A simple rapid colorimetric method for determination of creatine kinase activity

A colorimetric method for determining creatine kinase activity in serum and body fluids is described. This method is a modification of the Tanzer and Gilvarg method in which the "forward" creatine kinase reaction is coupled to the pyruvate kinase reaction. Instead of using the lactic dehydrogenase system as an indicator, the pyruvate produced is measured directly by utilizing 2,4-dinitrophenylhydrazine. The substrate concentrations have been studied and shown to be present in optimal amounts. A linear relationship between enzyme activity and enzyme concentration has been demonstrated in purified preparations. However, in serum, a heat stable inhibitor appears to be present. The effect of this inhibitor is reduced by dilution making the serum to substrate volumes important in assay of the enzyme. The importance of adding a sulfhydryl agent to the reaction mixture is confirmed. This leads to increased enzyme activity and stabilizes the activity in serum for at least 5 days when stored at refrigerator temperature. The sensitivity of the method is excellent and appears to be considerably greater than either a commercially available phosphate method or the Tanzer and Gilvarg method. The mean serum activity found in normal women was 12.8 I.U. and in normal men 17.6 I.U.