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isoenzymes

  1. 1. Enzymes-3 Isoenzymes Clinical enzymology RITTU CHANDEL 05-02-13
  2. 2. ISOENZYMES  Isoenzymes or isozymes are mutiple forms of same enzyme that catalyse the same chemical reaction  Different chemical and physical properties:  Electrophoretic mobility  Kinetic properties  Amino acid sequence  Amino acid composition 2
  3. 3. S. Property E.g. No 1 Electrophoretic Isoenzymes of Lactate dehydrogenase have mobility different electrophoretic mobility 2 Heat stability Alkaline phosphatase isoenzymes are either heat labile or stable 3 Inhibitor An inhibitor can inhibit only one isoenzyme of an enzyme eg. Acid phosphatase 4 Km Glucokinase and hexokinase 5 Cofactors Mitochondrial isocitrate dehydrogenase requires NAD+ , cytosolic form requires NADP+ 6 Tissue localisation LDH 1 is present in heart, LDH 5 in muscle 7 Antibodies For creatine kinase, each isoenzyme can be bound only by a specific antibody 3
  4. 4. Lactate dehydrogenase (LDH)  E.C – 1.1.1.27  L-lactate :NAD+ oxidoreductase:LDH  Molecular weight- 134 kDa  tetramer  M (A) -muscle –chromosome 11(basic)  H (B) -heart – chromosome 12(acidic) 4
  5. 5. Lactate dehydrogenase (LDH) Normal values Serum -100 -200 U/L CSF - 7 -30 U/L Urine - 40 -100 U/L 5
  6. 6. Isoenzyme Composition Electrophore Present in Elevated in name tic migration LDH 1 ( H 4) Fastest Myocardium, myocardial Heat moving RBC,kidney infarction resistant LDH2 (H3M1) Myocardium, Kidney Heat RBC,kidney disease,megalo resistant blastic anemia LDH3 (H2M2) brain Leukemia,malig nancy LDH4 (H1M3) Lung,spleen Pulmonary Heat labile infarction LDH5 (M4) Slowest Skeletal Skeletal muscle Heat labile moving muscle, Liver and liver Inhibited by diseases urea 6
  7. 7. Lactate dehydrogenase (LDH) This is an example in which two duplicated genes have become specialized to different tissues. The isozymes are also differentially expressed in different developmental stages. Before birth the heart is more anaerobic compared with adulthood. Indeed, before birth the main isozyme in the heart is the M4, and with time it switches to HM3 (at birth), to H2M2 and HM3 at 1 year after birth, and to H3M AND H4 after 2 years. My main LDH is HM3. Great! My main LDH is HM3 7
  8. 8. Atypical forms of LDH  sixth isoenzyme LDH- X  Seventh isoenzyme – LDH -6 8
  9. 9. Clinical significance of LDH  Myocardial infarction (LDH 1>LDH2)  Megaloblastic anemia (50 times upper limit of LDH 1 and LDH 2)  Muscular dystrophy (LDH 5)  Toxic hepatitis with jaundice (10 times more LDH 5)  Renal disease- tubular necrosis or pyelonepheritis  Pulmonary embolism LDH 3 (massive destruction of platelets) 9
  10. 10.  Leukemia (LDH 2 and LDH 3)  Malignancy (LDH 3)  Hodgkins disease  germ cell tumors  Urinary LDH-3 to 6 times normal: chronic glomerulonephritis Systemic lupus erythematosus Diabetic nephrosclerosis Bladder and kidney malignancies 10
  11. 11.  In CSF:  Bacterial meningitis – LDH 4 and LDH 5  Viral meningitis - LDH 1  Metastatic tumors - LDH 5  Neonatal cases of intracranial haemorrhage associated with seizures and hydrocephalus 11
  12. 12. LDH in starch gel The H(B) monomer is very negatively charged 12
