THE BIOCHEMICAL CHARACTERIZATION 7

OF A PECUIJAR KIND OF CITRULLINEMIA '
(AN INBORN ERROR 0F METABOLISM IN MAN):
A POSSIBLE NEW PATHWAY FOR UREOGENESIS

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
AIOVI B. SCOTT- EMUAKPOR

1970

L 1323/1 R ,
Michigan State
University

J

   

   

This is to certify that the

thesis entitled

THE BIOCHEMICAL CHARACTERIZATION OF A PECULIAR
KIND OF CITRULLINEMIA (AN INBORN ERROR OF METABOLISM IN MAN):
A POSSIBLE NEW PATI'MAY FOR UREOGENESIS

presented by

Ajovi B. Scott-Elmakpor

has been accepted towards fulfillment
of the requirements for

_Ph_-D_o_degree in Zoology

<_~/J “V ‘ 52:“)

Major professor
Herman M. Slatis

 

Date July 16; 1970

0-169

ABSTRACT

THE BIOCHEMICAL CHARACTERIZATION OF A PECULIAR
KIND OF CITRULLINEMIA (AN INBORN ERROR OF METABOLISM IN MAN):
A POSSIBLE NEW PATHWAY FOR UREOGENESIS

by

Ajovi B. Scott-Emuakpor

A mentally retarded middle aged white male, who has excessive
citrulline, homoarginine and lysine in his blood and excretes large
amounts of citrulline, homocitrulline, ornithine, homoarginine and
(occasionally) arginine in his urine, has been analyzed biochemically
for the purpose of characterizing his syndrome. The assumption has been
made that all of the aminoacidopathies found in this patient are the
result of a single genetic defect in his metabolism: the deficit or
complete absence of argininosuccinic acid synthetase, an enzyme that
catalyzes the conversion of citrulline to argininosuccinic acid. This
syndrome appears to be distinct from all other citrullinemics because
of his abnormal pattern of amino acids and because, unlike the
previously described citrullinemics, he has normal ammonia levels in
his serum. His serum citrulline levels are always more than 60 times
normal, and his urinary citrulline levels are always many thousandfold
above normal under various dietary conditions. Evidence from urea and
creatinine clearances show that his kidney function is normal. He
excretes normal amounts of urea. Both of his parents, his only sib

(a brother), and his two nieces and a nephew were available for analysis,

Ajovi B. Scott-Emuakpor

which failed to Show any similar defect in the family. Analysis of
the pedigree showed low likelihood of consanguinity.

An attempt to explain the patient's complex defects led to a
series of biochemical assays using rat liver. These assays have
revealed what could possibly be a minor secondary pathway for ammonia
diSposal and for urea synthesis. This pathway involves the enzymatic
conversion of lysine to homocitrulline in the presence of carbamyl
phosphate. Homocitrulline is then converted to homoarginine in an
enzymatic reaction that requires ATP and aspartic acid. The aspartic
acid dependence indicates that an unstable intermediary, homoarginino-
succinic acid, exists between homocitrulline and homoarginine. The
homoarginine is apparently then hydrolyzed to urea and lysine. Thus,

a lysine-urea cycle, with substrates that have an extra CH group

2
relative to substrates of the ornithine-urea cycle, is thought to exist.
With this cycle, it was possible to understand the biochemical
characteristic of this patient as well as several other previously
unexplained phenomena observed by other workers in patients manifesting
errors of the urea cycle. In the patient observed in this study, the

secondary pathway probably enables him to avoid serious continuing

ammonia intoxication.

THE BIOCHEMICAL CHARACTERIZATION
OF A PECULIAR KIND OF CITRULLINEMIA (AN INBORN
ERROR OF METABOLISM IN MAN):
A POSSIBLE NEW PATHWAY FOR UREOGENESIS

by
05

c

c"

0‘)
Ajovi B> Scott-Emuakpor

A Thesis

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology

1970

To my Family
and
To my Teachers
and Friends who have made my
academic endeavors

intellectually stimulating

ACKNOWLEDGMENTS

I thank my professor, Dr. Herman M. Slatis, for his help. I
have found his originality and his critical academic outlook an in-
spiration and a guide to my own scientific approach. I also wish to
thank Mrs. Gay Slatis for her interest and well-wishes.

Special thanks are due to Dr. James V. Higgins for his effective
supervision and encouragement throughout this work. Special thanks
are also due to Dr. Arthur F. Kohrman of the Department of Human
Development for the many hours he spent with me planning experiments
and executing them, for making his laboratory available for most of
the crucial work, and for his untiring advice and financial support of
this research. I also thank Dr. Richard L. Anderson of the Biochemistry
Department for patiently listening to my problems and providing very
adequate advice, while serving on my dissertation committee.

I thank Dr. and Mrs. C. S. Thornton for making my stay at Michigan
State University a very pleasant one.

I thank Dr. Isak Berker and Mr. John Secord of the Lapeer State
Home and Training School for their help and cooperation. I also thank
Beverly Showerman, Dr. Dorice Czajka Narins, Dr. Thomas Helmrath,

Dr. James Trosko and Jan Perreault for their excellent assistance.

For their help, friendship and encouragement, I specially thank

Dr. Francis J. Chlapowski of the Biological Laboratories, Harvard

University, and Michael Abruzzo, Larry Yotti, Nancy Noble, Pat Alvord,

ii

Pat Jolly, Patty Voss, Astrid Mack, Habib Fakhrai, Frankie Brown,
Lou Betty Richardson, Stella Cook, Terry Hassold, Carole Sack, Ron
Riemer, Maurice Borone, Allan Bancroft, and Rachel Rich.

Life in the U.S. would not have been the same but for the parental
gestures of Mr. and Mrs. Warren Fowler of Pontiac, Michigan. I am
grateful to the entire family for their love.

I am grateful to the University of Nigeria for encouraging me to
pursue graduate studies in the United States.

This work was partially supported by grants from the American
Health Education for African Development, Inc., and the Society of

the Sigma Xi.

iii

TABLE OF CONTENTS

Page
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . ii
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . vi
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . vii
LIST OF APPENDICES . . . . . . . . . . . . . . . . . . . . . . . ix
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . 1
REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . 14

Identification of Error . . . . . . . . . . . . . . . . . . 14
Test for Mucopolysaccharides . . . . . . . . . . . . . . . . 14
Test for Keto Acids . . . . . . . . . . . . . . . . . . 14
Test for Compounds with Disulfide Bonds . . . . . . . . . . 15
General Amino Acid Screen . . . . . . . . . . . . . . . . . 15
The Subject . . . . . . . . . . . . . . . . . . . . . . 18
Analysis of the Subject's Metabolism . . . . . . . . . 19
Amino Acid Determination . . . . . . . . . . . . . . . 19
Ammonia Determination . . . . . . . . . . . . . . . . . 20
Creatinine Determination . . . . . . . . . . . . . . . 22
Urea Nitrogen Determination . . . . . . . . . . . . . . 22
Enzyme Assays . . . . . . . . . . . . . . . . . . . . . . . 23
Ornithine Transcarbamylase . . . . . . . . . . . . . 26
Argininosuccinic Acid Synthetase and Argininosuccinase. 26
Arginase . . . . . . . . . . . . . . . . . . . . . . . 27
The Overall Reaction . . . . . . . . . . . . . . . . . 28
Radio-isotope Assay . . . . . . . . . . . . . . . . . . 28

RESULTS 0 O C C O O C O C O O O O O O O O O O O O O O O O O O O O 32

Generalized Metabolic Screens . . . . . . . . . . . . . . . 32
Amino Acid Screen . . . . . . . . . . 32
Dietary Manipulation of the Patient' 3 Metabolic Processes . 32
Normal Diet . . . . . . . . . . . . . . . . . . . . . . 35
Low Protein Diet . . . . . . . . . . . . . . . . . . . 42
High Protein Diets . . . . . . . . . . . . . . . . . . 42
Enzyme Studies . . . . . . . . . . . . . . . . . . . . . . 46
Lysine to Homocitrulline . . . . . . . . . . . . . . . 46
Homocitrulline Metabolism . . . . . . . . . . . . . . . 60
Radio-isotope Studies . . . . . . . . . . . . . . . . . 6O
Homoarginine Metabolism . . . . . . . . . . . . . . . 69

iv

DISCUSSION .

Hypothesis
The Back-up Urea Cycle .
Relationship of the Lysine-urea Cycle to the Patient' 3 Disorder
Reasons for Normal Urea Production
Evidence Concerning the Two-cycle Hypothesis
The Possible Role of the Lysine-urea cycle

0

The Course of Citrullinemia .

REFERENCES .

APPENDICES .

76

77
78
81
84
84
86
87

89

94

Table

10

ll

12

13

14

15

16

17

18

19

LIST OF TABLES

General metabolic screens

The patient's urea clearance

The patient's creatinine clearance
Urea levels in the urine of patient
The patient's ammonia levels

The patient's serum amino acids under conditions of
normal diet

The patient's urine amino acids under conditions of
normal diet

The patient's urine amino acids under conditions of
low protein diet

The patient's serum amino acid under conditions of
low protein diet

The patient's serum amino acids under conditions of
high protein diet without milk

The patient's serum amino acids under conditions of
high protein diet with milk

The patient's urine amino acids under conditions of
high protein diet without milk

The patient's urine amino acids under conditions of
high protein diet with milk

Lysine-ornithine competition experiment

The conversion of lysine-C14 to homoarginine-C14 as a
function of carbamyl phosphate concentration

Further studies showing lysine conversion to hbmoarginine
as a function of carbamyl phosphate concentration

Experiment showing the purity of the lysine-C14 used in
the previous radio-isotope experiments

Homoarginine formation as a function of carbamyl
phosphate concentration

Table comparing the rate of homoarginine and arginine
hydrolysis

vi

Page
33
36
37
38
4O

41

43

44

45

47

48

49

50

59

61

63

64

66

73

Figure

5a

5b

8a

8b

9a

9b

10

11a

11b

12a

LIST OF FIGURES

The ornithine-urea cycle

The pattern of separation of amino acids by descending
paper chromatography

The pattern of separation of amino acids by automatic
amino acid analyzer

Absorption curve for citrulline and homocitrulline
Paper chromatographic separation of the patient's urine
and serum amino acids compared with a normal subjects

(Ninhydrin stain)

Over-dip of chromatogram in Fig. 5a with Ehrlich's
reagent

Lysine conversion to homocitrulline as a function of
enzyme concentration

Ornithine conversion to citrulline as a function of
enzyme concentration

Homocitrulline formation as a function of lysine
concentration

Citrulline formation as a function of ornithine
concentration

Homocitrulline formation as a function of carbamyl
phosphate concentration

Citrulline formation as a function of carbamyl phosphate
concentration

The course of lysine-—e. homocitrulline reaction as a
function of time

Lineweaver-Burk plot for lysine._+, homocitrulline
reaction

Lineweaver-Burk plot for ornithine ——e.citru11ine
reaction

Lineweaver-Burk plot for carbamyl phosphate in
homocitrulline formation

vii

Page

17

21

25

34

34

52

52

53

53

54

54

55

56

56

57

12b

13

14a

14b

15a

15b

16a

16b

17
18

19

Lineweaver-Burk plot for carbamyl phosphate in citrulline
formation

Histogram showing homoarginine formation as a function
of carbamyl phosphate concentration

The course of homoarginine hydrolysis as a function
of enzyme concentration

The course of arginine hydrolysis as a function of enzyme
concentration

The course of homoarginine hydrolysis as a function of
incubation time

The course of arginine hydrolysis as a function of
incubation time

The course of homoarginine hydrolysis as a function of
homoarginine concentration

The course of arginine hydrolysis as a function of
arginine concentration

Lineweaver-Burk plots for arginine and for homoarginine
The back-up urea cycle (lysine-urea cycle}

The lysine-urea and the ornithine-urea cycles

viii

57

68

7O

7O

71

71

72

72

75

79

83

LIST OF APPENDICES

Appendix Page
1 The Pedigree 97
2 The Karyotype 99

ix

IN TRODU CT ION

 

Within the last few decades, our knowledge of the relationship
between biochemical errors and mental retardation has increased enormously.
There is considerable evidence that most of the biochemical errors are
inherited, hence the name "inborn errors of metabolism" is applied to
them collectively. Although we have made great advances in this area
of genetics, only a tiny fraction of the mentally retarded population
has problems that can be associated with known errors of metabolism, and
our knowledge of the exact relationship between most of the metabolic
errors and mental retardation is still not clear. Most of the enzymatic
processes that lead to these errors are still not properly delineated.
The relationship of the primary defects to other metabolic intermediaries
is still a matter of considerable speculation. The possibility of
back-up pathways in place of blocked primary pathways has not been
adequately explored. It is for these, and many more reasons, that the
study of biochemical genetics has attained its present level of interest.

Diseases of the urea cycle fall into this category of poorly
understood inborn errors of metabolism, perhaps most of which are
associated with mental retardation. We do not know a lot of things
that we should know if we are to help these individuals in adapting
to their own unusual internal environment. The urea cycle is believed
to be the only pathway for the biosynthesis of urea in man, and hence

for the disposal of ammonia, which would otherwise reach toxic levels.

A mentally retarded patient has been found who has an apparent
urea cycle disease known as citrullinemia (a block, partial or total,
in one of the steps in the urea pathway), and yet he has normal
ammonia levels in his serum and is able to synthesize normal amounts of
urea. Besides having enormously elevated amounts of citrulline in his
blood and urine, he has high levels of closely related amino acids.

