High performance liquid chromatography with TSK 5000 PW or TSK 6000 PW size exclusion columns combined with a 125I labelled hyaluronic acid binding protein assay was used to study the effects of oxygen derived free radicals on synovial fluid hyaluronate. A continuous flux of free radicals was generated by the xanthine oxidase/hypoxanthine system. When the free radical flux was generated with xanthine oxidase/hypoxanthine in the presence of the iron chelator desferrioxamine and the hydroxyl radical scavenger mannitol a 30-50% decrease in hyaluronate peak was detected, but the molecular weight of synovial fluid hyaluronate remained almost unchanged as a result of reaction with superoxide radicals and hydrogen peroxide. When trace amounts of iron and EDTA were present in the reaction mixture depolymerisation of synovial fluid hyaluronate occurred, and it reached a final molecular weight of about 13,500 daltons. These results suggest that superoxide and hydroxyl radicals may have a different mode of action on synovial fluid hyaluronate. Superoxide radicals and hydrogen peroxide do not induce depolymerisation but, rather, change the molecular configuration of synovial fluid hyaluronate.
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