liver enzymes.pdf

This is your presentation title LIVER ENZYMES Saint Louis University School of Nursing, Allied Health, and Biological Sciences DEPARTMENT OF MEDICAL LABORATORY SCIENCE  TOPIC OUTLINE ▪ Enumerate tissue sources of each enzyme. ▪ Discuss the clinical significance....

This is your presentation title LIVER ENZYMES Saint Louis University School of Nursing, Allied Health, and Biological Sciences DEPARTMENT OF MEDICAL LABORATORY SCIENCE  TOPIC OUTLINE ▪ Enumerate tissue sources of each enzyme. ▪ Discuss the clinical significance. ▪ Discuss patient preparation and specimen considerations. ▪ Enumerate and discuss methods of enzyme determination. 2 JDPA, RMT 2  1. ASPARTATE I. ASPARTATE AMINOTRANSFERASE AMINOTRANSFERASE ▪ Serum glutamic-oxaloacetic transaminase (SGOT) ▪ EC Code Number: _________ ▪ Coenzyme: pyridoxal phosphate 3 JDPA, RMT 3  1. ASPARTATE I. ASPARTATE AMINOTRANSFERASE AMINOTRANSFERASE ▪ MAJOR TISSUE SOURCES: ▪ Cardiac tissue ▪ Liver ▪ Skeletal muscle ▪ Other sources: kidney, pancreas, and erythrocytes 4 JDPA, RMT 4  1. ASPARTATE I. ASPARTATE AMINOTRANSFERASE AMINOTRANSFERASE ▪ ISOENZYME FRACTIONS: ▪ 1. Cytoplasmic isoenzyme ISOENZYME ANALYSIS is NOT ROUTINELY DONE ▪ 2. Mitochondrial isoenzyme 5 JDPA, RMT 5  CLINICAL SIGNIFICANCE ▪ Hepatocellular disorders ▪ Skeletal muscle involvement ▪ Acute Myocardial Infraction 6 JDPA, RMT 6  1. ASPARTATE CLINICAL SIGNIFICANCE AMINOTRANSFERASE 7 JDPA, RMT 7  1. ASPARTATE CLINICAL SIGNIFICANCE AMINOTRANSFERASE ▪ HIGHEST ELEVATION: 5 or more x ULN ▪ Acute hepatocellular disorders ▪ Myocardial infarction ▪ Circulatory collapse (Shock) ▪ Acute pancreatitis ▪ Infectious mononucleosis 8 JDPA, RMT 8  2. ALANINE CLINICAL SIGNIFICANCE AMINOTRANSFERASE ▪ MODERATE ELEVATION (3-5 x ULN): ▪ Biliary obstruction ▪ Cardiac arrhythmias ▪ Congestive heart failure ▪ Metastatic or primary tumor in the liver ▪ Muscular dystrophy 9 JDPA, RMT 9  2. ALANINE CLINICAL SIGNIFICANCE AMINOTRANSFERASE ▪ SLIGHT ELEVATION (up to 3 x ULN): ▪ Pericarditis ▪ Liver cirrhosis ▪ Pulmonary infarction ▪ Delirium tremens ▪ Cerebrovascular accident ▪ Potential hepatotoxic drugs 10 JDPA, RMT 10  1. ASPARTATE ASPARTATE AMINOTRANSFERASE (AST) AMINOTRANSFERASE ▪ METHODOLOGIES: ▪ Karmen Method ▪ Reitman-Frankel Method ▪ Diazonium Salt Reaction ▪ Chemiluminescence ▪ Chromatography 11 JDPA, RMT 11  KARMEN METHOD ▪ ____________________ method ▪ Basis for IFCC recommended method ▪ Addition of ___________ ▪ Optimum pH: _________ ▪ Rate of decrease in absorbance is directly proportional to the activity of AST (340nm) 12 JDPA, RMT 12  REITMAN-FRANKEL METHOD ▪ ___________ method ▪ Substrate: aspartic alpha-ketoglutarate ▪ Color developer (reagent): _____________________ ▪ Color intensifier: ______________ ▪ Final reaction: blue-colored product (505nm) NOT COMMONLY USED 13 JDPA, RMT 13  DIAZONIUM SALT REACTION ▪ Colorimetric assay ▪ Stabilized diazonium salt + oxaloacetic acid → Formation of diazonium derivative (_______) ▪ Most specific and more sensitive 14 JDPA, RMT 14  CHEMILUMINESCENCE ▪ HIGHLY SENSITIVE ▪ Inhibitors: N-nitro-L-arginine methyl Superoxide dismutase ester hydrochloride ▪ Direct detection of ___________ and ___________ 15 JDPA, RMT 15  CHROMATOGRAPHY ▪ Sensitive in measuring AST and ALT ▪ Can measure: As low as 0.1 IU/L 16 JDPA, RMT 16 SPECIMEN AND ASSAY CONSIDERATION ▪ Hemolysis ▪ Lipemic and Icteric sample ▪ Pyridoxal phosphate ▪ Pyruvate (>0.2 mmol/L) TG: >650 mg/dL ▪ Drugs: ▪ 1. Anabolic steroids ▪ 2. Chloramphenicol ▪ 3. Aspirin 17 JDPA, RMT 17 Bili: >19 mg/dL SPECIMEN AND ASSAY CONSIDERATION ▪ Preferred sample: Fresh, Un-hemolyzed SERUM ▪ PLASMA can be used 18 JDPA, RMT 18  2. ALANINE II. ALANINE AMINOTRANSFERASE AMINOTRANSFERASE ▪ Serum glutamic pyruvic transaminase (SGPT) ▪ EC Code Number: _________ Coenzyme: Pyridoxal phosphate 19 JDPA, RMT 19  2. ALANINE II. ALANINE AMINOTRANSFERASE AMINOTRANSFERASE ▪ MAJOR TISSUE SOURSES: ▪ Liver ▪ Kidney ▪ Other tissue sources: Heart and Skeletal muscle 20 JDPA, RMT 20  2. ALANINE CLINICAL SIGNIFICANCE AMINOTRANSFERASE ▪ LIVER DISEASES: ___________ EXCEPT in: HEPATIC CIRRHOSIS LIVER NEOPLASIA Acute hepatocellular injury 21 JDPA, RMT 21  2. ALANINE CLINICAL SIGNIFICANCE AMINOTRANSFERASE ▪ De Ritis Ratio: ▪ AST:ALT Quotient/Ratio ▪ >1 ▪ 3-4:1 22 JDPA, RMT 22  2. ALANINE CLINICAL SIGNIFICANCE AMINOTRANSFERASE ▪ HIGHEST ELEVATION: ___________________ ▪ Non-alcoholic, asymptomatic patients ▪ Mild elevation: Hepatitis C infection ▪ Used to screen donors ▪ More specific and sensitive screening test for _______________________________________ 23 JDPA, RMT 23  2. ALANINE II. ALANINE AMINOTRANSFERASE AMINOTRANSFERASE ▪ METHODOLOGIES: ▪ 1. Wroblewski and La Due: ▻ ___________________ reaction ▻ Basis for IFCC recommended method ▻ Addition of __________ ▻ Other methods: Reitman-Frankel Method, Diazonium Salt Reaction, Chemiluminescence, 24 JDPA, RMT Chromatography 24  III. ALKALINE PHOSPHATASE 3. ALKALINE PHOSPHATASE ▪ EC CODE NUMBER: 3.1.3.1 ▪ Requires _______________ 25 JDPA, RMT 25  III. ALKALINE PHOSPHATASE 3. ALKALINE PHOSPHATASE ▪ TISSUE SOURCE: ▪ ____________ ▪ ____________ ▪ Small Intestine ▪ Kidney Origins of ALP ISOENZYMES: ▪ Placenta Genetic Loci: Chromosome 1: Kidney, Liver, Bone Chromosome 2: Intestinal and Placental 26 JDPA, RMT 26  III. ALKALINE PHOSPHATASE 3. ALKALINE PHOSPHATASE ▪ ISOENZYMES: NORMAL ALP ISOENZYMES: Differentiated by: 1. Intestinal 2. Placental 1. Electrophoresis 3. Bone 2. Heat denaturation 4. Liver 3. Chemical inhibition 27 JDPA, RMT 27  III. ALKALINE PHOSPHATASE 3. ALKALINE PHOSPHATASE ▪ ABNORMAL ALP ISOENZYME FRACTIONS: CARCINOPLACENTAL ALP: 1. Regan 2. Nagao 3. Kasahara 28 JDPA, RMT 28  III. ALKALINE PHOSPHATASE 3. ALKALINE PHOSPHATASE ▪ ABNORMAL ALP ISOENZYME FRACTIONS: REGAN ISOENZYME: 1. Lung cancer 2. Breast cancer 3. Ovarian and gynecological cancers 4. Colon cancer 29 JDPA, RMT 29  III. ALKALINE PHOSPHATASE 3. ALKALINE PHOSPHATASE ▪ ABNORMAL ALP ISOENZYME FRACTIONS: NAGAO ISOENZYME: KASAHARA ISOENZYME: Metastatic carcinoma of Hepatoma pleural surfaces (Pleural cancer) Adenocarcinoma (pancreas Tumors (GIT) and bile duct) 30 JDPA, RMT 30  III. ALKALINE PHOSPHATASE 3. ALKALINE PHOSPHATASE ▪ ELECTROPHORESIS: ELECTROPHORETIC MOBILITY: 31 JDPA, RMT 31  III. ALKALINE PHOSPHATASE 3. ALKALINE PHOSPHATASE ▪ To improve separation of bone and liver isoenzymes: ▪ 1. Neuraminidase: Removes sialic acid ▪ 2. Wheat germ lectin: Bind other isoenzymes ▪ 3. High resolution electrophoresis: ▻ ________________ ▻ ________________ 32 JDPA, RMT 32  III. ALKALINE PHOSPHATASE ALKALINE PHOSPHATASE ▪ HEAT DENATURATION: HEAT STABILITY: 56 degrees Celsius MOST HEAT STABLE OF ALL ALP 10-15 minutes ISOENZYMES: REGAN ISOENZYME 33 JDPA, RMT 33  III. ALKALINE PHOSPHATASE Placental ALP vs Regan ALP Regan ALP vs Nagao ALP Electrophoretic mobility Electrophoretic mobility Heat stability Heat stability Phenylalanine inhibition Phenylalanine inhibition 34 JDPA, RMT ____________________  III. ALKALINE PHOSPHATASE 3. ALKALINE PHOSPHATASE ▪ CHEMICAL INHIBITION: L-phenylalanine Placental, Intestinal, Regan and Nagao ALPs Levamisol Liver and Bone ALPs L-homoarginine Liver and Bone ALPs 2M Urea Bone ALP L-leucine Nagao ALP 20% ethanol Denatures liver ALP rapidly than Bone ALP 35 JDPA, RMT 35  CLINICAL SIGNIFICANCE 3. ALKALINE PHOSPHATASE ▪ PRONOUNCED ELEVATION (5 or more x ULN): ▪ Bile duct obstruction ▪ Biliary cirrhosis ▪ Osteitis deformans (Paget’s disease) ▪ Osteogenic sarcoma ▪ Hyperparathyroidism 36 JDPA, RMT 36  CLINICAL SIGNIFICANCE 3. ALKALINE PHOSPHATASE ▪ MODERATE ELEVATION (3-5 x ULN): ▪ Granulomatous or Infiltrative liver diseases ▪ Infectious mononucleosis ▪ Metastatic tumors in bone ▪ Metabolic bone diseases: ▪ Ricket’s disease ▪ Osteomalacia 37 JDPA, RMT 37  CLINICAL SIGNIFICANCE 3. ALKALINE PHOSPHATASE SLIGHT ELEVATION (up to 3 X ULN): 1. Viral hepatitis Hepatocellular disorders 2. Cirrhosis 3. Healing fractures 4. Pregnancy Third trimester until labor 5. Normal growth (children) 6. Diet induced ALP elevation (Group O and B secretors) 38 JDPA, RMT 38  CLINICAL SIGNIFICANCE 3. ALKALINE PHOSPHATASE ▪ DECREASED LEVEL: ▪ Inherited condition of hypophosphatasia ▪ ___________ of bone isoenzyme 39 JDPA, RMT 39 SPECIMEN AND ASSAY CONSIDERATION 3. ALKALINE PHOSPHATASE ▪ Avoid hemolysis ▪ Process sample immediately: ▪ ALP activity in serum increases ______________ ▪ SERUM in contact with clot: _______________ ▪ _______________: ▪ 25% higher ALP values ▪ PLASMA should NOT be used 40 JDPA, RMT 40  III. ALKALINE PHOSPHATASE 3. ALKALINE PHOSPHATASE ▪ Bowers Mc Comb ▪ Electrophoresis ▪ Heat Stability Test/ Heat Denaturation/ Heat Fractionation ▪ Chemical Inhibition ▪ Other methods 41 JDPA, RMT 41  BOWERS Mc COMB 3. ALKALINE PHOSPHATASE ▪ _________________ –IFCC recommended method ▪ _________________ method ▪ Substrate: ____________________ ▪ Optimum pH: 10.5 at 30 ℃ ▪ Product measured: _____________ (405nm) ▪ Note: ____________________________________ 42 JDPA, RMT 42  OTHER METHODS 3. ALKALINE PHOSPHATASE ▪ Other methods: Substrate End product/s Bessey, Lowry, P-nitrophenyl P-nitrophenol or Brock phosphate