*2.5. Brain Oxidative Stress and Inflammatory Parameters*

Protein carbonyl groups were measured spectrophotometrically in the brain homogenate supernatant using 2,4-dinitrophenylhydrazine (DNPH) as previously described [34]. The molar extinction coefficient (ε) of 2.20 <sup>×</sup> <sup>10</sup><sup>4</sup> <sup>M</sup>−<sup>1</sup> cm−<sup>1</sup> was used in the calculations.

The levels of lipoperoxides were quantified by means of the thiobarbituric acid reactive substances (TBARS) assay [35]. The content in TBARS was calculated from the standard curve prepared with 1,1- ,3,3- -tetraethoxypropane.

The content of ROS was quantified via the 2- -7- -dichlorofluorescein-diacetate (DCFH-DA) assay [36], which quantifies the oxidation of DCFH to the fluorescent 2- ,7- -dichlorofluorescein (DCF) in

the presence of ROS. The formation of DCF was measured by spectrofluorimetry (Shimadzu RF-5301; 504 nm for excitation and 529 nm for emission). Although ROS have short half-lives, decomposition in frozen tissue is not very pronounced and the fractional loss and inactivations are independent of their concentrations, so that the proportions between the various conditions are preserved when they are assayed at the same time [9,17].

The rate of mitochondrial ROS production was estimated by measuring the linear fluorescence increase (504 nm for excitation and 529 nm for emission) due to DCF formation from DCFH via oxidation by H2O2 in the presence of horseradish peroxidase [37]. Briefly, intact mitochondria (0.5 mg) were suspended in 2 mL of a mixture containing 250 mM mannitol, 1.36 μM DCFA-DA, 10 mM HEPES buffer (pH 7.2), 10 μM rotenone and 10 mM succinate as respiratory substrate. Fluorescence was recorded during 10 min under agitation. The results were expressed as nmol min−<sup>1</sup> (mg protein)<sup>−</sup>1.

The production of NO was indirectly quantified by measuring the levels of nitrite plus nitrate in the brain homogenate supernatant. Nitrate was first converted into nitrite by adding nitrate reductase and the total nitrite was quantified by the Griess reagent [38].

Reduced (GSH) and oxidized (GSSG) glutathione were measured in the brain homogenate by spectrofluorimetry (excitation at 350 nm and emission at 420 nm) with the *o*-phthalaldehyde (OPT) assay [39]. The activities of catalase, superoxide dismutase (SOD), xanthine oxidase (XO) and myeloperoxidase (MPO) were assayed by spectrophotometry in the supernatant of the brain homogenate. The catalase activity was estimated spectrophotometrically at 240 nm using H2O2 as substrate [40]. The activity of SOD was estimated by the pyrogallol autoxidation method [41]. The activity (MPO) was measured by spectrophotometry with *o*-dianisidine [42]. The XO activity was estimated as the increase in absorbance at 295 nm due to uric acid formation [43].
