Clinical manifestations

Presentation and course

The classic presentation of carbamoyl phosphate synthetase 1 deficiency is a catastrophic illness in the first week of life (neonatal-/early-onset; > 70% of reported cases) (11).

Typically, the affected neonate is born after an uncomplicated full-term pregnancy, labor, and delivery with normal Apgar scores. Compared to distal urea cycle disorders (argininosuccinate synthetase deficiency and argininosuccinate lyase deficiency), subjects with carbamoyl phosphate synthetase 1 deficiency and ornithine transcarbamylase deficiency (OTCD) present earlier, usually within 24 to 72 hours of age, and with a higher initial peak-blood ammonia level (02). Symptoms resemble those of a neonatal sepsis-like picture with hyperventilation, respiratory distress, and temperature instability. In addition, poor sucking, vomiting, and hypotonia may be observed. Typically, symptoms rapidly progress from somnolence and lethargy to coma (28). Neurologic findings may include increased deep tendon reflexes, and papilledema. Convulsions may already be late complications and follow alterations in consciousness. A review highlights separately the clinical manifestation of various individuals with carbamoyl phosphate synthetase 1 deficiency along with their genetic background (41).

Cases of late-onset disease with partial deficiencies have been reported (50). Symptoms may develop from infancy to adulthood and are associated with weaning or switching from formula to cow's milk, high-protein diet (eg, barbecue, (family) feast, parenteral nutrition), or triggers for catabolic stress. Such triggers might be fever, infections, gastrointestinal bleeding, vomiting, decreased energy or increased protein intake, and surgery. Furthermore, drugs, especially steroids, valproate, haloperidol, and L-asparaginase/pegaspargase (06), as well as the postpartum period (due to catabolism and the involution of the uterus) are important triggers for late-onset hyperammonemia (22; 46; 28). Childbirth may result in postpartum hyperammonemia in previously asymptomatic women with partial enzyme deficiency presenting with initial symptoms of postpartum psychosis, a clinical picture that may lead to a delay in the diagnosis of the urea cycle disorder and even progress to death (22).

Acute hyperammonemic episodes may resemble or actually involve stroke-like episodes, hemiplegia may be evident, and MRI may reveal infarction (05; 78) or injury to the bilateral lentiform nuclei and the deep sulci of the insular and perirolandic regions (88), demonstrating a diffuse or focal pattern of cerebral MRI changes and brain edema (14; 26). However, the impact of acute cerebral changes on long-term outcome, and thus, the predictive value of acute MRI changes, is unclear. 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 (25). Published results from the UCDC consortium report neuroimaging and neurocognitive findings of more than 600 urea cycle disorder patients, including patients suffering from carbamoyl phosphate synthetase 1 deficiency. In some disorders, 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. Patients suffering from carbamoyl phosphate synthetase 1 deficiency (n = 10) tended to have global early developmental delays and their scores did not decline over time (93).

Unlike in ornithine transcarbamylase deficiency, late-onset disease manifestation in carbamoyl phosphate synthetase 1 deficiency is rare (11). Findings revealed that late-onset carbamoyl phosphate synthetase 1 deficiency corresponds to the minority of the respective disease manifestations (42; 11; 43).

Prognosis and complications

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, which is the brain, leading to intellectual disability, cerebral palsy seizure disorder, and visual deficits (11; 44). A study of long-term survivors demonstrated that approximately half of the patients had IQ scores above 85. Most of them 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 (91). A review and meta-analysis of observational studies spanning a period of more than 35 years demonstrated that early-onset patients, the most common presentation of carbamoyl phosphate synthetase 1 deficiency, have a high risk of permanent disease manifestation and early death (11). Normal outcome of carbamoyl phosphate synthetase 1 deficiency by the end of the first year was found for only 20% of surviving patients, and no improvement of survival was observed over a period of more than 30 years.

