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MB Rozanowska, A Pawlak, B Rozanowski, M Boulton, JM Burke, T Sarna, T Sarna, J Simon; Role of Chloroform Soluble and Insoluble Components in Photoreactivity of RPE Lipofuscin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):681.
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Purpose: Photoreactivity of RPE lipofuscin (LF) has been studied either on whole LF granules or on chloroform soluble components (ChS) obtained by chloroform/methanol extraction. The aim of this study was to compare the photoreactivity of ChS with chloroform insoluble components (ChNS), and to establish their roles in photoreactivity of lipofuscin granules. Methods: LF granules were isolated from human RPE cells and pooled into four age groups - below 40, 41-60, 61-80, and above 80 years of age. Chloroform/methanol extraction was performed to separate ChS and ChNS. Lipofuscin granules were counted in a hemocytometer and the content of ChS and ChNS per lipofuscin granule was determined by weighing dry mass. Photoreactivity was assessed by photo-induced oxygen uptake, measured by electron spin resonance (ESR) oximetry, and photogeneration of free radicals, measured by ESR spin trapping during irradiation with narrow band light from the range 338 - 542 nm. Results: Both ChS and ChNS mediated photo-induced oxygen uptake. Uptake rates, normalized to 1 mg of dry mass per ml, were up to three times higher for ChS than ChNS when excited with UV light, but became similar when excited with blue or green light. Photoexcitation of both ChS and ChNS led to generation of superoxide and oxidation of lipids and proteins. Neither ChS nor ChNS exhibited age related changes in photoreactivity when normalized to dry mass. ChS contents in lipofuscin granules did not significantly change with aging, while ChNS contents increased with aging by a factor of 1.8. Conclusion: Both ChS and ChNS exhibit substantial photoreactivity. An age-related increase in ChNS content is the main factor responsible for the age-related increase in photoreactivity of lipofuscin granules.
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