Arginine Metabolism: Enzymology, Nutrition,

and Clinical Significance

Clinical Consequences of Urea Cycle Enzyme Deficiencies and Potential

Links to Arginine and Nitric Oxide Metabolism1,2
Fernando Scaglia,* Nicola Brunetti-Pierri,* Soledad Kleppe,* Juan Marini,
Susan Carter,* ** Peter Garlick, Farook Jahoor, William OBrien,* and Brendan Lee* **

*Department of Molecular and Human Genetics and Pediatrics, Childrens Nutrition Research Center, Baylor
College of Medicine, and **Howard Hughes Medical Institute, Houston, TX 77030 and Department of Animal
Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801

KEY WORDS:

urea cycle

arginine

nitric oxide

The enzymes of the urea cycle (UC)4 serve 2 purposes: the

detoxification of ammonia into urea and the de novo biosynthesis of arginine (1). The UC disorders (UCD) are a group of
inborn errors of metabolism that affect the transfer of nitrogen

into urea. Deficiencies of all of the enzymes of the cycle were

identified, and, although each specific disorder results in the
accumulation of different precursors, hyperammonemia and
hyperglutaminemia are common biochemical hallmarks of
these disorders (2,3). Most of these disorders [N-acetyl glutamate synthase (NAGS) deficiency, carbamyl phosphate synthetase I deficiency (CPSID), argininosuccinate synthetase
(ASS) deficiency, argininosuccinic acid lyase (ASL) deficiency, and arginase 1 (ARG1) deficiency] are inherited in an
autosomal recessive fashion, whereas ornithine transcarbamylase deficiency (OTCD) is X-linked (4). These conditions are
estimated to have an overall prevalence of 1:8200 in the
United States (5). Infants with null activity of a UC enzyme,
other than an ARG1 deficiency, usually present with hyperammonemic encephalopathy in the neonatal period (6). Patients with partial activity show late-onset presentation at any
age, with hyperammonemic crises that are precipitated by
catabolic stress and that carry a significant risk of developmental disabilities (7). These patients may manifest with cerebral
palsy, developmental delay, psychiatric illness, hyperammonemia associated with valproate therapy, seizure activity, and
neurodevelopmental disabilities (3,8). Variable expressivity is

1
Prepared for the Symposium on Arginine held April 5 6, 2004, in Bermuda. The conference was sponsored in part by an educational grant from
Ajinomoto USA, Inc. Conference proceedings are published as a supplement to
The Journal of Nutrition. Guest editors for the supplement were Sidney M. Morris,
Jr., Joseph Loscalzo, Dennis Bier, and Wiley W. Souba.
2
The work was supported in part by the Baylor College of Medicine General
Clinical Research Center (RR00188), Mental Retardation and Developmental Disabilities Research Center (HD024064), the Child Health Research Center
(HD041648), and the NIH (DK54450).
3
To whom correspondence should be addressed.
E-mail: blee@bcm.tmc.edu.
4
Abbreviations used: ALI, acute lung injury; ARG, arginase; ASA, argininosuccinate aciduria; ASL, argininosuccinate lyase; ASS, argininosuccinate synthetase; BMT, bone marrow transplantation; CPS, carbamyl phosphate synthetase; CPSID, carbamoylphosphate synthetase I deficiency; GABA, gammaamino butyric acid; HA, homoarginine; HTN, hypertension; HVOD, hepatic venoocclusive disease; NAA, N-acetylarginine; NAGS, N-acetylglutamate synthase;
NMDA, N-methyl-D-aspartate; nNOS, neuronal nitric oxide synthase; NO, nitric
oxide; NO2, nitrite; NO3, nitrate; NOS, NO synthase; NOx, total NO metabolites;
OLT, orthotopic liver transplantation; OTCD, ornithine transcarbamylase deficiency; PVT, pulmonary vascular tone; UC, urea cycle; UCD, urea cycle disorder.

0022-3166/04 $8.00 2004 American Society for Nutritional Sciences.

