DIETARY EFFECTS ON THE ENZYME
ACTEVITIES OF THE HYPOTHESIZED
LYSiNE-UREA CYCLE IN RATS

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSUY
MICHAEL A. ABRUZZO
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ABSTRACT

DIETARY EFFECTS ON THE ENZYME ACTIVITIES OF

THE HYPOTHESIZED LYSINE - UREA CYCLE IN RATS

by

Michael A. Abruzzo

Rat livers were analyzed in giggg for the purpose
of studying the possible conversion of lysine to urea.
The data indicate that lysine is converted to homocitrulline
in a reaction dependent on carbamyl phosphate. Also,
it is indirectly shown that this homocitrulline is then
metabolized to homoargininosuccinate. Homoarginine
conversion to urea and lysine is also demonstrated.

The possible interrelationship of these reactions in a
"lysine - urea" cycle is discussed.

Rats were fed high protein and high lysine diets
to study the response of the above reactions and the
reactions of the Krebs-Henseleit urea cycle to these
conditions. It is shown that on the high protein diet
the reactions of both urea cycles increased in activity.

On the high lysine diet, the activity of ornithine

Michael A. Abruzzo

transcarbamylase and argininosuccinic acid synthetase
decreased, while arginase increased. The conversions
from homocitrulline to homoargininosuccinate and

from homoarginine to urea increased in activity in
rats on high lysine diets. The possible in 1132
stimulation of arginase by lysine, and the possible
role of the "lysine - urea" cycle in patients with

urea cycle disorders is discussed.

DIETARY EFFECTS ON THE ENZYME ACTIVITIES OF

THE HYPOTHESIZED LYSINE - UREA CYCLE IN RATS

by

\‘ ’.*.‘::
_\\xt

.\

Michael AX Abruzzo

A Thesis

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology

1973

To

Linda

Mike and Alison

ACKNOWLEDGMENTS

I wish to thank my professor, Dr. James V. Higgins
for his many suggestions and criticisms, not only through-
out this study, but during the many years I spent in his
laboratory.

Special thanks are due Dr. Arthur F. Kohrman for

the many hours he spent with me planning experiments,
for making his laboratory available for most of the
crucial work, and for his untiring advice and support.
I also thank Dr. James Asher and Dr. Herman Slatis for
their critical reading of the thesis. The many helpful
suggestions, not only for this work but also for future
studies, are invaluable.

A very special thanks are due Dr. Ajovi B. Scott-
Emuakpor for his friendship and advice in my first years
as a graduate student. His academic outlook was an
inspiration and a guide in my research and teaching.

I thank Dr. Dorice Narins for her assistance on the
development of the diets and feeding procedures. I also
thank Habib Fakhrai for his friendship and help in the
urea determinations. Thanks are also due Terry Hassold,
Larry Yotti, LouBetty Richardson, Diana Smith, and Chris

Liebrock for their friendship, help, and encouragement

ii

over the last five years. Life at M.S.U. would not
have been the same without them.

Thanks are also due Bruce Corigliono, Carlo Picione,
Bob Pandolfi, Dan Mankoff, and Cathy Blight for their
assistance throughout this work.

For their friendship, and encouragement, I thank
Ron and Carol Wilson, Dan Friderici, Gary Marsiglia,
Astrid Mack, Rachel Rich, Frankie Brown, and Michelle
Kipp.

This work was supported in part by a grant from

the Society of the Sigma Xi.

iii

TABLE OF CONTENTS

ACKNOWLEDGMENTS . . . . . . . . . . . . . .
List of Tables . . . . . . . . . . . . . .
List of Figures . . . . . . . . . . . . . .
Introduction . . . . . . . . . . . . . . .
Review . . . . . . . . . . . . . . . . . .
Materials and Methods . . . . . . . . . . .

Enzyme Assays . . . . . . . . . . . .
Ornithine transcarbamylase . . .
Argininosuccinic acid synthetase
Arginase . . . . . . . . . . . .
Radio-siotope assay . . . . . . .

Animal Feeding Experiments . . . . . .
Animals . . . . . . . . . . . . .
Diets . . . . . . . . . . . . . .
Experimental design . . . . . . .
Liver enzymes . . . . . . . . . .
Ammonia determination . . . . . .
Urea determination . . . . . . .

Results 0 O O O I O I O O O O O O O O O O 0

Enzyme Studies . . . . . . . . . . . .
Lysine to homocitrulline . . . .
Lysine to homoarginine . . . . .
Homoarginine to urea . . . . . .
Homocitrulline to urea . . . . .

Animal Feeding Experiments . . . . . .
Food consumption and growth . . .
Urea cycle enzyme activities . .
Serum determinations . . . . . .

iv

Page
ii
vi

vii

22

22
24
25
26

. 26

28
28
28
30
32
33
34

35

35
35
41
45
52

55
55
58

. 62

Discussion .

Enzyme Studies

Lysine to homocitrulline .

Homocitrulline to homoASA

Homoarginine to urea .

Animal Feeding Experiments . .
High protein diets .

High lysine diets
Evidence for the Lysine - urea cycle

Speculation on the Enzymes

References .

64
65
65
66
67
68
68
69
73
74

76

Table

LIST OF TABLES

Rat casein diets
Experimental design

Conversion of C14-1ysine to C14-

homocitrulline and Ola-homoarginine
Conversion of homocitrulline to urea

Food consumption and growth of rats on
three different diets

Urea cycle enzyme activities in rats on
three diets

Serum urea and ammonia

Page

29

31

36

54

57

59

63

Figure

10

11

12

13

14

15

16

LIST OF FIGURES

Ammonia formation and detoxication
Possible sites of ammonia toxicity

The Krebs-Henseleit urea cycle

A speculated pathway fbr urea synthesis

The initial reactions of the lysine -
urea cycle and the ornithine - urea cycle

Lysine conversion to homocitrulline as a
function of enzyme concentration

Ornithine conversion to citrulline as a
function of enzyme concentration

Lysine conversion to homocitrulline as a
function of time

Ornithine conversion to citrulline as a
function of time

Homocitrulline formation as a function
of lysine concentration

Citrulline formation as a function of
ornithine concentration

The final reactions in the lysine - urea
cycle and the ornithine - urea cycle

Homoarginine conversion to urea as a
function of enzyme concentration

Arginine conversion to urea as a function
of enzyme concentration

Homoarginine conversion to urea as a
function of time

Arginine conversion to urea as a function
of time

Page

11
13
18

37

38

39

40

42

43

44

46

47

48

49

50

l7

18

19

20

Urea formation as a function of
homoarginine concentration

Urea formation as a function of arginine
concentration

The speculated metabolism of homocitrulline

Changes in enzyme activity in rats on
high lysine diets

51

53

S6

61

INTRODUCTION

It has been known for many years that in
individuals with proven enzymatic errors in urea
synthesis normal or near normal urea production
occurs. The question is how does the biosynthesis
of urea occur in these individuals. There is much
speculation on the possible mechanisms for this
synthesis.

In 1969, studies in our laboratory on rat livers
indicated that there may be an alternate urea cycle.
The results of these earlier studies led to the
hypothesis of a cycle with reactions analagous to the
classic Krebs-Henseleit urea cycle. In this, it is
believed that lysine is converted to homocitrulline
which is subsequently metabolized to homoarginine
which is then hydrolyzed to urea and lysine, thus
completing the cycle. This lysine - urea cycle was not
totally demonstrated because of the inability to detect
homocitrulline metabolism. Speculation on the possible
adaptive role of this hypothesized cycle in a patient
‘with citrullinemia centered around the mechanisms or

environmental stimuli that could cause an increase in

the synthesis of urea by this pathway. It was
speculated that in patients with an enzymatic error
in the Krebs-Henseleit urea cycle, the activity of the
lysine - urea cycle increases and thus takes over the
function of urea production.

The purposes of this study are:

(1) to test for the ability of rat liver to
catalyze the postulated reactions of the
lysine - urea cycle, with emphasis on the
metabolism of homocitrulline which is the
only step in the cycle which has not been
demonstrated

(2) to study the response of the reactions
of the lysine - urea cycle to dietary
differences in rats

It is hoped that these studies will yield further information
on the basic question: how is urea produced in individuals

with inborn errors in the Krebs-Henseleit urea cycle.

