Introduction. Clinical manifestations

Transcription

1 Carbamyl phosphate synthetase I deficiency Roland Posset MD ( Dr. Posset of the University Center for Child and Adolescent Medicine in Heidelberg has no relevant financial relationships to disclose. ) Georg F Hoffmann MD ( Dr. Hoffmann of the University Center for Child and Adolescent Medicine in Heidelberg has no relevant financial relationships to disclose. ) Barry Wolf MD PhD, editor. ( Dr. Wolf of Lurie Children's Hospital of Chicago has no relevant financial relationships to disclose.) Originally released November 28, 1994; last updated December 15, 2017; expires December 15, 2020 Introduction This article includes discussion of carbamyl phosphate synthetase I deficiency, carbamylphosphate synthetase 1 deficiency (CPS1D), congenital hyperammonemia type 2, and CPS1 deficiency. The foregoing terms may include synonyms, similar disorders, variations in usage, and abbreviations. Overview Carbamyl phosphate synthetase I deficiency (CPS1D) is an inherited urea cycle defect that causes hyperammonemia, neurologic sequelae, and most importantly, intellectual disability and early death. Complete enzyme deficiency almost invariably results in hyperammonemic coma within the first days of life ( 28 days; neonatal-/early-onset), whereas partial deficiency can present with hyperammonemia at any age (> 28 days; late-onset). Biochemical markers include elevated plasma glutamine and reduced or absent L-arginine and L-citrulline concentrations on amino acid analysis. Diagnosis is established by enzyme analysis of liver tissue and/or genetic analysis. Treatment consists of a proteinrestricted diet, ammonia scavenger drugs, and substitution with L-citrulline or L-arginine. Liver transplantation cures recurrent hyperammonemic episodes, but will not restore irreversible neurologic sequelae. Currently, international networks for rare metabolic diseases (UCDC, E-IMD, JUCDC) aim to more completely describe the natural history, especially the initial and evolving clinical phenotype, of urea cycle disorders such as carbamyl phosphate synthetase I deficiency. Furthermore, they want to determine if the natural disease course can be favorably modulated by diagnostic and therapeutic interventions. These networks collect systematic data to improve the clinical knowledge, develop guidelines, and provide patients and professionals with reliable data on disease manifestation, complications, as well as long-term outcomes of urea cycle disorders. They include the Urea Cycle Disorders Consortium (UCDC), established in 2003, the European Registry and Network for Intoxication Type Metabolic Diseases (E-IMD), established in 2011, and the Japanese Urea Cycle Disorders Consortium (JUCDC), established in 2012 (Summar et al 2014). Key points Carbamyl phosphate synthetase I deficiency is a rare urea cycle disorder that causes hyperammonemia, neurologic sequelae, and intellectual disability. Disease manifestations occur most often within the first days of life (early onset 28 days) and less commonly after the neonatal period (late onset > 28 days). Neurologic outcome depends on noninterventional parameters, eg, intrinsic disease severity (reflected by onset type and initial peak blood ammonium concentration level during first metabolic decompensation). Therapy is based on principles of acute and long-term management involving diet and antihyperammonemic pharmacotherapy. Historical note and terminology Carbamyl phosphate synthetase 1 deficiency was first reported in 1962 (Russell et al 1962). The nomenclature distinguishes this mitochondrial urea cycle enzyme from carbamyl phosphate synthetase 2, which is cytosolic and involved in pyrimidine synthesis. Clinical manifestations