yellow nitrophenoxide Huggins and Phenolphthalein Phenolphthalein red Talalay diphosphate Moss Alpha naphthol Alpha-naphthol phosphate 43 JDPA, RMT 43  OTHER METHODS 3. ALKALINE PHOSPHATASE ▪ Other methods: Substrate End product/s Bodansky, Beta- Inorganic phosphate Shinowara, Jones, glycerophosphate + glycerol Reinhart Klein, Babson and Buffered Free Phenolphthalein Read phenolphthalein phosphate King and Phenyl phosphate Phenol 44 JDPA, RMT Armstrong 44  1. GAMMA-GLUTAMYL IV. GAMMA GLUTAMYL TRANSFERASE TRANSFERASE ▪ EC CODE NUMBER: ________ ▪ FUNCTIONS: ▪ Peptide and protein synthesis ▪ Regulation of tissue glutathione levels ▪ Transport of amino acids across cell membranes 45 JDPA, RMT 45  1. GAMMA-GLUTAMYL IV. GAMMA GLUTAMYL TRANSFERASE TRANSFERASE ▪ TISSUE SOURCE: ▪ KIDNEY ▪ LIVER ▪ Prostate ▪ Pancreas 46 JDPA, RMT 46  1. CLINICAL GAMMA-GLUTAMYL SIGNIFICANCE TRANSFERASE ▪ Hepatobiliary disorders BILIARY OBSTRUCTION: highest concentration of ALP and GGT DIFFERENTIATES source of ALP elevation ▪ Enzyme – inducing drugs Warfarin, Phenobarbital, and Phenytoin 47 JDPA, RMT 47  1. CLINICAL GAMMA-GLUTAMYL SIGNIFICANCE TRANSFERASE ▪ Alcoholic hepatitis Most sensitive marker of ALCOHOLISM (Acute alcoholic hepatitis) ▪ Acute pancreatitis and prostatic disorders ▪ Diabetes mellitus ▪ Myocardial infarction 48 JDPA, RMT 48  1. CLINICAL GAMMA-GLUTAMYL SIGNIFICANCE TRANSFERASE GGT ALP Skeletal disorders Pregnancy Hepatobiliary disorder Bone disorder 49 JDPA, RMT 49  1. GAMMA-GLUTAMYL IV. GAMMA GLUTAMYL TRANSFERASE TRANSFERASE ▪ METHODOLOGIES: ▪ 1. Szasz and Rosalki ▪ 2. Other methods: ▪ Persjin and Van der Silk Method ▪ Goldberg Method 50 JDPA, RMT 50  1. GAMMA-GLUTAMYL SZASZ AND ROSALKI TRANSFERASE ▪ IFCC recommended method ▪ Optimum pH: _____________ ▪ Substrate: _____________________________ ▪ Product measured: _____________ (405-420nm) ▪ 2. Other methods: ▻ Persjin and Van der Slik Method ▻ Goldberg Method 51 JDPA, RMT 51  1. GAMMA-GLUTAMYL OTHER METHODS TRANSFERASE ▪ PERSIJN AND VAN der SLIK METHOD: ▪ Advantage: direct chromogen production ▪ Substrate: L-GG-3-carboxy-4-nitroanilide ▪ GOLDBERG METHOD: ▪ Substrate: L-GG-beta naphthylamide ▪ Product measured: beta naphthylamide 52 JDPA, RMT 52  1. GAMMA-GLUTAMYL SPECIMEN AND ASSAY CONSIDERATION TRANSFERASE ▪ Preferred sample: SERUM (non-hemolyzed) ▪ PLASMA CAN BE USED: ___________ Heparin – produces turbidity Citrate, oxalate, and fluoride – decrease GGT activity by 10-15% ▪ Hgb (100-500 mg/dL) → _____________ in GGT ▪ Bilirubin (20 mg/dL) → ______________ in GGT 53 JDPA, RMT 53  1. GAMMA-GLUTAMYL VALUES TO REMEMBER TRANSFERASE AST ALT UV Kinetic Method: UV Kinetic Method: @30℃: up to 28 IU/L @30℃: up to 26 IU/L @37℃: up to 40 IU/L @37℃: up to 38 IU/L IFCC Recommended Method: IFCC Recommended Method: Up to 40 IU/L Up to 40 IU/L GGT Kinetic Method: ALP Male: 0-45 IU/L UV Kinetic Method: Female: 0-30 IU/L Adult: 25-123 IU/L IFCC Recommended Method: IFCC Recommended Method: Male: Up to 40 IU/L Adult: 30-130 IU/L Female: Up to 25 IU/L 54 54 JDPA, RMT