a r damage a r e a s s o c i a t e d with

its u s e i n humans. In a d d i t i o n fluroxene i s extremely flammable,
and i t i s primarily for this reason that i t has been removed
from c l i n i c a l u s e .
Enflurane i s tranformed t o unknown m e t a b o l i t i e s a n d f l u o r i d e
ion i n humans ( 5 ) .
CF H0CF CHC1F
2 2 -» Unknown M e t a b o l i t e s + F"
Although these fluoride ion levels a r e r o u t i n e l y low, i n some

patients they reach 80 μ m / l , w h i c h i s d a n g e r o u s l y close t o the
normal fluoride tolerance level (2).
Chronic exposures t o t r a c e s o f these fluorinated anesthetics
can be a h a z a r d . According t o a r e c e n t ASA s t u d y (7), operating

10. DENSON ET AL. Fluorinated Volatile Anesthetics 193
room p e r s o n n e l h a v e shown a n i n c r e a s e i n s p o n t a n e o u s a b o r t i o n ,
malformation of children, cancer i n female anesthesiologists,
liver d i s e a s e , and k i d n e y disease.
Synthesis of Fluorinated Ethers
While o n l y a few f l u o r i n a t e d ethers are i n actual clinical

application, o t h e r s have shown p r o m i s e . A n example o f a c l a s s
of cyclic diether anesthetics i s 4,5-dihalo-2,2-(bis)trifluoro-
methyl-1,3-dioxolanes, 1.
Gilbert (8) p a t e n t e d the use o f the parent 2,2-(bis)trifluoro-

methyl-l,3-dioxolane 1 (X = Y = H) a s a n i n h a l a t i o n a n e s t h e t i c
in 1967. Terrell a n d Moore (9) t h e n patented t h e u s e o f 4,5-
dihalosubstituted-2,2-(bis)trifluoromethy1-1,3-dioxolanes 1 in
1973. ~
Gilbert found h i s m a t e r i a l t o be more p o t e n t than halothane
(8). While no d e f i n i t i v e A n e s t h e t i c I n d e x d a t a a r e p r o v i d e d by
Terrell e t aJL., i t a p p e a r s that the halosubstituted materials
a r e more p o t e n t than the parent compound ( 9 ) ; 1,3-dioxolane
itself has l i m i t e d anesthetic properties (10).
Our experience i n the conversion o f carbonates to difluoro-
formals l e d us t o i n v e s t i g a t e the p o s s i b i l i t i e s of preparing
equally as potent, b u t more s t a b l e , cyclic diether inhalation
anesthetics. Our approach i s based on t h e f o l l o w i n g r e a c t i o n :
Cat.
R-0-CF -0-R
Δ
2
Because o f t h e ready availability of the starting material, the

ease o f p r e p a r i n g a host o f halogenated analogs, t h e apparent

success of the 4 , 5 - d i h a l o - 2 , 2 - ( b i s ) t r i f l u o r o m e t h y l - 1 , 3 - d i o x o l a n e s
as a n e s t h e t i c s ( 9 ) , we initiated our i n v e s t i g a t i o n using ethylene
carbonate, 2.
We extended the conversion of carbonates to difluoroformals to

include the p r e p a r a t i o n of 4,5-dihalo-2,2-difluoro-1,3-dioxolanes:
where X and Y a r e h a l o g e n s
Ethylene carbonate, 2, c a n be c h l o r i n a t e d t o g i v e 4-chloro,

3, and 4 , 5 - d i c h l o r o e t h y l e n e carbonate, 4.
2 3 4
Monochloroethylene carbonate, 3, c a n be d e h y d r o h a l o g e n a t e d to
vinylidene carbonate, 5, a s shown b e l o w ( 1 1 ) .
CI
Ο + (C H ) N
2 5 3 >= 0
*Ο *0'
3 5

10. DENSON E T A L . Fluorinated Volatile Anesthetics 195
Vinylidene carbonate, 5, c a n be c o n v e r t e d t o a number o f o t h e r

halogenated derivatives a s shown i n Scheme 1.
8 7
Compounds ^7, and £ are readily o b t a i n e d by t h e u s e o f

polyhydrogenfluoride/pyridine reagent, a s d e s c r i b e d by O l a h e t a l .
(12). Straightforward hydrobromination o r b r o m i n a t i o n (11)
affords compounds 10 and 11. A similar series of derivatives
c a n be p r e p a r e d from t h e d e h y d r o h a l o g e n a t i o n of 4,5-dichloro-
ethylene c a r b o n a t e , 4^.
The fluorination reactions of these carbonates have led to
much e x c i t i n g and u s e f u l chemistry. The f l u o r i n a t i o n o f 4,5-
dichloroethylene carbonate, 4, f o r example, gives three products:

14
The product mixture ratio i s extremely dependent on the c a t a -

lyst/SF 4 ratio. An increase of > 0.5 i n the c a t a l y s t / S F 4 ratio
results i n the formation of 4-chloro-2,2,5-trifluoro-l,3-
d i o x o l a n e , J^3, as the major p r o d u c t . The c h l o r i n a t e d - u n s a t u r a t e d
1,3-dioxolane, ^4, suggests that the formation of 4-chloro-2,2,5-
t r i f l u o r o - 1 , 3 - d i o x o l a n e , ^3, results from a dehydrochlorination
reaction f o l l o w e d by a hydrogen f l u o r i d e addition reaction. The
unsaturated-1,3-dioxolane, ΊΛ i s an important finding in this
r e a c t i o n a s we will see i n the fluorination of 4-chloroethylene
carbonate, 3.
B e c a u s e we observed halogen exchange i n the fluorination of
4,5-dichloroethylene carbonate, 4, we began o u r investigation of
the monochlorinated d e r i v a t i v e 3 u s i n g an H F / S F 4 ratio of 0.45.
The totally unexpected result was the formation of 4-chloro-
2,2,5-trifluoro-1,3-dioxolane, 13, as the o n l y i s o l a t e d product.
3 13

