Peroxidase-coupled method for kinetic colorimetry of total creatine kinase activity in serum

Abstract

We describe a peroxidase-coupled method involving a colorimetric indicator reaction for determining the total activity of creatine kinase (EC 2.7.3.2) in serum. The kinetically favorable reverse reaction is exploited to generate adenosine 5'-triphosphate, which is used in the glycerol kinase-catalyzed phosphorylation of glycerol. The glycerol 3-phosphate so generated is oxidized in the presence of α-glycerophosphate oxidase to produce hydrogen peroxide, which is reduced in the presence of peroxidase with the simultaneous oxidation and coupling of 4-aminoantipyrene and 2-hydroxy-3,5,-dichlorobenzenesulfonate to produce an intensely colored red chromogen. Results of the proposed method (y) correlate well with those of the Boehringer-Mannheim 'CK-NAC UV' method as applied to the Hitachi 705 chemistry analyzer (y = 1.025x - 18.1, r = 0.9985, n = 100, range = 19-4531 U/L). The sensitivity of the method, based on molar absorptiveness, is nearly fourfold that of procedures involving the reduction of NADP+.