LITERATURE REVIEW

INTRODUCTION

 

The first report of a metabolic error affecting the
urea cycle was in 1958. Since that time the relationships
of urea synthesis, ammonia toxicity, and ammonia detoxication
have been studied in detail. The possibility of alternate
mechanisms for urea synthesis arose because of the finding
of normal urea synthesis in individuals with urea cycle
errors. The metabolic basis of ammonia toxicity and the
regulatory mechanisms for the maintenance of normal blood
ammonia levels have now been brought to light, mainly due

to studies of urea cycle disorders.

AMMONIA FORMATION

The major source of ammonia nitrogen is dietary protein.
Ammonia is formed in many metabolic reactions within the
tissues and also by bacterial enzyme activity within the
intestine. Whatever the source of ammonia, it is rapidly
metabolized to maintain a very low level in blood.

There are two bacterial enzymes present in the intestine

of mammals which will form ammonia. The most important one

is urease which will split urea into ammonia and 002 (Galloway
etual., 1966). The urea is present in the intestine because
of its free diffusion from the blood. Ammonia is formed at

a much greater rate than urea diffusion and the formed ammonia
is reabsorbed along the entire length of the inteétine at a
high rate (Bourke £5 21., 1966; Summerskill, 1966). Most
authors believe that there is no endogenous urease in the
intestine (Levenson g£_§l,, 1959) but in a more recent study
activity was detected in the intestine after treatment with
antibiotics to eliminate the bacterial flora. If there is

any endogenous urease activity, it is minor when compared

to the bacterial enzyme (Summerskill and WOIpert, 1970).

The other bacterial enzyme present in the intestine is
glutaminase which splits glutamine (liberated by protein
hydrolysis) into glutamic acid and ammonia.

The ammonia formed by these two reactions then enters the
portal circulation and undergoes detoxication in the liver.
Bacterial activity accounts for 20 - 25% of the daily ammonia
production.

Many intracellular reactions in mammals form ammonia
as one of the products, the most important of which are
glutamate dehydrogenase and glutamine degradation. The
other reactions are only minor contributors to the ammonia
pool but when considered in totality they can be important

as possible causative factors in ammonia toxicity.

In the glutamate dehydrogenase system, amino acids
are first transaminated with -(-ketoglutarate to form
glutamic acid which is then converted back to ak-ketoglutarate
with the liberation of ammonia. NAD+ and NADP+ serve as
coenzymes. ADP and amino acids stimulate the reaction and
NADH and NADPH inhibit it (Frieden, 1963, Hershko and Kinder,
1966).

Oxidation deamination of amino acids can also lead to
ammonia formation (Levin, 1971) but most of the amino acid
oxidase activity is due to transamination and glutamate
dehydrogenase. More important than oxidative deaminations
are amine oxidases which utilize dopamine, histamine,
tyramine etc., as substrates (Colombo, 1971). There are also
non-oxidative deamination systems for certain amino
acids but these are very minor.

Nucleotide and Nucleoside deaminases also give rise to
ammonia. This source of ammonia is most important in muscle.
It has been shown that after muscular work, ammonia
production doubles and is due mainly to the deamination of
5' adenosine monophosphate (Parnas gt El-’ 1927, Schmidt,
1928). The amount of ammonia released depends on the type
and adaptive characteristics of the muscle that is exercised
(Gerez and Kirsten, 1965).

Folk and Cole (1965) have shown a transglutaminase

which splits ammonia from protein bound glutamine molecules.

Ammonia is also liberated in the synthesis of heme (Levin,
1971).

The production of ammonia in the kidney deserves
special attention because it is a major source of arterial
and urinary ammonia. Glutamine serves as the major precursor
of renal ammonia under conditions of both normal acid base
balance and mild ammonium chloride acidosis. The resulting
glutamic acid may also serve as a potential source of renal
ammonia. Other amino acids do not contribute appreciably
to renal ammonia production at normal plasma amino acid
levels (Owen and Robinson, 1963, Stone and Pitts, 1967).

From all of the above reactions, it is quite clear that
the body has the capability to produce large quantities of
ammonia. It is also evident however, that blood ammonia
levels are very slight, usually less than 45 ug/lOO ml
even though 70 grams or more of protein are ingested daily
by an adult (levin, 1971). These two facts indicate that

ammonia once formed is rapidly detoxified.

AMMONIA DETOXICATION MECHANISMS

Ammonia is normally detoxified by several reactions,
the most important of which is mitochondrial carbamyl
phosphate synthetase, the first reaction in the formation
of urea. Acetylglutamate activates this reaction and the

formation of acetylglutamate is stimulated by L - arginine

(Kim.g£”§l., 1972). Under high arginine conditions this
mechanism is capable of detoxifying a potentially lethal dose
of ammonium acetate and therefore points to the importance
of urea formation as an ammonia detoxication mechanism.
Carbamyl phosphate synthetase in the cytosol, is also
a mechanism for ammonia detoxication but unlike mitochondrial
carbamyl phosphate synthetase, the ammonia so fixed by
this enzyme does not enter into the structure of urea
but is incorporated into pyrimidines. This reaction system
functions only when pyrimidine synthesis is required and
therefore ammonia concentrations do not appreciably
stimulate this mechanism (Bresnick, 1963).
Glutamine synthetase serves as a detoxication reaction

in the liver and the brain. The reaction is:

Glutamate + NH3 + ATP._______4p glutamine + ADP + P1

The resulting glutamine can then be split by glutaminase I
which functions in the kidney and the liver. In the kidney,
the liberated ammonia is utilized as a urinary buffer and in
the liver it enters the urea cycle by way of mitochondrial
carbamyl phosphate synthetase (Colombo g£_§l,, 1967). The
formation of glutamine in the brain is the major detoxication
mechanism in this tissue. Figure I summarizes the ammonia

formation and detoxication reactions found in mammals.

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AMMONIA TOXICITY

 

The toxic effect of ammonia was first shown by Kirk
and Summer (1931, 1932). They injected urease into
rabbits and noted convulsions and death in less than
one hour. Since that time many studies have shown
that high levels of ammonia are toxic (Clark and Eiseman,
1958, Bessman, 1958, Hutchinson st 31., 1964, 1965,
Katanuma 25 21-: 1966, Schenker £3 31., 1967, Roberge
and Charbonneau, 1969, Walker and Schenker, 1970, Kim
£5,313, 1972). The actual mechanism of this toxicity
is not clearly understood but evidence seems to indicate
that the ammonia affects the energy metabolism of the
brain.

Bessman (1958) formulated the hypothesis that the
toxicity of ammonia was due to a depletion of essential
cerebral metabolites. The first direct evidence that

toxic doses of ammonia.ig_vivo acutely affects cerebral

energy metabolism came from Schenker gt 213(1967) who

found that ATP and phosphocreatinine levels dropped
significantly in the base of the brain in rats treated

with ammoniumracetate. There are many possible reasons

for reduced ATP. Studies with cat cortex mitochondria show
that ammonia may interfere with the entry of pyruvate

into the Krebs cycle and thus slow the cycle down (McKhann

and Tower, 1961). However, that theory is disputed by

10

Walker and Schenker (1970) who could detect no affect of
ammonia on pyruvate decarboxylase in the brain. Another
theory, which is based on in zi££g_studies in rats, suggests
that during detoxication of cerebral ammonia with the
formation of glutamic acid from w-ketoglutarate the supply
of available NADH, necessary for mitochondrial generation
of cerebral energy, is depleted (Worcel and Erecinska,
1962). The hypothesis which gains the widest acceptance
stipulates that ammonia toxicity depends on the

depletion of cerebral d-ketoglutarate resulting in
impairment of the Krebs cycle and a subsequent decrease

in ATP synthesis (Walker and Schenker, 1970). Decreased
IK-ketoglutarate has been demonstrated in the brain of
dogs and rats treated with ammonia (Bessman, 1961).

After the administration of ammonia chloride in rats,
brain glutamine increased (Nakazawa and Quastel, 1968).
Because of this the authors feel that the low ATP levels
are due to increased consumption through the glutamine
synthesis reaction. It has also been noted that ATPase
activity is stimulated in giggg by ammonia but this even
occurs at physiological concentrations of ammonia and therefore
is probably not significant. Whatever the actual mechanism
for depletion of cerebral energy, it appears that toxic
levels of ammonia affect the brain by disrupting energy

metabolism. Figure 2 summarizes the speculated sites of

11

Figure 2

POSSIBLE SITES OF AMMONIA TOXICITY

Glucose-6-phosphate

 

 

NAD NADH
Pyruvate Acetyl CoA
I
/ 0AA \
Succinate (ATP Formed) Citrate

\ /

W—Ket lutarate

NADH

II N113

 

IV
Glutamic Acidé—Q Glutamine
ATP

I. Impaired oxidative decarboxylation of pyruvate

II. Depletion in supply of NADH
111- Depletion 0f ot-Ketoglutarate—fi decreased ATP synthesis by Krebs cycle

IV. Increased consumption of ATP through glutamine synthesis

12

ammonia toxicity in the brain.

