RELATIONSHIPS BETWEEN GLYCATION AND OXIDATION RELATED FLUORESCENCES IN RAT COLLAGEN DURING AGING - AN IN-VIVO AND IN-VITRO STUDY

BACKGROUND: Glycation and oxidation are spontaneous chemical modificat ions of body proteins. Usually these reactions have been studied separ ately by assessing their fluorescent final products. Glycation of prot ein and its related fluorescence increases during aging, whereas the l evel of the fluorescence related to protein adducts from lipoperoxidat ion side products is unknown. Moreover, no data on the fluorescence, a t different wavelengths, connected to the two reactions in the same sa mple are available. Nevertheless recent in vitro studies support the p ossibility of an interaction between the two spontaneous reactions. EX PERIMENTAL DESIGN: In this study, we evaluated the modification of pro teins due to glycation and to lipoperoxidation side products, by measu ring their specific fluorescence levels in the collagen of 65 healthy Wister rats during the aging process. The relationships among the fluo rescence at different wavelengths were also reported. The fluorescence pattern of insoluble collagen was characterized by a tridimensional s tudy after the incubation of insoluble collagen with probable precurso rs of protein glycation (ribose) and oxidation (malondialdehyde and hy droxynonenal); the maximum peaks of fluorescence were recognized and c ompared. RESULTS: An increase of all fluorescence intensities was obse rved in rat collagen during aging: the glycation-related ones (y(370/4 40) = 28.3 e(0.08x), r = 0.808, p < 0.01; y(335/385) = 66.7 e(0.06x), r = 0.798, p < 0.01) and the hydroxynonenal adduct-related (y(356/460) = 44.3 e(0.06x), r = 0.0810, p < 0.01) were exponential, whereas that derived from MDA-adduct was almost linear (y(390/460) = 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 ag e-adjusted fluorescence intensities of both reactions, suggesting a cl ose relationship between glycation and oxidation, besides a mutual inf luence due to the broad spectrum area. The in vitro study confirmed a good specificity of collagen fluorescence after incubation with a redu cing sugar (ribose 0.5 M for 6 hours) for protein glycation, and after incubation with malondialdehyde (0.1 mM for 3 hours) for lipoperoxida tion adducts; surprisingly enough hydroxynonenal (0.5 mM for 3 hours) significantly increased the fluorescence related to pentosidine-like p roducts (335 nm excitation/385 nm emission) suggesting that this compo und 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 oxid ation, 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 hypot hesis is supported by the in vitro findings of this study.