For t h i s compound t o form, a hydrogen s u b s t i t u t i o n reaction must

be occurring. There i s no r e p o r t e d e v i d e n c e f o reither a direct
halogen exchange o r a d e h y d r o c h l o r i n a t i o n h y d r o g e n fluoride addi-
tion reaction. This result i s independent of cat/SF 4 concentra-
tion a n d o c c u r s w h e t h e r HF o r T i F 4 i s the catalyst. Heating the
precursor carbonate with either HF o r T i F 4 a t 150°C f o r 24 h r
results i n no r e a c t i o n . Heating 4-chloroethylene c a r b o n a t e , 3^,
with SF 4 i n t h e absence o f c a t a l y s t f o r 24 h r r e s u l t s i n recovery
of the s t a r t i n g carbonate, 3. Hydrogen s u b s t i t u t i o n by S F 4 has
b e e n r e p o r t e d by A p p l e q u i s t a n d S e a r l e ( 1 3 ) .
Experiments conducted a t lower temperatures resulted i n
either no r e a c t i o n o r i n t h e f o r m a t i o n o f 2 , 2 , 5 - t r i f l u o r o - 1 , 3 -
d i o x o l a n e , ^15, a s t h e m a j o r product.
Cl-,
= 0 + SF A
Cat./125°C
-0'
Hydroquinone 15
A similar result was o b t a i n e d i n attempts to fluorinate

ethylene carbonate, 2, The o b j e c t i v e o f t h i s experiment was t o
prepare 2,2-difluoro-1,3-dioxolane f o r c o m p a r i s o n w i t h 1,3-
dioxolane t o determine the contribution of the difluoroformal
moiety to biological activity. In these experiments, t h e HF/SF 4
r a t i o was a g a i n m a i n t a i n e d b e l o w 0.5. T h e f o r m a t i o n o f 2,2,4,5-

t e t r a f l u o r o - 1 , 3 - d i o x o l a n e , J^6, was i n d e p e n d e n t o f both tempera-
t u r e and c a t a l y s t concentration.
16
The hydrogen substitution reaction observed f o r the fluorination

of 4 - c h l o r o e t h y l e n e carbonate, 3, i s t h e p r e d o m i n a n t reaction i n
this case. We have been u n s u c c e s s f u l i n a l l o u r a t t e m p t s t o pre-
pare 2,2-difluoro-1,3-dioxolane. This reaction i s under further
investigation t o determine the f e a s i b i l i t y of preparing this
compound.

Structure-Activity Relationships
The major s t r u c t u r e - a c t i v i t y relationships for volatile

anesthetics a r e as f o l l o w s :
• Halogenation o f h y d r o c a r b o n s and ethers increase

in potency i n the order I > Br > Cl > F.
• Unsaturation increases potency.
• F l u o r i n e a d d i t i o n decreases potency, boiling
point and f l a m m a b i l i t y and increases stability
of adjacent halogen atoms.
• Increased potency i n a homologous s e r i e s follows
an increase i n molecular weight, boiling point,
and oil/gas coefficient.
• One o r more h y d r o g e n atoms a r e necessary for CNS
depression.
Since molecules c o n t a i n i n g only carbon, fluorine, hydrogen, and

oxygen are not u s u a l l y very potent a n e s t h e t i c s , we attempted to
introduce one chlorine into 2,2,4,5-tetrafluoro-1,3-dioxolane,
16. These experiments are summarized i n the following equations
16
Photochemical chlorination of 16 with one e q u i v a l e n t of c h l o r i n e

at room t e m p e r a t u r e results i n the formation of 4-chloro-2,2,4,5-
tetrafluoro-1,3-dioxolane, along with five products. The
maximum y i e l d of t h i s p r o d u c t , ^17, obtained to date i s 53%. Fur-
ther chlorination of the r e a c t i o n mixture results i n an increase
in the by-products, with no significant i n c r e a s e i n the desired
4-chloro-2,2,4,5-tetrafluoro-l,3-dioxolane, 17.
Photochemical chlorination of 2,2,4,5-tetrafluoro-1,3-di-
oxolane, 16, with excess chlorine results i n the formation of
4,4,5,5,-tetrachloro-2,2-difluoro-1,3-dioxolane, JL8, a s the major

product (>95%). This reaction i s under further s t u d y and w i l l

be reported i n a future publication. We do n o t b e l i e v e that
4,4,5,5-tetrachloro-2,2-difluoro-1,3-dioxolane, 18, w i l l be a
potent anesthetic since t h e r e a r e no h y d r o g e n atoms i n t h e m o l e -
cule. Generally one o r more h y d r o g e n s are required f o r CNS
depression.
The structure-activity relationships p r e s e n t e d above suggest
that a more p o t e n t member o f t h i s c l a s s would contain a bromine
atom. We have a t t e m p t e d t o maximize a n e s t h e t i c potency without
the i n c o r p o r a t i o n o f bromine, because i t i s a thermodynamically
weak bond i n metabolic environments. However i n a n e f f o r t t o
determine how w e l l our c l a s s of d i e t h e r s adheres t o the reported
structure reactivity r e l a t i o n s h i p s g i v e n above, we a t t e m p t e d t o
f l u o r i n a t e 4,5-dibromoethylene carbonate 11 The major product
from this reaction i
19.
11 19
As i n the case o f the f l u o r i n a t i o n of 4,5-dichloroethylene

