1. Disposal of ammonia, ammonia toxicity, pH regulation by the liver Ms. K. Rohini Lecturer – Biochem Unit

2. Sources of Ammonia and its disposal Importance of liver in acid-base regulation 2 LEARNING OBJECTIVES

3. Ammonia is produced in the body from nitrogen containing compounds Major nitrogen containing compounds in human cells are: – Amino acids – Amines – Purines INTRODUCTION 3

4. Sources Disposal Dietary protein Amino acid catabolism Glutamine synthesis Purine nucleotide metabolism Glutamate synthesis Pyrimidine nucleotide metabolism Ammonia from the intestine UREA synthesis Monoamine oxidase reactions NH 3 4

5. COO - +H3N+H3N C H R α Amino group The carbon skeleton of amino acids can be used in metabolism only if the α-amino group is removed Carbon skeleton AMMONIA FROM AMINO ACID CATABOLISM 5

6. 1.Non-oxidative Deamination 2.Oxidative Deamination 3.Transamination followed by L-glutamate dehydrogenase reaction 4.Glutaminase reaction FOLLOWING REACTIONS INVOLVED THE REMOVAL OF AMINO GROUP FROM AMINO ACID 6

7. Deamination The removal of amino group as ammonia from an amino acid Oxidative Non oxidative deamination deamination 7

8. Amino acid Imino acid Keto acids Amino acid oxidase FAD FADH 2 Oxidative Deamination The liberation of free ammonia from the amino group of amino acids coupled with oxidation to form ketoacids is known as oxidative deamination L-amino acid oxidase D-amino acid oxidase L-glutamate dehydrogenase 8 NH 3

9. Non oxidative Deamination a.Serine dehydratase b.Cysteine desulphydrase c.Histidase d.Asparginase Serine pyruvate [keto acids] +NH 3 +H 2 0 Cysteine pyruvate + NH 3 +H 2 S Serine dehydratase Desulphydrase 9

10. Transamination followed by L-glutamate dehydrogenase reaction  Major mechanism for removal of amino group  Transfer of amino group to α-keto acid is the first step in removal of the amino group  Transamination is the reaction for transferring amino groups to α-keto acid  α ketoglutarate is the predominant keto acid in transamination  α ketoglutarate receives amino groups from all amino acids and becomes L-glutamate  L-glutamate thus collects amino groups from all amino acids in the body 10

11. 11 L- glutamate undergoes oxidative deamination to release free ammonia TRANSDEAMINATION

12. Glutamine Glutaminase H2OH2O NH 3 Glutamate  Glutaminase is seen in kidneys, intestine  Active in kidneys during severe acidosis  Ammonia formed in kidney is used for bicarbonate generation to correct the acidosis 12 GLUTAMINASE REACTION

13. 13

14. Normally contributes to small amounts of NH 3 This source becomes clinically significant in renal patients Urea Urease NH 3 + H 2 O 14 INTESTINAL BACTERIA

15. Dopamine degradation Norepinephrine/epinephrine degradation AMP deaminase, adenosine deaminase Guanase 15 AMINE OXIDASE REACTION DEGRADATION OF PURINE NUCLEOTIDES

16. Highly toxic ammonia Urea is synthesized In the liver from ammonia and carbon dioxide and Excreted via the kidney Detoxified Produced by all cells Disposal by Brain: glutamine AMMONIA & UREA CYCLE 16

17. Even a slight increase in blood NH 3 levels is toxic to the CNS NH 3 from various organs is transported to liver where it is converted to urea (soluble and less toxic) Transport in blood is very essential, at the same time low level must be maintained in the blood 17 TRANSPORT OF AMMONIA IN THE BLOOD

18. From most of the tissues: Transported as glutamine Glutamate Glutamine synthetase NH 3 ATP ADP iP Glutamine 18 TRANSPORT OF AMMONIA IN THE BLOOD

19. From muscle: Transported as alanine L-glutamateα-ketoglutarate + NH 3 pyruvate α-ketoglutarate Alanine ALT 19 TRANSPORT OF AMMONIA IN THE BLOOD

20. In the liver, Glutamine and alanine are converted back to glutamate, ammonia is released Ammonia is converted to more soluble, less toxic urea Some glutamate undergoes transamination with oxaloacetate to form aspartate Aspartate is needed to detoxify ammonia to urea 20

21. Conversion of toxic NH 3 to less toxic, water soluble urea that is easily excreted in the urine Site: Liver Subcellular site: Mitochondria, cytosol Sources of atoms: NH 2 -CO-NH 2 Free NH 3 CO 2 Aspartate 21 UREA CYCLE

22. Mitochondria Cytosol 22 UREA CYCLE 5 steps 1.Formation of Carbomyl phosphate 2.Formation of Citrulline 3.Formation of Arginosuccinate 4.Formation of Arginine and Fumarate 5.Formation of Urea

23. HCO 3 - +NH 3 + 2ATP Carbamoyl phosphate Carbamoyl phosphate synthetase-1 N-acetyl glutamate 2ADP + iP Citrulline Ornithine transcarbamoylase Ornithine iP 23

24. Citrulline Ornithine Citrulline Arginino succinate synthase ATP Aspartate Argininosuccinate Fumarate Arginine Argininosuccinate lyase Arginase UREA Ornithine AMP PPi 24

