Clinical manifestations

Presentation and course

The classic presentation of citrullinemia type 1 is as a catastrophic illness in the first week of life (neonatal or early-onset). Typically, the affected neonate is born after an uncomplicated full-term pregnancy, labor, and delivery with normal Apgar scores. Unlike patients with proximal urea cycle disorders (carbamoyl phosphate synthetase 1 deficiency and ornithine transcarbamylase deficiency) presenting usually between the first 36 to 72 hours, subjects with citrullinemia type 1 and argininosuccinate lyase deficiency present usually between 72 hours and 1 week of age and with slightly lower initial peak-blood ammonia concentrations (01). Symptoms resemble a neonatal sepsis-like picture with hyperventilation, respiratory distress, and temperature instability. Poor sucking, vomiting, and muscular hypotonia may be present. Respiratory alkalosis is often seen. Typically, symptoms rapidly progress from somnolence and lethargy to stupor and coma with respiratory arrest (due to hyperammonemia). Increased intracranial pressure is usually evident, and subarachnoid hemorrhage has been reported (02). Acute hyperammonemic episodes demonstrate a diffuse or focal pattern of cerebral MRI changes and brain edema (19; 32; 95). However, the impact of (acute) cerebral changes on clinical long-term outcome, and thus, the predictive value of diagnostic imaging is yet to be determined and subject to future analyses (79; 95). An analysis of MRIs during a stable metabolic condition of individuals with ornithine transcarbamylase deficiency, another urea cycle disorder, showed structural, biochemical, and functional changes in the brain between symptomatic subjects and normal controls (31).

Cases of late-onset citrullinemia type 1 with partial enzyme deficiencies generally become symptomatic in early childhood, often associated with weaning or switching from formula to cow's milk or with intercurrent illnesses, but may also occur at a later age and include anorexia, (cyclic) vomiting, irritability, and behavioral changes, which often progress to stupor or coma. Intercurrent hyperammonemic episodes in affected individuals have been precipitated by protein-rich meals, infections, seizures, medications (especially steroids, valproate, haloperidol, and L-asparaginase/pegaspargase), menstruation, severe exercise, trauma or burns, and surgery. Citrullinemia type 1 can also present with acute liver failure (26). Importantly, the postpartum period (due to catabolism and the involution of the uterus) is an important trigger for late-onset citrullinemia type 1, typically presenting with an episode of acute hyperammonemia or acute liver failure (58; 33; 107; 10).

Citrullinemia type 1 usually presents as early-onset disease. Compilations of data from Japan and Europe highlight that early-onset argininosuccinate synthetase deficiency corresponds to approximately 75% of all incidences of argininosuccinate synthetase deficiency (45; 72; 54). For late-onset citrullinemia type 1, childhood (age 2 to 12 years) appears to be a later, vulnerable period for the first hyperammonemic crisis (99). In countries where extended newborn screening programs have included the determination of citrulline, mild variants are now being detected that mostly remain completely free of symptoms and do not need treatment (61). However, they may still be at risk for metabolic crisis.

Because the effect of genotypic variability on the clinical (neurologic) disease course was unknown, thus far, the phenotypic variability–ranging from lethal hyperammonemic encephalopathy to an asymptomatic disease course–was unpredictable based on the respective genotype. However, a newly established mammalian biallelic expression system was used to determine the residual enzymatic activity of ASS1 in individuals with citrullinemia type 1. Interestingly, residual enzymatic ASS1 activity reliably allows phenotypic severity prediction in citrullinemia type 1 because citrullinemia type 1-individuals with 8% of residual enzymatic ASS1 activity or less (severe phenotype) had more frequent and more severe hyperammonemic events and lower cognitive function than citrullinemia type 1-individuals above 8% of residual enzymatic ASS1 activity (attenuated phenotype) (113). Moreover, this new method allows the evaluation of current or future diagnostic and therapeutic interventions on clinical endpoints in a severity-adjusted manner (84).

Citrullinemia type 2 was first described in adults mainly from Japan but now has also been found in other countries, with high incidences also in China and Korea. Affected patients suffer from sudden disturbances in consciousness, abnormal behavior, disorientation, restlessness, and convulsions (56). Hyperammonemia can rapidly progress to coma and death due to brain edema. Patients may present with hepatic steatosis, hepatic fibrosis, or hepatocellular carcinoma. The liver shows a picture of nonalcoholic steatohepatitis (102; 36), and neuropathologic and imaging studies show characteristic cortical atrophy termed pseudoulegyria (92). Unlike patients suffering from other urea cycle disorders, these patients have a natural aversion to carbohydrates and favor protein and sometimes lipid-rich foods. Onset of significant and recurrent illness has been described to start from 11 to 79 years, with a mean of 34.4 years (108).

Citrullinemia type 2 can present with a distinctively different symptomatology in neonates as neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) (103; 97; 36). These patients often fail to thrive and can develop severe liver disease (83; 36). They may test positive on newborn screening with hypergalactosemia, hypermethioninemia, or hyperphenylalaninemia (77) and often have very high serum concentrations of alpha-fetoprotein. The neonatal intrahepatic cholestasis caused by citrin deficiency picture mostly resolves between 6 and 12 months of age, but the same patients may develop liver disease, failure to thrive, (recurrent) hypoglycemia, and dyslipidemia (FTTDCD), or may later manifest the above-mentioned adult presentation (citrullinemia type 2) (83; 04; 36; 44). Characteristic growth impairment of individuals with citrin deficiency suggests a significant role of citrin in growth, becoming evident especially during the vulnerable phase of growth development, ie, within the first 6 months of life and later in adolescence (76; 83; 04; 44).

