A fluorophotometric determination of serum creatinine and creatine using a creatinineamidohydrolase-creatineamidinohydrolase-sarcosine oxidase-peroxidase system and diacetyldichlorofluorescin.

Abstract

A sensitive method for the fluorophotometric assay of creatinine and creatine was established by using a novel enzyme system, creatinineamidohydrolase-creatineamidinohydrolase-sarcosine oxidase-peroxidase system. Diacetyldichlorofluorescin, which has been regarded as a stable storage form of dichlorofluorescin, was found to react readily with peroxide in the presence of peroxidase in a buffer, (pH 7.7) to give intense fluorescence. This reagent was employed for the estimation of hydrogen peroxide generated by the action of the above enzyme system on creatinine and creatine. The present method is highly sensitive and only 20 μl of serum is required for each assay. Ascorbic acid, bilirubin and uric acid, which often affect enzymatic assay methods that utilize peroxidase, did not interfere with the present method. Excellent recovery from human serum was observed for both creatinine and creatine. The results obtained by the present method showed a good correlation with those obtained by the Jaffe method.