  13. 13. CREATINE PHOSPHOKINASE  Adenosine triphosphate:creatine N- phosphotransferase  E.C-2.7.3.2  Dimeric enzyme (82 kDa)  4 -60 IU/L 13
  14. 14. 14
  15. 15.  Enzyme unstable in serum  Activity lost due to sulfhydryl group oxidation at active site  Dimer (each of 41000 Da)  B (brain) – chromosome 14  M (muscle) –chromosome 19 15
  16. 16. Isoenzy Compo me Present in Elevated in sition name CK-1 Brain,prostate,GI Fast BB tract,lung,bladder,uteru CNS diseases moving s,placenta CK-2 Acute myocardial 2% of MB Myocardium/ Heart infarction total CK-3 Skeletal muscle, Slow MM Myocardium moving All 3 in cytosol 16
  17. 17. 17
  18. 18. atypical forms of CK  Fourth form - CK-Mt (chromosome 15) severe illness Malignant tumors  macroCK  type 1- CK BB complexed with IgG  type 2-oligomeric CK-Mt 18
  19. 19. Clinical significance of CK  CK 1 elevated: very low birth weight newborns brain damage in neonates neurological injury –CK 1 rise in CSF >200 U/L –die 100 – 200 U/L – survive with neurological defecits <100 U/L – good chance of recovery 19
  20. 20. Elevated CK 1  Adenocarcinomas of GI tract  Carcinoma lung  Ca prostate,bladder,testes,kidneys,breast, ovaries,uterus,CNS,leukemia,lympho ma and sarcoma 20
  21. 21. Elevated CK 2: myocardial infarction head injuries subarachnoid haemorrhage exercise Elevated CK 3: muscular dystrophies(DMD- 10000 IU/L) myopathies hypothyroidism (5 fold more than normal value,also CK 2 is elevated) 21
  22. 22. Alkaline phosphatase (ALP)  E.C -3.1.3.1.  Orthophosphoric monoester phosphohydrolase  In mucosa of small intestine, proximal convoluted tubule, bone, liver, placenta  Catalyses alkaline hydrolysis of naturally occuring and synthetic substrates 22
  23. 23. Isoenzymes of ALP  Alpha 1 ALP-epithelial cells of biliary canaliculi  Alpha 2 heat labile ALP- hepatic cells  Alpha 2 heat stable ALP-not destroyed at 65˚C  inhibited by phenylalanine  placental  Pre beta ALP – bone,heat labile  Gamma ALP – intestinal cells  inhibited by phenylalanine  Leukocyte alkaline phosphatase –decreased in CML  increase in lymphoma ATYPICAL ISOENZYMES  Regan isoenzyme-heat stable,inhibited by L-phenylalanine  Nagao isoenzyme- variant of regan  inhibited by L-leucine 23
  24. 24. Clinical significance  Hepatobiliary disease  Hepatic carcinoma  Hepatic metastases  Pagets disease (10 – 25 times)  Bone cancer  Healing of bone fracture  Osteomalacia and rickets  Hyperparathyroidism  Ca of ovary,uterus-regan isoenzyme  Metastatic Ca of pleural surfaces –Nagao isoenzyme 24
  25. 25. Acid phospatases acid phosphatses E.C -3.1.3.2. Hydrolyse phosphoric acid ester at pH 5 -6 In lysosomes Extalysosomal- prostate,bone,spleen,platelet, liver,kidney (pH - 5) RBC (pH – 6) 0 – 0.6 U/L Extremly heat labile 25
  26. 26.  Isoenzyme of ACP inhibitor prostatic dextrorotatory tartarate ions Erythrocytic formaldehyde cupric ions Majorly the serum contains tartarate resistant ACP (originating in osteoclasts) 26
  27. 27. Clinical significance  To detect, monitor Ca prostate  Tartarate resistant ACP increase in pagets disease and bone cancer  Marker of bone disease-increases in: giant cell tumor of bone normal growing children Gauchers disease In high concentrations in semen 27