Prognosis and complications

Although mortality has somewhat decreased, morbidity in urea cycle disorders remains high in survivors of neonatal hyperammonemic coma. Frequent comorbidities in urea cycle disorders are associated with the most vulnerable organ (ie, the brain), leading to mental retardation, cerebral palsy, and seizure disorders (55). A study of urea cycle disorder long-term survivors demonstrated that approximately only half of the patients had IQ scores higher than 85, and most of those patients had late-onset disease manifestation or were diagnosed and treated prospectively, ie, before onset of clinical symptoms conceding poor outcome for early-onset disease manifestation (104). A review and meta-analysis of observational studies spanning a period of more than 35 years demonstrated that early-onset patients, among those with argininosuccinate synthetase deficiency, have an especially high risk of early death (13). Normal outcome for argininosuccinate synthetase 1 deficiency was only reported for 36% of surviving patients by the end of the first year. No significant improvement of survival was observed over more than 30 years for urea cycle disorders (13), which is in contrast to a study from Japan with the survival rate of early-onset individuals with citrullinemia type 1 highest with approximately 97% (48).

Neurocognitive outcome is independent of age at which the first symptoms occurred or age at diagnosis but does essentially depend on the initial as well as peak-blood ammonia concentration (05; 25). At the first hyperammonemic attack, peak-blood ammonia concentrations of less than 180 µmol/L are associated with good outcomes, and peak-blood ammonia concentrations of more than 360 µmol/L indicate a poor prognosis. Variable outcome is observed when peak-blood ammonia concentration is between 180 µmol/L and 360 µmol/L (104; 48). The intrinsic disease severity, which is reflected by the onset type and initial peak-blood ammonia concentration, affects the neurologic outcome of individuals with urea cycle disorders (88). Importantly, current clinical knowledge suggests that cognitive outcome at the last regular visit differs between patients with proximal (ie, carbamoyl-phosphate synthetase 1 deficiency, ornithine transcarbamylase deficiency) and distal defects (ie, argininosuccinate synthetase deficiency, argininosuccinate lyase deficiency); the latter group is more compromised despite lower initial peak blood ammonium concentrations (86).

Published results from the UCDC consortium reported on neuroimaging and neurocognitive findings of more than 600 urea cycle disorder patients, including patients suffering from citrullinemia types 1 and 2 (106). In citrullinemia type 1 (n=75), gross motor skills were significantly less well developed than expressive language skills, and adults performed less well than younger patients in neurocognition; however, it remains unclear whether this is due to decline throughout life or improvements in diagnostics (eg, introduction of newborn screening) and treatments in recent years. Individuals with citrullinemia type 2 tend to develop normally with regular employment status and no significant impairment in adulthood. Severe developmental disability was only reported in 2 of 17 adults in a Japanese cohort (44). However, given the low number of investigated individuals, additional studies will have to confirm this observation.

Furthermore, a study demonstrated no difference in movement disorders (ie, dystonia, spasticity, chorea, ataxia) between proximal and distal urea cycle disorders, but compared to patients with early-onset disease manifestations, patients with late-onset urea cycle disorders developed movement disorders and motor abnormalities less often (55; 88). A detailed overview of organ-specific disease manifestations and complications in patients suffering from urea cycle disorders (eg, citrullinemia type 1) has been provided by Kölker and colleagues (55). Patients with urea cycle disorders suffer from increased frequencies of mental disability and behavioral/emotional problems; interestingly, however, health-related quality of life of these patients is normal (41).

Patients with citrullinemia type 2 presenting with neonatal intrahepatic cholestasis caused by citrin deficiency usually recover completely by their first birthday with no developmental delay or neurologic sequels. However, some continue to have a progressive disease course, and some develop adult disease much later (97; 83; 04; 44). Adult citrullinemia type 2 is often progressive, possibly leading to hepatic fibrosis and cirrhosis, hepatocellular carcinoma, brain edema, and death (36; 44). The most effective treatment for this disorder is liver transplantation, although many patients can be managed conservatively. However, population genetics indicate that many homozygous individuals never experience symptoms. So far, there are no markers predicting which individuals will exhibit which specific clinical disease course in citrin deficiency.

Clinical vignette

Patient 1: Adult type citrullinemia type 2. The following is a case of a previously reported 32-year-old adult (78). At 32 years of age, the patient was admitted because of night delirium. Hepatic coma was suspected because of abnormal liver function tests and triphasic waves on EEG. He recovered in a few days, but at 42 years of age he presented with a second episode of disturbance of consciousness associated with mild hyperammonemia of 90 µM (control, less than 35 µM). Citrulline concentration was 227 µM (control, 28 to 40 µM), and arginine concentration was 187 µM (control, 64 to 97 µM). A final episode occurred two years later and progressed to coma with prominent pyramidal signs associated with an elevated ammonium concentration of 170 µM. He developed clonic convulsions and marked abnormalities of hepatic and renal function. Plasma ammonia increased to 370 µM, and he died in coma on the 30th hospital day. The clinical diagnosis was adult-type citrullinemia type 2 associated with liver cirrhosis.