carbonate, 3, we have o b s e r v e d h a l o g e n e x c h a n g e , but here this
conversion i s independent of cat/SF 4 concentration. We h a v e n o t
been a b l e t o i s o l a t e t h e u n s a t u r a t e d bromine c o n t a i n i n g 2,2-
d i f l u o r o - 1 , 3 - d i o x o l a n e , 20.
20
Experiments with T i F 4 a t 1 0 0 ° and 150°C resulted i n no

exchange i n t h e absence of SF . 4 Similarly, e x p e r i m e n t s w i t h SF,
in t h e absence of catalyst resulted i n no e x c h a n g e . Additional
experiments involving the f l u o r i n a t i o n o f b o t h 4,5-dibromo-
ethylene c a r b o n a t e , 11, and 4 - b r o m o - 5 - f l u o r o e t h y l e n e carbonate,
6 f are i n progress.

Physiological Evaluation
The potential anesthetic agents synthesized in this re-

search were t e s t e d i n m i c e by procedures similar to those
described by Burgison (14). A wide-mouth, s c r e w cap, glass jar
was flushed with o x y g e n f o r one minute, and a m e a s u r e d amount of
a synthesized compound i n a s y r i n g e was ejected from a syringe
onto the bottom of the jar. Initially the amount o f a t e s t sub-
stance t h a t would saturate the " a i r " i n the j a r was calculated
according to the methods r e p o r t e d by Ough and Stone (15).
Several concentrations of each t e s t compound t h a t w o u l d not
saturate the " a i r " were e v a l u a t e d . The j a r was closed quickly
and evaporation of the s u b s t a n c e was facilitated by gentle
rotation of the container. F i v e m i c e were q u i c k l y d r o p p e d into
the j a r , and the bottl
Every 15 seconds
time r e q u i r e d f o r each animal t o become a n e s t h e t i z e d (loss of
righting r e f l e x ) was noted. Since the occurrence of induction
after 0.5 min but no later t h a n 5 min i s considered to represent
the optimal i n d u c t i o n t i m e f o r known p o t e n t anesthetic agents,
the interval between t h e s e two time l i m i t s was u s e d as a crite-
r i o n of induction.
The m i c e were k e p t i n the j a r f o r 10 minutes, then removed
and placed on a pan. The time of recovery (to righting) was
noted f o r each animal. Postanesthetic a n a l g e s i a was tested by
pinching the base of each a n i m a l ' s t a i l every five minutes. A
s q u e a k on pressing the tail was considered as the end point. If
an aminal still showed a n a l g e s i c r e s p o n s e a h a l f an hour after
being removed from the j a r , the pinch test was administered
every 15 minutes. The s u b j e c t s were k e p t f o r twenty-four hours
to record any latent toxicity of the compounds.
The e v a l u a t i o n of the substances i n m i c e was conducted in
two stages. In the first stage, a dose-range experiment was
performed to determine the lethal concentration range, and three
appropriate concentrations were s e l e c t e d . Fifteen a n i m a l s were
tested at each of the three concentrations. In the second stage,
another dose-range study was conducted t o chose t h r e e lower con-
centrations. Fifteen a n i m a l s were t e s t e d a t each selected
concentration t o determine the medium e f f e c t i v e anesthetic
concentration. The anesthetic p o t e n c y and margin of safety of
e a c h compound were e x p r e s s e d as an anesthetic index.
The median a n e s t h e t i c c o n c e n t r a t i o n (AC 5 0 ) i s defined as an
estimated concentration by w h i c h 50% of the animals are expected
to be anesthetized. The median l e t h a l concentration (LC 5 0 ) is

10. DENSON E T AL. Fluorinated Volatile Anesthetics 201
an e s t i m a t e d c o n c e n t r a t i o n by w h i c h 5 0 % o f t h e a n i m a l s a r e
expected to die. The a n e s t h e t i c i n d e x (AI) i s the r a t i o of LC 5 0
to AC 5 0 . The l a r g e r the a n e s t h e t i c index t h e g r e a t e r margin of

safety. The lower the A C 5 0 , the lower the c o n c e n t r a t i o n o f drug
available f o r metabolism. However, a d r u g acting at lower con-
centration i s not n e c e s s a r i l y safer. As a l r e a d y d i s c u s s e d ,
methoxyflurane i s nephrotoxic, yet i t s A C 5 0 i s about half that
for halothane.
Before p r e s e n t i n g our p h y s i o l o g i c a l data, i t i s worthwhile
reviewing some d a t a a v a i l a b l e f o r other anesthetics. These data
are summarized i n Table 1 (2, 8 ) . While no h e p a t o c e l l u l a r o r
nephrotoxicity data are a v a i l a b l e for 2,2-(bis)trifluoromethyl-
1,3-dioxolane, i t i s interesting t o note that i t s AI i s s l i g h t l y
larger than halothane (8) T h i s molecule contains only carbon
fluorine, hydrogen, an
reported by T e r r e l l (9) halogenate analogs,
noteworthy that t h e r e was very little increase i n activity.
Table 1
ANESTHETIC POTENCY FOR FLUORINATED ANESTHETICS
B.P.
Anesthetic (°C) AC 5 0 LC 5 0
AI MAC
Halothane 50 0.78 2.74 3.50 0.78
Methoxyflurane 105 0.3 - - 0.23
Fluroxene 43 1.2-8 - - 6.0
Enflurane 57 2-4 - - 2.2
2,2 (Bis)trifluoro- 100 0.5 2.38 4.70 -

