Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS: An insight into the redox state of hematopoietic stem cells.
Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS: An insight into the redox state of hematopoietic stem cells.
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DOI：
10.1016/j.freeradbiomed.2016.05.005
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发表时间：
2016-08
影响因子：
7.4
通讯作者：
St Clair DK
中科院分区：
文献类型：
--
作者：
Carroll D;Howard D;Zhu H;Paumi CM;Vore M;Bondada S;Liang Y;Wang C;St Clair DK
细胞氧化还原平衡在造血干祖细胞（HSC/MPP）自我更新和分化的调控中起着重要作用。细胞氧化还原稳态的不受调节的变化与大多数血液学疾病的发生有关。然而，准确测量干细胞中的氧化还原状态是困难的，因为缺乏HSC/MPP。谷胱甘肽（GSH）构成了最丰富的细胞抗氧化剂库。因此，GSH代谢可能在血液病的发病和进展中起关键作用。研究HSC/MPPs中GSH代谢的一个主要限制是无法定量测量少量HSC/MPPs中的GSH浓度。目前用于测量GSH水平的方法不仅依赖于大量的细胞，而且还依赖于GSH的化学/结构修饰或酶促再循环，因此可能仅准确测量总谷胱甘肽含量。在这里，我们描述了一种灵敏的方法，用于直接和同时定量的氧化和还原型谷胱甘肽通过液相色谱法，然后串联质谱法（LC-MS/MS）从骨髓中分离的HSC/MPPs的验证。GSH和GSSG的定量下限（LLOQ）分别确定为5.0 ng/mL和1.0 ng/mL，两种谷胱甘肽物质的检测下限均为0.5 ng/mL。利用小鼠骨髓进行的标准加入分析表明，该方法灵敏度高，准确度高，分析物回收率可重现。该方法将简单提取与高通量分析平台相结合，允许有效测定小鼠HSC/MPP群体、细胞培养物内的化疗治疗条件和人类正常/白血病患者样品中的GSH/GSSG浓度。这些数据暗示了GSH/GSSG氧化还原对的调节在干细胞相关疾病中的重要性。
Cellular redox balance plays a significant role in the regulation of hematopoietic stem-progenitor cell (HSC/MPP) self-renewal and differentiation. Unregulated changes in cellular redox homeostasis are associated with the onset of most hematological disorders. However, accurate measurement of the redox state in stem cells is difficult because of the scarcity of HSC/MPPs. Glutathione (GSH) constitutes the most abundant pool of cellular antioxidants. Thus, GSH metabolism may play a critical role in hematological disease onset and progression. A major limitation to studying GSH metabolism in HSC/MPPs has been the inability to measure quantitatively GSH concentrations in small numbers of HSC/MPPs. Current methods used to measure GSH levels not only rely on large numbers of cells, but also rely on the chemical/structural modification or enzymatic recycling of GSH and therefore are likely to measure only total glutathione content accurately. Here, we describe the validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) in HSC/MPPs isolated from bone marrow. The lower limit of quantitation (LLOQ) was determined to be 5.0 ng/mL for GSH and 1.0 ng/mL for GSSG with lower limits of detection at 0.5 ng/mL for both glutathione species. Standard addition analysis utilizing mouse bone marrow shows that this method is both sensitive and accurate with reproducible analyte recovery. This method combines a simple extraction with a platform for the high-throughput analysis, allows for efficient determination of GSH/GSSG concentrations within the HSC/MPP populations in mouse, chemotherapeutic treatment conditions within cell culture, and human normal/leukemia patient samples. The data implicate the importance of the modulation of GSH/GSSG redox couple in stem cells related diseases.