Patient 2: Neonatal/-early onset citrullinemia type 1. A 3120 g full-term male, appeared well until the second day of life when he developed progressive lethargy and tremulousness, which proceeded to coma by 48 hours of age. He became unresponsive to painful stimuli and required tracheal intubation and artificial ventilation. The liver was palpable 7 cm below the right costal margin. Determination of liver functions on the sixth day of life included: serum glutamic oxaloacetic transaminase 996 IU, serum glutamic pyruvic transaminase 648 IU, prothrombin time 35 seconds (control, 10 seconds), partial thromboplastin time 125 seconds (control, 30 seconds), and plasma ammonia 700 µM. Cultures of the blood and stool were positive for Staphylococcus aureus, and the infant was treated with antibiotics. Despite institution of specific metabolic therapy with intravenous sodium benzoate, sodium phenylacetate, and L-arginine-HCl, the patient remained obtunded with opisthotonic posture and increased deep tendon reflexes. An electroencephalogram revealed generalized dysrhythmia. Plasma ammonia concentrations continued to rise, and the boy succumbed the following day. Plasma citrulline was 2700 µM, and the enzymatic defect of citrullinemia type 1 was confirmed by assaying argininosuccinate synthetase activity in cultured fibroblasts.

Biological basis

Etiology and pathogenesis

Citrullinemia type 1 is caused by a partial or complete deficiency of argininosuccinate synthetase, a cytosolic enzyme in the urea cycle that catalyzes the conversion of citrulline, aspartate, and adenosine triphosphate to argininosuccinate, adenosine monophosphate, and pyrophosphate (33). Citrullinemia type 2 is caused by the absence of a mitochondrial aspartate/glutamate carrier protein (SLC25A13) called citrin due to recessive mutations in the gene encoding this transporter (80; 81). The citrullinemia type 2 locus resides on chromosome 7q21.3 (53). Both citrullinemia type 1 and citrullinemia type 2 are inherited as autosomal recessive traits. In citrullinemia type 1, the deficient enzyme is expressed in multiple tissues, including liver, kidney, and cultured fibroblasts. The gene has been localized to the q34.11 region of chromosome 9, and both the nucleotide coding and the amino acid sequence are known (28). It is a cytoplasmic homotetramer with a subunit molecular weight of 46,400. Updates on mutations of patients with citrullinemia type 1 were given by Engel and colleagues and, more recently, by Diez-Fernandez and colleagues (24; 21). Mutations are distributed throughout the gene. Molecular genetic analysis is more often used to confirm diagnosis than measurement of enzymatic activity (87). Deletion of exon 7 (IVS6-2A-G) is responsible for approximately 50% of the alleles in Japan. However, the mutation spectrum is heterogeneous in other populations (24). A new biallelic expression system became available to reliably predict the phenotypic severity of individuals with a specific genotypic background (113). This might be an important tool for genetic and clinical counseling of individuals and their families with citrullinemia type 1 and serves as a basis for investigation of diagnostic and therapeutic interventions in a severity-adjusted manner (84).

Citrullinemia type 2 occurs frequently in China and Japan (where 2 mutations account for about 70% of mutant alleles) and is caused by mutations in a calcium-dependent mitochondrial membrane protein, citrin (53). This inner mitochondrial membrane transporter has been shown to transport aspartate and glutamate (80; 81).

Citrullinemia type 2 is also associated with decreased activity of argininosuccinate synthetase 1 activity (2% to 50%) and protein in the liver, but with normal concentrations of argininosuccinate synthetase in other tissues (52). Moreover, citrin deficiency leads also to glutamine synthetase impairment, which might be the main contributor to hyperammonemia (36). The link between citrullinemia type 2 and the finding of a partial deficiency of argininosuccinate synthetase 1 remains elusive.

In neonates with citrullinemia type 1 dying of hyperammonemic coma, neuropathologic changes involve prominent cerebral edema and generalized neuronal cell loss. The mechanisms of the ammonia-induced brain damage are only partially understood. Ammonia is normally detoxified in astrocytes by glutamate dehydrogenase and glutamine synthetase. The accumulation of ammonia and glutamine has a number of potentially toxic effects on the brain, including depletion of intermediates of cell energy metabolism and of organic osmolytes, altered amino acid and neurotransmitter concentrations, increased extracellular potassium concentrations (06; 118), potentially altered water transport through aquaporin 4 channels (60), and oxidative and nitrosative stress due to increased free radical production and increased nitric oxide synthesis (75). Glutamine is being trapped in the brain by polar glutamine transport across the blood-brain barrier (35), and accumulation of glutamine in the astrocyte is likely responsible for the cerebral edema associated with hyperammonemic coma. In survivors of prolonged neonatal hyperammonemic coma, changes observed on neuroimaging studies obtained months later include ventriculomegaly with increased sulcal markings, bilateral symmetrical low-density white matter defects, and diffuse atrophy with sparing of the cerebellum (19). The distribution of age-specific MRI lesions is thought to reflect hypoperfusion predominantly affecting brain regions with a high metabolic rate (30). Interestingly, a zebrafish model was used to analyze the effects of hyperammonemia, demonstrating strongly enhanced transamination-dependent formation of osmolytic glutamine and excitatory glutamate, thereby inducing neurotoxicity and death via synergistically overactivation of NMDA receptors and bioenergetic impairment induced by depletion of 2-oxoglutarate. Withdrawal of 2-oxoglutarate from the tricarboxylic acid cycle with consecutive tricarboxylic acid cycle dysfunction ultimately causes impaired oxidative phosphorylation with ATP shortage, decreased ATP/ADP-ratio, and elevated lactate concentrations. Interestingly, inhibition of ornithine aminotransferase is a promising and effective therapeutic approach for preventing neurotoxicity and mortality by hyperammonemia in zebrafish (112; 114).

