Fully Enzymatic Colorimetric Assay of Serum and Urine Creatinine Which Obviates the Need for Sample Blank Measurements

Abstract

Abstract A fully enzymatic method for the colorimetric determination of serum and urine creatinine is described which does not require sample blank measurements. It is based on the formation of hydrogen peroxide from creatinine in a reaction sequence catalyzed by creatinine iminohydrolase, ATP-dependent 1-methylhydantoinase, N-carbamoylsarcosine amidohydrolase and sarcosine oxidase. The hydrogen peroxide is quantitated with high sensitivity at 546 nm by a chromogenic system consisting of peroxidase, 2′-sulpho-2-methyl-benzthiazolinone hydrazone and 2,4,6-tribromo-3-hydroxy-benzoic acid. Only 20 μL of sample are needed for the assay, the total reaction being complete within 10 min at 25°. Within-run precision gave a CV of 3.1 and 1.6 % at serum creatinine concentrations of 79 and 160 μmol/L, respectively, and the standard curve is linear up to at least 1760 μmol/L. The assay yields results which agree well with those found by both an enzymatic UV-method and an alternate enzymatic colorimetric procedure necesitating sample blank measurements to correct for endogenous creatine.