Neurocognitive outcome does essentially depend on the initial and peak blood ammonium levels (04; 20). At the first hyperammonemic attack, a peak blood ammonium level of less than 180 µmol/L is associated with good outcome, and a peak blood ammonium level of more than 360 µmol/L is a marker for poor prognosis. Variable outcome is observed when peak blood ammonium level is between 180 and 360 µmol/L (91; 42). The intrinsic disease severity, as reflected by the onset type and the initial peak blood ammonium level, is of utmost importance for the neurologic outcome of urea cycle disorder patients (70). Importantly, current clinical knowledge suggests that cognitive outcomes 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 being more compromised despite lower initial peak blood ammonium concentrations (71). Furthermore, a study demonstrated no difference in movement disorders (ie, dystonia, spasticity, chorea, ataxia) with regard to proximal versus distal urea cycle disorders; however, compared to patients with early-onset disease manifestations, patients with a late-onset phenotype developed movement disorders less often (44). A detailed overview of organ-specific disease manifestations and complications in individuals suffering from urea cycle disorders (eg, carbamoyl-phosphate synthetase 1 deficiency) has been given by Kölker and colleagues (44). Patients with urea cycle disorders suffer from high frequencies of associated mental disability and behavioral/emotional problems. However, the health-related quality of life of these patients is within the normal range (35).

Clinical vignette

Patient 1: Neonatal-onset CPS1D. The female patient was delivered at 39 weeks’ gestation after an uncomplicated pregnancy, labor, and delivery to parents who had had a stillborn baby at 39 weeks’ gestation one year before. Apgar scores were 8 and 9 after one and five minutes, respectively. Birth weight was 3220 g. She did well for the first 24 hours, taking in some breast feedings. At about 36 hours of age, she was noted to be lethargic, limp, and without response to pain. A capillary blood gas showed respiratory alkalosis with a pH of 7.55 and pCO₂ of 21. Serum glucose, electrolytes, creatinine, and a complete blood count were all normal. Investigations for infection were negative. Within a few hours she developed hypothermia, respiratory distress, and seizures. The first blood ammonia concentration at 65 hours of age was 1296 µmol/L (normal 11 to 110), rising to 1496 µmol/L two hours later; this was associated with otherwise normal liver tests. After endotracheal intubation, she was transferred to a metabolic center where she was admitted at 75 hours of age. She was comatose with fixed and dilated pupils. She had jerking movements and hiccups, and her blood ammonia was 1880 µmol/L. Biochemical investigation on admission revealed undetectable plasma L-citrulline with an elevated concentration of plasma glutamine of 1575 µmol/L (normal less than 933 µmol/L) and alanine of 1214 µmol/L (normal less than 611 µmol/L). Urinary amino acids, orotic acid, and organic acid analyses were all normal.

Venous catheters were inserted into the internal jugular and femoral veins. Intravenous glucose as well as intravenous sodium benzoate, sodium phenylacetate, and L-arginine-HCl (10% solution) were given with a bolus and then continued at a constant infusion rate. Hemodialysis was initiated at 79 hours of age, at which time blood ammonia concentration was 2235 µmol/L. After three hours of hemodialysis, the blood ammonia concentration fell to 270 µmol/L, and the patient’s coma resolved. Hemodialysis was stopped. Despite rebound of the blood ammonia concentration on two occasions, one requiring reinstitution of hemodialysis, blood ammonia concentrations were eventually reduced and kept under control (< 220 µmol/L). Episodes of hyperglycemia were treated with continuous insulin infusions. The patient was extubated at 113 hours of age. Feeding started at 144 hours through a nasogastric tube, using a combination of cow’s milk formula and an essential amino acid mixture, and was gradually increased to supply a protein-restricted diet. Oral L-citrulline replaced intravenous L-arginine-HCl administration.

The patient was discharged from the hospital 13 days after admission in good condition. Molecular analysis confirmed the diagnosis of carbamoyl phosphate synthetase 1 deficiency. Since her discharge, she has had several episodes of mild hyperammonemia treated in the hospital with intravenous fluids and medications. At three years of age, psychomotor functions were close to normal, including motor skills and comprehension; however, her speech was markedly delayed (90). She underwent liver transplantation at seven years of age, which cured her hyperammonemia, but she continues to have developmental delay.

