Automated microanalysis of creatinine by coupled enzyme reactions

Hydrogen peroxide was generated from creatinine by the sequential enzyme reactions of creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase. Hydrogen peroxide was, in turn, used stoichiometrically for the condensation of 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine catalyzed by horse-radish peroxidase, resulting in the formation of quinone dye with maximum absorbance at 546 nm. The optimized assay conditions for the enzymatic determination of creatinine in a HITACHI 7250 autoanalyzer was established. This system, which requires less than 5 microliters of sample, was found to be the most economical for laboratories equipped with autoanalyzers.