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Tadeusz Sarna, Anna Pilat, Andrzej Zadlo, Shosuke Ito, Kazumasa Wakamatsu, Grzegorz Szewczyk, Aaron Kittell, Theodore Camenisch, Janice Burke, Christine Skumatz; Physicochemical evidence for oxidative modifications of human RPE melanin with aging and of bovine RPE melanin with in vitro photoaging. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1795.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously demonstrated that the content of human RPE melanin decreases with age. We postulated that chronic photooxidation of RPE melanin might be responsible for the observed phenomenon. The aim of the present study was to characterize chemical and physical changes that accompany in situ aging of human RPE melanin and in vitro photooxidation of bovine RPE melanosomes.
Human eyes from Caucasian donors, obtained from the Wisconsin Lion’s Eye Bank, were stored at -80 C until chemical analysis and electron paramagnetic resonance (EPR) examination. Purified melanosomes isolated from bovine RPEs were irradiated in phosphate buffer at 4 C with intense visible light, and progress of melanosome photobleaching was monitored by X-band EPR spectroscopy. The effect of aging on human RPE melanin and photobleaching on bovine RPE melanosomes was examined by high-frequency W-band EPR spectroscopy. Characteristic products of alkaline H2O2 oxidation of eumelanins in human RPE cells of different age, and in control and photobleached bovine RPE melanosomes were determined by HPLC.
Chemical analysis showed that the ratios of free pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) to total PTCA - a specific degradation product of 5,6-dihydroxyindole-2-carboxylic acid - were sensitive markers for oxidative degradation of the melanin structure. The free/total PTCA and even more PTeCA/PTCA increased upon photobleaching of bovine RPE melanosomes. Significantly, PTeCA/PTCA and to a lesser extent the free/total PTCA, determined in human RPEs, also increased with age of the donors, indicating a substantial oxidative modification of the melanin. These chemical changes were accompanied by remarkable changes in the W-band EPR spectra of human and bovine RPE melanin with aging and photobleaching, respectively.
This study provides the first direct evidence for oxidative modifications of human RPE melanin with aging. Age-related changes of RPE melanin can also be monitored by high-frequency EPR spectroscopy. Although, the biological impact of such changes of RPE melanin remains mostly speculative, it can be postulated that oxidatively modified melanin loses, at least partially, its antioxidant and photoprotective functions.
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