Citrullinemia type 2 is known to be linked with hepatocellular carcinoma, and a metabolic link between argininosuccinate synthetase and cancer cell proliferation has been demonstrated (16; 91). It can be shown that decreased activity of argininosuccinate synthetase in cancer supports proliferation by facilitating pyrimidine synthesis via CAD (carbamoyl-phosphate synthase 2, aspartate transcarbamylase, and dihydroorotase complex) activation. Partial argininosuccinate synthetase deficiency in cancer increases cytosolic aspartate concentrations, which increases CAD activation. Decreasing CAD activity decreases proliferation and, thus, may serve as a therapeutic strategy in cancers in which argininosuccinate synthetase is downregulated. These results support argininosuccinate synthetase downregulation as a novel mechanism supporting tumorigenesis through a metabolic link between the urea cycle and pyrimidine synthesis (70).

Epidemiology

The estimated cumulative prevalence of urea cycle disorders is 1 in 35,000 to 52,000 newborns (74).

Data provided by the UCDC and the newborn screening program calculated the overall incidence of argininosuccinate synthetase 1 deficiency in the United States as 1 in 250,000 people (94; 101). This incidence increases by extended newborn screening, mainly due to the detection of mild to asymptomatic individuals with citrullinemia type 1 (84).

The frequency of citrullinemia type 2 in Japan was calculated to about 1:100,000 (53); however, calculations from the frequency of heterozygous alleles estimate the frequencies of homozygotes to be 1 in 19,000 in Japan, 1 in 50,000 in Korea, and 1 in 17,000 in China (63). It is very rare in other ethnicities.

Prevention

No method is known for preventing citrullinemia type 1. However, prenatal diagnosis is available using chorionic villus or cultured amniocytes to measure argininosuccinate synthetase activity or detect mutations (42). According to a guideline for the diagnosis and management of urea cycle disorders, molecular genetic analysis is the preferred prenatal testing method for all urea cycle disorders (33). The first successful live birth following preimplantation genetic diagnosis for citrullinemia type 1 was reported in a Korean couple heterozygous for ASS1 mutation (18).

Newborn screening by tandem mass-spectrometry using blood spots now allows presymptomatic diagnosis of citrullinemia and early treatment (03; 61). Nevertheless, studies from the E-IMD consortium showed that newborn screening for citrullinemia type 1 is rarely performed in Europe. If newborn screening for citrullinemia type 1 was performed, results of the testing were often not available before the diagnosis was made by selective metabolic investigation for early-onset patients with argininosuccinate synthetase 1 deficiency (87). Screening programs have also identified a considerable portion of individuals with mild to asymptomatic citrullinemia type 1 who apparently have no risk for a (severe) disease course and might need no treatment (61); however, sufficient scientific evidence as to whether phenotypic mild to asymptomatic individuals with citrullinemia type 1 diagnosed by newborn screening need continuous dietary or medical treatment is lacking (34). Moreover, only mild elevations of citrulline concentrations in dried blood spots during newborn screening might lead to the identification of individuals with heterozygous citrullinemia type 1 carrier status (96). Because of the high detection rate of mild variants of questionable significance and heterozygous carriers, this practice has been discontinued in some European countries (eg, Germany). A severity-adjusted evaluation from the United States and Europe confirmed that individuals with attenuated disease courses were indeed overrepresented, whereas individuals with severe citrullinemia type 1 disease courses were underrepresented in the newborn screening group. However, newborn screening enables the attenuation of the initial hyperammonemic decompensation in all identified individuals but does not affect the frequency of subsequent metabolic decompensations. Future long-term studies will need to evaluate the clinical impact of this finding, especially with regard to mortality, as well as long-term cognitive outcome and quality of life of survivors (84).

Prenatal diagnosis of citrullinemia type 2 is also possible but appears questionable because of the less clear treatability and variable clinical manifestations. Elevations of citrulline are usually too low to be detected by extended newborn screening programs targeting this parameter. A molecular second-tier approach by testing common pathogenic variants of the SLC25A13 gene in cases of citrulline elevations improve detection rates and sensitivity (17), but this is most likely only efficiently applicable in countries with a high overall prevalence of citrullinemia type 2 and the presence of the respective variants in the general population.

Differential diagnosis

Confusing conditions

A number of inborn errors of metabolism can also have clinical presentations similar to citrullinemia type 1, especially in the newborn period. These include other urea cycle disorders and amino acidopathies, congenital lactic acidosis or mitochondriopathies, defects in fatty acid oxidation, and organic acidurias. In addition, a number of acquired conditions, including transient hyperammonemia of the newborn, sepsis, intracranial hemorrhage, and cardiorespiratory disorders, can present with a similar symptom complex. Another disorder to be included in the differential diagnosis is mitochondrial carbonic anhydrase VA deficiency (105). Differentiation depends on identifying hyperammonemia associated with citrullinemia. Occasionally, neonates with transient hyperammonemia will also have elevated plasma citrulline concentrations, especially after arginine supplementation. However, these elevations are transient, lasting normally less than 1 week, and are much less pronounced. There has also been a report of children with pyruvate carboxylase deficiency having elevated plasma citrulline concentrations (20); however, they also have markedly elevated plasma lactate concentrations, which are unusual in urea cycle disorders. In older children and adults, a number of acquired disorders can also present with hyperammonemia, including liver disease, Reye syndrome, drug toxicity, and hepatotoxins. Marked elevations of citrulline in blood and urine should make the diagnosis of argininosuccinate synthetase 1 deficiency obvious.

Citrullinemia type 2 has a broad differential diagnosis for neonatal cholestatic liver disease. Early differentiation between biliary atresia, a choledochal cyst, and “neonatal hepatitis syndrome” is most important. Metabolic differential diagnoses include alpha1-antitrypsin deficiency, cystic fibrosis, classical galactosemia, tyrosinemia type 1, Niemann-Pick type C, other urea cycle disorders, peroxisomal disorders, and bile acid synthesis defects. In adults, the differential diagnosis of fluctuating and varying neuropsychologic symptoms is even broader. An important clue to diagnosis is the recognition of additional liver pathology and hyperammonemia.

