Chemical inhibition used in a kinetic urease/glutamate dehydrogenase method for urea in serum

Abstract

We describe a fixed-time-interval, kinetic inhibition method, with use of a competitive inhibitor (/) of the urease/glutamate dehydrogenase reaction to increase the 'apparent' Michaelis constant by a factor of (1 = [I]/Ki). This allows greater flexibility in selecting an appropriate sample dilution for kinetic determinations of urea in serum (i.e., [S]/K(m) ratio). Nine compounds were screened as potential inhibitors for this study. Adding 5 mmol of hydroxyurea per liter increases the 'apparent' Michaelis constant for the coupled enzyme reaction by 10-fold. We used a sample ditlution dilution 21-fold vs. dilutions of 141-to 350-fold for previously reported kinetic methods. Mean analytical recovery with this method was 100.2%. Reaction rate vs. urea concentration was linear, and complete recovery extended to 30 mmol of urea per liter. Of 22 potential interferents, only fluoride (250 mmol/L) and bilirubin (1 mmol/L, or 580 mg/L) caused >5% interference. We discuss precision and effects of specimen dilution, and compare results for 100 specimens with those by a manual Berthelot-indophenol method, a manual diacetyl monoxime method, and a diacetyl monoxime method adapted to continuous-flow analysis.