Preparation of artificial ceroid/lipofuscin by UV-oxidation of subcellular organelles

E Nilsson, D Yin
Mechanisms of Ageing and Development 1997 December 1, 99 (1): 61-78
Recent studies have consistently shown that, during oxidative damage, glycation, and other oxygen stress-related reactions, various biomolecules are converted into ceroid- and lipofuscin-like fluorescent pigments. In this study, artificial ceroid/lipofuscin was produced by exposing rat liver fractions to UV-light overnight. Thiobarbituric acid reactive substances (TBARS) were formed in increasing amounts during the early stages of the process, but decreased as the material was later converted into a polymeric structure with few remaining peroxides. In the transmission electron microscope the artificial pigment showed lamellar structures and was osmiophilic. By energy-dispersive X-ray analysis the material was found to contain Ca and Fe in the same way as natural ceroid/lipofuscin. Moreover, it exhibited ceroid/lipofuscin-like, greenish-yellowish autofluorescence when assayed by microfluorometry, with a fluorescence maximum consistently found at 430 nm when excited at 350 nm. Identical fluorescence maxima were found for each fraction of rat liver that was used as the origin of the pigments, i.e. nuclei, mitochondria, lysosomes and microsomes. Extracts with either chloroform-methanol, or sodium dodecylsulphate, showed identical complex fluorescence. When the pigments were extracted by chloroform-methanol, five fluorescent bands were obtained after thin-layer chromatographic separation. Fibroblasts were found to endocytose the material, a process that converted them into lipofuscin-loaded cells of an aged phenotype as observed by light and electron microscopy. Similar fluorescence emission spectra were obtained from cells grown at 40% O2, in order to stimulate endogenous lipofuscin-formation, and from cells exposed to artificial ceroid/lipofuscin. The described technique for creating artificial ceroid/lipofuscin is relatively easy to perform and should provide a useful new tool to study the possible influences of ceroid/lipofuscin on lysosomal and cellular functions.

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