A universal biomarker of cellular ageing in eukaryotic postmitotic cells is the appearance over time of autofluorescent lysosomal residual bodies called age pigments or lipofuscin granules. Their role in the process of cellular ageing has been debated without resolution. Neither the identity nor mechanism of formation of the fluorophores has been definitively determined. A postmitotic cell type that accumulates large quantities of age pigments is the ocular retinal pigment epithelium. We have now identified the major orange-emitting fluorophore of these pigments using fast-atom bombardment tandem mass spectrometry with collisional activation analysis. It is an amphoteric quaternary amine that arises as a Schiff base reaction product of retinaldehyde and ethanolamine. This compound should display lysosomotropic detergent behaviour which would help explain many of the age-related changes shown in this cell. These results suggest a new role for Schiff base reaction products as lysosomotropic amines in the genesis of cellular age pigments.

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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.

Lipofuscin granules (age pigments) emit yellow light under ultraviolet excitation in the fluorescence microscope. The reported blue emission maximum of extracts of lipofuscin-laden cells may result from instrumental bias. The major fluorescent components that accumulate with age in these lysosomal residual bodies of human retinal pigment epithelium are yellow-emitting fluorophores. Different age-related fluorophores, which do emit blue light, are derived from other intracellular sources. A reevaluation of the connection between blue-emitting lipid peroxidation products and the age-related lipofuscin granules of classical pathology is necessary.