Introduction Enzymes are usually proteins that act as catalysts, compounds that increase the rate of chemical reactions. They bind specifically to a substrate, forming a complex. This complex lowers the activation energy in the reaction: –without the enzyme becoming consumed or –without changing the equilibrium of the reaction. A product is produced at the end of the reaction Mohammed Laqqan

M. Zaharna Clin. Chem. Lab General Properties of Enzymes Like all proteins 1°, 2°, 3°, and 4° structures Active site → cavity where substrate interacts –Often water-free site –Reacts with charge moieties Allosteric site –Another site on enzyme where co-factors or regulatory molecules interact Mohammed Laqqan

M. Zaharna Clin. Chem. Lab Isoenzyme Isoenzymes: are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. Now called “isoform” of an enzyme They have similar catalytic activity, but are different biochemically or immunologically and can be demonstrated by electrophoretic mobility, differences in absorption properties or by their reaction with a specific antibody Mohammed Laqqan

M. Zaharna Clin. Chem. Lab Cofactors Non-protein molecules required for enzyme activation  Inorganic Activators Chloride or magnesium ions, etc.  Organic coenzymes e.g. Nicotinamide adenine dinucleotide (NAD) Mohammed Laqqan

M. Zaharna Clin. Chem. Lab Enzyme classification Plasma vs. non-plasma specific enzymes a.Plasma specific enzymes have a very definite/specific function in the plasma 1) Plasma is normal site of action 2) Concentration in plasma is greater than in most tissues 3) Often are liver synthesized 4) Examples: plasmin, thrombin Mohammed Laqqan

M. Zaharna Clin. Chem. Lab B.Non-plasma specific enzymes have no known physiological function in the plasma 1) Some are secreted into the plasma 2) A number of enzymes associated with cell metabolism normally found in the plasma only in low concentrations. Source of non-plasma enzymes –From cells during the normal process of breakdown and replacement. –higher concentration following injury or death of tissue cells. –altered membrane permeability that may occur with inflammation An increased plasma concentration of these enzymes is associated with cell disruption or death Mohammed Laqqan

M. Zaharna Clin. Chem. Lab Classes of Enzymes International Union of Biochemistry (IUB) 1 = Oxidoreductases (Examples: LDH, G6PD) Involved in oxidation - reduction reactions 2 = Transferases (Examples: AST, ALT) Transfer functional groups 3 = Hydrolases (Examples: acid phosphatase, lipase) Transfer groups to -OH 4 = Lyases (Examples: aldolase, decarboxylases) Add across a double bond 5 = Isomerases (Example: glucose phosphate isomerase) Involved in molecular rearrangements 6 = Ligases Complicated reactions with ATP cleavage Mohammed Laqqan

M. Zaharna Clin. Chem. Lab Factors Affecting Enzyme Levels in Blood Entry of enzymes into the blood –Leakage from cells –Altered production of enzymes e.g. increased osteoblastic activity results in increase in enzymes in bone disease Clearance of enzymes –Half life vary from few hours to several days Mohammed Laqqan

Enzyme measurement Enzymes are not directly measured Enzymes are commonly measured in terms of their catalytic activity We don’t measure the molecule … We measure how much “work” it performs (catalytic activity) The rate at which it catalyzes the conversion of substrate to product The enzymatic activity is a reflection of its concentration Activity is proportional to concentration Mohammed Laqqan

Enzyme activity can be tested by measuring Increase of product Decrease of substrate Decrease of co-enzyme Increase of altered co-enzyme If substrate and co-enzyme are in excess concentration, the reaction rate is controlled by the enzyme activity. Photometric measurement of activity Mohammed Laqqan

M. Zaharna Clin. Chem. Lab NADH ( a common co-enzyme ), the reduced form, absorbs light at 340 NM NAD does not absorb light at 340 nm –Increased ( or decreased ) NADH concentration in a solution will cause the Absorbance (Abs) to change. Measuring enzyme activity Mohammed Laqqan

Measurement of Enzymatic Activity One of two general methods may be used to measure the extent of an enzymatic reaction 1.Fixed time –Measure at specified time (e.g. 0 and 60 seconds) –The reactants are combined the reaction proceed for a designated time –The reaction is stopped usually by inactivating the enzyme with a weak acid –And a measurement is made of the amount of reaction that has occurred Mohammed Laqqan

Measurement of Enzymatic Activity 2.Continuous monitoring or kinetic assay –Are recorded by the spectrophotometer –Measurements of absorbance change are made during the reaction either at: Measure at specific time intervals (usually every 0, 30, 60, 90, 120 seconds) Mohammed Laqqan

