Erythrocyte Phosphatase Activity in Hemolysed Sera and Estimation of Serum “Acid” Phosphatases

Abstract

Erythrocytes contain an apparently specific phosphatase 1 , 2 , 3 classified by Folley and Kay 4 as phosphomonoesterase A_4. This enzyme in laked red cell systems readily dephosphorylates monophenylphosphate, 2 the pH range of activity for the reaction showing an optimum at 5.8-6.0 but extending to well below pH 5.0. 2

In estimating the “acid” phosphatase activity of blood serum 5 , 6 by a recently described adaptation 7 of the King and Armstrong “alkaline” phosphatase method, 8 monophenylphosphate substrate is used with citrate buffer at pH 4.9. These conditions also permit of hydrolysis by such erythrocyte phosphatase as may be present if sufficiently hemolysed samples of blood serum are employed. The determination of serum “acid” phosphatases (which has certain clinical applications 9 , 10 , 11 ) therefore gives misleading results in markedly hemolysed blood samples. If the use of such samples is unavoidable, a correction can be made after hydrolysis in the presence of NaF, which in proper concentration differentially inhibits serum “acid” phosphatases.

Experimental. Effect of hemolysis on phosphatase activity at pH 5.0. Fresh, non-hemolysed, normal sera exhibit slight “acid” phosphatase activity: 1.4 and 1.6 units/100 cc respectively in the 2 subjects cited in Table I. Defibrinated whole blood samples from these normal subjects showed more than 100 times greater phosphatase activity when determined at the same pH (5.0) by the same method, because large amounts of erythrocyte phosphatase are present in the hemolysates. Fresh, non-hemolysed sera of patients with metastasizing prostatic carcinoma contain prostate “acid” phosphatase and show elevated “acid” phosphatase levels (Table I). Defibrinated whole blood samples from these patients exhibit further increases in phosphatase activity at pH 5.0, due to the added effect of erythrocyte phosphatase.