Measurement of serum malondialdehyde (MDA) levels

A thiobarbituric acid (TBA) reactive substances assay was performed, with a minor modification, as previously detailed by Van Ye et al (24). The modification was as follows: proteins were precipitated to remove the adverse effects of protein residues on the experiment. The sample was mixed with 20% (w/v) trichloroacetic acid and the precipitate was then centrifuged at room temperature for 10 min at 1,100 × g. The reaction with TBA at 90–100°C was used to determine the MDA level, as MDA or similar substances react with TBA and produce a pink pigment (25) that has an absorption maximum of 532 nm (26). To ensure protein precipitation, the sample is mixed with 4°C 20% (wt/vol) trichloroacetic acid and the precipitate is then centrifuged for 10 min at 1,100 × g and room temperature to form a pellet. An aliquot of the supernatant is then placed into an equal volume of 0.6% (wt/vol) TBA in a boiling water bath for 30 min. Following cooling, sample and blank absorbance were read at 532 nm and the results were expressed as nM/ml, based on a graph by our group where 1,1,3,3-tetramethoxypropane was used as the MDA standard (27).