1. FORMATION,TRANSPORT AND DETOXIFICATION OF AMMONIA.pptx

urea cycle

1. Male infant, 2.9 kg at birth, healthy Day 3 – seizures plasma NH 4 + = 240 uM (25-40 normal) hyperammonemia mild alkalosis (pH=7.5, normal 7.35-7.45)

2. Plasma AA gln = 2400 uM (350-650) ala = 750 uM (8-25) arg = 5 uM (30-125) cit = undetectable Urinary orotic acid = 285 ug/mg creatinine (0.3-10)

3. FORMATION,TRANSPORT AND DETOXIFICATION OF AMMONIA

4. Glycine

5. FORMATION: Transamination followed by Transdeamination. Oxidative deamination Non-Oxidative deamination Intestinal bacteria-Major source when there is intestinal obstruction Pyrimidine Catabolism 6. Hydrolytic deamination of AMP (enzyme – adenosine deaminase )

6. Transamination followed by T ransdeamination Definition: It is a reversible reaction in which NH 2 group of one amino acid is transferred to keto acid resulting in the formation of new amino acid and new keto acid. Amino acid + Keto acid New amino acid + New Keto acid

7. Aspartate α – Ketoglutarate Oxaloacetate Glutamic acid + + Alanine α – Ketoglutarate Pyruvate Glutamic acid + + AST ALT

8. TRANSDEAMINATION

9. NADP NADPH NAD NADH L-Glutamic acid α –Iminoglutaric acid L-glutamate dehyrogenase (requires Zn) α – Ketoglutarate + NH3

10. Deamination It is the process by which N(Nitrogen) of amino acid is directly removed as NH 3 . TYPES : Oxidative deamination Non oxidative deamination

11. a) Oxidative deamination L amino acid oxidase L - amino acids α - imino acid FP FPH 2 α – Keto acid NH 3 Does not play a major role in mammalian aminoacid catabolism and formation of NH 3

12. b) Non oxidative deamination 1. Amino acid dehydratase Serine B 6 H 2 O Imino acid NH 3 + Pyruvic acid 2.Deamination of Histidine Histidine Urocanic acid Histidase NH3

13. 3.Amino acid desulfhydrases : L-Cysteine Iminoacid-I desulfydrase H 2 O Pyruvic acid Iminoacid -II NH 3

14. First all aminoacids are transaminated to glutamate. Glutamate undergoes trandeamination to form ammonia. Some aminoacid undergoes oxidative and non oxidative deamination to form ammonia directly.

15. TRANSPORT OF AMMONIA

16. Peripheral Tissues Transport Nitrogen to the Liver Two ways of nitrogen transport from peripheral tissues (muscle) to the liver: 1. Alanine cycle. Glutamate is formed by transamination reactions Glutamate is not deaminated in peripheral tissues

17. Nitrogen is then transferred to pyruvate to form alanine , which is released into the blood. The liver takes up the alanine and converts it back into pyruvate by transamination. The glutamate formed in the liver is deaminated and ammonia is utilized in urea cycle .

18. 2. Nitrogen can be transported as glutamine. Glutamine synthetase catalyzes the synthesis of glutamine from glutamate and NH 4 + in an ATP-dependent reaction:

19. Glutamine is principal transport form of amino nitrogen The figure is from http://www.sbuniv.edu/~ggray/CHE3364/b1c25out.html (Dec 2006)

20. Two important transport form of ammonia from periphery to liver: 1. Alanine 2. Glutamine

21. Why ammonia is toxic? NH 3 NH 3 + α - Ketoglutarate Glutamate in α -ketoglutarate leads to decreased activity of TCA cycle Cellular respiration

22. NH 3 Conversion of glutamate to glutamine Brain cell pool of glutamate Formation of GABA which is an inhibitory amino acid

23. Glutamate is carried out from the brain cell by the same transporter that allows the entry of tryptophan into the cells. Concentration of tryptophan in brain cells Production of serotonin (neurotransmitter)

24. Urea cycle (Krebs – Henseleit cycle) Location Organ: Liver Organelles: Mitochondria, cytosol

25. Over all blue print Carbamoyl phosphate ornithine citrulline arginine urea Aspartic acid + + CO + CAA = U

26. STAGES : 1. Synthesis of carbamoyl – phosphate. 2. Synthesis of citrulline . 3. Synthesis of argininosuccinate . 4. Cleavage of argininosuccinate . 5. Cleavage of arginine to form ornithine.

27. Sources of C and N of urea N -Ammonia Aspartic acid C - Bicarbonate

28. Urea cycle - a cyclic pathway of urea synthesis first postulated by H.Krebs THE UREA CYCLE The sources of nitrogen atoms in urea molecule: aspartate ; NH 4 + . Carbon atom comes from CO 2 .

