Oxidation causes melanin fluorescence

PURPOSE. The goal of this study is the characterization of the strong yello w fluorescence of oxidized melanin in the retinal pigment epithelium (RPE) and the choroid. METHODS. Naturally occurring melanin in the human retina and choroid was ox idized by exposing fixed and plastic-embedded sections of a human eye to li ght and hydrogen peroxide. Synthetic melanin was also oxidized in vitro by exposure to light and hydrogen peroxide. The fluorescence of oxidized melan in was examined by absorption spectroscopy, fluorescence spectroscopy, and fluorescence microscopy. RESULTS. Naturally occurring melanin oxidized in situ exhibited a lipofusci n-like yellow fluorescence. Oxidation of melanin in vitro degraded the mela nin polymer, resulting in a fluorescent solution. Fluorescence spectroscopy gave an excitation maximum at approximately 470 nm and an emission maximum at approximately 540 nm for both natural and synthetic melanin. Increasing the time of exposure to light or hydrogen peroxide increased melanin fluor escence. CONCLUSIONS. The results indicate that the strong yellow fluorescence of me lanin in the RPE and choroid in situ is a property of oxidized melanin and is not due to contamination of the melanin by proteinaceous or lipid materi als. The data presented allow a reinterpretation of the results obtained fr om fluorescence investigations of melanin-containing tissue and suggest a l ink between melanin degradation and lipofuscin formation.