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ABSTRACT Urea cycle disorders (UCD) are human conditions caused by the dysregulation of nitrogen transfer
from ammonia nitrogen into urea. The biochemistry and the genetics of these disorders were well elucidated. Earlier
diagnosis and improved treatments led to an emerging, longer-lived cohort of patients. The natural history of some
of these disorders began to point to pathophysiological processes that may be unrelated to the primary cause of
acute morbidity and mortality, i.e., hyperammonemia. Carbamyl phosphate synthetase I single nucleotide polymorphisms may be associated with altered vascular resistance that becomes clinically relevant when specific
environmental stressors are present. Patients with argininosuccinic aciduria due to a deficiency of argininosuccinic
acid lyase are uniquely prone to chronic hepatitis, potentially leading to cirrhosis. Moreover, our recent observations suggest that there may be an increased prevalence of essential hypertension. In contrast, hyperargininemia
found in patients with arginase 1 deficiency is associated with pyramidal tract findings and spasticity, without
significant hyperammonemia. An intriguing potential pathophysiological link is the dysregulation of intracellular
arginine availability and its potential effect on nitric oxide (NO) metabolism. By combining detailed natural history
studies with the development of tissue-specific null mouse models for urea cycle enzymes and measurement of
nitrogen flux through the cycle to urea and NO in UCD patients, we may begin to dissect the contribution of
different sources of arginine to NO production and the consequences on both rare genetic and common
multifactorial diseases. J. Nutr. 134: 2775S2782S, 2004.

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Neurotoxicity in UCDs
The mechanism of neurotoxicity in UCDs was not completely elucidated. The neurotoxic effect of ammonia was well
recognized, although the manner by which it exerts its effects
upon the central nervous system is not very well known (18).
Its acute effects include increased blood brain barrier permeability, depletion of intermediates of cell energy metabolism,
and disaggregation of microtubules (18). The effects of
chronic, mildly elevated ammonia may include alterations of
axonal development and alterations in brain amino acid and
neurotransmitter levels (19,20). In models of brain edema,
where lethal doses of ammonia are administered, glial fibrillary
acidic protein is reduced (21) and glutamine is increased (22),
preceded by an increase in blood flow (23). It is not known
whether NO production plays a role in such an increase. The
arginine recycling enzymes are induced in astrocytes by ammonium, possibly originating NO via inducible NO synthase
or neuronal NO synthesis (nNOS) (24). The stimulated
nNOS might mainly produce O2, which combines with NO
to form the highly toxic peroxynitrites (25).
Although chronic hyperammonemia impairs the activation
of N-methyl-D-aspartate (NMDA) receptors and leads to reduced re-uptake of extracellular glutamate (26), acute hyperammonemia leads to excessive activation of NMDA receptors
and increased intracellular calcium, which bounds to calmodulin and stimulates NOS. The difference between acute and
chronic hyperammonemia might be explained by the neuromodulation of glutamate receptors by glutathione (27). There
is substantial evidence that creatine is essential for axonal
elongation (20,23). The exposure of brain cells to ammonia
may alter the recycling of arginine, which plays a key role in
the central nervous system, not only as a NO precursor but
also as a substrate for creatine synthesis. Exposure of brain-cell
aggregates to NH4Cl leads to a decrease of intracellular creatine and phosphocreatine concentrations (20). When creatine
is added to the aggregate cultures during NH4Cl exposure, it
seems to protect the axons from growth impairment (20).
Management of UCDs
The management in UCDs is achieved by restricting dietary protein, providing supportive management of catabolic
stress, and the use of compounds that remove excess nitrogen
compounds by alternative pathways (28,29). Alternative pathway therapy includes the use of sodium phenylbutyrate or
sodium benzoate to stimulate the excretion of nitrogen in the
form of phenylacetylglutamine and hippuric acid (30). Arginine becomes an essential amino acid in UCDs (29), except in
arginase deficiency, where arginine degradation is defective,
resulting in its accumulation. Citrulline or arginine are provided to fulfill the requirement for arginine and to provide
precursors for enzymatic steps upstream of the metabolic block.
Oral citrulline, in combination with ammonia scavengers,
is recommended for OTCD and CPSID (31). It was our
experience that use of the recommended dose of citrulline
(170 mg kg1 d1) in several patients with OTCD resulted
in a low serum arginine level, prompting us to use additional
supplementation with arginine to ensure optimal metabolic
control. Other groups (11) had a similar experience. Further
evaluations need to be performed to determine whether there
is any advantage in giving arginine for some OTCD patients.
Furthermore, there is evidence suggesting branched chain
amino acid depletion with the use of sodium phenylbutyrate in
UCD patients (32).
In the longer term, advances in liver transplantation and