POSSIBLE MECHANISMS FOR UREA PRODUCTION

Urea, the form of nitrogen waste excreted by mammals
has classically been considered to be synthesized exclusively
by the Krebs-Henseliet urea cycle (Figure 3). Ammonia
may enter the cycle in two places: through carbamyl
phosphate and through aspartate. This is believed to be
the major mechanism for the synthesis of urea and subsequent
ammonia detoxication. However, there are many observations
and some evidence which point to the possibility of
alternate mechanisms for urea synthesis.

In many individuals with inborn errors of the urea
cycle, urea production is normal. Such normal production
occurs even when direct assay of the hepatic enzyme in
question shows zero activity (for a review of urea cycle
errors, see Levin, 1971). When Ct-D, L—methyl aspartic
acid is added to a liver homogenate, it completely inhibits
argininosuccinic acid synthetase. However, the injection
of this compound into rats did not inhibit in xiyg urea
synthesis (Cedrangolo £3 21., 1962, Rowe and Miller, 1971).
What is interesting is that these authors also noted that
in the presence of glutamine as nitrogenous substrate
the conversion of citrulline to urea is inhibited by methyl
aspartate but there is no inhibition of urea synthesis.

Because of these two findings; urea production in individuals

13

THE KREBS-HENSELEIT UREA CYCLE

1 Mg'H'
NH3 + HCO3' + 2ATPL— % carbamyl phosphate + 2ADP + P1
acetyl glutamate

 

 

2
Carbamyl phosphate + omithineh—————9 citrulline + P 1
3
Citrulline + aspartate 4- ATP H- 2) argininosuccinate + AMP + P1
118

l.
Argininosuccinate————) arginine + fumarate

5
Arginine# a ornithine + urea
m-H-

 

l. Carbamyl phosphate synthetase I
2. Ornithine transcarbamylase

3. Argininosuccinate synthetase

4. Argininosuccinate 1yase

5. Arginase

Figure 3

14

with urea cycle errors, and continued urea production

in the presence of a urea cycle inhibitor, many studies
have been undertaken in a search for alternate mechanisms
for the synthesis of urea.

One possible explanation for urea synthesis in
individuals with urea cycle errors centers around the
belief that the enzymes involved are not totally inactive
and that the level of in 3132 activity is sufficient
for normal or near normal urea synthesis. This level
of activity is not, however, believed to be sufficient
for ammonia detoxication, thus leading to the usual
hyperammonemia. Levin (1967) believes that if this
theory is correct it can not be used to explain urea
production in the patient he studied. His patient
routinely produced 5 grams of urea per day and given
a maximum in yigg activity of 22, it would be impossible
to explain this quantity of urea (by way of the urea
cycle). Another theory based on in gigg activity of urea
cycle enzymes being different than in yigrg_determdnations
is discussed by Scriver (1969). Necessary assumptions
are that the patient has a mutant form of the enzyme
and that this mutant has a much higher Km. In this
way, the increased concentrations of urea cycle substrates
found in these patients would allow normal urea production

because the maximum rate of reaction would be reached at

15

the much increased substrate level. This theory gains
support from the study of Tedesco and Mellman (1967)

on skin fibroblasts of a citrullinemic patient. They
found that normal urea production occurred when citrulline
concentrations were raised above what was believed to

be the maximum substrate concentration. An apparent Km
shift could therefore explain normal urea production in
patients with urea cycle errors. A problem with this
hypothesis is that the patient studied by Tedesco and
Mellman does not have normal urea production. Another
problem with this theory is that the substrate accumulations
noted are probably due to the defective enzyme and
therefore can hardly be considered an adaptation to a

Km shift (Levin, 1971). Under normal circumstances,

urea cycle enzymes believed to be defective are assayed
with excess substrate and are still found to have a very
low, if not zero, activity and therefore refute or cast
doubt on the Km shift theory.

Another possible theory is that the enzymes of the
urea cycle exist as isozymes and although the liver
isozyme is defective, urea synthesis will occur in the
other tissues of the body. This theory is supported in
part by the study of Vidailhet 25 El. (1971). They
studied the tissues of a patient with citrullinemia (died

at 7 1/2 months) and found that argininosuccinic acid

l6

synthetase was completely absent in the liver while the
kidney assay showed normal activity. The problem with this
case is that the kidney arginase activity was zero and
therefore normal urea production could not occur in this
tissue. The authors could not, therefore, use the isozyme
theory to explain their patients normal urea production
without assuming a transport between tissues.

Some authors believe that urea production in the
presence of a defective Krebs-Henseleit cycle is due to
alternate metabolic cycle(s). This theory was first
suggested by Bach (1939). He believed that glutamine could
combine with ammonia and carbon dioxide to form glutamic
acid and urea. That observation was later shown to be in
error (Archibald, 1945)., The urea production was due to
arginine contamination. In 1961, Levin £5 21, suggested
that an alternate cycle may exist but did not speculate
on the possible mechanisms. Cedrangolo 35 a1, (1962, 1963)
suggested that alternate pathways of urea synthesis exist.
This is based upon their work with methyl aspartate. They,
like Levin, did not speculate on the possible pathways.

Cohen 25 El- (1968) suggested that urea could be
produced from guanidinosuccinic acid. They postulated two
possible pathways:

a) aspartate + arginine-—————€’ ornithine + guanidinosuccinate

guanidinosuccinate-———-—————4) urea + aspartate

17

b) arginosuccinate + NH3————————qp ornithine + carbamyl aspartate

carbamyl aspartate + NH3 :a. guanidinosuccinate

 

 

guanidinosuccinate ; urea + aspartate
The evidence for these pathways comes from their finding
of guanidinosuccinate in the urine of patients with uremia.
The reactions are only speculative and further work by Stein
ggugl. (1969) shows that guanidinosuccinate production
requires an intact urea cycle, the evidence being the
finding of no guanidinosuccinate in the urine of
individuals with urea cycle disorders. If this speculated
pathway exists, it would appear as though it is not
responsible for normal urea production in disorders of
the urea cycle.

Another pathway for urea biosynthesis was postulated
by Scott-Emuakpor (1970). This pathway (Figure 4) parallels
the classic ornithine urea cycle utilizing the homocompounds
(one additional methyl group in the carbon skeleton) of
the intermediates of the ornithine urea cycle as
substrates. There are many pieces of direct and indirect
evidence which support this hypothesis.

Vidailhet £5 31. (1971) described a patient with
elevated homocitrulline, homoarginine, lysine, ornithine,
and citrulline. Liver biopsy showed that there was zero
activity for argininosuccinic acid synthetase. Likewise,

these same amino acids were found to be elevated in a

18

A SPECULATED PATHWAY FOR UREA SYNTHESIS

 

 

 

(L-lysine)
Carbamyl
Phosphate

(Lphomoarginine) L-arginine L-citrulline (L-homocitrulline)
r /
\\~ .I
A
\\ I]? aspart ate
\\ HI e ATP
\ l'
\\ ASA ,’
x ,/
\
x‘ k
(homoASA)

—----- The hypothesized Lysine—Urea Cycle

The Ornithine-Urea Cycle

ASA.- argininosuccinic acid

L—lysine - homoornithine

‘ Figure 4

19

patient described by Scott-Emuakpor ggual. (1972).
The patient has no detectable activity of argininosuccinic
acid synthetase in either his skin fibroblasts (Scott-
Emuakpor 25h313, 1972) or lymphoblasts (Spector and Bloom,
1973). This patient, like most with urea cycle disorders,
has normal urea production butxunlike most also has
normal blood ammonia levels. -

Homoarginine and homocitrulline have been detected
in the urine of patients with hyperlysinemia and in
normals after lysine loading (woody, 1967, Armstrong and
Rabinow, 1967, Ghadimi g£_§1,, 1965, Colombo, 1971, Ryan
and Wells, 1964). In 1964, Ryan and Wells showed that
lysine could be converted to homocitrulline and homoarginine.
They injected C14 - lysine into rats and isolated €14 -
homocitrulline and C14 - homoarginine. The reaction mechanisms
were not known. In 1968, Ryal gtngl. showed that homo-
citrulline production from lysine was enzymatic. They,
however, could not show any production of homoarginine,
nor could they detect any further metabolism of homocitrulline.
They were also able to show that homoarginine could be
cleaved by arginase into lysine and urea. This was not
confirmed by Colombo(1971). Scott-Emuakpor (1970), however,
was able to demonstrate homoarginine conversion to urea
and lysine by both rat liver homogenate and commercial
arginase. The conversion of lysine to homoarginine was

shown by Ryan £5 a1. (1969) to occur in rat kidney by

20

transamidination.