methyl-1,3-dioxolane
We began o u r p h y s i o l o g i c a l e v a l u a t i o n s using halothane as a

standard. In these experiments we o b t a i n e d a n A I o f 4.75, which
is slightly h i g h e r t h a n t h e A I o f 3.50 previously reported (see
Table 1). At a n e s t h e t i c concentrations, halothane i s character-
ized during induction by p t o s i s accompanied by lacrimation.
Recovery was c h a r a c t e r i z e d by " f l a t tails." Once t h e a n i m a l r e -
gained the r i g h t i n g reflex, they maintained it. In a d d i t i o n ,
rapid r e c o v e r y t i m e s were o b s e r v e d , indicating rapid halothane
elimination. (Rapid e l i m i n a t i o n i s often associated with lower
lipophilicity.) At l e t h a l c o n c e n t r a t i o n s , dark eyes and b l a n c h e d
e a r s were o b s e r v e d i n addition t o p t o s i s and lacrimation.
T a b l e 2 summarizes our f i n d i n g s f o r halothane.

Table 2
EFFECTS OF HALOTHANE CF -CHBrCl, ON MICE 3
(AI = 4.75)
Cone, No of Induced Induced Mean Recovery
Vol % Animals before 30 sec before 5 min Time, min Deaths

0.78 0
0.94 15 5
0.83 0
1.00 15 7
1.06 15 - 12 1.03 0
4.00 15 8 15 7.72 3
5.00 15 13 15 8.13 9
5.50 15 14 15 8.83 12
At anesthetic concentration
dioxolane, 12, minimum h i n d l e g movement and p t o s i s were observed,
but no l a c r i m a t i o n o r c o n v u l s i v e b e h a v i o r . R e c o v e r y was charac
terized by t h e a n i m a l s running in circles. M i c e were w o b b l y and
had a bouncy g a i t . Recovery times f o r 1£ were much l o n g e r than
for halothane, a s shown i n T a b l e 3, s u g g e s t i n g t h a t , e v e n a t low
concentrations, elimination i s not n e a r l y as r a p i d as i t i s f o r
halothane. Slower e l i m i n a t i o n suggests a higher lipophilicity
for 4 , 5 - d i c h l o r o - 2 , 2 - d i f l u o r o - 1 , 3 - d i o x o l a n e , 12. L e t h a l concen
t r a t i o n s were c h a r a c t e r i z e d by i r r e g u l a r respiration and apparent
bradycardia. Ptosis a n d l a c r i m a t i o n were o b s e r v e d i n a few
animals.
Table 3
EFFECTS OF
:c> |
o
12
\ F„ ON MICE
/
(ΑΙ = 7.88)
Cone, No of Induced Induced Mean Recovery
Vol % Animals before 30 sec before 5 min time, min Deaths
0.25 15 0 - 0
0.30 15 2 0.63 0
0.35 15 - 13 10.60 0
2.00 15 - 15 60.08 0
2.50 15 - 15 60.47 6
3.00 15 1 15 120 14

Anesthetic concentrations of 4-chloro-2,2,5-trifluoro-1,3-

dioxolane, 13, were c h a r a c t e r i z e d by h y p o a c t i v i t y , s l i g h t convul-
siveness, ptosis, and o c c a s i o n a l l a c r i m a t i o n . During recovery the
animals often ran i n c i r c l e s and some l o s t their righting reflexes.
Recovery times, given i n Table 4, were a b o u t half those reported
for 4,5-dichloro-2,2-difluoro-1,3-dioxolane, 12 (see Table 3 ) .
Although t h e AI d e t e r m i n e d f o r4-chloro-2,2,5-trifluoro-1,3-
dioxolane, 13^, was a p p r o x i m a t e l y t h e same a s t h e A I d e t e r m i n e d for
halothane, recovery times were somewhat longer f o r 13. At lethal
concentrations, the animals' e y e s became e x t r e m e l y dark (exophthal-
mos) and deep j e r k y r e s p i r a t o r y response was f o l l o w e d by shallow
breathing and b r a d y c a r d i a .
Table 4
E F F E C T S OF
0<
F
13
(AI = 4.66)
Cone, No. o f Induced Induced Mean R e c o v e r y
Vol % Animals before 30 s e c before 5 min time, min Deaths
0.88 15 5 1.95 0
0.94 15 - 13 3.07 0
1.00 15 - 14 4.13 0
3.00 15 - 15 15.48 0
4.00 20 - 20 17.78 10
5.00 15 5 15 19.72 12
The preliminary evaluation of 2,2,4,5-tetrafluoro-1,3-

dioxolane, 1£, i s summarized i n T a b l e 5. Rapid hind l e g movement
ptosis and t a c h y p n e a were a s s o c i a t e d w i t h anesthetic concentra-
tions. During recovery, the animals ran i n c i r c l e s , and some
shivered f o r a few m i n u t e s . Recovery times were f a r more rapid
than those observed for either 4,5-dichloro-2,2-difluoro-1,3-
dioxolane, 1J2, o r 4 - c h l o r o - 2 , 2 , 5 - t r i f l u o r o - 1 , 3 - d i o x o l a n e , 1£.
During induction, lethal concentrations of 2,2,4,5-tetrafluoro-
1,3-dioxolane were c h a r a c t e r i z e d by t h e same o b s e r v a t i o n s made f o r

the anesthetic concentrations. During recovery, however, hind

l e g s were e x t e n d e d and d r a g g e d , and a n i m a l s crawled with their
front legs.
Table 5
Ο
F^°v
E F F E C T S OF F 2 ON MICE
0
16
Cone., No. o f Induced Mean R e c o v e r y

