Generation of induced pluripotent stem cells (UCLi024-A) from a patient with argininosuccinate lyase deficiency carrying a homozygous c.437G > A (p.Arg146Gln) mutation.

Argininosuccinic aciduria (ASA) is a rare inherited metabolic disease caused by argininosuccinate lyase (ASL) deficiency. Patients with ASA present with hyperammonaemia due to an impaired urea cycle pathway in the liver, and systemic disease with epileptic encephalopathy, chronic liver disease, and arterial hypertension. A human induced pluripotent stem cell (iPSC) line from the fibroblasts of a patient with ASA with homozygous pathogenic c.437G > A mutation of hASL was generated. Characterization of the cell line demonstrated pluripotency, differentiation potential and normal karyotype. This cell line, called UCLi024-A, can be utilized for in vitro disease modelling of ASA, and design of novel therapeutics.

Argininosuccinate lyase deficiency causes blood-brain barrier disruption via nitric oxide-mediated dysregulation of claudin expression.

Nitric oxide (NO) is a critical signaling molecule that has been implicated in the pathogenesis of neurocognitive diseases. Both excessive and insufficient NO production have been linked to pathology. Previously, we have shown that argininosuccinate lyase deficiency (ASLD) is a novel model system to investigate cell-autonomous, nitric oxide synthase-dependent NO deficiency. Humans with ASLD are at increased risk for developing hyperammonemia due to a block in ureagenesis. However, natural history studies have shown that individuals with ASLD have multisystem disease including neurocognitive deficits that can be independent of ammonia. Here, using ASLD as a model of NO deficiency, we investigated the effects of NO on brain endothelial cells in vitro and the blood-brain barrier (BBB) in vivo. Knockdown of ASL in human brain microvascular endothelial cells (HBMECs) led to decreased transendothelial electrical resistance, indicative of increased cell permeability. Mechanistically, treatment with an NO donor or inhibition of Claudin-1 improved barrier integrity in ASL-deficient HBMECs. Furthermore, in vivo assessment of a hypomorphic mouse model of ASLD showed increased BBB leakage, which was partially rescued by NO supplementation. Our results suggest that ASL-mediated NO synthesis is required for proper maintenance of brain microvascular endothelial cell functions as well as BBB integrity.

Natural history of epilepsy in argininosuccinic aciduria provides new insights into pathophysiology: A retrospective international study.

OBJECTIVE: Argininosuccinate lyase (ASL) is integral to the urea cycle, which enables nitrogen wasting and biosynthesis of arginine, a precursor of nitric oxide. Inherited ASL deficiency causes argininosuccinic aciduria, the second most common urea cycle defect and an inherited model of systemic nitric oxide deficiency. Patients present with developmental delay, epilepsy, and movement disorder. Here we aim to characterize epilepsy, a common and neurodebilitating comorbidity in argininosuccinic aciduria. METHODS: We conducted a retrospective study in seven tertiary metabolic centers in the UK, Italy, and Canada from 2020 to 2022, to assess the phenotype of epilepsy in argininosuccinic aciduria and correlate it with clinical, biochemical, radiological, and electroencephalographic data. RESULTS: Thirty-seven patients, 1-31 years of age, were included. Twenty-two patients (60%) presented with epilepsy. The median age at epilepsy onset was 24 months. Generalized tonic-clonic and focal seizures were most common in early-onset patients, whereas atypical absences were predominant in late-onset patients. Seventeen patients (77%) required antiseizure medications and six (27%) had pharmacoresistant epilepsy. Patients with epilepsy presented with a severe neurodebilitating disease with higher rates of speech delay (p = .04) and autism spectrum disorders (p = .01) and more frequent arginine supplementation (p = .01) compared to patients without epilepsy. Neonatal seizures were not associated with a higher risk of developing epilepsy. Biomarkers of ureagenesis did not differ between epileptic and non-epileptic patients. Epilepsy onset in early infancy (p = .05) and electroencephalographic background asymmetry (p = .0007) were significant predictors of partially controlled or refractory epilepsy. SIGNIFICANCE: Epilepsy in argininosuccinic aciduria is frequent, polymorphic, and associated with more frequent neurodevelopmental comorbidities. We identified prognostic factors for pharmacoresistance in epilepsy. This study does not support defective ureagenesis as prominent in the pathophysiology of epilepsy but suggests a role of central dopamine deficiency. A role of arginine in epileptogenesis was not supported and warrants further studies to assess the potential arginine neurotoxicity in argininosuccinic aciduria.

