Graig E. Eldred

Fluorophores of the human retinal pigment epithelium: Separation and spectral characterization

Ten fluorescent fractions originating from the chloroform extracts of retinal pigment epithelial (RPE) cells of human donor eyes (ages 52-98 yr) have been separated and characterized by UV-vis absorbance and corrected fluorescence spectroscopy. The semipurified fluorophores fall into four categories based upon their spectral properties: green-emitting fluorophores, a golden yellow-emitting fluorophore, yellow-green-emitting fluorophores and orange-red-emitting fluorophores. All share common absorbance peaks around 280- and 330 nm, and the orange-red-emitting fluorophores also exhibit a strong absorbance peak at 420 nm. No significant visible-emitting fluorophores were detected in the methanol-water phase of these extracts. While these fluorophores are abundant in extracts from adult-derived RPE, most of the fluorophores occur in much lower amounts in RPE extracts from human donors under 10 yr of age. Eyes from child human donors also have much less RPE lipofuscin than those from adult donors, suggesting that most of the fluorophores are lipofuscin derived. This interpretation is supported by the previous finding that all of the fluorophores from whole RPE are also present in extracts of purified lipofuscin granules. Characterization of the chromatographic and spectral properties of the chloroform-soluble fluorescent components from the human RPE provides an important tool for determining the mechanism of RPE lipofuscin fluorophore formation. The absorbance properties defined here are of significance to investigations into the photobiology of the RPE and to those using laser therapy in treatment of age-related retinal diseases.

PHOTOPHYSICAL STUDIES ON HUMAN RETINAL LIPOFUSCIN

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 caμs) 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.

Influence of early photoreceptor degeneration on lipofuscin in the retinal pigment epithelium

Experiments were conducted to evaluate the role played by photoreceptor cells in the accumulation of age pigment, or lipofuscin, in the retinal pigment epithelium (RPE). The age-related accumulation of RPE lipofuscin was compared between rats with hereditary photoreceptor degeneration (RDY) and congenic rats with normal retinas. In the RDY animals, the age-related increase in RPE lipofuscin content was substantially less than in normal controls. This suggests that the photoreceptor cells play a significant role in RPE lipofuscin deposition, although they may not be the sole contributors to RPE lipofuscin formation. Evidence that outer-segment components may be converted into lipofuscin fluorophores was provided by the discovery that in young RDY rats, fragments of outer segments from degenerating photoreceptor cells had fluorescence properties similar to those of RPE lipofuscin. Chloroform-methanol extraction of retina-RPE tissue from young normal and dystrophic rats, and analysis of the chloroform fractions by thin-layer chromatography, revealed three distinct fluorescent components associated with the lipofuscin-like fluorescence of the outer-segment fragments in the RDY rats.

Lipofuscin autofluorescence: evidence for vitamin A involvement in the retina.

A lipofuscin-like autofluorescence develops in the degenerating photoreceptor cells of the RCS rat, a strain with inherited retinal dystrophy. In animals with normal retinas, age-related lipofuscin accumulation in the eye is restricted to the retinal pigment epithelium (RPE). Previous investigations have established that RPE lipofuscin accumulation in the normal rat retina can be reduced by dietary vitamin A deficiency. In order to determine whether the photoreceptor-derived fluorescence in the RCS rat retina is related to RPE lipofuscin fluorescence, the influence of dietary vitamin A on the fluorophore content of the RCS rat retina was studied. Vitamin A deficiency substantially reduced the autofluorescence associated with degenerating photoreceptor cells of the RCS rat retina. A specific vitamin A-dependent fluorophore was isolated from these retinas using thin-layer chromatography (TLC). The mobility of this fluorophore on TLC differs from that of the major age-dependent fluorophore isolated from the RPE of normal rats. Thus, if the vitamin A-dependent fluorophores of the photoreceptors and RPE are related, it appears that the fluorophore generated in the photoreceptor cells must undergo chemical modification once it has been taken up by the RPE. The fact that both the RPE- and photoreceptor-associated fluorophores are vitamin A-dependent suggests that such a relationship between them is likely. These experiments indicate that the RPE is somewhat different from other lipofuscin-accumulating tissues in that a major precursor of RPE lipofuscin fluorophores originates in another cell type and enters the RPE via phagocytosis.

