IN-VITRO OXIDATION OF VITAMIN-E, VITAMIN-C, THIOLS AND CHOLESTEROL IN RAT-BRAIN MITOCHONDRIA INCUBATED WITH FREE-RADICALS

The kinetics of oxidation of endogenous antioxidants such as vitamins C and E and thiols as well as membrane cholesterol in isolated rat brain mitochondria were studied. Oxidation was induced by incubating the mitochondria at 37 degrees C with the free radical generators 2,2' azobis (2'-amidinopropane) dihydrochloride (ABAPH) and 2,2' azobis (2,4-dimethyl) valeronitrile (ABDVN) which undergo thermal decomposition to yield free radicals. An approximate order for the in vitro ease of oxidation was : ascorbate much greater than alpha-tocopherol > sulfhydryls much greater than cholesterol. However, small amounts of ascorbate were present in the mitochondria when oc-tocopherol and sulfhydryl compounds were getting oxidized. This observation is different from those with more homogeneous biological substrates like blood plasma or serum. The order of oxidation of the various compounds is a function of not only the redox potentials but also the (a) concentrations of the oxidized and reduced species, (b) compartmentation of the compounds and (c) enzymatic and nonenzymatic systems for the repair or regeneration of the individual antioxidants. Even though ascorbate levels are quite low within mitochondria this nutrient may play a major role as a first line of defense against oxidative stress. The lipid-soluble ABDVN was much more potent in oxidizing membrane cc-tocopherol and thiols than the water-soluble ABAPH. With both free radical generators the rate of oxidation of the antioxidants consisted of two phases. The initial phase, that is more rapid, may represent a pool of antioxidant that is involved in immediate antioxidant protection of the organelle with the slower compartment being responsible for replenishing the faster pool whenever needed. The observation that one antioxidant (e.g. vitamin E) is oxidized prior to the total depletion of a more easily oxidized compound (vitamin C) suggests that antioxidants of different structures and redox potentials can function simultaneously in biological systems. Many degenerative brain diseases such as Parkinson's disease have been associated with oxidative damage, Therefore, it is possible that novel synthetic antioxidants may find therapeutic use in these conditions by providing additional antioxidant protection and/or enhancing the activities of endogenous antioxidants.