2 Presentation and course The classic presentation of carbamyl phosphate synthetase I deficiency is a catastrophic illness in the first week of life (neonatal-/early-onset; > 70% of reported cases) (Burgard et al 2016). 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 carbamyl phosphate synthetase I 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 (Ah Mew et al 2013). 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 (Häberle et al 2012). Neurologic findings may include increased deep tendon reflexes, and papilledema. Convulsions may already be late complications and follow alterations in consciousness. Cases of late-onset disease with partial deficiencies have been reported (Lo et al 1993). 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 (Batshaw and Brusilow 1982; Verbiest et al 1992; Oechsner et al 1998), as well as the postpartum period (due to catabolism and the involution of the uterus) are important triggers for late-onset hyperammonemia (Fassier et al 2011; Häberle et al 2012; Langendonk et al 2012). 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 urea cycle disorder diagnosis and even progress to death (Fassier et al 2011). Acute hyperammonemic episodes may resemble or actually involve stroke-like episodes, hemiplegia may be evident, and MRI may reveal infarction (Batshaw et al 1975; Sperl et al 1997) or injury to the bilateral lentiform nuclei and the deep sulci of the insular and perirolandic regions (Takanashi et al 2003). Published results from the UCDC consortium report neuroimaging and neurocognitive findings of more than 600 urea cycle disorder patients, including patients suffering from carbamyl phosphate synthetase I 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 carbamyl phosphate synthetase I deficiency (n = 10) tended to have global early developmental delays and their scores did not decline over time (Waisbren et al 2016). However, unlike in ornithine transcarbamylase deficiency, late-onset disease manifestation in carbamyl phosphate synthetase I deficiency is rarer (Burgard et al 2016). Findings from Japan revealed that late-onset carbamyl phosphate synthetase I deficiency corresponds to approximately 10% of all manifestations of carbamyl phosphate synthetase I deficiency (Kido et al 2012). These data coincide with inquiries from Europe demonstrating that early-onset carbamyl phosphate synthetase I deficiency corresponds to the vast majority of carbamyl phosphate synthetase I deficiency cases (Burgard et al 2016; Kölker et al 2016a). Prognosis and complications Although mortality has 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 (Burgard et al 2016; Kölker et al 2016b) ie, the brain leading to intellectual disability, cerebral palsy seizure disorder, and visual deficits (Msall et al 1984). A study of urea cycle disorder long-term survivors demonstrated that approximately half of the patients had IQ scores greater than 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 earlyonset disease manifestation (Uchino et al 1998). A review and meta-analysis of observational studies spanning a period of more than 35 years demonstrated that early-onset patients, most of whom suffer from carbamyl phosphate synthetase I deficiency, have a high risk of permanent disease manifestation and early death (Burgard et al 2016). Normal outcome of carbamyl phosphate synthetase I deficiency by the end of the first year was found for only 20% of surviving patients, and no improvement of survival was observed over more than 30 years. Neurocognitive outcome does essentially depend on the initial and peak blood ammonium levels (Bachmann 2003; Enns et al 2007). At the first hyperammonemic attack, a peak blood ammonium level of less than 180 µmol/l is

3 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 (Uchino et al 1998; Kido et al 2012). The impact of noninterventional variables on the neurologic outcome was also emphasized by demonstrating that the intrinsic disease severity, which is reflected by the onset type and the initial peak blood ammonium concentration level, is of utmost importance for the neurologic outcome of urea cycle disorder patients (Posset et al 2016). Importantly, neurocognitive outcomes do not differ between patients with proximal (ie, CPS1D, OTCD) and distal defects (ie, argininosuccinate synthetase deficiency, argininosuccinate lyase deficiency) (Ah Mew et al 2013). Furthermore, a study demonstrated no difference in movement disorders (ie, dystonia, spasticity, chorea, ataxia) with regard to proximal or distal urea cycle disorders, but compared to patients with early-onset disease manifestations, patients with late-onset urea cycle disorders less often developed movement disorders (Kölker et al 2016b). A detailed overview of organ-specific disease manifestations and complications in patients suffering from urea cycle disorders (eg, CPS1D) has been given by Kölker and colleagues (Kölker et al 2016b). Furthermore, 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 (Jamiolkowski et al 2016). 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 1 year before. Apgar scores were 8 and 9 after 1 and 5 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 pco 2 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 35), rising to 1496 µmol/l 2 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 3 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 2 occasions, 1 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 carbamyl 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 3 years of age, psychomotor functions were close to normal, including motor skills and comprehension; however, her speech was markedly delayed (Tuchman et al 1992). She underwent liver transplantation at 7 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 3 after delivery. Her medical history was marked by 2 similar phases of agitation, confusion, and mystic delusions starting on day 3 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