Vol % Animals before 5 min Time, min Deaths
4.0 10 2 0.92 0
5.0 5
6.0 5 5 2.37 0
10.0 5.58
12.0
Statistical Analysis
The data obtained from the a d m i n i s t r a t i o n o f t h e lower con

c e n t r a t i o n s of halothane were a n a l y z e d by c a l c u l a t i n g the per
centage of animals t h a t were a n e s t h e t i z e d a t e a c h o f t h e t h r e e
lower c o n c e n t r a t i o n s . The p e r c e n t a g e s were p l o t t e d against con
c e n t r a t i o n on l o g a r i t h m i c p r o b a b i l i t y p a p e r a s shown i n Figure 1.
Similarly, t h e p e r c e n t a g e o f mice a n e s t h e t i z e d a t each o f t h e
three lower c o n c e n t r a t i o n s of 4,5-dichloro-2,2-difluoro-1,3-
dioxolane, 12, and t h e p e r c e n t a g e induced at each o f the three
lower c o n c e n t r a t i o n s of 4-chloro-2,2,5-trifluoro-1,3-dioxolane,
13^, were p l o t t e d . From t h e g r a p h , the A C 5 0 o f e a c h compound was
computed a c c o r d i n g t o t h e method o f L i t c h f i e l d and W i l c o x o n (16)
as shown i n Table 6. Since A C 5 0 of 4,5-dichloro-2,2-difluoro-
1,3-dioxolane, 12, i s s i g n i f i c a n t l y lower than that of halothane,
the former i s considered t o be more p o t e n t than the l a t t e r i n
inducing anesthesia.

0.2 0.4 0.6 1 Figure 1. Effects of inhalation

VOLUME PERCENT CONCENTRATION anesthetics on mice
2 4 6 10 20 Figure 2. Effects of high doses of

VOLUME PERCENT CONCENTRATION inhahtion anesthetics on mice

Table 6
ANESTHETIC POTENCY AND MARGIN OF SAFETY

STRUCTURE VS. ACTIVITY
Compound X Y
:t> ^0
AC 5 0 LC 5 0
AI
Halothane 0.99 4.7 4.75
12 CI Cl 0.33 2.6 7.88
13 CI F 0.88 4.1 4.66
16 F _ _
The results of the a d m i n i s t r a t i o n of high concentrations of

compounds were a n a l y z e d by computing the percentage of animals
that d i e d at each of the three high concentration levels of each
compound. The p e r c e n t a g e s were p l o t t e d a g a i n s t concentrations
as shown i n F i g u r e 2. From t h e three concentration-response
curves, the LC 5 0 o f e a c h compound was derived as listed in Table
6. Further a n a l y s i s showed t h a t t h e AI of 4,5-dichloro-2,2-
difluoro-1,3-dioxolane i s s u b s t a n t i a l l y higher than that of
halothane, indicating that the margin of s a f e t y of the new com-
pound i s considerably higher than that of halothane.
Summary
From t h e summary o f o u r data i n Table 6, we can see some

trends in structure-activity relationships. A l t h o u g h our d i -
o x o l a n e s do not contain b r o m i n e , good a n e s t h e t i c a c t i v i t y is
present. As could be predicted, the activity d e c r e a s e s as chlo-
rine i s replaced by fluorine. The lipophilicity appears to
d e c r e a s e as chlorine i s replaced by fluorine (partition coeffi-
cients will be determined and reported in a later publication).
Anesthetic activity increases as the boiling point of the series
increases.
A new c l a s s of potent volatile anesthetics has been devel-
oped. The structure activity r e l a t i o n s h i p s of t h i s c l a s s of com-
pounds a p p e a r t o f o l l o w t h o s e reported i n the literature. Other
compounds i n t h i s c l a s s are being prepared and evaluated, and
more d e t a i l e d p h y s i o l o g i c a l e x a m i n a t i o n s w i l l be conducted.

Acknowledgement s
The authors gratefully acknowledge t h e support of this re

search by I n s t i t u t e s of Health, General Medical Sciences
Division, under Grant Number 5-R01-GM-20082-02.
Discussion
Q: How l o n g d i d y o u o b s e r v e the animals f o r evidence of latent

