Spin-trapping of superoxide by 5,5-dimethyl-1-pyrroline N-oxide: application to isolated perfused organs.
Spin-trapping of superoxide by 5,5-dimethyl-1-pyrroline N-oxide: application to isolated perfused organs.
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5,5-二甲基-1-吡咯啉 N-氧化物自旋捕获超氧化物：应用于分离的灌注器官。
DOI：
10.1016/0003-2697(90)90202-k
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发表时间：
1990
影响因子：
2.9
通讯作者：
Rosen,GM
中科院分区：
文献类型：
--
作者：
Pou,S;Rosen,GM
在用于识别自由基的现有技术中，自旋捕捉提供了同时测量和区分各种重要的生物产生的自由基的独特机会。对于超氧化物和羟基自由基，最常用的是自旋陷阱5，5-二甲基-1-吡咯烷-1-氧化物(DMPO)。然而，这种硝酮有几个缺点。例如，它与超氧化物的反应很慢，二级速率常数在10M−1s−1左右。因此，为了观察到相应的自旋捕获加合物5，5-二甲基-2-羟基-1-吡咯烷二氧基，高浓度的DMPO是必不可少的。在某些情况下，这可能会导致细胞毒性。为了绕过这一严重的限制，有人建议使用间接方法来检测和识别由于缺血/再灌注损伤而产生的自由基。在直接(最常用的)方法中，首先在适当的实验条件下将自旋陷阱添加到孤立的灌流器官中。然后，将含有自旋陷阱加合物(S)的输液缓冲液放入石英平板池中，插入电子自旋共振光谱仪。在间接法中，将自旋陷阱添加到先前离开器官的灌流液中。因此，使用这种方法可以防止任何自旋陷阱介导的对隔离灌流器官的毒性。然而，由于超氧化物或羟基自由基催化自由基反应的速度非常快，用这种间接方法记录的ESR谱是否来自于自由基自旋捕获是值得怀疑的。在这份报告中，我们根据上面讨论的动力学因素对间接自旋捕获技术进行了评估。
Of the available techniques used to identify free radicals, spin-trapping offers the unique opportunity to simultaneously measure and distinguish among a variety of important biologically generated free radicals. For superoxide and hydroxyl radical, the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) is most frequently used. However, this nitrone has several drawbacks. For example, its reaction with superoxide is slow, having a second-order rate constant around 10 m−1s−1. Because of this, high concentrations of DMPO are essential in order to observe the corresponding spin-trapped adduct, 5,5-dimethyl-2-hydroperoxy-1-pyrrolidinyloxy. This may, in some cases, lead to cellular toxicity. In an attempt to circumvent this serious limitation, it has been proposed that an indirect approach be employed to detect and identify free radicals generated as a consequence of ischemia/reperfusion injury. In the direct (most frequently used) approach, the spin trap is first added to an isolated perfused organ under the appropriate experimental conditions. Then, the infusion buffer containing the spin-trap adduct(s) is placed into an quartz flat cell to be inserted into an ESR spectrometer. In the indirect method, the spin trap is added to the perfusate, which had previously exited the organ. Therefore, with this method one can prevent any spin-trap-mediated toxicities to the isolated perfused organ. However, because of the very rapid rate of free radical reactions catalyzed by either superoxide or hydroxyl radical, it is questionable whether ESR spectra recorded using this indirect method result from the actual spin-trapping of free radicals. In this report, we evaluated the indirect spin-trapping technique in light of the kinetic considerations discussed above.