Patient 2: Late-onset CPS1D. After giving birth to her third child, a 35-year-old mother was referred to the inpatient psychiatric unit for the management of acute postpartum psychosis. Initially, midwifes noticed an increased level of anxiety and obnubilation, as well as a rapidly fluctuating symptomatology consisting of confusion, agitation, and violence starting on day three after delivery. Her medical history was marked by two similar phases of agitation, confusion, and mystic delusions starting on day three after each previous delivery. The outcome of each episode had been favorable, with weaning from antipsychotics within several weeks after delivery and no residual psychiatric symptoms between episodes. Initial medical records indicated normal results on physical examination and laboratory tests (complete electrolyte panel, CBC, liver function tests, and ECG). On postpartum day 16, she presented with fever (40°C, 104°F) and was referred to the inpatient obstetric unit with the suspicion of endometritis. An antipsychotic and an antibiotic were administered to treat the postpartum psychosis and a possible infection. However, unlike her previous postpartum episodes, the patient’s status responded only partially to antipsychotic treatment. A psychiatric consultant delineated that the patient was more confused and less delusional than one would have expected in a typical postpartum psychosis. Furthermore, she complained about chronic headaches and a habitual reluctance to eat meat. Blood pressure and pulse were stable, no signs of severe sepsis were remarkable, and a neurologic examination was uneventful. The psychiatrist extended his spectrum of differential diagnosis and ordered immediate blood tests, including ammonia concentrations. Hyperammonemia (224 µmol/L; normal is below 50 µmol/L) and respiratory alkalosis were found. All other blood tests were normal. After consulting the internal medicine fellow, the psychiatrist decided to transfer the patient to the intensive care unit for immediate start of antihyperammonemic treatment. Intravenous sodium benzoate and sodium phenylbutyrate were given as a bolus and then continuously along with L-citrulline and a protein-free hypercaloric nutrition. CT imaging of the brain was unremarkable. Subsequently, the patient’s neuropsychiatric status improved and her ammonia concentration fell to 19 µmol/L the following day. The diagnosis of a urea cycle disorder was substantiated by amino acid analysis showing high glutamine and low concentrations of L-arginine and L-citrulline. Weaned from antipsychotics and asymptomatic, she was discharged 13 days after the diagnosis with an emergency card and instructions for a protein-reduced diet and long-term treatment with sodium benzoate, sodium phenylbutyrate, and L-citrulline. Molecular analysis found two mutations of the carbamoyl phosphate synthetase gene (p.P87S and p.R803C) confirming carbamoyl phosphate synthetase 1 deficiency (22).

Biological basis

Etiology and pathogenesis

This disorder is caused by a complete or partial deficiency of carbamoyl phosphate synthetase 1, the first enzyme in the urea cycle, which catalyzes the formation of carbamoyl phosphate from ammonia, ATP, and bicarbonate using N-acetylglutamate as a cofactor (28).

The gene encoding carbamoyl phosphate synthetase 1 has been sequenced and mapped to the long arm of chromosome 2q.34 (23; 86). Various mutations causing enzyme deficiency have been found in patients, many resulting in messenger RNA instability and decay and, therefore, absent or markedly reduced amounts of carbamoyl phosphate synthetase enzyme (19; 30). The C-terminal domain of the enzyme was suggested to have an important function on enzyme regulation depending on the allosteric activator of carbamoyl phosphate synthetase 1, N-acetylglutamate (16; 17).

Carbamoyl phosphate synthetase 1 deficiency is inherited as an autosomal recessive trait. This mitochondrial matrix enzyme, expressed in hepatocytes and intestinal mucosa epithelial cells (74), catalyzes the synthesis of carbamoyl phosphate from bicarbonate, ammonia, and ATP.

It is the most abundant protein in liver mitochondria, accounting for 20% of the mitochondrial matrix protein (51). The enzyme consists of a single polypeptide with a molecular weight of 165,000 and 1500 amino acid residues, respectively. Like many metabolic disorders, carbamoyl phosphate synthetase 1 deficiency does not arise from a common mutation, and numerous mutations have been identified (82; 30; 41). A study on the mutational spectrum of CPS1 found that approximately 90% of the reported mutations were “private” and only about 10% recurred in unrelated families (96). However, there is uncertainty about the impact of functional data on clinical long-term outcome variables due to a lack of systematic (functional) genotype-phenotype analyses of carbamoyl phosphate synthetase 1 deficiency. An analysis on five children with CPS1 suggested that mutations interfering with the catalytic sites, the internal tunnel, or the regulatory domain resulted in a severe phenotype (21).

Biochemically, the principal finding is hyperammonemia. Amino acid abnormalities include elevated concentrations of glutamate, as well as glutamine, alanine, and asparagine (the latter being storage forms of ammonia); decreased or absent citrulline (the product of ornithine transcarbamylase activity); and decreased arginine (an end product of the urea synthetic activities). Blood urea nitrogen is also low.

