Lipoprotein oxidation and measurement of thiobarbituric acid reacting substances formation in a single microtiter plate: its use for evaluation of antioxidants.

Transition metals catalyze free radical-mediated oxidation of lipids and lipoproteins. This process is currently studied because of its potential relevance to pathological processes like atherosclerosis. Formation of thiobarbituric acid-reacting substances from polyenoic fatty acids is frequently used to follow oxidation of lipids and plasma lipoproteins. We describe here how Cu(II)- and Fe(III)-catalyzed oxidation of human low density lipoprotein or soy bean phospholipids and the photometric evaluation of the thiobarbituric acid-reaching substances formed can be conducted in the same 96-well microtiter plate. The procedure showed a correlation of 0.98 with conventional two-stage fluorimetric and spectrophotometric methods and also showed better reproducibility. The plate method can handle up to one plate per hour with considerably less labor than the test tube assays. The plate procedure required small volumes of diluted samples of lipoproteins lipids and reagents. The method was suitable for testing the concentration-dependent antioxidant potency of substances like probucol, butylated hydroxytoluene, and alpha-tocopherol. The method can also be used to follow the kinetics of oxidation of lipoproteins.