The purpose of this study is to characterize this syndrome, to
try to explain the reason for normal blood ammonia levels and normal
urea production despite an apparent block in the pathway for ureogenesis,
and to try to answer some of the questions posed by the abnormally high

levels of some amino acids.

REVIEW

AMMONIA DISPOSAL

Unlike fats and carbohydrates, which are oxidized to water and
carbon dioxide that are easily excreted, man hydrolyzes proteins to
produce various nitrogenous compounds - amino acids. One of the
metabolic fates of the amino acids is deamination, with the consequent
production of ammonia.

Ammonia must be kept very low in the blood of man because of its
toxicity when sustained at levels higher than lOOpg/lOOml (Conway and
Cooke, 1939). Thus, ammonia has to be excreted as fast as it is
produced or else converted to some less toxic compound. In most
mammals, very little ammonia is excreted directly, but it is converted to
urea, which is less toxic and more readily soluble in water.

The liver of man contain an enzyme, arginase, which hydrolyzes
the amino acid arginine to urea with the formation of another amino
acid, ornithine. Ornithine can be converted through three steps back
to arginine, thus forming a complete cycle (Fig. l). Ammonia enters
this cycle at two points. One is in the formation of a fairly high
energy phosphate compound, carbamyl phosphate, which combines with
ornithine to form citrulline. The second is in the formation of glutamic
acid which, through aspartic acid, combines with citrulline to form
argininosuccinic acid in the presence of ATP (adenosine triphosphate).
Thus, this cycle constitutes an effective way of ammonia diSposal in

man.

 

 

 

1“"2
$30 NH3 + C02 + 2ATP
NH, NH2 [1]
C-NH UREA
32:: 'r'" rs: I
(ClH2)3 NH2 I'NIHZ
H’CflNH2 (C|H2)3 $=O
COOH H--f.‘.-NH2 OH PO4
Arginine COOH
(EOOH T Carbamyl phosphate
CH Ornithine
HE \[41 Pi
|
COOH 2]
Fumarate
COOH 'f'H2
NH CH E'0
I 2 I 2 NH
CBN—ClZ—I-I I
ITIH COOH (Cleh
H‘C-NH
i \[3] Citrulline
COOH
Argininosuccinic acid Glutamic e— NH
COOH / acid 3
AMP + PPi ATP I
Ch
H---C|'I--NH2
COOH
Aspartic acid

Fig. 1 :

[I] Carbamyl phosphate Synthetase
[2] Ornithine Transcarbamylase
[3] Argininosuccinato Synthetase

[4] Argininosuccinase
[fl Arginase

The Urea Cycle (Simplified)

( Ratner at al., 19530, l953b, 1953c)

INBORN ERRORS OF THE UREA CYCLE

Defects of the five enzymes known to take part in the process of
ammonia conversion to urea (see Fig. l) have been reported in man,
except for arginase (enzyme 5). Phenotypically, all four errors are
associated with ammonia intoxication; the blood ammonia being markedly
elevated, usually after a meal of protein, but sometimes even in the
fasting state (Efren, 1967). Affected patients are usually ataxic, often
with a history of seizures, and most have severe mental retardation. A
biochemical analysis is necessary for the purpose of distinguishing the
four types.

Deficiency of the enzyme carbamyl phosphate synthetase (enzyme 1)
has been suggested for patients with hyperglycinemia (Efron, 1967). As
would be expected, this deficiency leads to the accumulation of ammonia
in the blood and consequent ammonia intoxication. Of particular
interest is the excessive accumulation of glycine, which plays no known
role in the synthesis of carbamyl phosphate. As of now, only one
confirmed case of a hyperglycinemic patient with carbamyl phosphate
synthetase deficiency has been reported. However, because of the
hyperammonemia found in all hyperglycinemic patients, it has been
suggested that this is the enzyme defect present in all of the patients.

A famdly having three members (an identical twin set and their
cousin) with hyperammonemia and no amino acid abnormality was described
by Russell 25.31. (1962). They were found to be deficient in the enzyme
ornithine transcarbamylase (OCT) (enzyme 2). One member of the twin

set and the cousin were severely affected, and both died of this condition.

Enzyme assays done on liver biopsies from both of them gave very
perplexing results (Levin, 1967). One of the patients showed only

ten per cent OCT activity when compared with normal subjects. The other
showed two defects - five per cent of normal activity of OCT and twenty
per cent of normal activity of carbamyl phosphate synthetase. Of
particular interest is the lack of elevation of ornithine in the plasma
or urine of the patients. This is understandable because of the
possibility of ornithine conversion to glutamic acid and proline (Roloff
g£‘31., 1940). An increase in blood glutamine was found in these
patients, which could be due to the stimulation of glutamine production
because of ammonia accumulation in the blood (Tigerman and MacVicar,
1951).

Citrullinemia, which results from the deficiency in the activity
of argininosuccinic acid synthetase (ASA synthetase) (enzyme 3), has
been confirmed in two patients (McMurray g£_al,, 1962; Morrow gt al.,
1967). This is perhaps the best documented of all the defects of the
urea cycle. McMurray _£H_l. found the defect in a mentally retarded
child whose parents were first cousins. Citrulline levels were markedly
elevated in the blood, cerebrospinal fluid (CSF), and urine. There was
an insignificant elevation of some neutral amino acids which could be
the result of renal competition with citrulline for clearance (Webber,
1962). Urinary citrulline for this patient was over 1000 times that of
normal subjects. The patient's plasma citrulline was about 40 times
normal and his CSF citrulline level was about 50 times normal (Efron,

1966). Enzyme assays from a liver biopsy showed this patient to be

lacking in the enzyme ASA synthetase (McMurray e£_§l., 1963; Mohyuddin
_£__l,, 1967). The case described by Morrow 23 a1. (1967) was that
of a 21-month-old female who showed all the clinical symptoms of a
urea cycle disease. Upon the analysis of some urinary and plasma
constituents, it was found that the patient was a citrullinemic with the
high ammonia levels which are often found to be associated with all urea
cycle defects. Morrow's patient was interesting from the point of
view that she produces very little urea (the significance of this
comment will become obvious later). Her ASA synthetase was found to be
defective in cultured skin fibroblast cells (Tedesco and Mellman, 1967).
They found that at very high citrulline concentrations, the synthesis
of arginine was normal, but at low citrulline concentrations, arginine
synthesis was only tenper cent of normal. The Michaelis constant (Km\
for citrulline in her cell lines was between 25 and 250 times that in
normal cell lines. These results suggest very strongly that the defect
is only a partial block of the enzyme activity. There are no data
available on the activities of the urea cycle enzymes of a third
possible case from Northern Ireland (Carson and Neill, 1962). The
parents of this patient were thought to be first cousins (Efron, 1966).
A defect of the splitting enzyme, argininosuccinase (enzyme 4)
leads to the accumulation of argininosuccinic acid (ASA), resulting in
the disease argininosuccinicaciduria. This defect has been reported in
about thirteen patients, some of whom have shown very severe mental
retardation and hyperammonemia, while others show mild retardation and

one case approaches normal intelligence (Efron, 1966). The first cases

reported were by Allan 23.21. (1958). They described two siblings

in a family of six children who excreted very high levels of a
ninhydrin-positive compound in their urine. This compound was later
identified as argininosuccinic acid (Westall, 1958, 1960). Whereas
normal subjects do not excrete identifiable amounts of ASA, these
patients excreted about 3000 mg per day in their urine. The CSF concen-
trations of ASA in these patients were more than two times the plasma
level - about 4 mg per 100 cc in plasma and 9.5 mg per 100 cc in CSF
(Levin gtflgl., 1961). Other patients were subsequently described with
the same condition (Dent, 1959; Carson and Neill, 1962; Coryell gt 31.,
1964; Armstrong 25 31., 1964; Moser gt_al., 1967). One phenotypic
feature which has regularly been found in argininosuccinicaciduria

but not in the other urea cycle defects is that the patient's hair was
short, grew irregularly, gave a tufted appearance, and seemed to break
off easily (Efron, 1966). Scriver (1962) reported that Dent has identi-
fied the hair lesion histologically as "trichorexis nodosa."

The enzyme argininosuccinase, which has been found to be active
in the red blood cells of normal subjects, was found to be completely
absent in three argininosuccinicaciduric patients, and to be at about
half the normal level in both parents of two of these patients (Tomlinson
and Westall, 1964). When a citrulline or ornithine load was administered
to these patients, there was a corresponding increase in ASA excreted.
These results, therefore, justify the conclusion that this disease is

due to blockage of argininosuccinase.

EFFECT OF THESE DISEASES ON UREA PRODUCTION

All the cases of carbamyl phosphate synthetase deficiency, OCT
deficiency, and argininosuccinase deficiency that have been described
excrete normal amounts of urea. Of the two cases with ASA synthetase
deficiency that have been studied in detail, one excretes normal amounts
of urea and the other has defective urea production (Morrow g£_al., 1967).
In an attempt to explain the reason for normal blood urea in the presence
of an obvious deficiency of the enzyme that catalyzes one of the steps
of the known urea cycle, many investigators have proposed interesting
speculations.

One of these speculations is that the enzyme defect is a partial
one; and that urea synthesis is carried out along the normal pathway,
but that the rate of synthesis is insufficient to get rid of the ammonia
rapidly. This point seemed to have been supported by Tedesco and Mellman
when they found that at very high citrulline concentrations, the skin
fibroblasts from a citrullinemic patient are able to synthesize normal
amounts of urea; but at low citrulline concentrations, these same skin
fibroblasts only had ten percent efficiency. Their data on enzyme
kinetics is also in agreement with this point. They found that the
Km value for citrulline in citrullinemic patients was between 25 and
250 times as much as the value in normal subjects. This means that
fibroblasts from citrullinemic patients need between 25 and 250 times
more citrulline than those from normal subjects in order to have normal
urea synthesis. However, the hypothesis that normal urea production in

many urea cycle defects is due to partial enzyme activity has major

lO

weaknesses. One weakness is that the patients with argininosuccinase
deficiency whose red cell enzyme activity was investigated, have been
found to have zero activity (Tomlinson and Westall, 1964; Maser _£.a1.,
1967). This means that the enzyme block is complete and yet they synthe-
size normal amounts of urea. Another major weakness of this hypothesis
is the fact that it was formed to explain the results for the only
patient who has defective urea production, although he has more than

50 times the normal level of plasma citrulline.

Another speculation is that the enzymes of the urea cycle exist
in different organs as isoenzymes and that each isoenzyme is genetically
independent. By this hypothesis, each one of the biochemical defects
reviewed above exists in at least one organ, but not in all organs.
Indeed, it has been suggested that in argininosuccinicaciduria, two
genetically independent argininosuccinase isoenzymes exist: that one
exists in the brain and the other in the liver and that while the brain
enzyme is affected, the liver enzyme continues to function (Dent, 1959;
Westall, 1960). No experimental evidence was found which lends support
to this hypothesis.

A third speculation has to do with the presence of a completely
new pathway for ureogenesis. This speculation was made by Russell
._£._1. (1962) when they described a patient with OCT deficiency who has
normal levels of urea in his plasma. Since that time, it has been

found that homoarginine can be hydrolyzed to urea by arginase, the same

enzyme that hydrolyzes arginine to urea (Ryan gt 31,, 1968, 1969).

11

The same workers have found that the source of homoarginine as well as
homocitrulline is lysine. They found that kidney enzymes can convert
lysine to homoarginine by transamidation, but the mode of lysine
conversion to homocitrulline was not known. Thus, when rats, as well

as human subjects, are given a lysine load, there are corresponding
increases in the urinary homoarginine and homocitrulline levels (Ryan
and Wells, 1964). However, they were not able to demonstrate the
conversion of homocitrulline to homoarginine, and hence were not able to
prove the existence of a true cycle.

Support for the existence of an alternate cycle may be found in
lysinemia, a disease that sometimes has symptoms of the urea cycle de-
fects (Colombo 23 31., 1967). They described a patient with ammonia in-
toxication who had elevated lysine in both his blood and urine. Following
an oral load of lysine, there was an enormous elevation of the patient's
plasma ammonia. This elevation of the plasma ammonia level following
lysine ingestion was not observed in the patient's parents, a sibling,
and an unrelated child. During periods of high protein intake, this
patient's plasma lysine rose from 2.7 to 6.8 mg per cent. Associated with
lysine elevation was a rise in plasma arginine from 2.2 to 6.2 mg per cent.
Because of this apparent relationship between lysine and ammonia disposal,
all of the urea cycle enzymes were assayed from a liver biopsy taken from
the patient. All the enzymes showed normal activity. The first step
in the degradation of lysine in man is not very well known, but it is
thought to involve an NAD-dependent oxidation reaction catalyzed by lysine

dehydrogenase (or lysine: NAD oxido-reductase) (Rothstein and Miller,

12

1954a, 1954b). Buergi st 21. (1966) have found this enzyme to be
defective in their lysinemic patient, with one-fourth of the activity
found in normal subjects. The relationship between lysine and arginine
in their patient has led to the belief that lysine is a potent inhibitor
of arginase. The exact mode of this inhibition has not been worked out.
However, in the hyperlysinemic patient described by Ghadimi _£__1,
(1964, 1967) there was no ammonia intoxication. Only 58 per centof the
lysine administered is metabolized by the patient compared with more
than 99 per cent for controls. Homoarginine and homocitrulline were
found in some of this patient's plasma samples, implying that the

pathway(s) for converting lysine to these compounds is (are) intact.

Unfortunately, they did not report the arginine levels of their patient.

INHERITANCE

The family described by Russell gt _1. (1962) with three apparent
cases with OCT deficiency leads to the presumption that the condition,
being familial, is an inherited disease. The mode of inheritance is
still obscure since enzyme assays were not done on any unaffected
relatives.