Neuropathologic changes in neonates dying of hyperammonemic coma involve prominent cerebral edema and generalized neuronal cell loss (18). Survivors of prolonged hyperammonemic coma show changes on neuroimaging studies obtained months later (ventriculomegaly with increased sulcal markings, bilateral symmetrical low-density white matter defects, and diffuse atrophy with sparing of the cerebellum) (14). Further MRI changes, despite liver transplantation, were reported to involve hyperintensities of the insular cortices and deep frontal gyri as well as caudate nucleus and putamen (65).

The mechanisms of the ammonia-induced brain damage are only partly 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 (102; 48), potentially altered water transport through aquaporin 4 channels (48), and oxidative and nitrosative stress due to increased free radical production and increased nitric oxide synthesis (64). 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 acting overactivation of NMDA receptors and bioenergetic impairment induced by depletion of 2-oxoglutarate (98; 99). Marked elevation of glutamate in plasma has been reported in a cohort of individuals with CPS1 deficiency and poor neurocognitive outcome and/or early death (15). 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. A constitutive neonatal mouse model of CPS1 deficiency has been developed mimicking the clinical picture of early-onset individuals as well as key biochemical features of this disorder (37). Moreover, a liver-humanized mouse model for CPS1 deficiency was generated by repopulation of the mouse liver with CPS1-deficient human hepatocytes aiming to assess the impact of various (emerging) therapeutic interventions (ie, gene-editing technologies) on human hepatocytes for a long period of time providing safety and efficacy data (79). These mice models might be helpful for developing new therapeutic strategies for individuals with carbamoyl phosphate synthetase 1 deficiency in the future. Utilization of patient-derived pluripotent CPS1-deficient stem cells, which can be differentiated to hepatocyte-like cells, might be another approach for the development and assessment of novel targeted therapies. However, CRISPR-mediated addition of human codon-optimized CPS1 with constitutive expression in CPS1-deficient stem cells failed to efficiently restore ureagenesis and lower ammonia concentrations after differentiation to hepatocyte-like cells when compared to their unedited counterparts, which was partially due to transcriptional silencing at the stem cell stage (63). Future research is needed to assess the efficacy of different genome editing techniques to restore CPS1 function for the treatment of CPS1 deficiency.

Interestingly, neonatal pulmonary hypertension was found to correlate with reduced plasma concentrations of arginine, nitric oxide, and polymorphisms in the CPS1 gene (67; 85). Pulmonary artery pressure is regulated by endogenous NO, which is derived from arginine supplied by the urea cycle. A combination of four single nucleotide polymorphisms (SNPs), including one in the CPS1 gene, had a 70% predictive value for lack of response to asthma therapy in a cohort of African-American patients with asthma (56). Furthermore, carbamoyl phosphate synthetase 1 (CPS1) has been suggested to play a role in tumor genesis, as demonstrated by findings in liver kinase B1-inactivated lung adenocarcinoma. Carbamoyl phosphate synthetase 1 knockdown may reduce cell growth and metabolite concentrations and may contribute, in combination with other chemotherapy agents, to a new approach in treating specific neoplasms (13). With recent advances in basic and translational studies, appreciation of the CPS1 enzymatic function in various other medical fields, eg, its function in cardiovascular disease, cancer, obesity, and major depressive disorders, is emerging and may play a relevant role for future treatment of these disorders (61).

Epidemiology

The estimated cumulative prevalence of urea cycle disorders is 1 in 35,000 to 52,000 newborns (60). Data provided by the UCDC (76) calculated the overall incidence of carbamoyl phosphate synthetase 1 deficiency in the United States as 1 in 1,300,000 people (87).

Prevention

No method is known for preventing carbamoyl phosphate synthetase 1 deficiency. Prenatal diagnosis is available using molecular methods (82; 29). Furthermore, preimplantation genetic diagnosis in couples at risk is possible (27). 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 (28).

There is still no satisfactory method of newborn screening that enables the attenuation or even prevents the initial hyperammonemic decompensation in many identified individuals. A promising approach combines the determination of citrulline and orotate (80). Future long-term studies will be needed to evaluate the clinical impact of this finding, especially with regard to mortality and long-term cognitive outcome and quality of life of survivors.

