Thiobarbituric acid tba

The liposomes (1 mM), mitochondria (1 mg/mL), or liver homogenates (1 mg/mL) were incubated with or without different Ro concentrations at 37 °C for 30 min, and with 50 μM Fe(NH₄)₂(SO)₄ (plus 2.0 mM sodium citrate) or 0.6 mM t-BOOH as inducers of oxidative stress. Following incubation, for a thiobarbituric acid reactive substances (TBARS) assay, 1% thiobarbituric acid (TBA, Sigma-Aldrich, USA) was prepared by adding 50 mM NaOH, 15 μL of 10 M NaOH, and 75 μL of 20% H₃PO₄ to each sample, followed by further incubation for 20 min at 85 °C. The MDA-TBA complex was extracted with 2 mL of n-butanol, and the absorbance was measured at 535 nm. The TBARS were calculated from ε = 1.56 × 10⁵ M⁻¹.cm⁻¹, as described in [53 (link)]. Lipid hydroperoxides (LOOH) were also quantified in isolated mitochondria using xylenol orange, as previously described in [54 (link)]. When applied, the percentages of inhibition by Ro were calculated in relation to positive controls (t-BOOH or Fe²⁺) which were considered to achieve 100% inhibition.

Lipid peroxidation was assessed via the measurement of malondialdehyde (MDA) production employing the TBARS assay, adapted for a 96-well plate and ELISA reader. Samples were treated with 5% sodium dodecyl sulfate (SDS; Sigma-Aldrich, St. Louis, MO, USA) and exposed to 0.53% thiobarbituric acid (TBA; Sigma-Aldrich, St. Louis, MO, USA) dissolved in 20% acetic acid adjusted with NaOH (Centralchem, Bratislava, Slovakia) to pH 3.5, and afterwards boiled at 90–100 °C for an hour. Subsequently, the samples were placed on ice for 10 min to stop the reaction. The samples were subjected to centrifugation (1750× g for 10 min), and the obtained supernatant was analyzed for the concentration of MDA. The analysis was performed on Multiskan FC microplate photometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 530–540 nm [38 (link)]. The content of MDA is expressed as μmol/g protein.