Spectrophotometric method for simultaneous detection of nitrate and nitrite

Concentration of nitric oxide can be measured by variety of methods. Its short half life and values of low detectability decrease clinical importance of these methods. Deficiencies of methods used for NO measurement can be eliminated by measurement of stable NO products such as nitrites and nitrates. In this study we have modified a method for simultaneous evaluation of nitrate and nitrite concentrations. Human sera were collected from blood donor volunteers. Before testing the samples were deproteinized. Reduction of nitrate was achieved with vanadium(III). Nitrite concentration was measured by Griess reaction. The nitrate concentration was calculated as difference of NOx (nitrites and nitrates), determinate in presence of vanadium (III) and nitrites concentration. This assay has shown sensitivity to 0,5μM NO3 and is useful in variety of fluids including serum, plasma and cell culture media. We have examined the influence of various factors on detection of NOX, such as reagent composition, volume, and temperature. The method has shown higher sensitivity when Griess reagent with HCl was used, compared to use of Griess reagent with H3PO4. It has been also noticed that regardless of which Griess reagent composition was used, sensitivity of this reaction was higher when samples were incubated at 37°C instead at 25°C. AU incubations lasted 30 minutes.