Associated or underlying disorders

Clinically, individuals suffering from neonatal-onset argininosuccinate synthetase 1 deficiency or from other early-onset urea cycle disorders are hardly distinguishable. Importantly, biochemical investigation will provide more insight and help differentiating these disorders.

Diagnostic workup

Plasma ammonia, acylcarnitines, and amino acids as well as a urine sample to determine amino acids, organic acids, and orotic acid should be obtained in suspected cases, ie, especially all neonates with unexplained, overwhelming, or progressive disease, particularly after normal pregnancy and birth (38).

Biochemically, the principal findings in citrullinemia type 1 are hyperammonemia and high plasma citrulline along with low arginine concentrations. Plasma ammonia concentrations are highly elevated during hyperammonemic crises. Citrulline concentrations are generally 50- to 100-fold elevated (normal is less than 50 µmol/L) (07). Additional amino acid abnormalities include elevated concentrations of glutamine (a storage form of ammonia) and decreased arginine (an end product of urea synthetic activity). Urinary orotic acid and citrulline excretion may also be increased.

In adult-onset citrullinemia type 2, plasma ammonium concentrations are “only” about 5- to 10-fold elevated during acute episodes, and citrulline concentrations are approximately 20-fold elevated. Interestingly, arginine concentrations are normal to mildly increased rather than decreased (51) because argininosuccinate synthetase is also expressed in the small intestine and the kidney. These two organs are the main sources of arginine synthesis and are not affected in citrin deficiency (16). It has been noted that serum pancreatic secretory trypsin inhibitor is also increased and may be useful as a diagnostic marker (50).

Management

For a detailed discussion see the Suggested Guidelines for the Diagnosis and Management of Urea Cycle Disorders (33).

Management of acute hyperammonemia in citrullinemia type 1. In the newborn period, management of acute hyperammonemia may be anticipatory or reactive. In families who have had an index patient, the birth of an at-risk or prenatally diagnosed infant provides the opportunity for prospective management. Within hours after birth, the child can be placed on oral therapy with appropriate ammonia scavengers and amino acids as subsequently described. The same applies for infants diagnosed by newborn screening.

For newborns or infants who have been diagnosed during hyperammonemia or coma, therapy must not be delayed because coma duration of less than 1.5 days (82) and timely start of treatment are important determinants of outcome. In fact, current knowledge proposes that noninterventional variables such as disease onset and initial peak-blood ammonia concentration are of utmost importance for the neurologic outcome of individuals with urea cycle disorders (88). Large ongoing studies from the E-IMD and UCDC consortia aim to investigate the effect of (early) diagnosis and current treatment principles on the neurologic and cognitive outcome of affected individuals. Specialized pediatric hospitals should have first-line medications and consensus-based treatment-protocols and must act according to the following principles:

Suggestions for a consensus-based treatment protocol, following above outlined principles, are depicted in Tables 1 and 2. Each (specialized) pediatric hospital should be able to adapt these recommendations to facility-specific conditions to provide best care medicine for their patients and prevent delay of treatment.

Table 1. Management of Acute Hyperammonemia of Citrullinemia Type 1: Protein, Liquid, and Glucose Management

Consensus-based treatment protocol for pediatric (specialized) hospitals treating argininosuccinate synthetase 1 deficient patients with acute hyperammonemia according to Suggested Guidelines for the Diagnosis and Management of Urea Cycle Disorders (33).

Table 2. First-line Medication for Citrullinemia Type 1 ****

Consensus-based treatment protocol for pediatric (specialized) hospitals treating argininosuccinate synthetase 1 deficient patients with acute hyperammonemia according to Suggested Guidelines for the Diagnosis and Management of Urea Cycle Disorders (33).

Ammonia scavengers (sodium benzoate, sodium phenylacetate/-butyrate) provide alternate pathways to eliminate waste nitrogen (12). A few studies demonstrate that treatment with sodium benzoate and sodium phenylbutyrate is safe and effective for the treatment or prevention of hyperammonemia in urea cycle disorders (39; 09). Sodium benzoate is conjugated with glycine to form hippurate, and sodium phenylbutyrate is conjugated with glutamine to form phenylacetylglutamine, both of which are cleared by the kidneys. Glutamine contains two nitrogen atoms. Thus, two moles of waste nitrogen are removed for each mole of phenylacetate or phenylbutyrate administered. Theoretically, on a mole-per-mole basis, nitrogen-disposing efficacy of sodium phenylbutyrate should be twice that of sodium benzoate and although biochemical superiority of sodium phenylbutyrate has been demonstrated (71), no systematic studies regarding the effects of long-term pharmacotherapy on neurologic or cognitive outcome, as defined by clinical endpoints, exists thus far.