  28. 28. SERUM AMYLASE(calcium metalloenzyme)  E.C -3.2.1.1.  Molecular weight -54 -62 kDa  From salivary gland and pancreas  Enzymes are products of 2 closely linked loci on chromosome 1  macroamylases 28
  29. 29. 29
  30. 30. Serum aldolase  Tetramer  Catalyses interconversion of fructose- 1,6-bi-phosphate and triose phosphate  5 isoenzymes  Subunits A B 1- 7.5 U/L Skeletal muscle,liver,brain,heart 30
  31. 31. Clinical significance of serum aldolase  Elevated in:  Progressive muscular dystrophy particularly high in DMD  Viral hepatitis  Advanced cancer of prostate 31
  32. 32. SOURCES PLASMA CELL DERIVED/PLASM DERIVED/PLASM A SPECIFIC A NON SPECIFIC BLOOD COAGULATION ENZYMES FERROXIDASE LIPOPROTEIN LIPASE PSEUDOCHOLINESTERA SE SECRETOR METABOLIC Y 32
  33. 33. Mechanisms responsible for abnormal levels Increased serum decreased serum level level Increased Impaired Decreased Enzyme release excretion formation inhibition Cell Increased necrosi permeabilit genetic acquired s y 33
  34. 34. 34
  35. 35. Enzymes in blood Cell death Defects in cellular membrane Release of cytoplasmic enzymes initially In infarctions 35
  36. 36. Elevation of enzymes in blood No.of cell Gradient of injured cell/plasma Rate of Rate of enzyme clearance entry in from plasma plasma 36
  37. 37. Serum enzyme assay in clinical practice  In diagnosis  In differential diagnosis  In prognosis  Early detection of disease 37
  38. 38. Serum transaminases 38
  39. 39. Serum transaminases  Catalyse interconversion of aminoacids to ketoacids by transfer of amino group  AST-aspartate aminotransferases(SGOT) 10-30 U/L  ALT-alanine aminotransferases (SGPT) 10-40 U/L Both present in plasma,bile,CSF,saliva 39
  40. 40. Gamma glutamyl transpeptidases (GGT)  E.C -2.3.2.2.  Γ – glutamyl – peptide:amino acid Γ – glutamyl transferases  In proximal convoluted tubule,liver,pancreas,intestine  Clinical significance  hepatobiliary disease  neoplasms  heavy drinkers 40
  41. 41. cholinesterase  Hydrolyse acetylcholine  Types-  acetylcholinestarase -3.1.1.7  pseudocholinesterase – 3.1.1.8 Clinical significance insecticide poisoning atypical form of enzymes who are at risk to muscle relaxants sensitive indicator of synthetic capacity of liver 41
  42. 42. Glucose – 6 –phosphate dehydrogenase  Dimer with identical subunits  In HMP - for production of NADPH  G-6-P + NADP+ 6-PG + NADPH + H+  hemolytic anemia  prolonged neonatal jaundice  conditions are directly related to the inability of specific cell types to regenerate reduced nicotinamide adenine dinucleotide phosphate (NADPH) 42
  43. 43. Serum lipase  E.C – 3.1.1.3.  Molecular weight -48 kDa  Hydrolyses glycerol esters of long chain fatty acids  Pancreas, intestinal and gastric mucosa  0.2 – 1.0 U/L 43
  44. 44. Serum enzymes in cardiac diseases Why enzyme diagnosis? Enzyme assays Creatine phosphokinase (CPK) Aspartate transaminases (SGOT OR AST) Lactate dehydrogenase (LDH) γ-Glutamyl transpeptidase (GGTP) Histaminase Pseudocholinesterase 44
  45. 45. Cardiac biomarkers in myocardial infarction 45
  46. 46. Onset peak duration 3-6 hrs 18-24 hrs 36-72 CK-MB hrs 4.5-20% of total Troponins 4-10hrs days 18-24 8-14 LDH 6-12hrs 24-48 hrs 6-8 days Flipped pattern 24-36 hrs 4-5days 10-12 AST d Myoglobin 1-4hrs 6-7hrs 24hrs 46
