ENZYMES IN MEDICINE

1. ENZYMES IN MEDICINE

2. Diagnostic indicators – the activities of many enzymes are routinely determined in plasma ( rarely in tissue biopsies) for diagnostic purposes in diseases of the heart, liver, skeletal muscle, pancreas and other tissues - enzyme diagnostics • Therapeutic agents – several enzymes are used as drugs; new approach - enzymotherapy • Diagnostic tools – use as chemicals in clinical laboratory assays

3. ENZYMES IN CLINICAL DIAGNOSIS secretory - produced by tissues (namely liver), acting in plasma – prothrombin, plasminogen, cerruloplasmin, choline esterase; lipoprotein lipase Enzymes intracellular – function intracellulary, have no physiological use in plasma - membrane bound – ALP, GMT - cytosolic – ALT, AST, LD, MDH - mitochondrial – AST, GMDH - lysosomal - ACP - tissue specific – glucose-6-phosphatase – liver amylase – pancrease LD1 – heart

4. Healthy individuals - levels of intracellular enzymes fairly constant, low – the rate of enzyme release from damaged cells into plasma balanced by the rate of removal of enzyme protein from plasma  Physiological enzyme levels reference values of the enzyme activities (determined in clinical laboratory – each lab has its own reference values) • Elevated enzyme activity in the plasma – reflecttissue damageaccompanied by increased release of intracellular enzyme Skeletal muscle during exertion – physiologicallyelevated levels of muscle enzymes in plasma • Many diagnostically important enzymes = isoenzymes – pattern of isoenzymesin plasma (determined electroforetically) – a means ofidentifying the damaged tissue

5. ALTERATION OF ENZYME PLASMA LEVELS Increased values – increased cell membrane permeability anoxia, disturbances of energy metabolism  cytosolic enzymes – ALT, LD, CK - cell necrosis  membrane-bound enzymes – ALP, GMT mitochondrial enzymes – AST, GMDH - induction of the enzyme synthesis drugs – ALP, GMT Decreased values –inhibition of the activity drugs - inhibition of the synthesis cell damage, drugs

6. Examples of enzymes commonly assayed for diagnostic purposes Enzyme Location Cause of elevated plasma level Acid phosphatase - ACP Prostate Prostatic cancer Alkaline phosphatase – ALP Bone, liver Rickets, hypoparathyroidism, osteomalacia, obstructive jaundice, cancer of bone/liver Alanine aminotransferase – ALT Liver (muscle, Hepatitis, jaundice, circulatory heart, kidney) faillure with liver congestion Aspartate aminotransferase – AST Heart, muscle, Myocardial infarction, muscle red cells, liver damage, anemia, hepatitis, circulatory faillure with liver congestion Amylase - AM Pancres Acute pancreatitis, peptic ulcer -Glutamyl transferase – GMT Liver, kidney, Hepatitis, alcoholic liver pancreas damage, cholestasis

7. Examples of isoenzymes commonly assayed for diagnostic purposes Enzyme Location Cause of elevated plasma level Creatine kinase – CK CK-MB Heart Myocardial infarction CK-MM Skeletal muscle Muscular dystrophy Lactate dehydrogenase – LD LD1> LD2Heart, kidney, Myocardial infarction, kidney blood cells disease, megaloblastic anemia, leukemia LD2, LD3 Leukemia LD5 Liver, muscle Liver disease, muscle damage

8. ENZYMES IN THERAPY • Substitution of missing production of digestive enzymes – digestive enzymes – pepsin trypsin… • Removal of deposits of death tissue or fibrin (e.g. in lungs, eyes), treatment of skin defects – proteinases, nucleases, collagenase • Acceleration of fibrinolysis in lungs embolization(activation of plasmin and plasminogen) – streptokinase, urokinase

9. ENZYMOTHERAPY Orally administered enzymes – treatment of a variety disorders - digestive, gastrointestinal, pancreatic - inflammatory diseases, edema - immune and autoimmune diseases(arthritis, multiple sclerosis) - viral diseases (herpes, AIDS) - cancer Mixtures of enzymes of plant and/or animal origin - proteinases, amylase, lipase - administered as acidoresistent tablets • Pancreatin – trypsin, chymotrypsin, lipase, amylase • Wobenzym – pancreatic and plant proteolytic enzymes – trypsin, chymotrypsin, papain (Carica papaya), bromelain (ananas) = combination of enzymes with different specificity, pH optimum, stability, interaction with inhibitors and antiproteinases  multiple action

10. Mechanism of resorption(transport of large macromoleculesacross the intestinal barrier) – paracellular transport, receptor mediated endocytosis and transcytosis • Mechanism of action – interaction with plasma antiproteinases – 1-antitrypsin, 2-macroglobulin complexes direct proteolytic action, degradation of adhesive molecules, secretion of cytokins (tranforming growth factor TGF-), modulation of receptor function not fully clarified ?

