ENZYMATIC RATE ASSAY OF CREATININE IN SERUM AND URINE

A two-step method for assaying creatinine in serum and urine samples, suitable with automated analyzers, is reported. Reagent 1, for the fir st step, contains a blanking system [creatine amidinohydrolase (CRTase ), urease, glutamate dehydrogenase, NADPH, and 2-oxoglutarate] and a N ADPH-regenerating system [Mg2+-dependent isocitrate dehydrogenase (ICD ), MgCl2, and excess isocitrate]. Reagent 2, for the second step, cont ains the metal-chelating reagent trans-1,2-cyclohexanediamine-N,N,N'N' -tetraacetic acid (CyDTA) and a trigger system [creatinine amidohydrol ase (CRNase)]. When a specimen is mixed with reagent 1, all the creati ne, urea, and NH3 present are removed by the blanking and NADPH system s. On adding reagent 2, CyDTA inactivates ICD to inhibit the NADPH sys tem. Simultaneously, the creatinine (1 mol) in the specimen is hydroly zed into creatine by CRNase, and then releases NADP+ (2 mol) through t he blanking system. Our optimized method can determine creatinine line arly up to 500 mg/L, with within-day CVs <1.2% and day-to-day CVs <2.7 %.