The first case of citrullinemia (ASA synthetase deficiency)
described was of a child of a consanguineous marriage (Mohyuddin g; 31.
1967). One could postulate an autosomal recessive inheritance because
of this consanguinity. HOwever, preliminary enzyme studies of a
patient in Oregon have shown the enzyme to be deficient in skin fibro-

blasts of one parent and near normal activity in the other parent (Buist,

1970). This could be an indication of dominant inheritance.

l3

Argininosuccinicaciduria (argininosuccinase deficiency) has
been reported among siblings in four families (Allen _£‘_l., 1958;
Carson and Neil, 1962; Armstrong 25.31., 1964; Moser gt al., 1967).

Each one of these cases had zero activity of the enzyme and in each case
investigated both parents had below normal function of the same enzyme.
The familial occurrence of this error is consistent with an inherited
disease, and the subnormal level of enzyme activity in the parents
suggests that they are heterozygous for the allele responsible for

this error. It seems, therefore, that argininosuccinicaciduria is
inherited as an autosomal recessive trait.

The inheritance of lysinemia seems to be the best documented of
all. Ghadimi gt a}, (1964) reported a familial case with no parental
consanguinity. Whody (1964a) described two siblings with the syndrome
who are offspring of an incestuous mating (father-daughter). Another
family with two affected siblings and an affected cousin was also
described, and each one of the affected patients in this case had
consanguineous parents (WOody, 1964b; Woody 25.21'9 1966; Ghadimi gt 31.,
1967). we therefore have strong evidence that this is a genetic error
that is inherited as an autosomal recessive trait.

Careful studies of enzyme abnormalities of the type that lead to
urea cycle defects have almost always indicated that they are recessively
inherited in man. Carriers have reduced enzyme activity, when this can

be measured accurately.

MATERIALS AND METHODS

IDENTIFICATION OF ERROR

Starting in 1967, patients in the state institutions for the
mentally retarded in Michigan have been screened for metabolic errors.
A random sample of urine was collected from each patient and blood
flowing from a finger prick was collected in two capillary tubes. In
this section, methods that are fully described in the cited reference
will not be given in detail; but those that have been modified from
any printed description will be more fully described. Each urine sample

was analyzed for the following:

(1) Mucopolysaccharides:

Two methods for the detection of mucopolysaccharides
were used. The first is a modification of Renuart's method
(1966). One ml of clear urine (centrifuged if cloudy) was
placed in a test tube at room temperature and 1 m1 of cetyl-
trimethylammonium bromide (CTAB) reagent was added. The urine
was allowed to stand at room temperature for 5 minutes. A
cloudy precipitate constituted a positive test. The CTAB
reagent was prepared by dissolving 5 grams of the salt in 100 ml
of l M citrate buffer, pH 6. The second method used is the
Toluidine Blue Spot Test as described by Berry and Spinanger
(1960). various mucopolysaccharidoses are detectable by these

1.288118.

(2) Keto acids and most of their derivatives:
The ferric chloride test described by Renuart (1966) was

used. This test gives positive reactions (usually a greenish

15

color) with urines from patients who have phenylketonuria (PKU),
maple syrup disease, homogentisicaciduria, and tyrosinosis

The 2,4-dinitrophenylhydrazine test was also used for identifying
keto compounds. The technique employed was described by

Penrose and Quastel (1937).

(3) Compounds with disulfide bonds:

The test used was the sodium nitroprusside (sodium
nitroferricyanide) test as described by Carson 35 a1. (1963)
and Knox (1966). Cystinuria and homocystinuria are the most

common diseases detected by this test.

(4) General amino acids:

A simple one dimension chromatographic procedure was
used for the detection of various amino acid abnormalities.
The method employed was a slight modification of that described
by Smith (1960a) and Efron g£_§1. (1964). Between 5 and 70
microliters of each urine (depending on creatinine content)
was spotted along one edge of a 23 x 57 cm Whatman 3 mm paper.
This size paper takes eight samples at a time. The chromatograms
were developed in a descending fashion using butanol:acetic acid:
'water (12:3:5) solvent at room temperature for approximately
16 hours. The chromatograms were then dried in a fume hood for
one hour, after which they were dipped through a ninhydrin/isatin
stain (2.5 grams ninhydrin, 0.1 gram isatin, 1000 cc acetone),

and heated in an oven at 80-900C for 10 minutes. The pattern of

l6

separation of the amino acids is represented in Fig. 2.
Many deviations from the normal pattern are readily identified.
After eye-scanning the ninhydrin positive spots for ab-
normalities, each chromatogram was cut into two just above the
histidine spot. The bottom part was dipped into Pauly's reagent
(described by Scriver, 1964) for evidence of a histidine
abnormality while the top half was dipped into Ehrlich's reagent
(Scriver, 1964) for evidence of proline, hydroxyproline,
citrulline and homocitrulline disorders. The urea content of
each sample can also be crudely analyzed from the Ehrlich's

reaction.

The blood sample was analyzed only for general amino acid disorders.
The capillaries were spun in a centrifuge to separate red cells from
serum and about 10 microliters of serum were spotted on Whatman 3 mm

paper and chromatographed as described for urine amino acids.

17

Fig. 2: The pattern of separation of amino acids by descending paper chromatography

Rf
Scale

68‘

60I

281

20

12

04.

ORIGIW

 

O

i

\.

 

 

00mm

-‘o--.-—*--- 0L--'-’4-¢-‘-&-‘u---.'--’-J--_-J--__-d

Leucine, Isoleucine

Phenylalanine

Tyrosine, Valine
Methionine

Beta—Amino Isobutyric acid (BAIB)
Tryptophane

Alpha-Amino Butyric acid

Proline

Alanine

Glutamic acid, Threonine, Homocitrulline
Hydroxyproline
Glycine

Glutamine, Citrulline, Serine, Homocystine

Argininosuccinic acid
Histidine
Lysine, Arginine, Ornithine

Cystine

 

 

SOLVENT:- Butanol:Acetic ocid:Water (12:3:5)
SEPARATION TIME:- 16 - 18 Hours

18

THE SUBJECT

With the above screening method, a patient was discovered with
a rare aminoacidopathy - citrullinemia.

The patient, R.D., was born in Warren, Michigan, on March 15,
1937. His mother reports that he was a full-term product of an undis-
turbed pregnancy and normal delivery. His growth and development were
apparently normal until, at the age of one, he suffered from what a
doctor diagnosed as encephalitis and spinal meningitis. The symptoms
that led to the diagnosis were attacks of vomiting and profound lethargy.
He was hospitalized in a semi-comatose state. Upon "recovery" he was
no longer able to talk and did not resume talking until the age of
2 1/2, according to his mother's report. A few other attacks followed
this recovery, but only lasted a few hours. Two of these subsequent
attacks followed a breakfast of eggs, toast and milk. The pattern of
attack in each of these two cases was similar, with the patient standing
motionless, growing increasingly lethargic and reaching for support,
his eyes rolling backwards, with mild convulsions following.

A complete physical examination has been given to this patient
in many instances, and each time no physical anomaly was detected.

The patient's IQ was tested in 1967 with the following results:

verbal IQ 53
performance IQ 54
full scale IQ 50

Wais, Bender-Gestalt test

19

The patient is thus classified as operating within moderate range
of retardation.

The patient's maternal grandfather is of Swiss extraction and his
maternal grandmother is of German extraction. On the paternal side of
the patient, his grandfather is of German and Austrian extraction, and
his paternal grandmother is of Polish extraction. Although both the
patient's maternal grandmother and his paternal grandfather have a
similar ancestry, there is no knowledge of any blood relationship between
them. Close consanguinity has been ruled out.

The patient has one brother who was available for biochemical

analysis and was found to be normal. (See complete pedigree in appendix.)

ANALYSIS OF THE SUBJECT'S METABOLISM
To gain information that would lead to an understanding of the
patient's metabolism, he was hospitalized for three weeks and maintained

under each of the following conditions for at least four days:

(1) Normal institution feeding conditions
(2) Very low protein diet conditions
(3) Very high protein diet conditions, with milk

(4) Very high protein diet conditions, without milk

Serum and 24-hour urine samples were collected daily during each

test, and each sample was subjected to the following analysis:

(a) Amino acid determination
The qualitative and quantitative determination of amino acids was

done with the aid of a two-channel automatic amino acid analyzer

20

(Technicon Model). The elution of amino acids was carried out with
0.05M sodium citrate buffer, pH 2.875, pH 3.8, and pH 5.0 (Fig. 3
shows the pattern of amino acid separation). The urine was not pre-
treated before use. 0.2 ml of the urine was applied on the column for
analysis. It was necessary to dilute the urine (1:10) in order to
quantitate citrulline and to apply 1 ml of undiluted urine to
quantitate homoarginine. The serum, however, had to be deproteinized.
About 10 cc of venous blood was drawn in heparin tubes and centrifuged
for about 15 minutes at 300 rpm. The serum was decanted into a
centrifuge tube and 65 mg of sulfosalicylic acid was added for every
ml of serum. After thorough mixing, it was centrifuged for 15 minutes
at 3000 rpm. The supernatant is a protein-free filtrate and 0.5 ml of
this was applied to the column for chromatography. It was necessary to
apply 2.5 ml of serum to quantitate homoarginine.

When these tests were applied to control subjects, the blood was
drawn without anticoagulants and allowed to coagulate before centri-

fugation.

(b) Ammonia determination

Only serum ammonia was determined. This was done daily two hours
following lunch. On the last day of each feeding condition, ammonia
was determined six times - immediately before lunch, then at 30, 60, 90,
120, and 180 minutes later.

Two methods for ammonia determination were employed. The micro-
diffusion method of Conway (1950) involves the liberation of ammonia
with alkali (potassium carbonate), the diffusion of ammonia into an
acidic indicator (mixed indicator containing 0.066% methyl red and

0.033% bromocresol green in alcohol), and the subsequent titration of

A].

Absorbence at 570 nanometers

Absorbence at 440 nanometers

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Fig. 3: The pattern of separation of amino acids by automatic amino acid analyzer

22

the now basic indicator with a very weak acid (0.001 N H01).

The other method of ammonia determination used in this work is
the Betherlot color reaction (Chaney and Marback, 1962) as modified
by Caraway (1966). By this method, proteins were precipitated with
sulfate and sodium tungstate. The ammonium ions in the protein—free
filtrate were coupled with phenol in the presence of hypochlorite to
form an indophenol which gave a blue color in an alkaline solution after
10 minutes of incubation at 37°C. This color can be read in a
spectrophotometer at 630 nanometers. Because of the reaction of EDTA
with copper to form a blue supernatant, EDTA was never used as an

anticoagulant. Blood was therefore drawn in heparin tubes.

(c) Creatinine determination

Creatinine determination was by a method which is essentially that
of Folin and Wu as described by Caraway (1966). This method is based
on the fact that in the presence of creatinine, alkaline picric acid
will form a reddish-brown color which has maximum absorbence in a
spectrophotometer at 520 nanometers. Serum and urine samples were
analyzed in essentially the same way, but the urine sample was diluted

1:100 before use.

(d) Urea nitrogen determination

Blood urea nitrogen (BUN) was determined by a modification of the
method of Gentzkow (1942). The principle involves the hydrolysis of
urea by urease to ammonium carbonate, precipitation of proteins with
sodium tungstate, and the colorometric determination of ammonia using a

simple stabilizing reagent - Nessler's reagent (Connerty 35 21., 1955).

23

The Nessler's reagent was prepared in the following way:
Reagent l: 15 grams of potassium iodide were dissolved in 10
m1 of water. 20 grams of mercuric iodide were added,
stirred to dissolve, and made up to 100 ml with
water.
Reagent 2: 2.50 N carbonate-free sodium hydroxide
Reagent 3: To 80 m1 of Reagent 1, 390 ml of Reagent 2 were added
slowly with mixing. It was allowed to stand for a
day, and the clear supernatant was decanted and
stored as Nessler's reagent.
To 1 m1 of blood, 4 drops of urease solution (prepared according
to Gentzkow's method) were added. 0.5 m1 of disodium phosphate buffer
(3.55 gm anhydrous Na HPO

2 4

added and allowed to stand at room temperature for 20 minutes. Then

in 500 m1 water)and 1.5 ml of water were

5 ml of water, 1 ml of 0.66 N sulfuric acid and 1 ml of 10% aqueous
solution of sodium tungstate were added, thoroughly mixed and centrifuged.
0.5 m1 of the supernatant was taken, 9 m1 of water and 0.5 m1 of
Nessler's reagent were added, allowed to stand for 5 minutes, and read
in a spectrophotometer at 500 nanometers.

Urine urea was determined in the same way as serum urea except
that the 24-hour urine sample had to be diluted 1:10 or 1:100 depending

on its volume.

ENZYME ASSAYS
Liver biopsy specimens were unavailable from this patient.

Appropriate enzyme assays were performed using liver specimens from

24

inbred colonies of Sprague-Dawley and Long-Evans strains of rats. They

were routinely maintained on wayne laboratory block diet until used.
For an experiment, one rat was sacrificed by decapitation, the liver
was quickly removed and placed in a beaker surrounded by ice. One
gram of the liver was weighed out and homogenized in 19 cc of ice cold
distilled water for about 1 minute. Enzyme analyses were made

within the following few minutes. This homogenate gave a reasonable
protein concentration for the assays (Schimke, 1962).

With the exception of argininosuccinase, commercial reagents were
employed. Argininosuccinase was partially purified from cow liver
according to the method of Ratner (1955). All assays were performed
in duplicate, and each assay had its own set of standards.