toxicity?
A: In a l l cases f o r at least 24 h o u r s .
Q: A r e t h e compounds s t a b l e t o base?
A: Y e s ! You c a n c o n v e r t t h e d i f l u o r o f o r m a l group back t o t h e
carbonate using concentrated sulfuric acid. T h e s e compounds
are also stable t o mild acid.
Q: I s t h e r e any s p l i t t i n
A: No, a t l e a s t we hav
Q: The p o s s i b i l i t y exists f o r other isomers. C a n y o u comment
on that?
A: We c a n n o t predict w h e t h e r we a r e s t u d y i n g one a c t i v e isomer
or d e a l i n g with unknown m i x t u r e s . We c a n s a y t h a t i f other
isomers a r e present, we a r e n o t o b s e r v i n g an a c u t e lethal
effect.
Literature Cited
1. Cascorbi, H. F., "Anesthesia Toxicity," in 1974 Annual
Refresher Course Lectures, American Society of Anesthesi
ologists Annual Meeting, Washington, D. C., October 12-16,
1974, Lecture number 227 and references cited therein.
2. Larsen, Ε. R., "Fluorine Compounds in Anesthesiology," in
Fluorine Chemistry Review, P. Tarrant, Vol. 3, pp. 1-44
(1969) and references cited therein.
3. Robbins, J. H., J. Pharmacol. Exptl. Therap. (1946), 86 197.
4. Brown, B. R., Jr., "Enzymes and Anesthesia," in 1974 Annual
Refresher Course Lectures, American Society of Anesthesiolo
gists Annual Meeting, Washington, D.C., October 12-16, 1974,
Lecture Number 225 and references cited therein.
5. Van Dyke, R. A. and Chenoweth, Μ. Β., Anesthesiology (1965),
26, 348.
6. Tucker, W. K., Rackstein, A. D., and Munson, E. S. Brit. J.
Anaesth., (1974) 46, 392.
7. Cohen, E., et al., Anesthesiology (1974), 41, 321.
8. Gilbert, Ε. E., (to Allied Chemical Co.), U.S. Pat. 3,314,850
(1967).

9. Terrell, R. C., and Moore, G. L., (to Airco, Inc.), U.S. Pat.
3,749,791 (1973).
10. Virtue, R. W., Proc. Soc. Exptl. Biol. Med., (1950), 73, 259.
11. (a) Newman, M. S., and Addor, R. W., Amer. Chem. Soc. (1953)
75 1263.
(b) Newman, M. S., and Addor, R. W., J. Amer. Chem. Soc.,
(1955), 77, 3789.
12. Olah, G., Nojima, Μ., and Kerekes, I., Synthesis, (1973) 779.
13. Applequist, D. E., and Searle, R., J. Org. Chem. (1964), 29,
987.
14. Burgison, R. Μ., "Animal Techniques for Evaluating Anesthetic
Drug," in Animal and Clinical Pharmacologic Techniques in
Drug Evaluation, J. H. Nodine and P. E. Siegler, eds.,
Yearbook Medical Publishers Inc, Chicago, Ill. (1964),
pp. 369-372.
15. Ough, C. S., and Stone, , , (1961), ,
16. Litchfield, J. T., Jr., and Wilcoxon, F. Α., J. Pharm. Exptl.
Ther. (1949), 96, 99.

INDEX
A Analgesia 190
Analogs, amino acid 37
AC 200
Anesthesia, surgical 190
8 0
ACD 121
Anesthetic(s)
N-Acetylserotonin 39 concentration, median 200
iV-Acetyltransferase 39 cyclic diether 193
adrenergenic induction of 41 fluorinated volatile 190-206
cyclic A M P induction of 43 hazards of fluorinated 191
pineal 3
induction of 4
N-Acetyltryptamine 3 3,4-Anhydro-D-galactose 104
Acid(s) Antilipolytic drug 91
amino, analogs 37 Antiviral activities of fluorohistidines 29
electrophoresis of fluorocitric 10 A T P synthesis 17
erythrofluorocitric 8
fluoroacetic 8 Β
fluorocarboxylic 1-19
fluoro-dicarboxylic 2 Benz[a]anthracene induction 43
fluorourocanic, effects on urocanase 31 Biochemistry of ring-fluorinated
inhibitory properties of F-carboxylic 3 imidazoles 23-33
monofluorocitric 7 Biological disposition of R F C 181
isomers of 9 Bonds, phosphate 38
nicotinic 90,91 Brain, amphetamine in rat 81
perfluoro-octanoic 129 Brain, /?,/?-difluoroamphetamine in rat 81
substrate properties of F-carboxylic 3 Brominated fluorocarbons 171-186
Aconitase, cytoplasmic 12 Bronchography with R F C emulsions 180
Actinomycin 28
Activities of fluorohistidines, antiviral 29 C
Activity, ornithine decarboxylase 46
Adenosine 3',5'-monophosphate 39 F-Carboxylic acids
Adipose tissue 91 inhibitory properties of 3
substrate properties of 3
Adrenalin 41
Catalysis of M D H 2
Adrenergenic agents 41
Catecholamines 41
Adrenergenic induction of N-acetyl-
CF dUMP
3 66
transferase 41
Chlorination, photochemical 198
AI 201
p-Chloroamphetamine 88
5-AICAR 32,33
Chloroform 191
Alditol Ill Cholecystography 186
Aliphatic fluorine 77 Chymotrypsin 38
Alphamethyldopa 37 Cis-trans perfluorodecalin emul
Alveolographic studies with R F C 178 sions 135-167
Amethopterin 57 Citrate-isocitrate exchange 6
Amine drugs 77 Complexes, Michaelis-Menten 2
Amino acid analogs 37 Cyanosis 190
p-Aminobenzoylglutamate 60 Cyclic A M P induction of N-acetyl
3-Aminomethyl-pyridine 90,91 transferase 43
A M P , cyclic 43 Cyclic diether anesthetics 193
Amphetamine 78 Cycloheximide 28
in rat brain 81 N-Cyclopropyl amines 92
A M P induction of N-acetyltransferase, Cytochalasin Β 101
cyclic 43 Cytoplasmic aconitase 12
211

