Nitrate determination in biological fluids by an enzymatic one-step assay with nitrate reductase

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 41; no. 6; pp. 904 - 907
• Main Authors: Bories, PN, Bories, C
• Format: Journal Article
• Language: English
• Published: Washington, DC Am Assoc Clin Chem 01.06.1995; American Association for Clinical Chemistry
• Subjects: Adult; Analysis of complex biological substances; Analytical, structural and metabolic biochemistry; Aspergillus - enzymology; Biological and medical sciences; Blood and biological fluids; Female; Flavin-Adenine Dinucleotide - metabolism; Fundamental and applied biological sciences. Psychology; Humans; Male; Middle Aged; NADP - metabolism; Nitrate Reductase; Nitrate Reductases - metabolism; Nitrates - blood; Nitrates - urine; Oxidation-Reduction; Reference Values; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Human; NADPH; Urine; Biological fluid; Enzyme; Interference; Nitrates; Method; Nitrate reductase; Clinical biology; Nitrogen monoxide; Serum; Oxidoreductases; Quantitative analysis
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1093/clinchem/41.6.904

We report a simple, enzymatic, end point method for determining nitrate in serum and urine with use of nitrate reductase from Aspergillus sp. (EC 1.6.6.2). The decrease in absorbance at 340 nm as a result of the oxidation of beta-NADPH is measured. We used FAD as a supplementary electron carrier and added an internal standard to avoid interference from possible inhibitors of the enzymatic reaction. The method was linear from 5 to 200 mumol/L nitrate in serum. Within-run (n = 12) and total (n = 14 days) imprecisions (CV) were 5.1-7.7% and 6.2-9.8%, respectively, at 20-90 mumol/L nitrate in serum. Recoveries of added nitrate were 80-106%. The median (range) concentration in serum of 20 healthy subjects was 16 (0-42) mumol/L.