Differential diagnosis

Confusing conditions

A number of inborn errors of metabolism can have similar clinical presentations to carbamoyl phosphate synthetase 1 deficiency, especially in the newborn period. These include other urea cycle disorders and amino acidopathies, 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. Differentiation between these diagnostic possibilities depends both on typical clinical signs and on identifying hyperammonemia together with specific pathological patterns of amino acids, acylcarnitines, or organic acid abnormalities. In addition, mitochondrial carbonic anhydrase VA deficiency should be included in the differential diagnosis (92).

In older children and adults, a number of acquired disorders can also present with hyperammonemia, including liver disease, Reye syndrome, drug toxicity, and hepatotoxins. Historical information, prothrombin time, a urinary toxic screen, and plasma amino acid pattern should help to differentiate these disorders.

Associated or underlying disorders

Clinically as well as biochemically, individuals suffering from carbamoyl phosphate synthetase 1 deficiency or N-acetylglutamate synthase deficiency are not distinguishable. Thus, conditions depriving the N-acetylglutamate synthase enzyme of its substrates acetyl-CoA or glutamate, direct inhibition of N-acetylglutamate synthase, or competition with N-acetylglutamate at the carbamoyl phosphate synthetase 1 binding site, might correspond to (partial) carbamoyl phosphate synthetase 1 deficiency. For a more thorough description of such conditions, please see the chapter “Associated or underlying disorders” from the article “N-acetylglutamate synthase deficiency.”

Diagnostic workup

In carbamoyl phosphate synthetase 1 deficiency, the principal biochemical feature is hyperammonemia, with highly elevated plasma concentrations (range: 250 to 5000 µmol/L; normal is less than 110 µmol/L in a newborn, less than 50 µmol/L after the neonatal period). The most common inborn errors of metabolism that can present as a catastrophic illness in the newborn period are urea cycle disorders, organic acidemias, fatty acid oxidation disorders, mitochondriopathies, and maple syrup urine disease.

Of these, only maple syrup urine disease is consistently associated with normal plasma ammonia concentrations. In organic acidemias (methylmalonic acidemia, propionic acidemia, isovaleric acidemia, glutaric acidemia type 2, and multiple carboxylase deficiency), there is usually a marked metabolic acidosis, ketosis, and an increased anion gap. Acylcarnitine profiles by tandem mass spectrometry from simple blood spots collected on a Guthrie card or plasma identify disease-characteristic acylcarnitines, gas chromatography-mass spectrometry of urine diagnostic organic acids.

Fatty acid oxidation defects present with hypoketotic hypoglycemia, decreased ketones, and increased free fatty acids. Acylcarnitine analyses reveal disease-specific profiles. Urinary organic acid analysis typically shows dicarboxylic aciduria.

Congenital lactic acidoses or mitochondriopathies can be the result of a genetic defect in pyruvate metabolism or the mitochondrial respiratory chain. The principal biochemical finding is lactic acidosis. In primary defects of pyruvate metabolism, the ratio of lactate to pyruvate is usually maintained at between 10:1 and 15:1, whereas in secondary lactic acidosis (shock, sepsis, heart failure) and mitochondrial defects this ratio is significantly increased. These findings are in contrast to inborn errors of urea synthesis, such as carbamoyl phosphate synthetase 1 deficiency, where the urinary organic acid profile is normal and plasma lactate concentration is normal or only mildly increased.

Plasma amino acid patterns are distinct in the urea cycle disorders, with elevated concentrations of glutamine, alanine, and asparagine, and low concentrations of citrulline and arginine. Citrulline is the product of carbamoyl phosphate synthetase 1 and ornithine transcarbamylase activity, and the substrate for argininosuccinic synthetase.

