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.