Thiobarbituric Acid Reactive Substance (TBARS) Assay

Thiobarbituric acid reactive substance (TBARS) assay was conducted according to the method of Oakes et al.46 A molecule of malondialdehyde (MDA) forms an adduct with two TBA molecules to produce a red chromogen that absorbs ultraviolet-light at 532 nm. This assay was done by measuring the level of MDA spectrophotometrically. Primarily, 1 mL of the liver homogenate was mixed with 1 mL TCA (10% w/v) and centrifuged at 3000 rpm for 10 min. Then, 1 mL of TBA (0.67% w/v) was added to an equal volume of supernatant and incubated at 95°C for 60 min. The mixture was allowed to cool to room temperature in an ice bath for 10 min. One mL of n-butanol was added to precipitate the MDA-TBA complex. Then, n-butanol was removed and the precipitate was dissolved in water. Absorbance was recorded at 532 nm against an appropriate blank without the sample. The content of TBARS in the samples was calculated and the results were expressed as MDA equivalents in nmol/g of liver tissue.