The reaction mechanism for the conversion of lysine
to homocitrulline was demonstrated by Scott-Emuakpor (1970).
Lysine was converted to homocitrulline by rat liver
homogenate in a reaction that was linear in relation to
protein, carbamyl phosphate, and lysine concentrations,
and time of reaction. The transcarbamylation of lysine
was not found to occur when human ornithine transcarbamylase
was the enzyme source (Colombo, 1971). Strandholm £5 .31.
(1971) were able to show the enzymatic conversion of
homoargininosuccinic acid to homoarginine and fumarate
in pig kidney. This conversion is one of the speculated
reactions of the lysine - urea cycle.

In summary, the finding of elevated levels of the
homocompounds and normal urea cycle substrates in patients
with citrullinemia and hyperlysinemia is indirect
evidence for the existence of the lysine - urea cycle.
Lysine loading tests also support this hypothesized cycle.
Specific reactions of the lysine - urea cycle have been
shown to occur by three independent researchers but two
of these reactions have not been confirmed by one researcher
and the third reaction has not been subjected to complete
kinetic studies. The metabolism of homocitrulline, a
necessary prerequisite of the postulated cycle has not
been demonstrated. No evidence as to the nature of the

enzymes which catalyze the speculated reactions has been

21

reported.

This cycle, although not confirmed, has substantial
evidence in its favor. It is because of this evidence
and because of the conflicts in the literature concerning
lysine metabolism that the following experiments were
performed. It is hoped that these experiments will
illustrate the mechanism or mechanisms which allow
normal urea synthesis in patients with inborn errors

of the Krebs-Henseliet urea cycle.

MATERIALS AND METHODS

ENZYME STUDIES

 

The animals used in these experiments were from
colonies of adult Sprague-Dawley rats obtained from
Spartan Research Animals. They were routinely maintained
on a Wayne laboratory block diet containing 24% protein.

A rat was selected at random, killed by decapitation,

and the liver was quickly removed and placed on ice.

A one gram slice of liver was homogenized in either

19 ml or 9.0 m1 of ice cold water depending on the assay
to be run. The remaining liver was frozen at -80°C.

The activities of the enzymes assayed from the frozen
livers were not significantly different from those for
fresh liver. This is in agreement with Hutchinson g£_§l,
(1964).

The assays were based on the colorimetric determination
of urea, citrulline and homocitrulline, by a modification
of the method of Archibald (1944). The color reagent used
for all determinations was diacetyl monoxime (0.75% in

water). For color development, an aliquot of the reaction

23

tube was mixed with 1 ml of the color reagent and 5 ml
of acid reagent prepared as follows: commercial grade
sulfuric acid (concentrated), phosphoric acid (85%), and
water in a ratio of 1:3:6. The tubes were boiled for

15 minutes and cooled in a room temperature water bath
for 10 minutes. The color was read at 490 nm for
citrulline and homocitrulline and at 480 nm for urea.
The color was stable for 2 hours when tubes were kept

in the dark.

The methods used for the assays of ornithine
transcarbamylase and arginase are patterned after those
of Schimke (1962). The unit of activity for these two
enzymes is expressed as uMoles of product formed per
hour per gram wet weight of liver. The method used for
argininosuccinic acid synthetase is a modification of
the method of Wixom g£_§l. (1972). The unit of activity
for this enzyme is expressed as uMoles of substrate
depleted per hour per gram wet weight of liver. Protein
was determined by the method of Lowry g£_§l, (1951).

Because the assays were performed on rat liver homo-
genates the actual enzymes that catalyze the lysine to
homocitrulline, homocitrulline to homoargininosuccinate,
and homoarginine to urea reactions are not known. The
methods for these assays are described under the methods

for ornithine transcarbamylase, argininosuccinic acid

24

synthetase and arginase for simplicity. It is not to

be inferred that these enzymes are catalyzing the

reactions of the lysine - urea cycle. The methods described
only test the ability of a rat liver homogenate to catalyze

the lysine - urea cycle reactions.

Ornithine Transcarbamylase

 

This enzyme activity was determined as the rate of
citrulline formation. The assay medium contained SOmM
glycyl—glycine buffer, 20mM dilithium carbamyl phosphate,
15mM L-ornithine, and 50 ul of a 1:20 liver homogenate.
The final volume of the medium was 1.0 ml and the pH 8.3.
The medium was prepared immediately before the assay
was performed and kept on ice because of the instability
of the carbamyl phosphate. To start the reaction, the
tubes were removed from the ice bath and incubated for
15 minutes at 37.5"C. The reaction was stopped with the
addition of 1.0 m1 of 30% perchloric acid, and centrifuged
for 10 - 20 minutes at 2000 rpm. 50 ul of the supernatant
were used for color development. When homocitrulline
production was assayed L-lysine was substituted for ornithine
in the same concentration, but the liver homogenate volume
was 500 ul. The final volume of the medium was 1.0 ml and
the pH 8.3. 1000 ul of the supernatant was used for the

color reaction.

25

Argininosuccinic acid synthetase

 

The activity of this enzyme was measured as the depletion
of citrulline or homocitrulline, depending on the substrate
used in the assay. The assays were performed by two
different methods; one with an ATP regenerating system
and one without. The assay medium with an ATP regenerating
system contained ZOOmM tris buffer, SmM aspartate, SmM
magnesium sulfate, 2mM L-citrulline or L-homocitrulline,
lmM ATP, 2mM phospheonolpyruvate (PEP), 2 ug pyruvate
kinase, 0.5 mg urease, and 500 ul of 1:20 liver homogenate
for citrulline as substrate or 500 ul of 1:10 liver
homogenate for homocitrulline as substrate. The final
volume was 1.0 m1 at a pH of 7.6. The reaction medium
without an ATP regenerating system was the same except
that ATP was increased to a SmM concentration and
neither PEP nor pyruvate kinase added. The mediums were
prepared shortly before the assays and kept on ice. The
reaction was started by removing the tubes from the ice
bath and incubating at 37.5"C for 30 minutes (citrulline
as substrate) or for 120 minutes (homocitrulline as
substrate). The reaction was stopped by boiling for five
minutes and the tubes were cooled and centrifuged for 30
minutes at 2000 rpm. For color development, 50 ul of the
supernatant was used for either homocitrulline or

citrulline determinations.

26

Arginase

This reaction was measured as the production of urea.
The assay medium contained L-arginine or L-homoarginine in
a concentration of 0.250 M, pH 9.7, and 0.001 M MhSOa.
For the arginine medium 20 ul of a pre-treated liver homogenate
was added and for the homoarginine medium 100 ul of this
homogenate was added. The final volume of the reaction
medium was 0.5 ml. To prepare the treated liver homogenate
a 1:10 homogenate was incubated with an equal volume of
0.1 M MnSOA for 5 minutes at 55°‘C. The tubes were then
centrifuged for five minutes at 2000 rpm and the supernatant
used as the enzyme source. The reaction tubes were kept
in an ice bath until the reaction was started by incubating
the tubes at 37.5. C for 15 minutes. The reaction was
stopped by the addition of 1.0 ml of 30% perchloric acid.
The tubes were centrifuged for 10 - 20 minutes at 2000

rpm and 50 ul (arginine as substrate) or 500 ul (homoarginine

as substrate) are used for color development.

Radio - Isotope Assay

 

In an attempt to show the conversion of L-lysine
to urea, a radio-isotope assay using C14 - lysine was
developed. The reaction medium contained 0.05 M
potassium phosphate (KH2 P04) buffer, 0.003 M MgSO4,
0.01 M L-aspartate, 0.03 M L-lysine, 0.004 M ATP,

0.002 M PEP, 0.5 ug pyruvate kinase, arginase (1 mg/ml),

27

dilithium carbamyl phosphate (in one of four concentrations:
0.0, 0.04 M, 0.08 M or 0.120 M), 50 ul of 1:20 liver
homogenate, and l uC of L-lysine - C14 (U.L.) in a final
volume of 300 ul at a pH of 7.5. All reaction tubes were
prepared in an ice bath and the reaction was started by
incubating in a water bath at 37.5. C. Tubes were incubated
for one hour and the reaction was stopped by boiling for
five minutes. The tubes were centrifuged for 30 minutes
at 2000 rpm and the supernatant was used for the
isolation of homocitrulline and homoarginine.

