Dr. Saidunnisa Hans Krebs Associate Professor Department of Biochemistry Ammonia metabolism and Urea Cycle

Learning Objectives At the end of the session student shall be able to: 1.List the sources of ammonia and explain the reactions of the urea cycle with regulation and Energetics. 2.Interpret the various hereditary disorders of urea cycle with biochemical explanation for its treatment. 3.Explain biochemical basis of Hyperammonemia in renal failure. 4.Integrate urea cycle with TCA cycle. 5.Explain the biochemical basis for ammonia toxicity.

Introduction Urea cycle was the first metabolic cycle that was elucidated by Hans Krebs and Kurt Henseleit cycle hence called krebs- Hanseleit cycle. It is a cyclical process because ornithine is regenerated in the last reaction which acts as a catalytic agent and repeats the cycle again. This cycle takes place in the liver.

Sources of Ammonia 1.Transamination 2.Oxidative and Non-oxidative deamination. 3.Absorption of NH 3 from the gut. 4.From dietary proteins 5.Pyrimidine catabolism (small fraction)

Amino acids taking part in Urea cycle 1.Ornithine 2.Citrulline 3.Glutamic acid 4.Aspartic acid 5.Arginine

Urea cycle

Sources of nitrogen One nitrogen of the urea molecule is supplied by free NH 3, and the other nitrogen by aspartate. [Note: Glutamate is the immediate precursor of both ammonia (through oxidative deamination by glutamate Dehydrogenase) and aspartate nitrogen (through transamination of oxaloacetate by AST)] The carbon and oxygen of urea are derived from CO 2. Urea is produced by the liver, and then is transported in the blood to the kidneys for excretion in the urine.

Urea has two amino groups one derived from NH 3 and the other from aspartate. Carbon atom is supplied by CO 2. Urea synthesis is five step cyclic process, with five distinct enzymes. The first two enzymes are present in mitochondria while the rest three are localized in cytosol.

Step-1 Synthesis of Carbamoyl phosphate Carbamoyl phosphate synthase I (CPS I) of mitochondria catalyses the condensation of NH 4 + ions with CO 2 to form Carbamoyl phosphate. This step consumes 2 ATP molecules. It is irreversible reaction. Rate limiting reaction. Requires NAG (N acetyl glutamate) for its activity.

Step-2 Formation of Citrulline Carbamoyl phosphate reacts with ornithine to form citrulline this reaction is catalyzed by OTC (ornithine trans carbamoylase). Citrulline produced in this reaction is transported to cytosol by a transporter system. Ornithine is regenerated and used in urea cycle. Therefore its role is compared to oxaloacetate in citric acid cycle. Ornithine and citrulline are basic amino acids they are never found in protein structure due to lack of codons.

Step-3 synthesis of Arginosuccinate Citrulline condenses with aspartate to produce Arginosuccinate this reaction is catalyzed by Arginosuccinate synthase. The second amino group of urea is incorporated in this reaction by aspartate. This step requires ATP which is cleaved to AMP and PPi.

Step-4 Cleavage of Arginosuccinate Arginosuccinase cleaves Arginosuccinate to give Arginine and fumarate. Arginine is an immediate precursor for urea Fumarate liberated here provides a connecting link with TCA cycle.

Step-5 Formation of Urea Arginase is the fifth and final enzyme that cleaves Arginine to yield urea and ornithine. Arginase is activated by CO 2 + and Mn 2 +. Ornithine so regenerated enters mitochondria for its reuse in the urea cycle.

Energetics 1.Urea cycle is irreversible and consumes 4 ATP. 2.Two ATP are utilized for the synthesis of Carbamoyl phosphate. 3.One ATP is converted to AMP and PPi to produce Arginosuccinate which equals to 2ATP.

Regulation 1.Coarse regulation: related to protein content in diet. During starvation, the activity of urea cycle enzymes is elevated due to increased protein catabolism. 2.Fine regulation: The first reaction catalyzed by CPS I is rate limiting reaction. It is allosterically activated by N-Acetyl glutamate (NAG). The rate of urea synthesis in liver is correlated with the concentration of NAG. 3. Compartmentalization of enzyme location.

Formation of NAG N-Acetylglutamate is synthesized from acetyl coenzyme A and glutamate by N- acetylglutamate synthase, in a reaction for which Arginine is an activator.

