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Y.P. Jang, K. Nakanishi, Y. Itagaki, J.R. Sparrow; Characterization of a Highly Fluorescent RPE Fluorophore . Invest. Ophthalmol. Vis. Sci. 2005;46(13):255.
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Purpose: Constituents of RPE lipofuscin include the bisretinoid fluorophore A2E, iso–A2E and other less abundant isomers, photooxidation products of A2E and conjugates of all–trans–retinal dimer (ATR–dimer). Here we report results from further analysis of the composition of RPE lipofuscin Methods: Chloroform/methanol extracts of human RPE isolated from donor eyes were analysed by HPLC with UV and fluorescence detection and by LC– and high resolution mass spectrometry. Results: By reverse phase HPLC using a solvent gradient and monitoring at 430 nm, a sharp peak attributable to a more polar compound was observed in addition to A2E and its photoisomers. This compound has a UV–visible absorbance maximum at 400 nm. Comparison of peak intensities at 430 nm revealed that the ratio of this unknown fluorophore to A2E was 0.37:1. However, the fluorescence intensity of this unknown fluorophore was considerably greater. With excitation at 430 nm and detection of emission at 600 nn, the ratio of the unknown fluorophore to A2E in terms of fluorescence intensity was 18.4:1. Conclusions: We have begun to characterize a fluorescent pigment that is extractable from human RPE. The absorbance of this compound is blue–shifted relative to A2E and the pigment is considerably more fluorescent than A2E.
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