assays

• forward method for total CK

  • Uses CK to convert ATP to ADP
  • Uses CK to convert NADH to NAD (OXIDATION)
• backward method for total Ck assay

  • Uses CK to convert ADP to ATP
  • Uses CK to convert NADP to NADPH (REDUCTION)
• • Forward assay of LD converts lactate to pyruvate; slower of the two assays
• forward assay of LDH has an Optimal pH of 8.3 to 8.9
• • Backward converts pyruvate to lactate and has an optimal pH of 7.1 to 7.4
• Assay for AST is called teh Karmen method, it is a coupled enzyme reaction that uses malate dehydrogenase to oxidize NADH
• AST assay has an optimal pH of 7.3-7.8
• the assay for ALT is a coupled enzymatic reaction, which uses lactate dehydrogenase to oxidize NADH
• alkaline phosphatase assay: Uses p-nitrophenylphosphate - in pH of 10.2, ALP will liberate phosphate group, resulting in p-nitrophenol and phosphate ion, turning solution yellow
• GGT assay includes transferring GC (gamma-glutamyl) groups to chromagen at pH of 8.2 
• four amylase assays: saccharogenic, amyloclastic, chromogenic, and coupled enzymatic
• • Saccharogenic - starch is broken down into reducing sugars by amylase in sample 
• in saccharogenic assay, the concentration of reducing sugars is measured
• in saccharogenic assay, Starch in reagent, amylase in sample, determine concentration of reducing sugar
• Amyloclastic assay uses Iodine bound to starch (starts dark blue); Amylase breaks down starch, iodine is released, and color changes: blue lightens
• • Chromogenic assay of amylase: Starch-dye complex is added to sample and the dye is released when the starch is hydrolyzed; solution will turn a color
• • Coupled enzymatic assay of amylase Uses continuous monitoring; Not used a lot
• coupled enzymatic assay of amylase Uses NADH formation; NAD reduced to NADH; looking for change of absorbance 
• First assay of lipase was tee cherry crannell method, which took 24 hours to run
• the cherry crannell method involved measuring fatty acids that were liberated from olive oil
• Current colorimetric method of lipase involves triolium broken down to oleic acid; uses peroxidase with color indicator
• General assay of G6PD uses glucose-6-phosphate and NADP; uses glucose-6-phosphate dehydrogenase in the sample to yield 6-phosphogluconate and NADPH