Chemical inhibition used in a kinetic urease/glutamate dehydrogenase method for urea in serum.
We describe a fixed-time-interval, kinetic inhibition method, with use of a competitive inhibitor (l) of the urease/glutamate dehydrogenase reaction to increase the "apparent" Michaelis constant by a factor of (1 + [l]lKl). This allows greater flexibility in selecting an appropriate sample dilution for kinetic determinations of urea in serum (i.e., [S]lKm 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 dilution of 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 greater than 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.
D001322:Autoanalysis; D001663:Bilirubin / Q000097:blood; D003124:Colorimetry; D005459:Fluorides / Q000494:pharmacology; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D008722:Methods; D014508:Urea / Q000097:blood*; D014510:Urease / Q000378:metabolism
D014508:Urea; D005969:Glutamate Dehydrogenase; D014510:Urease; D005459:Fluorides; D001663:Bilirubin; D006918:Hydroxyurea