A new fluorometric method for the estimation or detection of total and fractionated alkaline phosphatase.

A highly sensitivefluorometric method for the assayof total alkaline phosphataseand the detection of its components is described. The commercially available substrate naphthyl AS-MX phosphate, combined in 1 M 2-amino-2-methyl-1-propanol buffer, pH 9.8, is cleaved to the highly fluorescent naphthol AS-MX. Fluorometry requires filters passing405 m primary and 505 mi. secondary.As little as 20 1d. of normal serum per 3 ml. of reaction mixture can be assayed.Alkaline phosphatasecomponents, separatedby vertical starch gel (or other methodsof) electrophoresis,produce yellow fluorescence under ultraviolet light when incubated at 37#{176} with the same buffersubstrate. After vertical starch gel electrophoresis,all normal serums exhibit at least one component (f-globulin region), but six distinct areas of activity have been located. These correspond to Taswell and Jeffers’ origin, beta-lipoprotein, alpha-2, alpha-beta, and beta (5). Few serums contain all of these; rather there appears to be a correlation between the ones present, their relative activity, and the disease state.