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the rule in OTCD females because of a random X-inactivation

pattern in the liver (9). Morbidity and mortality in these
disorders correlate with the duration and severity of hyperammonemic episodes (2).
While the neonatal presentation of UCD is relatively uniform, late-onset presentations are due to hypomorphic mutations whose clinical presentation can be quite varied. The
different UCDs have unique clinical features that are only now
becoming recognized as the natural history of each disease
becomes apparent with a longer-lived patient population.
Moreover, the clinical and biochemical consequences of our
therapeutic interventions themselves lent insight into unique
genenutrient environment interactions related to nitrogen
transfer. Much of these emerging data, however, are retrospective and/or anecdotal, and there is a dire need for coordinated
natural history studies. In spite of this, the clinical and laboratory observations provide important clues that led to emerging hypotheses that can be tested in appropriate animal models
and in the human subjects themselves. Similar observations
were derived from our diagnostic and therapeutic approaches
to these patients.
Clinical and laboratory diagnoses of UCDs may be difficult.
Metabolic parameters such as hyperammonemia, plasma
amino acid profile, or orotic aciduria are late indicators of
metabolic derangement and may be inadequate for predicting
clinical severity (10). It is not known whether circulating
glutamine correlates well with brain glutamine (11). Although
the molecular basis for each of the UCDs was determined, a
particular genotype and/or in vitro enzyme activity does not
necessarily predict clinical severity (10). Moreover, the detection of OTCD carriers can represent a diagnostic dilemma.
Although DNA testing is recommended, in 20% of the
cases, no mutations can be found (12), and the allopurinol
loading test is not completely specific or sensitive (13).
We previously developed stable isotope approaches to determine in vivo rates of total body urea synthesis and UCspecific nitrogen transfer to evaluate phenotypic severity in
patients with a variety of UCDs (14). The ratio of isotopic
enrichments of 15N-urea/15N-glutamine allowed us to distinguish normal control subjects from patients with neonatal and
late presentations. As expected, patients demonstrated a relatively greater glutamine flux in association with decreased
urea synthesis. This study demonstrated that the 15N-urea/
15
N-glutamine ratio, i.e., fractional transfer to urea, is a sensitive index of in vivo UC activity and correlates with clinical
severity (14). Moreover, we used this approach in an integrated manner to aid in the diagnosis and the prospective
treatment of an OTCD female (15). Potentially, this stable
isotope protocol could provide a sensitive tool to aid in the
diagnosis and the management of UCD patients and, more
specifically, OTCD females. These same studies may also lend
insight into the transfer of 15N via the UC into arginine and
ultimately nitric oxide (NO) as explored in a previous study (16).
Using a stable isotope approach and by comparing the
transfer of 15N from intravenous vs. oral 15NH4Cl into urea,
we investigated whether OTCD females rely on alternative
pathways to compensate for the reduced urea synthetic activity
observed in this disorder to maintain good dietary protein
tolerance (17). OTCD females seemed to retain good dietary
protein tolerance by maintaining higher ammonia appearance
rates, expanding the plasma ammonia pool, and clearing excess ammonia via glutamine synthesis in the perivenous hepatocytes (17). The conclusions of the study showed the importance of preventing sudden changes in nitrogen metabolism in
UCD patients by minimizing their exposure to conditions that
increase peripheral protein catabolism.

UREA CYCLE ENZYME DEFICIENCIES

Observations on arginine metabolism from the study and

from the natural history of UCDs
CPSID. CPSID (MIM 237300) is a rare UCD that results
from the deficiency of CPSI (EC 6.3.4.16). CPSI is thought to
be the rate-limiting enzyme catalyzing the first step of the UC.
Expression of CPSI is limited to high levels in the liver and
smaller amounts in the intestinal mucosa. This enzymatic step
is part of the intramitochondrial component of the UC. CPSID directly affects the removal of waste nitrogen compounds,

TABLE 1
Amino acid levels pre- and post-OLT in UCD subjects
Subject1

Diagnosis2

Pre-OLT
arginine3

Post-OLT
arginine3

Post-OLT
citrulline4

M
1
2
3

CPSID
OTCD
CPSID

63 9
49 6
101 19

33 6
20 3
32 7

81
41
84

1 Pre-OLT subjects were supplemented with citrulline (200 mg/kg/

d). Posttransplantation subjects were not supplemented.
2 Means with standard deviations are shown. N for each mean 5.
3 Pre- and post-OLT arginine levels are significantly different (P
0.03), normal range for arginine (0 1 y range): 42132 M.
4 Citrulline levels are low normal, even post-transplantation; normal
range for citrulline (0 1 y range): 2 41 M.

as well as the production of citrulline, arginine, and urea.

