Automated enzymatic determination of urinary nitrates in humans

Urinary nitrate determinations are used to evaluate the in vivo synthesis of nitric oxide, the major endothelial-derived relaxing factor, In the present study, the enzymatic method using the Aspergillus nitrate reductase was applied to an autoanalyzer to determine urinary nitrate in 17 healthy men. This method was fast and required a small amount of biologic sample (7 mu L). Linearity of the assay was between 0.062 mu mol/mL and 1.00 mu mol/mL; accuracy, measured as percentage of nitrate recovered. ranged from 96% to 118%: between-day imprecision, assessed as coefficient of variation, ranged from 3.5% to 4.5%. The mean urinary nitrate concentration was 0.73 +/- 0.29 mu mol/mL (range, 0.26 to 1.26 mu mol/mL). To carefully quantify nitrate production, urinary nitrate excretion rates were also measured. Rates averaged 0.71 +/- 0.18 mu mol/min (range, 0.44 to 0.98 mu mol/min). Nitrate excretion rates were much less variable than nitrate concentrations. Nitrate excretion rates, but not concentrations, differed significantly in smokers (n = 7, 0.54 +/- 0.08 mu mol/min) and nonsmokers (n = 10, 0.85 +/- 0.11 mu mol/min). In conclusion, (1) the nitrate reductase assay applied to an autoanalyzer is an accurate, fast, and convenient method for the determination of urinary nitrates in humans; (2) urinary nitrate excretion rate is less variable than nitrate concentration; and (3) cigarette smoking is associated with a diminished nitrate excretion rate, Further studies are needed to assess the nitric oxide generation rate in these subjects.