  47. 47. Serum enzymes in GI tract diseases Serum amylase A cute pancreatitis-4 -6 fold increase in 2 -12 hrs , maximum level 12 -72 hrs , normal in 3 - 4 day Urinary amylase - increased on 1st day and remains elevated till 8- 10 day Ca pancreas- amylase in ascitic and pleural fluid Cholecystitis – 4 fold elevation 47
  48. 48. Serum lipase Acute pancreatitis: 2 -50 times in 4 -8 hrs,peaks at 24 hrs,decreases in 8 -14 days 48
  49. 49. Serum enzymes in liver disease Severe toxic hepatitis Extrahepatic cholestasis Cirrhosis (AST>ALT) Serum transaminases Hepatic carcinoma (5 -10 fold rise) Hepatobiliary disease Extrahepatic obstruction (10- 15 times) Serum alkaline phosphatases 49
  50. 50. • Toxic hepatitis with jaundice (10 times more Serum LDH LDH 5) Extrahepatic and intrahepatic causes (2 -6 fold incre 5’nucleotidase Early infectious hepatitis Alcoholic cirrhosis and alcoholics Gamma glutamyl Hepatic carcinoma transferase 50
  51. 51. Serum enzymes in muscle diseases Serum aldolase Progressive muscular dystrophy muscular dystrophies CPK myopathies hypothyroidism (5 fold more than normal value,also CK 2 is elevated) SGOT/SGPT Muscular dystrophy and dermatomyositis 51
  52. 52. Serum enzymes in bone diseases • Pagets disease (10 – 25 times) • Bone cancer Alkaline • Healing of bone fracture phosphatase • Osteomalacia and rickets • Marker of bone disease-increases in: Acid • giant cell tumor of bone phosphatase • normal growing children 52
  53. 53. As tumor markers  Aldolase-liver  ALP – bone,liver,leukemia,sarcoma  Placental ALP – ovarian,lung,hodgkins  Amylase – pancreatic  CPK BB – prostate,lung,breast,colon,ovarian  GGT – liver  LDH – liver,lymphoma,leukemia  Neuron specific enolase – tumors of neuroendocrine origin 53
  54. 54. Prostate specific antigen (PSA or semenogelase)  From secretory epithelium of prostate gland  32 kDa glycoprotein  Mild Serine protease activity  1- 5 μg/L Levels between 4 -10 μg/L –increased risk of prostate cancer >10 μg/L - suggestive of Ca prostate >20 μg/L - Ca prostate with metastases 54
  55. 55. Enzymes-therapeutic agent 1.Streptokinase plasminogen streptokinase plasmin fibrin soluble product 2.urokinase 55
  56. 56. 3. Bacterial asparginase in leukemia 4.α- chymotrypsin-extraction of lens 5.Chymotrypsin,papain –anti inflammatory 6.Collagenase – debridment of dermal ulcers 7.Fibrinolysin – venous thrombosis, pulmonary embolism 8.Hyaluronidase – rapid absorption of drugs injected subcutaneously 56
  57. 57. 9.Lysosome –antibacterial (eye infections) 10.Trypsin – clean wounds treatment of acute thrombophlebitis 57
  58. 58.  Analytical use of enzymes  as reagents  as labels 58
  59. 59. Alcohol Ethanol dehydrogenase Lactate Lactate dehydrogenase Glucose Glucose oxidase and peroxidase Uricase Uric acid Urease Urea Cholesterol oxidase and peroxidase cholesterol 59
  60. 60. As labels  In immunoassays  for determining concentration of drugs, hormones  Glucose -6- phosphate dehydrogenase  Alkaline phosphatase  Beta galactosidase  peroxidase 60
  61. 61. BIBLOGRAPHY  T.B. OF BIOCHEMISTRY:  SATYANARAYAN  VASUDEVAN  PANKAJA NAIK  RANA SHINDE  TEITZ  HARRISON-INTERNAL MEDICINE THANK YOU 61

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