Quantifying Nitrite Levels in Rat Plasma

After the functional parameter measurements, all rats were deeply anesthetized by an overload of xylazine hydrochloride and ketamine hydrochloride (Anasedan^®, 20 mg/kg and Dopalen^®, 160 mg/kg, i.v., respectively, Ceva Sante Animale, Paulínea, SP, Brazil) and euthanized by decapitation. Blood samples were collected in heparinized vacutainers immediately after the euthanasia and centrifuged at 4,000 rpm for 5 min for analysis of nitrite concentration as previously published (Jacomini et al., 2017 (link)). In summary, proteins were quantified using automated biochemistry equipment (model A-15, Biosystems S/A, Barcelona, Spain) to normalize the calculation of nitrite concentration. Nitrites (NO₂^–), metabolites of NO, were determined in plasma using Griess reagent in which a chromophore with a strong absorbance at 540 nm is formed by the reaction of nitrite with a mixture of naphthyl ethylenediamine (0.1%) and sulfanilamide (1%). Samples were analyzed in duplicate, and plasma results are expressed as nmol/mg of protein.