A COUPLED-ENZYME EQUILIBRIUM METHOD FOR MEASURING UREA IN SERUM - OPTIMIZATION AND EVALUATION OF THE AACC STUDY-GROUP ON UREA CANDIDATE REFERENCE METHOD

Abstract

A coupled-enzyme equilibrium method is described for measuring urea in serum, which is performed on supernates prepared by treating each specimen with Ba(OH)2 and ZnSO4 (Somogyi reagent). Analytical recovery of [14C]urea added to a variety of matrices was essentially complete (mean, 100.6%) for the supernates after precipitation. Nine variables were univariately examined in arriving at the reaction conditions for the method: glutamate dehydrogenase (EC 1.4.1.2), urease (EC 3.5.1.5), 2-oxoglutarate, ADP, Tris .cntdot. HCl, NaDH, EDTA, pH and temperature. The reagent is stable for at least 48 days at -20.degree. C and for 23 days at 4.degree. C. Mean analytical recovery of urea (14 mmol/l) added to 7 different specimens (3 different matrices) was 100.8%. The analytical linear range of the method extends to 30 mmol of urea/l. Of 22 potential interferents, only bilirubin at 1 mmol/l (580 mg/l), Hb at 10 g/l, and hydroxyurea at 6 mmol/l showed more than 2% interference. Precision and effects of specimen dilution are discussed, and the results for 100 human serum specimens are compared with those measured for the same specimens with 4 other urea methods. The effects of measuring a blank, consisting of sample and reagent without urease, with each specimen were examined.