Release of SMP30 in Extracellular Vesicles under Conditions of Ascorbic Acid Deficiency Is Involved with Acute Phase Response in ODS Rat.

Senescence marker protein-30 (SMP30) is a senescence marker molecule that exhibits lactonase activity in the ascorbic acid (AsA) biosynthesis pathway, except in primate mammals, including humans. Although numerous studies have shown that hepatic AsA deficiency causes acute-phase responses, details of the relationship between SMP30 expression and acute-phase responses in AsA-deficient conditions remain to be elucidated. Here, we investigated the effects of AsA deficiency on the relationship between SMP30 and acute liver injury in osteogenic disorder Shionogi (ODS) rats, which have a hereditary defect in AsA biosynthesis. Male-ODS rats (4 wk old) were pair-fed an AsA-free diet with distilled or 0.1% AsA-dissolved water for 14 d. Under AsA-deficient conditions, hepatic SMP30 protein level was decreased and liver injury markers, the serum aspartate aminotransferase/alanine transaminase ratio and cytokine-induced neutrophil chemoattractant-1 (CINC-1) concentration, were elevated. In contrast, SMP30 protein level in extracellular vesicles (EVs) was significantly increased in addition to the positive acute proteins haptoglobin and asialoglycoprotein receptor 1 (ASGPR1), hepatic-derived specific markers expression under AsA-deficient conditions. AsA deficiency also activated signal transducer and activator of transcription 3 (STAT3) which is linked to EVs release in the liver. These results suggest that the release of SMP30 in EVs by AsA deficiency is involved with acute-phase responses.

Cutting Edge: l-Arginine Transfer from Antigen-Presenting Cells Sustains CD4+ T Cell Viability and Proliferation.

Metabolomics analyses suggest changes in amino acid abundance, particularly l-arginine (L-ARG), occur in patients with tuberculosis. Immune cells require L-ARG to fuel effector functions following infection. We have previously described an L-ARG synthesis pathway in immune cells; however, its role in APCs has yet to be uncovered. Using a coculture system with mycobacterial-specific CD4+ T cells, we show APC L-ARG synthesis supported T cell viability and proliferation, and activated T cells contained APC-derived L-ARG. We hypothesize that APCs supply L-ARG to support T cell activation under nutrient-limiting conditions. This work expands the current model of APC-T cell interactions and provides insight into the effects of nutrient availability in immune cells.

Nitric oxide modulates bone anabolism through regulation of osteoblast glycolysis and differentiation.

Previous studies have shown that nitric oxide (NO) supplements may prevent bone loss and fractures in preclinical models of estrogen deficiency. However, the mechanisms by which NO modulates bone anabolism remain largely unclear. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing arginine, the sole precursor for nitric oxide synthase-dependent (NOS-dependent) NO synthesis. Moreover, ASL is also required for channeling extracellular arginine to NOS for NO production. ASL deficiency (ASLD) is thus a model to study cell-autonomous, NOS-dependent NO deficiency. Here, we report that loss of ASL led to decreased NO production and impairment of osteoblast differentiation. Mechanistically, the bone phenotype was at least in part driven by the loss of NO-mediated activation of the glycolysis pathway in osteoblasts that led to decreased osteoblast differentiation and function. Heterozygous deletion of caveolin 1, a negative regulator of NO synthesis, restored NO production, osteoblast differentiation, glycolysis, and bone mass in a hypomorphic mouse model of ASLD. The translational significance of these preclinical studies was further reiterated by studies conducted in induced pluripotent stem cells from an individual with ASLD. Taken together, our findings suggest that ASLD is a unique genetic model for studying NO-dependent osteoblast function and that the NO/glycolysis pathway may be a new target to modulate bone anabolism.

Severity-adjusted evaluation of newborn screening on the metabolic disease course in individuals with cytosolic urea cycle disorders.

