1 Enzymes in Clinical Biochemistry 2 You are supposed to know … •Enzymes – main features, properties •Coenzymes – structures, functions •Enzyme kinetics •Enzyme activity • 3 Isoenzymes – General Features •Genetically determined differences in primary structure •Catalyze the same reaction •May have different subcellular distribution (cytoplasm × mitochondria) •May have different tissue distribution •May be combined from more subunits (quarternary structure) •May differ in kinetic properties (KM) •Usually are determined by electrophoresis • 4 Q. 2 (p. 27) Explain the terms: proenzyme, isoenzyme, isoform 5 A. 2 •Proenzyme (zymogen) – inactive form of enzyme that becomes active after partial proteolysis example: pepsinogen ® pepsin •Isoenzyme – see previous page •Isoform – more general term, includes true isoenzymes and pseudoisoenzymes (posttranslational variations) • 6 Lactate dehydrogenase (LD) •Tetramer •Two different chains (H - heart, M - muscle) •Five isoenzymes: LD1 (H4), LD2 (H3M), LD3 (H2M2), LD4 (HM3), LD5 (M4) •Widely distributed in body •Total activity determination – nonspecific finding • LD1 + LD2 ….. marker of myocardial infarction (MI) •Today is LD assay considered out-of-date 7 Creatine kinase (CK) •Dimer •Two different chains (M – muscle, B – brain) •Three isoenzymes: MM (muscle), MB (heart), BB (brain) •Major isoenzyme in blood is MM (95 %) •MB form in blood: 0 – 6 % •BB in blood: traces (BB cannot pass across blood-brain barrier) •MB isoenzyme …. excellent marker of myocardial infarction 8 Enzymes in Blood Feature Plasmatic enzymes Secretory enzymes Intracellular enzymes Example Source organ Function in coag. factors liver blood amylase, lipase pancreas GIT AST, … various cells Enzyme activity in blood after source organ damage ? ? ? 9 Enzymes in Blood Feature Plasmatic enzymes Secretory enzymes Intracellular enzymes Example Source organ Function in coag. factors liver blood amylase, lipase pancreas GIT AST, … various cells Enzyme activity in blood after source organ damage 10 Q. 6 Why are low activities of cellular enzymes detected even in serum of healthy people? 11 A. 6 •Low activities of intracelular enzymes •in extracelular fluid (blood plasma, serum) •are the consequence •of physiological cell disintegration. 12 Main Tissue Distribution of Enzymes AST ALT LD CK GMT ALP ACP AMS LPS CHS liver, myocard liver not specific myocard, muscles liver biliary tract, bones prostate pancreas pancreas liver 13 Intracellular Location of Enzymes Intracellular Location Enzymes Cytoplasm Mitochondria Golgi complex, ER Lysosome Membrane LD, ALT, 30 % AST 70 % AST CHS, AMS ACP GMT, ALP 14 Consider the AST/ALT ratio •AST/ALT > 1 ……… severe liver damage • • • •AST/ALT < 1 ……… mild liver damage • • 15 Enzymes of Clinical Significance Enzyme Source of blood elevation ALT AST GMT ALP ACP CK AMS LPS CHS hepatopathy MI, hepatopathy hepatopathy (alcohol, drugs) biliary tract diseases, bone diseases prostatic cancer MI (CK-MB), muscle diseases pancreatitis pancreatitis hepatopathy (alcohol, drugs) – decreased 16 Catalytic concentration of some enzymes Enzyme Reference values (serum) ALT AST LD CK 0.1 - 0.9 mkat/l 0.1 - 0.7 mkat/l up to 7.5 mkat/l up to 4 mkat/l see also the lab manual 17 Q. 7 What enzymes might appear in blood a)In mild hepatocellular damage b) In serious hepatocellular damage 18 A. 7 a) Mild hepatocellular damage: enzymes from cytoplasm and/or membrane are released into ECF – ALT, GMT, ALP b) Severe hepatocellular damage: enzymes from mitochondria are released into ECF – AST 19 Q. 8 Write equations of reactions catalyzed by: ALT AST LD 20 ALT Reaction •alanine + 2-oxoglutarate D 21 ALT Reaction •alanine + 2-oxoglutarate D pyruvate + glutamate 22 AST reaction •aspartate + 2-oxoglutarate D 23 AST reaction •aspartate + 2-oxoglutarate D oxaloacetate + glutamate 24 LD reaction •lactate + NAD+ D 25 LD reaction •lactate + NAD+ D pyruvate + NADH + H+ 26 Q. 9 The levels of most blood enzymes are increased in newborns and infants. What enzyme persists elevated till puberty? 27 A. 9 •ALP – the bone isoenzyme activity persists till puberty 28 Biochemical Diagnostic of MI Enzyme / Protein Half-time (hrs) Myoglobin Troponine T cardial form CK-MB AST LD12 0,25 2 13 17 110