Kinetic assay of serum and urine for urea with use of urease and leucine dehydrogenase

Abstract

We describe a new kinetic assay for determining urea in serum or urine with use of urease (EC 3.5.1.5) and leucine dehydrogenase (EC 1.4.1.9). The latter enzyme is suitable for the kinetic assay of NH4+ because its K(m) value for NH4+ at pH 8.75 is large (~500 mmol/L). Interference from endogenous NH4+ in serum or urine is obviated by subtraction of the assayed endogenous NH4+ value in a sample blank. For serum, within-assay CVs (n = 10) were 0.39-0.58%; day-to-day CVs (n = 10) were 1.56-2.30%. In urine, within-assay CVs (n = 10) were 0.86-1.15%. Analytical recovery of urea (0.893-71.4 mmol/L) added to patients' sera (urea 6.14 mmol/L) was 99.2- 105.2%. The calibration curve for serum was linear through zero for urea concentrations up to 142.9 mmol/L and for urine up to 714.3 mmol/L. No influences of added ammonium ion, bilirubin, hemoglobin, ascorbic acid, or Intralipid were observed. The regression equations for this method (y) and conventional methods (x = Deierminer-LUN for serum assays, Serotec UUR-R for urine) were: y = 1.016x - 0.12 mmol/L (r = 0.999, S(y/x) = 0.34 mmol/L, n = 100) for sera, and y - 1.070x - 12.6 mmol/L (r = 0.998, S(y/x) = 7.41 mmol/L, n = 100) for urine.