May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
HPLC–ESI/MS Analysis of A2E and Its Relation With Carotenoids in Overlying Retina
Author Affiliations & Notes
  • B. Serban
    Moran Eye Center, University of Utah, Salt Lake City, UT
  • P. Bhosale
    Moran Eye Center, University of Utah, Salt Lake City, UT
  • P.S. Bernstein
    Moran Eye Center, University of Utah, Salt Lake City, UT
  • Footnotes
    Commercial Relationships  B. Serban, None; P. Bhosale, None; P.S. Bernstein, None.
  • Footnotes
    Support  NIH Grant EY11600;Research to Prevent Blindness;Steinbach Foundation
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 2885. doi:
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      B. Serban, P. Bhosale, P.S. Bernstein; HPLC–ESI/MS Analysis of A2E and Its Relation With Carotenoids in Overlying Retina . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2885.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : 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 and correlated them with the amounts of macular carotenoid pigments present in overlying retinal and macular tissues.

Methods: : RPE and retina samples were collected from donor eyes free of ocular pathology. A solvent–based extraction protocol was used to obtain A2E 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 a positive ion electron spray ionization (ESI) mode. Ocular carotenoids in overlying retinal tissues were measured by HPLC–MS with atmospheric pressure chemical ionization (APCI) and were correlated with the levels of A2E.

Results: : 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) +. The limit of quantification and detection for A2E at a signal–to–noise ratio of 10 was observed to be 1 ng and 100 pg respectively, as shown by single ion monitoring (SIM) at m/z 592.7 (M) +. MS peaks at 608 and 624, monofuranoids and monoperoxides of A2E, were detected exclusively in aged donors. We observed a statistically significant increase in the total A2E levels in the RPE with age (N=66, P<0.0001). A2E levels from 8 mm punches of macula and peripheral retina were inversely correlated with the carotenoid levels from overlying retinal regions (N=27, P=0.00473). We observed that A2E levels in the macular region (2.88 ± 3.03 ng/ 8 mm punch, N=35) were approximately three times less than in peripheral retinal region (8.25 ± 4.6 ng/ 8 mm punch, N=35).

Conclusions: : The sensitivity and specificity of HPLC coupled with in–line mass spectral detection was found to be suitable for further investigation of the physiological relationships between A2E and carotenoids in human. Our results suggest that carotenoids in the overlying retina protect against the formation of potentially toxic A2E in the RPE.

Keywords: carotenoids/carotenoid binding proteins • macular pigment • retinal pigment epithelium 

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