Degradation of Hyaluronic Acid, Poly- and Mono-Saccharides, and Model Compounds by Hypochlorite: Evidence for Radical Intermediates and Fragmentation

Degradation of hyaluronic acid by oxidants such as HO· and HOCl/ClO− is believed to be important in the progression of rheumatoid arthritis. While reaction of hyaluronic acid with HO· has been investigated extensively, reaction with HOCl/ClO− is less well defined. Thus, little is known about the site(s) of HOCl/ClO− attack, the intermediates formed, or the mechanism(s) of polymer degradation. In this study reaction of HOCl/ClO− with amides, sugars, polysaccharides, and hyaluronic acid has been monitored by UV-visible (220–340 nm) and EPR spectroscopy. UV-visible experiments have shown that HOCl/ClO− reacts preferentially with N-acetyl groups. This reaction is believed to give rise to transient chloramide (R—NCl—C(O)—R′) species, which decompose rapidly to give radicals via either homolysis (to produce N· and Cl·) or heterolysis (one-electron reduction, to give N· and Cl−) of the N—Cl bond. The nature of the radicals formed has been investigated by EPR spin trapping. Reaction of HOCl/ClO− with hyaluronic acid, chondroitin sulphates A and C, N-acetyl sugars, and amides gave novel, carbon-centered, spin adducts, the formation of which is consistent with selective initial attack at the N-acetyl group. Thus, reaction with hyaluronic acid and chondroitin sulphate A, appears to be localized at the N-acetylglucosamine sugar rings. These carbon-centered radicals are suggested to arise from rapid rearrangement of initial nitrogen-centered radicals, formed from the N-acetyl chloramide, by reactions analogous to those observed with alkoxyl radicals. The detection of increasing yields of low-molecular-weight radical adducts from hyaluronic acid and chondroitin sulphate A with increasing HOCl/ClO− concentrations suggests that formation of the initial nitrogen-centered species on the N-acetylglucosamine rings, and the carbon-centered radicals derived from them, brings about polymer fragmentation.