Urea Synthesis: Case Report & Diagnosis

• The text provides a case report, diagnosis, plasma amino acids, urine orotic acid, enzymology, mutation analysis, and biochemical perspectives in relation to inborn errors of urea synthesis.

Case Report

• A male infant born at term developed respiratory distress and was poorly fed.
• No evidence of infection was found despite antibiotic treatment.
• The infant became increasingly unresponsive with respiratory alkalosis.
• Plasma ammonium was significantly elevated (1250 µmol/L, normal 5-30).
• The infant was treated with intravenous glucose, L-arginine, sodium benzoate, and sodium phenylacetate, along with hemodialysis.
• Mutation analysis revealed the infant carried two separate, previously described, mutations in the carbamoyl phosphate synthetase I (CPSI) gene.
• Subsequent issues included global developmental delay, reduced head growth, and a seizure disorder.
• Prenatal diagnosis was performed via molecular analysis of chorionic villus in a subsequent pregnancy, resulting in a healthy baby.

Diagnosis

• The case report describes the classical presentation of a patient with a urea cycle disorder (UCD).
• Neonatal hyperammonemic encephalopathy is a common and severe presentation of UCDs.
• Arginase (ARG-1) deficiency results in progressive spasticity of the lower limbs.
• Citrin and ornithine transporter 1 (ORNT-1) deficiencies result in adult-onset encephalopathy and recurrent hyperammonemia with protein aversion in infancy and childhood, respectively.
• Organic acidurias, fatty acid oxidation defects, amino acidopathies, and mitochondrial respiratory chain disorders can cause neonatal hyperammonemia.
• UCDs can be distinguished by routinely available clinical chemistry tests such as blood gases, acid/base balance, plasma glucose, ammonium, or lactate.
• Urea production is decreased in UCDs.
• Respiratory alkalosis is an important diagnostic clue that should prompt measurement of plasma ammonium.
• Increased intracerebral ammonium or glutamine accumulation can cause alkalosis (hyperventilation).
• Hyperammonemia associated with an increased anion gap suggests an organic acidemia, while concomitant hypoketotic hypoglycemia suggests a defect in fatty acid oxidation.
• Urine organic acids, plasma carnitine levels, and a plasma acylcarnitine profile help distinguish these disorders and often establish a definitive diagnosis.
• Measurement of plasma ammonium seldom exceeds 100 µmol/L.

Plasma Amino Acids

• Quantitation of plasma amino acids, especially citrulline, is the first step in determining the precise enzyme or transport protein defect in patients with a UCD.
• Low plasma citrulline concentration suggests a defect in N-acetylglutamate synthetase (NAGS), CPSI, or OTC.
• Marked hypercitrullinemia (>2000 µmol/L) is seen in argininosuccinate synthetase (AS) deficiency.
• Moderate increases (>200 µmol/L) are found in argininosuccinate lyase (AL) and citrin deficiencies.
• In AL deficiency, argininosuccinic acid and its anhydrides are present.
• Elevated plasma concentrations of glutamine and alanine often occur in parallel with ammonium.
• Arginine concentrations are low because the urea cycle is the only synthetic route for arginine in humans.
• In ARG-1 deficiency, the arginine concentration is elevated.
• Hyperornithinemia and homocitrullinuria are characteristic of hyperammonemia, hyperornithinemia, and homocitrullinuria (HHH) syndrome caused by a defect in the ornithine transporter (ORNT-1).

Urine Orotic Acid

• Orotic acid is an intermediate in pyrimidine synthesis.
• Defects in ureagenesis causing accumulation of intracellular carbamoyl phosphate provide substrate for orotic acid synthesis, leading to orotic aciduria.
• Detection of elevated orotic acid helps differentiate between OTC deficiency and CPSI- or NAGS-deficient patients.

Enzymology

• A definitive diagnosis of a UCD can be made by specific enzyme assays.
• CPSI or NAGS activity is established by liver biopsy.
• Random X inactivation in females with OTC deficiency may result in normal enzyme activity.
• AS is assayed in skin fibroblasts and liver.
• AL is assayed in various tissues.
• ARG-1 activity is measured in liver or red blood cells.

Mutation Analysis

• The genes for the six enzymes and two transporters involved in the urea cycle have been identified.
• Disease-causing mutations in these genes have been reported.
• Most mutations are private, with no common mutation accounting for a large proportion of affected individuals.
• Mutation testing requires screening for mutations in the entire coding region and, in some cases, for larger gene rearrangements or regulatory region mutations.

