4.6. TBARS Assay

The TBARS assay was performed according to the method previously reported by Kikugawa [50], with some modifications. This was carried out to measure lipid peroxidation in the liver tissues. First, the TBARS reaction mixture was made by adding the following solutions to one bottle: 2 mL of 5.2% sodium dodecyl sulfate (SDS) in Milli-Q water, 15 mL of a 0.8% solution of thiobarbituric acid (TBA) in Milli-Q Water, 19.125 mL of Milli-Q water alone, 500 µL of a 0.8% solution of butylated hydroxytoluene (BHT) in glacial acetic acid, and 8 mL of a 0.1 M acetate buffer (pH 3.5). The total volume of the mixture was then 42.75 mL. After that, the TBARS mixture and liver tissues were mixed at a ratio of 9:1 (TBARS mixture µL:liver mg) in a micro-tube, and the tissues were homogenized under a bead crusher with zirconia beads. We transferred 500 µL of the homogenate to a new micro-tube, and the samples were incubated at 4 °C for one hour, followed by incubation at 95 °C for 1 h. After cooling the samples to room temperature, 500 μL of 15:1 v/v 1-butanol and pyridine was added to the chilled tubes, after which they were shaken. The mixtures were centrifuged at 3000 rpm for 10 min at 4 °C. The fluorescence of the supernatant was measured at 540 nm excitation and 590 nm emission. The TBARS concentrations (nmol/mL) were calculated from a regression equation plotted with a 1,1,3,3-tetraethoxypropane standard (200 to 0.4 nmol/mL). Finally, the quantified values were converted to TBARS nmol/g liver. This assay was conducted in duplicate measurements.