The assays were based on the colorimetric determination of urea
and citrulline by the method of Archibald (1944) as modified by Ratner
(1955). Homocitrulline was determined colorimetrically the same way as
citrulline with the same reagents. Figure 4 shows that the maximum
absorption of homocitrulline, like citrulline, is at 490 nanometers.
The color reagents were l-phenyl-l, 2-propanedione-2-oxime for urea
determination and 2,3-butanedione-2-oxime for citrulline and homo-
citrulline determinations. The methods used for the assays were
patterned after those of Brown and Cohen (1959) as modified by Schimke
(1962). The unit of activity of the enzymes was expressed as uMoles

of products formed per hour per gram wet weight of liver.

OPTICAL DENSITY

25

Fig. 4: Absorption curve For citrulline and homocitrulline

 

 

 

 

 

1.6 1.
/'k
‘0 4T * ” \\
’ \
I s
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I, ‘\
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x—-— -. "x Homocitrulline

26

ORNITHINE TRANSCARBAMYLASE

This enzyme activity was determined as the rate of citrulline
formation. Whenever lysine was used as a substrate instead of ornithine,
the enzyme activity was measured as the rate of homocitrulline formation.
The assay medium contained 20 pMoles of L-ornithine or L-lysine, pH 8.0
(Sigma Chemical Company), 90 pMoles of glycyl-glycine buffer, pH 8.3,
and the liver homogenate. The reaction was started with 20 uMoles of
dilithium carbamyl phosphate (Sigma Chemical Company). The final
volume was 2.0 ml containing 1.0 ml of the homogenate. Incubations
were at 37.50C for 15 minutes, at the end of which the reactions were
stopped with 2 ml of 30% perchloric acid. A medium treated with
perchloric acid before incubation served as zero time control. For
the ornithine reactions, 150 pLiters of the incubated medium were taken
for citrulline color development; and for the lysine reactions, 1.5 ml
of the incubated medium were taken for homocitrulline color development.
ARGININOSUCCINIC ACID SYNTHETASE (CONDENSING ENZYME) AND ARGININOSUCCINASE

(SPLITTING ENZYME)

The activities of both of these enzymes were assayed together
using two different assay methods. One method contained ATP regenerating
system and the other did not. The assay medium with ATP regenerating
system contained 1.25 pMoles of citrulline or homocitrulline, pH 7.8;
1.25 pMoles of aspartate, pH 7.8; 12.5 pMoles of potassium phosphate
buffer, pH 7.8; 0.5 pMoles of ATP, pH 7.8; 2.5 pGrams of pyruvate kinase;
0.75 uMoles of magnesium sulfate; 2.5 pMoles of phosphoenol pyruvic acid

(PEP); 20 units of arginase; and 50 pLiters of liver homogenate. The

27

final volume was 250 pLiters, and the incubation time was one hour at
37.50C. The reaction was stopped at the end of incubation by the addition
of 1 ml of 15% perchloric acid. A medium treated with perchloric acid
before incubation served as a zero blank. 1 ml of reaction medium was
taken for urea color development. This assay condition was modified from
Schimke (1962).

The assay medium without ATP regenerating system contained
50 pMoles of potassium phosphate buffer, pH 7.0; 5 uMoles of L-Citrulline or
L-homocitrulline, pH 7.0; 5 uMoles of L-aSpartate, pH 7.0; 5 uMoles of ATP,
pH 7.0; 5 pMoles of magnesium sulfate; and 20 units of arginase. The
volume of the above medium was 0.5 ml and 0.5 ml of liver homogenate was
added to start the reaction. Incubation was for 1 hour at 37.50C. At
the end of incubation, 1 ml of 30% perchloric acid was added to stop the
reaction. The zero blank was a medium treated with perchloric acid before
incubation. 1 ml of the reaction medium was taken for urea color deve10p-

ment. This assay technique was modified from that of Brown and Cohen (1959).

ARGINASE

The assay medium contained 250 pMoles of L-arginine or L-homoarginine,
pH 9.8 and 1 pMoles of manganese sulfate (MnSOA) in a volume of 1.0 m1.
In order to increase the activity of the enzyme, 50 pMoles of Mn804 were
added for every ml of homogenate, and this was preincubated for 5 minutes
at 55°C. This gave about an 80 to 125 per cent increase in enzyme

activity (it was not necessary to treat commercially prepared enzyme the

same way). 20 pLiters of preincubated homogenate were added to 1 ml of

28

assay medium and incubated for 15 minutes at 37.500. At the end of the
incubation, 2.5 ml of 15% perchloric acid were used in stopping the
reaction. A medium treated with perchloric acid before incubation served
as zero blank. For the reaction with arginine as substrate, 0.1 ml of
reaction medium was used for urea color development, while for that with
homoarginine as substrate, 1.0 m1 of reaction medium was used for urea

color development.

THE OVER-ALL REACTION

The activities of three enzymes, ornithine transcarbamylase,
argininosuccinate synthetase (condensing enzyme), and argininosuccinase
(splitting enzyme), were measured as the amount of urea produced using
a coupled system that contained excess arginase. The assay system contained
90 uMoles of glycyl glycine buffer, pH 8.0; 20 uMoles of L-ornithine or

L-lysine, pH 8.0; 5 uMoles of ATP, pH 7.8; 2.5 uMoles of MgSO 10 uMoles

4;
of L-aspartate, pH 7.8; and 1 mg of arginase per ml of assay medium.

The reaction was started with 20 uMoles of dilithium carbamyl phOSphate.
Incubation was carried out for one hour in 2 ml of the medium that
contained 1 m1 of liver homogenate. At the end of incubation, the reaction
was stopped with 2 ml of 15% perchloric acid. Zero blanks were gotten

the same way as in the other assay systems.

ATP regenerating system was not used because between 5 and 10

uMoles of ATP proved to be equally as satisfactory.

RADIO-ISOTOPE ASSAY
Because the conversion of L-lysine to urea is inefficient,
the colorimetric quantification of urea was not possible. Therefore,

for the overall reaction, L-lysine-C14 was used as substrate.

29

Activities of the enzymes were measured as amount of homoarginine produced.

Homoarginine was separated by low-voltage paper electrophoresis as

described by Smith (1960b) using 0.53 per cent sodium carbonate buffer,

pH 11.5.

By this method, lysine and homocitrulline moved toward the

anode, and homoarginine moved a short distance from the origin toward the

cathode.

The preparation of reagents and the procedure for the radio-isotope

overall reaction were as follows:

Reagents
(l)
(2)
(3)

(4)

(5)

_ <6)

(7)

100 mM potassium phOSphate (KHZPO ) buffer, pH 7.8;

4
450 mM glycyl glycine buffer, pH 8.0;

150 mM aSpartate, pH 7.8, prepared in Reagent 1

(10 pLiters of this solution contain 1.5 uMoles of aspartate);

100 mM ATP, pH 7.8, 400 mM KCl, and 24 mM MgSO all prepared

4,
in Reagent 1. (10 pLiters of this solution contain 1 pMole

of ATP, 4 uMoles of KCl, and 0.2 pMoles of MgSOA);

200 mM phosphoenol pyruvic acid (PEP) and 50 pGrams/ml

pyruvate kinase, prepared in water. 1 mg of arginase was

added for each ml of assay medium where necessary. (10 pLiters
of this solution contain 2 pMoles of PEP and 0.5 pg of pyruvate
kinase. Where arginase is used, it contains 10 pg);

40 mM carbamyl phosphate prepared in water. (50 pLiters of
this solution contains 2 uMoles of carbamyl phosphate);

300 mM L-lysine, pH 8.0. 548.1 mg of L-lysine were dissolved

in 10 ml of Reagent 2 and adjusted to pH 8.0;

30

(8) Before use, 100 pLiters of Reagent 7 were added to 100
pLiters of L-lysine-Cla (New England Nuclear). (20 pLiters
of this mixture contain 3 pMoles of L-lysine and l uCurie

of L-lysine-Cla).

Procedure
0.5 gm of liver was homogenized in 10 m1 of Reagent 1. 100 uLiters
of the homogenate were added to the reaction tube (5 ml test tube),
followed by 10 pLiters of Reagent 3, 10 uLiters of Reagent 4,
20 pLiters of Reagent 8, and 10 pLiters of Reagent 5. 50 pLiters

of Reagent 6 were added to start the reaction.

The entire reaction medium was incubated for an hour at 37.50C
after which the reaction was stopped by boiling. Preboiled medium served
as zero blank.

After the reaction was stopped, the tubes were centrifuged for
10 minutes and 100 pLiters of medium were spotted on Whatman no. 4 paper
for an electrophoretic run. The dimensions of the paper were 30 x 18 cm
and the electrical current was 3 volts per cm of paper.

Column chromatography was also used for clearly isolating
homoarginine. Technicon resin (chromobeads) were used in a 150 cm column
and 0.05 M sodium citrate buffer of pH 6.8 was used as eluent. One
hundred microliters of incubated medium were chromatographed and at a
flow rate of 0.5 ml per minute, all amino acids were eluted by the
eighth hour. The last amino acid, homoarginine, was eluted between 7 1/2

and 8 hours. Authentic homoarginine was added as a carrier, and a

31

ninhydrin color reaction was allowed to take place before collection, so
that the smallest possible volume that contained homoarginine was
collected. Between 30 and 50 cc volume was required to collect all of the
homoarginine, and 5 cc of the total eluent was added to 15 cc of the
counting fluid (aqueous and room temperature counting fluid) to be

counted.

RESULTS

GENERALIZED METABOLIC SCREENS

The results of the generalized metabolic screens are given in
Table 1. There was no evidence that the patient excretes mucopoly-
saccharides as the CTAB and toluidine blue spot tests were negative.
The ketone tests, ferric chloride and 2,4-dinitropheny1 hydrazine, were
negative, implying that the patient is free of errors like PKU, branched
chain ketonuria, homogentisicaciduria and tyrosinuria, to name the more
common ketone diseases. No common sulfur disease, such as cystinuria and
homocystinuria, was present in the patient, as indicated by the negative

cyanide-nitroprusside test.

AMINO ACID SCREEN

Figure 5a shows the amino acid pattern of the urine and plasma
compared with a normal pattern. The position occupied by citrulline is
indicated. Migrating to the same position as citrulline are glutamine and
serine, but it is easy to identify citrulline (Fig. 5b) because with
Ehrlich's reagent only citrulline will stain from yellow to pink, depending
on its concentration. Urea will stain bright yellow with Ehrlich's
reagent, hence a crude estimate of how much urea is excreted could be made

from paper chromatography.

DIETARY MANIPULATION OF THE PATIENT'S METABOLIC PROCESSES
The patient was hospitalized for three weeks to permit a series of
studies of his responses to specialized diets. For the first seven days

(Monday through Sunday) he was maintained on a normal diet. On the

33

 

Test Result Comments

1. CTAB test No precipitate Patient normal for
mucopolysaccharides

2. Toluidine Blue test Negative Patient normal for
mucopolysaccharides

3. Ferric chloride test Negative Patient normal for
ketonurias

4. 2,4-dinitrophenyl No precipitate Patient normal for

hydrazine test ketonurias
5. Sodium.cyanide - Negative Patient is normal for

Sodium nitroprusside

test

most of the known
sulfur diseases

 

Table 1:

General metabolic screens

34

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eighth day (Monday) he was placed on a low protein diet, which was
maintained for a total of five days (through Friday). One day was then
spent on a normal diet (Saturday), and the next eight days (Sunday through
Sunday) were on a high protein diet, with the first four days without

milk and the last four days with milk. Milk was omitted from the first
four days on the high protein diet because of the large amounts of

citrulline and homocitrulline that it contains.

Urea and creatinine clearances

Each of these two tests was performed on three successive days
while the patient was hospitalized, but on a normal diet. The tests
checked the patient's kidney function to see if any of his biochemical
anomalies could be attributed to poor kidney function. Tables 2 and 3
give the clearance values for urea and creatinine, reSpectively, for each
of the three successive days. The urea clearance values ranged from 91 to
148 ml per minute (normal range is described by Caraway, 1966, as 75 to
125 ml per minute) with an average clearance of 125.6 ml per minute. This
mean value is acceptable as approximately normal. The creatinine clearance
levels ranged from 121.4 to 161.6 ml per minute (normal range is described
by Caraway, 1966, as 100 to 125 ml per minute) with an average clearance
of 142.8 ml per minute. This mean value is by no means unusual. Based
on the urea and creatinine clearances, the patient's kidney can be

assumed to be functioning normally.

Normal diet
The urea levels in the patient's urine are given in Table 4 for

each day of his hospitalization. The patient's average urea excretion,

36

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Vol. of 24-hour Sample Grams per day*

1160 12.18

1705 9.20

1340 8.04

Normal Diet 870 9.57
785 9.03

1600 10.48

740 9.25

850 8.67

1620 8.42

Low Protein Diet 2225 6.68
1620 5.35

1365 4.64

720 normal diet 2'11

1390 9.48

High Protein Diet 1745 8.55
(without milk) 1240 6.08
1305 7.70

1080 6.90

High Protein Diet 1285 8.60
(with milk) 1060 8.05
1430 11.20

 

*Normal values for urine urea - 10 - 15 gm per day

Table 4: Urea levels in the urine of patient

39

9.67 grams per day, is close to the generally accepted normal values
of 10 to 15 grams per day.

The ammonia levels in the patient's serum are given in Table 5.
There are no data for the last two days on the normal diet and for the
day on normal diet following the low protein diet. It appears that on a
normal diet, his ammonia level is generally within an acceptable range
(30 to 80 pg per cent, according to Conway, 1950), although one value
below and several values above this range are also observed.