THE PHOTOCHEMISTRY OF HUMAN RETINAL LIPOFUSCIN AS STUDIED BY EPR

Fluorescent material generated in the human retina accumulates within lipofuscin (HLF) granules of the retinal pigment epithelium (RPE) during aging. We have been investigating the possible light‐induced contribution of these fluorophores to various diseases including age‐related macular degeneration. Our studies have shown that some of the fluorescent components of HLF are products of the reaction of retinaldehyde with ethanolamine and that synthetic mixtures of this reaction can serve as a useful model for photophysical studies. Previous research by us has demonstrated that irradiation of either natural or synthetic lipofuscin resulted in the formation of a triplet state and possibly a free radical. Here EPR studies were performed to verify the formation of that radical. The UV irradiation of either synthetic or natural human retinal lipofuscin extracts in oxygen‐free methanol led to the formation of a 5,5‐dimethylpyrroline‐N‐oxide (DMPO) spin‐trapped carbon‐centered radical resulting from either hydrogen atom or electron abstraction from solvent molecules. In the presence of oxygen superoxide was formed, which was observed as a DMPO adduct. It is concluded that certain components of the chloroform‐soluble fluorophores of human RPE lipofuscin granules and the fluorescent reaction products of retinaldehyde and ethanolamine are photophysically similar but not the same. Electron or hydrogen abstraction from a substrate by these fluorophores in vivo and the resulting radical products may contribute to the age‐related decline of RPE function and blue light damage in the retina.

The autofluorescent products of lipid peroxidation may not be lipofuscin-like

The fluorescent molecules of cellular age pigment granules (lipofuscin) are commonly thought to be end products of membrane lipid autoxidation. Lipofuscin fluorophores of the retinal pigment epithelium (RPE) appear to be derived from photoreceptor outer segment membranes. Experiments were therefore conducted to determine whether the in vitro oxidation of retinal homogenates would generate fluorophores similar to the naturally occurring lipofuscin fluorophores of the RPE. Neural retina and RPE-choroid homogenates from young (2-3 month old) albino rats were subjected to an iron-ascorbate-air pro-oxidant reaction medium, and compared to unoxidized control samples from young age-matched animals as well as senescent (24 month old) rats. In addition, neural retina and RPE-choroid homogenates from 3 month old albino rats were subjected to a 100% oxygen atmosphere to test whether the fluorescent products of autoxidation differ substantially from those generated in the pro-oxidant medium. The chloroform-soluble fluorophores of chloroform-methanol sample extracts were analyzed by corrected fluorescence spectroscopy and thin-layer chromatography (TLC). In vitro pro-oxidation of both the neural retina and the RPE from young rats produced blue-emitting fluorophores which differed from the orange- and yellow-emitting fluorophores extracted from the RPE of senescent rats. Corrected fluorescence spectroscopy of aged tissue extracts revealed vitamin A-related fluorescence (330 nm excitation maximum; 515 nm emission maximum) and a spectrally resolvable age-related fluorescence (420 nm excitation maximum; 600 nm emission maximum). Only the vitamin A-related fluorescence could be measured in the control of young samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Retinoid Reaction Products in Age Related Retinal Degeneration

Great strides are being made toward defining the gene defects in retinitis pigmen-tosa1–3, Best’s vitelliform dystrophy4, and animal models of retinal degeneration5–7. Yet, little progress has been made in identifying the biochemical bases of the leading cause of blindness in the elderly: age related macular degeneration (AMD)8. Truly age-related retinal pathologies may well be due to causes other than identifiable point mutations in well defined genes and/or gene products.

Questioning the Nature of Age Pigment (Lipofuscin) in the Human Retinal Pigment Epithelium and its Relationship to Age-Related Macular Degeneration

Second only to cataracts, age-related macular degeneration (AMD) is a leading cause of visual impairment in the elderly. Incidence of AMD has been estimated at between 1.3% and 4% of the population in the 65–74 year age groups (18, 29). With both the number and proportion of people over the age of 65 continuing to increase (27), AMD is likely to become an increasingly significant health problem. There is reason to suspect that age-related changes in the retina may be at the root of this disease (45,46,10). In the following discussion, the reasoning, the resulting theory, a discrepancy in that theory, and current experimental progress into the resolution of this discrepancy will be described.