Abstract
Purpose: :
To determine the structures of the main components of the hydrophobic, higher molecular weight fraction of human RPE lipofuscin using high pressure liquid chromatography-mass spectroscopy (LC-MS) with PDA detection. This fraction is ca 10 times more abundant than A2E itself.
Methods: :
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). 1H NMR spectra were recorded with a Bruker Avance 500 MHz NMR.
Results: :
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 losses of M+- 106, 190, 174 and/or 150 amu and the formation of fragments of ca. 592 amu. These have discrete molecular weights of 800-900 m/z, 970-1080 m/z and above 1200 m/z regions. The majority of the mixture consists of relatively hydrophobic components corresponding to derivatized A2E; most likely formed by the polymerization reaction with aldehydes formed by the oxidation of other A2E moieties. Detailed mass spectral analysis and NMR structural analysis of these compounds allowed for the tentative prediction of specific molecular structures.
Conclusions: :
Lipofuscin and its reaction products harvested from human RPE are detected readily by LC-MS and are structurally related to A2E. Aging of RPE lipofuscin results from the auto and/or photooxidation of A2E to form aldehydes which then further react with A2E to give a series of polymers that are much more hydrophobic than A2E. This increases log P (hydrophobicity factor) and induces the sequestering of these derivatives into granules with a concomitant diminution in reactivity. These reactions also trap resultant aldehydes which would oxidize cellular components with concomitant cellular damage..
Keywords: aging • ipofuscin • oxidation/oxidative or free radical damage