isoenzymes

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  • 1. Enzymes-3IsoenzymesClinical enzymology RITTU CHANDEL 05-02-13
  • 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. S. Property E.g.No1 Electrophoretic Isoenzymes of Lactate dehydrogenase have mobility different electrophoretic mobility2 Heat stability Alkaline phosphatase isoenzymes are either heat labile or stable3 Inhibitor An inhibitor can inhibit only one isoenzyme of an enzyme eg. Acid phosphatase4 Km Glucokinase and hexokinase5 Cofactors Mitochondrial isocitrate dehydrogenase requires NAD+ , cytosolic form requires NADP+6 Tissue localisation LDH 1 is present in heart, LDH 5 in muscle7 Antibodies For creatine kinase, each isoenzyme can be bound only by a specific antibody 3
  • 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. Lactate dehydrogenase (LDH) Normal values Serum -100 -200 U/L CSF - 7 -30 U/L Urine - 40 -100 U/L 5
  • 6. Isoenzyme Composition Electrophore Present in Elevated inname tic migrationLDH 1 ( H 4) Fastest Myocardium, myocardialHeat moving RBC,kidney infarctionresistantLDH2 (H3M1) Myocardium, KidneyHeat RBC,kidney disease,megaloresistant blastic anemiaLDH3 (H2M2) brain Leukemia,malig nancyLDH4 (H1M3) Lung,spleen PulmonaryHeat labile infarctionLDH5 (M4) Slowest Skeletal Skeletal muscleHeat labile moving muscle, Liver and liverInhibited by diseasesurea 6
  • 7. Lactate dehydrogenase (LDH)This is an example in which two duplicated genes havebecome specialized to different tissues.The isozymes are also differentially expressed indifferent developmental stages. Before birth the heartis 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 H2M2and HM3 at 1 year after birth, and to H3M AND H4after 2 years. My main LDH is HM3. Great! My main LDH is HM3 7
  • 8. Atypical forms of LDH sixth isoenzyme LDH- X Seventh isoenzyme – LDH -6 8
  • 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.  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.  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. LDH in starch gel The H(B) monomer is very negatively charged 12
  • 13. CREATINEPHOSPHOKINASE Adenosine triphosphate:creatine N- phosphotransferase E.C-2.7.3.2 Dimeric enzyme (82 kDa) 4 -60 IU/L 13
  • 14. 14
  • 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. Isoenzy Compome Present in Elevated in sitionnameCK-1 Brain,prostate,GIFast BB tract,lung,bladder,uteru CNS diseasesmoving s,placentaCK-2 Acute myocardial2% of MB Myocardium/ Heart infarctiontotalCK-3 Skeletal muscle,Slow MM Myocardiummoving All 3 in cytosol 16
  • 17. 17
  • 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. 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. 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. Elevated CK 2: myocardial infarction head injuries subarachnoid haemorrhage exerciseElevated CK 3: muscular dystrophies(DMD- 10000 IU/L) myopathies hypothyroidism (5 fold more than normal value,also CK 2 is elevated) 21
  • 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. 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. 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. Acid phospatasesacid phosphatsesE.C -3.1.3.2.Hydrolyse phosphoric acid ester at pH 5 -6In lysosomesExtalysosomal- prostate,bone,spleen,platelet, liver,kidney (pH - 5) RBC (pH – 6)0 – 0.6 U/LExtremly heat labile 25
  • 26.  Isoenzyme of ACP inhibitor prostatic dextrorotatory tartarate ionsErythrocytic formaldehyde cupric ionsMajorly the serum contains tartarate resistant ACP (originating in osteoclasts) 26
  • 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 childrenGauchers diseaseIn high concentrations in semen 27
  • 28. SERUM AMYLASE(calciummetalloenzyme) 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
  • 30. Serum aldolase Tetramer Catalyses interconversion of fructose- 1,6-bi-phosphate and triose phosphate 5 isoenzymes Subunits A B1- 7.5 U/LSkeletal muscle,liver,brain,heart 30
  • 31. Clinical significance of serumaldolase Elevated in: Progressive muscular dystrophy particularly high in DMD Viral hepatitis Advanced cancer of prostate 31
  • 32. SOURCES PLASMA CELL DERIVED/PLASM DERIVED/PLASM A SPECIFIC A NON SPECIFIC BLOOD COAGULATION ENZYMES FERROXIDASE LIPOPROTEIN LIPASEPSEUDOCHOLINESTERA SE SECRETOR METABOLIC Y 32
