Properties of Protonated Schiff Base Conjugates in RPE Lipofuscin

Abstract

Purpose:: Amongst the bis-retinoid compounds that accumulate as RPE lipofuscin, all-trans-retinal dimer-phosphatidylethanolamine (atRAL dimer-PE) and all-trans-retinal dimer-ethanolamine (atRAL dimer-E) are protonated ~ 510 nm absorbing species that form by Schiff base reaction with all-trans-retinal dimer (atRAL dimer). We have further characterized these pigments.

Methods:: Human RPE cells and Abcr-/- mice eyecups were extracted with CHCl3/methanol and analyzed by HPLC. atRAL dimer-PE was incubated with phospholipase D and lysosomal fractions; UV/visible absorbance and fluorescence spectroscopy was performed, singlet oxygen phosphorescence spectra were recorded and time-dependent density functional theory (TDDFT) was employed to calculate absorbances.

Results:: Phospholipase D (PLD)-mediated phosphate hydrolysis of atRAL dimer-PE generated atRAL dimer-E but at a slower rate than PLD-mediated cleavage of A2PE, the precursor of A2E. With excitation at 500 nm the atRAL dimer-conjugates exhibited weak fluorescence. Unconjugated atRAL dimer fluorescence was greater when excited at 430 nm. With irradiation at 430 nm, chemiluminescence at 1270 nm, typical of singlet oxygen phosphorescence, was more pronounced with unconjugated atRAL dimer than with A2E or atRAL dimer-E. Since the observed pKa of atRAL dimer-E was relatively low (~ 3.5), a proportion of the atRAL dimer-conjugates would be expected to deprotonate and hydrolyze in the acidic environment of lysosomes. It is thus potentially significant that in VMD2-/- and VMD2 knock-in mice, unconjugated atRAL dimer was present at elevated levels and atRAL dimer-E and atRAL dimer-PE were reduced. All-trans-retinal dimer-PE and atRAL dimer-E were elevated in eyecups of Abcr-/- mice as compared to wild-type, levels in murine eyes varied with the Rpe65 Leu450Met polymorphism and the compounds exhibited little age-related increase in human and mouse eyes. Calculation of the UV/visible absorbance by TDDFT gave theoretical spectra for atRAL dimer-E that agreed with experimentally derived spectrum as did the wavelength difference between the two absorbance maxima (227 nm). For A2PE-H2 , an unstable intermediate in the A2E biosynthesis pathway, TDDFT predicted a difference between absorbance maxima of 150 nm; thus A2PE-H2 is unlikely to be the pigment previously reported to exhibit maxima at 260 and 510 nm.

Conclusions:: Insight into the properties of individual RPE lipofuscin pigments is fundamental to our understanding of blinding retinal disorders associated with abnormal accumulations of RPE lipofuscin.

Keywords: age-related macular degeneration • retinal pigment epithelium