Table 6 shows the values for the patient's serum amino acids. His
serum citrulline level was 1.474 uMoles per ml, compared with a normal
level of 0.015 uMoles per ml (thus, his level is about 100 times that of
normal). His homocitrulline levels varied by a factor of two, and were
generally above those observed among the controls (one control individual
is a high excretor of homocitrulline, valine, leucine, and isoleucine,
and he has disproportionately contributed to high values for the means
and standard deviations for these four amino acids). There is a wide
variation in the patient's ornithine levels, but the mean value of 0.096
uMoles per ml of serum appears to be within the normal range. The
patient's serum lysine level of 0.379 pMoles per ml and serum arginine
level of 0.133 pMoles per m1 are generally above the values observed
among the controls. Homoarginine was not detected in any control subjects,
but the patient had values ranging from 0.024 to 0.041 pMoles per ml.
This indicates that the patient's homoarginine levels are extremely

elevated.

40

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41

Serum Amino Acids
uMoles per ml
Normal values*

The Patient's Serum Amino Acids
uMoles per ml
(Normal Institution Diet Condition)

 

 

Mean S.D Mean S.D
Aspartic Acid 0.026 0.021 0.025 trace trace trace trace tr.-0.025 -
Threonine 0.502 0.047 0.470 0.404 0.340 0.280 0.240 0.346 0.092
Serine 0.202 0.041 0.958 0.159 0.148 0.130 0.127 0.144 0.015
Glutamic Acid 0.133 0.091 0.100 0.095 0.103 0.170 0.114 0.116 0.030
Citrulline 0.015 0.013 1.400 1.400 1.570 1.570 1.430 1.474 0.088
Proline** 0.271 0.052 - - - - 0.290 0.290 -
Glycine 0.331 0.085 0.207 0.200 0.201 0.240 0.264 0.222 0.028
Alanine 0.498 0.111 0.420 0.530 0.560 0.480 0.558 0.509 0.059
Homocitrulline 0.022 0.036 0.020 0.036 0.043 0.035 0.040 0.034 0.008
Valine 0.312 0.131 0.220 0.260 0.237 0.238 0.280 0.247 0.023
1/2 Cystine 0.099 0.024 0.130 0.130 0.148 0.134 0.160 0.140 0.013
Methionine 0.027 0.010 0.050 0.040 0.034 0.041 0.060 0.045 0.010
Isoleucine 0.103 0.062 0.080 0.063 0.059 0.092 0.084 0.080 0.014
Leucine 0.207 0.110 0.120 0.113 0.119 0.143 0.150 0.129 0.016
Tyrosine 0.069 0.017 0.100 0.100 0.110 0.112 0.093 0.103 0.007
Phenylalanine 0.073 0.022 0.080 0.070 0.090 0.075 0.074 0.077 0.007
Ornithine 0.124 0.036 0.106 0.103 0.093 0.055 0.127 0.096 0.026
Lysine 0.275 0.069 0.330 0.360 0.392 0.440 0.375 0.379 0.040
Histidine 0.130 0.027 0.120 0.116 0.140 0.130 0.160 0.133 0.017
Arginine 0.104 0.018 0.145 0.109 0.137 0.130 0.146 0.133 0.015
Homoarginine 0.000 - 0.031 0.024 0.041 0.030 0.037 0.032 0.006

 

*Normal values were determined from data on 6 men and 4 women who work in the Human
Genetics Laboratory

**The patient's proline peak was obscured by his excessive amounts of citrulline.

Table 6:

Patient's serum amino acids under conditions of normal diet

42

The patient's urinary amino acids under the normal diet conditions
are reported in Table 7. His urinary citrulline is many thousandfold
higher than normal. Homocitrulline, ornithine, arginine and homoarginine
are strikingly higher than normal. His lysine level is in the high range
of normal excretion. Except for ornithine, the amino acids elevated in

the urine appear to be elevated in the serum.

Low protein diet

The patient's urine urea level became progressively lower each day
on the low protein diet, and even continued to drop on the day when he was
returned to a normal diet (Table 4). This result is not unexpected
because of the dependence of urea production on the level of protein
metabolism. 'Like the urine urea, the serum ammonia level dropped on
this diet, until, on the fifth day, levels were too low to be measured
except shortly after eating (Table 5).

Urinary citrulline, homocitrulline, and homoarginine levels dropped
sharply on the low protein diet, but they remained much higher than the
values for normal individuals (Table 8). The urinary lysine, ornithine,
and arginine levels are well within the range set by the normals. In
the patient's serum, only citrulline appears to be unusually high

(Table 9).

High protein diets
On a high protein diet, the normal urine urea levels for this
patient were rapidly restored, but they did not increase to compensate

for the unusually high levels of ingested nitrogen (Table 4). Serum

43

Urine Amino Ac ids
uMoles per day
Normal Values*

The Patient's Urine Amino Acids
uMoles per day
(Normal Institution Diet Condition)

 

 

Range Mean S.D
Aspartic Acid tr.-l74.7 trace trace trace trace trace trace trace tr. -
Threonine 129.6-1558.4 764.5 681.4 702.5 231.0 645.2 721.3 742.4 641.1 185.0
Serine 129.6—1387.1 403.1 473.5 788.6 254.6 321.1 549.5 452.8 463.3 173.6
Glutamic acid tr.-230.4 trace trace trace trace trace trace trace tr. -
Citrulline 0.000-trace 14178 13629 17819 14293 12110 14052 14448 14361 1717.6
Praline 0.000 - - - - - - - - -
Glycine 576.0-3493.6 1911 1679 2285 803.3 2644 2041 2128 1927 580.3
Alanine 129.6-894.0 299.5 323.4 399.8 301.4 483.7 293.6 496.0 371.0 88.8
Homocitrulline tr.-57.6 340.5 246.0 401.8 377.3 499.7 370.9 420.0 379.4 77.4
Valine tr.-72.0 67.4 56.6 56.1 45.2 62.6 31.4 42.9 51.7 12.5
1/2 Cystine 28.8-334.6 143.8 139.7 185.8 197.4 370.9 113.8 97.9 178.4 92.0
Methionine 16.6-61.7 38.2 30.4 26.6 37.0 30.2 28.1 31.0 31.6 4.3
Isoleucine tr.-57.6 134.1 70.5 66.7 41.7 58.9 49.9 51.0 67.5 31.0
Leucine 28.8-85.6 71.6 38.6 47.7 51.8 43.7 75.3 40.8 52.7 14.8
Tyrosine 67.1-232.2 166.8 100.5 226.8 117.0 280.1 153.0 219.2 180.4 64.4
Phenylalanine tr.-100.8 124.4 84.6 92.1 85.7 98.1 121.7 308.8 130.7 80.1
Ornithine tr.-52.7 159.8 83.2 156.4 34.8 182.0 192.3 115.2 131.9 57.1
Lysine 28.8-537.7 410.3 280.7 494.5 252.1 678.6 265.3 364.8 392.3 153.6
Histidine 216.0-2613.3 2641 2056 3395 1628 3793 2433 2672 2660 742.2
Arginine tr.-288.0 686.6 418.1 886.6 220.4 873.5 537.7 816.0 634.1 252.9
Homoarginine 0.000 32.0 51.4 34.1 190.3 103.8 71.8 59.9 77.6 55.3

 

*The "normal values" were estimated from data on two control subjects.
subjects, samples were collected under conditions of very low protein diet and very

high protein diet.

Table 7:

From one of the

Patient's urine amino acids under conditions of normal diet

Urine Amino Ac ids

uMoles per day
Normal Values*

44

uMoles per day

The Patient's Urine Amino Acids

(Condition of Low Protein Diet)

 

 

Range Mean S.D
Aspartic acid tr.-174.7 trace trace trace trace trace trace trace -
Threonine 129.6-1558.4 332.6 216.8 210.6 182.4 353.2 135.1 238.4 86.1
Serine 129.6-1387.1 189.8 200.8 226.8 298.1 152.3 226.6 215.7 48.8
Glutamic acid tr.-230.4 trace trace trace trace trace 196.6 tr.-196.6 -
Citrulline 0.000-trace 6781 5525 4779 4984 4666 4054 5164.7 1009.1
Proline 0.000 - - - - — - - -
Glycine 576.0-3493.6 747.0 714.0 844.0 1024 1047 1066 906.9 158.3
Alanine 129.6-894.0 145.0 164.9 132.1 111.3 184.7 214.3 158.7 37.2
Homocitrulline tr.-57.6 252.2 136.0 128.0 100.9 165.2 82.5 144.1 60.1
Valine tr.-72.0 trace trace trace trace trace trace trace -
1/2 Cystine 28.8-334.6 88.1 51.3 95.2 59.7 66.4 49.8 68.4 19.1
Methionine 16.6-61.7 trace trace trace trace trace trace trace -
Isoleucine tr.-57.6 trace trace trace trace trace trace trace -
Leucine 28.8-85.6 trace trace trace trace trace trace trace -
Tyrosine 67.1-232.2 trace trace trace trace trace trace trace -
Phenylalanine tr.-100.8 trace trace trace trace trace trace trace -
Ornithine tr.-52.7 trace 83.9 32.7 trace trace 46.1 tr.-83.9 -
Lysine 28.8-537.7 164.5 102.0 156.3 148.9 110.2 116.0 132.9 26.6
Histidine 216.0-2613.3 1280 994.5 1170 1023 1597 1102 1194.4 222.7
Arginine tr.-288.0 218.1 162.4 184.7 100.2 220.3 213.6 183.2 46.5
Homoarginine 0.000 31.9 25.6 trace trace trace trace tr.-31.9 -

 

*The "normal values" were estimated from data on two control subjects.
subjects, samples were collected under conditions of very low protein

high protein

Table 8:

diet.

From one of the

diet and very

Patient's urine amino acids under condition of low protein diet

45

The Patient's Serum Amino Acids
uMoles per m1
(Condition of Low Protein Diet)

Serum Amino Acids
uMoles per m1
Normal Values*

 

 

Mean S.D Mean S.D

Aspartic acid 0.026 0.021 trace trace trace 0.019 trace tr.-0.019 -

Threonine 0.502 0.047 0.368 0.170 0.146 0.203 0.366 0.250 0.108
Serine 0.202 0.041 0.033 0.086 0.052 0.138 0.108 0.083 0.042
Glutamic acid 0.133 0.091 0.175 0.064 0.058 0.132 0.220 0.129 0.061
Citrulline 0.015 0.013 1.722 0.972 0.500 1.050 1.300 1.108 0.448
Proline** 0.271 0.058 - - - 0.240 - 0.240 -

Glycine 0.331 0.085 0.232 0.159 0.108 0.240 0.245 0.196 0.060
Alanine 0.498 0.111 0.630 0.408 0.300 0.680 0.686 0.540 0.176
Homocitrulline 0.022 0.036 0.035 0.013 trace 0.011 trace tr.-0.035 -

Valine 0.312 0.131 0.210 0.123 I 0.078 0.180 0.153 0.148 0.051
1/2 Cystine 0.099 0.024 0.144 0.096 0.065 0.140 0.132 0.115 0.033
Methionine 0.027 0.010 0.031 0.014 0.020 0.026 0.015 0.021 0.007
Isoleucine 0.103 0.062 0.059 0.036 0.031 0.047 0.046 0.043 0.010
Leucine 0.207 0.110 0.121 0.070 0.048 0.099 0.076 0.082 0.028
Tyrosine 0.069 0.017 0.194 0.055 0.039 0.065 0.043 0.079 0.064
Phenylalanine 0.073 0.022 0.062 0.038 0.035 0.048 0.061 0.048 0.012
Ornithine 0.124 0.036 0.090 0.062 0.122 0.041 0.053 0.073 0.032
Lysine 0.275 0.069 0.372 0.190 0.075 0.255 0.233 0.225 0.107
Histidine 0.130 0.027 0.170 0.117 0.066 0.105 0.107 0.113 0.037
Arginine 0.104 0.018 0.110 0.070 0.058 0.102 0.128 0.093 0.028
Homoarginine 0.000 - 0.023 trace 0.000 0.000 0.000 0.000-0.023 -

 

*Normal values were determined from data on 6 men and 4 women who work in the
Human Genetics Laboratory.

**The patient's proline peak was obscured by the excessive amounts of citrulline.

Table 9:

Patient's serum amino acids under condition of low protein diet

46

ammonia levels were restored more gradually (Table 5). While on a low
protein or normal diet, the peak serum ammonia level appears to be within
90 minutes of eating, on the high protein diets the ammonia levels rose
continually for 180 minutes, the last observation in each series.

The serum citrulline and homoarginine remained high on both high
protein diets (Tables 10 and 11). The serum lysine is above average on
the high protein diet without milk and increases when milk is added.

The other amino acids appear to be within an expected range, except that
arginine, inexplicably, became quite low on the high protein diet with
milk.

The urinary citrulline, homocitrulline and homoarginine levels are
very high on the high protein diets (Tables 12 and 13). Ornithine is
above the normal range and lysine is in the high normal range. Lysine,
citrulline, homocitrulline, and ornithine may have increased somewhat
during the course of the high protein diet with milk, as if in response
to a progressive change in the patient's metabolism, and, at the same
time, the patient's arginine level appeared to drop. As noted in the
previous paragraph, there is no apparent explanation of the drop in

arginine on this diet.