Classic CPSID with null activity usually presents in the neonatal period with hyperammonemia, with little effect from the
environment. A delayed onset form also was described
(40,41). Diagnosis is based on the plasma amino acid profile
with elevated glutamine and low or absent citrulline and urine
orotic acid. These findings cannot discriminate this disorder
from a NAGS deficiency (MIM 237310). To establish a correct
diagnosis, enzymatic analysis of the liver tissue or identification of
mutations in the CPS1 gene is required. Prenatal diagnosis in
affected families is feasible, based on mutation analysis or identification of disease-related CPS1 haplotypes (42).
NO was identified as the active factor in the endothelialderived relaxing factor in the 1980s (43,44), and it was the
focus of hundreds of investigations regarding its role in the
regulation of systemic and pulmonary vascular resistance.
There are increasing data showing that NOS may play a role
in oxidative injury and that NOS may generate free radicals
that can result in cellular damage. Therefore, the relationship
between NO and L-arginine, an intermediate of the UC and a
precursor of NO, raised the question as to whether polymorphisms in the UC genes may play a role in the pathophysiology
of neonatal pulmonary hypertension, postcardiac surgery pulmonary hypertension, and bone marrow transplantation
(BMT) complications, and whether patients with a classic
UCD may have abnormal blood pressure regulation. It was
hypothesized (45,46) that CPSI polymorphisms may affect the
function of the enzyme in conjunction with certain environmental stressors such that the availability of the NO substrates
may become clinically relevant. Summar et al. (47) evaluated
a known CPS1 polymorphism to determine if it influences NO
metabolite concentrations or NO dependent vasodilatation in
adults. A C A (cytosine adenosine) transversion at base
4340 in exon 36 of the CPS1 gene results in the substitution
of asparagine for threonine in the critical region for N-acetylglutamate binding (T1405N). The Thr1405 variant is known
to be associated with lower enzymatic activity (30 40% lower
activity), when compared with the Asn1405 variant (45). This
CPS1 polymorphism was shown to significantly influence venous NO concentrations as well as NOS dependent and independent vasodilatation (47). These results support the hypothesis that the CPS1 genotype affects the L-citrulline/Larginine cycle and subsequent NO production. Also, this
hypothesis was compatible with the fact that CPS1 genotype
did not affect the response to the NO-independent tissue-type
plasminogen activator response to bradykinin (47).
It was hypothesized that this functional polymorphism in
the CPS1 gene might limit the availability of precursors for
NO synthesis and consequently might influence pulmonary
vascular resistance, resulting in persistent pulmonary hypertension of the newborn (48). When compared with the general population, the infants in the study (65 near-term neonates with respiratory distress) had a significantly skewed
distribution of the genotypes for the CPS variants at the amino
acid position 1405 (P 0.005). None of the infants with
pulmonary hypertension were homozygous for the T1405N
polymorphism. When compared with infants without pulmonary hypertension, infants with pulmonary hypertension had
lower mean plasma concentrations of arginine (P 0.001)
and NO metabolites (P 0.05). These data, when combined
with previous observations (49), suggest that arginine deficiency might precede and lead to impaired NO synthesis in
infants with persistent pulmonary hypertension.
Regarding postcardiac-surgeryrelated pulmonary hypertension, arginine levels in patients with evidence of increased
pulmonary vascular tone (PVT) were compared with all

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hepatocyte gene therapy offer the best hope for UCD patients
(33,34). The outcome for orthotopic liver transplantation
(OLT) in children continues to improve. The metabolic effect
of OLT was consistent; however, the disorder is not completely corrected. Most patients with OTCD or carbamyl
phosphate synthetase I deficiency (CPSID) deficiency have
low plasma citrulline concentrations, because the deficiency is
not corrected in the gut by OLT (35,36). We also observed
that besides low plasma citrulline concentrations, arginine
concentrations are significantly low (P 0.05) in patients
with OTCD and CPS deficiency who were transplanted and
who were not receiving L-arginine supplementation when
compared with the pre-OLT levels observed in the same
patients (Table 1). Here, correction of the hepatic deficiency
does not relieve the net arginine requirement, because de novo
arginine synthesis is initiated in the gut with the synthesis of
citrulline and is completed in the kidney with the conversion
of citrulline into arginine. However, most patients do not have
problems with a deficiency as long as they maintain their
dietary intake of arginine. Gene therapy trials in OTCD with
an adenoviral vector did not result in a useful increase in the
enzyme activity (37), and the studies now were discontinued
because of severe complications resulting in the death of a
patient (38). Hepatocytes transplantation may provide an
alternative therapeutic approach (39).
In clinical practice, the first goal when treating hyperammonemic crises is to abate the nitrogen load and subsequent
hyperammonemia. For the outpatient management of UCD
patients, a depletion of arginine and other essential amino
acids can be prevented using a special mixture of essential
amino acids to improve the quality of the limited natural
protein ingested. In summary, several of the findings described
above argue in favor of L-arginine supplementation in sufficient amounts to provide for adequate brain creatine, protein,
and NO synthesis in UCD patients.

