Ammonia Transport & Urea Cycle

AMMONIA TRANSPORT & . UREA CYCLE

TRANSPORT OF NITROGEN TO LIVER AND KIDNEY

Muscle protein responds to conditions such as: starvation, trauma, Burns, and septicemia by undergoing massive degradation. - The most percentage of body protein is in skeletal muscle. - Under conditions of energy need, this protein is degraded and amino groups from amino acids are transferred to produce glutamine and alanine. -Finally, these amino acids “glutamine and alanine” are transported to Liver and kidney to produce urea and ammonia.

3 (3) AMMONIA TRANSPORT (Transport of ammonia to the liver)

Two mechanisms are available in humans for the transport of ammonia from the peripheral tissues to the liver for its ultimate conversion to urea.

THE FIRST, found in MOST TISSUES, uses glutamine synthetase to combine ammonia (NH3) with glutamate to form glutamine—a nontoxic transport form of ammonia . Two mechanisms THE FIRST

The main destination of glutamine in the blood is the liver, where ammonia is released by glutaminase, and glutamate dehydrogenase.

Glutamate dehydrogenase not only releases ammonia but also produces NAD and α- ketoglutarate as source of energy. THE SECOND transport mechanism, used primarily by MUSCLE, involves transamination of pyruvate (the end product of aerobic glycolysis) to form alanine. THE SECOND transport mechanism …..

Alanine is transported by the blood to the liver, where it is converted to pyruvate, again by transamination. In the liver, the pathway of gluconeogenesis can use the pyruvate to synthesize glucose, which can enter the blood and be used by muscle— a pathway called the glucose- alanine cycle. liver muscle

glucose glucose

gluconeogenesis glycolysis

pyruvate pyruvate transamination transamination transport in blood alanine alanine 10 AMMONIA IS RELEASED IN LIVER The main destination of glutamine and alanine in the blood is the liver, where ammonia is released by alanine aminotransferase, glutaminase, and glutamate dehydrogenase.

Glutamate dehydrogenase not only releases ammonia but also produces NAD and α-keto-glutarate as source of energy. The liver has only small quantities of glutaminase; however, levels of the enzyme are high in the intestine where the ammonium ion from deamination can be sent directly to the liver via the portal blood and used for urea synthesis. The intestinal bacteria and glutamine from dietary protein contribute to the intestinal ammonia entering the portal blood). Two mechanisms are available …… NOTES

•The first, found in most tissues, uses glutamine synthetase to combine ammonia (NH3) with glutamate to form glu•tamine—a nontoxic transport form of ammonia (Glutamine Synthetase:Most tissues, including muscle, have glutamine synthetase, which captures excess nitrogen by aminating glutamate to form glutamine. The reaction is irreversible. Glutamine, a relatively nontoxic substance, is the major carrier of excess nitrogen from tissues). The glutamine is transported in the blood to the liver where it is cleaved by glutaminase to produce glutamate and free ammonia (Glutaminase. The kidney contains glutaminase, allowing it to deaminate glutamine arriving in the blood and to eliminate the amino group as ammonium ion in urine. The reaction is irreversible. Kidney glutaminase is induced by chronic acidosis, in which excretion of ammonium may become the major defense mechanism. The liver has only small quantities of glutaminase; however, levels of the enzyme are high in the intestine where the ammonium ion from deamination can be sent directly to the liver via the portal blood and used for urea synthesis. The intestinal bacteria and glutamine from dietary protein contribute to the intestinal ammonia entering the portal blood). UREA CYCLE The urea cycle is the major mechanism for utilization and excretion of ammonia (excretion of ammonia in form of urea in the kidneys). In urea cycle: urea will be produced. Since humans cannot metabolize Urea, it is transported to the kidneys for filtration and excretion.

14 MECHANISM OF AMMONIA TOXICITY

Ammonia is a very toxic substance, and usually excreted in the form of urea. Metabolic disorders that arise from abnormal function of enzymes Of urea synthesis (urea cycle) are fatal and cause coma “due to ATP depletion”, specially when ammonia concentration is high. High concentration of ammonia sequesters α- ketoglutarate in the form of Glutamate, leading to: - depletion of TCA cycle intermediates - and reducing ATP production

15 . REACTIONS OF THE UREA CYCLE Urea cycle: location and source of atoms

a. Urea synthesis takes place mostly in the liver. b. One N atom of urea comes from Asp (blue). c. One N atom comes from NH4+ (green). d. One C atom comes from CO2 (red). The first two reactions leading to the synthesis of urea occur in the mitochondria, whereas the remaining cycle enzymes are located in the cytosol.