25. Carbamoyl phosphate Synthase I is the key enzyme and is regulated by NAG Mitochondria Cyotplasm 25

26.  Carbamoyl phosphate synthetase-I reaction : 2 ATPs used  Argininosuccinate synthetase reaction : 2 ATPs used  Total 4 ATPs used up for synthesis of 1 molecule of urea 26 ENERGETICS

27. In kidney failure, urea accumulates in blood, greater transfer to intestine, more ammonia formation, hence hyperammonemia Diffuses from liver, transported in blood, to kidneys, excreted in urine A small portion diffuses from blood to intestine and is acted upon by bacterial urease 27 FATE OF UREA

28. Regulatory enzyme: carbamoyl phosphate synthetase I Activator: N-acetyl glutamate GlutamateAcetyl CoA N-acetyl glutamate synthetase acetate CoA arginine + hydrolase 28 REGULATION OF UREA CYCLE BY AMINO ACIDS AVAILABILITY

29. Protein breakdown results in generation of bicarbonate from the carboxyl groups of amino acids Urea cycle is also a mechanism to correct the transient alkalosis by binding the bicarbonate to ammonia and excrete it as urea When pH in ECF decreases, rate of urea synthesis also decreases; the bicarbonate that is retained will correct the fall in pH Decrease in ECF bicarbonate produces a similar effect 29 REGULATION OF UREA CYCLE BY PH

30. pH control of urea cycle depends on: – Ammonia/ammonium ratio – pH dependent transport of amino acids into liver cells – Activity of hepatic glutaminase – Activity of carbonic anhydrase Acidosis shifts ammonia detoxification from urea synthesis to glutamine synthesis 30 REGULATION OF UREA CYCLE BY PH

31. Excess ammonia is sequestered into glutamine Hyperammonemia due to inhibition of urea cycle in acidosis is prevented by increased activity of renal glutaminase With respect to bicarbonate and ammonia homeostasis, liver and kidney act as a team 31 REGULATION OF UREA CYCLE BY PH

32. Features Vomitting Irritability Tremors Seizures Blurring of vision Slurring of speech Coma Death 32 HYPERAMMONEMIA

33.  Hepatic encephalopathy is the occurrence of confusion, altered levels of consciousness and coma caused by liver failure  The major reason of encephalopathy in liver failure is hyperammonemia  Ammonia crosses blood brain barrier and enters brain cells 33 HEPATIC ENCEPHALOPATHY IN HYPERAMMONEMIA

34.  α-ketoglutarate in the brain cells react with ammonia to form glutamate  Brain cells are starved of α-ketoglutarate, so no TCA cycle, no ATP; therefore encephalopathy  Increased glutamate is converted to GABA (gamma amino butyric acid) which is a inhibitory neurotransmitter, another reason for encephalopathy 34 HEPATIC ENCEPHALOPATHY IN HYPERAMMONEMIA

35.  Increased ammonia increases entry of aromatic amino acids across blood brain barrier; – more tryptophan in brain; increased synthesis of serotonin, a neurotransmitter. – Loss of balance between neurotransmitters - another reason for encephalopathy 35 HEPATIC ENCEPHALOPATHY IN HYPERAMMONEMIA

36. Two types: 1. Acquired: a. Acute processes like viral hepatitis, ischemia, hepatotoxins b. Cirrhosis of liver (caused by alcoholism, hepatitis, biliary obstruction) - Results in formation of collateral circulation, portal blood bypasses liver, directly reaches systemic circulation 36 HYPERAMMONEMIA

37. 2. Hereditary: Genetic deficiencies of urea cycle enzymes 37 HYPERAMMONEMIA DisorderDefective enzyme Hyperammonemia type ICarbamoyl phosphate synthetase I Hyperammonemia type IIOrnithine transcarbamoylase CitrullinemiaArgininosuccinate synthase Argininosuccinic aciduriaArgininosuccinate lyase HyperargininemiaArginase

38. 1. Low protein diet 2. Frequent protein meals in small amounts 3. Benzoic acid 4. Phenylbutyrate 5. L-arginine (for defects in enzymes 3 & 4 of urea cycle) 6. Neomycin 7. Lactulose (6 & 7 are to manage hyperammonemia in patients with hepatic insufficiency) 38 TREATMENT

39. Hippuric acid Glycine Benzoic acid CO 2 NH3 Soluble, excreted in urine 39 BENZOIC ACID

40. Phenylacetyl glutamine Glutamine Phenylbutyrate is a prodrug; metabolised in the body to α-ketoglutarate NH3 Soluble, excreted in urine phenylacetate Glutamate 40 PHENYL BUTYRATE

41. Patients suffer from hypoalbuminemia, so must take minimum maintenance levels of proteins These patients are managed with neomycin or lactulose Neomycin is a non absorbable antibiotic which kills urease producing bacteria, decreases ammonia production Lactulose increases the growth of enterobacteria which produce acids; decrease in pH converts ammonia to ammonium which is excreted 41 MANAGEMENT OF HYPERAMMONEMIA IN HEPATIC INSUFFICIENCY

42. 42 LEARNING OUTCOMES  List the sources of ammonia  Explain the biochemical basis of ammonia toxicity  Describe the urea cycle  Explain the role of urea cycle in acid-base regulation

43. 43 14 Nov 2011Rohini K FoM 43 Thank You