Measuring Lipid Peroxidation by Modified TBARS Assay

A modified thiobarbituric acid-reactive species (TBARS) assay [14 (link)] was used to measure the lipid peroxide formed, using egg-yolk homogenates as lipid-rich media [15 (link)]. Malondialdehyde (MDA), a secondary product of the oxidation of polyunsaturated fatty acids, reacts with two molecules of thiobarbituric acid (TBA), yielding a pinkish red chromogen with an absorbance maximum at 532 nm [16 (link)]. Egg homogenate (250 μL, 10% in distilled water, v/v) and 50 μL of extract were mixed in a test tube and the volume was made up to 500 μL, by adding distilled water. Finally, 25 μL “FeSO₄” (0.07 M) was added to the above mixture and incubated for 30 min, to induce lipid peroxidation. Thereafter, 750 μL of 20% acetic acid (pH 3.5) and 750 μL of 0.8% TBA (w/v) (prepared in 1.1% sodium dodecyl sulphate) and 25 μL 20% TCA were added, vortexed, and then heated in a boiling water bath for 60 min. After cooling, 3.0 mL of 1-butanol was added to each tube and centrifuged at 3000 rpm for 10 min. The absorbance of the organic upper layer was measured against 3 mL butanol at 532 nm. For the blank 50 μL of distilled water was used in place of the extract.