OBJECTIVE: The implementation of newborn screening (NBS) programs for citrullinemia type 1 (CTLN1) and argininosuccinic aciduria (ASA) is subject to controversial debate. The aim of this study was to assess the impact of NBS on the metabolic disease course and clinical outcome of affected individuals. METHODS: In 115 individuals with CTLN1 and ASA, we compared the severity of the initial hyperammonemic episode (HAE) and the frequency of (subsequent) HAEs with the mode of diagnosis. Based on a recently established functional disease prediction model, individuals were stratified according to their predicted severe or attenuated phenotype. RESULTS: Individuals with predicted attenuated forms of CTLN1 and ASA were overrepresented in the NBS group, while those with a predicted severe phenotype were underrepresented compared to individuals identified after the manifestation of symptoms (SX). Identification by NBS was associated with reduced severity of the initial HAE both in individuals with predicted severe and attenuated phenotypes, while it was not associated with lower frequency of (subsequent) HAEs. Similar results were obtained when including some patients diagnosed presymptomatically (i.e. prenatal testing, and high-risk family screening) in this analysis. CONCLUSION: Since one of the major challenges of NBS outcome studies is the potential overrepresentation of individuals with predicted attenuated phenotypes in NBS cohorts, severity-adjusted evaluation of screened and unscreened individuals is important to avoid overestimation of the NBS effect. NBS enables the attenuation of the initial HAE but does not affect the frequency of subsequent metabolic decompensations in individuals with CTLN1 and ASA. Future long-term studies will need to evaluate the clinical impact of this finding, especially with regard to mortality, as well as cognitive outcome and quality of life of survivors.

From genotype to phenotype: Early prediction of disease severity in argininosuccinic aciduria.

Argininosuccinic aciduria (ASA) is an inherited urea cycle disorder and has a highly variable phenotypic spectrum ranging from individuals with lethal hyperammonemic encephalopathy, liver dysfunction, and cognitive deterioration, to individuals with a mild disease course. As it is difficult to predict the phenotypic severity, we aimed at identifying a reliable disease prediction model. We applied a biallelic expression system to assess the functional impact of pathogenic argininosuccinate lyase (ASL) variants and to determine the enzymatic activity of ASL in 58 individuals with ASA. This cohort represented 42 ASL gene variants and 42 combinations in total. Enzymatic ASL activity was compared with biochemical and clinical endpoints from the UCDC and E-IMD databases. Enzymatic ASL activity correlated with peak plasma ammonium concentration at initial presentation and with the number of hyperammonemic events (HAEs) per year of observation. Individuals with ≤9% of enzymatic activity had more severe initial decompensations and a higher annual frequency of HAEs than individuals above this threshold. Enzymatic ASL activity also correlated with the cognitive outcome and the severity of the liver disease, enabling a reliable severity prediction for individuals with ASA. Thus, enzymatic activity measured by this novel expression system can serve as an important marker of phenotypic severity.

Intrahepatic Administration of Human Liver Stem Cells in Infants with Inherited Neonatal-Onset Hyperammonemia: A Phase I Study.

Previous studies have shown that human liver stem-like cells (HLSCs) may undergo differentiation in vitro into urea producing hepatocytes and in vivo may sustain liver function in models of experimentally induced acute liver injury. The aim of this study was to assess the safety of HLSCs intrahepatic administration in inherited neonatal-onset hyperammonemia. The study was approved by the Agenzia Italiana del Farmaco on favorable opinion of the Italian Institute of Health as an open-label, prospective, uncontrolled, monocentric Phase I study (HLSC 01-11, EudraCT-No. 2012-002120-33). Three patients affected by argininosuccinic aciduria (patient 1) and methylmalonic acidemia (patients 2 and 3) and included in the liver transplantation list were enrolled. In all patients, HLSCs were administered by percutaneous intrahepatic injections (once a week for two consecutive weeks) within the first months of life. The first patient received 125,000 HLSCs x gram of liver/dose while the other two patients received twice this dose. No immunosuppression was administered since HLSCs possess immunomodulatory activities. None of the patients experienced infections, hyperammonemia decompensation, or other adverse events during the whole observation period. No donor specific antibodies (DSA) against HLSCs were detected. Patients were metabolic stable despite an increase (~30%) in protein intake. Two patients underwent liver transplantation after 19 and 11 months respectively, and after explantation, the native livers showed no histological alterations. In conclusion, percutaneous intrahepatic administration of HLSCs was safe in newborn with inherited neonatal-onset hyperammonemia. These data pave the way for Phase II studies in selected inherited and acquired liver disorders.

