Background: Glycation and oxidation are spontaneous chemical modifications of body proteins. Usually these reactions have been studied separately by assessing their fluorescent final products. Glycation of protein and its related fluorescence increases during aging, whereas the level of the fluorescence related to protein adducts from lipoperoxidation side products is unknown. Moreover, no data on the fluorescence, at different wavelengths, connected to the two reactions in the same sample are available. Nevertheless recent in vitro studies support the possibility of an interaction between the two spontaneous reactions.
Experimental design: In this study, we evaluated the modification of proteins due to glycation and to lipoperoxidation side products, by measuring their specific fluorescence levels in the collagen of 65 healthy Wistar rats during the aging process. The relationships among the fluorescence at different wavelengths were also reported. The fluorescence pattern of insoluble collagen was characterized by a tridimensional study after the incubation of insoluble collagen with probable precursors of protein glycation (ribose) and oxidation (malondialdehyde and hydroxynonenal); the maximum peaks of fluorescence were recognized and compared.
Results: An increase of all fluorescence intensities was observed in rat collagen during aging: the glycation-related ones (y370/440 = 28.3 e0.08x, r = 0.808, p < 0.01; y335/385 = 66.7 e0.06x, r = 0.798, p < 0.01) and the hydroxynonenal adduct-related (y356/460 = 44.3 e0.06x, r = 0.810, p < 0.01) were exponential, whereas that derived from MDA-adduct was almost linear (y390/460 = 17.7 + 4.1x, r = 0.661, p < 0.01). A different accumulation rate might explain this result. Significant correlation coefficients were found within the age-adjusted fluorescence intensities of both reactions, suggesting a close relationship between glycation and oxidation, besides a mutual influence due to the broad spectrum area. The in vitro study confirmed a good specificity of collagen fluorescence after incubation with a reducing sugar (ribose 0.5 M for 6 hours) for protein glycation, and after incubation with malondialdehyde (0.1 mM for 3 hours) for lipoperoxidation adducts; surprisingly enough hydroxynonenal (0.5 mM for 3 hours) significantly increased the fluorescence related to pentosidine-like products (335 nm excitation/385 nm emission) suggesting that this compound might be the precursor of products with a fluorescence similar to pentosidine or of pentosidine itself.
Conclusions: The in vivo results of this study confirm that nonenzymatic reactions, glycation and oxidation, significantly modify collagen fluorescence during aging and can play a role in tissue damage related to age. The close relationships among fluorescences may be due to a reciprocal interconnection rather than to a parallel increase of both reactions during aging; this hypothesis is supported by the in vitro findings of this study.