Light-induced generation and toxicity of docosahexaenoate-derived oxidation products in retinal pigmented epithelial cells.

Oxidative cleavage of docosahexaenoate (DHA) in retinal pigmented epithelial (RPE) cells produces 4-hydroxy-7-oxohept-5-enoic acid (HOHA) esters of 2-lysophosphatidylcholine (PC). HOHA-PC spontaneously releases a membrane-permeant HOHA lactone that modifies primary amino groups of proteins and ethanolamine phospholipids to produce 2-(ω-carboxyethyl)pyrrole (CEP) derivatives. CEPs have significant pathological relevance to age-related macular degeneration (AMD) including activation of CEP-specific T-cells leading to inflammatory M1 polarization of macrophages in the retina involved in "dry AMD" and TLR2-dependent induction of angiogenesis that characterizes "wet AMD". RPE cells accumulate DHA from shed rod photoreceptor outer segments through phagocytosis and from plasma lipoproteins secreted by the liver through active uptake from the choriocapillaris. As a cell model of light-induced oxidative damage of DHA phospholipids in RPE cells, ARPE-19 cells were supplemented with DHA, with or without the lipofuscin fluorophore A2E. In this model, light exposure, in the absence of A2E, promoted the generation HOHA lactone-glutathione (GSH) adducts, depletion of intracellular GSH and a competing generation of CEPs. While DHA-rich RPE cells exhibit an inherent proclivity toward light-induced oxidative damage, photosensitization by A2E nearly doubled the amount of lipid oxidation and expanded the spectral range of photosensitivity to longer wavelengths. Exposure of ARPE-19 cells to 1 µM HOHA lactone for 24 h induced massive (50%) loss of lysosomal membrane integrity and caused loss of mitochondrial membrane potential. Using senescence-associated ß-galactosidase (SA ß-gal) staining that detects lysosomal ß-galactosidase, we determined that exposure to HOHA lactone induces senescence in ARPE-19 cells. The present study shows that products of light-induced oxidative damage of DHA phospholipids in the absence of A2E can lead to RPE cell dysfunction. Therefore, their toxicity may be especially important in the early stages of AMD before RPE cells accumulate lipofuscin fluorophores.

Isolevuglandins and mitochondrial enzymes in the retina: mass spectrometry detection of post-translational modification of sterol-metabolizing CYP27A1.

We report the first peptide mapping and sequencing of an in vivo isolevuglandin-modified protein. Mitochondrial cytochrome P450 27A1 (CYP27A1) is a ubiquitous multifunctional sterol C27-hydroxylase that eliminates cholesterol and likely 7-ketocholesterol from the retina and many other tissues. We investigated the post-translational modification of this protein with isolevuglandins, arachidonate oxidation products. Treatment of purified recombinant CYP27A1 with authentic iso[4]levuglandin E(2) (iso[4]LGE(2)) in vitro diminished enzyme activity in a time- and phospholipid-dependent manner. A multiple reaction monitoring protocol was then developed to identify the sites and extent of iso[4]LGE(2) adduction. CYP27A1 exhibited only three Lys residues, Lys(134), Lys(358), and Lys(476), that readily interact with iso[4]LGE(2) in vitro. Such selective modification enabled the generation of an internal standard, (15)N-labeled CYP27A1 modified with iso[4]LGE(2), for the subsequent analysis of a human retinal sample. Two multiple reaction monitoring transitions arising from the peptide AVLK(358)(-C(20)H(26)O(3))ETLR in the retinal sample were observed that co-eluted with the corresponding two (15)N transitions from the supplemented standard. These data demonstrate that modified CYP27A1 is present in the retina. We suggest that such protein modification impairs sterol elimination and likely has other pathological sequelae. We also propose that the post-translational modifications identified in CYP27A1 exemplify a general mechanism whereby oxidative stress and inflammation deleteriously affect protein function, contributing, for example, to cholesterol-rich lesions associated with age-related macular degeneration and cardiovascular disease. The proteomic protocols developed in this study are generally applicable to characterization of lipid-derived oxidative protein modifications occurring in vivo, including proteins bound to membranes.

Serum vitamin E and oxidative protein modification in hemodialysis: a randomized clinical trial.

BACKGROUND: Patients with end-stage renal disease have increased circulating concentrations of oxidatively modified circulating proteins. Therefore, we examined the ability of vitamin E alpha (alpha-tocopherol) to alter levels of these modified proteins. STUDY DESIGN: Randomized clinical trial. SETTING & PARTICIPANTS: 27 clinically stable patients treated by means of hemodialysis in 4 freestanding outpatient dialysis units. INTERVENTION: Oral administration of 800 IU of vitamin E alpha or placebo daily. OUTCOMES & MEASUREMENTS: Plasma levels of alpha- and gamma-tocopherol and oxidative protein modifications reflecting 2 pathways for protein-oxidant damage. The advanced glycation end product pentosidine reflects glycoxidation. The lipid peroxidation products iso[4]-levuglandin E(2), (E)-4-hydroxy-2-nonenal, and (E)-4-oxo-2-nonenal are formed through covalent adduction. RESULTS: Circulating levels of all oxidative protein modifications were increased in patients with end-stage renal disease. Supplementation with alpha-tocopherol caused alpha-tocopherol levels to rise (13.2 +/- 3.7 to 27.3 +/- 14 mug/mL), but gamma-tocopherol levels to decrease (4.1 +/- 1.6 to 3.5 +/- 1.1 mug/mL). Control values were unchanged. There was no effect on oxidative protein modifications (placebo versus treatment; mean for pentosidine, 15.6 +/- 11.4 (SD): 95% confidence interval (CI), 8.2 to 23.1 versus 21.3 +/- 9.0 pg/mg protein; 95% CI, 16.1 to 26.6; iso[4]-levuglandin E(2), 8.31 +/- 2.55; 95% CI, 6.77 to 9.85 versus 8.46 +/- 2.37 nmol/mL; 95% CI, 7.09 to 9.84; (E)-4-hydroxy-2-nonenal, 0.51 +/- 0.11; 95% CI, 0.45 to 0.57 versus 0.51 +/- 0.08 nmol/mL; 95% CI, 0.46 to 0.56; (E)-4-oxo-2-nonenal, 189 +/- 44; 95% CI, 162 to 215 vs 227 +/- 72 pmol/mL; 95% CI, 183 to 271). LIMITATIONS: Sample size was adequate to show changes in alpha- and gamma-tocopherol levels in response to treatment. However, power was insufficient to show an effect on oxidative protein modifications. CONCLUSIONS: Intervention of oral supplementation with alpha-tocopherol did not result in changes in circulating oxidative protein modifications. A larger study may be required to show an effect in this clinical setting.