Clinical Chemistry, Vol 15, 108-123, Copyright © 1969 by the American Association for Clinical Chemistry

A New Fluorometric Method for the Estimation or Detection of Total and Fractionated Alkaline Phosphatase

¹ Division of Clinical Laboratories, Scripps Clinic and Research Foundation, 476 Prospect St., La Jolla, Calif. 92037.

A highly sensitive fluorometric method for the assay of total alkaline phosphatase and 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 passing 405 mµ primary and 505 mµ secondary. As little as 20 µl. of normal serum per 3 ml. of reaction mixture can be assayed. Alkaline phosphatase components, separated by vertical starch gel (or other methods of) electrophoresis, produce yellow fluorescence under ultraviolet light when incubated at 37° with the same buffer-substrate. After vertical starch gel electrophoresis, all normal serums exhibit at least one component (-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.