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Masahiro Kono, John E. Oatis, Zsolt Ablonczy, Patrice W. Goletz, Joe G Hollyfield, Rosalie K Crouch; Components of human lipofuscin. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2381. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
In the aging human eye, fluorescence in the retinal pigment epithelium (RPE) increases due to accumulation of lipofuscin, which is thought to be toxic to the RPE and to contribute to the pathology of age-related macular degeneration. To date, the best-characterized component of lipofuscin is the bisretinoid A2E. However, recent studies indicated that areas of highest fluorescence in human RPE do not correlate with A2E levels. Thus, other fluorescent compounds must be responsible for a significant proportion of lipofuscin. The purpose of this study is to identify new fluorophores in human lipofuscin, with the ultimate goal of understanding their toxicity.
On the premise that established purification methods have been optimized for bisretinoids, we are developing new extraction and separation conditions. RPE samples were brushed from the inner eye wall after removal of the retina from human donor eyes. Molecules were extracted from the RPE homogentate with various organic solvent/aqueous buffer mixtures and separated by thin layer chromatography (TLC). Bands were isolated from the plate and subjected to mass spectrometry (MS) with MS/MS information collected on prominent MS peaks. Pure samples of A2E and retinyl palmitate served as TLC controls.
Fluorescent compounds that did not co-migrate with either A2E or retinyl palmitate were resolved by TLC. The brightest bands displayed multiple peaks by MS, but dominant peaks did not correspond to A2E. MS-MS of these samples fragmented well. Analysis from previously obtained MALDI-MS imaging of human RPE tissue indicated that the abundances of at least four of these newly identified compounds correlate spatially with lipofuscin fluorescence.
Fluorescent compounds other than A2E are extractable from human RPE. They have distribution patterns that match those of lipofuscin. This study may provide new strategies into determining the relative toxicity of the multiple fluorophores present in lipofuscin associated with aging and age-related macular degeneration.
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