Photophysical studies of A2-E, putative precursor of lipofuscin, in human retinal pigment epithelial cells

With age, human retinal pigment epithelial cells accumulate lipofuscin that can absorb photons in the visible range leading to light-induced damage an d impaired vision. A putative precursor of lipofuscin, 2-[2,6-dimethyl-8-(2 ,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octa-tetrenyl]-1-(2-hydroxyet hyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl ]-pyridinium (A2-E), has recently been isolated and characterized from aged human retinal pigment epithelial cells. We have found that A2-E inhibits t he growth of human retinal pigment epithelial cells at concentrations great er than 1 mu M. Time-resolved fluorescence measurements of 1 mu M A2-E in s olution, performed under 413 nm excitation, showed that fluorescence wave f orms integrated across the spectrum (450-600 nm) were best-fitted with thre e decay times in the nanosecond and subnanosecond time scale: 6.6, 1.9 and 0.33 ns. Untreated retinal pigment epithelial cells were characterized by t hree fluorescence lifetimes: 6.3, 1.7 and 0.35 ns. In retinal pigment epith elial cells treated with 1 mu M A2-E, the fluorescence decay was significan tly faster, with the marked presence (congruent to 30%) of a fourth short l ifetime (0.12 ns). These fluorescence decay times for A2-E bound to human r etinal pigment epithelial cells are similar to those of lipofuscin granules isolated from aged human retinal pigment epithelial cells. This similarity supports the hypothesis that A2-E is a precursor of lipofuscin and suggest s that A2-E may play a role in the overall light damage associated with age -related retinal diseases.