IN-VITRO OXIDATION OF VITAMIN-E, VITAMIN-C, THIOLS AND CHOLESTEROL INRAT-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 bra in mitochondria were studied. Oxidation was induced by incubating the mitochondria at 37 degrees C with the free radical generators 2,2' azo bis (2'-amidinopropane) dihydrochloride (ABAPH) and 2,2' azobis (2,4-d imethyl) valeronitrile (ABDVN) which undergo thermal decomposition to yield free radicals. An approximate order for the in vitro ease of oxi dation was : ascorbate much greater than alpha-tocopherol > sulfhydryl s much greater than cholesterol. However, small amounts of ascorbate w ere present in the mitochondria when oc-tocopherol and sulfhydryl comp ounds 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 oxid ized 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 l ow 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 t he water-soluble ABAPH. With both free radical generators the rate of oxidation of the antioxidants consisted of two phases. The initial pha se, that is more rapid, may represent a pool of antioxidant that is in volved in immediate antioxidant protection of the organelle with the s lower compartment being responsible for replenishing the faster pool w henever needed. The observation that one antioxidant (e.g. vitamin E) is oxidized prior to the total depletion of a more easily oxidized com pound (vitamin C) suggests that antioxidants of different structures a nd redox potentials can function simultaneously in biological systems. Many degenerative brain diseases such as Parkinson's disease have bee n associated with oxidative damage, Therefore, it is possible that nov el synthetic antioxidants may find therapeutic use in these conditions by providing additional antioxidant protection and/or enhancing the a ctivities of endogenous antioxidants.