Abstract
Abstract: :
Purpose:To determine the signatures of lipofuscin (the main absorbing species within RPE) and its reaction products using high pressure liquid chromatography–mass spectrometry (LC–MS), and determine whether or not these products are present within the inner aspects of human Bruch’s membrane Methods:Two preparations were analyzed: (1) Human RPE lipofuscin granules isolated as described (Feeney–Burns, Am. J. Ophthalmol. 1980;90:783) from donor globes (Midwest Eye Banks and Transplantation Centers). (2) Explants of human Bruch’s membrane isolated by removing the native RPE with ammonium hydroxide and isolating the inner aspects of Bruch’s membrane as described (Tezel, Del Priore, Kaplan. IOVS 1999;40:467).The organic soluble portion was obtained from both samples by extraction with equal amounts of CHCl3:CH3OH:H2O, and the extract analyzed by LC–MS (Thermo Finnigan, LCQ Advantage, Surveyor; Surveyor LC with PDA detector, quadrupole ion trap mass analyzer, electrospray ion source). Results:An absorbing species (> 400 nm) in RPE lipofuscin has been identified previously as a bis–retinoid pyridinium compound referred to as A2E. We present here that most of the remainder of the chromophores in RPE lipofuscin are structurally related to A2E and consist of 1) oxidation products in which molecular oxygen is added to A2E forming furanoxides, 2) reduction products, where A2E gains 2 hydrogen atoms, and 3) a series of relatively hydrophobic components representing A2E derivatized with fatty acids, most likely at the free hydroxyl group on the pyridinium ring. Analyses of deposits from the Bruch's membrane failed to reveal these components in significant amounts. Conclusions:Lipofuscin and its reaction products harvested from human RPE are detected readily by LC–MS and determined to be oxidation products and derivatives of A2E. Unless structural modifications are present that alter their spectroscopic characteristics, it does not appear as if deposits that originate within the inner aspects of human Bruch’s membrane contain a significant amount of RPE lipofuscin
Keywords: drusen • retinal pigment epithelium • retinal degenerations: cell biology