A novel protocal to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO and DMDO: LC/ESR, LC/MS, and dual spin trapping combinations
A novel protocal to identify and quantify all spin trapped free radicals from in vitro/in vivo interaction of HO and DMDO: LC/ESR, LC/MS, and dual spin trapping combinations
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DOI：
10.1016/j.freeradbiomed.2004.09.024
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发表时间：
2005-01-01
影响因子：
7.4
通讯作者：
Mason, RP
中科院分区：
文献类型：
--
作者：
Qian, SY;Kadiiska, MB;Mason, RP
当二甲基亚砜（DMSO）通过羟基自由基（HO*）氧化时，它形成甲基自由基（* CH 3），可以通过电子自旋共振（ESR）进行自旋捕获和检测。这种ESR自旋捕获技术已被广泛用于许多生物系统中，以指示体内HO* 的形成。然而，我们最近报道，* CH 3可能不是唯一的碳中心的自由基，被捕获和检测的EsR从体内DMSO氧化。在本研究中，新开发的组合技术，包括双自旋捕获（自由基捕获的规则和氘代α-[4-吡啶基]-N-叔丁基硝酮，d（0）/d（9）-POBN），然后LC/ESR和LC/MS被用来表征和量化所有POBN捕获的自由基从HO* 和DMSO的相互作用。除了从这种相互作用中识别出两种众所周知的自由基'CH 3和 * OCH 3之外，我们还表征了另外两种自由基 * CH 2 OH和 * CH 2S（O）CH 3。与ESR不同，它只能测量POBN加合物的自由基形式，LGMS确定和量化所有三种氧化还原形式，包括ESR活性自由基加合物和两种ESR沉默形式，硝酮加合物（氧化加合物）和羟胺（还原加合物）。在用DMSO和POBN处理的大鼠胆汁中，未检测到POBN/* CH 3的ESR活性形式。然而，随着LC/MS技术的加入，我们发现类似于0.75 μ M POBN/* CH 3羟胺，这代表了自由基检测灵敏度和可靠性的极大提高。这种新的协议提供了一个全面的方式来表征和量化在体外和体内自由基的形成，并将在生物学研究中有许多应用。(C)2004年爱思唯尔公司All rights reserved.
When dimethyl sulfoxide (DMSO) is oxidized via hydroxyl radical (HO*), it forms methyl radicals (*CH3) that can be spin trapped and detected by electron spin resonance (ESR). This ESR spin trapping technique has been widely used in many biological systems to indicate in vivo HO* formation. However, we recently reported that *CH3 might not be the only carbon-centered radical that was trapped and detected by EsR from in vivo DMSO oxidation. In the present study, newly developed combination techniques consisting of dual spin trapping (free radicals trapped by both regular and deuterated alpha-[4-pyridyl]-N-tert-butyl nitrone, d(0)/d(9)-POBN) followed by LC/ESR and LC/MS were used to characterize and quantify all POBN-trapped free radicals from the interaction of HO* and DMSO. In addition to identifying the two well-known free radicals, 'CH3 and *OCH3,, from this interaction, we also characterized two additional free radicals, *CH2OH and *CH2S(O)CH3. Unlike ESR, which can measure POBN adducts only in their radical forms, LGMS identified and quantified all three redox forms, including the ESR-active radical adduct and two ESR-silent forms, the nitrone adduct (oxidized adduct) and the hydroxylamine (reduced adduct). In the bile of rats treated with DMSO and POBN, the ESR-active form of POBN/*CH3 was not detected. However, with the addition of the LC/MS technique, we found similar to0.75 muM POBN/*CH3 hydroxylamme, which represents a great improvement in radical detection sensitivity and reliability. This novel protocol provides a comprehensive way to characterize and quantify in vitro and in vivo free radical formation and will have many applications in biological research. (C) 2004 Elsevier Inc. All rights reserved.