Free-radical degradation of high-molar-mass hyaluronan induced by Weissberger's oxidative system: potential antioxidative effect of bucillamine.
OBJECTIVES: Hyaluronan (HA), one of the main components of extracellular matrix, is a glycosaminoglycan composed of repeating disaccharide units of N-acetyl-d-glucosamine and d-glucuronic acid linked by β-(1→4) and β-(1→3) glycoside bonds. High-molar-mass HA was used as a model for studying its oxidative degradation. In the present paper protective effects of bucillamine against the free-radical degradation of HA were investigated. The HA fragments generated were characterized as well.
METHODS: To induce free-radical-mediated degradation of high-molar-mass HA under aerobic conditions, we applied Weissberger's oxidative system, comprising biogenic compounds in relevant pathophysiological concentrations, i.e. 100 µM ascorbate plus 1 µM Cu(II). Time-dependent decreases of dynamic viscosity of the HA solutions were recorded by rotational viscometry. Electron donor behaviors of bucillamine were studied by a standard ABTS test method and a chemiluminescence (CL) assay. Ability of incorporation of generated bucillamine thiyl radicals into the biopolymer was verified by Fourier-transform infrared spectroscopy (FT-IR) and size exclusion chromatography with a multi-angle light scattering photometer (SEC-MALS).
RESULTS: Decrease of HA viscosity reflected HA degradation. The drug tested was applied in two arrangements: to prevent •OH radical generation (1) and ROO• type radicals propagation (2). Bucillamine, which acted as an efficient •H donor, is also a proper electron donor, as proved by ABTS and CL assays. FT-IR and SEC-MALS methods showed that the drug tested did not incorporate into the biopolymer chains.
CONCLUSION: Bucillamine significantly protected high-molar-mass HA against free-radical degradation in vitro, and supposedly this positive action of the drug may be involved in its beneficial effect observed in clinical practice....