After pre-incubation, [U-14C] glucose (0.055 μCi) plus 5.0 mM of unlabeled glucose or 0.055 μCi [1-14C] acetate plus 1.0 mM of unlabeled acetate were added to the incubation medium. The flasks were gassed with a O2/CO2 (95:5) mixture and sealed with rubber stoppers Parafilm M. Glass center wells containing a folded 60 nm/4 nm piece of Whatman 3 filter paper were hung from the stoppers. After 60 min incubation

at 35 °C in a metabolic shaker (90 oscillations min−1), 0.2 mL of 50% trichloroacetic acid was supplemented to the medium and 0.1 mL of benzethonium selleck hydroxide was added to the center of the wells with needles introduced through the rubber stopper. The flasks were left to stand for 30 min to complete CO2 trapping and then opened. The filter paper were removed and added to vials containing scintillation fluid, and radioactivity was counted ( Assis et al., 2004). Results were calculated as pmol CO2 h−1 g tissue−1. Citrate synthase activity was measured according to Srere (1969), by determining DTNB reduction at λ = 412 nm. The activity of the enzyme aconitase was measured according to Morrison (1954), following the reduction of NADP+ at wavelengths of excitation and emission of 340 and 466 nm, respectively. Isocitrate dehydrogenase Wortmannin chemical structure activity was accessed by the method of Plaut (1969), by following NAD+ reduction at wavelengths of excitation and emission of 340 and 466 nm, respectively. The activity

of α-ketoglutarate dehydrogenase complex was evaluated according to Viegas et al. (2009). The reduction of NAD+ was recorded in a Hitachi F-4500 spectrofluorometer at wavelengths of excitation and emission of 340 and 466 nm, respectively. The activity of succinate dehydrogenase was determined as described by Fischer et al. (1985). Fumarase activity was measured according to O’Hare and Doonan (1985), measuring

the increase of absorbance at λ = 250 nm. Malate dehydrogenase activity was measured according to Kitto (1969) by following the reduction of NADH at wavelengths of excitation and emission of 340 and Niclosamide 466 nm, respectively. The activities of the CAC enzymes were calculated as nmol min−1 mg protein−1, mmol min−1 mg protein−1 or μmol min−1 mg protein−1. The activities of succinate-2,6-dichloroindophenol (DCIP)-oxidoreductase (complex II) and succinate/cytochrome c oxidoreductase (complex II–III) were determined according to Fischer et al. (1985). The activity of NADH/cytochrome c oxidoreductase (complex I–III) was assayed according to the method described by Schapira et al. (1990) and that of cytochrome c oxidase (complex IV) according to Rustin et al. (1994). The methods described to measure these activities were slightly modified, as described in details in a previous report ( Silva et al., 2002). The activities of the respiratory chain complexes were calculated as nmol min−1 mg protein−1 or mmol min−1 mg protein−1.