Abstract
Purpose: :
We have presented two biosynthetic pathways by which all-trans-retinal can form RPE lipofuscin pigments, one leading to A2E formation and the other generating the all-trans-retinal dimer (atRAL dimer) series of compounds, including unconjugated all-trans-retinal dimer (atRAL dimer), atRAL dimer-PE and atRAL dimer-E (Fishkin et al., PNAS 2005; Kim et al., PNAS 2007). We showed that atRAL dimer is a more efficient generator of singlet oxygen than is A2E and in Abcr-/- mice levels of atRAL dimer-PE exceed that of A2E. While A2E is a pyridinium salt with a quaternary amine nitrogen that does not deprotonate/reprotonate, the imine nitrogen of atRAL dimer-E and atRAL dimer-PE protonates/deprotonates in a pH-dependent fashion. Here we report additional studies related to the photooxidation of these bisretinoid compounds.
Methods: :
atRAL dimer, atRAL dimer-E and atRAL dimer-PE and oxidized-atRAL dimer (oxo-atRAL dimer) were synthesized. Reverse-phase HPLC analysis was performed with UV-visible and fluorescence detection. Mouse eyecups were extracted with chloroform/methanol for analysis. Complement activation was monitored using enzyme immunoassay to measure the C3 split product iC3b in undiluted serum incubated (37°C) in wells precoated with atRAL dimer, peroxy-atRAL dimer and furano-atRAL dimer. iC3b generation in normal human serum (NHS) was compared to that in factor B-depleted human serum.
Results: :
Oxo-atRAL dimer exhibited more pronounced fluorescence (excitation, 430 nm; emission, 600 nm) than atRAL dimer. In a comparison of pigmented and albino Abcr-/- mice, no differences in levels of atRAL dimer (pmoles/eye) were observed; however, oxo-atRAL dimer was increased in albino mouse eyecups relative to pigmented mice . Oxo-atRAL dimer was also more abundant than atRAL dimer. iC3b was also increased in serum incubated in wells pre-coated with atRAL dimer and oxo-atRAL dimer(peroxy- atRAL dimer and furano- atRAL dimer). Substitution of NHS with factor B-depleted human sera containing 20 mM calcium and magnesium abrogated the increase in iC3b.
Keywords: age-related macular degeneration • retinal pigment epithelium • ipofuscin