Searching for the Cause of the Increased Photoreactivity of Aged Human RPE Melanosomes

Abstract

Abstract: : Purpose: To identify key consituents responsible for the increased photoreactivity of aged human RPE melanosomes by analyzing photochemical properties of chloroform–soluble and chloroform–insoluble fractions. Methods: RPE melanosomes from donors: 10 – 20, 45 – 55 and 80 – 89 year old, and from porcine eyes, were purified by ultracentrifugation in a discontinuous succrose gradient. Electron spin resonance (ESR) oximetry and ESR spin trapping were used to determine oxygen uptake and generation of superoxide anion in suspension of melanosomes irradiated with blue light. Aerobic photoreactivity of intact melanosmes was compared with that of the melanosomes treated with Triton X–100 and/or with chloroform/methanol. Photoreactivity of chloroform–soluble fraction of melanosomes (CSM) was analyzed in liposomes, made of peroxidizable and non–peroxidizable phospholipids and cholesterol, by measuring oxygen photo–uptake and photo–generation of cholesterol hydroperoxides using ESR oximetry and HPLC–EC(Hg) detection, respectively. Results: Melanosomes treated with chloroform/methanol exhibited significantly reduced photoreactivity, while melanosomes treated with Triton X–100 had comparable photoreactivity with that of the native melanosmes. CSM from human melanosmes, unlike from porcine melanosomes, was photoreactive and such a photoreactivity increased with age. When irradiated with blue light, human CSM generated singlet oxygen as evidenced by the detection of singlet oxygen–specific cholesterol hydroperoxides. An age–related increase of the photoreactivity was also observed in the chloroform–insoluble fraction of human melanosomes. Conclusions: Our study suggests that the increased photochemical reactivity of aged RPE melanosomes and their higher phototoxic potential, result from an accumulation of chloroform–soluble photosensitizing compounds and chemical modifications of the melanin and/or proteinaceous constituents of the pigment granules.

Keywords: oxidation/oxidative or free radical damage • retinal pigment epithelium • aging