4 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 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 2 mutations of the carbamyl phosphate synthetase gene (p.p87s and p.r803c) confirming carbamyl phosphate synthetase I deficiency (Fassier et al 2011). Biological basis Etiology and pathogenesis This disorder is caused by a complete or partial deficiency of carbamyl phosphate synthetase 1, the first enzyme in the urea cycle, which catalyzes the formation of carbamyl phosphate from ammonia, ATP, and bicarbonate using N- acetylglutamate as a cofactor (Häberle et al 2012). The gene encoding carbamyl phosphate synthetase 1 has been sequenced and mapped to the long arm of chromosome 2q.34 (Fearon et al 1985; Summar et al 2003; Funghini et al 2003). 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 carbamyl phosphate synthetase enzyme (Eeds et al 2006; Häberle et al 2011). The C- terminal domain of the enzyme was suggested to have an important function on enzyme regulation depending on the allosteric activator of carbamyl phosphate synthetase 1, N-acetylglutamate (de Cima et al 2015; Díez-Fernández et al 2015). Carbamyl phosphate synthetase 1 deficiency is inherited as an autosomal recessive trait (McReynolds et al 1981). This mitochondrial matrix enzyme, expressed in hepatocytes and intestinal mucosa epithelial cells (Ryall et al 1985), catalyzes the synthesis of carbamyl phosphate from bicarbonate, ammonia, and ATP. It is the most abundant protein in liver mitochondria, accounting for 20% of the mitochondrial matrix protein (Lusty 1978). The enzyme consists of a single polypeptide with a molecular weight of 165,000 and 1500 amino acid residues, respectively (Haraguchi et al 1991). Neonatal-onset cases have less than 5% normal activity in liver, whereas residual activity is higher in late-onset cases (Qureshi et al 1986). Like many metabolic disorders, carbamyl phosphate synthetase 1 deficiency does not arise from a common mutation, and numerous mutations have been identified (Finckh et al 1998; Summar 1998; Funghini et al 2003; Häberle et al 2011). Biochemically, the principal finding is hyperammonemia. Amino acid abnormalities include elevated concentrations of glutamine, alanine, and asparagine (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 (Ebels 1972). 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) (Msall et al 1984). Further MRI changes, despite liver transplantation, were reported to involve hyperintensities of the insular cortices and deep

5 frontal gyri as well as caudate nucleus and putamen (Nunley and Ghosh 2015). 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 (Butterworth et al 1987; Zwingmann et al 2004; Butterworth 2007; Lichter-Konecki 2008), potentially altered water transport through aquaporin 4 channels (Lichter-Konecki et al 2008), and oxidative and nitrosative stress due to increased free radical production and increased nitric oxide synthesis (Norenberg et al 2007). Although there is no clinical sign of liver damage, hepatic pathology studies show diffuse microvesicular steatosis, periportal nuclear glycogen, and variable portal fibrosis with occasional portal to portal bridging (Badizadegan and Perez-Atayde 1997). Interestingly, neonatal pulmonary hypertension was found to correlate with reduced plasma concentrations of arginine, nitric oxide, and polymorphisms in the CPS1 gene (Pearson et al 2001; Summar et al 2004). Pulmonary artery pressure is regulated by endogenous NO, which is derived from arginine supplied by the urea cycle. A combination of 4 single nucleotide polymorphisms (SNPs), including 1 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 (Moore et al 2009). Furthermore, carbamyl 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. Carbamyl 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 (Celiktas et al 2016). Epidemiology" Data provided by the UCDC (Seminara et al 2010) calculated the overall incidence of carbamyl phosphate synthetase I deficiency in the United States as 1 in 1,300,000 people (Summar et al 2013). Prevention No method is known for preventing carbamyl phosphate synthetase I deficiency. Prenatal diagnosis is available using molecular methods (Kamoun et al 1995; Summar 1998; Häberle and Koch 2004). Furthermore, preimplantation genetic diagnosis in couples at risk is possible (Hanson and Hamberger 1997). 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 (Häberle et al 2012). Differential diagnosis A number of inborn errors of metabolism can have similar clinical presentations to carbamyl 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 associated with specific pathological patterns of amino acids, acylcarnitines, or organic acid abnormalities. Mitochondrial carbonic anhydrase VA deficiency is included in the differential diagnosis (van Karnebeek et al 2014). 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. Diagnostic workup In carbamyl phosphate synthetase I deficiency, the principal biochemical feature is hyperammonemia, with highly elevated plasma concentrations (range: 250 to 5000 µmol/l; normal is less than 30 µmol/l). 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.