Carbamoyl phosphate and citrulline, the first two intermediates of urea cycle are synthesized in mitochondria, while the rest of the reactions in the cytosol. Reactions of the cycle • The first two reactions leading to the synthesis of urea occur in the mitochondria, whereas the remaining cycle enzymes are located in the cytosol. • [Note: Gluconeogenesis and heme synthesis also involve both the mitochondrial matrix and the cytosol • The precursors of urea cycle: ammonium ion and bicarbonate, Both will form carbamoyl phosphate. • Carbamoyl phosphate and citrulline, the first two intermediates of urea cycle are synthesized in mitochondria, while the rest of the reactions in the cytosol. 1 Formation of carbamoyl phosphate: • Formation of carbamoyl phosphate by carbamoyl phosphate synthetase I is driven by cleavage of two molecules of ATP. Ammonia incorporated into carbamoyl phosphate is provided primarily by the oxidative deamination of glutamate by mitochondrial glutamate dehydrogenase. Ultimately, the nitrogen atom derived from this ammonia becomes one of the nitrogens of urea. Carbamoyl phosphate synthetase I requires N- acetylglutamate as a positive allosteric activator. 2.Formation of citrulline:

• The carbamoyl portion of carbamoyl phosphate is transferred to ornithine by ornithine transcar•bamoylase (OTC) as the high-energy phosphate is released as Pi. The reaction product, citrulline, is transported to the cytosol. Ornithine is regenerated with each turn of the urea cycle(6) much in the same way that oxaloacetate is regenerated by the reactions of the citric acid cycle . 3. Synthesis of argininosuccinate:

• Argininosuccinate synthetase combines citrulline with aspartate to form argininosuccinate. The α- amino group of aspartate provides the second nitrogen that is ultimately incorporated into urea. The formation of argininosuccinate is driven by the cleavage of ATP to adenosine monophos•phate (AMP) and pyrophosphate. This is the third and final molecule of ATP consumed in the formation of urea. 4. Cleavage of argininosuccinate:

• Argininosuccinate is cleaved by argininosuccinate lyase to yield arginine and fumarate. The arginine formed by this reaction serves as the immediate precursor of urea. Fumarate produced in the urea cycle is hydrated to malate, providing a link with several metabolic pathways. For example, the malate can be transported into the mitochondria via the malate shuttle, reenter the tricarboxylic acid cycle, and get oxidized to oxaloacetate (OAA), which can be used for gluconeogenesis . Alternatively, the OAA can be converted to aspartate via transamination , and can enter the urea cycle. Cleavage of arginine to ornithine and urea: • Arginase cleaves arginine to ornithine and urea, and occurs almost exclusively in the liver. Thus, whereas other tissues, such as the kidney, can synthesize arginine by these reactions, only the liver can cleave arginine and, thereby, synthesize urea. For example, the malate can be transported into the mitochondria via the malate shuttle, reenter the tricarboxylic acid cycle, and get oxidized to oxaloacetate (OAA), which can be used for gluconeogenesis . Alternatively, the OAA can be converted to aspartate via transamination , and can enter the urea cycle.

Fate of urea: Urea diffuses from the liver, and is transported in the blood to the kidneys, where it is filtered and excreted in the urine. A portion of the urea diffuses from the blood into the intestine, and is cleaved to CO2 and NH3 by bacterial urease. This ammonia is partly lost in the feces, and is partly reabsorbed into the blood. Fate of urea:

• In patients with kidney failure, plasma urea levels are elevated, promoting a greater transfer of urea from blood into the gut. The intestinal action of urease on this urea becomes a clinically important source of ammonia, contributing to the hyperammonemia often seen in these patients. Oral administration of NEOMYCIN reduces the number of intestinal bacteria responsible for this NH3 production. BENEFIT OF GIVING NEOMYCIN TO HEPATO COMROMISED PATIENT

Oral administration of NEOMYCIN reduces the number of intestinal bacteria responsible for this NH3 production. Regulation of the urea cycle • N-Acetylglutamate is an essential activator for carbamoyl phosphate synthetase I—the rate-limiting step in the urea cycle . • N-Acetylglutamate is synthesized from acetyl coenzyme A and glutamate by N-acetylglutamate synthase ,in a reaction for which arginine is an activator. Therefore, the intrahepatic concentration of N-acetylglutamate increases after ingestion of a protein-rich meal, which provides both a substrate (glutamate) and the regulator of N-acetylglutamate synthesis. This leads to anincreased rate of urea synthesis.

Acetyl CO A Acetate GLUTAMATE a.as esp arginine Synthetase ++

N- ACETYL GLUTAMATE CO A

ALLOSTERICALLY ++ carbamoyl phosphate synthetase 1 Overall stoichiometry of the urea cycle

• Aspartate + NH3 + CO2 + 3 ATP + H2O ~ • urea + fumarate + 2 ADP + AMP + 2 Pi + Ppi • Three high-energy phosphate bonds are consumed in the synthesis of each molecule of urea; therefore, the synthesis of urea is irreversible. [Note: The ATP is replenished by oxidative phosphorylation.] PNEUMONIC FOR UREA CYCLE Word Enzyme Word Molecule

Orange Ornithine Carbamoyl Carbamoyl Can Phosphate Colored Synthetase 1 Phosphate Cats Citrulline Ornithine Our Transcarbamoylase Aspartate (enters Always the cycle) Argininosuccinate Aunts Synthetase Ask Argininosuccinate Argininosuccinate Aim Fumarate (leaves Lyase For the cycle)

Accurately Arginase 1 Awesome Arginine

Urea (leaves the Umbrellas cycle) TRANSPORT OF AMMONIA Ammonia transporter (glutamine)

Ammonia generated from N2 is assimilated into low molecular weight metabolites such as glutamate or glutamine 50% of circulating amino acid molecules are glutamine. The amide group of glutamine is a nitrogen donor for several classes of Molecules including: purine bases, and the amino group of cystine. Glutamate and ammonia are the substrates for glutamine synthetase. Removal of the amide group is catalyzed by glutaminase