New amylase substrate and assay procedure.

This report describes a new procedure for serum amylase assay. A novel substrate, dyed amylopectin, is used that combines the advantages of saccharogenic and amyloclastic methods. Serum amylase hydrolyzes the dyed amylopectin into ethanol-soluble fragments, which are quantified colorimetrically after serum proteins and unhydrolyzed substrate are precipitated with alcoholic tannic acid. The substrate is prepared by coupling Reactone Red 2B to amylopectin in alkaline solution. Unreacted dye is removed by gel filtration. The clear red solution of dyed amylopectin is buffered and diluted to a standard concentration; it can be preserved indefinitely by lyophilization. The assay procedure is the following: 0.2 ml serum is added to 1 ml substrate at 37#{176}C. After incubation for 10 mm, 5 ml of alcoholic tannic acid are added, and the mixture is centrifuged. Absorbance of the supernatant solution at 540nm is a linear function of amylase activity. Serum blanks are not required. The results correlate well with the saccharogenic assay of Somogyi.