6 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 to 1 and 20 to 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 carbamyl phosphate synthetase I deficiency, where the urinary organic acid profile is normal and plasma lactate concentration is normal or 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 (Brusilow and Maestri 1996). Citrulline is the product of carbamyl phosphate synthetase 1 and ornithine transcarbamylase activity, and the substrate for argininosuccinic synthetase. Thus, its concentration is absent or markedly reduced in carbamyl phosphate synthetase I 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 carbamyl phosphate synthetase I 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 carbamyl phosphate synthetase I deficiency. A deficiency of N-acetylglutamate synthase (NAGSD), the enzyme needed for the production of the cofactor (N-acetylglutamate) for carbamyl phosphate synthetase 1 has the same constellation of metabolites as carbamyl phosphate synthetase I deficiency. Very similar is finally the mitochondrial carbonic anhydrase VA deficiency which is also associated with low-normal orotic acid excretion (van Karnebeek et al 2014). Diagnosis of carbamyl phosphate synthetase I 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 greater than 500 µmol/l, and normal when the patient is clinically stable. Citrulline and arginine concentrations are often low-normal in partial carbamyl phosphate synthetase I 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, carbamyl phosphate synthetase I, 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 (Tuchman et al 1989; Haraguchi et al 1991; Martín-Hernández et al 2014). CPS1 activity assay can be faster for confirmation of carbamyl phosphate synthetase I deficiency than molecular genetics but it requires invasive techniques to gain biological tissue and is only available in very few research laboratories worldwide (Häberle et al 2012; Kido et al 2012). An algorithm for the differential diagnosis of N-acetylglutamate synthase deficiency and carbamyl phosphate synthetase I deficiency has been provided (Häberle et al 2012). In vivo stable isotope dilution assays of urea synthetic capacity have been developed (Yudkoff et al 1998; Opladen et al 2016). 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 (Häberle et al 2012). Management of acute hyperammonemia in CPS1D.

7 Table 1. Protein, Liquid, and Glucose Management Escalation level NH 3 (µmol/l) Protein Liquid IV (ml/kg/d) Glucose IV (mg/kg/min) 1 < 100 Stop* ** 10*** i.n.**** Insulin Comments***** Stop* ** 10*** i.n.**** Inform metabolic clinic Stop* ** 10*** i.n.**** Inform dialysis clinic 4 > 500 Stop* ** 10*** i.n.**** Hemodialysis Consensus-based treatment protocol for pediatric (specialized) hospitals treating CPS1D patients with acute hyperammonemia according to Suggested guidelines for the diagnosis and management of urea cycle disorders (Häberle et al 2012). *Stop protein intake for 24 hours (maximum 48 hours). **Liquid management might be adapted to hospital- and age-specific requirements. ***Glucose management might be adapted to age-specific requirements. ****If necessary (i.n.) give 0.05 units/kg/h. *****Hyperglycemia can be dangerous, monitor every hour; monitor blood ammonia levels every 3 hours; monitor electrolytes, blood gases, and lactate regularly, eg, every 3 hours. Table 2. First-Line Medication for CPS1D**** Escalation level NH 3 Sodium benzoate (µmol/l) IV*** Bolus Mainten-ance Bolus (mg/kg) (mg/kg/d)** (mg/kg) in 90 in min min Sodium benzoate/- phenylacetate (Ammonul ) IV*** Maintenance (mg/kg/d)** L-Arginine hydrochloride 21% IV*** Bolus Maintenance (mg/kg) (mg/kg/d) in min 1 < 100 / / / / / / / g/m 2 /d* g/m 2 /d* 4 > g/m 2 /d* Carbamyl-glutamate by mouth Consensus-based treatment protocol for pediatric (specialized) hospitals treating CPS1D patients with acute hyperammonemia according to Suggested guidelines for the diagnosis and management of urea cycle disorders (Häberle et al 2012). *If patient > 20 kg body weight **If on dialysis maintenance doses should increase to 350 mg/kg/d. ***First-line medications must be diluted in 10% glucose prior to IV application. (Caution: L-Arginine-HCl may cause metabolic acidosis and extravasation may lead to tissue necrosis.) ****Control electrolytes due to possible danger of hypernatremia and hypokalemia. 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 (Picca et al 2001) and timely start of treatment are the most important determinants of outcome. In fact, an analysis showed that noninterventional variables (eg, disease onset and initial peak blood ammonium level) are of utmost importance for the neurologic disease outcome (Posset et al 2016). (Specialized) pediatric hospitals should have first-line medications, consensus-based treatment-protocols, and must act according to the following principles: (1) Stop protein intake (see Table 1) (2) Start intravenous fluid and glucose substitution (see Table 1) (3) Start first-line medication (see Table 2)