ENZYME STUDIES
Lysine to homocitrulline

Figure 6 indicates that lysine conversion to homocitrulline is an
enzyme catalyzed reaction. Homocitrulline formation increased approximately
linearly with the protein (enzyme) concentration up to about 80 pGrams

of protein per m1 of homogenate. The response to enzyme concentration

47

The Patient's Serum Amino ACids
uMoles per m1
(Condition of High Protein Diet Without Milk)

Serum Amino Acids
uMoles per m1
Normal Values*

 

 

Mean S.D Mean S.D

Aspartic acid 0.026 0.021 trace trace trace 0.029 tr.-0.029 -

Threonine ‘0.502 0.047 0.444 0.232 0.250 0.280 0.301 0.097
Serine 0.202 0.041 0.136 0.086 0.114 0.153 0.122 0.028
Glutamic acid 0.133 0.091 0.178 0.128 0.214 0.240 0.190 0.048
Citrulline 0.015 0.013 1.280 0.860 1.140 1.510 1.197 0.271
Proline** 0.271 0.052 - - - - - -

Glycine 0.331 0.085 0.266 0.156 0.202 0.252 0.219 0.050
Alanine 0.498 0.111 0.580 0.420 0.606 0.580 0.546 0.085
Homocitrulline 0.022 0.036 0.022 0.038 0.022 0.034 0.029 0.008
Valine 0.312 0.131 0.260 0.214 0.286 0.302 0.265 0.038
1/2 Cystine 0.099 0.024 0.132 0.084 0.118 0.144 0.119 0.025
'Methionine 0.027 0.010 0.060 0.042 0.048 0.024 0.043 0.015
Isoleucine 0.103 0.062 0.110 0.084 0.106 0.084 0.096 0.013
Leucine 0.207 0.110 0.166 0.137 0.162 0.156 0.155 0.012
Tyrosine 0.069 0.017 0.102 0.076 0.095 0.101 0.093 0.012
Phenylalanine 0.073 0.022 0.086 0.058 0.075 0.066 0.071 0.012
Ornithine 0.124 0.036 0.060 0.043 0.058 0.088 0.062 0.018
Lysine 0.275 0.069 0.366 0.260 0.340 0.384 0.337 0.054
Histidine 0.130 0.027 0.144 0.104 0.140 0.147 0.133 0.020
Arginine 0.104 0.018 0.130 0.090 0.110 0.128 0.114 0.018
Homoarginine 0.000 - 0.024 0.048 0.050 0.064 0.046 0.016

 

*Normal values were determined from data on 6 men and 4 women who work in the
Human Genetics Laboratory.

**The patient's proline peak was obscured by the excessive amounts of citrulline.

Table 10:

Patient's serum amino acids under condition of high protein diet without milk

Serum Amino Acids

uMoles per ml

Normal Values*

48

uMoles per m1
(Condition of High Protein Diet With Milk)

The Patient's Serum Amino Acids

 

 

Mean S.D Mean S.D
Aspartic acid 0.026 0.021 trace trace 0.012 0.016 tr.-0.016 -
Threonine 0.502 0.047 0.320 0.356 0.325 0.358 0.339 0.020
Serine 0.202 0.041 0.081 0.102 0.078 0.122 0.095 0.020
Glutamic acid 0.133 0.091 0.188 0.230 0.178 0.191 0.196 0.022
Citrulline 0.015 0.013 1.120 1.167 1.150 1.268 1.176 0.064
Proline** 0.271 0.052 - - 0.271 0.297 0.274 -
Glycine 0.331 0.085 0.153 0.162 0.179 0.163 0.164 0.010
Alanine 0.498 0.111 0.400 0.451 0.378 0.498 0.431 0.053
Homocitrulline 0.022 0.036 0.020 0.022 0.022 0.026 0.022 0.002
Valine 0.312 0.131 0.220 0.213 0.203 0.219 0.213 0.007
1/2 Cystine 0.099 0.024 0.096 0.082 0.088 0.092 0.089 0.005
‘Methionine 0.027 0.010 0.042 0.047 0.038 0.040 0.041 0.003
Isoleucine 0.103 0.062 0.094 0.055 0.065 0.065 0.069 0.016
Leucine 0.207 0.110 0.138 0.130 0.125 0.119 0.128 0.064
Tyrosine 0.069 0.017 0.071 0.085 0.079 0.067 0.075 0.008
Phenylalanine 0.073 0.022 0.050 0.053 0.023 0.039 0.041 0.013
Ornithine 0.124 0.036 0.066 0.037 0.028 0.041 0.043 0.016
Lysine 0.275 0.069 0.420 0.402 0.381 0.393 0.400 0.016
Histidine 0.130 0.027 0.119 0.109 0.126 0.122 0.119 0.007
Arginine 0.104 0.018 0.092 0.060 0.079 0.041 0.068 0.022
Homoarginine 0.000 - 0.055 0.050 0.048 0.052 0.051 0.002

 

*Normal values were determined from data on 6 men and 4 women who work in the
Human Genetics Laboratory.

**The patient's proline peak was obscured by the excessive amounts of citrulline.

Table 11:

Patient's serum amino acids under condition of high protein diet with milk

49

Urine Amino Acids The Patient's Urine Amino Acids
prles per day uMoles per day
Normal Values* (Condition of High Protein Diet Without Milk)

 

 

Range Mean S.D
ASpartic acid tr.-174.7 250.2 trace trace trace trace tr.-250.2 -
Threonine 129.6-1558.4 478.2 145.4 492.1 465.0 783.0 472.7 225.7
Serine 129.6-1387.l 490.3 219.2 326.3 427.8 690.0 430.7 177.7
Glutamic acid tr.-230.4 431.6 304.8 trace trace trace tr.-431.6 -
Citrulline 0.000-trace 13010 5814 10051 11160 18812 11769.4 4741.3
Proline 0.000 - - - - - - -
Glycine 576.0-3493.6 4436 1788 2530 2396 3895 3009.1 1108.6
Alanine 129.6-894.0 442.4 217.8 289.7 483.6 549.4 396.5 138.2
Homocitrulline tr.-57.6 297.8 187.2 266.7 261.4 407.8 284.1 80.1
Valine tr.-72.0 trace trace 36.4 49.9 91.2 tr.-9l.2 -
1/2 Cystine 28.8-334.6 203.0 117.6 97.7 90.5 184.7 138.7 51.7
Methionine 16.6-61.7 trace trace trace 48.9 46.7 tr.-48.9 -
Isoleucine tr.-57.6 63.2 67.8 56.7 142.8 137.3 93.5 42.6
Leucine 28.8-85.6 trace trace 55.1 136.4 139.0 tr.-139.0 -
Tyrosine 67.1-232.2 156.4 43.4 141.4 91.8 182.7 123.1 55.5
Phenylalanine tr.-100.8 52.1 38.6 84.3 88.0 94.4 71.4 24.5
Ornithine tr.-52.7 347.7 95.1 63.8 47.2 223.8 155.5 127.8
Lysine 28.8-537.7 235.3 122.4 309.7 240.0 632.9 308.0 193.5
Histidine 216.0-2613.3 3059 1527 2134 1942 2858 2304.3 640.0
Arginine tr.-288.0 172.7 194.4 385.6 294.6 573.7 324.2 163.3
Homoarginine - 0.000 58.9 67.8 54.5 31.0 90.2 60.4 21.4

 

*The "normal values" were estimated from data on two control subjects.
subjects, samples were collected under conditions of very low protein diet and very
high protein diet.

Table 12;

From one of the

Patient's urine amino acids under condition of high protein diet without milk

50

The Patient's Urine Amino Acids
uMoles per day
(Condition of High Protein Diet With Milk)

Urine Amino Acids
uMoles per day
Normal Values*

 

 

Range Mean S.D
Aspartic acid tr.-174.7 trace trace trace 80.8 tr.-80.0 -
Threonine 129.6-1558.4 266.8 388.7 428.0 870.5 488.5 263.7
Serine 129.6-1387.1 179.8 244.1 156.2 332.8 228.2 78.9
Glutamic acid tr.-230.4 trace trace trace trace trace -
Citrulline 0.000-trace 5189.4 9091.4 12114 18229 11156.0 5501.6
Proline 0.000 - - - - - -
Glycine 576.0-3493.6 950.4 1333 2385 3368 2008.9 1090.0
Alanine 129.6-894.0 140.4 194.7 189.1 329.4 213.4 81.0
Homocitrulline tr.-57.6 99.1 273.1 466.4 712.5 387.7 263.3
Valine tr.-72.0 41.0 38.6 63.7 73.8 54.2 17.2
1/2 Cystine 28.8-334.6 37.0 38.2 65.2 146.6 71.4 51.8
Methionine 16.6-61.7 36.4 31.4 37.8 39.0 36.1 3.3
Isoleucine tr.-57.6 30.3 30.1 37.4 57.9 38.9 13.0
Leucine 28.8-85.6 24.0 39.2 28.4 32.9 31.1 6.4
Tyrosine 67.1-232.2 89.6 98.3 90.0 168.0 111.3 37.9
Phenylalanine tr.-100.8 89.0 73.1 69.4 49.3 70.2 16.3
Ornithine tr.-52.7 109.1 142.0 378.8 664.9 323.7 257.2
Lysine 28.8-537.7 215.5 478.7 475.4 736.5 476.5 212.7
Histidine 216.0-2613.3 853.2 1279 1838 2587 1639.0 749.5
Arginine tr.-288.0 214.4 108.5 90.2 64.1 119.3 65.9
Homoarginine 0.000 86.4 82.3 94.1 98.7 90.3 7.3

 

*"Normal values" were estimated from data on two control subjects.

From one of

the subjects, samples were collected under conditions of very low protein diet
and very high protein diet.

Table 13:

Patient's urine amino acids under condition of high protein diet with milk

51

in the ornithine to citrulline reaction (Fig. 7) is very similar.

The formation of homocitrulline from lysine is not only dependent
on lysine concentration (Fig. 8a), but it is also dependent on the
concentration of carbamyl phosphate (Fig. 9a). In the experiments
summarized in Fig. 8, the carbamyl phosphate concentration was held
constant at 10 uMoles per m1 and lysine or ornithine concentration was
varied from 2.5 to 15 uMoles per ml. For the carbamyl phosphate dependent
reaction, the lysine or ornithine concentration was held constant at 10
uMoles per m1 and carbamyl phosphate concentration was varied from 1.25 to
10 pMoles per m1 (Figs. 9a and 9b).

The ratio of citrulline to homocitrulline formed in the ornithine
or lysine dependent reactions was consistently about 20 at each concentration
of substrate. But in the carbamyl phosphate dependent reactions, this
same ratio ranged from 50 at the lowest concentration to about 130 at
the highest concentrations. The reason for this obvious discrepancy is
not clear.

The course of the lysine reaction as a function of time is presented
in Fig. 10. Fig.11a shows a Lineweaver-Burk plot for lysine. From this

plot, the Km for lysine in this reaction was estimated as 2.23 x 10.3 M

and the Km for ornithine was estimated from Fig. 11b as 1.86 x 10.3 M.
Two Km values were calculated for carbamyl phosphate. For the

lysine reaction (Fig. 12a), the Km was 1.50 x.1d‘2 M and for the ornithine

reaction (Fig. 12b), the meas 2.30 x 10.2 M. Thus, the following

52

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58

reaction may exist:

 

L-lysine + Carbamyl Phosphate Homocitrulline + Pi
T“2
1an N. T=°
(CH ) 2 NH
' 3 4 + l I + Inorganic Phosphate
"0 x ( H )
H-f-NHZ f» 7 i 2 4
°HP03 HC-NHZ
COOH
COOH

This reaction, in all essential respects, is similar to the
ornithine to citrulline reaction.

Experiments have been carried out to determine if there is any
competition between lysine and ornithine for an enzyme. In the
experiments, ornithine concentration was held constant at Km and various
concentrations of lysine were added. The colorimetric method used in
these experiments do not distinguish between citrulline and homocitrulline.
The experimental results indicate that the total product produced
(citrulline plus homocitrulline) decreases as the amount of lysine
increases (Table 14). Considering the difference in Vmax with ornithine
and lysine, these results are consistent with one enzyme (presumably

OCT) acting on both substrates.

59

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coaumuuaooaoo ocammq

60

In Fig. 12, it appears that the rate of lysine conversion to
homocitrulline is substantially increased at small carbamyl phosphate
concentrations. For ornithine conversion to citrulline, it appears
that the reverse is the case (that is, the rate of citrulline production
drops sharply at low carbamyl phosphate concentrations). There is no

apparent explanation for these observations.

Homocitrulline Metabolism

In order to find out if homocitrulline can be metabolized, an attempt
was made to convert it to urea. Using liver homogenate as the enzyme source,
preliminary results indicated that at the substrate concentrations used,
homocitrulline could be converted to urea with the same efficiency as
citrulline conversion to urea. The unfortunate aspect of these experiments
(which were done fifteen times) is that the optical density readings of
the urea color reactions were less than 0.1 for both citrulline and
homocitrulline. Thus the optical density values were in an unreliable
range and little confidence can be placed in these results.

Radio-isotope studies were carried out in order to test for
homocitrulline conversion to homoarginine. Because of the commercial un-
availability of labelled homocitrulline, labelled lysine was used in these
experiments. The test was for the conversion of lysine-C14 to homoarginine-C14
as a function of carbamyl phosphate concentration. Tableljishows the results
obtained. The tests were run in duplicate (El and E2), and the counts were
made after paper electrophoresis, which would adequately separate lysine

from homoarginine.

