SALICYLATE HYDROXYLATION AS AN INDICATOR OF HYDROXYL RADICAL GENERATION IN DEXTRAN SULFATE-INDUCED COLITIS

Reactive oxygen and nitrogen species have been implicated as mediators of mucosal injury in inflammatory bowel disease. This study investiga ted hydroxyl radical ((OH)-O-.) generation in the inflamed colon of de xtran sulfate sodium (DSS)-induced colitis by measuring the (OH)-O-.-s pecific product of salicylate hydroxylation, 2,3-dihydroxybenzoic acid (DHB). Colitis was induced in 6-7 week old CBA/H male mice by supplem enting the drinking water with 5% DSS for 7 days. On the last day of d extran exposure, mice were injected with salicylate (SAL) (100 mg/kg i .p.) 60 min before sacrifice, and mucosal homogenates were assayed for SAL and 2,3-DHB by HPLC with fluorescence and electrochemical detecti on. Mucosal 2,3-DHB levels in mice exposed to 5% DSS were increased by 83% (p <.005); however, SAL levels were also elevated by 182% (p <.00 1). This translated to a 34% decrease in the ratio 2,3-DHB:SAL in infl amed mucosa, possibly indicating greater catabolism or decreased produ ction of 2,3-DHB. In vitro investigation of the stability of DHBs and SAL in the presence of oxidants of inflammatory lesions revealed that 2,3-DHB and 2,5-DHB were rapidly degraded by hypochlorous acid (HOCl), with initial decomposition rates of 190 and 281 nmol/min, respectivel y (100 mu M DHB with 200 mu M HOCl). Methionine prevented decompositio n of DHBs in vitro; however, in mice with 5% DSS-induced colitis, wher e mucosal myeloperoxidase activity was ten-fold control levels (p <.00 1), administration of methionine (up to 200 mg/kg i.p.) with SAL was i neffective at increasing the ratio 2,3-DHB:SAL. SAL was also degraded in vitro by HOCl (4.7 nmol/min) resulting in the formation of new fluo rescent species which may be useful as indicators of HOCl-mediated inj ury. Salicylate hydroxylation was unable to provide conclusive evidenc e supporting a role for (OH)-O-. in the tissue injury of DSS-induced c olitis, as metabolic disturbances in the diseased animals other than c hanges in (OH)-O-. generation may have altered 2,3-DHB levels. This pr oblem is relevant to any study involving the in vivo use of trapping m olecules. In particular, the susceptibility of 2,3-DHB to degradation by HOCl brings into question the usefulness of salicylate hydroxylatio n for measurement of (OH)-O-.-generation in any neutrophilic inflammat ory lesion. (C) 1998 Elsevier Science Inc.