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Zsolt Ablonczy, Dan Higbee, Danielle B. Gutierrez, David M. Anderson, Anne M. Hanneken, Yiannis Koutalos, Kevin L. Schey, Rosalie K. Crouch; Lipofuscin Does Not Correlate With A2E In The Human RPE. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3916.
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The development of age-related macular degeneration (AMD) has been associated with the accumulation of lipofuscin, the orange fluorescent pigment of the retinal pigment epithelium (RPE) lysosomes. The best characterized component of lipofuscin is A2E but a direct correlation of A2E with lipofuscin has not been possible because fluorescence is not specific enough to differentiate chemically similar compounds. In this study, we utilize the molecular specificity of MALDI imaging to spatially identify A2E and other bis-retinoids in the RPE.
RPE-choroid preparations from human donors (fetal eyes to age 80) were flattened onto ITO-coated slides. Lipofuscin was imaged with a color camera and spectra determined in a confocal microscope using a dry 20× lens (NA=0.7), 488 nm excitation and 565-725 nm emission. Tissue fluorescence was quantitated in a Xenogen IVIS 200 imaging system. Thereafter, the tissues were covered with 10 mg/mL DHA in 80% ethanol using spray-deposition, and analyzed in a Bruker Autoflex III mass spectrometer. Mass spectra were acquired in 250 µm steps in the 250-2500 m/z range. Synthetic A2E and other retinoids were used as standards.
Lipofuscin fluorescence develops early (before age 11) in the human RPE. The distribution of this fluorescence was uniform, with a less than order of magnitude increase towards the center of the eye. With age, the level of lipofuscin and A2E increased across the RPE until the age of 70. However, the distribution of A2E and its oxides was peripheral rather than central or uniform. Interestingly, neither the fluorescence nor the A2E profile changed significantly with age.
There is an apparent lack of correlation between lipofuscin fluorescence and A2E levels in the human eye. This implies that compared to murine models, lipofuscin fluorescence is not a good measure of A2E accumulation in the human RPE. Moreover, lipofuscin components other than A2E and its oxides could play a more significant role in the development of AMD. Other bis-retinoids and small molecules were also detected in the same experiments, indicating the mechanism of A2E generation in the RPE. This methodology will be invaluable for determining the components of lipofuscin, as well as the rate and mechanism of their generation.
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