In vitro Testing of Antioxidants and Biochemical End-Points in Bovine Retinal Tissue

Abstract

Lipid peroxidation in aliquots of bovine retina (without rod outer segments, ROS), purified ROS and retinal pigment epithelium (RPE) was initiated with 5 mM ferric iron and 80 mM ADP. After 30 min of oxidation at 37°C, the concentration of thiobarbituric-acid-reacting substances (TBARS) which 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 aqueous antioxidants (AOX) from natural or synthetic sources, including five flavonoids, were evaluated for their ability to inhibit the oxidative reaction. Palm-oil-derived vitamin E showed significant protection in retina, ROS and RPE (64, 68 and 74%), respectively. Of the flavonoids tested, good protection in the retina was found at 10^–5M for epigallocatechin gallate (50%) and at 50 ng/ml for pycnogenol (61%) and catechin (52%). When catechin and palm oil vitamin E, catechin and coenzyme Q₁₀ or coenzyme Q₁₀ and pycnogenol were combined, the individual effect was enhanced. TBARS as an indirect measure of LHP level and hemoglobin-methylene blue determination for direct LHP were used as alternative end-point determinations of lipid peroxidation. These measure different aspects of AOX reactions. The results demonstrate the usefulness of an in vitro model system that can rapidly and accurately determine the capacity of a single AOX against lipid peroxidation or be used to show synergistic efficacy.