Automated microanalysis of creatinine by coupled enzyme reactions
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Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice
Published on: August 9, 2013
K Asano1, H Iwasaki, K Fujimura
1Central Research Laboratories, Kobayashi Pharmaceutical Co., Ltd., Osaka, Japan.
Abstract:
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.
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