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SUPPLEMENT

Genetics, Baylor College of Medicine, and B. Lee, Howard

Hughes Medical Institute, personal observation, unpublished
results). We currently follow 2 patients, of 19 and 9 y of age,
affected with ASA and high blood pressure in which secondary causes of hypertension were excluded. In one case, systolic
blood pressure values range from 114 to 165 mm Hg and
diastolic blood pressure from 72 to 134 mm Hg. In the second
case, the range for systolic blood pressure was 106 to 160 mm
Hg, and the range for diastolic blood pressure was 69 to 115
mm Hg. Hypertension was not reported as a long-term complication so far, although few follow-up studies of patients with
ASA are available (51,54). Hypertension thus far was reported
in one patient with ASA who developed high blood pressure
during the newborn period (55). The pathogenesis of hypertension in this condition is open to speculation, but the
potential downregulation of endothelial NO production secondary to intracellular deficiency of L-arginine is intriguing.
The more mundane explanation is that ASA may be toxic to
hepatocytes. Interestingly, NO was associated with the pathogenesis of liver cirrhosis (56), and this may potentially contribute to liver dysfunction. Specifically, it was proposed that
reduced NO production may lead to impaired blood pressure
regulation in the liver, resulting in hepatic fibrosis (56). A low
intracellular arginine flux may contribute to alternative generation of free radicals, leading to cellular damage. These
mechanisms may contribute to the significant liver dysfunction observed in ASA. However, the contribution of plasma
arginine to the intracellular arginine pool leading to NO
production may confound this mechanism. On the other hand,
the contribution of de novo, intracellular arginine production
via ASL to NO production in different cell types is unknown.
In all cases, these patients offer a unique opportunity to study
the in vivo human consequences when intracellular contribution of de novo arginine production via ASL is clearly abolished due to the underlying genetic defect.
Using our cohort of UCD and control subjects studied via
our 15N glutamine/18O 13C urea primed constant infusion
protocol (14), we further measured the 15N enrichment in
total NO metabolites or NOx [nitrite (NO2) plus nitrate
(NO3)] and steady-state NOx levels. Interestingly, no remarkable differences were noted in the plasma concentrations of
NOx, NO2, NO3, arginine, and ASA in 2 ASL deficient
subjects, one of whom had essential hypertension (Table 2).
However, both were being treated with arginine (500
mg kg1 d1) at the time. In an unrelated male subject
with ASA, we were able to perform a stable isotopic infusion
before and after OLT. As expected, the subject had a markedly
increased urea flux (140 mol kg1 h1 pre-OLT vs. 294
TABLE 2
Plasma amino acid, NO2, and NO3 levels in ASL deficiency
Subject1

NOx2,3

NO22

NO32

Arginine4

ASA5

81
78

127
106

mol/L
1 (w/HTN)
2 (w/o HTN)

48
41

13
10

35
31

1 Subject 1 is a 9-y-old male with ASA and essential hypertension,

and subject 2 is a 32-year-old female with ASA, who is normotensive.
2 NO2, NO3, and NOx values are shown. Comparable levels can be
seen for all metabolites.
3 NOx was measured as previously described (16).
4 Normal range for arginine (218 y range) is 18 127 mol/L.
5 Normal range for ASA: 0 mol/L.

