OXIDATION INDUCED MEMBRANE DAMAGE IN CATARACTOGENESIS

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DESCRIPTION: Oxidative modifications of lens proteins and lipids occur in maturity onset human cataract. Yet, its relation to cataractogenesis remains questionable. Oxidation of membrane proteins precedes that of cytosolic protein in normal human lenses of the elderly; lipids are also more prone to oxidation than proteins, in spite of being critical to maintenance of structural and functional integrity of proteins. Phospholipid hydroperoxides (LOOH) and their aldehydic breakdown products can cause both protein oxidation and increased membrane permeability. The overall objective of the proposed research is to establish peroxidation of membrane lipids as a precataractous event, with the goal of identifying early reactions which occur during cataract progression sequentially, by detecting membrane lipid-derived oxidant species such as conjugated dienes, LOOH, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), by UV spectrophotometry and HPLC in normal lenses of human donor eyes as a function of age. Aminophospholipid MDA (or 4-HNE), protein-NH2-MDA aminophospholipid adducts produced by Schiff-base conjugation of carbonyl groups with NH2 groups, and protein carbonyls produced by Michael type addition reactions of 4-HNE with NH2 groups of lysine residues, histidyl residues and cysteinyl -SH groups of proteins will be measured by TLC, HPLC and spectrofluorometry in normal human lenses. The data may yield definitive evidence of membrane damage due to lipid peroxidation in the precataractous condition. As additional indication of membrane damage due to peroxidation of lipids, analyses of other functionally important membrane proteins such as major intrinsic proteins and less prevalent glycoproteins will be done by SDS-PAGE and western blotting. Correlative studies will be performed using precataractous lenses of Emory mice exposed to in vivo photochemical insult, a condition analogous to man exposed to sunlight. The proposed work could clarify initial mechanisms of cataract progression in man, and be useful in modeling and testing of anticataract drugs.