Cerebral cortex was homogenized in 10 volumes of 0.32 mM sucrose solution containing 5.0 mM HEPES and 1.0 mM EDTA. Membranes were prepared according to the method of Jones and Matus (1974) PF-562271 datasheet using a discontinuous sucrose density gradient consisting of successive layers of 0.3, 0.8 and 1.0 mM. After centrifugation at 69,000 × g for 2 h, the fraction at the 0.8–1.0 mM sucrose interface was taken as the membrane enzyme preparation. TBA-RS levels were measured according to the method described by Yagi (1998) with slight modifications. Briefly, 200 μL of 10% trichloroacetic acid and 300 μL of 0.67% TBA in 7.1% sodium sulfate were added to 100 μL of

tissue supernatant and incubated for 2 h in a boiling water bath. The mixture was allowed to cool on running tap water for 5 min. The resulting pink-stained complex was extracted with 400 μL of butanol. Fluorescence of the organic phase was read at 515 and 553 nm as excitation and emission wavelengths, respectively. MS-275 mw Calibration curve was performed using 1,1,3,3-tetramethoxypropane and subjected to the same treatment as supernatants.

TBA-RS levels were calculated as nmol TBA-RS/mg protein. This assay is based on the reduction of 5,5′-dithio-bis (2-nitrobenzoic acid; DTNB) by thiols, generating a yellow derivative (TNB), whose absorption is measured spectrophotometrically at 412 nm (Aksenov and Markesbery, 2001). Briefly, 30 μL of 10 mM DTNB and 980 μL of PBS were added to 50 μL of cerebral cortex supernatants. This was followed by 30-min incubation at room temperature

in a dark room. Absorption was measured at 412 nm. Results are reported as nmol TNB/mg protein. Protein carbonyl content formation, a marker of oxidized proteins, was measured spectrophotometrically according to Levine et al., 1994 and Reznick and Packer, 1994. One hundred microliters of the aliquots from the incubation was treated with 400 μL of 10 mM 2,4-dinitrophenylhidrazine (DNPH) dissolved in 2.5 N HCl or with 2.5 N HCl (blank control) and left in the dark for 1 h. Samples were Ribose-5-phosphate isomerase then precipitated with 500 μL 20% TCA and centrifuged for 5 min at 10,000 × g. The pellet was then washed with 1 mL ethanol/ethyl acetate (1:1, v/v) and re-dissolved in 550 μL 6 M guanidine prepared in 2.5 N HCl. Then, the tubes were incubated at 37 °C for 5 min to assure the complete dissolution of the pellet and the resulting sample was determined at 365 nm. The difference between the DNPH-treated and HCl-treated samples was used to calculate the carbonyl content. The results were calculated as nmol of carbonyls groups/mg of protein, using the extinction coefficient of 22,000 × 106 nmol/mL for aliphatic hydrazones. Nitrate and nitrite concentrations were determined according to Miranda et al. (2001).