When the activity of a pure LDH preparation was measured in a reaction system consisting of NAD, lactate, an electron carrier and 2-(p-lodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium Chloride (INT) a tetrazolium salt, and estimated from the amount of formazan formed, the activity values gained were much lower than those determined by the UV-method. On the other hand, when the activity of the pure LDH was determined in the colorimetric assay system, to which either Triton X-100 or heated human serum was added, formazan formation was markedly increased and the activity value estimated from its amount was consistent with that determined by the UV-method. This action of Triton X-100 and heated human serum on the formazan formation was investigated using 9-dimethylaminobenzo-α-phenazoxonium chloride (Meldola's Blue, MB), phenazine methosulfate (PMS) and 1-methoxy-5-methylphenazinium methylsulfate (1-M-PMS) as electron carriers. It was found that when INT was omitted from the colorimetirc LDH assay system with or without either Triton X-100 or heated human serum, a large amount of hydrogen peroxide (H2O2) was produced. This finding indicates that in the formazan formation based on the reduction of INT by NADH via electron carrier, the electron carrier reduced by NADH reacted not only with INT, but also easily with molecular oxygen (O2). It is assumed, therefore, that Triton X-100and heated human serum enhance the reactivity of redu ced electron carriers with tetrazolium salts by changing the redox potentials of the tetrazolium salts, resulting in a marked increase in formazan formation.