Homocitrulline and homoarginine were isolated by
paper chromatography on Whatman 3MM paper. 50 ul of
the supernatant was spotted (3 ul portions). Lysine,
homocitrulline, and homoarginine standards were spotted
for identification purposes. The chromatograms were run
in a descending direction for 24 hours in a solvent of
butanol, acetic acid, and water (12:3:5). The chromatograms
were then dried in a fume hood and the standards selectively
stained with ninhydrin reagent. Those areas correSponding
to homocitrulline and homoarginine, utilizing the stained
standards as a guide, were then cut out and placed in
counting vials. 20 m1 of dioxane counting fluid was
added to each vial and allowed to stabilize in the dark
for 24 hours before counting. Counting was performed

on a Beckman room temperature liquid scintillation counter.

28

ANIMAL FEEDING EXPERIMENTS

To investigate the possible activation of the
hypothesized alternate urea cycle and the enzymes
involved in the reactions, an experiment was designed
in which controlled groups of animals were fed three
different diets. These animals were then killed and
their blood and liver analyzed for specific enzyme
activities and blood substances that could be considered

related to the overall problem of the biosynthesis of urea.

Animals
The animals used in this experiment were taken from
six litters of highly inbred colonies of Sprague-Dawley
strain rats. They were raised from the day of weaning
until the start of the experiment on Wayne Lab Blox
(crude protein content, 24%). The animals were separated
by sex before reaching maturity and were placed in
similar cages and housed together in an animal care facility.

All animals were the same age on the day of killing.

2222s.
Table 1 shows the ingredients of the control and

experimental diets. The casein, dextrine, salt mix,

vitamin mix and agar were purchased from Nutritional

Biochemicals, Cleveland, Ohio. The corn oil used was

29

 

Table 1

RAT CASEIN DIETS

 

 

22% Protein 60% Protein 22% Protein + 5% Lysine
Casein (g) 660 1800 660
Dextrose (g) 555 150 555
Sucrose (g) 441 147 441
Dextrine (8) 7.591 150 591
Corn Oil (g) 450 450 450
Harper Salt Mix (g) 120 120 120
Vitamin Mix (g) 66 66 66
Lysine (g) --- --- 150
Water (ml) 3000 3000 3000

Agar (g) 105 105 105

 

30

Mazola brand. It should be noted that the 60% protein
diet replaces carbohydrates with protein and that the
high Lysine diet is exactly the same as the 22% protein
diet except lysine is added. The diets were developed

in this manner so that they would be isocaloric. The
diet mixes were cut into blocks, weighed and placed
directly into the cages. To determine intake, the uneaten
portion of the diets was removed daily and weighed again.
Because of this technique, the food consumption is only

an approximation to the actual consumption.

Experimental Design_

The experiment was structured as a completely
randomized block design. There were nine blocks in the
experiment formed as follows: blocks 1 through 6
contained males, three males from the same litter per
block. The males were selected at random from each litter.
Blocks 7, 8 and 9 were formed in the same manner except
females were used. There was no blocking for weight. The
weight differences were blocked as a secondary result of
blocking for sex. The three treatments were the diets;
22% Protein (control), 60% protein, and 22% protein + 5%
lysine. The treatments were assigned at random to the
animals in a block. Table 2 is a summary of the blocks.

The results from all of the determinations made on these

 

 

31

 

 

 

 

 

 

 

 

 

aoxmo mums
mamaficm gowns aoum xooao
osu uaommummu mumnasclla

m nouuwa scum moamaom m m

m.m.n o.m q.m ~.H ocamzq Nn + N nouufia scum moamaom m m

dwououm NNN H HouuHH aouw moamamw m m

o nouuaa aoum moans m o

¢.m.m o.m <.m ~.H cwououm woo m umuuaa aouw moans m m

a e Houuaa aoum moans m e

m umuuaa scum moans m m

m.m.n o.m q.m N “a :Hououm NNN m nouufia aoum moans m N

H Houufia aoum moans m H

momma Hmawn< muoaQ mamaaa< mxooam
OZHQmmm use UZHMUOAm may

 

zuHmma A<HZMZHMmmxm

N mam<H

 

32

rats were analyzed by an Analysis of Variance for
completely randomized blocks design. The contrasts
tested were: 22% protein diet vs 60% Protein and 22%
protein diet vs 22% protein + 5% lysine. These contrasts
were non orthogonal and were therefore tested using
Bonferroni's t-test (Dunn, 1961).

All the animals were fed the 22% protein diet
for seven days (time required to stabilize consumption
rate) before the start of the experiment. At the end
of the seven days, each treatment group (9 rats/treatment)
was placed on the diet for that group for an additional
ten days. During that period, each rat was weighed
every two days and food consumption was determined daily.
At the end of the ten days the rats were anaesthesized with
ether and 5.0 to 10.0 ml of blood was collected from the
vena cava. (There was no difference in the enzyme activity
between the decapitated or ether killed rats). The livers

of each animal were rapidly removed and frozen at -80"C.

Liver Enzymes

The activities of ornithine transcarbamylase,
argininosuccinic acid synthetase and arginase were
determined for each liver. In addition the ability of
each liver homogenate to metabolize lysine, homocitrulline,

and homoarginine was determined. These enzymes were

33

assayed as previously described except that each liver
homogenate was prepared from 0.5 g in 9.5 ml of ice cold
water. Each assay was run in duplicate along with
appropriate blanks. All assays were run within three days

from the date of killing of the animal.

Serum Ammonia Nitrogen Determination

The method for ammonia determination is a modification
of that described by Chaney and Marback (1962). The freshly
collected blood was refrigerated for fifteen minutes
to allow clotting to occur. It was then centrifuged
for ten minutes at 2000 rpm to separate the serum. The

reagents for the determination are:

l) phenylnitroprusside solution - 50 grams of
reagent grade phenol and 0.25 grams of
sodium nitroprusside are dissolved in distilled
deionized water.
2) alkaline hypochlorite solution - in 600 m1
of distilled deionized water dissolve 25 grams
of NaOH pellets, let cool, then add 40 m1
of NaOCl (Clorox) and bring up to 1000 ml
with distilled deionized water.
For the reaction add 0.5 ml of serum and 1.0 ml of reagent
and mix. Then add 1.0 ml of reagent 2 and mix; stopper

the test tube and incubate for five minutes at 37.5‘ C.

34

To each tube add 3.5 m1 of distilled deionized water and
read at 630 nm. Each determination was completed within
thirty minutes from the time of death. With each set of

determinations, ammonia standards were run.

Blood Urea Nitrogen

 

Blood urea nitrogen was determined using an
automated technique. The determinations were done on
a technicon 2 channel auto analyzer by the method of

Marsh g£_§l, (1965).

RESULTS

ENZYME STUDIES

 

Lysine to Homocitrulline

 

Table 3 gives the result of the radioisotope
assay using 014 - Lysine. This table shows that the
conversions of lysine to homocitrulline only occurs
in the presence of carbamyl phosphate. In this
experiment, lysine concentration was held constant at
0.030 M.