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other patients (PVT) to determine if the development of

PVT was associated with a decrease in substrate for NO
production (45,50). Arginine levels were significantly lower in
the PVT patients (P 0.05). Patients with the AA genotype were less likely to develop increased postoperative PVT.
The overrepresentation of CCs and underrepresentation of
AAs in the PVT patients made this group significantly
different from the general population (P 0.022). Similar
observations were noted in hepatic veno-occlusive disease
(HVOD) after BMT (45). The presence of post-BMT complications [acute lung injury (ALI), and HVOD] was correlated
with the T1405N genotypes. All patients with the CPSI
T1405N genotype, even those who developed ALI, had longer
survival and did not develop HVOD (P 0.05). In this study,
suboptimal production of arginine and NO correlated with
increased morbidity and mortality after BMT (45).
The data from CPSI polymorphisms suggest that subtle
functional polymorphisms could play a major role in common
diseases, with availability of intracellular arginine derived from
nitrogen transfer via CPSI being the common pathogenetic
link. These studies could better determine the risk of outcomes
for patients undergoing cardiac surgery or chemotherapy trials.
In the future, the application of stable isotope tracers could
confirm the in vivo effects of these polymorphisms. Controlled
trials, however, are needed to determine whether the supplementation of the precursor metabolites for NO could modify
the incidence of toxicity in BMT and cardiac surgery patients.
Argininosuccinic acid lyase deficiency. Argininosuccinic
aciduria (ASA) deficiency (MIM 207900) results from ASL
(EC 4.3.2.1) deficiency. The reported incidence of this disease
is about 1 in 70,000 live births in the United States, and thus
it is the second most common heritable disorder of the UC.
Patients with this disorder typically present in the neonatal
period or during infancy with vomiting, lethargy, developmental delay, and hyperammonemia. A characteristic finding in
these patients is trichorrexis nodosa, which consists of very
friable hairs. The biochemical hallmarks of this disease consist
of the elevations of argininosuccinic acid and citrulline, and
low levels of plasma L-arginine (1). Patients identified through
a newborn screening program and who were initiated on a
protein restriction diet and arginine supplementation before
the onset of symptoms developed normally (51). However,
despite treatment and metabolic control, some patients may
exhibit intellectual impairment and delayed motor skills (B.
Lee, Howard Hughes Medical Institute, personal observation).
These sequelae are mainly thought to arise from initial hyperammonemia, although the degree of impairment appears out of
proportion to the degree of recorded hyperammonemia.
Hence, it is possible that morbidity may also be associated with
a cell autonomous deficiency of ASL, perhaps contributing to
a compartmentalized UC intermediate deficiency. However, if
this is the case, it is still not clear why similar anecdotal
observations were not reported for upstream deficiencies such
as ASS deficiency, especially because ASS and ASL are
thought to be coexpressed. Alternative possibilities include 1)
differential regulation of ASS and ASL, 2) differential in vivo
expression of ASS and ASL, or 3) the existence of as yet
undescribed functions for ASL outside of the UC.
The most compelling clinical evidence is the more complex
clinical phenotype in ASA. Patients with ASA can suffer from
progressive hepatic disease (36,52,53), which may ultimately
evolve to cirrhosis. Importantly, this is independent of the risk
and control of hyperammonemia. In addition, our experience
suggests that these patients may be at risk for the development
of previously unrecognized systemic hypertension (N. Brunetti-Pierri, F. Scaglia, Department of Molecular and Human

UREA CYCLE ENZYME DEFICIENCIES

(59), whose pathogenesis is not very well understood. Those

patients who were ascertained and treated at birth with protein restriction and essential amino acid supplementation
seem to remain asymptomatic, with the oldest patients being
35 y of age (60). This suggests that chronically elevated
levels of arginine directly contribute to the neuropathology. In
symptomatic patients whose treatment was begun at diagnosis,
there is ample evidence that effective therapy stops the progressive neurological degeneration. While the lower-extremity
spasticity may progress slightly, it can be effectively treated
with botox or tendon-release procedures.
It is unlikely that elevated plasma ammonia is the main
neuropathogenic agent in argininemia, because hyperammonemia seldom occurs in this condition, and the severe
spasticity observed in this disease does not commonly occur in
other UC defects (61). Guanidino compounds accumulate in
other conditions, such as uremia and epilepsy, and there is
evidence suggesting that these compounds might contribute to
the neurological dysfunction observed in these disorders
(62,63). It was postulated that these compounds might be
involved as well in the neuropathology of argininemia (64
66). The guanidino compounds that accumulate in hyperargininemic patients [N-acetylarginine (NAA), homoarginine
(HA), and arginic acid] were found to have epileptogenic properties (67 69). These guanidino compounds may affect neurotransmission or membrane fluidity (70,71). Certain guanidino
compounds accumulating in argininemia might affect (GABA)ergic neurotransmission, by inhibiting gamma-amino butyric acid
(GABA) and glycine responses in cultured neurons (67). Regarding the potential for guanidino compounds to modulate membrane fluidity, it was shown that methylguanidine inhibits brain
Na, K, ATP-ase (72), an enzyme embedded in the cell membrane. NAA, HA, and AA significantly inhibit Na, K, ATP-ase
activity in the cerebral cortex of rats (73).
Arginine is known to modulate neuronal survival through
its ability to serve as a substrate for NOS (74). Subsequent
studies showed that the protective effects of arginase were
related to depletion of arginine. In one study, it was demonstrated that arginine, HA, NAA, and AA induced free-radical
production and decreased cellular antioxidant defenses in vitro
(75). The guanidino compounds were found to inhibit the
activities of catalase, superoxide dismutase, and glutathione
peroxidase, which are considered to be the main enzymatic
defenses in the brain against free radicals. Although the concentration of guanidino compounds used in the previously
described animal experiments were similar to those observed

