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X. Gu, K. Shadrach, M. Sun, K.A. West, L. Shan, S. Hazan, R.G. Salomon, J.G. Hollyfield, J.W. Crabb; Identification of Lipid Oxidation Products and Proteins in Bruch’s Membrane from Normal and AMD Donor Eyes . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1622.
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Purpose: To identify lipid oxidation products and proteins in Bruch’s membrane possibly associated with the pathogenesis of age-related macular degeneration (AMD). Methods: Bruch’s membrane was isolated free of adjacent tissues and in the presence of antioxidants from 5 normal and 4 AMD donor eyes. Lipids were extracted with chloroform and methanol and analyzed by LC MS. Proteins were subjected to SDS-PAGE, blotted to PVDF membranes and/or gel bands were excised and proteins identified by LC MS/MS. Western analyses were used to screen for oxidative protein modifications. Results: Lipid oxidation products from docosahexaenoyl phosphatidylcholine (DHA-PC), arachidonoyl (AA)-PC, and linoleyl (LA)-PC were detected in relatively greater amounts from AMD than age matched healthy donor eyes. Common drusen proteins were also detected in Bruch’s membrane, including tissue inhibitor metalloproteinase-3, clusterin, vitronectin and serum albumin. Carboxyethylpyrrole protein adducts generated from the oxidation of DHA containing lipids were more abundant in Bruch’s membrane from AMD donors. Conclusions: These observations are consistent with recent drusen analyses (2002 Proc. Natl. Acad. Sci. USA 99: 14682) and support a role for lipid oxidation products in Bruch’s membrane changes associated with AMD.
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