8 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 (Brusilow et al 1980). A published retrospective study from France showed that intravenous sodium benzoate treatment is safe and effective for the treatment or prevention of hyperammonemia in urea cycle disorders (Husson et al 2016). 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 2 nitrogen atoms. Thus, 2 moles of waste nitrogen are removed for each mole of phenylacetate/-butyrate administered. Over 40% of total waste nitrogen can be excreted as phenylacetylglutamine (Brusilow 1991). 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 g/kg 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 below ageadapted recommendations. Carbohydrates were the primary, if not sole, energy source, whereas fat was often omitted from initial emergency treatment (Posset et al 2016). 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 (Hoffmann et al 2017). 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 (planned and organized earlier, ie, at levels > 400 µmol/l) in neonates or children with ammonia concentrations of greater than 500 µmol/l (see Table 1) or at lower levels if response to medical treatment is inadequate. Note, even though CVVHDF is by far the most efficient method for extracorporal ammonia elimination (Picca et al 2001), 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. 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 (Häberle et al 2012). 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 or failure to thrive (Hoffmann et al 2017). 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 hospital (Zschocke and Hoffmann 2011; Hoffmann et al 2017). 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

9 Sodium benzoate by mouth (in mg/kg/d)** Sodium phenylbutyrate by mouth* < 20 kg (in mg/kg/d)** > 20 kg (in g/m 2 /d)** L-Arginine by mouth*** < 20 kg (in mg/kg/d) > 20 kg (in g/m 2 /d) L-Citrulline by mouth*** Carbamyl-glutamate by mouth / Dose / Maximum 12 g/d 12 g/d 6 g/d 6 g/d / Consensus-based long-term treatment protocol for pediatric (specialized) hospitals treating patients with CPS1D according to Suggested guidelines for the diagnosis and management of urea cycle disorders (Häberle et al 2012). *Second choice that should be given together with sodium benzoate in patients in which benzoate alone is not enough. **In some patients higher doses may be needed (< 20 kg: mg/kg/d and > 20 kg: g/m 2 /d). ***L-Citrulline may be preferable; when given no need for concomitant use of L-arginine Long-term management of carbamyl phosphate synthetase I deficiency relies on the goals of preventing recurrent hyperammonemia, neurologic sequelae, and improving quality of life by the following principles (Häberle et al 2012; Hoffmann et al 2017): (1) Long-term medication (see Table 3) (2) If individually necessary, avoidance of catabolism (3) Suitable emergency regimens in intercurrent illness (see Tables 1 and 2) 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, lowprotein 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 upon minimizing the nitrogen load on the urea cycle. The FAO/WHO/UNO 2007 report (Häberle et al 2012) can be used as age- and genderdependent recommendations for energy intakes. 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 include glutamine) and blood ammonia may permit adjustment of therapy before clinical symptoms appear. Long-term medication comprises the use of sodium benzoate and/or sodium phenylbutyrate as well as the essential amino acid 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 (Scaglia et al 2004; Häberle et al 2012; Burrage et al 2014). To avoid complications, eg, mucositis or gastritis, sodium benzoate and sodium phenylbutyrate should be administered several times daily during meals with abundant fluids (Häberle et al 2012). 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 (Batshaw et al 1982; Praphanphoj et al 2000). Supplementations of L-arginine or L-citrulline aim at maximizing ammonia excretion through the urea cycle (Brusilow 1984; Leonard and Morris 2002). 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 responsive carbamyl phosphate synthetase 1 deficiency patients (Posset et al 2016).