Because the radioisotope experiments consistently
showed a carbamyl phosphate dependence, this reaction
(Figure 5) was assayed colorimetrically in an attempt to
demonstrate linear response to protein and time. Figure
6 shows the results of the reaction when lysine (15mM)
and carbamyl phosphate (20mM) concentrations were held
constant and protein was increased. Figure 7 gives the
results of the same reaction with ornithine as substrate.
When the two figures are compared, it is seen that
approximately 35 times as much protein is needed for

the lysine reaction. Figure 8 shows that the conversion

36

 

omo.om
Ame.mm
AoN.mH

mam.a

ocficwwuooaom

mea.ao
ssn.nn
e~o.ms
Nao.~

ounsfia Hoe muaooo
onaaaouuwooaom

z o~H.o
z omo.o
z occ.o

0.0

coaumuucooaoo
moanewonm Heamnumu

 

unanwmquEOMI Ho can
onwaasuuaooaomlqao ou moan Aleao mo cowmuo>aoo

m” mamdy

 

37

m muowfim

 

 

mafiaasnufiu
moow
mmzuunm
mAwmov .A
am + _ 5! «mongoose Hmamnumo
mz
.
ono
_
Nmz
wcaaaapofiooaom
EOOQ
mmzlunm oumnmmocn Hmamnumu
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m + _ .I _
m2 ono
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on Nmz
_
mmz

macro <NMDINZHIHHsz NEH Q24

mquwo <mmalmsz>A nah mo monHo<mm A<HHHZH mmH

mcfieuacno
moou
mmzuwum
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mfiamhg

mooo
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sake

mz

38

ea

coauouunooaoo oshuco
we coauoaom m mm onwaanuufiooaoe ou nowmuo>ooo ocamma

no ouowfim

oumcowoaon no as 000H\m0H x cwououm no w:

«a

NH

0H

m

0

q

 

«.0

«.0

0.0

0.0

lnoq/aurttninroomoq go satown

39

acaumuunoonoo maenao
mo coauooow m we osuaaouuwo ou :oamuo>aoo measuasuo

oumamwoaos no as 0m\aaououm wo wo

00¢ 00m 00m 00H

an muowfim

 

 

0H

0N

0m

0w

Jnoq/BUIIIHJQIO JO satoNn

40

mean 00 coauocom m on mafiaaouufiooaos ou coemum>aoo mafimkg

monocfia cw mEHu cowumnooaH

00 0h 00 on 00 0m 0m

“0 muowfim

0H

 

 

0.q

0.0

0.~H

0.0a

JaArt go mBJS/aurtjniqroomoq go sanNn

41

of lysine to homocitrulline increases linearly with
time. The lysine concentration was 15mM and the
carbamyl phosphate concentration was 20mM. When
ornithine is used as substrate (Figure 9) it is seen
that maximum activity is reached at 40 minutes.

Figure 10 is a graph of the results of an experiment
with increasing concentrations of lysine. The carbamyl
phosphate concentration was 20 mM. The reaction ran
for 30 minutes with 400 ul of 1:20 liver homogenate.
Figure 11 shows the results of the same experiment
when ornithine is substrate. Again, as with time and
protein, it is evident that the rate of conversion of
lysine to homocitrulline is very low compared to the
ornithine to citrulline reaction. The uMoles of
citrulline produced per hour with ornithine as substrate
is 21.38; with lysine as substrate 0.223 uMoles of
homocitrulline are produced. The rate of activity,
therefore, when lysine is substrate is approximately

1/100 that of ornithine.

Lysine to Homoarginine

 

Table 3 shows the results of an experiment where
the conversion of C14 - Lysine to C14 Homoarginine was
measured. It can be seen from the data that this conversion

is dependent on carbamyl phosphate concentration. It, however,

42

oEHu mo coauocom m mm mafiaashuuu ou dogmuo>aoo moanuaauo

00

On

wwufiga d5” 05%“ COfiuflDfiUa—H

00

on

00

00

ON

"a ouowam

0H

 

 

000m

0000

0000

0000

JaArI go mezS/aurttninro JO saronn

43

cOHumuucmocoo oCHmzH mo oOHuoasw m we aoHumau00 ooHHHsuuHoosom "0H muome

Ha\o:HmmH mo moHozo

0N 0H NH 0 q

 

m.~

0.0

0.5

a 0.0H

m.~H

0.mH

 

iaArI go mezB/inoq/aurttnlnroomoq JO satown

44

COHomuucmocoo moHSOHsuo mo oOHuocnm m mm GOHumauow ooHHHouuHo “HH muomHm

He\mcH£ucho mo mmHoz:

0N 0H NH 0 q

 

000m

0000

0000

. 0000

0000H

 

JBAII go mBJB/lnoq/BUIIIDJJIO JO satown

45

was not possible to demonstrate a dependence on protein
concentration. Because there is no ureido carbon label
in lysine the complete cycle from lysine to urea cannot
be demonstrated by this assay method. Therefore, assays
using colorimetric methods were established in an
attempt to demonstrate an enzymatic conversion of

homoarginine to urea (Figure 12).

Homoarginine to Urea

 

Figure 13 shows the results of an experiment in
which homoarginine concentration was held constant (250mM)
and protein was increased. The results indicate a
linear dependence of this conversion on protein. When
these results are compared with the results in Figure
14, it is seen that the rate of conversion of homoarginine
to urea is approximately 1/30 of that when arginine is
substrate. Figure 15 shows that the conversion of homo-
arginine to urea increases linearly with time. The
concentration of homoarginine was 0.250 M and each reaction
tube contained 100 ul of a 1:20 liver homogenate. Figure
16 is the same reaction with arginine as substrate (0.250 M).
The reaction tubes contained 20 ul of a 1:20 liver homogenate.
These results again show that the conversion of homoarginine
to urea is very slight when compared to the conversion of
arginine to urea. Figure 17 is a graph of the results

obtained when protein and time were held constant and urea

46

NH muomHm

 

 

ochH0u<
manucho 0000
N _
000% 02:01:
_
mmznolm + mop: AMI, mANmov
_ _
mANmov mz
_ _
Nmz 0
.
~02
oaHnHmumoaom
ochmg 0000
moan _
N _ mz .
:Zuonm + _ A e Ammuv
_ one _
«Awmuv _ mz
N _ NE _
:z mzno
_
Nmz

macro <mmnlmszHHzmo mmH nz<

mgowo <mmblmszwH mmH zH monHo<mm H<sz mmH

47

00H

oumcmmoaon 00 Ho 0~\:Hmuouo mo 0:

0NH

nOHumuuamoaoo oshuam
mo coHuocom o no mow: ou :OHmuo>aoo ooHanumosom

00

00

00

"MH ouome

 

 

«.0

0.0

0.0

0.0

0.H

inoq/eain go satown

48

mo GOHuooow m we won: ou aonno>noo maHaH0u<

00H

:oHumuuaoonoo mahuao

oumaowoao: 00 H: 0N\aHououn mo 0:

0NH

00

00

00

"0H muome

 

 

0H

0H

0N

mm

00

00

inoq/sain go satown

49

oaHu mo GOHuoc=0 m mm no»: on oonum>ooo ooHnkumoaom "0H muomHm

mononHa :H oEHu GOHumnnocH

00 on 00 00 00 00 ON 0H

 

000

000

000

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000H

000H

00HN

 

JaArI go mBJS/eain go satoun

50

mEHu mo cOHuucom m mm won: on GOHmum>coo mcHaH0u< "0H ouome

mmchHB GH mEHu GOHumnaocH

00 on 00 00 00 00 ON 0H

 

00

A 02

00H

00m

00m

 

JBAII go m913/g01 x earn go satown

51

GOHomuucmocoo mcHanumoaon mo GOHuoaom m on GOHumEuow mop: ”NH ouome

Ha\oaHaH0umoaon mo moHozo

00m 00m 00H 00H 00

 

00m

000

000

000

000H

00~H

 

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000H

 

JBAII go mBJS/Jnoq/ealn go satoun

52

production was determined for increasing concentrations
of homoarginine. It is seen that maximum velocity is
approximately reached at 0.250 M concentration of
homoarginine. Figure 18 shows the results of this

same experiment when arginine is substrate. Here it

is evident that the maximum velocity is reached at a
concentration of 0.200 M. When velocities are compared
for the two substrates, it is again shown that the speed

of the reaction is approximately 1/30 of that of arginine.

Homocitrulline to Urea

 

Table 4 demonstrates the conversion of homocitrulline
to urea. This conversion was measure by observing
homocitrulline depletion and the formation of urea was
confirmed by detecting the difference in duplicates
with and without urease. The results are shown for two
concentrations of homocitrulline. (These results were
obtained using the ATP regeneration system.) It is
evident that there is a significant depletion of
homocitrulline in the presence of protein. There was
only slight or no depletion measurable when urease
was not present which indicates that urea was interfering
with the colorimetric determination of homocitrulline.