Energy is supplemented via oral, nasogastric, or intravenous routes by 20% to 100% above the recommended daily requirements using carbohydrate (such as glucose orally or dextrose 20% orally or glucose intravenously) and fat (intralipid 20%) starting at 1 and up to 3 g/kg per day. However, a multicenter study showed that during the first 24 hours of emergency treatment, caloric intake was lower than during maintenance treatment and was below age-adapted recommendations (88). Carbohydrates were the primary, if not sole, energy source, whereas fats were often omitted from initial emergency treatment. Soluble insulin is provided to avoid hyperglycemia and to support intracellular glucose uptake. The intake of natural protein is stopped for 24 to maximally 48 hours and is then reintroduced gradually as tolerated (38). In the event that ammonia concentrations do not respond to this management and biochemical or clinical symptoms worsen, continuous veno-venous hemodiaflitration should be started immediately (already planned and organized earlier, ie, the latest at concentrations greater than 400 µmol/L) in neonates or children with ammonia concentrations of greater than 500 µmol/L (see Table 1) or at lower concentrations if response to medical treatment is inadequate (82; 64). Note, even though continuous veno-venous hemodiafiltration is by far the most efficient method for extracorporal ammonia elimination (82), prognosis is not related to dialysis modality, but primarily to the duration of coma before start of treatment confirming the necessity for rapid and aggressive management. In fact, a retrospective data analysis of 202 published cases revealed that current practice in dialysis does not affect outcome (37). Only 20% of all investigated individuals had a normal clinical outcome. In conclusion, dialysis along with conservative pharmacotherapeutic treatment is recommended to be initiated as early as possible and at even lower concentrations of ammonium (37). However, long-term data analyzing these recommendations is not available.

The dietary aim is to minimize external protein (and, thus, nitrogen) intake and at the same time to prevent endogenous protein catabolism by meeting high-energy demands of the patient. The initial dietary emergency regimen should be protein-free, but protein or essential amino acids must be reintroduced after 24 to 48 hours or once blood ammonia concentration has fallen below 100 µmol/L (33). In addition to close meshed control of laboratory parameters like ammonia, electrolytes, glucose, etc., plasma amino acids must be determined and the results available daily to safely modify such management. The reduction of protein intake must be carefully monitored to prevent over-restriction. A diet with inadequate intake can impair protein synthesis and lead to catabolic metabolic decompensation or failure to thrive (38).

An oral dietary emergency regimen might be applicable to treat a hyperammonemic episode as long as the patient is not at risk of developing catabolism due to insufficient energy supply as a consequence of vomiting, loss of appetite, or diarrhea. Protein, liquid, and glucose management is identical to the protocol in Table 1.

Patients and families might start with an oral emergency dietary regimen at home. Antipyretic measurements must be consequently performed if temperature exceeds 38°C; however, patients and parents must be aware that these measures must not postpone or replace adequate emergency treatment in hospital (38; 117).

Patients should be supplied with an emergency card, letter, or bracelet containing instructions for emergency measures and phone numbers. Logistics of rational therapeutic measures should be repeatedly evaluated by the specialist team with the family and the primary care physicians.

Long-term management of citrullinemia type 1. Long-term management of citrullinemia type 1 relies on the goals of preventing recurrent hyperammonemia, neurologic sequelae, and improving quality of life by the following principles (33; 38):

Table 3. Long-term Management of Citrullinemia Type 1

Catabolism must be avoided as much as possible. In addition to intercurrent illnesses, especially if associated with high fever and decreased intake of food and fluids, very dangerous triggers are severe exercise, seizures, trauma or burns, steroid administration, chemotherapy, and gastrointestinal hemorrhage.

Dietary treatment is an essential anchor point of long-term management and requires the knowledge of a specialist metabolic dietitian. For infants and older children with citrullinemia type 1, nutritional management involves the use of a high-caloric, low-protein diet supplemented with essential amino acids and, if necessary, vitamins and minerals. This is most readily accomplished by using small amounts of natural protein, an essential amino acids formula, and supplemental calories provided by a formula that does not contain protein. The goal of long-term management is to minimize the nitrogen load on the urea cycle. The FAO/WHO/UNO 2007 Report can be used as age- and gender-dependent recommendations for energy intakes (33). Especially in young infants and children, fasts should be avoided and snacks given to reduce the possibility of (overnight) catabolism. Dietary measures are very important for safely steering the conservative management in urea cycle disorders; however, growth impairment is a common and considerable burden for affected individuals (48). Thus far, it was unknown if and to which extent a protein-controlled or protein-reduced diet iatrogenically contributes to growth retardation in urea cycle disorders. A study showed that growth impairment was determined by disease severity and associated with reduced or borderline plasma branched-chain amino acid (BCAA) concentrations but was not associated with the degree of natural protein restriction, which is important for the consultation and reassurance of the affected urea cycle disorder individual (85).

Periodic measurement of plasma amino acids, which include glutamine and blood ammonia, may permit adjustment of therapy before clinical symptoms appear. Long-term medication comprises the use of sodium benzoate or sodium phenylbutyrate as well as the essential amino acid L-arginine. A detailed overview is provided in Table 3. Drugs may not be well tolerated by the child or family. Sodium phenylbutyrate tastes and smells unpleasantly and may be irritating to the stomach. Glycerol phenylbutyrate (RAVICTI®) has the same mechanism of action as sodium phenylbutyrate but is a sodium- and sugar-free prepro-drug of phenylacetic acid that has little odor or taste. In addition, phenylbutyrate may deplete branched chain amino acid concentrations and cause menstrual dysfunction or amenorrhea in up to 25% of postpubertal females (14; 33). To avoid complications, eg, mucositis or gastritis, sodium benzoate and sodium phenylbutyrate should be administered several times daily during meals with abundant fluids (33). Supplementation of L-arginine aims at maximizing ammonia excretion through the urea cycle (11; 59).

Plasma carnitine deficiency may occur in urea cycle disorder patients who are on a low-protein diet or receive treatment with ammonia scavengers. Neomycin and metronidazole have formerly been put forth as a means of decreasing intestinal ammonia in hepatic encephalopathy; however, there is still no good evidence to support this use and pharmacological gastrointestinal decontamination is only sporadically used (88; 33). Furthermore, the benefit of vaccinations outweighs the risk of decompensations. They are recommended at the same schedule as for healthy children (68; 33).

