Luminometric single step urea assay using ATP-hydrolyzing urease

An automatic enzyme kinetic luminometric method for determination of small quantities of urea in biological fluids and in microdialysates is presented. The method is based on the ATP-hydrolyzing urease reaction [urea amidohydrolase (ATP-hydrolyzing); EC [3.5.1.45][1]], monitored by a luciferin-luciferase ATP reaction. The assay range is 100 pmol to 50 nmol with a detection limit of 5 μmol/L in the sample, compared with detection limits of 0.1 mmol/L in earlier spectrophotometric methods. To reduce the non-urea-dependent ATPase activity ( v blank) and to increase the urea-dependent activity, 1,2-propanediol was included. Assay conditions were optimized by multivariate analysis. Recoveries of urea added to blood dialysate and plasma were 96–103%. No analytical interference of common metabolites, drugs, or other additives was observed. The total CVs (6 days and six concentrations, 1.2–21.8 mmol/L) were 3.6–8.5%. The results obtained with the present assay were highly correlated for dialysate ( r = 0.979) and for plasma ( r = 0.978) with those obtained by a spectrophotometric kit method with slopes of 1.02–1.03 and intercepts of 0.08–0.23 mmol/L. [1]: pending:yes