TABLE 3
15N

Subject1

NOx2
(m 1)

transfer from glutamine to arginine and NOx in ASL deficiency

Arg2 (m 1)
(m 2)
MPE

ASA
pre-OLT w/Arg
post-OLT
Control
Control

0.073
0.036
0.13
0.16

15NOx/15NArg

NOx

NO2

NO3

47.1
47.4
15.3
32

ASA3

NH33

231 43
88 63
0
0

44 10
17 2
22 6
18 4

mol/L

%
0.155
0.076
0.85
0.50

Arg3

72
64
57
53

16
6
8
20

57
58
49
33

22 54
63 4
84 4
88 34

1 Subjects were admitted and stabilized on a restricted protein intake (0.4 g kg1 d1). On d 3, primed coinfusions of 15N-glutamine and 18O
13C urea were performed for 8 h as previously described (14). Blood sampling was performed at 0, 4, 6, and 7.5 h.
2 15N enrichment shown as mole percent excess is shown at the 7.5-h time point and 15N Arginine and 15NOx were measured as previously

described (16).
3 ASA normal range: 0 mol; Arg: arginine, normal range for 218 y: 18 127 mol; NH3 normal range: 22 48 mol.
4 For multiple samples (N 3), means with standard deviations are shown.

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mol kg1 h1 post-OLT) after liver transplantation, in spite

of being on the same protein intake (1.1 g/kg/d). However, the
glutamine flux was not markedly different (388 mol kg1
h1 vs. 410 mol kg1 h1). Interestingly, the fractional transfer of 15N from arginine into NOx at 7.5 h of infusion was not
dramatically affected by liver transplantation (Table 3). In
both experiments, 15N content of NOx and arginine was lower
in the ASL deficiency subject compared with control subjects,
even though the controls were on the lower protein intake
(0.4 g kg1 d1), indicating impaired transfer of glutamineamide nitrogen to NO. This suggests that hepatic arginine via
nitrogen transfer through ASL and the UC is not a major
contributor to total body NO production. In fact, the fractional
transfer of 15N from arginine into NOx was identical pre- vs.
post-OLT. As expected, citrulline and ASA levels were still
elevated after liver transplantation (Table 3). These data support
the hypothesis that de novo hepatic sources of arginine synthesis
do not contribute significantly to NOx production. These data are
limited by the fact that citrulline flux or NO3 flux were not
independently measured in these studies. Hence, the actual
flux of the NO pathway could not be derived. In the future, the
measurement of citrulline and/or NO3 flux, in conjunction
with an independent measure of total plasma arginine flux,
would be informative in dissecting the contributions of de
novo production of arginine from the UC vs. exogenous contribution of arginine into total body NO flux.
Unfortunately, the mouse mutant for ASL was not helpful
with these studies. Asl null mice die in the perinatal period
secondary to hyperammonemia. There do not appear to be
differences in NO2 level, muscle and liver creatine, and urinary cyclic guanosine monophosphate (cGMP) levels (57).
Ultimately, the generation of tissue-specific null mutants that
can bypass the neonatal lethality attributable to hyperammonemia may enable better physiological studies of nitrogen
transfer into arginine in different tissues.
ARG1 deficiency. Argininemia (MIM 207800) is a rare
autosomal recessive UCD caused by a defect in the final step
of the UC, the hydrolysis of arginine to urea and ornithine by
the enzyme ARG1 (EC 3.5.3.1). This is a rare disorder, for
example, with an estimated incidence of 1 in 1,000,000 live
births in Quebec (58). This condition results in elevated
plasma arginine and ammonia levels, with elevated tissue
levels of arginine and other guanidino compounds (1). Patients affected with this condition usually present with a
neurological syndrome that consists of a variable degree of
cognitive deficits, epilepsy, and progressive spastic diplegia

