ENZYMES IN MEDICINE • 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 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 • 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 – reflect tissue damage accompanied by increased release of intracellular enzyme Skeletal muscle during exertion – physiologically elevated levels of muscle enzymes in plasma • Many diagnostically important enzymes = isoenzymes – pattern of isoenzymes in plasma (determined electroforetically) – a means of identifying the damaged tissue 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 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, heart, kidney) Hepatitis, jaundice, circulatory faillure with liver congestion Aspartate aminotransferase – AST Heart, muscle, red cells, liver Myocardial infarction, muscle damage, anemia, hepatitis, circulatory faillure with liver congestion Amylase - AM Pancres Acute pancreatitis, peptic ulcer -Glutamyl transferase – GMT Liver, kidney, pancreas Hepatitis, alcoholic liver damage, cholestasis Examples of isoenzymes commonly assayed for diagnostic purposes Enzyme Creatine kinase – CK CK-MB CK-MM Lactate dehydrogenase – LD LD1 > LD2 LD2, LD3 LD5 Location Cause of elevated plasma level Heart Skeletal muscle Myocardial infarction Muscular dystrophy Heart, kidney, blood cells Myocardial infarction, kidney disease, megaloblastic anemia, leukemia Leukemia Liver disease, muscle damage Liver, muscle 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 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 • Mechanism of resorption (transport of large macromolecules across 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 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 NUCLEOTIDES Structure, Function NUCLEOTIDE STRUCTURE Nucleotides nitrogenous base + pentose + purine ribose pyrimidine deoxyribose other (nicotinamide) Nucleosides phosphate group(s) 1-3 THE NITROGENOUS BASES Purine bases NH2 | C N O || C adenine N C HN CH HC C N C CH C N H N guanine H2N C N H N Pyrimidine bases O || C NH2 | C O N CH C CH N H cytosine O O || C CH3 HN C C CH N H thymine HN CH C CH O N H uracil NUCLEOSIDE • A sugar - base combination. Base N O HOCH2 H Sugar In this case deoxyribose H H OH H H -N-glycosidic linkage thymine O HN C C H HN CH N O OH H H H H O H C N CH C CH H H OH OH N O H H OH OH CH N NH2 | C deoxythymidine C O HOCH2 HOCH2 H C O HOCH2 H O CH3 C O uracil H cytidine cytosine H uridine NH2 | C N adenine O N C C HN CH HC C H2N C C N O HOCH2 H C H OH H H H deoxyadenosine OH | C N C N H H OH OH hypoxanthine CH HC C N ! O HOCH2 H N H H OH OH H inosine N O HOCH2 H N CH N N guanine H guanosine NUCLEOTIDES 5’-OH on the sugar of a nucleoside is converted into a phosphate ester. NH2 | C deoxyadenosine monophosphate (dAMP) N Each is named based on sugar and base name and then the number of phosphates is indicated. C N CH HC C O || N -O-P-O-CH | O- - O 2 H N H H OH H H ATP - adenosine triphosphate adenine phosphate chain O - O P O- O O P O- NH2 O O N P O O- CH2 N O OH AMP ADP ATP ribose OH N N NUCLEOTIDE FUNCTION • Precursors of 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 NAD+ reactive site O C OO P O CH2 O O OH P O O - ribose CH2 nicotinamide N+ NH2 OH N O NH2 N O OH OH N N adenine FAD O H3C N H3C reactive site NH N N H C H H C OH H C OH H C OH H C H O riboflavin NH2 N O O P O O - ribose CH2 N O OH OH N N adenine Coenzyme A phosphorylated ADP pantothenate unit NH2 O O H CH3 C-CH2-CH2-N-C-C-C-CH2 H HO CH3 H-N O O N P O P O O- O- CH 2 O N CH2-CH2 S H N Sulfhydryl group O O P OO- OH N CH2 OH CH2 OH H H OH O H H OH H OH -D-glucose H O H OH O OH H H H CH2 OH OH -D-glucose H O H OH H H OH OH H OH H O H C O C O H C O C O 2 O P -O- CH2 - O Non-polar tail cAMP – cyclic adenosine monophosphate cAMP NH2 | C N C N CH HC C N O CH2 - O O P H H O N H H OH O- cAMP – cyclic adenosine monophosphate -intracellular mediator, second messenger of hormonal signal tranduction via adenylate cyclase cascade - mechanism of action: allosteric effector O O O O CH2-O-C C-O-CH SCoA SCoA HO HO HO HO CH2-O - P ~ P HO HO