A new enzymatic serum creatinine measurement based on an endogenous creatine-eliminating system

We describe a new colorimetric determination of serum creatinine which does not require a blank to correct for endogenous creatine. In the first reaction, creatinase (creatinase amidinohydrolase EC 3.5.3.3) and sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating) EC 1.5.3.1) were used in the enzymatic hydrolysis of endogenous creatine to produce hydrogen peroxide. In the presence of horseradish peroxidase, 2,4-dichlorophenolsulfonate (2,4-DCPS) was converted to a colorless polymer by hydrogen peroxide. In the second reaction, creatininase (creatinine amidohydrolase EC 3.5.2.10) and 4-aminoantipyrine (4-AA) were added, and only the creatine generated from creatinine by creatininase was hydrolyzed sequentially by creatinase and sarcosine oxidase to produce hydrogen peroxide. This newly-formed hydrogen peroxide was measured at 510 nm in a coupled reaction catalyzed by peroxidase, with 2,4-DCPS/4-AA as a chromogen. The standard curve was linear up to 20 mg/dl; 40 microliter of serum and 20 min were required for determination. Analytical recovery of creatinine added to either normal or abnormal sera averaged 98.5%. Within-day and day-to-day studies gave CV values of less than or equal to 2.9% and less than or equal to 4.8%, respectively. No significant interferences were observed with the proposed method. The results obtained by the present method correlated well with those obtained by the Jaffé procedure.