11. ENZYMES - USE IN LABORATORY ASSAYS Enzymes isolated from different sources - used for determination of various substances in the blood, plasma/serum and urine enzyme methods much more specific than chemical methods, the presence of relative substances with similar chemical properties does not hinder Components of commercial kits or diagnostic strips - determination of glucose - glucose oxidase, peroxidase cholesterol - cholesterol esterase, cholesterol oxidase peroxidase, urea – urease, ……. in blood, plasma, serum - proof of glucose (glucose oxidase), …….. in blood or urine (strips) Markes in the immunochemical analysis - ELISA (=enzyme-linked immunoadsorbent assay) – peroxidase, alkaline phosphatase

13. NUCLEOTIDESStructure, Function

14. NUCLEOTIDE STRUCTURE Nucleotides nitrogenous base + pentose + phosphate group(s) purine ribose 1 - 3 pyrimidine deoxyribose other (nicotinamide) Nucleosides

15. NH2 | O || C C N N N C N C H CH CH H C C C C N H N H H2N N N NH2 | O || O || CH3 C C C N H C N H C H N C H C C H C C H C C H O O N O N N H H H THE NITROGENOUS BASES Purine bases adenine guanine Pyrimidine bases cytosine thymine uracil

16. O HOCH2 H H H H OH H NUCLEOSIDE • A sugar - base combination. Base N -N-glycosidic linkage Sugar In this case deoxyribose

17. O O O NH2 | H H H H H H H H H H H H CH3 C C C OH OH OH H N N H H C C N C H C C C C H H C H C O O O N N N thymine uracil O O H HOCH2 HOCH2 OH uridine deoxythymidine cytosine HOCH2 OH cytidine

18. NH2 | OH | O C C C N N N N N C C N H C CH CH CH H H C C C C C C N N N H2N N N N O O O H H H H H H H H H H H H OH OH OH H guanine adenine HOCH2 HOCH2 OH guanosine deoxyadenosine hypoxanthine ! HOCH2 inosine OH

19. NH2 | C N N C CH H C C N O || N O -O-P-O-CH2 | O-- H H H H OH H NUCLEOTIDES 5’-OH on the sugar of a nucleoside isconverted into a phosphate ester. deoxyadenosine monophosphate (dAMP) Each is named based on sugar and base name and then the number of phosphates is indicated.

20. adenine phosphate chain NH 2 O O O N N - O P O P O P O N - - - N O O O CH 2 O OH OH ribose ATP - adenosine triphosphate AMP ADP ATP

21. NUCLEOTIDE FUNCTION • Precursorsof DNA, RNA - NTPs • Energy transport - ATP • Allosteric effectors of enzymes – ATP, ADP, AMP • Covalent modification of enzymes – ATP • Intracellular mediators (= second messengers) – cAMP, cGMP • Coenzymes – NAD+, NADP+, FAD, CoA-SH • Activated precursors of polysaccharaides, glycoproteins, proteoglycans, phospholipids, glycolipids – UDPG, UDPGA, UDPGal…, CDP-choline, CDP-diacylglycerol… • Active groups (group transport) – SAM, PAPS

22. NAD+ reactive site nicotinamide adenine ribose

23. reactive site riboflavin adenine ribose FAD

24. phosphorylated ADP pantothenate unit NH 2 O O O CH3 H O N N C-CH2-CH2-N-C-C-C-CH2 P O P O O N - - N O O CH HO CH3 H H-N 2 O CH2-CH2 Sulfhydryl group S OH O H - P O O - O Coenzyme A

25. CH CH OH OH 2 2 O O OH H H H H H H H OH OH O H OH OH OH H H -D-glucose -D-glucose CH OH 2 H O H H H OH OH OH OH H

26. 2 H O H C O C O H C O C O -O- O P CH2 - O Non-polar tail

27. cAMP – cyclic adenosine monophosphate cAMP

28. NH2 | C N N C CH H C C N N O - H H H H H CH2 O O O P O O- • cAMP – cyclic adenosine monophosphate • intracellular mediator, second messenger of hormonal signal tranduction • via adenylate cyclase cascade • - mechanism of action: allosteric effector

29. ~ P O CH2-O-C O C-O-CH CH2-O - P