Development of a high-sensitivity double kinetic assay for creatinine using the enzymatic cycling method and its application to serum samples

Abstract

Estimated glomerular filtration rate (eGFR) is often used as a measure of renal function in clinical practice owing to its simplicity. Serum creatinine levels are essential for eGFR calculation. Although the Jaffé method is widely used for creatinine measurement, it exhibits low specificity and sensitivity. Development of various enzyme methods has increased its specificity; however, its sensitivity is still insufficient for accurate eGFR calculation. To overcome this issue, we developed a highly sensitive assay for creatinine detection using enzymatic cycling reaction in this study. Our method consisted of two steps: Endogenous ammonia elimination and creatinine analysis. NADH derived from creatinine changed the water-soluble tetrazolium salt-8 color in the presence of the electron carrier, 1-methoxy-5-methylphenazinium methylsulfate. The cycling oxidation–reduction of 1-methoxy-5-methylphenazinium methylsulfate to NADH facilitated the coloration of water-soluble tetrazolium salt-8, aiding in creatinine measurement with high sensitivity. The within-run reproducibility of the developed method was good (<1.76 % at each concentration tested), with a detection limit of 1.00 μmol/L, making it approximately 9 times more sensitive than the Jaffé method. Notably, its correlation with the high-performance liquid chromatography method was excellent (r = 0.976). Overall, this study successfully developed a new, rapid, simple, and highly sensitive method for creatinine analysis.