Transdeamination (Deamination of L-Glutamic Acid) The enzyme L-glutamate dehydrogenase catalyzes the deamination of L-glutamate to form α- iminoglutaric acid, which on addition of a molecule of water forms NH3 and α-keto glutarate.

NH3 Transport NH3 sources: •NH3 formed in the tissues • NH3 is produced in the gut by intestinal bacteria flora, both *From dietary proteins *From urea present in fluids secreted into the GI tract. This NH3 is absorbed from intestine into blood. Under normal conditions of health, liver promptly removes the NH3 from the portal blood so that leaving the liver is virtually NH3 -free .This is essential since even small quantities of NH3 are toxic to CNS.

Normal Blood Ammonia Level blood NH3: 40 -70 µg/100ml Free NH4 : ˂20 µg/100ml Hyperammonaemia Two type: 1- Acquired hyperammonaemia: the result of Cirrhosis of the liver. 2-Inherited hyperammonaemia: results from genetic defects in the urea cycle enzyme

Features of NH3 intoxication: The symptoms of NH3 intoxication include: - a peculiar flapping tremor - slurring of speech - blurring of vision - and in severe cases follows to coma and death. These resemble those of syndrome of hepatic coma, where blood and brain NH3 levels are elevated.

Why NH3 is toxic? • Increased NH3 concentration enhances amination of α-ketoglutarate, an intermediate in TCA cycle to form glutamate in brain.This reduces mitochondrial pool of α-ketoglutarate consequently depressing the TCA cycle, affecting the cellular respiration. • Increased NH3 concentration enhances "glutamine" formation from glutamate and thus reduces" brain-cell" pool of Glutamic acid. Hence there is decreased formation of inhibitory neurotransmitter "GABA"( γ- amino butyric acid)

• Rise in brain glutamine level enhances the outflow of glutamine from brain cells. Glutamine is carried "out" by the same "transporter" which allows the entry of "tryptophan" into brain cells. Hence "tryptophan" concentration in brain cells increases which leads to abnormal increases in synthesis of " serotonin", a neurotransmitter.

Metabolic fate of NH3 in the body Three main fates: 1-mainly NH3 is converted to urea (urea cycle) 2-formation of glutamine. 3-amination of α-keto acid to form α-amino acid.  

Urea Formation (Krebs-Henseleit cycle) Ammonia is highly toxic to the central nervous system It is converted to urea, which is much less toxic, water soluble and easily excreted in urine. The liver is the site of Urea biosynthesis.Urea biosynthesis occurs by urea cycle (Krebs Hensleit cycle) in five steps by five enzymes. Any defect in one of these enzymes leads to ammonia intoxication The first 2 steps occur in mitochondria, while the last 3 steps occur in cytoplasm

Note Other Organs • Kidneys: Urea cycle operates in a limited extent. Kidney can form up to ariginine but cannot form urea, as enzyme arginase is absent in kidney tissues. • Brain: Brain can synthesise urea from citrulline, but lacks the enzyme for forming citrulline from ornithine. Thus, neither the kidneys nor the brain can form urea in significant amounts.   • Location of enzymes: It is partly mitochondrial and partly cytosolic. • One mol. of NH3 and one mol. of CO2 are converted to one mol. of urea for each turn of the cycle and orinithine is regenerated at the end, which acts as a catalytic agent. • The overall process in each turn of cycle requires 3 mols of ATP.

Steps of Urea Biosynthesis 1-Biosynthesis of carbamoyl phosphate In this reaction, HCO3–, NH4+ and phosphate derived from ATP reacts to form carbamoyl-P (also called Carbamyl-P).The reaction is catalysed by the mitochondrial-enzyme Carbamoyl phosphate synthetase 1. There are 2 types of the enzyme: • Carbamoyl synthetase I: Occurs in mitochondria of Liver cells. It is involved in urea synthesis. • Carbamoyl synthetase II: Present in cytosol of liver cells which is involved in pyrimidine synthesis (Refer to metabolic fate of carbamoyl-P).  

2-Formation of citrulline This step occurs in mitochondria. It is catalyzed by ornithine transcarbamoylase

3-Formation of argininosuccinate This step occurs in cytoplasm. It is catalyzed by agininosuccinate synthetase . It utilizes one ATP

4- Cleavage of argininosuccinate This step occurs in cytoplasm. It is catalyzed by argininosuccinase enzyme Argininosuccinate is cleaved into arginine and fumarate Fumarate produced is used to regenerate aspartic acid again

5-Cleavage of arginine This step occurs in cytoplasm It is catalyzed by arginase enzyme Arginine is cleaved to urea and ornithine  

Regulation of urea cycle 1. Achieved by linkage of mitochondrial glutamate dehydrogenase with carbamoyl-P-synthetase I. 2. Carbamoyl-P-synthetase I is thought to act in conjunction with mitochondrial glutamate dehydrogenase to channelise nitrogen from glutamate and,therefore, from all amino acids as NH3 and then through carbamoyl-P and thus finally to urea. 3. Though the equilibrium constant of the glutamate dehydrogenase reaction favours glutamate formation rather than formation of NH3, but removal of NH3 by the carbamoyl-P-synthetase I reaction and oxidation of α-ketoglutarate by TCA cycle favours the glutamate catabolism. 4. The above effect is favoured by the presence of ATP, which in addition to being a requirement for carbamoyl-P-synthetase I reaction, it also stimulates Glutamate dehydrogenase activity unidirectionally in the direction of NH3 formation

Relationship Between Urea Cycle and Citric Acid Cycle 1-CO2 needed for urea synthesis is mostly produced from citric acid cycle 2-Ammonia used for carbamoyl phosphate synthesis (in mitochondria) is derived from glutamic acid by glutamate dehydrogenase enzyme 3-Fumarate produced by urea cycle can be converted into oxalacetic acid by citric acid cycle 4-Aspartic acid used in urea cycle is formed from oxalacetic by transamination with glutamic acid by AST (GOT) enzyme 5-ATP needed for urea cycle is derived from citric acid cycle

Clinical significance of urea 1-Normal level: the normal concentration of blood plasma in healthy adult ranges from 20-40 mg/dl 2- Increase levels Increases in blood urea may occur in a number of diseases in addition to those in which the kidneys are primarily involved. The causes can be classified as: • Prerenal, • Renal, and • Postrenal

Prerenal Most important are conditions in which plasma vol / body-fluid are reduced: • Salt and water depletion, • Severe and protracted vomiting as in pyloric and intestinal obstruction, • Severe and prolonged diarrhea, • Pyloric stenosis with severe vomiting, • Haematemesis, • Haemorrhage and shock; shock due to severe burns, • Ulcerative colitis with severe chloride loss, • In crisis of Addison’s disease (hypoadrenalism).

(b) Renal The blood urea can be increased in all forms of kidney diseases like: • In acute glomerulonephritis. • In early stages of type II nephritis (nephrosis) the blood urea may not be increased, but in later stages with renal failure, blood urea rises. • Other conditions are malignant nephrosclerosis, chronic pyelonephritis and mercurial poisoning. • In diseases such as hydronephrosis, renal tuberculosis; small increases are seen but depends on extent of kidney damage.  

c) Postrenal Diseases These lead to increase in blood urea, when there is obstruction to urine flow.   Causes: • Enlargement of prostate, • Stones in urinary tract, • Stricture of the urethra, • Tumors of the bladder affecting urinary flow. Note Increase in blood urea above normal is called uraemia.

3- Decreased levels: are rare, but may be seen in: • some cases of severe liver damage. • physiological condition: blood urea is lower in pregnancy than in normal non pregnant women.