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B. Serban, P. Bhosale, F. Ahmed, P. S. Bernstein; HPLC-ESI/MS and Fluorimetric Analysis of A2E From AMD and Age-Matched Normal Donors. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5640.
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A2E and its isomer, iso-A2E, the major fluorophores of lipofuscin in human retinal pigment epithelium (RPE), are thought to be important mediators of light-induced oxidative damage associated with aging. Understanding the biochemical mechanisms of their formation and function may provide helpful insight into the pathogenesis of age-related macular degeneration (AMD). In this study, we have measured the levels of A2E and iso-A2E from human RPE from AMD donors and age matched normal donors.
RPE samples from peripheral region (8-mm punches) were collected from donor eyes free of ocular pathology (n=19) and from AMD donors (n=20). A solvent-based extraction protocol was used to obtain A2E and related compounds from RPE tissue (Parish et al. Proc Natl Acad Sci 1998). The amounts of A2E, iso-A2E, and their oxidation products were determined by reverse-phase HPLC coupled with an in-line single quadrupole mass spectrometer (MS) in positive ion electron spray ionization (ESI) mode. The fluorophores were also characterized for their fluorescent properties using an inline-fluorimeter.
Using ESI in a positive full scan mode between 350-800 m/z, major mass spectral ion peaks were observed corresponding to A2E and iso-A2E at m/z 592.7 (M) +.A2E and iso-A2E from AMD donors displayed significantly higher levels of monofuranoids, monoperoxides and other oxidation products in comparison to age-matched donors. Interestingly, AMD donor eyes actually had lower levels of A2E (3.6 ± 3.1 ng/8mm RPE tissue) and iso-A2E ( 0.7 ±1.1 ng/8mm RPE tissue) as compared to age matched donors (6.8 ± 4 ng/8mm RPE tissue; 1.5 ± 0.9 ng/8mm RPE tissue, respectively, P<0.01); however, AMD donors uniquely displayed an unknown peak with m/z 580 with unusually high fluorescent properties. We are currently in the process of identifying this unknown fluorophore.
A2E and iso-A2E are generally considered to be the major lipofuscin fluorophores involved in AMD oxidative damage; however, our results indicate the presence of other fluorophores in the RPE of AMD donors which may be better AMD biomarkers.
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