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Isolation and structural studies of native RPE and BM fluorophores
Author(s) -
GAILLARD E,
DILLON JP,
MURDAUGH L
Publication year - 2011
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2011.3311.x
Subject(s) - lipofuscin , fluorescence , chemistry , retinal , analytical chemistry (journal) , photochemistry , biophysics , chromatography , optics , biochemistry , biology , physics
Abstract Purpose To determine the chemical structure, fluorescence spectra and lifetimes and spatial distribution of the major retinal lipofuscin and Bruch’s membrane fluorophores and to correlate their relative amounts with disease. Methods Human RPE lipofuscin granules and Bruch’s membrane explants were isolated from donor globes (Midwest Eye Banks and Transplantation Centers). The organic soluble portion was obtained by extraction with equal amounts of CHCl3:CH3OH:H2O, and the extract was analyzed by LC‐MS (Thermo Finnigan, LCQ Advantage, Surveyor; Surveyor LC with fluorescence and PDA detectors, quadrupole ion trap mass analyzer, electrospray ion source). Fluorescence lifetimes were measured with a PTI Timemaster Lifetime analyzer. MALDI spatial distribution maps were imaged with an Applied Biosystems Voyager‐DE Biospectrometry workstation. Results Several derivatives, including higher molecular weight condensation products, of A2E have been isolated and structurally and spectroscopically characterized. A Bruch’s membrane specific biomarker, nitro‐A2E, has also been characterized and found to increase in concentration with both age and progression of AMD. These materials have unique fluorescence emission maxima and lifetimes and can be used with non‐invasive diagnostic methods. The spatial distribution of these molecules yields information as to their origin. Conclusion Knowledge of the fluorescence maxima and lifetimes of identified retinal fluorophores may allow for a more detailed interpretation of fundus autofluorescence. Of particular importance are the trends in concentration as a function of disease state.