10 Carnitine deficiency may be present in urea cycle disorder patients that 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 (Häberle et al 2012; Posset et al 2016). The benefit of vaccinations outweighs the risk of decompensations. They are recommended at the same schedule as for healthy children and should include influenza (Morgan et al 2011; Häberle et al 2012). A number of patients with various urea cycle disorders have received partial or total orthotopic liver transplants to provide enzyme replacement therapy (Hasegawa et al 1995; Inui et al 1996; Whittington et al 1998; Mazariegos et al 2014). 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. 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 (Tuchman 1989). Ideally, orthotopic liver transplantation should be performed between (3-) 6 and 12 months of age before irreversible neurologic damage has occurred, in patients with severe neonatal-onset disease, and patients suffering from recurrent severe decompensations despite intensive medical treatment (Häberle et al 2012). In a study, however, it was demonstrated that urea cycle disorder children receiving a liver transplant were mainly between 1 to 6 years of age. Only 25% received a liver below 1 year of age (Kido et al 2012). 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, 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. New trends and emerging therapies include the use of hypothermia in neonatal hyperammonemia. Mild systemic hypothermia was used in the treatment of neonatal hyperammonemic coma, with the rationale of lowering the enzymatic rate of ammonia production (Whitelaw et al 2001; Lichter-Konecki et al 2013). Hepatocyte transplantation is an interesting therapeutic bridging option in patients with urea cycle disorder awaiting liver transplantation (Meyburg et al 2009a; Meyburg et al 2009b) and is investigated in studies. At present, there appears to be no place yet for gene or enzyme replacement therapy in the treatment of urea cycle disorders (Häberle et al 2012). Outcomes Prior to the development of alternate pathway therapy using ammonia scavengers (eg, sodium benzoate, sodium phenylacetate/-butyrate), virtually all children with neonatal carbamyl phosphate synthetase I deficiency died in the newborn period or during infancy. Between the 1980s and mid-1990s, the 1-year survival rate for children with earlyonset type urea cycle disorder was approximately 50% and worse for early-onset carbamyl phosphate synthetase I deficiency (Uchino et al 1998); 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 1-year survival rate for early-onset and late-onset urea cycle disorder patients has substantially improved, including results for carbamyl phosphate synthetase I deficiency (Kido et al 2012). In contrast, a review and meta-analysis showed less convincing results suggesting that no improvement of survival for urea cycle disorders was observed over more than 3 decades between 1978 and 2014 (Burgard et al 2016). Some studies showed that noninterventional variables reflecting disease severity are associated with the highest risk of mortality and poor neurologic outcome (Enns et al 2007; Posset et al 2016). 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 (Krivitzky et al 2009). These data were confirmed by a more recent study including 103 subjects with neonatal-onset urea cycle disorders (Ah Mew et al 2013). However, uncertainty with regard to the impact of peak-blood ammonia level on neurocognitive outcome in urea cycle disorders exists. Whereas Bachman suggests that neurocognitive outcome does essentially depend on the initial and peak-blood ammonia level (Bachmann 2003), surprisingly Ah Mew and coworkers could not correlate peak blood ammonium level with poor cognitive outcome (Ah Mew et al 2013).

11 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 (Wong et al 1994). 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 (Langendonk et al 2012), as outlined in the clinical vignette (Fassier et al 2011). Because lateonset manifestations appear to occur in more than 50% of all urea cycle disorders (Nassogne et al 2005; Summar et al 2008; Kido et al 2012; Nakamura et al 2014; Kölker et al 2016a) 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 (Fassier et al 2011). A severe 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 (Häberle et al 2012; Langendonk et al 2012). 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 (Langendonk et al 2012). Anesthesia There has been 1 report of hyperammonemia induced by enflurane in argininosuccinic aciduria (Asai et al 1998). 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 carbamyl 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 perioperatively 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 2 siblings with severe ornithine transcarbamylase deficiency (Schmidt et al 2006). 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 (Häberle et al 2012). 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 (Gharavifard et al 2014). References cited Ah Mew N, Krivitzky L, McCarter R, et al. Clinical outcomes of neonatal onset proximal versus distal urea cycle disorders do not differ. J Pediatr 2013;162(2): PMID Asai K, Ishii S, Ohta S, Furusho K. Fatal hyperammonaemia in argininosuccinic aciduria following enflurane anaesthesia. Eur J Pediatr 1998;157: PMID Bachmann C. Outcome and survival of 88 patients with urea cycle disorders: a retrospective evaluation. Eur J Pediatr 2003;162(6): PMID Badizadegan K, Perez-Atayde AR. Focal glycogenosis of the liver in disorders of ureagenesis: its occurrence and diagnostic significance. Hepatology 1997;26: PMID Batshaw M, Brusilow S, Walser M. Treatment of carbamyl phosphate synthetase deficiency with keto analogues of essential amino acids. N Engl J Med 1975;292: PMID Batshaw ML, Brusilow SW. Valproate induced hyperammonemia. Ann Neurol 1982;11: PMID Batshaw ML, Brusilow S, Waber L, et al. Treatment of inborn errors of urea synthesis: activation of alternative pathways of waste nitrogen synthesis and excretion. N Engl J Med 1982;306: PMID