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R. K. Crouch, P. Goletz, L. Blakeley, Y. Koutalos, A. Grey, K. L. Schey, Z. Ablonczy; Mass Spectrometric Imaging of A2E in RPE-Choroid Tissue. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1216.
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Lipofuscin accumulates in RPE cells with age and in some inherited retinal degenerations. Its composition is complex and includes bis-retinoid pyridinium pigments, the best characterized of which is A2E. We report here the spatial distribution of A2E in RPE using imaging technology based on mass spectrometry which allows the localization of individual molecular ions.
Synthetic A2E was used as a standard. Under dim red light, posterior eyecups were flattened onto ITO coated slides, dried, and sprayed with 20 mg/mL sinnapinic acid in 70% ethanol. After drying, tissues were analyzed in a Bruker Autoflex III TOF-TOF mass spectrometer; mass spectral and structural information was obtained in 50 (250 for human tissue) micron steps across the tissue surface in the 400-2600 m/z range. Lipofuscin fluorescence from flattened eyecups was measured in a Leica SP2 confocal microscope with a dry 20× lens (NA=0.7), 488 nm excitation and 565-725 nm emission. Tissue drying had no major effect on the fluorescence signal.
A2E (m/z 592) was detected in all analyzed tissue. ABCR-/- mice had more A2E than age-matched wt Sv129 mice. C57Bl6 mice had the least A2E. Overall, there were marked differences in distribution, with accumulation mostly in the central area in 2 month old mice, and strong accumulation across the RPE including the periphery in 9 month old ABCR-/- mice. The central area size showing accumulation and the relative abundance of A2E increased with age. A2E distribution across the RPE was also observed in a 60 year human eye. There was a lack of correlation of the fluorescence data with the A2E distribution, which is consistent with multiple products contributing to the fluorescence of lipofuscin.
Our data provide new evidence on the distribution of A2E in the RPE. These data contain mass spectral information on various lipid-soluble small molecules in the 500-700 m/z range corresponding to the various A2E derivatives and other lipofuscin components. It is expected that with this methodology we will be able to characterize the distribution of components of lipofuscin.
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