212 BIOCHEMISTRY INVOLVING CARBON—FLUORINE BONDS
D Fluoro-dicarboxylic acids 2
Fluorohistidines, antiviral activities of 29
Deaminase, liver microsomal 79 Fluorohistidines, effects on histidine
Deamination, oxidative 82 ammonia-lyase 30
Defluorination of fluorocitrate 14 2-Fluorohistidine 26
4- Deoxy-D-glucose 104 4-Fluorohistidine 30
Deoxyfluoro-D-glucoses 101 2-Fluoro-L-histidine 37
Deoxyfluoro-monosaccharides 99-113 Fluoroimidazole(s) 26
Dephosphorylation 38 ribosides 33
Dibutyryl adenosine 3',5'-monophos- 4-Fluoroimidazoles, synthesis of 24
phate 43 Fluorourocanic acids, effects on
Dibutyryl cyclic AMP 43 urocanase 31
Dibutyryl adenosine 3',5'-monophos- Fluroxene 191,192
phate 43
Dibutyryl cyclic AMP 43
Diether anesthetics, cyclic 193 G
Difluoro-oxalacetate 2 D-.Galactose 104
0,/?-Difluoro substitution 79-80 Gastroenerography 173
β,/^Difluoroamphetanune 83,86 GI tract, RFC in the 175
in rat brain 81
Drug, antilipolytic 9
Drugs, amine 7 Gluconeogenesi
dUMP 57 Glucose-6-phosphatase 38
Gregaria, Schistocerca 109
£
H
E. coli, effect offluorocompounds on 29
Efflux, inhibition of isocitrate 16 Halothane 191
Efflux, rates of isocitrate 15 effects on mice 202
Electrophoresis offluorocitricacid 10 Hazards offluorinatedanesthetics .... 191
Emulsions Hepatocytes 143
bronchography with RFC 180 Hepatography 185
cis-trans perfluorodecalin 135-167 Histidine 28
of RFC 180 ammonia-lyase, effects of fluoro
Enflurane 191,192 histidines on 30
Enzymatic probes 1-19 in enzymes 37
Enzyme induction 37, 43 incorporation into protein 50
Enzymes, histidine in 37 Human erythrocyte membrane 99
Erythrocyte membrane, human 99 Human plasma 117-133
Erythrofluorocitrate 11 HydTOxylindole-O-methyltransferase. 50
Ervthrofluorocitric acid 8 Hyperlipoproteinemia 90
Etners, synthesis of fluorinated 193 Hyperthermia 83
Ethylamine 77 Hypotensive effect 137
Ethylene carbonate 194
Exchange, citrate-isocitrate 6
I
F Imidazole(s) 26
biochemistry ofring-fluorinated..23-33
FdUMP 59 pharmacology of ring-fluorinated 23-33
5- FICAR 32,33 Incorporation of ( H) 2-fluoro-L-
3
Fluorinated volatile anesthetics 190-206 histidine into protein 52

Fluorine, aliphatic 77 Incorporation of histidine into protein 50
Fluoroacetic acid 8 Induction
Fluoro compounds, effect on E. coli 29 of N-acetyltransf erase, adrenergic .. 41
Fluorocarbons, brominated 171-186 of Ν-acetyltransferase, cyclic AMP 43
Fluorocarbons of high vapor pressure 176 benz [ a ] anthracene 43
Fluorocarboxylic acids 1-19 enzyme 37,43
Fluorocitrate 7 of pineal N-acetyltransferase 41
defluorination of 14 steroid 43
Fluorocitric acid, electrophoresis of 10 Inhibition of isocitrate efflux 16
5-Fluoro-2'-deoxyuridylate 59 Inhibition of monoamine oxidase 94

INDEX 213
Inhibitory properties of F-carboxylic Ρ

acids 3
Pancreatography 186
Intraabdominal surgery 190
Para-chlorophenylalanine 37
Isocitrate efflux, inhibition of 16
Para-fluorophenylalanine 37
Isocitrate efflux, rates of 15
Perfluorochemicals 135
Isomers of monofluorocitric acid 9
Perfluorodecaline 135,147
Isoproterenol 41,55
emulsions, cis-trans 135-167
Perfluorohexylbromide 185
L Perfluoro-octanoic acid 129
Peritoneal contamination 175
Lactobacillus casei 57,72 PFC 135
LC 5 0 200 PFD 135
Lethal concentration, median 200 Phagocytosis of R F C 176
Lipolysis 90 Pharmacology of ring-fluorinated
Lipophilicity 201 imidazoles 23-33
Liver microsomal deaminase 79 Phenethylamine 84
Locusta migratoria 109 C-Phenethylamine
14
86
Lymphography 181 Phosphate bonds 38
Phospholipid 147
M
Pineal N-acetyltransferase 39
M D H , catalysis of 2 induction of . 41
Median anesthetic concentration 200 Pineal gland 39
Median lethal concentration 200 Polyols HI
Melatonin 39 Potency for fluorinated anesthetics 201
Membrane, human erythrocyte 99 Pressure, fluorocarbons of high vapor 176
Metabolic probes 1-19 Probes, enzymatic 1-19
Metabolism, sorbital Ill Probes, metabolic 1-19
Methoxyflurane 191,192,201 Processes, multienzymatic 1
N-Methyltransferase 79 Protein, incorporation of ( H ) 2 -
3
Mice, halothane effects on 202 fluoro-L-histidine into 52

