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J. P. Dillon, L. Murdaugh, E. R. Gaillard; Composition Studies of Human Rpe Lipofuscin. Invest. Ophthalmol. Vis. Sci. 2008;49(13):217.
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To determine the spectroscopic characteristics of the main components of the hydrophobic fraction of human RPE lipofuscin using high pressure liquid chromatography-mass spectroscopy (LC-MS) with PDA and fluorescence detection
Human RPE lipofuscin granules were isolated as described (Feeney-Burns, 1980) from donor globes (Midwest Eye Banks and Transplantation Centers). 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, LCQ Advantage, Surveyor; Surveyor LC with fluorescence and PDA detectors, quadrupole ion trap mass analyzer, electrospray ion source).
A visible absorbing species in RPE lipofuscin has been identified previously as a bis-retinoid pyridinium compound referred to as A2E. Most of the remainder of the chromophores in RPE lipofuscin are structurally related to A2E as determined by their fragmentation pattern with loses of M+- 190, 174 and/or 150 amu and the formation of fragments of ca 592 amu. In addition to oxidation and reduction products, the majority of the mixture consists of relatively hydrophobic components corresponding to derivatized A2E; most likely formed by covalent modification at the free hydoxyl group of A2E. Analysis of lipofuscin from various donors indicates that the extracts consist of at least three and up to fifteen of these hydrophobic components which are also observed to form spontaneously in vitro over extended periods of time.
Lipofuscin and its reaction products harvested from human RPE are detected readily by LC-MS and are structurally related to A2E. The majority of modifications are hydrophobic which would aid in the sequestering of A2E into granules with the concomitant diminution of its reactivity
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