Measurement of Nitric Oxide Levels

Nitric oxide production (total concentrations of nitrate and nitrite) was measured using a QuantiChrom Nitric Oxide Assay Kit (Bioassay Systems, Hayward, CA, USA), according to the Griess method [24 (link),25 (link)]. A sodium nitrite standard curve was prepared (0–150 μM). Deproteination of the plasma was accomplished by mixing 150 μl of plasma with 8 μl ZnSO₄, followed by mixing with 8 μl NaOH, vortexing, and centrifuging at 12,000 × g at 4°C for 10 min. Subsequently, 100 μl of each supernatant and the standard were added to 200 μl of the working reagent and incubated at 37°C for 60 min. After centrifugation of the reacted samples at 6,200 × g at 4°C for 5 min, the supernatant was diluted 2-fold with distilled water. Optical density was measured at 550 nm using an Eppendorf BioPhotometer Plus (Eppendorf, Hamburg, Germany). The total nitrite concentrations (μM) were determined according to the sodium nitrite standard curve.