In the presence of urease, the color production was
reduced and an accurate measure of homocitrulline depletion

(free from urea interference) was obtained. The

53

SCH UGHUGQUGOU

00w

ochHmum mo aoHuoodw m mm aoHumauom mom: “0H ouome

00N

Ha\oaH0um mo moHoz:

00H

00H

00

 

 

 

00

00H

00H

00N

00N

laAII go mBJS/Jnoq/ OI x vain go satoun

54

 

 

NH.H mq.H 0.0 0.0 moHozd 0.0

00.0 00.H q.~ N.m mmHozn 0.0
mGHHHDHuHooEom
oumuum0m< oz 094 oz mommy: uoonuHS mummy: nuHB museumnom

Ho>HH mo uszo3 um3 emH0\.H:\0oumHmo0 mmHozd

 

NQHD Cu mfiHHHSHUfiUOEOW MO COfimH0>GOU

q mHm<H

 

55

table also shows that when ATP or aspartate is removed
from the medium only a slight depletion in homocitrulline
was detectable. Unfortunately, due to the lack of
sensitivity in the assay, it was not possible to obtain

a response to increasing protein. It appears as though

depletion is only measurable at maximum activity.

ANIMAL FEEDING EXPERIMENTS

 

Food Consumption and Growth

 

The effects of the two treatment diets on growth
and food consumption in adult rats are given in Table 5.
The food consumed is a mean calculated from nine rats
in each group. It is a total measure for the 10 days
on the diets. The rats in the 60% protein diet
consumed less food than either the control (22% protein)
group or the high lysine group. The high protein diet
had a much reduced sugar content and its consistency
was very dry compared to the other diets. It can be
seen that although the high protein group consumed less
food, it did have an increased protein intake. The final
weight of the animals in the three groups was similar.

This weight is tabulated as a mean weight per rat.

56

aH shaman

 

 

QCHanumosom
WOOD wumhgfih
Nmznwum mooo
q Amie—00 + mom A 1
.1 a
mzuw 000%
a
0Hom oHaHooomochHwnmoao: ocHHHsuuHooaom
moou . mumnumam< moon
Ham + Az< + mmznwum zoom Nmznwnm
«Ammwv “A ma< + Nmznwnm + «Ammbv
mooo m“ New my
muw.||.zuw meow ouw
a... a. a.
meow

mzHHHDMHHUQZOS ho ZmHHom<Hmz QMH<HDUmm0 mmH

57

 

2.2. w mmm 3.: a Om 3.8 u. mmm 3 ”.8 use 332., H22

 

0H.m 00.0H 0H.0 hm0\umu\mamu0
oxmucH aHououm
HNHN M. Nmm 2.3 fl mmm HH.0H M. 00m ammo oH\umu\mamuw
vasomaou 000m
0 m m wumm mo nonasz
oaHmmH N0 + chuoum NNN aHououm N00 cHououm NNN

 

mumHQ oomu000H0 ooHnH so mumm mo nu30u0 0am GOHuQESmaoo 000m

0 MHmflH.

 

58

Urea Cycle Enzyme Activities

 

The effects of the high protein diet on the activity
of the reactions of the ornithine urea cycle and the
lysine urea cycle are listed in Table 6. The results
are tabulated according to the substrate used in the
assay. All assays were run using liver homogenates,
therefore, the activities listed only represent the
ability of a liver homogenate to catalyze the reactions.
Lysine, homocitrulline, and homoarginine are not
necessarily to be considered as substrates of ornithine
transcarbamylase, argininosuccinic acid synthetase,
or arginase. The high protein diet significantly
increased the activity of ornithine transcarbamylase
when ornithine was the substrate. The difference from
the 22% protein group was significant at P < .01.

On the high protein diet when lysine was the substrate,

the ability of the liver homogenate to convert lysine

to homocitrulline also was significantly increased

(P < .01) when compared to the 22% protein group. The
increases detected are proportionately the same for both
substrates. The conversion of citrulline to argininosuccinate
also showed a significant (P< 0.01) increase in rats on

the high protein diet. This increase is proportionately

the same as those for ornithine and lysine. There was no

significant increase in the ability of the liver homogenate

59

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60

to convert homocitrulline to urea. The arginine to
urea reaction is significantly increased (P< 0.01)
in rats on the high protein diet. When homoarginine is
the substrate, the activity is also significantly
increased (P< 0.01). These increases are again
proportionate to each other and to those detected
for the other substrates.

The effects of the high lysine diet on the activity
of these same reactions are also listed inTable 6.
When considering ornithine urea cycle substrates, the
level of activity drops significantly for both the
ornithine and citrulline reactions, while the level
of activity for the arginine reaction increases.
All of these findings are significant at P<: 0.01.
The ability of the liver homogenate to convert the
homocompounds of the urea cycle increases significantly
or remains at the control level for all three homo-
compounds. There is no significant decrease for any
of these three assays as there was for two of the
ornithine urea cycle substrates. The increase for
homocitrulline is significant at P< 0.05 and for
homoarginine at P < 0.01. The increase seen for lysine
is not significant at P<L0.05 but the change in activity
is in line with both homocitrulline and homoarginine.
Figure 20 graphically depicts these results as the

percentage of the difference from the control.

61

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62

Serum Determinations

 

Serum urea nitrogen and ammonia nitrogen were
determined on all three treatment groups (Table 7).
There were no significant changes in blood ammonia
nitrogen in either the high protein or high lysine
groups. It is interesting to note that the blood
urea nitrogen significantly increased in both
treatment groups. The increase in the high lysine
group is significant at P4: 0.05.

Protein determinations on the livers of all the
animals were found not to show any significant

differences between the treatment groups.

63

 

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DISCUSSION

In early 1969, as the result of a screening
program of all the institutionalized mentally retarded
in the State of Michigan, an individual was detected
who excreted an excessive amount of citrulline. This
patient has citrullinemia, an inborn error of urea
biosynthesis (Scott-Emuakpor'ggngl., 1972). This
patient has normal urea and ammonia concentrations
in the blood, and normal urea excretion, in the
presence of an enzymatic error in the Krebs-Henseliet
urea cycle. These findings, along with the blood and
urine amino acid patterns, were the basis for
speculating about an alternate pathway of urea
synthesis that utilizes the homocompounds of the
substrates of the classic urea cycle in reactions
parallel to those of the ornithine urea cycle. The
results of the experiments of Scott-Emuakpor (1970)
supported the existence of this alternate pathway. The
present research was undertaken in an effort to gain
further insight on this hypothesized mechanism of urea

production.

65

ENZYME STUDIES

 

An attempt was made to demonstrate the overall
lysine - urea cycle by demonstrating the conversion of
C14 - lysine to 014 - homoarginine. Although the
results of that assay were supportive of the cyclic
nature of the speculated reactions, the assay system
was not sensitive enough to demonstrate the individual
reactions. Because of this, each individual reaction
was studied using colorimetric assay techniques. In

this way, the existence of the overall cycle can be shown

by demonstrating each individual reaction.

Lysine to homocitrulline

 

Gerritsen g£_§L, (1963) believed that homocitrulline
was not synthesized by the body or formed by intestinal
bacteria. The results reported here demonstrate the
production of homocitrulline from lysine. The reaction
was shown to be linearly dependent on protein, carbamyl
phosphate and lysine concentrations. These data are in
agreement with the findings of Scott-Emuakpor (1970).

The data indicate that the enzymatic conversion of
lysine to homocitrulline demonstrated by Ryan g£_§l, (1968)
probably proceeds by a transcarbamylation. Ryan EE El:

did not demonstrate the carbamyl phosphate dependence of

66

this reaction in their studies. Marshall and Cohen (1963)
found a very minor activity for ornithine transcarbamylase
with lysine as substrate but Colombo (1971) could not
detect any activity for OTC with lysine as substrate.

The studies by Colombo were done on human liver while those
reported here were done on rat liver. It is, therefore,
possible that the human liver does not have the capability
of transcarbamylating lysine to homocitrulline. Colombo
did not describe his technique for the assay and therefore
it is possible because of the minor conversions noted in
these experiments that his technique may not have been

sufficiently sensitive to detect the activity.

Homocitrulline to Homoargininosuccinic Acid

 

This reaction has not previously been demonstrated
in any tissue of any organism. Ryal E£Héi° (1968) could
not detect any metabolism of homocitrulline in rat liver.
The conversion of homocitrulline to homoargininosuccinic
acid is indirectly shown by the results of the experiments
reported here. The conversion is assayed as the depletion
of homocitrulline and the formation of urea is inferred
by a depletion of color after the addition of urease. In
addition, evidence that this reaction proceeds by way of
homoargininosuccinic acid is the finding of Strandholm it. E-

(1971) that homoargininosuccinic acid is formed in an

67

enzymatic reaction from fumarate and homoarginine. The
reaction demonstrated by Strandholm 2E.fll: is the third

reaction in the postulated lysine - urea cycle.

