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.
D001205:Ascorbic Acid; D002021:Buffers; D002733:Chlorophenols; D003124:Colorimetry; D003404:Creatinine / Q000097:blood*; D006735:Horseradish Peroxidase; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008722:Methods; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase; D013997:Time Factors; D014512:Ureohydrolases
D002021:Buffers; D002733:Chlorophenols; C028879:sulfonated 2,4-dichlorophenol; D003404:Creatinine; D006735:Horseradish Peroxidase; D010089:Oxidoreductases, N-Demethylating; C494349:PIPOX protein, human; D050879:Sarcosine Oxidase; D014512:Ureohydrolases; C019944:creatinase; D001205:Ascorbic Acid