29. The free ammonia is coupling with carbon dioxide to form carbamoy l phosphate Two molecules of ATP are required Reaction takes place in the matrix of liver mitochondria Enzyme: c a rbamoyl phosphate synthetase (20 % of the protein of mitochondrial matrix)

30. C arbamoyl phosphate donates carbamoyl group to ornithine The product - citrui l line Enzyme: ornithine carbamoy l transferase Reaction takes place in the mitochondrial matrix C itrulline leaves the matrix and passes to the cytosol

31. In the cytosol c itrulline in the presence of ATP reacts with aspartate to form arg in inosuccinate Enzyme: argin ino succinate synthetase

32. A rgininosuccinate is cleaved to free arginine and fumarate Enzyme: argininosuccinate lyase T he fumarate enters the tricarboxylic acid cycle

33. Arginine is hydrolyzed to generate urea and ornithine Enzyme: arginase (present only in liver of ureotelic animals) Ornithine is transported back into the mitochondrion to begin another cycle Urea is excreted (about 40 g per day)

34. Relationship between urea cycle and TCA cycle NH 3 + CO 2 + H 2 O 2ATP CPS-1 2ADP+Pi Carbamoyl phosphate OTC Ornithine Citrulline Aspartic acid urea ATP AST Arginase AMP+Pi ASS Oxaloacetate H 2 O Arginine Argininosuccinate Malate dehydrogenase Fumarate Malate ASL Fumarase TCA cycle Urea cycle

35. The Linkage between Urea Cycle, Citric Acid Cycle and Transamination of Oxaloacetate Fumarate formed in urea cycle enters citric acid cycle and is converted to oxaloacetate. Fates of oxaloacetate: transamination to aspartate, conversion into glucose, condensation with acetyl CoA to form citrate, conversion into pyruvate.

36. Comments on urea cycle in kidneys and brain Kidneys: Enzyme arginase is absent in kidney tissues . Brain : Enzyme ornithine transcarbomylase is absent in brain .

37. Clinical Case Study Male infant, 2.9 kg at birth, healthy Day 3 – seizures plasma NH 4 + = 240 uM (25-40 normal) hyperammonemia mild alkalosis (pH=7.5, normal 7.35-7.45)

38. Clinical Case Study Plasma AA gln = 2400 uM (350-650) ala = 750 uM (8-25) arg = 5 uM (30-125) cit = undetectable Urinary orotic acid = 285 ug /mg creatinine (0.3-10)

39. Resolution of Clinical Case Diagnosis of neonatal hyperammonemia symptoms blood ammonium concentration Defect in urea cycle elevated glutamine and alanine low or absent arginine and citrulline

40. Resolution of Clinical Case Genetic deficiency of ornithine transcarbamoylase urinary orotic acid CP spills into cytosol where enters pyrimidine biosynthetic pathway, orotic acid an intermediate in the pathway

41. Relationship between urea cycle and pyrimidine metabolism Urea cycle Co 2 + NH 4 Carbamoyl Phosphate Ornithine Citrulline Arginine Arginosuccinate Carbomaylphosp-hate synthase-I Ornithine transcarbomylase Pyrimidine metabolism Co 2 + Glutamine Carbamoyl Phosphate + Aspartic acid Orotic acid OMP UMP TMP pi Orate phosphoribosyl transferase Orotidylic acid decarboxylase Carbomaylphosphate synthase -II

42. Clinical Case Study Treatment Oral therapy essential amino acids arginine sodium benzoate @7 days clinically well normal NH 4 +

43. Resolution of Clinical Case: Treatment Essential Amino Acids Arginine w/o urea cycle, becomes essential Benzoic acid conjugates with glycine and excreted in urine as hippuric acid glycine in equilibrium with ammonia Glycine synthase CO 2 + Me-THF + NADH + NH 3 => glycine removal results in reducing ammonia levels

44. Sodium phenylacetate : Phenylacetate conjugates with glutamate to form phenylacetylglutamine. Sodium Benzoate and Phenylacetate combination should be used in conjunction with a low protein diet.

45. Resolution of Clinical Case: Genetics Gene for OTC found on X-chromosome Women are carriers usually asymptomatic may experience migraines, vomiting, lethargy when eating high protein meals (meat) OTC deficiency most common (but rare) disorders of the urea cycle (1: 20-80,000)

46. A baby born after normal pregnancy, developed difficulty in respiration 36 hours after birth. After two days she had severe vomiting and unresponsive stimuli. Her blood investigations are given below. Normal range pH = 7.5 7.35 - 7.45 Urea = 11mg% 15 - 40mg/dl Serum Ammonia = 500microgram/dl 20 -30micro gram/dl Serum glutamine and alanine were also increased. What would be the probable diagnosis ? b ) What is the enzyme defect ? c ) What would be the treatment?

47. A 2year old girl was seen in the emergency room for vomiting and tremors. P lasma ammonia concentration was 200micro Molar/L. Metabolic screening of blood and serum were ordered and were remarkable for an elevation in the amino acid arginine in serum . Based on the history and lab results What c ould be the Probable diagnosis ? b) Name the enzyme that could be defective ? c) What type of diet will you advice to this patient ? d) What is the Normal range of ammonia?

48. A 7week old female infant developed intermittent vomiting . Initial Diagnosis included sepsis and pyloric stenosis. However vomiting continued, subsequently seizures, Hyperammonemic coma developed and she was rescued from Hyperammonemia Coma within 30 hours. Significant evaluation of citrulline and L-Glutamine were demonstrated she was discharged in excellent condition.   What is your probable diagnosis ? b) Name the enzyme defect in this condition?

49. A 40year old male was admitted in casualty with previous history of chronic alcohol consumption . O/E patient was confused , irritable, ascites, jaundice & hematemesis . Biochemical investigations are as follows urea - 35 mg/dl serum ammonia - 60 microgram/dl   a) What is your probable diagnosis? justify? b) Mention the reference range for blood ammonia & urea c) What is the role of lactulose administration in this condition ?