Homoarginine to Urea

 

Arginase has been shown to cleave homoarginine
to urea and lysine (Ryan g£_sl,, 1968, Scott-Emuakpor,
1971). This reaction occurs in rat liver and also with
commercial arginase (bovine). Colombo was unable to
demonstrate this conversion in human liver. The results
of the experiments described here confirm the ability
of rat liver to metabolize homoarginine to urea.

It appears that from the results of these experiments
and those published in the literature that all of the
hypothesized reactions of the lysine - urea cycle can
be shown to occur. Because the evidence gained supports
this alternate mechanism of urea synthesis, animal feeding
experiments were designed to gain further information
on the enzymes and adaptive characteristics of the lysine -

urea cycle.

68

ANIMAL FEEDING EXPERIMENTS

High Protein Diets

 

The results obtained in the studies presented here
show that the reactions of the ornithine - urea cycle
increased in activity in rats on the 60% protein diet.
The activities of two of the lysine - urea cycle
reactions also increased although the homocitrulline
reaction showed no significant change. This is the
first reported experiment on the effect of high protein
diets on the speculated lysine - urea cycle reactions.
The effect of high protein diets on the ornithine -
urea cycle reactions has previously been reported
(Schimke, 1962, Nazum and Snodgrass, 1971) and the
results found in the experiments here compare favorably.
The noted response of the lysine - urea cycle enzymes
may be an indication that the individual reactions are
indeed cyclic. Although the activities were determined
22 giggg, the findings of increased blood urea nitrogen
and normal serum ammonia are indications that the EEHXEEEQ.
activity is a measure of the 1§_giyg activity. The
increased blood urea nitrogen can be assumed to reflect
increased urea production because in animals on high

protein diets the glomerular filtration rate is increased,

69

thereby, eliminating the possibility of increased blood

urea nitrogen due to decreased filtration rate.

High Lysine Diets

Relationship of lysine and arginase

 

It is known that lysine inhibits arginase ig_yi££g_
(Hunter and Downs, 1945). However, the results of the
experiments presented here indicate that arginase
activity is increased in rats fed high lysine diets.

That this increased activity occurs iguyiyg is in doubt
because the increased blood urea nitrogen may not be

due to increased urea production but to impaired renal
function. There is no evidence in rats on the effects

of high lysine on renal function. However, in patients
with hyperlysinemia it has been shown that renal

function is normal (Colombo g£_§l,, 1967) giving evidence
that blood urea nitrogen may be indicative of increased
urea production. If this is assumed, then the experiments
presented here show an Lgflyiyg_stimulation of arginase

by lysine, which would seem to be in conflict with the
findings of Colombo (1971). He described a patient

with hyperlysinemia, high arginine, and hyperammonemia
and explained these findings by showing an i§_ylyg
inhibition of arginase. His patient, however, was

described as having an abnormally functioning arginase.

70

Although this is the first study in rats on the activity
of arginase after lysine feeding, studies on chick livers
have shown that arginase activity increases when the diet

is high in lysine.

Significantly Increased Blood Urea Nitrogen

The increased concentration of urea noted in the
blood of rats fed the high lysine diet occurred in the
presence of significantly decreased ornithine
transcarbamylase and argininosuccinic acid synthetase
activities. This increased urea concentration could
have arisen from one or a combination of the following:

1) The lysine diet may have impaired renal
function, thereby causing increased blood
urea nitrogen. There are no data available
in rats to indicate that this is or is not
the case. If it is assumed that the lysine
diet did not affect the kidney function,
then the increased blood urea nitrogen
is a measure of urea production and may
be explained by the following (2,3, and 4).

2) The enzymes of the ornithine - urea cycle
which are significantly reduced in activity
13 yiggg are still capable of producing
normal levels of urea because of increased

substrate forcing the reactions or due to

71

the increased arginase activity balancing

the reduced activity of the other enzymes.

It has been found that ornithine is indeed

increased when the diet is high in lysine

(Shu-heh Wang SE 3L., 1973).

3) The direct transamidination of lysine to

homoarginine by argine - lysine amidinotransferase.

This reaction is present in rat kidney (Ryan

ggngL., 1969). The homoarginine is then

cleaved to lysine and urea by arginase which

was shown to have a significantly increased

activity for this substrate, in the

experiments reported here.

4) Lysine is converted to homocitrulline

which is in turn metabolized to homoarginine.

The homoarginine thus produced is cleaved

to urea and lysine. The experiments reported

here have shown an ignyiggg increase in

activity for homocitrulline and homoarginine

metabolism.
Assuming that renal function is normal, an increase in urea
production per day per rat of 368 mg must be explained.
Making calculations from the activities of the reactions of
these mechanisms, at most 100 mg of this increased urea can
be explained by the above. Because of this, it appears as
though the noted increase in urea is due to a combination of

these mechanisms or a mechanism as yet unknown.

72

Normal blood ammonia in the presence of reduced

 

activities in the Ornithine - Urea Cycle.

 

This finding could have arisen due to any of the

following mechanisms or any combinations thereof:

1)

2)

3)

Ammonia could be'detoxified by any of

the other reactions as discussed in the
Literature Review.

The ornithine - urea cycle although reduced
in activity is still capable of detoxifying
ammonia. It is known that there is a
greater than usually necessary capability
of the urea cycle enzymes to detoxify
ammonia (Hutchinson and Labby, 1965),
therefore, a partial reduction in urea
cycle enzyme activities will not lead to
hyperammonemia.

Ornithine could be excreted in a higher
concentration than normal due to its
possible buildup because of reduced
ornithine transcarbamylase activity.

This in itself could serve as an ammonia
detoxication mechanism. As noted
previously, ornithine is increased in the

blood when lysine is increased in the diet.

4)

73

In the presence of a defective ornithine

urea cycle, the lysine - urea cycle could
enhance in activity and therefore function

as an ammonia detoxication mechanism. The
results here did show an increase in the
activity of two of the reactions of the lysine
urea cycle and at the same time decreased
activities in ornithine transcarbamylase

and argininosuccinic acid synthetase.

EVIDENCE FOR THE LYSINE - UREA CYCLE

 

From the previous sections of the discussion it is

obvious that there is substantial evidence supporting

the existence of the lysine - urea cycle. The following

is a summary of that evidence.

1)

2)

3)

The demonstration of the ability of a

rat liver homogenate to catalyze the
individual reactions of the hypothesized
cycle.

The demonstration of the concerted response
of these reactions when lysine is fed to

rats in higher than normal concentrations.
This coupled with (1) indicates the possible
interrelationship of the individual reactions.
The demonstration of an increased

concentration of blood urea nitrogen when

74

the activity of the ornithine urea cycle
is decreased.

4) The demonstration of normal blood ammonia
when the activity of the urea cycle is
decreased.

5) The findings associated with patients who
have a defect in the ornithine - urea
cycle (This is discussed in the Literature

Review).

SPECULATION ON THE ENZYMES

 

Although no conclusive evidence as to the enzymes
that catalyze the reactions of the lysine - urea cycle
has been found, the results of these studies do lend
themselves to some interesting speculation. The data
suggest that at least a portion of the lysine - urea
cycle is catalyzed by distinct enzymes other than those
for the ornithine - urea cycle. This is suggested
because of the increased activity for lysine and
homocitrulline when the decreased activity for
ornithine and citrulline when rats are fed high lysine
diets. It is possible that the same enzymes are
catalyzing the reactions but that there are different
active sites for the two substrates or the two substrates

are competing for one site. The data also suggest that

75

the two cycles join at a common point, namely arginase.
When rats are fed high lysine diets increased activity
for arginine as well as homoarginine occurs. Whatever
the case, it is evident that lysine is capable of
stimulating the reactions of the lysine urea cycle.
Although these data only represent evidence for
this alternate pathway of urea biosynthesis in the rat
liver, it should initiate studies on these reactions
in human tissues. It is possible that this pathway,
although very minor in the rat, is capable of
maintaining normal blood ammonia and of synthesizing
normal amounts of urea in patients with urea cycle
disorders. It is possible that given the right
environmental stimulus, this minor pathway could be
enhanced. Indeed, it has been shown in these experiments
that lysine stimulates the activity of these reactions
in a very short period of time. This, therefore, could
be a possible treatment for individuals with inborn
errors of the urea cycle. This points to the need for
gaining further knowledge of the lysine - urea cycle

in humans.

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