A significant number of patients with various urea cycle disorders have received partial or total orthotopic liver transplants to provide enzyme replacement therapy (67). Overall survival of 111 patients was 93% at a median follow-up of five years. In all successful cases, this has cured the hyperammonemia and permitted a normal protein intake. However, its effectiveness is hampered by expense, limited availability of donor organs, and significant morbidity and mortality from complication of transplantation or immunosuppression. After transplantation, hyperammonemia is controlled, but citrulline concentrations remain elevated, and arginine concentrations remain low. Thus, long-term arginine supplements may still be required following liver transplantation (90).

Ideally, orthotopic liver transplantation should be carried out between 6 and 12 months of age, before irreversible neurologic damage has occurred, in patients with severe neonatal-onset disease, and patients with progressive liver disease or patients suffering from recurrent severe decompensations despite intensive medical treatment (33). A retrospective survey study investigated if patients would benefit from liver transplantation by preventing further recurrent decompensations. Individuals with urea cycle disorders had an improved neurocognitive outcome if they received a liver graft, suggesting that they should undergo liver transplantation after comparatively low maximum ammonium concentrations in order to protect the patient’s neurocognitive abilities (47; 46). However, children with urea cycle disorder receive liver transplantation usually between 1 to 6 years of age (45; 67). Evidence indicates that if individuals with ASS1-deficiency suffer from severe (recurrent) hyperammonemic episodes that are not adequately manageable by conservative management, liver transplantation should be performed at an early age to avoid further neurodevelopmental deficits (62). An analysis evaluating the outcome of pediatric and adult urea cycle disorder patients who underwent a liver transplantation showed that approximately two thirds were transplanted before 5 years of age (111). Overall 1-, 5-, and 10-year survivals were excellent, with 93%, 89%, and 87% survival, respectively. Three analyses, including one study from the United Network for Organ Sharing database comprising all pediatric urea cycle disorder candidates receiving liver transplantation between 2002 and 2020, uniformly found that delayed transplantation was associated with a long-term risk of cognitive delay and that early liver transplantation might prevent progressive neurologic injury and optimize cognitive outcomes (86; 67; 115). However, the impact of liver transplantation on cognitive outcome in individuals with urea cycle disorders still remains elusive. Recent data from a nationwide study in Japan reporting the outcome of 70 individuals posttransplant showed positive effects of liver transplantation on long-term survival, sufficient prevention of recurrent hyperammonemic decompensations, but only limited effect, if at all, on the neurodevelopmental outcome in comparison to medical management, especially in individuals with a severe phenotype. Of note, individuals with citrullinemia type 1 tended to survive without cognitive impairment if liver transplantation was performed early during the disease course (49).

Some medications are contraindicated in urea cycle disorders because of secondary inhibition of the urea cycle, most importantly valproic acid and systemic steroids. Even in well controlled and managed patients, peri-acute deadly coma can occur. Less often, but also to be considered, is the potential development of hyperammonemic crises by treatment with carbamazepine, the use of asparaginase or 5-fluorouracil in cancer therapy or bladder, uterine, or joint irrigation with glycine solution during surgery.

Hepatocyte transplantation is an interesting therapeutic bridging opinion in urea cycle disorder patients awaiting liver transplantation and is being investigated in ongoing studies (66). At present, there appears to be no place yet for gene or enzyme replacement therapy in the treatment of urea cycle disorders (33). In vitro studies have suggested that mutations mapping in or near the substrate-binding site of the ASS1 gene are potential kinetic mutations whose decreased enzyme activities could be rescued by substrate supplementation, eg, by oral oxaloacetate. These suggestions need translation into practice, but might be a potential new therapeutic approach for patients with citrullinemia type 1 due to specific mutations with decreased affinity for aspartate (22).

A new interesting approach for treatment of acute hyperammonemia is inhibition of ornithine aminotransferase leading to transamination-dependent decrease of glutamate and glutamine. Mammalian transfer will provide further insight to determine if this approach is indeed a potential new therapeutic principle for individuals with urea cycle disorders (112). Lately, it was suggested that autophagy cooperates with the urea cycle in ammonia homeostasis. Selective activation of hepatic autophagy might, therefore, be used to treat hyperammonemia due to acquired or inherited diseases. However, it remains to be elucidated if autophagy also plays an important role during hyperammonemic conditions in the brain in order to determine if autophagy is a suitable target for treatment of hyperammonemic conditions (98). Moreover, gene therapy applying a recombinant adeno-associated viral vector harboring the ASS1 gene under control of a liver-specific promotor in combination with nitrogen scavenger pretreatment led to a normalization of ammonia concentration and reduction of citrulline concentrations in blood of 3-week-old citrullinemia type 1 mice with 100% survival rate up to the age of 7 months (08). Whether these promising results of this gene therapy can be translated to humans needs to be investigated.

Management of citrullinemia type 2. Citrullinemia type 2 is treated very differently from all other urea cycle disorders. In the infantile presentation, patients have improved on feeding with formula containing medium-chain triglycerides or lactose-free formula only. Breastmilk, which is relatively low in protein and high in carbohydrates, is not suitable for such patients. Sometimes they treat themselves with a diet high in protein and low in carbohydrates and also with a preference for beans, peas, and peanuts and avoidance of rice, other vegetables, and sweets. The preferred foods are high in arginine. A carbohydrate-restricted, high-protein diet and supplementations with L-arginine and sodium pyruvate are advocated as beneficial to these patients (40; 23; 69). Exogenous administration of sodium pyruvate can reduce the NADH//NAD+ ratio facilitating ureagenesis in the absence of citrin (16). This is antithetical to all other urea cycle disorders. Identically, dietary treatment is an essential anchor point of long-term management and also requires the knowledge of a specialist metabolic dietitian.