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TABLE 4
Nitrogen transfer in ARGI deficiency
Subject1

Gln Ra2

Urea Ra2

mol kg1 h1
ARGI
145 female
146 female

291
274

11
29

15NOx

NOx

NO2

Arg

15Nurea/
15Ngln

91 153
277 17

0.02
0.06

mol/L

MPE
0.121
0.221

NO3

52
48

11
13

40
35

1 Subjects were admitted and stabilized on a restricted protein intake (0.4 g kg1 d 1). On d 3, primed coinfusions of 15N-glutamine and 18O
13C urea were performed for 8 h as previously described (14). Blood sampling was performed at 0, 4, 6, and 7.5 h. 15N enrichment, shown as mole
percent excess, is shown at the 7.5-h time point. 15N arginine and 15NOx was measured as previously described (16). For multiple samples, means

with standard deviations are shown. Control values for Gln Ra and Urea Ra are as previously reported (14).
2 Gln Ra, glutamine appearance rate; Urea Ra, urea appearance rate.
3 For multiple samples (n 3), means with standard deviations are shown.

Conclusions
Human deficiencies of UC enzymes offer unique clinical
and experimental opportunities to study and to understand the
effects of intracellular dysregulation of arginine production, as
well as consequences of exogenous elevations of arginine.
Unique genotypephenotype correlations are emerging in
both these rare diseases and in more common pathophysiologic processes. The clinical observations raise important ques-

tions and hypotheses about the contribution of dysregulated

NO metabolism to the pathogenesis of these conditions. At
the same time, UCD patients constitute a unique resource for
answering these same questions.
Specifically, human and animal models of UCDs are unique
examples of genenutrient interactions centered on nitrogen
transfer between dietary vs. peripheral sources. They offer
potentially important tools for dissecting extracellular vs. intracellular contributions of arginine to the production of NO,
free radicals, polyamines, and creatine. Genetic polymorphisms in CPSI, the gene encoding the rate-limiting enzyme of
the UC, may correlate with altered arginine availability and
endothelial dysfunction during specific times of environmental
stress. ASL deficiency is a window into the consequences of
deficient de novo production of intracellular arginine. In contrast, ARG1 deficiency will provide insight into the consequences of chronic elevations of extracellular arginine as well
as the role of ARG1 as a direct intracellular regulator of NO
flux. The therapeutic approaches to UCD patients may further
offer models for understanding the effects of exogenous arginine supplementation, the contribution of liver to nitrogen
transfer in patients who have undergone liver transplantation,
and the effects of diverting flux away from UC nitrogen
transfer using alternative route therapy, i.e., sodium phenylbutyrate.
ACKNOWLEDGMENTS
The authors thank Olivia Hernandez for administrative assistance,
and the excellent nursing staff at the Texas Childrens Hospital
General Clinical Research Center. This review is dedicated to the
memory of Peter Reeds.

LITERATURE CITED
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in patients with argininemia (67), it would be difficult to

extrapolate these findings to an in vivo condition. However, it
is tempting to think that these mechanisms may contribute to
the pathogenesis of the neurological dysfunction found in
ARG1 deficiency. The transfer of nitrogen through the UC
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arginineimidino nitrogen into NOx in the cell. However, as
in the ASL-deficient patients, further studies focused on measuring the actual flux of NO metabolites will be required to
elucidate the effect of arginine levels in regulating NO production. Clearly, the respective contributions of chronic elevation of plasma arginine vs. the intracellular deficiency of
ARG1 on the regulation of intracellular NO production can
be addressed by studying this unique patient population. This
has direct relevance to understanding the neuropathology
associated with chronic arginine elevations.
Similar to the animal model in ASL deficiency, both Arg1
and Arg2 null mice were generated (76,77). The Arg1 null
mice succumb later than the Asl null mice, at about 10 12 d,
secondary to hyperammonemia. The mice have mild liver
disease and elevated arginine, with low proline and ornithine
levels. Again, the generation of tissue-specific nulls will be
important in performing steady-state studies in an adult animal.

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