Simultaneous determination of reduced and oxidized glutathione in tissues by a novel liquid chromatography-mass spectrometry method: application in an inhalation study of Cd nanoparticles
Simultaneous determination of reduced and oxidized glutathione in tissues by a novel liquid chromatography-mass spectrometry method: application in an inhalation study of Cd nanoparticles
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DOI：
10.1007/s00216-014-8033-z
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发表时间：
2014-09-01
影响因子：
4.3
通讯作者：
Hilscherova, K.
中科院分区：
文献类型：
--
作者：
Blahova, L.;Kohoutek, J.;Hilscherova, K.
本文介绍了一种先进的提取和快速分析LC MS/MS方法，同时分析还原型和氧化型谷胱甘肽（GSH和GSSG，分别）在不同的动物组织。GSH和GSSG的同时测定是至关重要的，因为GSH和GSSG的量和比例可能会改变氧化应激反应，毒性的一个重要机制。该方法使用GSH中游离巯基的衍生化。其性能在小鼠中较少探索的组织（肺、脑和肝）中得到证实。组合提取和分析方法具有非常低的变异性和良好的重现性，运行内和运行间分析的最大变异系数低于8%，定量限低; GSH和GSSG分别为0.2 nM（0.06 ng/mL）和10 nM（6 ng/mL）。该方法的性能进一步证明了在模型实验中的GSH和GSSG浓度的变化，在急性72小时和慢性13周暴露于CdO纳米粒子的小鼠肺。吸入染毒导致肺组织GSH浓度升高。GSSG水平一般不受影响;只有在较长的13周暴露期后才发生不显著的抑制。所开发的用于在非常低的组织质量中灵敏地检测GSH和GSSG的方法使这些参数能够在只有少量样品可用的情况下进行研究，即在小生物体或少量组织中。
The paper presents the development of an advanced extraction and fast analytical LC MS/MS method for simultaneous analyses of reduced and oxidized glutathione (GSH and GSSG, respectively) in different animal tissues. The simultaneous determination of GSH and GSSG is crucial because the amount and ratio of both GSH and GSSG may be altered in response to oxidative stress, an important mechanism of toxicity. The method uses the derivatization of free thiol groups in GSH. Its performance was demonstrated for less explored tissues (lung, brain, and liver) in mouse. The combined extraction and analytical method has very low variability and good reproducibility, maximum coefficients of variance for within-run and between-run analyses under 8 %, and low limits of quantification; for GSH and GSSG, these were 0.2 nM (0.06 ng/mL) and 10 nM (6 ng/mL), respectively. The performance of the method was further demonstrated in a model experiment addressing changes in GSH and GSSG concentrations in lung of mice exposed to CdO nanoparticles during acute 72 h and chronic 13-week exposures. Inhalation exposure led to increased GSH concentrations in lung. GSSG levels were in general not affected; nonsignificant suppression occurred only after the longer 13-week period of exposure. The developed method for the sensitive detection of both GSH and GSSG in very low tissue mass enables these parameters to be studied in cases where only a little sample is available, i.e. in small organisms or in small amounts of tissue.