Enzymatic assay for determination of bicarbonate ion in plasma using urea amidolyase

Abstract

Background: One of the important buffering systems to maintain blood pH is carbonic acid–bicarbonate. Together with other clinical tests, the measurement of bicarbonate ion concentrations is widely used for the diagnosis of the acid–base balance. We developed a kinetic assay for measurement of bicarbonate ion in plasma using urea amidolyase (EC 3.5.1.45) from yeast species. We evaluated the analytical performance of the present enzymatic method and examined the relationship between bicarbonate ion concentrations by present method and with ABL 520 blood gas system. Methods: Urea amidolyase catalyzes the reaction of bicarbonate ion with urea to rise to allophanate. We eliminated endogenous ammonium ion by the use of glutamate dehydrogenase (EC 1.4.1.4), and then monitored the production of ammonium ion in the presence of urea amidolyase, urea, ATP, potassium, and magnesium ions. Ammonium ion was produced proportional to the bicarbonate ion concentration and was determined by adding glutamate dehydrogenase to produce NADP + in the presence of 2-oxoglutarate and NADPH, and the change of absorbance at 340 nm was monitored. Results: The within-assay and day-to-day assay coefficient variations (CVs) of the present method were 1.3–2.8% and 3.1–5.4%, respectively. The analytical recoveries were 90–110%. The presence of ascorbic acid, bilirubin, hemoglobin, lipemic material, hydrogen phosphate, dihydrogen phosphate, ammonium, or calcium ion did not affect this assay. The correlation coefficient between the values obtained by present method ( y) and Radiometer ABL 520 blood gas system ( x) was 0.983 ( y=1.029 x−0.737 mmol/l, Sy/ x=0.764, n=100), with a mean difference of 0.03±0.77 mmol/l [(values by reference method−that of present method)±S.D.] using the Bland–Altman technique.