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Masahiro Kono, Zsolt Ablonczy, Patrice W. Goletz, Joe G Hollyfield, Rosalie K Crouch; A2E adducts in the human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1737. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To identify new fluorophores in human retinal pigment epithelial (RPE) cells.
Human RPE was brushed from donor eyes, homogenized, and extracted with a series of organic solvents. Phase separated fractions were collected and run on thin layer chromatography (TLC) plates. Prominent fluorescent bands were isolated and eluted off the silica. These samples were then subjected directly to mass spectrometry (MS), and the major peaks were further fragmented (MS-MS) to gain structural information.
Two TLC bands fluoresced orange. One correlated to the bisretinoid A2E. The other migrated farther on the TLC plate, indicative of compounds more hydrophobic than A2E. The MS of this orange band included peaks with mass/charge (m/z) of 970 and 999. Fragmentation of both compounds gave a primary peak corresponding to A2E (m/z = 592). Closer examination of the MS-MS spectra indicated fragments below 592 mass units are identical to A2E fragmentation, and those above 592 are consistent with A2E containing a phosphoglycerol adduct with monoacyl chains.
Additional A2E adducts are being isolated and identified from the RPE of human donor eyes upon a re-examination of extractable, fluorescent components. Our long-term goal is to identify and map the spatial distribution of additional fluorescent compounds within the human RPE and to understand the relative toxicity of the fluorescent compounds that accumulate in human lipofuscin granules of the RPE during aging.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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