Fluorometric assay of serum acid or alkaline phosphatase, either in solution or on a semisolid surface

We describe enzymatic fluorometric methods for determining activities of serum alkaline phosphatase and of serum acid phosphatase in solution and on silicone rubber pads. 4-Methylumbelliferone phosphate is used as substrate, in either tris(hydroxymethyl)aminomethane or citrate buffer. In solution, the reaction is measured at 37 degrees C in a 3-ml Pyrex cuvette. Measurements on the pads are also made at 37 degrees C, after establishing a stable substrate film by lyophilizing all reagents on the surface of the pads. Only 20 to 30 mul of substrate solution, 50 mul of buffer solution, and 1 to 10 mul of blood are necessary, making a total volume of 51 to 60 mul for each assay. The rate of appearance of the fluorescent 4-methylumbelliferone liberated from 4-methylumbelliferone phosphate by the enzymatic action is measured and equated to enzyme activity. Calibration plots of the change in fluorescence per minute vs. enzyme activity for measurements in solution and on pads show a good proportionality in the range of 30.8 to 633 U/liter for alkaline phosphatase and in the range of 0.265 to 5.3 King-Armstrong units for acid phosphatase, indicating the usefulness of these methods in the clinical laboratory.