LUMINOMETRIC SINGLE-STEP UREA ASSAY USING ATP-HYDROLYZING UREASE

An automatic enzyme kinetic luminometric method for determination of s mall 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], monitored by a luciferin-luciferase ATP reaction. The assay range is 100 pmol to 50 n mol with a detection limit of 5 mu mol/L, in the sample, compared with detection limits of 0.1 mmol/L, in earlier spectrophotometric methods . To reduce the non-urea-dependent ATP(ase) activity (v(blank)) and to increase the urea-dependent activity, 1,2-propanediol was included. A ssay 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 obse rved. The total CVs (6 days and six concentrations, 1.2-21.8 mmol/L) w ere 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 t hose obtained by a spectrophotometric kit method with slopes of 1.02-1 .03 and intercepts of 0.08-0.23 mmol/L.