ENZYMES

• Alanine Aminotransferase (ALT): enzyme used to ------- post transfusion hepatitis.
• DNPH: color developer in Reitman-Frankel
• Tanzer Gilvarg Assay: forward method of CK.
• p-nitrophenylphosphate (PNPP): substrate in Bowers-McComb
• normal in serum: LD 2>1>3>4>5>6
• normal in CSF: LD 1>2>3>4>5>6
• serum AMI: LD 1>2>3>4>5>6
• CSF bacterial meningitis: LD 6>5>4>3>2>1
• Instestinal: slowest ALP to migrate
• Acid Phosphatase (ACP): enzyme used to check rape cases
• Roy: reference method for ACP
• glucose: measured in saccharogenic amylase.
• starch: measured in amyloclastic amylase
• iodine: reagent in chromogenic amylase
• olive oil: substrate in lipase
• aspartate aminotransferase (AST) is greatly increased during viral hepatitis.
• Karmen Method - incorporates a coupled enzymatic reaction using MD as indicator enzyme and monitors the change in absorbance at 340 nm.
• Reitman-Frankel: Reaction with DNPH - ketoacids formed after the catalyzed reaction are reacted with 2,4 to form ketoacidhydrazones which produce intense color in the presence of measured at
• Coupled Enzyme Reaction Substrate: L-alanine Indicator Reaction: LD as indicator enzyme catalyze reduction of pyruvate to lactate with simultaneous oxidation of NADH monitored at 340 nm
• Reaction with DNPH- keto-acid formation Principle: ketoacids formed after the catalyzed reaction are reacted with 2,4 DNPH to form ketoacid hydrazones which produce intense blue color in the presence of NaOH measured at 505 nm
• LD 3 found in lymphocytes
• Wacker Method (most common) - Utilizes lactate to pyruvate reaction with formation of NADH to NAD, (blue-purple color) - not affected by product inhibition.
• Wroblewski Ladue - (reverse/ indirect)- pyruvate to lactate; NADH serves as co-substrate and consumed during the entire reaction.
• Shinowara-Jones- Reinhart substrate: Beta-glycerophosphate long incubation time; high blank values
• King-Armstrong

  substrate: Phenylphosphate End point; requires protein removal
• Bassey-Lowry- Brock (color dev: 0.02 NaOH)

  substarate: p-nitrophenylphosphate End point or kinetic; rapid
• Bowers-McComb substrate: p-nitrophenylphosphate - hydrolyzed to p-nitrophenol @ 405 nm (yellow) Uses phosphate- accepting buffer; reference method
• Bowers-McComb: reference method for ALP
• Bodansky; substrate: B-glycerophosphate; time consuming non-specific
• Gutman, King Armstrong; substrate: phenylphosphate; non-specific
• Babson and Reed; substrate: A-naphthylphosphate; ncomplicated, less sensitive
• Roy: substrate: thymolphthalein monophosphate; reacts only with prostatic ACP or more specific for prostatic form
• Hudson: substrate: p-nitrophenolphosphate; Rapid, non- specific
• Reitz & Guilbalt: substrate: 4methylumbelliferonephosphate; fluorescent, some with improved sensitivity
• P- nitrophenyl phosphate; ● Yields an appearance of Yellow color in alkaline solution (pH>10)- phenolate anion formation in basic solution ● Disadvantage: sample size (0.1 ml), incubation time of 30 mins and kinetic determination is not possible (since pH must be > 1
• thymolphthalein monophosphate; ● Specific for prostatic ACP ● Reaction is carried out in a citrate buffer @pH 6.0 and incubation for 30 minutes ● Interference: Hgb&Bilirubin absorb strongly @410-450 nm
• AMYLOCLASTIC: measures the disappearance of the starch substrate. reduction of intensity of color blue. classic reference method expressed in somogyi units.
• SACCHAROGENIC: measures the appearance of the product. starch (substrate); CuSO4 (rgt); Phosphomolybdic acid (color developer). reported in Somogyi units (mg of glu released in 30 minutes at 37’C)
• CHROMOGENIC/IODOMETRIC: measures the increasing color from production of product coupled with a chromogenic dye. Starch(substrate); Iodine (rgt); Blue (color
• COUPLED ENZYME REACTION TEST: continuous monitoring technique where change of absorbance of NAD+ at 340 nm is measure