Determination of N-retinylidene-N-retinylethanolamine (A2E) levels in central and peripheral areas of human retinal pigment epithelium

Abstract

SUMMARY The bis-retinoid N-retinylidene-N-retinylethanolamine (A2E) is one of the major components of lipofuscin, a fluorescent material that accumulates with age in the lysosomes of the retinal pigment epithelium (RPE) of the human eye. Lipofuscin, as well as A2E, exhibit a range of cytotoxic properties, which are thought to contribute to the pathogenesis of degenerative diseases of the retina such as Age-related Macular Degeneration. Consistent with such a pathogenic role, high levels of lipofuscin fluorescence are found in the central area of the human RPE, and decline toward the periphery. Recent reports have however suggested a surprising incongruence between the distributions of lipofuscin and A2E in the human RPE, with A2E levels being lowest in the central area and increasing toward the periphery. To appraise such a possibility, we have quantified the levels of A2E in the central and peripheral RPE areas of 10 eyes from 6 human donors (ages 75–91 years) with HPLC and UV/VIS spectroscopy. The levels of A2E in the central area were on average 3–6 times lower than in peripheral areas of the same eye. Furthermore, continuous accumulation of selected ions (CASI) imaging mass spectrometry showed the presence of A2E in the central RPE, and at lower intensities than in the periphery. We have therefore corroborated that in human RPE the levels of A2E are lower in the central area compared to the periphery. We conclude that the levels of A2E cannot by themselves provide an explanation for the higher lipofuscin fluorescence found in the central area of the human RPE.

Visualizing Retinal Pigment Epithelium Phenotypes in the Transition to Geographic Atrophy in Age-Related Macular Degeneration

“…Mass spectrometric and chromatographic analysis of normal human and monkey eyes revealed that the well-studied lipofuscin component A2E and its major oxidation products are lower in macula than in periphery. 100–103 Thus, the relevance of experimental A2E loading of cultured RPE cells to achieve AMD milestones such as cell death 104 and complement dysregulation 105 can be questioned. Because the lipofuscin phototoxicity hypothesis underlies use of visual cycle modulators for AMD 106 and blue- light blocking intraocular lenses for cataract, 107, 108 our conclusions if sustained could have wide-ranging impact on therapeutic strategies for age-related eye disease.…”

Metabolomics and Age-Related Macular Degeneration

“…There is also evidence which suggests that the link between lipofuscin accumulation in the RPE and AMD progression is more tenuous. Although A2E is known to be produced in the RPE, it has been shown that, in humans, A2E is preferentially located in the RPE cells of the peripheral retina, rather than the central area of the retina containing the macula [57]. Therefore, in fundus autofluorescence (FAF) imaging, the higher levels of lipofuscin fluorescence associated with the central area of the RPE cannot be exclusively attributed to the concentration of A2E.…”

Quantifying Retinal Pigment Epithelium Dysmorphia and Loss of Histologic Autofluorescence in Age-Related Macular Degeneration

“…The main fluorophores responsible for FAF imaging are visual cycle byproducts of the bisretinoid family that originate in photoreceptor outer segments and enter RPE via daily phagocytosis 7,12. N-retinylidene-N-retinylethanolamine (AE2) is one prominently investigated molecule13 recently shown to be abundant in peripheral retina14–19; bisretinoid discovery and characterization are ongoing 20…”