Michaelis-Menten complexes 2 Protein, incorporation of histidine
Microsomal deaminase, liver 79 into 50
Migratoria, Locusta 109 Ps. aeruginosa 107
Mitochondrial monoamine oxidase .... 80 Ps. fluorescens 107
Monoamine oxidase, inhibition of 94 Pseudomonad 107
Monochloroethylene carbonate 194 Ptosis 203
Monodeoxy-D-glucoses 99 Pyrimidines 67
Monodeoxymonofluoro-D-glucoses 99-100
Monofluorocitric acid 7
isomers of 9
R
Monofluoro-malate 5
Radiopaque fluorocarbon 173
Mosquito 111
Rat brain, amphetamine in 81
Multienzymatic processes 1
Rat brain, /?,^difluoroamphetamine in 81
Muscle relaxation 190
Rates of isocitrate efflux 15
Relaxation, muscle 190
RFC 173
Ν
alveolographic studies with 178
Narcosis 190 biological disposition of 181
Nicotinic acid 90, 91 emulsions 180
Norepinephrine 83,92 bronchography with 180
in the GI tract 175
phagocytosis of 176
Ο submersion in 176
Ribavirin 32
Organic fluoro compounds 117-133 Ring-fluorinated imidazoles
Ornithine decarboxylase activity 46 biochemistry of 23^33
Oxidase, inhibition of monoamine 94 pharmacology of 23-33
Oxidative deamination 82 RNAse 38

S Thymidylate synthetase 57-74

Tissue, adipose 91
Schistocerca gregaria 109
TMP 57
Serine 38
Toxicity of 3-deoxy-4-fluoro-D-glucose 109
Silk worm Ill
Triethyl fluorocitrate, N M R of 10
Sorbitol dehydrogenase Ill
β,β,β-Trifluoroethylamine 77
Sorbitol metabolism Ill
5-Trifluoromethyl-2'-deoxyuridylate .. 66
Splenography 185
Tryptamine 39
Steroid induction 43
Submersion in R F C 176
Substrate properties of F-carboxylic
acids 3 U
Surgery, intraabdominal 190
Swallowing reflex 190 Urocanase, effects of fluorourocanic
acids on 31
Synthesis
Urography 186
ATP 17
of fluorinated ethers 193
of 4-fluoroimidazoles 24
Synthetase, thymidylate 57-74 V
Τ
Tachypnea 203 Ventriculomyelography 185
Threonine 38 Vinylidene carbonate 195


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Biochemistry Involving Carbon-Fluorine Bonds Filler (ACS 1976)

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Biochemistry Involving

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

Illinois Institute of Technology

the Divisions of Fluorine

and Biological Chemistry

at the 170th Meeting of the

American Chemical Society,

Chicago, Ill., Aug. 26, 1975.

ACS SYMPOSIUM SERIES 28

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

Biochemistry involving carbon-fluorine fonds.

PRINTED IN T H E UNITED STATES O F AMERICA

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

Illinois Institute of Technology ROBERT FILLER

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

University of California, San Francisco, Department of Pharmacology, and the

The development of site specific reagents necessarily

regulated by properties of enzymatic components alone but more

(3) but the majority of biochemical texts have not incorporated

A series of chemical and enzymological experiments (5,6,7,

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

LEGEND TO TABLE I: MDH = malate dehydrogenase; GOT = glutamate oxalacetate amino­

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

COOH COOH COOH COOH

COOH COOH COOH

COOH COOH COOH

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

bivalent metal ion catalyzed decarboxylation to fluoro pyruvate.

system containing both aspartate and the fluoro-acid (b), repre­

of the inner mitochondrial membrane, as shown in Figure 2 (cf.

Figure 2. Spectrophotometric assay of citrate

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

of i s o c i t r a t e is monitored by an externally added NADP + Mg + + 2+

isocitrate dehydrogenase i s o c i t r a t e detector system in the dual

Substrate . ..... . . . _. . . Apparent

μπιοί x g" x miu

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

European Journal of Biochemistry

i n h i b i t gluconeogenesis from both precursors by 76-80% without

Studies with fluorocitrate

a.)Structure of the inhibitory isomer. In contrast to

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

This mechanism appears to be incompatible with the results of

tory" and "non-inhibitory

more details ). Elucidation of the chemical structure of the

reaction was followed by appearance of D P N H .

'The Citric Acid Cycle"

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

COO" coo" coo" COO"

fi/S System (2S.3R)- (2R.3S)- (2R.3R)- (2S.3Sh

Racemates ^s^gLg" 0 L .LgC^-

erythro-L - $ erythro-0 - $ threo-Oj- threo-L,-

Racemates erythro-D L - $ $ fhreo-O L - e f

"The Citric Acid Cycle"

Figure 4. The isomers of monofluorocitric acid *

Suggested N e w N o m e n c l a t u r e f o r the Isomers o f Isocitric A c i d , " / . Biol. Chan.

In Biochemistry Involving Carbon-Fluorine Bonds; Filler, R.;

Journal of Biological Chemistry

Figure 5. Electrophoresis of fluorocitric acid from enzymatic and

6£ 5Ό 40 3.0 2.0 ΙΌ 0 ppm ( δ )

Journal of Biological Chemistry

Figure 6. Nuclear magnetic resonance spectrum of triethyl

LEGEND TO TABLE I: MDH = malate dehydrogenase; GOT = glutamate oxalacetate amino

system containing both aspartate and the fluoro-acid (b), repre

experimental foundation. One of the - until recently - unrecog

Purification Total Total acon Specific

111,000. considerably higher than purified preparations requir

Figure 7. Competitive inhibition of cy

chondria agreed with in v i t r o enzyme kinetics (43). Fluoroci

10 mM c i t r a t e - T r i s , 0.5 mM malate-Tris separately or simul