In case of metabolic deteriorations, it is again important to act antithetically to all other urea cycle disorders, ie, to provide extra protein and reduce carbohydrates. Intravenous fluids with high dextrose concentrations should be avoided as they can further deteriorate the neurologic condition. L-arginine supplementation is highly effective in hyperammonemic episodes (16). Nevertheless, in case of rapid progression or end-stage disease, liver transplant may become the last therapeutic option (43) and is the only curative treatment for citrin deficiency at present (16). Yazaki and colleagues reported a 25-year-old man with type 2 citrullinemia who had complete neurologic recovery after partial liver transplantation using a graft from his father (110). Moreover, supplementation with medium-chain triglycerides (MCT) might also be beneficial for individuals with citrin deficiency (83). These results are corroborated by a nationwide study in Japan, where the clinical course and outcome of 218 individuals with citrullinemia type 2 suggest that early intervention by implementation of a low carbohydrate diet and MCT supplementation is associated with improved long-term outcome (44).

New mRNA-based principles are currently under investigation (15). Moreover, application of nicotinamide riboside corrected impaired glycolysis and fatty acid beta-oxidation by reducing cytoplasmic NAD:NAD+ concentrations in a citrin knockout HepG2 cell line and, therefore, might comprise a novel treatment strategy in citrullinemia type 2 in the future (109). A detailed overview on the metabolic basis and treatment of citrin deficiency is provided separately (83; 36).

Outcomes

Prior to the development of alternate pathway therapy using ammonia scavengers (eg, sodium benzoate, sodium phenylacetate or phenylbutyrate), virtually all children with neonatal citrullinemia type 1 died in the newborn period or during infancy. Today, the survival rates for early-onset citrullinemia type 1 patients correspond to approximately 97% (48). In contrast, a review and meta-analysis showed less convincing results suggesting that no improvement of survival of urea cycle disorders was observed over more than three decades, between 1978 and 2014 (13). Furthermore, studies have demonstrated that noninterventional variables reflecting disease severity, such as disease onset and initial peak-blood ammonia concentration, are associated with the highest risk of mortality and poor neurologic outcome (25; 88).

Long-term morbidity, however, is still a major problem in urea cycle disorder patients. It could be shown that 50% of patients with urea cycle disorders suffer from intellectual disability (57). These data were confirmed by a study including 103 subjects with neonatal-onset urea cycle disorders (01). Moreover, growing evidence with regard to the impact of high peak-blood ammonia concentrations on poor neurocognitive outcome in urea cycle disorders arises (25; 88; 86; 48); however, further pathomechanistic concepts and therapeutic options are pivotal to avoid or reduce long-term morbidity in survivors of hyperammonemic decompensations.

Special considerations

Pregnancy

Women carrying affected offspring have not routinely experienced complications during pregnancy, and the children appear well at term. It also seems that maternal citrullinemia type 1 is not teratogenic (89). Langendonk and colleagues studied a series of pregnancies in women with inherited metabolic disease and suggested that care must be taken not to confuse behavioral changes of hyperammonemia for symptoms of postpartum psychosis or depression (58). Because late-onset disease manifestation of urea cycle disorders appears to comprise more than 50% of all urea cycle disorders (73; 99; 72; 54) and because there is a high possibility for missing cases presenting with symptoms of postpartum psychosis, depression, or severe mood disorder, routine monitoring of plasma ammonia concentrations in those women was suggested (27). The greatest risk period for acute hyperammonemic decompensation is between 3 to 14 days postpartum. The relative metabolic stress during this episode is thought to be due to changes of the puerperium and an increased protein load following involution of the uterus (33; 58). Additionally, nausea and vomiting during pregnancy might lead to severe problems because of catabolism and, thus, should always be taken seriously and treated effectively with antiemetics. Ideally, pregnancy should be planned in women with inherited metabolic disease (58). Pregnancy has not been reported to be a risk factor for citrullinemia type 2.

Anesthesia

It is prudent to use anesthetics with low toxicity to the liver. Surgery requires stopping of oral medication and may be associated with a catabolic condition, both of which may induce hyperammonemia in citrullinemia type 1. Therefore, it is important to continue alternate pathway therapy intravenously until the patient is able to accept oral medication. The patient should also receive adequate glucose to prevent catabolism. One report describes the perioperative uneventful use of midazolam, s-ketamine, fentanyl, and isoflurane with local injection of ropivacaine, along with intravenous infusion of glucose and alternate pathway drugs in two siblings with severe ornithine transcarbamylase deficiency (93). In general, surgery should only be performed in centers prepared for dealing with acute hyperammonemic episodes. After surgery, close monitoring of the clinical status and ammonia and glutamine concentrations as well as shifting to oral medications and diet are required (33).

A 3.5-year-old child with citrullinemia type 1 receiving general anesthesia (sevoflurane, propofol, fentanyl, cisatracurium) who showed postoperative prolonged unconsciousness of four hours was reported. Regaining consciousness, the patient was unable to maintain his head posture and seemed ataxic and gradually recovered his complete health after 10 days in the intensive care unit. Postoperative ammonia concentration was reported to be elevated, but a clear reason for delayed awakening could not be determined. This case emphasizes, that appropriate guidelines for the pre- and postoperative care of patients with inherited metabolic diseases of the urea cycle are indispensable (29).