Thus, its concentration is absent or markedly reduced in carbamoyl phosphate synthetase 1 deficiency and ornithine transcarbamylase deficiency and markedly elevated in argininosuccinate synthetase deficiency and argininosuccinate lyase deficiency. This contrasts with transient hyperammonemia of the newborn or hyperammonemia due to other inherited metabolic diseases (see above), which are not associated with a primary urea cycle defect and have normal glutamine, arginine, and citrulline concentrations. For distinguishing carbamoyl phosphate synthetase 1 deficiency from ornithine transcarbamylase deficiency, it is crucial to determine urinary orotic acid, which is elevated in ornithine transcarbamylase deficiency and decreased or normal in carbamoyl phosphate synthetase 1 deficiency. A deficiency of N-acetylglutamate synthase (NAGSD), the enzyme needed for the production of the cofactor (N-acetylglutamate) for carbamoyl phosphate synthetase 1 has the same constellation of metabolites as carbamoyl phosphate synthetase 1 deficiency. Very similar is the mitochondrial carbonic anhydrase VA deficiency, which is also associated with low-normal orotic acid excretion (92). Mutation analysis is required to confirm the diagnosis.

Diagnosis of carbamoyl phosphate synthetase 1 deficiency in older children and adults with partial deficiencies may be less straight forward than in neonates. During symptomatic episodes, plasma ammonia concentrations may be in the range of 150 to 250 µmol/L, rather than above 500 µmol/L, and normal when the patient is clinically stable. Citrulline and arginine concentrations are often low-normal in partial carbamoyl phosphate synthetase 1 deficiency, rather than absent-trace. Definitive diagnosis in these cases is best attempted by molecular testing to detect specific mutations. If the answer cannot be readily obtained by this method, the genes for ornithine transcarbamylase deficiency, carbamoyl phosphate synthetase 1, N-acetylglutamate synthase deficiency, and mitochondrial carbonic anhydrase VA should be investigated. Measurement of CPS1 activity in hepatocytes or intestinal mucosa cells is nowadays rarely utilized (52). CPS1 activity assay can be faster for confirmation of carbamoyl phosphate synthetase 1 deficiency than molecular genetics but it requires invasive techniques to gain biological tissue and is only available in very few research laboratories worldwide (42). Molecular genetic analysis is nowadays more often used in North America and Europe to confirm diagnosis than measurement of enzymatic activity (Posset et 2019a). In addition, investigation by RNA sequencing can be a good tool to evaluate unclear cases (34). An algorithm for the differential diagnosis of N-acetylglutamate synthase deficiency and carbamoyl phosphate synthetase 1 deficiency has been provided (28).

In vivo stable isotope dilution assays of urea synthetic capacity have been developed (66). This involves the oral or intravenous administration of a stable isotope and the subsequent measurement by mass spectrometry of isotope enrichment in labeled urea.

Management

For a detailed discussion see “Suggested guidelines for the diagnosis and management of urea cycle disorders” (28).

Management of acute hyperammonemia in CPS1D.

Table 1. Protein, Liquid, and Glucose Management

Table 2. First-Line Medication for CPS1D****

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 described.

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 (68) and timely start of treatment are important determinants of outcome. In fact, current knowledge proposes that noninterventional variables (eg, disease onset and initial peak blood ammonium level) are of utmost importance for the neurologic outcome of individuals with urea cycle disorders (70). 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 (71). Specialized pediatric hospitals should have first-line medications and consensus-based treatment-protocols available, 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.

Ammonia scavengers (sodium benzoate, sodium phenylacetate/-butyrate) provide alternate pathways to eliminate waste nitrogen (09). 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 (33; 08). Sodium benzoate is conjugated with glycine to form hippurate and sodium phenylbutyrate, which 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/-butyrate 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, no systematic studies regarding the effects of long-term pharmacotherapy on neurologic or cognitive outcome, as defined by clinical endpoints, exists thus far for CPS1-deficiency (58).

Retrospective studies and patient reports suggest that N-carbamylglutamate (Carbaglu®) may be effective in reducing ammonium concentrations and, thus, allowing improvement of metabolic control in carbamoylphosphate synthetase 1 deficiency (97; 36; 81). Currently, a multicenter, randomized, double-blind, placebo-controlled trial of Carbaglu® in North America investigates if this drug might be helpful to those patients with regard to (1) improved neurocognitive outcome, and (2) more rapid reduction of ammonium concentration, improved function, and shortened hospitalization compared to standard therapy alone (01).

