Nitrate determination in biological fluids by an enzymatic one-step assay with nitrate reductase.
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.
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Laboratoire Central, Hôpital Albert Chenevier, Créteil, France.
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D000328:Adult; D001230:Aspergillus / Q000201:enzymology; D005260:Female; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D009249:NADP / Q000378:metabolism; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D009566:Nitrates / Q000097:blood* / Q000652:urine*; D010084:Oxidation-Reduction; D012016:Reference Values; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D009566:Nitrates; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D009565:Nitrate Reductases; D050901:Nitrate Reductase