Enzymic creatinine assay: a new colorimetric method based on hydrogen peroxide measurement

Abstract

We describe a new colorimetric method for measuring creatinine in serum and urine. Creatinine hydrolysis is catalyzed by creatinine amidohydrolase, and the creatine so produced is assayed in reactions catalyzed sequentially by creatine amidinohydrolase and sarcosine oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is measured at 510 nm in a reaction catalyzed by horseradish peroxidase, with 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone as the chromogen. This series of reactions is complete in 30 min at room temperature. A blank sample measurement corrects for endogenous creatine. The standard curve is linear for creatinine concentrations as great as 2.21 mmol/L. Analytical recovery of creatinine in human sera and urine averaged 99.8%. Within-run and between-run precision studies gave CVs of less than or equal to 3.3 and less than or equal to 4.3% for a serum with a creatinine concentration of 69 mumol/L. Results by this method agree well (r greater than 0.99) with those by both the enzymic ultraviolet method of Wahlefeld and the fuller's earth/Jaffé method.