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FC Delori, D Goger, JR Sparrow; Spectral Characteristics of Retinal Autofluorescence in Aging and of A2E in Cultured RPE Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):677.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Fundus autofluorescence (AF) has been shown to originate principally from RPE lipofuscin, a collection of fluorophores of which A2E is a dominant and possibly deleterious component. We investigated the influence of age on the spectra of retinal AF, and compared these spectra to those of A2E. Methods: AF spectra were measured in 174 normal subjects (ages: 20-80) by spectrofluorometry. All data were individually corrected for absorption by the ocular media and for choroidal reflection of emitted fluorescence. Cultured human adult RPE cells (ARPE-19) were incubated with 20 uM A2E for 24 to 192 hours, and their fluorescence measured with the same instrumentation. Results: AF increases with age for all excitation wavelengths (p<0.0001). AF is emitted in a broad band of =190 nm halfwidth. Wavelengths of maximum AF emission are 604±10, 611±9, and 627±5 nm for excitations at 430, 470, and 510 nm, respectively. For each excitation wavelength, the peak emission shifts to shorter wavelengths with increasing age (p<0.01), by =3 nm/decade. Peak excitation occurs at 493±6 nm (emission: 620 nm), increasing with age by =1.5 nm/decade (p<0.0001). The emission spectra of A2E are narrower (halfwidth: =160 nm) and peak at 593±3 and 615±6 nm (excitations: 430 and 510 nm, respectively), exhibiting a similar red-shift with increasing excitation wavelength. The excitation of A2E is maximal at 456±1 nm, and thus at a substantially shorter wavelength than in-vivo (A2E spectrum also narrower). On the basis of computations performed thus far, intraocular absorbers do not explain the differences in the excitation spectra. Conclusion: The red-shift in the emission spectra for longer wavelength excitation is characteristic of both AF and A2E in cultured cells and may be related to microenvironmental effects in the RPE cells. While the similarity of emission spectra and their red-shift in both AF and A2E suggests that A2E is detected in vivo, the difference in excitation spectra of AF and A2E is difficult to reconcile. This difference could be attributable to an additional RPE fluorophore that must also accumulate with age (the shape of the AF excitation spectra is not strongly dependent of age). The age-related shifts in AF spectra might reflect small differences in the accumulation rates of this fluorophore and A2E, or the influence of other non-RPE fluorophores. The excitation peak of the unknown fluorophore would be at 490-515 nm and its emission spectra would be similar to that of A2E. Whether this fluorophore is related to A2E or to another lipofuscin constituent will be of interest in our interpretation of in-vivo AF measurements.
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