A METHOD FOR THE RAPID DETERMINATION OF ALKALINE PHOSPHATASE WITH FIVE CUBIC MILLIMETERS OF SERUM

Abstract

The alkaline phosphatase of the serum increases early and markedly in rickets and returns completely to normal only after healing is complete. Because of this fact, serum phosphatase is the most satisfactory index now known for the detection of this deficiency. The phosphatase activity of serum is not strictly specific in this respect and has also proved clinically useful in a number of other pathological states; e.g., Paget’s disease, hyperparathyroidism, liver disease, etc. In connection wit.h nutritional studies on large groups of population, it became necessary to have a rapid method for the determination of this enzyme on small amounts of serum. By the use of a new substrate (pnitrophenyl phosphate) a method has been devised which requires only 5 c.mm. of serum (0.005 ml.) and which permits 50 to 100 analyses to be made in 2 hours. The simplicity and speed of the method recommend it for macroas well as microdeterminations and for either alkaline or acid phosphatase. A number of methods have been described for the determination of the phosphatase content of serum and other biological materials, all of which depend upon the principle of measuring the rate of hydrolysis of various phosphate esters under specified conditions of temperature and pH. The two most widely used methods are those of Bodansky (1) and King and Armstrong (2) in which glycerol phosphate and phenyl phosphate respectively are employed as substrates. While these methods are satisfactory for many uses, they are rather time-consuming when large numbers of determinations are needed; furthermore, they require larger samples of serum than is convenient for the purpose of dietary surveys. The substrate, p-nitrophenyl phosphate, was studied by King and Delory (3) and has been used for phosphatase estimations by Ohmori (4) and by Fujita (5). The compound is colorless, but upon splitting off the phosphate group, the yellow salt of p-nitrophenol is liberated (absorption maximum, 400 mp). Hence the substrate is itself an indicator of the amount of splitting and thus a measure of phosphatase activity. It is only necessary to incubate serum with the buffered reagent, stop the reaction