Thiobarbituric acid reactive substances assay

Thiobarbituric acid reactive substances assay (TBARS) was performed according to Barraza-Garza et al. [21] with slight modifications. After treatment, 100 μL of venom-treated erythrocytes were mixed with 400 μL of thiobarbituric acid reagent (20% trichloroacetic acid; 0.5% thiobarbituric acid and 2.5 N HCl) and the mixture was heated for 1 h at 75 °C. After cooling, the solution was centrifuged at 380 g for 10 min and the absorbance of supernatant was measured at 532 nm in a Helios Omega UV–vis spectrophotometer (Thermo Scientific, USA) using the reaction mixture as a blank. Results were expressed as concentration (μM) of malondialdehyde (MDA) per sample. A calibration curve was prepared with tetramethoxypropane (which is equivalent to the same concentrations of MDA) with concentrations ranging from 1.25 to 20 μM.