ENZYME MARKERS OF TOXICITY Why do we need enzyme markers In vivo monitoring Serial sampling Early detection of metabolic changes Detection of organ specific effects Establishment of NO EFFECT level Determination of toxic mechanism Is required by regulatory agencies Why enzyme markers ENZYMES highly specialized proteins that facilitate biochemical reactions that otherwise would proceed at a much lower rate Are usually confined to a specific cellular membrane cytosol mitochondria and or organ location Sensitive to membrane integrity changes in metabolism excretion inactivation The magnitude of response often correlates with the severity of damage Where do enzyme markers fit BIOMARKERS molecular biochemical or cellular alterations that are measurable in biological samples tissues cells or fluids Where do enzyme markers fit Markers of internal dose blood urine levels fat concentrations exhaled breath metabolites in urine Markers of biologically active dose DNA protein adducts both in cells and in body fluids Markers of early biological effect genetic alterations in target and reporter genes nuclear aberrations altered enzymatic activities Markers of altered structure function enzyme markers proliferation cell differentiation differential expression of genes cellular tissue changes From Kensler T W SOT 1992 AM 2 Laboratory evaluation of organspecific toxicity IMPORTANT ISSUES TO REMEMBER Cell types differ in susceptibility to toxic agents One organ many cell types Cellular injury vs organ function impairment Oxygen concentration gradients Metabolizing enzymes e g Cyt P450 concentration gradients K C P C I C LiverCancer com EM Atlas of Mammalian Tissues in the Internet Universit t Mainz Germany Rashmil Saxena LIVER TOXICITY Central Vein Portal triad Localization of damage Centrilobular zone 3 Most hepatotoxicants CCl4 APAP Less oxygen high P450 concentration 3 2 1 Periportal zone 1 Phosphorus aflatoxin allyl alcohol High oxygen highest dose at the site Midzonal zone 2 beryllium Massive necrosis iproniazid MAO inhibitors LIVER TOXICITY Cholestatic Injury Cytotoxic Injury Disturbances of hepatic function clearance Evaluation of liver toxicity in vivo Serum enzyme tests Hepatic excretory tests Alterations in chemical constituents of the liver Histological analysis of liver injury LIVER TOXICITY Zimmerman classification of serum enzymes to monitor liver injury 1 Cholestatic Injury ALP 5 NT GGT 2 Cytotoxic Injury A Somewhat non specific enzymes AST LDH B Enzymes that are found mainly in liver ALT C Enzymes that are found only in liver OCT SDH 3 Enzymes relatively insensitive to hepatic injury e g creatine phosphokinase 4 Enzymes that demonstrate reduced serum activity in liver disease cholinesterase from ANIT BSP Hayes A W Principles and Methods of Toxicology 4th Edition Taylor Francis 2000 naphtylisothiocyanate sulfobromophthalein LIVER TOXICITY I Markers of cholestatic injury A Enzymatic Alkaline Phosphatase AP ALP membrane Hydrolyzes phosphate esters e g ATP at pH 7 0 Normal circulating levels contributed by intestine bone rat intestine bone liver placenta humans Many isoforms humans 3 rats 2 Affected by diet age pregnancy and other factors Not a very reliable marker in rat studies diet strain LIVER TOXICITY I Markers of cholestatic injury A Enzymatic 5 Nucleotidase 5 NT membrane Hydrolyzes nucleoside 5 monophosphates Normally present in kidney intestinal mucosa etc Many isoforms humans 3 rats 2 Is made soluble from membranes by a detergent or bile acids released during cholestasis LIVER TOXICITY I Markers of cholestatic injury A Enzymatic Glutamyl Transpeptidase GGT membrane Participates in the transfer of amino acids across the cellular membrane and in glutathione metabolism High concentrations are found in the liver and kidney GGT is measured in combination with other tests ALP is increased in hepatobiliary disease and bone disease GGT is elevated in hepatobiliary disease but not in bone disease LIVER TOXICITY I Markers of cholestatic injury B Non enzymatic markers Total Serum Bile Acids Synthesized in the liver important for digestion and absorption of lipids and lipid soluble vitamins Relatively sensitive early marker of cholestasis Could be affected by altered enterohepatic circulation and impaired hepatic function LIVER TOXICITY I Markers of cholestatic injury B Non enzymatic markers Plasma Bilirubin Direct and Total Heme biliverdin bilirubin conjugated bilirubin Cholestasis direct conjugated is 50 total bilirubin Hemolysis direct conjugated is 50 total bilirubin LIVER TOXICITY II Markers of hepatocellular injury A Somewhat non specific enzymes Aspartate aminotransferase AST cytosol mitochondria L aspartate 2 oxoglutarate oxaloacetate glutamate a k a serum glutamate oxaloacetate transaminase SGOT Normally present in a wide variety of tissues skeletal muscle heart muscle liver etc AST in serum is stable RT 3 days frozen 30 days Red blood cells are loaded with AST be careful hemolysis LIVER TOXICITY II Markers of hepatocellular injury A Somewhat non specific enzymes Lactate Dehydrogenase LDH cytosol pyruvate L lactate Normally present in a wide variety of tissues Five isoenzymes isoenzyme profile may help identify specific tissue origin LDH 5 liver LDH 1 2 kidney LIVER TOXICITY II Markers of hepatocellular injury B Enzymes found mainly in liver Alanine aminotransferase ALT cytosol L alanine ketoglutarate pyruvate glutamate a k a serum glutamate pyruvate transaminase SGPT Greatest activity is found in the liver Activity can be found in serum and CSF but not in urine Stable at RT frozen and refrigerated Hemolysis has a negligible effect on ALT activity LIVER TOXICITY II Markers of hepatocellular injury C Enzymes almost exclusively located in liver Ornithine carbamyl transferase OCT mitoch ornithine citrulline Is found in liver 97 and sm intestine 2 Activity increases in both acute and chronic liver disease Diagnostically useful in all species As sensitive as histopathological examination of the liver Is elevated after acute obstruction of bile flow LIVER TOXICITY II Markers of hepatocellular injury C Enzymes almost exclusively located in liver Sorbitol dehydrogenase SDH cytosol D sorbitol D fructose Is found in liver and testes Greatest activity is found in the liver Diagnostically useful in all species Sensitive enzyme marker for liver necrosis but shall be combined with measurements of ALT or other enzymes Is elevated after acute obstruction of bile flow LIVER TOXICITY III Enzymes