Abstract
Purpose: :
We previously characterized an RPE lipofuscin fluorophore, all–trans–retinal dimer–phosphatidylethanolamine (ATR dimer–PE), with UV–visible absorbance maxima at 285 and 506 nm. It forms in outer segments when two all–trans–retinal condense and then form a Schiff base with PE. Pigments with UV–visible absorbances corresponding to ATR dimer–PE were detected in eye cups of Abca4/Abcr mice and human RPE. We report the detection of the related compounds – ATR dimer and ATR dimer–ethanolamine (ATR dimer–E).
Methods: :
Human RPE cells and ABCR –/– mice eyecups were homogenized, extracted with CHCl3/methanol and analyzed by HPLC using photodiode array and fluorescence detection and coinjection of authentic samples.
Results: :
ATR dimer (max: 285, 430 nm) and ATR dimer–E (max: 285, 503 nm) were detected in reaction mixtures prepared for biomimetic synthesis of A2E. A2E formation was favored, the amounts of A2E generated being approximately 10 fold greater than ATR dimer and ATR dimer–E. ATR dimer and ATR dimer–PE were identified in RPE from elderly human donors and in eyecups of ABCR –/– mice. ATR dimer–E, the hydrolysis product of ATR dimer–PE was also identified in ABCR –/– mice eye cups. With reverse phase columns, ATR dimer–E exhibited a retention time that was very similar to iso–A2E, a cis–isomer of A2E. As a result, the iso–A2E and ATR dimer–E peaks overlapped. Due to their lipophilicity, ATR dimer and ATR dimer–PE exhibited retention times that were considerably longer than A2E. ATR dimer and conjugates did not reach detectable levels in young human eyes. Interconversion between ATR dimer and its ethanloamine and PE conjugates, likely depends on the protonation/deprotonation capacity of the solvent. ATR dimer and its conjugates were not detected in young human eyes.
Conclusions: :
Lipofuscin fluorophores that accumulate in RPE cells form in large part from reactions of visual cycle retinoids. In addition to A2E, iso–A2E, minor cis–isomers of A2E and photooxidation products of A2E, other peaks in HPLC profiles generated to analyze RPE lipofuscin, are attributable to ATR dimer, ATR dimer–PE and its phosphate cleavage product ATR dimer–E.
Keywords: age-related macular degeneration • retinal pigment epithelium • retinoids/retinoid binding proteins