Measurement of NO serum levels
Blood samples for serum levels of NO were collected on day one, i.e., within 24 h of enrollment in the study. The NO level in whole blood is determined by measuring nitrite and nitrate (NO32- u NO22-) production using the classical colorimetric reaction (Griess). Blood samples for the determination of NO concentration were diluted 1:1 (vol/vol) with 0.9% saline, protein-precipitated using 30% ZnSO4, 0.05 ml per ml of blood and centrifuged at 700 g for 10 minutes and frozen at -20°C. Conversion of NO32- into NO22- was done with nitrate reductase elementary zinc. NO22-concentration in serum was determined by classic colorimetric Griess reaction. Briefly, equal volumes of samples and Griess reagent (sulfanilamide and naphthalene-ethylene diamine dihydrochloride) were mixed at room temperature. After 5 min, the absorbance was measured at 546 nm using spectrophotometer. The concentration of nitrite was determined by a standard curve prepared with sodium nitrite.