The colorimetric determination of phosphatases in human serum.

There are a number of methods currently available for the quantitative estimation of acid and alkaline phosphatase activity in biologic materials (l-5). In all the techniques, a phosphoric acid ester serves as a substrate and calorimetric determinations are made either of the inorganic phosphate or of the organic moiety of the substrate released by enzymatic hydrolysis. A description of a new method for the quantitative estimation of acid and alkaline phosphatase activity of human serum forms the basis of this report. The substrate consists of sodium /3-naphthyl phosphate (6). The calcium salt of this ester was used in a method for the histochemical localization of alkaline phosphatase (7,8). The Q! isomer was used for the histochemical localization of acid phosphatase (9), and, based on this work, the CY isomer was used in methods for demonstrating semen on clothing in cases of suspected rape (lo), and for estimating urinary acid phosphatase activity (11). The new method is in general similar to the method described for estimating esterase and lipase activity in serum and in urine with P-naphthyl laurate as the substrate (12-14). The /3 isomer, rather than the c11 isomer, is used for acid phosphatase in the present method, because the azo dye formed from fl-naphthol is, in the presence of protein, much more readily extracted into ethyl acetate for measurement of color density than is the case with the azo dye formed from a-naphthol. The method is applicable to any source of the enzyme such as serum, plasma, whole blood, tissue homogenates, tissue sections, semen, gastric juice, and urine. In essence, the method consists of incubation of the enzyme source with a solution of sodium /3-naphthyl phosphate buffered to an appropriate pH (4.8 for acid phosphatase and 9.1 for alkaline phosphatase) at 37.5” for a standard period of time (2 hours and 1 hour respectively). 2 molecules of