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(Investigative Ophthalmology and Visual Science. 2004;45:1052-1060.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0277
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Age-Related Changes in the Photoreactivity of Retinal Lipofuscin Granules: Role of Chloroform-Insoluble Components

Malgorzata Rózanowska,1,2 Anna Pawlak,1 Bartosz Rózanowski,1,2,3 Christine Skumatz,4 Mariusz Zarba,1 Mike E. Boulton,2 Janice M. Burke,4 Tadeusz Sarna,1 and John D. Simon5

1From the Department of Biophysics, Faculty of Biotechnology, Kraków, Poland; the 2School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom; the 3Department of Cytology and Genetics, Institute of Biology, Pedagogical Academy, Kraków, Poland; the 4Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, Wisconsin; and the 5Departments of Chemistry and Ophthalmology, Duke University, Durham, North Carolina.

PURPOSE. Lipofuscin accumulates in human retinal pigment epithelium (RPE) cells with age and may be the main factor responsible for the increasing susceptibility of RPE to photo-oxidation with age. As the composition, absorption, and fluorescence of lipofuscin undergo age-related changes, the purpose of this study was to determine whether photoreactivity of lipofuscin granules also changes with the donor age.

METHODS. To determine whether the photoreactivity of lipofuscin itself is age related, lipofuscin granules were isolated from human RPE and pooled into age groups. Photoreactivity was assessed by measuring action spectra of photo-induced oxygen uptake and photogeneration of reactive oxygen species. Separation of chloroform-soluble (ChS) and -insoluble (ChNS) components by Folch’s extraction was used to determine the factors responsible for the age-related increase in lipofuscin photoreactivity.

RESULTS. The observed rates of photo-induced oxygen uptake and photo-induced accumulation of superoxide-derived spin adducts indicated that when normalized to equal numbers of lipofuscin granules, aerobic photoreactivity of lipofuscin increased with age. Both ChS and ChNS mediated photogeneration of singlet oxygen, superoxide radical anion, and photo-oxidation of added lipids and proteins. Although both ChS and ChNS exhibited substantial photoreactivities, neither exhibited significant age-related changes when normalized to equal dry mass. In contrast, ChNS contents in lipofuscin granules significantly increased with aging.

CONCLUSIONS. Aerobic photoreactivity of RPE lipofuscin substantially increases with aging. This effect may be ascribed to the increased content of insoluble components.






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