A Spectrophotometric Assay for Nitrate Using NADPH Oxidation by Aspergillus Nitrate Reductase

An assay based on the oxidation of NADPH during the enzymatic conversion of nitrate to nitrite by Aspergillus nitrate reductase [EC 1.6.6.2.] was developed for specific quantification of nitrate. This spectrophotometnc method was used to measure nitrate present in human urine, human serum, and tissue culture medium. Used as a kinetic assay, the method exhibited (1) linearity over a range of 1.25 to 40 μM nitrate, (2) an upper sensitivity of 20 μM, (3) a lower sensitivity of 1.25 μM nitrate, and (4) intraday and interday variability ranging from 0.6 to 6.1%. To judge the acceptability of this method as a kinetic assay, we determined the K m for Aspergillus nitrate reductase to be 199 μM. The K m was based on analyzing three separate lots of commercially purified enzyme. Mean nitrate content of eight urine specimens analyzed by this assay (1111 μM) was not significantly different from that determined by a chemiluminescence method (1144 μM). Analysis of serum using the two methods showed mean nitrate concentrations of 23 and 36 μM, respectively. Based on serial dilutions of serum, the lower nitrate content of serum observed with nitrate reductase assay could not he explained by the presence of inhibitors. Rat pulmonary alveolar macrophages were induced to produce nitric oxide which oxidizes to nitrite and nitrate. Nitrite and nitrate present in tissue culture medium of unactivated and activated macrophages were in proportion to total nitrogen oxides (NO x) determined by the chemiluminescence method. We conclude that the Aspergillus nitrate reductase assay is an accurate spectrophotometric method for determining nitrate content of human urine and tissue culture supernatants.