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E. R. Gaillard, L. S. Murdaugh, S. Mandal, J. P. Dillon, J. D. Simon; Molecular Modifications of Human RPE Lipofuscin in vivo. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1837.
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To determine the molecular mechanisms of modifications to A2E found in human RPE lipofuscin.
Human RPE lipofuscin granules were isolated as described (Feeney-Burns, 1980) from donor globes (Midwest Eye Banks). The organic soluble portion was obtained by extraction with equal amounts of CHCl3:CH3OH:H2O, and the extract was analyzed by LC-MS (Thermo Finnigan, Surveyor LC with fluorescence and PDA detectors, LCQ Advantage ion trap mass analyzer, electrospray ion source).
In addition to A2E, human RPE lipofuscin contains numerous compounds that are structurally related to A2E as determined by their fragmentation pattern (losses of M+ -190, -174 and/or -150 amu and the formation of fragments of 592 amu). The majority consists of relatively hydrophobic components corresponding to derivatized A2E with discrete molecular weights of 800-900 m/z, 970-1080 m/z and above 1200 m/z regions. In order to determine the mechanism of these modifications, A2E was chemically modified by; 1) esterification 2) reactions with specific aldehydes and 3) allowed to spontaneously auto-oxidize. The reactions with aldehydes yielded nearly identical products as found in vivo while esterification yielded very different structures as determined mass spectrometrically.
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