Abstract
XTT (3′-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis(methoxy-6-nitro)benzenesulfonic acid hydrate) was reduced by incubated glucosamine hydrochloride. The XTT reducibility by incubated glucosamine was linearly related with the DNA-breaking activity. In order to elucidate the reaction mechanism, the glucosamine derivatives formed during the incubation process were separated by HPLC, and the compound responsible for the reduction was analyzed. Among the incubated products, fructosazine and deoxyfructosazine were identified by LC-MS, FAB-MS, and 1H- and 13C-NMR. These products showed no XTT reducibility, but an unstable intermediate with a molecular weight of 322 displayed reducibility. Since the intermediate gave fructosazine by oxidation with XTT and was a precursor of deoxyfructosazine, we conclude that the intermediate could have been dihydrofructosazine. Therefore, the XTT reducibility by incubated glucosamine was based on dihydrofructosazine formed by the condensation of two molecules of glucosamine.
