In vitro testing of antioxidants and biochemical end-points in bovine retinal tissue

Lipid peroxidation in aliquots of bovine retina (without rod outer segments , ROS), purified ROS and retinal pigment epithelium (RPE) was initiated wit h 5 mM ferric iron and 80 mM ADP. After 30 min of oxidation at 37 degrees C , the concentration of thiobarbituric-acid-reacting substances (TBARS) whic h approximates lipid hydroperoxide (LHP), increased in the ROS from 2.0 +/- 3.6 to 90.2 +/- 34.5 nmol malondialdehyde (MDA)/mg protein and in the RPE from 0.54 +/- 0.2 to 51.5 +/- 15.8 nmol MDA/mg protein. Sixteen lipid and a queous antioxidants (AOX) from natural or synthetic sources, including five flavonoids, were evaluated for their ability to inhibit the oxidative reac tion. Palm-oil-derived vitamin E showed significant protection in retina, R OS and RPE (64, 68 and 74%), respectively Of the flavonoids tested, good pr otection in the retina was found at 10(-5) M for epigallo-catechin gallate (50%) and at 50 ng/ml for pycnogenol (61%) and catechin (52%). When catechi n and palm oil vitamin E, catechin and coenzyme Q(10) or coenzyme Q(10) and pycnogenol were combined, the individual effect was enhanced. TEARS as an indirect measure of LHP level and hemoglobin-methylene blue determination f or direct LHP were used as alternative end-point determinations of lipid pe roxidation. These measure different aspects of AOX reactions. The results d emonstrate the usefulness of an in vitro model system that can rapidly and accurately determine the capacity of a single AOX against lipid peroxidatio n or be used to show synergistic efficacy.