N 1 naphthyl ethylenediamine ned

The level of NO_x in blood samples was determined by a modified method of Miranda et al [13 (link)]. As the first step, blood serum sample deproteinization was achieved by adding equal volumes of 0.3 M NaOH to 100 µL of blood serum. It was mixed well and incubated for 5 min at room temperature. Then 100 µL of 5% ZnSO₄ was added, mixed well and incubated for additional 5 min at room temperature. After the incubation, the mixture was centrifuged at 3,000 rpm at 4 °C for 15 min. An aliquot of 100 µL of the clear supernatant was then mixed with 200 µL of Griess reagent.
Griess reagent was prepared just prior to the assay and contained 0.25% VCl₃, 0.1% sulfanilamide and 0.05% N-(1-naphthyl)-ethylenediamine (NED) (Sigma-Aldrich) in 0.5 M HCl. Reagent blank was the same but contained 100 µL of distilled water instead of the blood serum sample. The mixture was incubated for 30 min at 37 °C and absorbance was measured at 540 nm with a microplate reader (Multiclan GO, Thermo Fisher Scientific, Finland). The standard curve for NaNO₂ was used to calculate total NO_x concentration in the samples [13 (link)].