Factors influencing laboratory tests Enzyme assays in clinical chemistry Seminar No. 2 Q. 1 A. 1 • No physical activity • Fasting 10-12 hours • The day before blood collection - limited fats in diet • Ample drinking simple water (prevention of dehydration) • No smoking • No alcohol • No drugs if possible • No stress if possible Q. 2 A. 2 after the change of position: lying ® sitting intravasal fluid (= ECF) moves partially to interstitial space as the consequence of gravitation TH the ECF becomes more concentrated TH protein-bound analytes exhibit higher concentration Q. 3 A. 3 • Dehydration TH concentrated blood  elevated Prot + Hb • Hypoglycemia – most probably from not sufficient food intake before match • Insufficient supply of oxygen into muscles  anaerobic glycolysis  production and export of lactate  accumulation of lactate in ECF  acidosis A. 4 A. 4 The albumin concentration decreases Q. 5 A. 5 Hemolysis = disintegration of erythrocytes Causes: • Improper needle size • Rapid evacuation of syringe • Shaking blood • Moisture and/or detergents in test tube • Improper centrifugation Q. 9 A. 9 Confident interval = x ± 2 s = 40 ± 2 × 5 = 40  10 Confident interval = 30 - 50 Q. 10 A. 10 The same as the unit of quantity measured Q. 11 A. 11 The range: 55 – 36 = 19 g/l The mean: 45 g/l 95 % confident interval: x ± 2 s = 45  2 × 6.3 = 32.4 – 57.6 g/l Q. 13 A. 13 Data are ordered from minimal value to maximal value: 3.8, 3.9, 4.1, 4.5, 5.4, 5.5, 5.6, 6.1, 8.2 If odd number of data – the middle value is median If even number of data – the average of two middle values Q. 14 A. 14 Q. 15 calculator at www.cskb.cz Q. 16 A. 16 The parameter CV[i ]Intra-individual biological variation varies the most Q. 17 A. 17 7 ............... 100 % 1.33 .......... 19 % = CD Significant difference: 7 ± 19 % = 7  1.33 = 5.67 - 8.33 mmol/l The cholesterol concentration over 8.33 mmol/l or lower than 5.67 mmol/l means a clinical change. Q. 18 A. 18 2,8 mmol/l ............... 100 % x ..................................26 % x = CD = 0,73 the difference between two tests: 3,2 – 2,8 = 0,4 Conclusion: 0,4 < 0,73 (CD) TH no change in clinical status Enzymes of clinical significance You are supposed to know • Enzymes – main features, properties, coenzymes • Enzyme kinetics, enzyme activity Q. 19 A. 19 Indirect determination • catalytic concentration • μkat/l • product of enzyme reaction is determined • most enzymes (ALT, AST) Direct determination • mass concentration • μg/l • enzyme molecules are determined as antigens (immunochemical assays) • only few enzymes, e.g. tumour markers (PSA) Q. 21 A. 21 • Temperature • pH • The presence of all necessary activators • The absence of inhibitors • Concentration of substrate – 0. order kinetics, the great excess of substrate so that the enzyme is saturated – the reaction proceeds with maximal velocity How do you set constant pH 7.4 of enzyme solution at laboratory conditions? • Hydrogen phosphate buffer is the best choice • Corresponds to physiological conditions • Solutions of Na[2]HPO[4] and NaH[2]PO[4] of the same concentration (e.g. 0.1 mol/l) are mixed together • Their ratio is calculated from H.-H. equation: Enzymes in Blood Enzymes in Blood Q. 24 Why are low activities of cellular enzymes detected even in serum of healthy people? A. 24 Low activities of intracellular enzymes in extracellular fluid (blood serum) are the consequence of physiological cell disintegration. Tissue distribution of important enzymes Q. 26 A. 26 Isoenzymes • 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 (K[M]) • Usually are determined by electrophoresis • Elevated blood values – specific markers of tissue damages Intracellular location of enzymes Q. 28 What enzymes might appear in blood: • in mild hepatocellular damage b) in serious hepatocellular damage A. 28 Q. 29 A. 29 • AST/ALT > 1 ……… severe liver damage • AST/ALT < 1 ……… mild liver damage Q. 31+32 The levels of most blood enzymes are increased in newborns and infants. What enzyme persists elevated till puberty? A. 31+32 ALP – the bone isoenzyme activity persists till puberty Q. 34 Write equations of reactions catalyzed by: ALT AST LD ALT Reaction alanine + 2-oxoglutarate D pyruvate + glutamate AST reaction aspartate + 2-oxoglutarate D oxaloacetate + glutamate LD reaction lactate + NAD^+ D pyruvate + NADH + H^+ Q. 35 Lactate dehydrogenase (LD) • Tetramer, two different chains (H - heart, M - muscle) • Five isoenzymes: LD[1] (H[4]), LD[2] (H[3]M), LD[3] (H[2]M[2]), LD[4] (HM[3]), LD[5] (M[4]) • Widely distributed in body • Total activity determination – nonspecific finding • LD[1] + LD[2] ….. marker of myocardial infarction (MI) • Today is LD assay considered out-of-date 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 …. marker of myocardial infarction Q. 37 A. 37 Amylase (AMS) Q. 38 A. 38 • Osteoblasts – ALP • Osteoclasts – ACP • Prostate – ACP • Cardiomyocytes – CK-MB • Liver – ALT, CHS, GMT Enzymes of Clinical Significance