Photophysical Studies on Human Retinal Lipofuscin

Abstract

Fluorescent material generated in the human retina accumulates within lipofuscin granules of the retinal pigment epithelium (RPE) during aging. Its presence has been suggested to contributed to various diseases including age-related macular degeneration. Because this material absorbs light at wave lengths as long as 550 nm, photophysical studies were performed to determine whether lipofuscin could contribute to light damage and to determine if its composition is similar to a synthetically prepared lipofuscin. Time-resolved experiments were performed to monitor (1) fluorescence decay, (2) the UV-visible absorption of longer-lived excited states and (3) the formation and decay of singlet oxygen at 1270 nm. Steady-state and time-resolved fluorescence studies indicate that human and synthetic lipofuscin have fluorophores in common. Time-resolved absorption experiments on human retinal lipofuscin and synthetic lipofuscin showed the presence of at least two transient species, one absorbing at 430 nm (lifetime ca 7 microseconds) and a second absorbing at 580 nm, which decays via second order kinetics. In addition, there is a third absorbing species stable to several hundred milliseconds. The transient species at 430 nm is quenched by oxygen, suggesting that it is a triplet state. Subsequent studies showed the formation of singlet oxygen, which was monitored by its phosphorescence decay at 1270 nm. These studies demonstrate that lipofuscin can act as a sensitizer for the generation of reactive oxygen species that may contribute to the age-related decline of RPE function and blue light damage.

Mechanisms involved in A2E oxidation

“…Uptake of oxygen has been measured in both suspensions of RPE cells and in lipofuscin granules with efficiency being greatest using short-wavelength visible light (Pawlak et al, 2002;Rozanowska et al, 1995;Rozanowska et al, 2002;Rozanowska et al, 2004). It is reported that photoexcited pigments in lipofuscin granules form triplet states with both singlet oxygen and superoxide anion being produced (Gaillard et al, 1995;Reszka et al, 1995;Rozanowska et al, 1995;Rozanowska et al, 1998). Using ESR (electron spin resonance) with DMPO as a spin trapping agent, superoxide anion production by irradiated A2E has previously been detected (Pawlak et al, 2002) while photosensitization studies have shown that photoexcitation of A2E also leads to singlet oxygen production Cantrell et al, 2001;Kanofsky et al, 2003;Lamb et al, 2001;Pawlak et al, 2003;Ragauskaite et al, 2001;Reszka et al, 1995).…”

Fluorescence Spectroscopy of the Retina for Diagnosis of Transmissible Spongiform Encephalopathies

“…[42][43][44][45][46][47][48] The spectral differences in scrapie-positive retinas compared to scrapie-negative retinas can be attributed to the result of altered or increased lipofuscin owing to prion infection. Given the pronounced structural differences observed in the spectra of the scrapie-positive tissue, additional control experiments were undertaken to ensure that these data were not an experimental artifact, since spectra were collected from solid samples, which may have contributed to excessive light scattering.…”

Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress

“…RPE cells phagocytose and digest photoreceptor outer segments, which provide an additional oxidative burden to the cells since outer segments are extremely rich in polyunsaturated fatty acids (PUFA) [62]. Thus, ROS can be generated in RPE cells by oxidation of PUFAs [63,64], the process of phagocytosis [48,65], and RPE photosensitizers exposed to intense visible light [66,67]. The production of excess ROS by normal metabolic processes and photochemical injury often lead to cumulative oxidative damage to the RPE [35][36][37], an event believed to be an underlying cause in some visual disorders.…”