Biochemical Perspectives

• Animals lack a mechanism to store nitrogen.
• Amino acids are used for protein synthesis and other nitrogenous compounds.
• Excess amino acids are deaminated, and their carbon skeletons are used for energy or gluconeogenesis/ketogenesis.
• All animals require a mechanism to excrete excess nitrogen.
• Humans excrete urea and are called ureotelic organisms.
• Birds and reptiles are uricotelic (excreting uric acid), and marine animals are ammoniatelic (excreting ammonium).
• Urea is synthesized via the urea cycle including Krebs and Henseleit.
• Ornithine stimulates urea synthesis without stoichiometric consumption.
• Nitrogen from both intrahepatic and extrahepatic sources incorporates into urea.
• One originated from ammonium, and it is derived from aspartic acid.
• Glutamate dehydrogenase generates ammonium in liver hepatocytes from the oxidative deamination of glutamic acid.
• Extrahepatic organs involved in urea synthesis are muscle, small bowel, and kidney.
• Muscle is an vital source of alanine which feeds hepatic urea and gluconeogenesis.
• As a net producer of glutamine, muscle contributes to the production of ammonium, alanine, and citrulline in the small intestine.
• Renal glutaminase activity may contribute ammonium to the hepatic urea cycle.

Carbamoyl Phosphate Synthetase I

• CPSI facilitates the formation of carbamoyl phosphate from bicarbonate, ammonium and ATP
• The first urea cycle step, occurs in the mitochondrial matrix, assimilates the first nitrogen atom.
• CPSI is a homodimer.
• N-Acetylglutamate (NAG) is a CPSI activator, and magnesium ions are required.
• Cytosolic CPSII occurs in mammals as part of the CAD trifunctional protein, which begins pyrimidine synthesis.
• CPSII has no role in ureagenesis.
• Defects in CPSI present with neonatal acute hyperammonemic encephalopathy.
• Both plasma citrulline and urine orotic acid concentrations are low.
• Liver tissue enzyme assay, or mutation analysis, confirms diagnosis.

N-Acetylglutamate Synthase

• NAG is an essential allosteric activator of CPSI activity, produced in the mitochondrial condensation of acetyl-CoA and glutamate catalyzed by NAGS.
• NAG plays a pivotal role regulating the urea cycle, production is upregulated by arginine while it's transport to a cytosolic compartment for degradation is inhibited by glucagon.

Ornithine Transcarbamoylase

• The second intramitochondrial step of urea cycle is the reversible condensation of phosphate and ornithine, catalyzed by OTC.
• OTC deficiency is the defective urea cycle.

Ornithine-Citrulline Transporter

• The inner mitochondrial membrane exchanges citrulline for ornithine, catalyzed by ORNT-1.
• ORNT-2 may attenuate HHH (Hyperammonemia, Hyperornithinemia, Homocitrullinuria) disease due to ORNT-1 deficiency.
• Impaired OTC substrate causes Hyperornithinemia.

Argininosuccinate Synthetase

• Argininosuccinic acid forms, with citrulline in the cytosol and apartate through condensation.

Argininosuccinate Lyase

• AL exists in liver, kidney, skin fibroblasts, and parts of the brain.
• Its main function is to cleave argininosuccinic into forming arginine and fumarate acid.

Arginase

• Final step of urea, is cleavage of arginine, which is catalyzed by orthinine.

Aspartate-Glutamate Transporter (Citrin)

• Defects in Citrin cause type citrullinemia

Therapy

• Treatment is split by: acute management of hyperammonemic and long term control.

Acute Management of Hyperammonemic Encephalopathy

• Hemodialysis is effective
• Stop protein intake and provide adequate calories.

Long-Term Management of Urea Cycle Disorders

• Reduce ammonia with dietary protein restriction.
• Recognize one can provide an alternate pathway.
• Protein is reduced and provide and alternate homeostosis.
• Changing growth rates, illness, and inadvertent protein intake all contribute to recurrent hyperammonemia.
• Monitor growth (e.g., height, weight, head circumference) and of adequate nutrition (blood concentrations of hemoglobin, prealbumin, vitamins, and trace elements), and monitoring of disease control.
• Requires restricting daily nitrogen to a specific amount.
• Increase supplements with nitrogen during periods of accelerated growth.
• Citrulline has two advantages: Contains 3(atoms)
• Contains less(4atoms)

Phenylbutrate

• Sodium benzoate and sodium phenylactetate provides a similar treatment.

Questions

• The text provides questions with topics including metabolic fates of the amino acid arginine in the human, carbamoyl phosphate, OTC deficiency, molecular genetics and location of the gene that encodes OTC on the X chromosome.