Analysis of Oxidation and Antioxidants Using Microtiter Plates

Measurement of thiobarbituric acid reacting substances (TBARS), diene derivatives, and hydroperoxides are probably the most widely used procedures for evaluation of free radical-mediated oxidation of biological or model systems containing polyunsaturated fatty acids (PUFA). It is possible to write a sequence of rational reactions in which dienes formation comes first, followed by lipid hydroperoxides (LOOHs) production, and that terminates with fragmentation of the PUFA chains to carbonyl compounds that are TBARS. However, it appears that after the first few min, species of the three types co-exist even in simple suspensions of linoleic acid (1 ,2 ). These compounds are very reactive, especially in biological systems, and follow different rates of formation and conversion. Therefore, it is more informative to follow their kinetics of appearance and disappearance than to measure single time-points (3 –5 ).