2.2.6. Thiobarbituric Acid Reactive Substances Assay

This assay was performed for the evaluation of the lipid peroxidation level by quantifying plasmatic MDA concentration, its major final product [21,22]. Plasma samples were mixed with equal volumes of 5% TCA and Tris-HCl (0.2 M) pH = 4.7 (v/v) and incubated 10 min at RT. Next, 0.55 M thiobarbituric acid (TBA) solution in sodium sulphate (2 M) was added and the mixture was incubated at 90 °C for 45 min [24,25]. After ice cooling, a 3 min centrifugation (15,000× g) was carried out with the same refrigerated centrifuge. During this procedure, a coloured product between MDA and TBA is formed, with its absorbance at 532 nm being measured with the same UV/VIS spectrophotometer Kruss. The molar extinction coefficient of MDA (1.55 × 105 M⁻¹cm⁻¹) supported our calculation of TBARS concentration, expressed as μmol/L. Almost all the reagents were provided from Sigma-Aldrich, Germany, except TBA, obtained from Fluka.