Abstract
Abstract: :
Purpose: In human retinal lipofuscin, a large percentage of the A2E is in the form of hydrophobic derivatives rather than molecular A2E. The purpose of this study is to determine the origin and quantity of these derivatives. Methods:A2E and A2PE were synthesized and purified according to standard methods. Human (ARPE–19) and calf RPE cells were maintained at confluence in DMEM/F12, 10% FBS at 37oC and 3% CO2. The cells were fed 10 µM A2PE four times at three day intervals. Six hours after the last addition of A2PE, the media was removed and the cells were washed with serum free/phenol red free media. The cultures were irradiated for different times with two Philips "Special Blue" 20W bilirubin bulbs through ¼" plexiglass sheet. After irradiation, the cells were trypsinized, homogenized and extracted with CHCl3:CH3OH (2:1 v:v). The extracts were analyzed by LC/ESI/MS (ThermoElectron, Surveyor HPLC, LCQ Advantage ion trap MS). Results:The chromatogram for the extract from irradiated cells shows a series of eight major derivatives eluting from 45 to 70 minutes that absorb at wavelengths longer than 400 nm. They are determined to be related to A2E based on the MS/MS fragmentation patterns where the fragment corresponding to A2E (m/z = 592) or the A2E furanoid oxide (m/z = 608) is observed and fragmentation of these ions yield losses characteristic of A2E (e.g. M–190, M–150, M–172) or the furanoid oxide. The molecular weights of these derivatives range from ca. 740 to 860. At least three of these derivatives (m/z = 763, 788 and 860) are also observed in extracts of human retinal lipofuscin. Interestingly, these derivatives are not observed if the cells are incubated with only A2E. Conclusions: A series of relatively hydrophobic derivatives of A2E are observed to form when RPE cells are incubated with A2PE either in the dark or irradiated. These compounds are most likely fatty acid derivatives of the ethanolamine side chain. Their presence in vivo may serve to help sequester A2E and therefore minimize its ability to cause damage.
Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • radiation damage: light/UV