Luminometric single step urea assay using ATP-hydrolyzing urease

Abstract

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], 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 ATP(ase) activity (υ(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.