Resting energy expenditure in argininosuccinic aciduria and in other urea cycle disorders.

No data are available on the specific energy needs of patients affected with Urea Cycle disorders (UCD) and especially argininosuccinic aciduria (ASA). In our experience, ASA patients tend to develop central adiposity and hypertriglyceridemia when treated with apparently adequate energy intake, while the other UCD do not. The aim of this study was to evaluate anthropometric parameters, body composition, risk of metabolic syndrome (MS) and resting energy expenditure (REE), both by indirect calorimetry (IC) and predictive equations, in UCD patients. Hypertension (5/13), pathological waist circumference-to-height ratio (WtHr) (6/13), hypertriglyceridemia (12/13), reduced HDL cholesterol (12/13), and MS (5/13) were found in ASA group. In the ASA cohort, the mean and median IC-REE were 88% of what was predicted by Food and Agriculture Organization of the United Nations and Harris-Benedict equations. The "other UCD" cohort did not show hypertension, dyslipidaemia nor MS; IC-REE was similar to the REE predicted by equations. A significant difference was seen for the presence of hypertension, dyslipidaemia, pathological WtHr, MS and IC-REE/predictive equations-REE in the two cohorts. ASA patients have a risk of overfeeding if their energy requirement is not assessed individually with IC. Excessive energy intake might increase the cardiovascular risk of ASA patients. We suggest to test ASA individuals with IC every year if the patient is sufficiently collaborative. We speculate that most of the features seen in ASA patients might depend on an imbalance of Krebs cycle. Further studies are needed to verify this hypothesis.

Argininosuccinate neurotoxicity and prevention by creatine in argininosuccinate lyase deficiency: An in vitro study in rat three-dimensional organotypic brain cell cultures.

The urea cycle disorder (UCD) argininosuccinate lyase (ASL) deficiency, caused by a defective ASL enzyme, exhibits a wide range of phenotypes, from life-threatening neonatal hyperammonemia to asymptomatic patients, with only the biochemical marker argininosuccinic acid (ASA) elevated in body fluids. Remarkably, even without ever suffering from hyperammonemia, patients often develop severe cognitive impairment and seizures. The goal of this study was to understand the effect on the known toxic metabolite ASA and the assumed toxic metabolite guanidinosuccinic acid (GSA) on developing brain cells, and to evaluate the potential role of creatine (Cr) supplementation, as it was described protective for brain cells exposed to ammonia. We used an in vitro model, in which we exposed three-dimensional (3D) organotypic rat brain cell cultures in aggregates to different combinations of the metabolites of interest at two time points (representing two different developmental stages). After harvest and cryopreservation of the cell cultures, the samples were analyzed mainly by metabolite analysis, immunohistochemistry, and western blotting. ASA and GSA were found toxic for astrocytes and neurons. This toxicity could be reverted in vitro by Cr. As well, an antiapoptotic effect of ASA was revealed, which could contribute to the neurotoxicity in ASL deficiency. Further studies in human ASL deficiency will be required to understand the biochemical situation in the brain of affected patients, and to investigate the impact of high or low arginine doses on brain Cr availability. In addition, clinical trials to evaluate the beneficial effect of Cr supplementation in ASL deficiency would be valuable.

Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer.

Argininosuccinate lyase (ASL) belongs to the hepatic urea cycle detoxifying ammonia, and the citrulline-nitric oxide (NO) cycle producing NO. ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia, multiorgan disease and neurocognitive impairment despite treatment aiming to normalise ammonaemia without considering NO imbalance. Here we show that cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress independent of hyperammonaemia. Intravenous injection of AAV8 vector into adult or neonatal ASL-deficient mice demonstrates long-term correction of the hepatic urea cycle and the cerebral citrulline-NO cycle, respectively. Cerebral disease persists if ammonaemia only is normalised but is dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This correlates with behavioural improvement and reduced cortical cell death. Thus, neuronal oxidative/nitrosative stress is a distinct pathophysiological mechanism from hyperammonaemia. Disease amelioration by simultaneous brain and liver gene transfer with one vector, to treat both metabolic pathways, provides new hope for hepatocerebral metabolic diseases.

A retrospective biochemical, molecular, and neurocognitive review of Saudi patients with argininosuccinic aciduria.

