FORMATION OF LIPOFUSCIN-LIKE FLUOROPHORES BY REACTION OF RETINAL WITHPHOTORECEPTOR OUTER SEGMENTS AND LIPOSOMES

During the aging process the retinal pigment epithelium (RPE) accumula tes autofluorescent lysosomal storage bodies (lipofuscin). Data from p revious studies led to the hypothesis that at least one of the fluores cent components of RPE lipofuscin is formed by reaction of vitamin A a ldehyde with phosphatidylethanolamine (PE) in the photoreceptor outer segments. Experiments were performed to test this hypothesis. All-tran s retinaldehyde was incubated with isolated bovine photoreceptor outer segments and with synthetic liposomes. Liposomes were made with two d ifferent lipid compositions. One type of liposome consisted of a mixtu re of lipids, including phosphatidylcholine (PC), none of which contai ned a primary amine. The other liposome type was identical in composit ion except that some of the PC was replaced with an equimolar amount o f phosphatidylethanolamine (PE). After incubation of the samples, aliq uots were examined with fluorescence microscopy to assess whether any lipofuscin-like fluorescence had developed. Lipids were extracted from additional aliquots of the samples and analyzed with thin layer chrom atography. Photoreceptor outer segments incubated with retinaldehyde d eveloped an intense golden yellow fluorescent emission when illuminate d with 395-440 nm light. Similar fluorescence developed in the liposom es containing PE, whereas the liposomes lacking PE or any other primar y amine did not develop any detectable fluorescence. The development o f fluorescence in the samples in situ correlated with the appearance o f an orange colored component in the lipid extracts that displayed a w eak red emission upon ultraviolet light illumination. Incorporation of this component into liposomes resulted in the appearance of a golden yellow fluorescent emission. The results of these experiments suggest that retinal, generated during visual pigment bleaching, can react wit h PE in the photoreceptor outer segments to form a fluorophore, a deri vative of which subsequently accumulates in RPE lipofuscin. An RPE lip ofuscin fluorophore was previously shown to be identical to a reaction product of retinal and ethanolamine. This fluorophore is probably der ived from the reaction product of outer segment PE and retinal. (C) 19 96 Elsevier Science Ireland Ltd.