The Estimation of Serum Inorganic Phosphate and “acid” and “alkaline” Phosphatase

In 1930, Kay (6, 7) introduced a method for the quantitative determination of “alkaline” phosphatase activity of blood, employing sodium P-glycerophosphate as the substrate. Of the many modifications which have been introduced in which this substrate is employed, Bodansky’s (3, 4) method seems to have found the greatest usage because of its short incubation period and its accuracy. For our studies certain fundamental changes in the technique for the quantitative estimation of serum phosphatase activity and inorganic phosphate were necessary. Presented in this communication are procedures for the simultaneous determination of serum inorganic phosphate and ‘(acid” and “alkaline” phosphatase activity. The estimation of all three tests may be made on 0.40 cc. of serum in the macrotechnique, 0.06 cc. in the microtechnique. Only one photometric calibration curve or one set of phosphorus standards for visual calorimetry is required. The unit of phosphatase activity is defined here as follows: Each unit of serum phosphatase activity is equivalent to 1 mg. of phosphorus as phosphate ion liberated during 1 hour of actual incubation at 37” with a substrate containing sodium fl-glycerophosphate, hydrolysis not exceeding 10 per cent of the substrate; and at