Hepatic nitric oxide formation: spin trapping detection in biliary efflux.
Hepatic nitric oxide formation: spin trapping detection in biliary efflux.
复制标题
肝一氧化氮形成：胆汁流出中的自旋捕获检测。
作者：
L. A. Reinke;D. Moore;Y. Kotake
开发了一种新的自旋捕获方法来连续监测大鼠肝脏体内一氧化氮（NO）的形成。该方法基于 NO 与 N-甲基葡糖胺二硫代氨基甲酸酯 (MGD-Fe) 的铁螯合物反应，从而形成室温稳定的具有 EPR 活性的 MGD-Fe 亚硝酰基络合物。给大鼠注射不同剂量的脂多糖（LPS）以诱导NO合酶活性，然后用异氟烷麻醉。胆管插管后，通过静脉注射施用 MGD-Fe，并收集胆汁样本进行 EPR 分析。 LPS 预处理大鼠的胆汁 EPR 谱包含 MGD-Fe 复合物捕获的 NO 特征性三线信号，而未用 LPS 处理的对照大鼠的胆汁不包含类似的 EPR 信号。该方法的检测限估计为 5 microM。在这些条件下，血浆或尿液中只能检测到来自 NO 的微弱信号，这表明胆汁 NO 加合物并非起源于肝外组织。通过给 LPS 处理的大鼠施用 NO 合酶诱导抑制剂 α-苯基-N-叔丁基硝酮 (PBN) 或 NO 合酶抑制剂 N-硝基-L-精氨酸甲酯 (L-NAME) 验证了该方法的可靠性。 PBN 和 L-NAME 均显着减少了胆汁中检测到的 NO，这与使用先前建立的 NO 形成方法进行的研究获得的结果一致。
A new spin trapping method has been developed to continuously monitor nitric oxide (NO) formation in rat liver in vivo. The method is based on the reaction of NO with iron chelates of N-methylglucamine dithio-carbamate (MGD-Fe), resulting in the formation of room-temperature stable EPR-active nitrosyl complexes of MGD-Fe. Rats were injected with various doses of lipopolysaccharide (LPS) to induce NO synthase activity and were later anesthetized with isoflurane. After cannulation of the bile duct, MGD-Fe was administered by iv injection, and samples of bile were collected for EPR analyses. The EPR spectra of bile from LPS-pretreated rats contained characteristic three-line signals of NO trapped by the MGD-Fe complex, while bile from control rats that were not treated with LPS did not contain similar EPR signals. The detection limit of this method was estimated to be 5 microM. Only weak signals from NO could be detected in plasma or urine under these conditions, suggesting that the biliary NO adducts did not originate in extra-hepatic tissues. The reliability of this method was verified by administering an inhibitor for NO synthase induction, alpha-phenyl-N-t-butylnitrone (PBN), or the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) to LPS-treated rats. NO detected in bile was significantly decreased by both PBN and L-NAME, which is consistent with results obtained from studies using previously established methods for NO formation.