Free radical-induced oxidation of docosahexaenoate lipids and protein modification in retina

This thesis research has identified a new family of lipid products from in vitro free-radical-induced peroxidation of docosahexaenoic acid (DHA) phosphatidylcholine (PC) ester. One of these products, 4-hydroxy-7-oxo-hept-5-enoate-PC (HOHA-PC), gives rise to carboxyethylpyrrole (CEP) modifications by reacting with the epsilon amino group of lysyl residues of proteins. CEP modification was shown to be a stable protein adduction product that could serve as a marker for DHA associated oxidative injuries.; Polyclonal and monoclonal antibodies were raised against immunogens containing CEP modifications. Both antibodies exhibited high structural selectivity. Immunoassays utilizing anti-CEP antibodies were employed to analyze animal and human tissues, and to probe the relevance of CEP modification to the pathogenesis of age-related macular degeneration (AMD).; CEP immunoreactivity was generated in vitro by the incubation of HOHA-PC with protein, and by in vitro oxidation of DHA-PC in the presence of protein. In both cases, CEPs formed were in their PC ester form. Base hydrolysis transformed these esters to their free acid form that can be recognized by anti-CEP antibodies.; Strong CEP immunorecognition was present in photoreceptor outer segment and retinal pigment epithelium (RPE) of a mouse retina, where oxidative stress is intense. A significantly elevated level of CEP immunoreactivity in human retina was associated with AMD compared with age-and-gender matched normal controls. Human plasma was found to contain putative CEP adducts. Levels of CEP adducts in AMD plasma are significantly higher than in plasma from non-AMD controls. Interestingly, CEP immunoreactivity in plasma was strongly correlated with atherosclerosis (AS). An elevated level of anti-CEP autoantibody was detected in the plasma of AMD patients compared with age-matched controls.; Taken together, this study provides evidence that free radical-induced DHA peroxidation plays an important role in the etiology and pathology of AMD. Since DHA is especially abundant in retina and brain, CEP modifications are unique indicators of oxidative injuries of these tissues. Lipid products and protein adducts derived from DHA oxidation may have biological activities that promote the development of AMD. The detection and quantification of CEP modifications in plasma may provide a minimally invasive diagnostic tool for neurodegenerations and retinal disease associated with oxidative injuries.