Assay validation for determining nitrites and nitrates in biological fluids

• Published in: Revista de investigacion clinica Vol. 55; no. 6; pp. 670 - 676
• Main Authors: Muñoz-Fuentes, Rosa María, Vargas, Florencia, Bobadilla, Norma A
• Format: Journal Article
• Language: Spanish
• Published: Mexico 01.11.2003
• Subjects: Body Fluids - chemistry; Humans; Nitrates - analysis; Nitrites - analysis; Reference Values
• ISSN: 0034-8376

Nitric oxide is an important modulator of numerous physiological and pathophysiological processes. An indirect form to detect NO production has been the quantification of its stable end products, nitrites and nitrates (NO2- + NO3-). These metabolites can be detected with a commercial kit, but it is somewhat expensive and not accessible to some laboratories in our country. To validate an easy, accessible and less expensive assay for detecting nitrates and nitrites in biological fluids. In this study we determined nitrates and nitrites by reducing nitrates enzymatically with nitrate reductase, followed by nitrites quantification using the Griess reagent. To validate the assay, NO3- concentration was evaluated in aliquots with known amounts of sodium nitrate, also NO2- + NO3- concentrations were detected in plasma containing known amounts of sodium nitrate, finally NO2- + NO3- levels were evaluated in plasma (n = 17) and ascites (n = 11) samples of cirrhotic patients. In samples of patients, NO2- + NO3- levels were also detected by using a commercial kit. The assay that we describe here detects nitrates in the range between 25 to 400 microM/L and nitrites between 25 to 100 microM/L. When specific concentrations of nitrates were added to plasma samples, the recovery percentage in most cases was greater than 95%. In plasma samples of cirrhotic patients, average concentrations of NO2- + NO3- was 44.6 +/- 22.4 microM (mean +/- SD), similar to that found using the commercial kit, 40.9 +/- 18.3 microM/L (p = 0.107). In ascitis samples, similar results using both methods were seen, 64.5 +/- 42.0 vs. 58.2 +/- 39.3 microM/L (p = 0.172) respectively. Our results suggest that the method described here could be considered as an alternative instead of commercial kits to determine NO2- + NO3- in plasma and ascites samples. In addition, this assay results more attractive because, it does not, require special equipment, it is very accessible to most laboratories, it may be use to assay one or more samples at any given time, but the most important advantage, is its cost effectiveness; thus each sample determination is about one fifth of the cost using the commercial kit.