Disposal of Urea Urea produced in the liver freely diffuses and is transported in blood to kidneys, and excreted. A small amount of urea enters the intestine where it is broken down to CO 2 and NH 3 by the bacterial enzyme urease. This ammonia is either lost in the feces or absorbed into blood.

In Renal failure The blood urea level is elevated (Uremia) resulting in diffusing of more urea into intestine and its breakdown to NH 3. Hyper ammonia is commonly seen in patients of kidney failure. For these patients, oral administration of antibiotics (neomycin) to kill the bacteria is advised.

Disorders of urea cycle Urea cycle enzyme deficiency results in hyperammonemia. When the block is in the earlier steps, the condition is severe since ammonia itself accumulates. Deficiencies of later enzymes result in accumulation of intermediates which are less toxic and symptoms are less.

Symptoms 1.Hyperammonemia 2.Encephalopathy 3.Respiratory Alkalosis 4.Vomiting, irritability, lethargy, severe mental retardation. Brain is very sensitive to ammonia.

Diagnosis Enzyme defects can be detected by tandem mass spectrometry.

Treatment 1.Low protein diet with sufficient arginine and energy 2.Frequent meals can minimize brain damage. Since ammonia levels donot increase. 3.Breast milk contain Citruline to be avoided. 4.Attempts to eliminate the amino nitrogen in other forms like hippuric acid, phenyl acetyl glutamine. 5.Gene therapy.

Phenyl butyrate mechanism Treatment includes administering compounds that bind covalently to amino acids, producing nitrogen-containing molecules that are excreted in the For example, phenyl butyrate given orally is converted to phenylacetate. This condenses with glutamine to form phenylacetylglutamine, which is excreted in urine.

Hereditary Hyperammonemia: Genetic deficiencies of each of the five enzymes of the urea cycle have been described, with an overall prevalence estimated to be 1:30,000 live births. Ornithine transcarbamoylase deficiency, which is X-linked, is the most common of these disorders, predominantly affecting males. All of the other urea cycle disorders follow an autosomal recessive inheritance pattern.

Urea cycle disorders

UCDEnzyme DeficiencySymptoms/Comments Type I HyperammonemiaType I Hyperammonemia, CPSD Carbamoylphosphate synthetase I Measurement of serum ammonia shows increased levels. Treatment with arginine which activates N- acetylglutamate synthetase Type 2 HyperammonemiaType 2 Hyperammonemia, OTCD Ornithine transcarbamoylase Most commonly occurring increased serum orotic acid due to mitochondrial carbamoylphosphate entering cytosol and being incorporated into pyrimidine nucleotides. treat with high carbohydrate, low protein diet, ammonia detoxification

Urea cycle disorders UCDEnzyme DeficiencySymptoms/Comments Classic CitrullinemiaClassic Citrullinemia, ASD Argininosuccinate synthetase Treat with arginine administration to enhance citrulline excretion, also with sodium benzoate for ammonia detoxification Argininosuccinic aciduriaArgininosuccinic aciduria, ALD Argininosuccinate lyase (argininosuccinase) elevated plasma and cerebral spinal fluid argininosuccinate: treat with arginine and sodium benzoate HyperargininemiaHyperargininemia, ADArginase rare UCD, ammonia and arginine high in cerebral spinal fluid and serum, treatment includes diet if EAA excluding arginine and low protein diet.

Acquired hyperammonemia 1.Liver failure leads to hepatic coma and death. 2.Management of the condition: Low protein diet, Intestinal disinfection, Withholding hepatotoxic drugs, Maintenance of electrolyte and acid base balance.

Urea level in blood Indicator of renal function Plasma-20-40mg/dl Urinary excretion 15-30g/day

Integration between urea and TCA cycle

The production of fumarate in urea cycle is the most integrating point with TCA cycle. Fumarate is converted to malate and then to oxaloacetate in TCA cycle. Oxaloacetate undergoes transamination to produce aspartate which enters urea cycle and combines with citrulline to produce Arginosuucinate.

SDL-Case Discussion 1.What will happen to urea cycle during starvation? 2.If there is defect in the urea cycle before the synthesis of Arginosuccinate would you prefer to manage the case with amino acid conjugates like benzoic acid or Arginine administration.

What will happen to urea cycle during starvation?-SDL The delivery rate of ammonia is increased because of muscle protein breakdown. This will activate the enzymes of urea cycle. There may be increased excretion of urea in the initial phase.