Nitric oxide bioavailability

The method consisted of simultaneous evaluation of nitrate and nitrite concentrations. The principle of this assay is reduction of nitrate by vanadium (III) combined with detection by the acidic Griess reaction. This assay is sensitive to 0.5 μM NO₃⁻ and is useful in plasma. After deproteinization, 100 μL of supernatants were distributed to 96-well plates in triplicate and 100 μL of Griess reagent (1% sulfanilamide, 0.1% N-(1-Naphthyl)ethylenediamine dihydrochloride and 2.5% H₃PO₄) (Sigma-Aldrich, St Louis, MO, USA) was added. The plates were incubated at room temperature and optical density was read at 540 nm. The NO concentration was calculated from a standard curve generated with NaNO₂ ranging from 0 to 200 μM.25