 

0000000000000 000000000

00E00000 00 00000000 0 m0 000-000000000600 o0 000-000000 00 000m00>000 000 0003000 00009 ”00 0000B

.000000000000000 000000> 300 an 003 000000000600 00 0000000000 "0902

 

61

 

0000 000m 0000 00000 00N00 2N0.o
000N 0000 N000 0N00 qu00 00000 2N0.o
0000 000m 000m 00N00 00000 2N0.o
0000 0000 0000 0000 NNOn 200.0
0000 0000 0000 0000 0000 0N00 200.0
0000 0000 0000 0000 0000 200.0
000 00NN N000 000N 0000 0000 200.0
NO0N NO0N 000N 000m 0000 200.0
000 000N N0~N 0m0N 0N0m 0mNm 200.0
000N mOmN 00mN 0000 0000 200.0
000 quN ~00N ~00N nmNm 000N 200.0
a 0000 0000 N000 0000 0000 200.0
00000 002 mH< oz .000 oz 0000000 0809 0000 N 0 0000000000000
00000>< N 000000000 00600000
00-00

 

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Il'lllll'lllll'hllil1lllli‘IVl
Ill‘"ll.

62

As checks, a zero time control using pre-boiled enzyme, a control
omitting aspartate, and a control omitting ATP were also run. All three
of these checks gave similar results, and all agreed in that the counts
increased substantially with increasing carbamyl ph03phate concentrations.
These data also indicate that aspartate and ATP are both required for
this reaction.

The increase in control counts with increasing carbamyl phosphate
level suggests that some non-enzymatic conversion is taking place. If
this conversion is in the first step of the sequence, lysine +
carbamyl phosphate homocitrulline, the homocitrulline might
have interfered with the counts for homoarginine because these two
amino acids migrate only a short distance from the origin, although
in opposite directions.

Because the electrOphoresis employed in the preceding experiment
might not have given satisfactory separation, column chromatography
was used in clearly isolating homoarginine from every other amino
acid (Table 16). Counts for the zero time control are still very large,
but they remain relatively constant for all three concentrations of
carbamyl phosphate. This supports the postulate that some homocitrulline
was counted with the homoarginine in the earlier experiments. However,
the remaining large counts in the zero time control were still unexplained,
as these counts should have been close to zero.

Experiments were, therefore, carried out to determine the purity

of the lysine-C14 (Table 17). It can be seen that although lysine

63

 

 

Homoarginine*
Counts per Minute
Carbamyl phosphate E Z Average
Concentration 1 2 Zero Time Control Net Count
0.00M 1042 1121 1003 78.5
0.04M 1790 1795 1101 691.5
0.06M 2101 2113 1132 980.0

 

*Homoarginine was separated by column chromatography

Table 16: Further studies showing lysine conversion to homoarginine as a
function of carbamyl phosphate concentration

lll’lllll'l III. I'llll.l1‘l‘|l{".trl

64

 

 

COMPOUND .TREATMENT MEAN CPM
14 ‘
1pc Lysine-C None 965700
14 _
1pc Lysine-C Chromatographed 378/20 _
and Lysine Peak é
Eluted r
14
1pc Lysine-C Chromatographed 1416

and Homoarginine
Peak Eluted

 

378720
965700

Per cent Recovery 0.392 or 39.2%

Table 17: Table of experiment showing the purity of the lysine-CM
used in the previous radio-isotope experiments

6S

elutes about four hours before homoarginine, the homoarginine eluted
contained radio-activity of about the same magnitude as the zero blanks
in Table 16. This suggests that the lysine-C14 used in the preceding
experiments contained some homoarginine or some other compound that
elutes with homoarginine.

For confirmatory purposes, fresh lysine-C14 was purchased and the
assays were repeated. The radioactivity counts in this set of assays
were extremely low, indicating excessive quenching. It was possible
that the quenching could be attributed to the salt content of the
buffer, which was 0.05 M sodium citrate and also 0.6 M sodium chloride.
Therefore, the eluents had to be desalted.

For desalting, 10 m1 of the eluent were passed through a
2.5 m1 resin bed in a short glass column. 30 m1 of water (pH adjusted
to about 6) were used in washing the resin, and 15 m1 of 28 per cent
ammonium hydroxide solution were used in eluting the amino acids. The
clution was done directly into counting vials, following which the
vials were evaporated to dryness in an oven. 15 m1 of dioxane
counting fluid (room temperature) were added before counting.

Table 18 shows the number of counts as a function of carbamyl
phosphate concentration. In order not to exceed the maximum capacity of
the resin, 10 ml of eluent were taken for desalting, and the counts were
corrected for the total amount of eluent observed (31 to 40 ml\ by
multiplying the count by total m1 of eluent divided by 10.

The zero time blanks should regularly have given values of zero
homoarginine (since they were done with preboiled enzyme), but gave

variable counts, all of which happened to be greater than those

 

6
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67

observed for the lowest runs made with arginase present. The unusually

long chromatographic runs needed to extract homoarginine suggest that

most of the count in the zero time blank is due to this amino acid,

and the drop in the count in the presence of arginase confirms this.

The homoarginine might be present due to non-enzymatic conversion

and/or to contamination of the lysine-014. There is no evidence for

a dependence of these processes on carbamyl phosphate, as the

homoarginine counts do not increase with increasing carbamyl phosphate.
The assays for homoarginine show a general increase with in-

creasing carbamyl phosphate concentration, suggesting that there is an

enzymatic conversion of lysine to homoarginine in the presence of

carbamyl phosphate. The relationship is roughly linear (Fig. 13). The

data of Fig. 13 are corrected by subtraction of the count for the zero

time blank from the total count. Because of the variability of the zero

time blanks, and perhaps other variation within the system that cannot

be identified, the data do not fall into as neat a pattern as would be

desired. Nevertheless, the general relationship between carbamyl

phosphate concentration and homoarginine level is readily apparent.

That this is due largely or completely to homoarginine is evidenced by

the fact that the same assays with arginase added give much lower counts

at all concentrations of carbamyl phosphate.

 

Fig. 13:

 

 

 

 

 

68

Histogran showing homoarginine formation as a function of
carbamyl phosphate concentration

Homoarginine counts for any: without Arginase

Homoarginine counts for assays with Arginase

 

 

 

 

 

 

 

 

 

 

5000‘ f
/
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69

Homoarginine Metabolism

It has been found by Ryan, Barak and Johnson (1968) that the enzyme
arginase is capable of hydrolyzing homoarginine to urea. It was earlier
found that arginase is a non-specific enzyme because of its ability to
hydrolyze arginic acid, canavanine, clupein, alpha-N-methyl-arginine,
and octopin (Sumner and Somers as cited by Ryan gt 31., 1968).

Figure 14a shows the relationship between protein (enzyme) concen-
tration and homoarginine hydrolysis using rat liver homogenate. This
relationship appears to be linear up to a concentration of about 180 uGrams
of protein per 20 uLiters of homogenate. Figure 14b shows a similar
response of arginine to enzyme concentration.

The amount of urea produced from homoarginine and arginine as a
function of incubation time is represented in Figs. 15a and 15b,
respectively.

Figure 16a shows urea formation as a function of homoarginine
concentration. The optimum substrate concentration appears to be about
250 uMoles per m1. It appears that there may be a substrate inhibition
above the optimum concentration, as shown in the figure. This apparent
substrate inhibition was not observed for urea formation as a function
of arginine concentration (Fig. 16b).

Both authentic arginase and liver homogenates were used for the
experiments summarized in Table 19. This table gives the ratio of the
amount of urea formed from arginine to the amount of urea formed from
homoarginine. This ratio varied from 40 (when authentic arginase was used

and the substrate was at optimum concentration) to 70 (when the

 

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74

concentration of substrate was 50 uMoles per ml and liver homogenate
was used). The value of 40 is probably the most accurate one because
of the possibility of interference from several several endogenous

factors present in liver homogenate.

From Table 19, Lineweaver-Burk plots were constructed as shown

2 II’T‘ -.

in Fig. 17. The Km value for homoarginine was estimated as 12.7 x 10- M

and that for arginine was estimated as 4.60 x 10.2 M.

Thus, the hydrolysis of homoarginine to urea apparently is an

enzyme catalyzed reaction.

 

"—

 

imz
NH
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Homoarginine NHZ Urea

75

 

 

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w" - - -X Lineweaver-Burk plot for homoarginine ,’I
[*I
1.5‘ [X
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/ —
1,0' ’1’ x Km for Homoarginine = 12.7 x 10 2M
I
>> .
’I
‘. 1’
i I,
. .r'
.---4
l/
4 I
I, i/ = 0.47
,’l Vm Km/V = slope = 5.55x iO-BM
0.5‘ ’ m
.” . Km for Arginine = 4.60 x iO-ZM

 

0004' 0008‘ 0.012 0.016 0.020
V51 mM

Fig. 17: Lineweaver-Burk plots for arginine and for homoarginine

DISCUSSION

 

This research was undertaken to determine the nature of the
anomaly of a particular mentally retarded patient, R.D. The patient
was first recognized as biochemically unusual because of the large
amount of citrulline detected by chromatography in his urine, and
further studies indicated that he also excretes unusually large amounts
of ornithine, homocitrulline, and homoarginine in his urine and lysine,
citrulline, and homoarginine in his blood. Excessive arginine was
occasionally excreted by the patient. He appeared to have a unique
syndrome because none of the two carefully described cases of
citrullinemia had this large variety of amino acid abnormalities. The
other described cases did not seem to represent instances of inadequate
analysis because they had been described as having large amounts of
ammonia in their blood and as suffering from ammonia intoxication,
which is not true for this patient.

It could be assumed that all of the unusual characteristics of
the patient should be ascribed to a primary defect in his metabolism.
The most probable defect appears to be a deficit or complete absence
of argininosuccinic acid synthetase (condensing enzyme), which would
explain the large amounts of citrulline in his blood and urine. Analysis
of both of the patient's parents, his only sib (a brother), and his
two nieces and a nephew has failed to indicate any similar defects
within the family. The patient shows normal creatinine and urea

clearances, which are indicative of a normal kidney function.

til|||lillu i ll“ {“iii‘r i

will ( [I .(.I.l.l

77

HYPOTHESIS

This analysis of the patient leads to the hypothesis that his
major defect is the deficit or complete absence of ASA synthetase. This
results in citrullinemia and the citrullinemia causes the other amino
acid defects that have been observed in him.

The following constitute an explanation of the biochemical and
physiological relationships of all of the patient's defects (see Fig. 1):

(1) As a result of the enzyme defect, citrulline accumulates in

the serum to a level of over 60 times normal. This citrulline

 

is cleared by the kidney, giving rise to the profound
citrullinuria noticed in the patient (always many thousandfold
above normal).

(2) Because of citrulline accumulation in the serum, the synthesis
of more citrulline from ornithine is greatly inhibited.
The level of serum ornithine remains approximately normal,
which suggests that this excess ornithine is rapidly cleared
by the kidney to produce the patient's hyperornithinuria
and some ornithine may also be metabolised through other
pathways (Roloff _£._l°' 1940).

(3) Ornithine competes with lysine (which is homoornithine) for
renal clearance (Scriver, 1967). The apparent hyperlysinemia
observed in the patient could therefore be due to the

excessive amounts of ornithine he clears at the expense of

lysine.

78

(4) Ryan and Wells (1964) have shown that when normal subjects
are given a lysine load, excessive homocitrulline and
homoarginine are excreted in the urine. In the series of
experiments reported here, it has been shown that lysine
can be enzymatically converted first to homocitrulline and
further to homoarginine. These enzymatic reactions have
been found to be dependent on the presence of carbamyl
phosphate, ATP and aspartate. Ryan.g£.§l. (1969) have
shown that there is a direct transamidation of lysine to
homoarginine. Thus, the large amounts of homoarginine in
the patient's serum probably can be accounted for as the
result of two separate processes. Apparently, serum
homocitrulline levels are maintained near normal by a
combination of clearance by the kidneys and metabolism to
homoarginine.

(5) The occasional hyperargininuria could be due to the com-
petition for arginase by homoarginine at the expense of
arginine. The resulting excess serum arginine is thus

rapidly cleared by the kidney to give rise to hyperargininuria.

THE BACK-UP UREA CYCLE

In this study, experiments with rat liver have suggested that an
alternate cycle for urea synthesis exists, adding evidence to an
hypothesis that has been in the literature for several years. The proposed
cycle (Fig. 18), lysine-homocitrulline-homoargininosuccinic acid-
homoarginine-lysine, would be parallel to the well-known ornithine-urea

cycle (Fig. l).

79

NH + C0 + 2ATP

   
  

 

 

 

 

 

THZ 3 2
NH 2 f: O
I NH2
Kreb's (f’ N“ UREA NH2
cycle irlH l
C=O
l (CH2)4 NH '
| 2 OHPO4
H"C'~NH2 ( H2)4
l l Carbamyl phosphate
Homoarginine
$00“ COOH Pi
EH Lysine = HomoomlthmeL
H NH
0 ' 2
OOH $30
Fumarate . ———T llflH
COOH
| (CH2)4
II\IH2 C|IH2 l
C|I= N' (II-H
NH COOH
l <—
(CiH2l4
H-c-NH2 $00“
I AMP + PPi (IIHZ
COOH
L— H-C'NH
. . . . 2
"Homoargumnosuccnmc" ? l
acid COOH
Aspartic acid

Glutamic acid

1

NH3

Fig: 18 THE BACK-UP UREA CYCLE

80

It has been shown in these experiments that lysine can be
converted to homocitrulline in an enzymatic reaction that is dependent

on carbamyl phosphate concentration. Thus,
Lysine + Carbamyl phosphate-—————9» Homocitrulline

Studies with radio-isotopes have suggested that lysine, in the
presence of carbamyl phosphate, ATP and aspartate, can be converted to
homoarginine. This reaction is distinct from the previously described

transamidation of lysine to homoarginine (Ryan t 1., 1969). Thus,

Lysine + Carbamyl phosphate + ATP + ASpartate-——~<::::; Homoarginine

Fumarate

The fact that aspartate is required for this reaction implies that
an intermediary in addition to homocitrulline exists before homoarginine
is reached. This intermediary would presumably be homoargininosuccinic
acid (HASA), a compound that would be similar to argininosuccinic acid

but with one extra CH group. The assumption that fumarate is produced

2
in the process is based on our knowledge of the chemical behavior of
argininosuccinic acid.

