Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario L8S 4M1, Canada.
Anal Chem. 2009 Aug 15;81(16):7047-56. doi: 10.1021/ac901174g.
Glutathione metabolism plays a fundamental role in maintaining homeostasis and regulating the redox environment of a cell. Despite the widespread interest in quantifying glutathione metabolites in oxidative stress research, conventional techniques are hampered by complicated sample handling procedures to prevent significant oxidation artifacts generated during sample collection, sample pretreatment, and/or chemical analysis. In this report, a simple and validated method for glutathione analysis from filtered red blood cell (RBC) lysates was developed using capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in conjunction with fingerprick microsampling and ultrafiltration. About a 3-fold improvement in precision with nanomolar detection limits was achieved when using online sample preconcentration with CE-ESI-MS via a modified injection sequence, which permitted accurate determination of the intracellular reduced/oxidized glutathione ratio (GSH/GSSG), as well as other glutathione species, including protein-bound glutathione mixed disulfide (PSSG), free glutathione mixed disulfides (GSSR) and glutathione thioether conjugates (GSX). In this work, the redox status of filtered hemolysates was determined by the equilibrium half-cell reduction potential for glutathione (E(GSSG/2GSH)), whereas its intrinsic antioxidant capacity was assessed by the apparent rate of metal-catalyzed oxidation of glutathione. In-vitro incubation studies of intact RBCs with 1-chloro-2,4-dinitrobenzene (CDNB) and N-acetyl-L-cysteine (NAC) were found to significantly alter E(GSSG/2GSH) and/or glutathione oxidation kinetics (e.g., k(GSSG)) relative to normal controls based on their function as a toxic electrophilic compound and a competitive free radical scavenging/reducing agent, respectively. Differential rates of glutathione oxidation (DIRGO) using CE-ESI-MS offers a novel strategy for global assessment of the impact of intrinsic metabolite constituents (i.e., metabolome) and/or extrinsic perturbants on cellular redox status that is relevant to improved understanding of aging and the pathogenesis of acute or chronic disease states.
谷胱甘肽代谢在维持细胞内环境平衡和调节氧化还原环境方面起着至关重要的作用。尽管人们广泛关注在氧化应激研究中定量测定谷胱甘肽代谢物，但传统技术受到复杂的样本处理程序的限制，这些程序旨在防止在样本收集、预处理和/或化学分析过程中产生显著的氧化假象。在本报告中，开发了一种使用毛细管电泳-电喷雾电离-质谱（CE-ESI-MS）结合指尖微采样和超滤从过滤的红细胞（RBC）裂解物中分析谷胱甘肽的简单且经过验证的方法。通过修改的进样序列，使用 CE-ESI-MS 进行在线样品预浓缩，可以将精度提高约 3 倍，同时将检测限降低至纳摩尔水平，这使得可以准确测定细胞内还原/氧化型谷胱甘肽比（GSH/GSSG）以及其他谷胱甘肽物质，包括与蛋白质结合的谷胱甘肽混合二硫化物（PSSG）、游离谷胱甘肽混合二硫化物（GSSR）和谷胱甘肽硫醚轭合物（GSX）。在这项工作中，通过谷胱甘肽的半细胞还原电位（E（GSSG/2GSH））来确定过滤后的溶血物的氧化还原状态，而其内在抗氧化能力则通过谷胱甘肽的金属催化氧化的表观速率来评估。用 1-氯-2,4-二硝基苯（CDNB）和 N-乙酰-L-半胱氨酸（NAC）对完整 RBC 进行体外孵育研究发现，相对于正常对照，它们的功能分别为有毒的亲电化合物和竞争性自由基清除/还原试剂，E（GSSG/2GSH）和/或谷胱甘肽氧化动力学（例如，k（GSSG））发生显著变化。使用 CE-ESI-MS 的谷胱甘肽氧化差异率（DIRGO）提供了一种新策略，用于全面评估内在代谢物成分（即代谢组）和/或外在干扰物对细胞氧化还原状态的影响，这对于更好地理解衰老和急性或慢性疾病状态的发病机制具有重要意义。
