No std

Heparinized blood samples were centrifuged at 3000 rpm for 10 min at 4°C, and the plasma obtained was stored at −80°C until use. The plasma was mixed with methanol (1:1, v/v) and centrifuged at 10,000 rpm for 10 min at 4°C. The resulting supernatant was used for the following experiments.
For the analysis of nitrite and nitrate, the supernatant was diluted with the mobile phase (NOCARA, Eicom) and injected into an ENO‐20 NOx Analyzer (Eicom). Nitrite and nitrate levels were calculated by comparison with the results obtained from standard solutions (NO‐STD, Eicom).
In the analysis of the thiobarbituric acid‐reactive substances (TBARS), the supernatant was incubated for 45 min at 65°C with thiobarbituric acid (TBA) under acidic conditions. The malondialdehyde (MDA)‐TBA adduct was measured colorimetrically at 532 nm. The TBARS levels were calculated by interpolation of a standard curve of MDA. An assay kit (FR40, Oxford Biomedical Research, Inc.) was used for these measurements.

Plasma was deproteinized by mixing with an equal volume of methanol. The resulting mixture was then centrifuged at 10,000 × g for 10 minutes at 4°C; the supernatant obtained was stored at −80°C for later analysis. NOx concentration was measured using a high-performance liquid chromatography-Griess system (ENO-20, Eicom, Kyoto, Japan). Briefly, the supernatant (10 μL) was diluted with the mobile phase (NOCARA, Eicom) and injected into the system, wherein the analysis was conducted by measuring the absorbance of azo dye formed through a “diazotization-coupling” reaction. Nitrite and nitrate levels were determined through comparison with the results obtained using standard solutions (NO-STD, Eicom).