Abstract
Purpose: :
To determine the molecular mechanisms of modifications to A2E found in human RPE lipofuscin.
Methods: :
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).
Results: :
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.
Keywords: aging • retinal pigment epithelium • oxidation/oxidative or free radical damage