2.5. Plasma Thiobarbituric Acid Reactive Substances (TBARS)

This test is based on the reaction between malondialdehyde (MDA) and thiobarbituric acid (TBA) (0.11 mol/L) (Sigma, St. Louis, MO, USA) in phosphoric acid (H₃PO₄) (0.2 mol/L) (Sigma, St. Louis, MO, USA) which results in formation of the adduct TBA-MDA by oxygen removal and thereby produces a pink pigment with absorption at 535 nm. Possible amplification of the peroxidation during the assay is prevented by the addition of the antioxidant butylated hydroxytoluene (BHT) (Sigma, St. Louis, MO, USA) (12.6 mM) [14]. A mix of 200 µL of plasma, 25 µL of BHT, 200 µL of H₃PO₄, and 25 µL of TBA were incubated for 45 min at 90 °C. To stop the reaction, the mix was cooled in ice and later 500 µL of butanol (Sigma, St. Louis, MO, USA) were added alongside 50 µL of a saturated solution of sodium chloride (NaCl) (Sigma, St. Louis, MO, USA). The absorbance was read at 535 nm and 572 nm to correct for baseline absorption. The quantification was done using a calibration curve.