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TJ Sarna, M Zareba, JM Burke, M Wrona, W Korytowski; Age-dependent Increase In Photo-induced Peroxidation of Lipids in Human RPE Cells In Vitro: Role of RPE Melanin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):682.
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Purpose:To determine whether aging modifies susceptibility of the human RPE to oxidative stress induced by blue light, and to evaluate the antioxidant and photoprotective efficiency of melanin from the aging RPE. Methods:RPE cells were form donors 11 to 95 year old, and bovine RPE cells were isolated from the tapetal and non-tapetal areas of the fundus. Melanosomes from human RPE cells of donors of different age, were purified by ultracentrifugation in a sucrose density gradient. Liposomes, made of peroxidizable and non-peroxidizable phospholipids and cholesterol, were subjected to photosensitized oxidation using methylene blue and red light. Lipid peroxidation in RPE cells, induced by blue light irradiation at 4°C, was monitored by measurement of oxygen uptake, using ESR oximetry, or by detection of phospholipid hydroperoxides and cholesterol hydroperoxides (ChlOOH), using the idometric assay and HPLC-EC(Hg), respectively. Antioxidant efficiency of RPE melanin was tested by measuring its ability to protect liposomal lipids against peroxidation. Results:A significant increase in the photoformation of both the free radical-dependent 7α/7ß-ChlOOH and the singlet oxygen specific 5α-ChlOOH in RPE cells, from donors eighty years and older, was observed upon in vitro irradiation of the cells with blue light. Control experiments, using pigmented and non-pigmented bovine RPE cells, revealed that the melanin-containing cells are more resistant to oxidative stress. The inhibitory role of RPE melanin on peroxidation of lipids was also confirmed in model systems using liposomes and purified melanin. A noticeable reduction in the antioxidant efficiency of RPE melanin was detected in melanin from donors over 80. Conclusion:Higher susceptibility of RPE cells from older donors to oxidative stress, may result from age-dependent decrease in the RPE melanin content and changes in antioxidant properties of the remaining melanin.
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