1 Methods for the determination in serum and urine Dr. Essam H. Aljiffri

2 Types of enzymes - The International Union of Biochemistry (IUB) in 1964 has suggested that enzymes are arranged in groups according to their functional catalytic activities.

3 ExamplesTypeGroup No. i) lactate dehydogenase ii) glucose oxidase iii) tyrosinase Oxidoreductase i) anaerobic dehydrogenase ii) aerobic oxidase iii) aerobic dehydrogenase 1 Alanine amino transfesrase (ALT) Aspartate amino transferase (AST) Creatine Kinase (CK) Transferases 2 Lipase Cholinesterase Hydolases3 Pyruvate decarboxylaseLyases4 Triphosphate isomeraseIsomerase5 Acetyl CoA synthetaseLigases6

4 Types of enzymes Enzyme Commision (EC) International enzyme numbering system 4 figures separated by dotes e.g Explanation Group no: group acted on: subgroup acted on: the individual enzyme

5 General consideration in enzyme assays in the clinical laboratory In enzyme assays the activity of the enzyme and not the enzyme concentration is measured Clinically important enzymes, i.e. Enzymes of diagnostic value, are those whose activities are reflective of the condition of a certain function in the body or an organ and the determination of their activity will assist in the diagnosis or the management of diseases in the patient.

6 General consideration in enzyme assays in the clinical laboratory Serum is the preferred specimen type for enzyme assays Avoid haemolysis; –RBCs contain high concentration of some enzymes such as LDH, transferases and G6PD –Haemoglobin may interfere with some assays especially those which include color production

7 General consideration in enzyme assays in the clinical laboratory Never shake the serum or the reaction mixture vigorously as this may denature the enzyme, mix the serum and reagent gently. Avoid using NaF/ K oxalate tube as NaF is a enzyme inhibitor Check if the patient is taking drugs that effect enzyme activity

8 General consideration in enzyme assays in the clinical laboratory Avoid prolonged application of tourniquet as it effect some enzymes such as LDH Note the physical condition of the patient (e.g. exercise or long walk may effect CK activity) Some enzymes are sex related i.e. higher or present in one sex type (e.g. prostatic ACP) Some enzymes are age related (e.g. ALP)

9 General consideration in enzyme assays in the clinical laboratory Some enzymes catalyze both direction of the reaction while others catalyze one direction only Many enzymes exit as isoenzymes (different forms in different organs), such enzymes have a good diagnostic value, the isoenzyme related to the organ should be analyzed together with the total enzyme activity; e.g. CK & CK-MB

10 General consideration in enzyme assays in the clinical laboratory Because the enzyme activity is measured, many conditions affect such reactions and include; Substrate type and concentration Product type and concentration Amount of enzyme present Buffer type and pH Activators and Coenzymes Temperature of the reaction Specificity of the enzyme to substrate Presence of inhibitors Direction of reaction (forward or reverse direction)

11 General consideration in enzyme assays in the clinical laboratory Due to the effects of various conditions on enzyme activity, each lab must determine its normal range for the enzymes in question and not rely on published data. The effect of the various conditions on enzyme activity make enzyme assays less precise than other smaller analytes so a coefficient of variation (cv) of up to 10% is acceptable.

12 General consideration in enzyme assays in the clinical laboratory Kinetic enzyme (rate of reaction ) assays are to be used instead of end point (two-point) assays because they provided better accuracy.

13 Some enzymes of diagnostic importance. 1) Creatine kinase (creatine phosphokinase) (CK) (EC: ) Activity: CK Catalyzes the reaction ATP + creatine ADP + creatine phosphate CK isoenzymes and clinical importance: Isoenzymetissue or organ% present CK-BB (CK-1)brain98 % CK-MB (CK-2)heart muscle20 % CK-MM (CK-3)muscle96 %

14 1) Creatine kinase (creatine phosphokinase) (CK) (EC: ) Method of analysis Continuous monitoring (kinetic) method (Scandinavian Committee on Enzyme, 1979) (Association of Clinical Biochemists, UK, 1980) Specimen Fasting serum (preferred) 50µmol/L N-acetylcysteine is added to serum immediately after separation (activator for CK) Storage at 4 0 C for up to 2 days Storage at – 20 0 C for up to 30 days Avoid haemolysis Never repeat thaw-freeze

15 1) Creatine kinase (creatine phosphokinase) (CK) (EC: ) Principle This is a rate kinetic method based on the reverse reaction of the enzyme and coupled to other enzyme reactions. CK 1.Creatine phosphate + ADP ATP + creatine hexokinase 2.ATP + glucose ADP + Glucose-6-phosphate G-6-P Dehydrogenase 3.G-6-P + NADP + 6-phosphogluconate + NADPH + H +

16 1) Creatine kinase (creatine phosphokinase) (CK) (EC: ) Principle The rate of formation of ATP is monitored using the increase in absorbance at 340 nm of NADPH formed by the coupled reactions.

17 Some enzymes of diagnostic importance. 2) Lactate dehydrogenase (LD) (EC ) This is a universal enzyme occurring in almost all tissues of the body with higher concentration in cardiac muscle, skeletal muscle, liver, kidney & rbc Activity: Catalyzes the reaction: LD Lactate + NAD + Pyruvate + NADH + H +

18 2) Lactate dehydrogenase (LD) (EC ) Iso-enzymes of LD and clinical importance Clinical significantSite % in circulationSubunitLD iso-enzyme MI Haemolytic anaemia Haemolyzed sample Acute renal failure Heart RBC Renal cortex % % HHHH HHHM LD1 LD2 Pulmonary pneumonia Lymphocytosis Acute pancreatitis Lung Lymphocyte Pancrease %HHMMLD3 Hepatic carcinoma Hepatic necrosis Skeletal muscle injury Liver Skeletal muscle 8-16 % HMMM MMMM LD4 LD5

19 2) Lactate dehydrogenase (LD) (EC ) In normal adult circulation LD2 > LD1, MI LD1 > LD2 and the LD1 > LD2 ratio is >1. This called “flipped LD” pattern. Method of analysis Continuous monitoring (kinetic) method (Scandinavian Committee on Enzymes, 1974) Specimen Fasting serum (preferred) Avoid haemolysis Never repeat thaw-freeze

20 2) Lactate dehydrogenase (LD) (EC ) Principle This is a kinetic method based on the reverse reaction of the enzyme LD Pyruvae + NADH + H + Lactate + NAD + The rate of reaction is monitored as pyruvate is converted to lactate by observing the decrease in absorbance at 340 nm as NADH is oxidized to NAD + This is faster than the forward reaction Less expensive than the forward reaction It requires less concentration of reagents