  • 33. Mechanisms responsible for abnormal levels Increased serum decreased serum level level Increased Impaired Decreased Enzyme release excretion formation inhibition Cell Increasednecrosi permeabilit genetic acquired s y 33
  • 34. 34
  • 35. Enzymes in blood Cell death Defects in cellular membrane Release of cytoplasmic enzymes initially In infarctions 35
  • 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. Serum enzyme assay in clinicalpractice In diagnosis In differential diagnosis In prognosis Early detection of disease 37
  • 38. Serum transaminases 38
  • 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/LBoth present in plasma,bile,CSF,saliva 39
  • 40. Gamma glutamyltranspeptidases (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. cholinesterase Hydrolyse acetylcholine Types- acetylcholinestarase -3.1.1.7 pseudocholinesterase – 3.1.1.8Clinical significance insecticide poisoning atypical form of enzymes who are at risk to muscle relaxants sensitive indicator of synthetic capacity of liver 41
  • 42. Glucose – 6 –phosphatedehydrogenase 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. 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. Serum enzymes in cardiacdiseases Why enzyme diagnosis? Enzyme assays Creatine phosphokinase (CPK) Aspartate transaminases (SGOT OR AST) Lactate dehydrogenase (LDH) γ-Glutamyl transpeptidase (GGTP) Histaminase Pseudocholinesterase 44
  • 45. Cardiac biomarkers inmyocardial infarction 45
  • 46. Onset peak duration 3-6 hrs 18-24 hrs 36-72 CK-MB hrs 4.5-20% of totalTroponins 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 dMyoglobin 1-4hrs 6-7hrs 24hrs 46
  • 47. Serum enzymes in GI tractdiseasesSerum amylaseA cute pancreatitis-4 -6 fold increase in 2 -12 hrs ,maximum level 12 -72 hrs , normal in 3 - 4 dayUrinary amylase - increased on 1st day and remainselevated till 8- 10 dayCa pancreas- amylase in ascitic and pleural fluidCholecystitis – 4 fold elevation 47
  • 48. Serum lipaseAcute pancreatitis:2 -50 times in 4 -8hrs,peaks at 24hrs,decreases in 8 -14 days 48
  • 49. Serum enzymes in liverdisease Severe toxic hepatitis Extrahepatic cholestasis Cirrhosis (AST>ALT) Serumtransaminases Hepatic carcinoma (5 -10 fold rise) Hepatobiliary disease Extrahepatic obstruction (10- 15 times)Serum alkalinephosphatases 49
  • 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 alcoholicsGamma glutamyl Hepatic carcinoma transferase 50
  • 51. Serum enzymes in musclediseasesSerum aldolase Progressive muscular dystrophy muscular dystrophiesCPK myopathies hypothyroidism (5 fold more than normal value,also CK 2 is elevated)SGOT/SGPT Muscular dystrophy and dermatomyositis 51
  • 52. Serum enzymes in bonediseases • Pagets disease (10 – 25 times) • Bone cancer Alkaline • Healing of bone fracturephosphatase • Osteomalacia and rickets • Marker of bone disease-increases in: Acid • giant cell tumor of bonephosphatase • normal growing children 52
  • 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. Prostate specific antigen (PSA orsemenogelase) From secretory epithelium of prostate gland 32 kDa glycoprotein Mild Serine protease activity 1- 5 μg/LLevels 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. Enzymes-therapeutic agent1.Streptokinase plasminogen streptokinase plasmin fibrin soluble product2.urokinase 55
  • 56. 3. Bacterial asparginase in leukemia4.α- chymotrypsin-extraction of lens5.Chymotrypsin,papain –anti inflammatory6.Collagenase – debridment of dermal ulcers7.Fibrinolysin – venous thrombosis, pulmonary embolism8.Hyaluronidase – rapid absorption of drugs injected subcutaneously 56
  • 57. 9.Lysosome –antibacterial (eye infections)10.Trypsin – clean wounds treatment of acute thrombophlebitis 57
  • 58.  Analytical use of enzymes as reagents as labels 58
  • 59. Alcohol Ethanoldehydrogenase Lactate Lactatedehydrogenase Glucose Glucose oxidase and peroxidase Uricase Uric acid Urease Urea Cholesterol oxidase and peroxidase cholesterol 59
  • 60. As labels In immunoassays for determining concentration of drugs, hormones Glucose -6- phosphate dehydrogenase Alkaline phosphatase Beta galactosidase peroxidase 60
  • 61. BIBLOGRAPHY T.B. OF BIOCHEMISTRY: SATYANARAYAN VASUDEVAN PANKAJA NAIK RANA SHINDE TEITZ HARRISON-INTERNAL MEDICINE THANK YOU 61