4.5. Lipid Peroxidation (MDA) Assay

Determination of malondialdehyde (MDA) levels was evaluated using the commercial kit “MDA Assay Kit” (Biovision, Milpitas, CA, USA), which measures the reaction of MDA with thiobarbituric acid (TBA) and the MDA-TBA adduct formation. To accomplish this, human whole blood aliquots (100 μL) were resuspended in 200 μL MDA lysis buffer with 2 μL butylated hydroxytoluene (BHT) (0.1 mM), sonicated and then centrifuged at 13,000× g for 10 min. The supernatants (200 μL) from each sample were added to 600 μL of TBA and incubated in a water bath at 95 °C for 60 min. Samples were cooled down on ice for 10 min, and 300 μL of n-butanol (Sigma-Aldrich, St. Louis, MO, USA) were added to create an organic phase in which the MDA molecules were to be placed. Samples were centrifuged for 10 min at 13,000× g at room temperature and 200 μL of the supernatants (upper organic phase) were collected and dispensed into a 96-well microplate for spectrophotometric measurement at 532 nm. MDA supplied in the kit was used as a standard, and MDA levels were determined by comparing the absorbance of samples with that of the standards. To prevent further formation of MDA during the preparation of the sample or during the heating step, an antioxidant was used. BHT, which is one of the most widely-used compounds, was applied when performing this assay. The protein content of the samples was determined by following the bicinchoninic acid (BCA) protein assay kit protocol (Sigma-Aldrich) and by using serum albumin (BSA, Sigma-Aldrich, USA) as the standard. Results were expressed as nmol of MDA per mg of protein.