Blackburn A C, Doe W F, Buffinton G D
Division of Molecular Medicine, John Curtin School of Medical Research, Australian National University, Canberra, ACT.
Free Radic Biol Med. 1998 Aug;25(3):305-13. doi: 10.1016/s0891-5849(98)00068-9.
Reactive oxygen and nitrogen species have been implicated as mediators of mucosal injury in inflammatory bowel disease. This study investigated hydroxyl radical (.OH) generation in the inflamed colon of dextran sulfate sodium (DSS)-induced colitis by measuring the .OH-specific product of salicylate hydroxylation, 2,3-dihydroxybenzoic acid (DHB). Colitis was induced in 6-7 week old CBA/H male mice by supplementing the drinking water with 5% DSS for 7 days. On the last day of dextran 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 detection. 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 < .001). This translated to a 34% decrease in the ratio 2,3-DHB:SAL in inflamed mucosa, possibly indicating greater catabolism or decreased production 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, respectively (100microM DHB with 200microM HOCl). Methionine prevented decomposition of DHBs in vitro; however, in mice with 5% DSS-induced colitis, where mucosal myeloperoxidase activity was ten-fold control levels (p < .001), administration of methionine (up to 200 mg/kg i.p.) with SAL was ineffective 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 fluorescent species which may be useful as indicators of HOCl-mediated injury. Salicylate hydroxylation was unable to provide conclusive evidence supporting a role for .OH in the tissue injury of DSS-induced colitis, as metabolic disturbances in the diseased animals other than changes in .OH generation may have altered 2,3-DHB levels. This problem is relevant to any study involving the in vivo use of trapping molecules. In particular, the susceptibility of 2,3-DHB to degradation by HOCl brings into question the usefulness of salicylate hydroxylation for measurement of .OH-generation in any neutrophilic inflammatory lesion.
活性氧和氮物种被认为是炎症性肠病中黏膜损伤的介质。本研究通过测量水杨酸羟基化的.OH特异性产物2,3 - 二羟基苯甲酸（DHB），研究了葡聚糖硫酸钠（DSS）诱导的结肠炎炎症结肠中羟基自由基（.OH）的产生。通过在6 - 7周龄的CBA/H雄性小鼠饮用水中补充5% DSS 7天来诱导结肠炎。在葡聚糖暴露的最后一天，小鼠在处死前60分钟腹腔注射水杨酸（SAL）（100 mg/kg），并通过具有荧光和电化学检测的高效液相色谱法测定黏膜匀浆中的SAL和2,3 - DHB。暴露于5% DSS的小鼠黏膜2,3 - DHB水平增加了83%（p <.005）；然而，SAL水平也升高了182%（p <.001）。这导致炎症黏膜中2,3 - DHB:SAL的比值下降了34%，可能表明2,3 - DHB的分解代谢增加或生成减少。对炎症损伤氧化剂存在下DHBs和SAL稳定性的体外研究表明，2,3 - DHB和2,5 - DHB被次氯酸（HOCl）快速降解，初始分解速率分别为190和281 nmol/min（100μM DHB与200μM HOCl）。蛋氨酸在体外可防止DHBs分解；然而，在5% DSS诱导的结肠炎小鼠中，黏膜髓过氧化物酶活性是对照水平的10倍（p <.001），腹腔注射蛋氨酸（高达200 mg/kg）与SAL联合使用并不能有效提高2,3 - DHB:SAL的比值。SAL在体外也被HOCl降解（4.7 nmol/min），导致形成新的荧光物种，这些物种可能作为HOCl介导损伤的指标。水杨酸羟基化无法提供确凿证据支持.OH在DSS诱导的结肠炎组织损伤中的作用，因为患病动物中除了.OH生成变化之外的代谢紊乱可能改变了2,3 - DHB水平。这个问题与任何涉及体内使用捕获分子的研究相关。特别是，2,3 - DHB对HOCl降解的敏感性使得水杨酸羟基化在任何嗜中性粒细胞炎症损伤中用于测量.OH生成的实用性受到质疑。