Energy should be 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 g/kg and increased up to 3 g/kg per day. However, a multicenter study showed that during the first 24 hours of emergency treatment, caloric intake was generally lower than during maintenance treatment and below age-adapted recommendations. Carbohydrates were the primary, if not sole, energy source, whereas fat was often omitted from initial emergency treatment (70). Soluble insulin is provided to support intracellular glucose uptake and to avoid hyperglycemia. The intake of natural protein is stopped for 24 to maximally 48 hours and is then reintroduced gradually as tolerated (32). In the event that ammonia concentrations do not respond to this management and biochemical or clinical symptoms worsen, continuous veno-venous hemodiafiltration (CVVHDF) should be started immediately in neonates or children with ammonia concentrations of greater than 500 µmol/L (see Table 1) (planned and organized earlier, ie, at levels greater than 400 µmol/L) or at lower levels if response to medical treatment is inadequate. Note, even though continuous veno-venous hemodiafiltration is by far the most efficient method for extracorporal ammonia elimination (68), 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 impact outcome, and only 20% of all investigated individuals had a normal clinical outcome (31). Therefore, dialysis along with conservative pharmacotherapeutic treatment is recommended to be initiated as early as possible and even at lower ammonium concentrations; however, long-term data analyzing these recommendations are 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 to less than 100µmol/L (28). 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 adjust such management. The reduction of protein intake must be carefully monitored to prevent overrestriction. A diet with inadequate intake can impair protein synthesis and lead to catabolic metabolic decompensation as well as failure to thrive (32).

According to the physician’s advice, 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 the hospital (32; 101).

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 CPS1D.

Table 3. Long-Term Medication for CPS1D

Long-term management of carbamoyl phosphate synthetase 1 deficiency relies on the goals of preventing recurrent hyperammonemia, neurologic sequelae, and improving quality of life by the following principles (32; 28):

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, 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 based on minimizing 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 (28). Especially in young infants and children, fasts should be avoided and snacks given to reduce the possibility of (overnight) catabolism.

Periodic measurement of plasma amino acids (which includes 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, or both, as well as the essential amino acids L-arginine or L-citrulline. A detailed overview is provided in Table 3. Drugs may not be well tolerated by the child or family. Sodium phenylbutyrate tastes and smells unpleasant 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. Phenylbutyrate may deplete branched-chain amino acids concentrations and cause menstrual dysfunction/amenorrhea in up to 25% of postpubertal females (12; 28). To avoid complications, eg, mucositis or gastritis, sodium benzoate and sodium phenylbutyrate should be administered several times daily during meals with abundant fluids (28). Acute toxicity of benzoate and phenylbutyrate has been rare, but severe overdoses (2 to 10 times recommended) have led to symptoms that may be clinically mistaken for hyperammonemic episodes, including lethargy, hyperventilation, metabolic acidosis, cardiopulmonary collapse, and death (07; 72). Supplementations of L-arginine or L-citrulline aim at maximizing ammonia excretion through the urea cycle (47). For long-term medication, sodium benzoate and sodium phenylbutyrate as well as L-arginine and L-citrulline are used. N-carbamylglutamate (Carbaglu®) is rarely used as an off-license drug for partially carbamoyl-phosphate synthetase 1 deficiency patients (70; 97; 81).

Carnitine deficiency may be present in urea cycle disorder patients that are on a low-protein diet or receive treatment with ammonia scavengers (28). The benefit of vaccinations outweighs the risk of decompensations. They are recommended at the same schedule as for healthy children and should include influenza (57; 28).

By now a significant number of patients with various urea cycle disorders have received partial or total orthotopic liver transplants to provide enzyme replacement therapy (94; 53; 55). 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 from complication of transplantation or immunosuppression. Long-term survival is over 95% in the proximal urea cycle disorders. Liver transplantation does not normalize citrulline concentrations, which are primarily produced in the gut; thus, following transplantation, supplementation with citrulline or arginine may be needed to be continued (89).

Ideally, orthotopic liver transplantation should be performed between 6 and 12 months of age before irreversible neurologic damage has occurred in patients with severe neonatal-onset disease and in patients suffering from recurrent severe decompensations despite intensive medical treatment (28). A retrospective survey study investigated if patients would profit 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 already after comparatively low maximum ammonium concentrations liver transplantation to protect the patient’s neurocognitive abilities (39; 40). However, data suggest rather limited impact of liver transplantation on the neurodevelopmental outcomes of individuals with a severe disease burden (38).

