New enzymatic assay for serum urea nitrogen using urea amidolyase

Abstract

We established an enzymatic assay for measurement of serum urea nitrogen using urea amidolyase (EC 3.5.1.45) from yeast species. The method is based on hydrolysis of urea by the enzyme. In this assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (EC 1.4.1.4). Then in the presence of urea amidolyase, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion was produced proportionally to urea concentration in serum. The concentration of ammonium ion formed was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH. We then monitored the change of absorbance at 340 nm. The inhibitory effect of calcium ion on this assay was eliminated by adding glycoletherdiamine-N, N, N′, N′-tetraacetic acid to the reaction system. The within-assay coefficient of variations (CVs) of the present method were 1.80-3.76% (n = 10) at 2.8-19.0 mmol/L, respectively. The day-to-day CVs were 2.23-4.59%. Analytical recovery was 92-115%. The presence of ascorbic acid, bilirubin, hemoglobin, lipemic material, ammonium ion, or calcium ion did not affect this assay system. The correlation between values obtained with the present method (y) and those by another enzymatic method (x) was 0.997 (y = 1.02x - 0.10 mmol/L, Sy/x = 0.841, n = 100), with a mean difference of -0.18 ± 0.86 mmol/L [(values by reference method - that of present method) ± SD] using the Bland-Altman technique. © 2003 Wiley-Liss, Inc.