Enzyme Activity

1. Enzyme Activity MALIK ALQUB MD. PHD.

2. Information from enzymes measurements in serum • Presence of disease • Organs involved • Aetiology /nature of disease: differential diagnosis • Extent of disease-more damaged cells-more leaked enzymes in blood • Time course of disease

3. Enzymes routinely measured

4. ALANINE TRANSAMINASE (ALT) AND ASPARTATE TRANSAMINASE( AST) - Oxoglutarate + L-alanine - Oxoglutarate + L-aspartate Alanineaminotransferase (ALT) Aspartateaminotransferase (AST) L- glutamate + oxaloacetate L - glutamate + pyruvate • Alaninetransaminase (ALT) and Aspartatetransaminase (AST) enzymes are the most abundantly present in the liver and is elevated in blood as a result of leakage from damaged cells • Measurement of these transaminases is useful for the diagnosis of liver diseases • In viral hepatitis the enzyme levels are increased 20-50 times above the upper limit of the normal range • Alaninetransaminase (ALT) increase is specific for liver damage involving hepatocellular damage • Aspartatetransaminase (AST) is moderately increased in Muscular dystrophy and acute myocardial infarction

5. ALKALINE PHOSPHATASE (ALP) • Is a group of enzymes that have maximal activity at a high pH 9.0-10.5 • Widely distributed throughout the body • High levels are seen is liver, bone, placenta and intestine and useful to assess hepatobiliary and bone diseases • In hepatobiliaryobstruction,hepatocytes lining the biliary ducts induces the ALP synthesis. • High levels of ALP is indicative of extrahepatic obstruction rather than intrahepatic obstruction • In bones, the enzyme is derived from osteoblasts. Hence increased in bone diseases like rickets, osteomalacia, neoplastic diseases with bone metastates and healing fractures

6. Isoenzymes • catalysesame reactions but are formed from structurally different polypeptides. • They perform the same catalytic function. • Different isoenzymes may arise from different tissues and their specific detection may give clues to the site of pathology. • Various isoenzymes of an enzyme can differ in three major ways: • - enzymatic properties • - physical properties (e.g heat stability) • - biochemical properties such as amino acid composition and immunological reactivities.

7. CREATINE KINASE (CK) Creatine + ATP phosphocreatine + ADP (Phosphocreatine – serves as energy reserve during muscle contraction) • Creatinekinase is a dimer made of 2 monomers occurs in the tissues • Skeletal muscle contains M subunit, Brain contains B subunits • Three different isoenzymes are formed

9. LACTATE DEHYDROGENASE (LDH) Pyruvate Lactate (anaerobic glycolysis) • LDH is elevated in myocardial infarction, blood disorders • It is a tetrameric protein and made of two types of subunits namely H = Heart, M = skeletal muscle • It exists as 5 different isoenzymes with various combinations of H and M subunits

12. Methods for Enzyme measurement • Fixed time methods • the reactants are combined, • the reaction proceeds for a designated time, • the reaction is stopped (usually by inactivating the enzyme with a weak acid), • a measurement is made of the amount of reaction that has occurred. • The reaction is assumed to be linear over the reaction time; the larger the reaction, the more enzyme is present. • Possible problems with extremely high enzyme levels

13. Methods for Enzyme measurement • Continuous-monitoring methods • multiple measurements, usually of absorbance change, are made during the reaction, • either at specific time intervals (usually every 30 or 60 seconds) • or continuously by a continuous-recording spectrophotometer. • These assays are advantageous over fixed-time methods because the linearity of the reaction may be more adequately verified.

14. Methods for Enzyme measurement • Continuous-monitoring methods (Kinetic assay) • If absorbance is measured at intervals, several data points are necessary to increase the accuracy of linearity assessment. • Continuous measurements are preferred because any deviation from linearity is readily observable.

15. Calculation of Enzyme Activity • Common unit is International unit or IU/L • Conditions to be maintained • pH • Temperature • Substrate

16. Reporting Enzyme Activity • Enzyme concentration is best measured by its activity or its rate of activity by observing • Substrate depletion • Product production • Increase/decrease in cofactor/coenzyme

17. 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

18. Measurement of Enzyme Mass • Immunoassay methodologies that quantify enzyme concentration by mass are also available and are routinely used for quantification of some enzymes. • Immunoassays may overestimate active enzyme as a result of: • possible cross-reactivity with inactive enzymes, • inactive isoenzymes, • or partially digested enzyme.

19. Measurement of Enzyme Mass • The relationship between enzyme activity and enzyme quantity is generally linear but should be determined for each enzyme. • Enzymes may also be determined and quantified by electrophoresis techniques which provide resolution of isoenzymes.

20. LDH • Colorimetric • Spectrophotometric method makes use of NADH to NAD coenzyme change in reaction

21. LDH

22. AST • Coupled reaction • Measured at 340 nm • L-aspartate + alpha- oxoglutarateoxaloacetate + glutamate • Oxaloacetate + NADH + H+malate +NAD+ AST

23. AST