A retrospective review was compiled of 54 patients with argininosuccinic aciduria who were either identified through the Saudi National Newborn Screening Program or diagnosed clinically from January 2000 to December 2015. The duration of follow-up is from 2 to 19 years. The majority of patients (65%) originated from the central province of Saudi Arabia. The mean patient age at review was 10 years (2-19 years), 92% received an early diagnosis (<28 days of age) and most were symptomatic at the time of the diagnosis (n = 34). Normal ammonia at diagnosis was reported in 30% of patients, who were detected under the newborn metabolic screen (n = 5/16). A very high rate of consanguinity was observed in our cohort (98%). Developmental delay was the most detectable long term neurocognitive consequence followed by seizure disorder; 90.7% (n = 49) and 62.9% (n = 34) respectively. As expected, the severe neonatal form was the major presentation. The most common variant identified in this cohort was the previously reported founder c.1060C > T; p.(Gln354*) nonsense mutation in the ASL gene. In addition, the frequency of hyperammonemia was higher in patients homozygous for c.1060C > T; p.(Gln354*) compared to the other mutations. Interestingly, frequent thrombocytosis with the mean level of 717 × 109/L (range = 457-1169 × 109/L) was observed in 96% of the patients with no clear explanation.

Histopathological findings in livers of patients with urea cycle disorders.

BACKGROUND: Urea cycle disorders (UCD) are caused by genetic defects in enzymes that constitute the hepatic ammonia detoxification pathway. Patients may present with variable clinical manifestations and with hyperammonaemia. Liver abnormalities have been associated with UCD, but only a few reports on the histopathological findings in the liver of UCD patients have been published. METHODS: We conducted a retrospective review of liver biopsies, ex-planted livers and livers at post-mortem of patients with UCD. A single pathologist reviewed all specimens. RESULTS: There were 18 liver samples from 13 patients with confirmed UCD: four ex-planted livers from patients with Ornithine Transcarbamylase (OTC) (n=3) and Carbamoyl Phosphate Synthetase 1 (CPS 1) (n=1) deficiencies, eight post-mortem samples from patients with CPS 1 (n=2), OTC (n=4), Argininosuccinate Synthetase (ASS) (n=1) and Argininosuccinate Lyase (ASL) (n=1) deficiencies, and six liver biopsies, three of which came from one patient with ASL deficiency. The other three liver biopsies were from patients who subsequently received liver transplantation. Histopathological findings in samples from neonates were non-specific. Samples from three late onset OTC deficient and one ASL deficient patients showed thin fibrous septa with portal to portal bridging fibrosis and focal marked enlargement and pallor of the hepatocytes due to accumulation of glycogen particles, resembling glycogenosis and resulting in a prominent nodular pattern. Serial liver biopsies in four UCD patients with interval between samples ranging from 1 year 2 months to 17 years showed progression in fibrosis in one OTC and one ASL deficient patients. Moderate fatty changes to no progression in liver disease were noted in the two patients (OTC=1 and CPS=1). A variety of non-specific features such as fatty change, mild inflammation, cholestasis and focal necrosis were seen in the other UCD patients. CONCLUSIONS: Histopathological changes in livers from neonates with UCD are non-specific. Older patients with UCD seem to show variable hepatic fibrosis compared to those who died early. Some of these patients also show focal and superficial resemblance to a glycogen storage disorder and cirrhosis. However, progression of these changes seems to be slow. To clarify the long term consequence of these changes, more extensive periods of follow up in a larger population series is needed.

Argininosuccinic aciduria: from a monogenic to a complex disorder.

In the early 1930s, phenylketonuria was among the first metabolic diseases to be defined. In the following years, multiple attempts to correlate genotype and phenotype in several inherited metabolic diseases, including phenylketonuria, were encountered with difficulties. It is becoming evident that the phenotype of metabolic disorders is often more multifaceted than expected from the disruption of a specific enzyme function caused by a single-gene disorder. Undoubtedly, revealing the factors contributing to the discrepancy between the loss of a single enzymatic function and the wide spectrum of clinical consequences would allow clinicians to optimize treatment for their patients. This article discusses several possible contributors to the unique, complex phenotypes observed in inherited metabolic disorders, using argininosuccinic aciduria as a disease model.Genet Med 2013:15(4):251-257.

Optimizing therapy for argininosuccinic aciduria.

