Reinke L A, Moore D R, Kotake Y
College of Pharmacy, Department of Pharmacology and Toxicology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Anal Biochem. 1996 Dec 1;243(1):8-14. doi: 10.1006/abio.1996.0476.
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.
一种新的自旋捕获方法已被开发出来，用于在体内连续监测大鼠肝脏中一氧化氮（NO）的生成。该方法基于NO与N-甲基葡糖胺二硫代氨基甲酸盐（MGD-Fe）的铁螯合物的反应，从而形成室温稳定的MGD-Fe的EPR活性亚硝酰配合物。给大鼠注射不同剂量的脂多糖（LPS）以诱导NO合酶活性，随后用异氟烷麻醉。在胆管插管后，通过静脉注射给予MGD-Fe，并收集胆汁样本进行EPR分析。来自LPS预处理大鼠的胆汁的EPR光谱包含被MGD-Fe配合物捕获的NO的特征性三线信号，而未用LPS处理的对照大鼠的胆汁则不包含类似的EPR信号。该方法的检测限估计为5微摩尔。在这些条件下，仅在血浆或尿液中检测到来自NO的微弱信号，这表明胆汁中的NO加合物并非起源于肝外组织。通过向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均使胆汁中检测到的NO显著降低，这与使用先前建立的NO生成方法所获得的研究结果一致。
