Mass spectrometry provides accurate and sensitive quantitation of A2E

Abstract

Summary Orange autofluorescence from lipofuscin in the lysosomes of the retinal pigment epithelium (RPE) is a hallmark of aging in the eye. One of the major components of lipofuscin is A2E, the levels of which increase with age and in pathologic conditions, such as Stargardt disease or age-related macular degeneration. In vitro studies have suggested that A2E is highly phototoxic and, more specifically, that A2E and its oxidized derivatives contribute to RPE damage and subsequent photoreceptor cell death. To date, absorption spectroscopy has been the primary method to identify and quantitate A2E. Here, a new mass spectrometric method was developed for the specific detection of low levels of A2E and compared to a traditional method of analysis. The new mass spectrometry method allows the detection and quantitation of approximately 10,000-fold less A2E than absorption spectroscopy and the detection and quantitation of low levels of oxidized A2E, with localization of the oxidation sites. This study suggests that identification and quantitation of A2E from tissue extracts by chromatographic absorption spectroscopyoverestimates the amount of A2E. This mass spectrometry approach makes it possible to detect low levels of A2E and its oxidized metabolites with greater accuracy than traditional methods, thereby facilitating a more exact analysis of bis-retinoids in animal models of inherited retinal degeneration as well as in normal and diseased human eyes.

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

“…We do not know the identity of the earliest peak, but its spectrum is similar to that of A2E. As has been shown previously, other compounds with similar spectra may co-elute with A2E 35 . We have not attempted to identify the compounds responsible for the other peaks in the chromatogram, their spectra however are consistent with bis -retinoids 2 .…”

“…The slight disagreement may be due to variation across human donors and samples, as well as differences in analytical procedures. HPLC absorbance-based measurements may overestimate the levels of A2E by as much as a factor of 2, as several compounds with similar spectra may co-elute and contribute to the absorbance signal 35 . Imaging mass spectrometry (IMS) has been used previously to display the spatial localization of A2E in retina tissues from animal models 37, 38 and from human donor eyes 32 .…”

Lack of Correlation Between the Spatial Distribution of A2E and Lipofuscin Fluorescence in the Human Retinal Pigment Epithelium

“…1 Currently, A2E levels usually are determined by ESI-MS/MS from whole tissue homogenates after extraction from the tissue with organic solvents and subsequent HPLC separation. 21 The methods used to detect A2E are highly sensitive and molecularly selective, but preclude spatial information unless small sections are assayed and compared, as was done by the Bernstein group. 26 Our goal was to determine the spatial distribution of A2E across the human RPE and to correlate this distribution with lipofuscin fluorescence, which is not specific for molecular identification.…”

“…The identities of these products were confirmed by tandem mass spectrometry. 21 For comparison of the distribution patterns, the signal intensities in the panels of Figure 4 were normalized to the ion of interest in the panel; however, the measured levels of oxidized A2E products always were lower (<10%) than those of A2E. The addition of more than two oxygen atoms could not be confirmed in any of the human RPE tissues studied, although higher oxidation levels have been reported previously from in vitro experiments.…”

Norbixin Protects Retinal Pigmented Epithelium Cells and Photoreceptors against A2E-Mediated Phototoxicity In Vitro and In Vivo

“…Mass spectrometry provides accurate and sensitive quantification of A2E [58]. HPLC-MS/MS analysis was performed on an Agilent 1100 in-line triple quadrupole mass spectrometer (API365 or API3200, Applied Biosystems, Les Ulis, France) operated in MRM positive-ion mode.…”