Isolation and characterization of a novel RPE fluorophore:An all–trans–retinal dimer.

Abstract

Abstract: : Purpose:To determine whether bis–retinoid fluorophores other than A2E accumulate in retinal pigment epithelial (RPE) cells as a constituent of lipofuscin. Methods:Extracts of human RPE and neural retina isolated from donor eyes and RPE and neural retina obtained from abcr –/– mice were analysed by HPLC, FAB–mass spectrometry, CD spectroscopy and NMR. Results:Authentic ATR dimer was synthesized as a racemate via base catalyzed cyclization (NaH) of retinal and as an enantiomerically enriched product using ethanolic (L)–proline and diisopropylethylamine as catalyst. This molecule was found to form natively in bleached rod outer segments as well as at elevated retinal concentrations. Chiral HPLC (ChiralCel OD) separation of the two enantiomers combined with NMR and CD analyses revealed that the ATR–dimer formed in 13% ee favoring the 13R configuration. Incubation of ATR dimer with phosphatidylethanolamine (PE) generated a compound with a UV spectrum exhibiting 2 peaks at lmax 285 and 506 nm. HPLC analysis of RPE and neural retina isolated from abcr –/– mice and human donor eyes revealed the presence of a pigment with the same UV absorbance and retention time as the synthetic ATR dimer–PE conjugate. Incubation of ATR dimer with ethanolamine generated a protonated Schiff base conjugate which when treated with 100 mN HCl generated A2E. In addition, exposure of the ATR dimer to blue light initiates a photo–oxidation process, leading to oxygen insertion in the double bonds on both sidearms of the molecule. These poly epoxide adducts, characterized by FAB–MS and NMR, are analogous to those found upon blue light irradation of A2E. Conclusions:ATR dimer exists as a condensation product of two molecules of all–trans–retinal. It likely forms by divergence from the A2–PE biosynthetic pathway with a PE–all–trans–retinal Schiff base conjugate (N–retinylidene–phosphatidyl–ethanolamine; NRPE) being a precursor common to both but with ATR dimer formation involving the elimination of the amino group of PE. The ATR dimer subsequently forms a protonated Schiff base conjugate with various amines including PE and is deposited in RPE. Demonstration of the formation of photo–oxidative products of the ATR dimer may also suggest a potential role for this molecule, or derivatives, in the light induced damage of RPE cells.

Keywords: age–related macular degeneration • retinal pigment epithelium • retinoids/retinoid binding proteins