Assays carried out to determine the metabolic fate of homo-
citrulline have suggested the possibility of its conversion to urea in
what appears to be an enzymatic reaction that is dependent on ATP.

It has been known for some time (and confirmed here) that the
hydrolysis of homoarginine by the enzyme arginase yields urea. Following

the hydrolysis of arginine, urea and ornithine are produced. By the same

token, it would be proper to assume that when homoarginine is hydrolized,

81

lysine (which is homoornithine) is produced as well as urea. Thus,
Homoarginine Urea
Lysine

Thus, the experiments reported here appear to confirm the
existence of the complete cycle, lysine to homocitrulline to HASA to
homoarginine and back to lysine. Since the synthesis of carbamyl
phosphate is the major first reaction in the disposal of excess blood
ammonia, this cycle can serve in ammonia removal from the system.

This hypothesized alternate pathway implies that homoornithine
(lysine) is a secondary substrate in place of ornithine, homocitrulline
in place of citrulline, HASA in place of argininosuccinic acid, and
homoarginine in place of arginine. By comparing reactions of the
ornithine-urea cycle to those of this lysine-urea cycle, it appears that
the efficiency of the latter is at most 5 per cent that of the former.
This creates serious doubts as to the physiological significance of the

lysine-urea cycle.

RELATIONSHIP OF THE LYSINE-UREA CYCLE TO THE PATIENT'S DISORDER

The interrelationships of the various aminoacidopathies reported
in the patient have been explained. There is evidence from his mother's
report, that this patient may have suffered from ammonia intoxication as a
child. The absence of excessive amounts of ammonia in the patient's blood
suggests that he became adapted to his condition by developing ways to
regulate his ammonia levels. With the data at our disposal, it is

possible to advance some theories about the ammonia regulatory mechanism

82

of this patient (see Fig. 19).

(l)

(2)

(3)

The data suggest that the defective enzyme is ASA
synthetase (3a in Fig. 19). It is possible that the
secondary substrates are acted upon by the same enzymes
that act upon the primary substrates. If this is the case,
then the reaction catalyzed by enzyme 3b should also be
defective. This means that we cannot hypothesize that
this patient is getting rid of his ammonia via the
lysine-urea cycle, but rather by spilling large amounts of
citrulline and homocitrulline in his urine. Indeed, this
patient excretes more citrulline in his urine than any
previously described citrullinemic.

Another possibility is that, although substrates of both
cycles are acted upon by the same enzymes, the inability
to utilize one substrate may not directly inhibit the
ability to utilize the other substrate. In this case, it
would be the lysine-urea cycle that has carried the major
burden of regulating his ammonia levels. This would implv
that the same enzyme has different reaction sites or
properties for the two substrates, and that although the
enzyme is defective for one substrate, it continues to
function for the other.

A third possibility is to postulate different sets of enzymes
for the ornithine-urea cycle and the lysine-urea cycle,
although the enzymes of each cycle may be capable of some

activity on the substrates of the other. If this is true,

83

Ammonia

[u
v
Carbamyl, phosphate

  
 
  

T

 
 
     
  

   
   

[4o]

 

 
 

W
Citrulline Homocitrulline

 

  

.-- --‘_-— - l- — -

Argininosuccinic
acid
I: Homoargininosuccinid
acid I / ,,,,,,, Aspartote
V“ [ah] I .' ’ .— ” ’
\\______-________.___..-_-——./“"’ '
Glutamine
l
l
i
’l
Ammonia

------- - - Lysine-Urea cycle (Back-up cycle)

Ornithine-Urea cycle

Fig. 19: The lysine-urea and the ornithine-urea cycles

I ‘IIII l A . A

84

then we can hypothesize that this patient is using the
lysine-urea cycle which has become efficient because of a
new steady state for the substrates, and that the early
problems experienced by the patient occurred before he
acquired this steady state. The experiments on rat liver
indicated very little activity for the enzymes of the
postulated secondary cycle. It is possible that they do
occur or are induced in appreciable amounts in human liver,

or that they exist in some other tissue, such as muscle.

REASONS FOR NORMAL UREA PRODUCTION

The normal levels of urea could be gotten from a combination of

the following:

(1)

(2)

(3)

Direct transamidation of lysine to homoarginine and the
subsequent hydrolysis of homoarginine to urea.

The very high concentration of citrulline could lead to the
conversion of some citrulline to arginine and the subsequent
hydrolysis of arginine to urea.

Conversion of lysine to homoarginine via homocitrulline and
HASA, and the subsequent hydrolysis of the homoarginine to

urea .

EVIDENCE CONCERNING THE TWO-CYCLE HYPOTHESIS

Several lines of evidence suggest that there are two separate

cycles for the production of urea and the disposal of ammonia.

(1) Defects of the ornithine-urea cycle usually do not lower urea

production in affected individuals. Alternate hypotheses can explain

this fact, so that it has been hypothesized that the defective enzymes

llll Ill-Ill I 'll III I: ' III. II All! I ‘ I ll. ll'l l.

85

are functional because of the higher concentrations of substrates,

that some non—enzymatic conversion has carried the substrate through

the blocked path, or that transamidation of lysine to homoarginine has
led by a separate path to urea. These hypotheses are tenable, and may
either be correct or may supplement the lysine-urea cycle. However, it
is difficult to assume that any one of these hypothesized sources of
urea is a good substitute for the normal ornithine-urea cycle, and thus,
it is questionable that they explain the large amounts of urea in most

of the persons with ornithine-urea cycle defects.

(2) Defects that increase the concentration of a substance in the
ornithine-urea cycle may also cause increases in the concentration of
substances in the hypothesized lysine-urea cycle. Thus, hyperornithinemia
is accompanied by homocitrullinuria (Shih fig 21., 1969). Citrullinemia,
as in the patient reported here, is accompanied by hyperlysinemia,

homocitrullinuria, and high blood and urine levels of homoarginine.

(3) What may be an enzyme defect of the lysine-urea cycle leads to

ammonia intoxication. Thus, a hyperlysinemic patient without homo-
citrullinuria (suggesting a block in the enzyne that converts lysine

to homocitrulline, was found to have ammonia intoxication (Colombo gt _l.,
1967). On the other hand, hyperlysinemia with homocitrullinuria
(suggesting a block in lysine metabolism other than lysine to homo-
citrulline) is not associated with ammonia intoxication (Ghadimi st 31.,
1967). These facts suggest that the conversion of lysine to homocitrulline

may be a step in the removal of ammonia from the body, as one would

expect if the lysine-urea cycle actually exists.

..V 1.71 ~q!
U.

 

A

 

ll l 4‘ Ill 1 ll ‘ III III: I (I. [I III III‘ ‘ 1'. ill El 0 ‘ .

86

If the two cycles do exist, there is evidence that they are

independent in part but, in one step, share an enzyme.

(1) There appear to be separate defects for the enzymes controlling

the step from ornithine to citrulline and that from lysine to homo-
citrulline. Ornithine transcarbamylase deficiency (Russell _£H_l., 1962)
has been reported in some persons, while an apparent deficiency in the
transcarbamylation of lysine has been reported in one individual

(Colombo g£_gl,, 1967). However, experiments reported here indicated
strong competition between lysine and ornithine for the utilization of

OCT, but there is no evidence of whether the lysine was actually being

converted, or was merely interfering with the conversion of ornithine.

(2) Arginase is known to be able to hydrolyse both arginine and

homoarginine, suggesting that only one enzyme functions for both cycles.

(3) The only defect of the ornithine-urea cycle that has not been
reported in man is a deficiency of arginase. With the above assumption
that arginase catalyzes the homologous reactions in both cycles, it
could be assumed that a serious deficiency of this enzyme would be
lethal, as it would block both the ornithine-urea and the lysine-urea
cycles. This then strengthens the suspicion that in all of the other
steps, separate enzymes are needed to catalyze homologous reactions of

the two cycles.

THE POSSIBLE ROLE OF THE LYSINE-UREA CYCLE
One would assume that the ornithine-urea cycle could adequately

account for the disposal of ammonia by the body and that a second cycle

87

is not needed. However, as noted above, an apparent defect of the
lysine-urea cycle leads to severe impairment of the affected individual,
suggesting that the two functioning cycles are utilized by all normal
persons. Despite the fact that both cycles appear to be needed, there is
no obvious reason for this.

From an evolutionary standpoint, one might suppose that a
single cycle would eventually assume the entire function of ammonia
disposal and that most species would possess only one of these cycles.
The evidence and data presented here is that both cycles are functional
both in man and in the rat. In our present state of knowledge, there is
at least one other example of an alternate genetic pathway for an
apparently adequate system: the delta chain of hemoglobin has no known
function not adequately performed by the beta chain in those primates
in which it is found (new world monkeys and anthropoids, including man).
Either mutation and selection have not efficiently destroyed the delta
chain, except in old world monkeys, or our knowledge of primate
hemoglobins is still incomplete. These same considerations may apply

to the two urea cycles.

THE COURSE OF CITRULLINEMIA

There are two citrullinemic patients adequately described in the
literature plus the individual who is described here, and each presents
a distinct set of biochemical abnormalities. Because they have different
biochemical characteristics, they may represent separate types of
mutations of the gene for ASA synthetase. However, they might all have

similar (complete or leaky) defects of the enzyme, but may represent

88

the stages of a series in which the patient of Morrow ££__l, (1967)
presents the earliest stage, with ammonia intoxication and low urea
production. The patient of McMurray g£_§l. (1962) has ammonia intoxication
but produces normal amounts of urea. The patient described here has no
ammonia intoxication (but a history suggestive of it in the past) and
normal urea production. The hypothesis that is put forth is that the
primary defect is in the production of ASA synthetase, which blocks the

ornithine-urea cycle. Early in life, ammonia builds up in the blood

to toxic levels and urea production is negligible. As the lysine—urea

 

”spill
l

cycle becomes more efficient (presumably because its high efficiency
occurs only if there is low efficiency of the ornithine-urea cycle) first
urea production proceeds to the normal range and then serum ammonia
levels fall below the toxic threshhold. The damage wrought by early
ammonia intoxication apparently is not fully repaired. Perhaps, if
diagnosed early enough in life, a low protein diet would minimize the
damage to the brain. The assumption of efficient urea production by the
lysine-urea cycle would depend on whether its late efficiency appears
naturally in all persons or is dependent on induction by its substrates

in those with ornithine-urea cycle defects.

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Woody, N. C. Hyperlysinemia. Am. J. Dis. Child. 108:543, 1964b.

Woody, N. C., J. Hutzler and J. Dancis. Further studies of hyperlysinemia.
Am. J. Dis. Child. 112:577, 1966.

APPENDICE S

II.

III.

(1)
(2)
(3)

(1)
(2)
(3)
(4)
(5)
(7)
(9)
(10)
(11)
(13)

(16)

(1)
(2)
(3)
(7)
(8)

(9)

(10)

(11)

95

THE PEDIGREE (See Appendix 1)

Wilomina P, died age 60 of cancer; Polish extraction

Gusta D, died age 60 of pneumonia; German and Austrian extraction

Louise S, born 1876, died 'natural' death; Swiss extraction

Died

Died

Died

Died

at age 4 of diphtheria
at age 43; was married but had no children
at age 16 of peritonitis

at age 28 of Diabetes mellitus

Adopted

Was married and has no children

Father of propositus

Mother of propositus

Was married but has no children

Died

at age 9 of peritonitis

Was married but has no children

Very
Same
Same
Same
Same

Same

mildly retarded (was reported as slow)
as 1; also has peritonitis

as l

as l

as 1; very nervous and never married

as 1; has speech impediment; never completed grade school;

complains of headaches

Suffers from TB; had a spleenolectomy and has ulcers of the stomach

Only

sib of propositus; tested biochemically and found to be normal

(12)

(38)

IV. (6)
(14)
(18)
(19)
(22)
(23)
(26)
(27)
(39)
(42)
(44)
(47)
(48)

(49)

NOTE:

(1)

(2)

PROPOSITUS

96

Has congenital hip dislocation

Has crossed eyes and other eye lesions

Has crossed eyes

Very short in stature; much less than 5 ft at 18

Same as 18

5 normal children (their
3 normal children (their
Has brain tumor

2 normal children (their

Very mildly retarded; in

sexes were not

sexes were not

sexes were not

special school

3 normal children (no sexes were given)

1 normal child (no sex was given)

Tested biochemically and was normal

Same as 47

Same as 47

given)

given)

given)

for reading problems

The number of miscarriages in the maternal part of the propositus.

There were 7 out of 69 births for a frequency of about 10 per cent.

The number of recognizable anomalies in the paternal half of the

propositus. There are 11 out of 45 births for a frequency of

about 25 per cent.

97

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m3.._m0..0¢.. I
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_

II.

000.. 31?? 30.. 00m 300% 00.0%

   

 

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mMMOHQmm mm... nda<

98

THE KARYOTYPE

Chromosome analysis of the patient's leukocytes was done (Appendix 2)
using the method described by the Grand Island Biological Company (Gibco).
There was no detectable evidence of chromosomal abnormality in the

patient as can be seen from the karyotype in Appendix 2.

 

 

 

 

 

 

 

 

 

 

 

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