Some medications are contraindicated in urea cycle disorders because of secondary inhibition of the urea cycle or inducing catabolism, most importantly valproic acid and systemic steroids. Even in well controlled and managed patients, peracute deadly coma can occur. Less often, but also to be considered, is the potential development of hyperammonemic crises by the 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 option in patients with urea cycle disorder awaiting liver transplantation and is investigated in studies (54). At present, there appears to be no place yet for gene or enzyme replacement therapy in the routine treatment of urea cycle disorders (28). However, first promising results of split AAV-mediated gene therapeutic approaches in a murine model of carbamoyl-phosphate synthetase 1 deficiency were published (62). A new interesting approach for treatment of acute hyperammonemia is inhibition of ornithine aminotransferase leading to transamination-dependent decrease of glutamate and glutamine. If this approach is a potential new therapeutic-principle for individuals with urea cycle disorders, mammalian transfer will provide more insight (98). 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 might also play an important role during hyperammonemic conditions in the brain in order to determine if autophagy might be a suitable target for treatment of hyperammonemic conditions (77).

Outcomes

Prior to the development of alternate pathway therapy using ammonia scavengers (eg, sodium benzoate, sodium phenylacetate/-butyrate), virtually all children with neonatal carbamoyl phosphate synthetase 1 deficiency died in the newborn period or during infancy. Between the 1980s and mid-1990s, the 1-year survival rate for children with early-onset type urea cycle disorders was approximately 50% and worse for early-onset carbamoyl phosphate synthetase 1 deficiency (91); this, however, has changed with the widespread availability of ammonia measurement in hospitals, growing knowledge about the disease, and the use of alternate pathway therapy. Currently, the one-year survival rate for early-onset and late-onset urea cycle disorder patients seems to have substantially improved, including results for carbamoyl phosphate synthetase 1 deficiency (42). In contrast, a review and meta-analysis showed less convincing results suggesting that no improvement of survival for urea cycle disorders can be observed over more than three decades between 1978 and 2014 (11). Some studies showed that noninterventional variables reflecting disease severity are associated with the highest risk of mortality and poor neurologic outcome (20; 70).

Long-term morbidity is still substantial in urea cycle disorder patients. It could be shown that 50% of patients with urea cycle disorders suffer from intellectual disability (45). A study on individuals with ornithine transcarbamylase deficiency showed that intellectual impairment is global rather than domain-specific (10). Bachmann suggests that neurocognitive outcome does essentially depend on the initial and peak-blood ammonia level (04). Lately, a transatlantic study with more than 500 individuals suggested that the concentration of initial peak blood ammonium inversely correlates with cognition in proximal (including carbamoyl-phosphate synthetase 1 deficiency) rather than distal urea cycle disorders (69). More work is needed in order to evaluate the pathomechanistic basis for this finding and to study further predictors of good and poor neurocognitive outcome in individuals with urea cycle disorders.

Special considerations

Pregnancy

Unlike in ornithine transcarbamylase deficiency, heterozygous women carrying affected offspring have not experienced complications during pregnancy, and the children appear well when born at term. However, homozygous women with previously undiagnosed partial defects could suffer lethal hyperammonemic coma postpartum (95). Langendonk studied a series of pregnancies in women with inherited metabolic disease and suggested that special care must be taken not to confuse behavioral changes of hyperammonemia for symptoms of postpartum psychosis or depression (46), as outlined in the clinical vignette patient 2 above (22). Because late-onset manifestations appear to occur in more than 50% of all urea cycle disorders (59; 83; 42; 43) 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 (22). A severe risk period for acute hyperammonemic decompensation is between three 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 (46; 28). 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. Pregnancy in women with inherited metabolic disease should, therefore, be monitored and escorted in close contact with a metabolic center (46).

Anesthesia

There has been one report of hyperammonemia induced by enflurane in argininosuccinic aciduria (03). It is prudent to use anesthetics with low toxicity to the liver. However, surgery requires the stopping of oral medication and may be associated with a catabolic condition, both of which may induce hyperammonemia in carbamoyl phosphate synthetase 1 deficiency. It is important to continue alternate pathway therapy intravenously until the patient is able to tolerate oral medication. The patient should also receive adequate glucose infusion to prevent catabolism. A 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 (75). 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 (28).

A case was reported emphasizing that appropriate guidelines for the pre- and postoperative care of patients with inherited metabolic diseases of the urea cycle are indispensable (24).