Measurement of Plasma and Tissue Nitrate and Nitrite Levels.

Blood samples were collected from anesthetized animals by cardiac puncture into 1-mL syringes containing 0.08 mL 3.8% (wt/vol) sodium citrate and centrifuged at 4 °C, 13,000 × g for 5 min and the plasma collected. Tissue and RBC samples were collected, snap-frozen, and stored at −80 °C. Samples were homogenized in the presence of a protease inhibitor mixture containing 4-(2-Aminoethyl)benzenesulfonyl fluoride (1 mg/mL), antipain, aprotinin, benzamidine, leupeptin, and pepstatin A, all at a concentration of 10 μg/mL, using a Precellys homogenizer at 4 °C and the homogenate centrifuged at 10,000 × g, 5 min, 4 °C, and the supernatant collected. Plasma and tissue supernatant were filtered using Sartorius Vivaspin 500 3,000 molecular weight cut-off PES (Sartorius Stedim Biotech) at 4 °C, 14,000 × g for 60 min (plasma) or 90 min (tissue). Before use, filters were washed twice with low NO_x containing 18 MΩ dH₂O. For RBC determination, the compacted pellet was resuspended 1:4 in “nitrite preserving solution” containing potassium ferriccyanide (0.8 mol/L; Sigma) and N‐ethylmaleimide (0.1 mol/L; Sigma) and then deproteinated using ice‐cold methanol. Briefly, to determine total NO_x concentration, samples were added to 0.1 mol/L vanadium (III) chloride in 1 mol/L hydrochloric acid refluxing at 95 °C under N₂. Nitrite concentration was determined by addition of samples to 0.09 mol/L potassium iodide in glacial acetic acid under nitrogen at room temperature. Nitrate concentration was calculated by subtraction of the nitrite concentration from the total NO_x.