Argininosuccinic aciduria (ASA) is a urea cycle disorder with a complex phenotype. In spite of a lower risk for recurrent hyperammonemic episodes as compared to the proximal disorders of ureagenesis, subjects with ASA are at risk for long-term complications including, poor neurocognitive outcome, hepatic disease and systemic hypertension. These complications can occur in spite of current standard therapy that includes dietary modifications and arginine supplementation suggesting that the presently available therapy is suboptimal. In this article, we discuss the natural history of ASA and the recent mechanistic insights from animal studies that have shown the requirement of argininosuccinate lyase, the enzyme deficient in ASA, for systemic nitric oxide production. These findings may have therapeutic implications and may help optimize therapy in ASA.

Requirement of argininosuccinate lyase for systemic nitric oxide production.

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases.

Argininosuccinate lyase deficiency-argininosuccinic aciduria and beyond.

The urea cycle consists of six consecutive enzymatic reactions that convert waste nitrogen into urea. Deficiencies of any of these enzymes of the cycle result in urea cycle disorders (UCD), a group of inborn errors of hepatic metabolism that often result in life threatening hyperammonemia. Argininosuccinate lyase (ASL) is a cytosolic enzyme which catalyzes the fourth reaction in the cycle and the first degradative step, that is, the breakdown of argininosuccinic acid to arginine and fumarate. Deficiency of ASL results in an accumulation of argininosuccinic acid in tissues, and excretion of argininosuccinic acid in urine leading to the condition argininosuccinic aciduria (ASA). ASA is an autosomal recessive disorder and is the second most common UCD. In addition to the accumulation of argininosuccinic acid, ASL deficiency results in decreased synthesis of arginine, a feature common to all UCDs except argininemia. Arginine is not only the precursor for the synthesis of urea and ornithine as part of the urea cycle but it is also the substrate for the synthesis of nitric oxide, polyamines, proline, glutamate, creatine, and agmatine. Hence, while ASL is the only enzyme in the body able to generate arginine, at least four enzymes use arginine as substrate: arginine decarboxylase, arginase, nitric oxide synthetase (NOS) and arginine/glycine aminotransferase. In the liver, the main function of ASL is ureagenesis, and hence, there is no net synthesis of arginine. In contrast, in most other tissues, its role is to generate arginine that is designated for the specific cell's needs. While patients with ASA share the acute clinical phenotype of hyperammonemia, encephalopathy, and respiratory alkalosis common to other UCD, they also present with unique chronic complications most probably caused by a combination of tissue specific deficiency of arginine and/or elevation of argininosuccinic acid. This review article summarizes the clinical characterization, biochemical, enzymatic, and molecular features of this disorder. Current treatment, prenatal diagnosis, diagnosis through the newborn screening as well as hypothesis driven future treatment modalities are discussed.

Argininosuccinic aciduria: clinical and biochemical phenotype findings in Malaysian children.

Argininosuccinic aciduria is an inborn error of the urea cycle caused by deficiency of argininosuccinate lyase (ASL). ASL-deficient patients present with progressive intoxication due to accumulation of ammonia in the body. Early diagnosis and treatment of hyperammonemia are necessary to improve survival and prevent long-term handicap. Two clinical phenotypes have been recognized--neonatal acute and milder late-onset form. We investigated patients with hyperammonemia by a stepwise approach in which quantitative amino acids analysis was the core diagnostic procedure. Here, we describe the clinical phenotypes and biochemical characteristics in diagnosing this group of patients. We have identified 13 patients with argininosuccinic aciduria from 2003 till 2009. Ten patients who presented with acute neonatal hyperammonemic encephalopathy had markedly elevated blood ammonia (> 430 micromol/L) within the first few days of life. Three patients with late-onset disease had more subtle clinical presentations and they developed hyperammonemia only during the acute catabolic state at two to twelve months of age. Their blood ammonia was mild to moderately elevated (> 75-265 micromol/L). The diagnosis was confirmed by detection of excessive levels of argininosuccinate in the urine and/or plasma. They also have moderately increased levels of citrulline and, low levels of arginine and ornithine in their plasma. Two patients succumbed to the disease. To date, eleven patients remained well on a dietary protein restriction, oral ammonia scavenging drugs and arginine supplementation. The majority of them have a reasonable good neurological outcome.