Molecule-Specific Imaging and Quantitation of A2E in the RPE
Lipofuscin accumulates in the aging retinal pigment epithelium (RPE). It is identified by its fluorescence; however, lipofuscin is a complex mixture, and fluorescence is not specific enough to identify its individual components. Utilizing matrix-assisted laser desorption-ionization imaging, we have recently determined the spatial distribution of lipofuscin components across the RPE. One of the most abundant signals was that of the bis-retinoid A2E, a byproduct of the visual cycle. To better understand the accumulation of A2E, we studied wild-type (wt), Rpe65 −/− , and Abca4 −/− mice. A2E was not found in Rpe65 −/− animals. In wt animals, A2E was most abundant in the center of the RPE and diminished toward the periphery. In contrast, the A2E signal was more intense and uniformly distributed in Abca4 −/− mice. The oxidized forms of A2E were also spatially localized. Furthermore, a highly sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was utilized to quantitate A2E. A2E oxidation sites were determined both in organic extracts and directly from the tissue. The ability to image a specific retinoid and its modified products from fresh tissue suggests wide applicability in the research and potential treatments of retinal degeneration.
KeywordsA2E Lipofuscin Retinal pigment epithelium Mass spectrometry MALDI imaging Quantitation Stargardt disease Age-related macular degeneration
The study was supported by NIH grants EY004939 (RKC), EY020661 (ZA/RKC), and an unrestricted award from Research to Prevent Blindness (RPB). RKC is an RPB Senior Scientific Investigator. The work was performed in the MUSC mass spectrometry facility. Experimental animals were housed in a facility constructed with support from NIH grant C06 RR015455.
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