Formation of lipofuscin-like fluorophores by reaction of retinal with photoreceptor outer segments and liposomes

Abstract

During the aging process the retinal pigment epithelium (RPE) accumulates autofluorescent lysosomal storage bodies (lipofuscin). Data from previous studies led to the hypothesis that at least one of the fluorescent components of RPE lipofuscin is formed by reaction of vitamin A aldehyde with phosphatidylethanolamine (PE) in the photoreceptor outer segments. Experiments were performed to test this hypothesis. All- trans retinaldehyde was incubated with isolated bovine photoreceptor outer segments and with synthetic liposomes. Liposomes were made with two different lipid compositions. One type of liposome consisted of a mixture of lipids, including phosphatidylcholine (PC), none of which contained a primary amine. The other liposome type was identical in composition except that some of the PC was replaced with an equimolar amount of phosphatidylethanolamine (PE). After incubation of the samples, aliquots 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 chromatography. Photoreceptor outer segments incubated with retinaldehyde developed an intense golden yellow fluorescent emission when illuminated with 395–440 nm light. Similar fluorescence developed in the liposomes containing PE, whereas the liposomes lacking PE or any other primary amine did not develop any detectable fluorescence. The development of fluorescence in the samples in situ correlated with the appearance of an orange colored component in the lipid extracts that displayed a weak 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 with PE in the photoreceptor outer segments to form a fluorophore, a derivative of which subsequently accumulates in RPE lipofuscin. An RPE lipofuscin fluorophore was previously shown to be identical to a reaction product of retinal and ethanolamine. This fluorophore is probably derived from the reaction product of outer segment PE and retinal.