M. Zaharna Clin. Chem. Lab Measurement Units Reported as “activity” not concentration –IU = amount of enzyme that will convert 1 μmol of substrate per minute in specified conditions –Usually reported in IU per liter (IU / L) SI unit = Katal = mol/sec moles of substrate converted per second –enzyme reported as katals per liter (kat / L) –1 IU = 17nkat Mohammed Laqqan

M. Zaharna Clin. Chem. Lab

Creatine Kinase (CK) Action of this enzyme is associated with the regeneration and storage of high energy phosphate (ATP). The enzyme catalyzes the conversion of Creatine to Creatine Phosphate The enzyme also catalyzes the reversible reaction Found in skeletal muscle, cardiac muscle, and brain  CK is especially useful to diagnose AMIs Skeletal muscle diseases ( Muscular Dystrophy ) Mohammed Laqqan

M. Zaharna Clin. Chem. Lab CK has 3 isoenzymes Each isoenzyme is composed of two different polypeptide chains (M & B) –CK - BB (CK1)Brain –CK - MB (CK2)Cardiac –CK - MM (CK3) Muscle Skeletal muscle CK is 99% CK-MM Cardiac muscle CK is 80% CK-MM and 20% CK-MB BB migrates fastest to anode then MB & MM MM is highest in serum in healthy patients MB trace to <6% total, BB 0-trace Mohammed Laqqan

M. Zaharna Clin. Chem. Lab Because of CK – MB’s association with cardiac tissue, increased CK – MB ( > 6% of the total CK activity ) is a strong indication of AMI Post AMI CK-MB –CK-MB increases 4 – 8 hours post AMI –Peaks at hours post AMI –Returns to normal hours later

Specimen: Serum, heparin plasma or EDTA plasma. CK assays are often coupled assays CK In the example below, the rate at which NADPH is produced is a function of CK activity in the first reaction. Hexokinase and G6PD are auxiliary enzymes Reverse reaction most commonly performed in clinical laboratory methods

Interference RBCs lack CK, but hemolyzed RBCs release Adenylate Kinase (AK) into the plasma, AK reacts with ADP to produce ATP which is then available to participate in the reaction causing falsely increased CK activity The interference can occur with hemolysis greater than 200 mg/dl of hemoglobin CK should be stored in the dark place because CK is inactivated by daylight Mohammed Laqqan

Lactate Dehydrogenase (LDH) Catalyzes interconversion of lactic and pyruvic acids NAD as coenzyme High activities in heart, liver, muscle, kidney, and RBC Lesser amounts: Lung, smooth muscle and brain Elevated with diseases of the above (Liver disease, AMI & Hemolytic diseases) Mohammed Laqqan

M. Zaharna Clin. Chem. Lab LDH Isoenzymes Because increased total LDH is relatively non-specific, LDH isoenzymes can be useful 5 isoenzymes composed of a cardiac (H) and muscle ( M ) component LD - 1 ( HHHH )Cardiac, RBCs LD - 2 ( HHHM )Cardiac, RBCs LD - 3 ( HHMM )Lung, spleen, pancreas LD - 4 ( HMMM )Hepatic and skeletal LD - 5 ( MMMM )Hepatic and skeletal Mohammed Laqqan

M. Zaharna Clin. Chem. Lab In healthy patients –LD-2 is in highest quantity then LD-1, LD-3, LD-4 and LD-5 Heart problems 2-10 x (Upper Limit of Normal) ULN in acute MI –If problem is not MI, both LD1 and LD2 rise, with LD2 being greater than LD1 –If problem is MI, LD1 is greater than LD2. This is known as a flipped pattern The highest levels of LD are seen in pernicious anemia and hemolytic disorders LD-3 with pulmonary involvement LD-5 predominates with liver & muscle damage Mohammed Laqqan

M. Zaharna Clin. Chem. Lab Assay for Enzyme activity The reaction can proceed in either a forward or reverse direction Lactate + NAD + Pyruvate + NADH + H+ The optimal pH: –for the forward reaction is 8.3 – 8.9 –For the reverse reaction 7.1 – 7.4 LD Mohammed Laqqan

Specimen: Serum, heparin plasma or EDTA plasma Measurements & Sources of error RBCs have 100+ times the amount of LD Hemolysis ruins sample for testing LD unstable during storage – test within 48 hours LD-5 most labile store at 25oC not 4oC In AMI, LDH levels begin to rise within 12h to 24h. Reach peak within 48h to 72h. Remain elevated for 10 days. Mohammed Laqqan

Clinical Significance The measurement of the serum concentration of LD has proven to be useful in the diagnosis of myocardial infarction. The LD enzyme activity in serum does not rise as much as CK or AST after myocardial infarction, but it does remain elevated for a much longer period of time. This is quite important when the patient does not see a physician for 3 or 4 days following an infarct. In hepatocellular disease, the serum activity of LD rises, but the measurement of this enzyme is much less useful than that of AST or ALT because the test is less sensitive. Mohammed Laqqan