The thiobarbituric acid (TBA) assay was developed to quantitatively determine lipid peroxidation for aldehydic compounds in biological matrices. Kohn and Liversedge introduced this methodology in 1944 ( 1 , 2 ). Since its introduction, the TBA assay has generated widespread interest in providing valuable information in the assessment of free radical-mediated damage owing to various disease pathologies as well as peroxidation of fatty acids, foods from plant and animal sources, cell membranes ( 2 – 4 ), and rat-liver microsomes ( 3 – 5 ). A biological marker that indicates oxidative stress with respect to lipid peroxidation in body fluids or cells is malondialdehyde (MDA). MDA is a byproduct of the arachidonate cycle, as well as lipid peroxidation ( 6 – 8 ) and is detectable in quantifiable amounts employing the TBA assay. TBA and MDA react to form a schiff base adduct (illustrated in Fig. 1 ) under high temperature/acidic conditions to produce a chromogenic/fluorescent product that can be easily measured employing various analytical techniques such as spectrophotometric ( 7 , 9 – 11 ) or fluorometric methods ( 6 , 12 – 14 ). Incorporating HPLC with ultraviolet (UV)/fluorometric detection or gas chromatography-mass spectrometry (GC-MS) have also been used previously to determine TBA-MDA adducts ( 2 – 3 , 5 , 15 – 17 ) but those methodologies are beyond the scope of this investigation. Our laboratory has developed a quick, simple, and reliable bioanalytical assay using a fluorescence microplate reader in the detection, as well as quantification of the TBA-MDA adduct (lipid peroxidation product) in rat liver microsomes, which we describe here. Fig. 1. The chemical reaction between TBA and MDA to yield the TBA-MDA adduct as discussed in the text.
