Institute of Clinical Chemistry and Laboratory Medicine, University Hospital and Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
Department of Medicine III, University Hospital and Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Apr 1;1236:124062. doi: 10.1016/j.jchromb.2024.124062. Epub 2024 Feb 27.
Reduced and oxidized glutathione play an important role for the intracellular detoxification of reactive oxygen species. The iron-dependent formation of such reactive oxygen species in conjunction with the inhibition of the redox-balancing enzyme glutathione peroxidase 4 underlie an imbalance in the cellular redox state, thereby resulting in a non-apoptotic form of cell death, defined as ferroptosis, which is relevant in several pathologies.
Here we present a rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based method providing the accurate quantification of 12 glutathione pathway metabolites after in situ derivatization with N-Ethylmaleimide (NEM). The method was validated regards linearity, recovery and accuracy as well as precision. The assay includes glutathione and its oxidized form glutathione disulfide. Furthermore, the related precursors cysteine, cystine, glutamic acid, γ-glutamylcysteine and cysteinylglycine, biomarkers of protein crosslinking such as cystathionine and lanthionine, as well as metabolites of the transsulfuration pathway, methionine, homocysteine and serine are simultaneously determined.
Twelve glutathione pathway metabolites were simultaneously analyzed in four different human cell line extracts within a total LC run time of 5.5 min. Interday coefficients of variation (1.7 % to 12.0 %), the mean observed accuracy (100.0 % ± 5.2 %), linear quantification ranges over three orders of magnitude for all analytes and sufficient metabolite stability after NEM-derivatization demonstrate method reliability. Immediate derivatization with NEM at cell harvesting prevents autooxidation of glutathione, ensures accurate results for the GSH/GSSG redox ratio and thereby allows interpretation of cellular redox state.
The described UPLC-MS/MS method provides a sensitive and selective tool for a fast and simultaneous analysis of glutathione pathway metabolites, its direct precursors and related compounds. Assay performance characteristics demonstrate the suitability of the method for applications in different cell cultures. Therefore, by providing glutathione related functional metabolic readouts, the method enables investigations in mechanisms of ferroptosis and alterations in oxidative stress levels in several pathophysiologies.
还原型和氧化型谷胱甘肽在细胞内解毒活性氧物质中发挥着重要作用。铁依赖性活性氧物质的形成以及还原平衡酶谷胱甘肽过氧化物酶 4 的抑制导致细胞内氧化还原状态失衡，从而导致一种非凋亡形式的细胞死亡，即铁死亡，这在几种病理学中具有重要意义。
本文介绍了一种快速超高效液相色谱-串联质谱（UPLC-MS/MS）方法，该方法通过原位与 N-乙基马来酰亚胺（NEM）衍生化，可对 12 种谷胱甘肽途径代谢物进行准确的定量分析。该方法对线性、回收率和准确性以及精密度进行了验证。该测定法包括谷胱甘肽及其氧化形式二硫化物谷胱甘肽。此外，还同时测定了相关前体半胱氨酸、胱氨酸、谷氨酸、γ-谷氨酰半胱氨酸和半胱氨酰甘氨酸、蛋白质交联的生物标志物如胱硫醚和高半胱氨酸以及转硫途径的代谢物蛋氨酸、同型半胱氨酸和丝氨酸。
在总 LC 运行时间为 5.5 分钟内，对四种不同的人细胞系提取物中的 12 种谷胱甘肽途径代谢物进行了同时分析。日内变异系数（1.7%-12.0%）、平均观察准确度（100.0%±5.2%）、所有分析物的线性定量范围跨越三个数量级、NEM 衍生化后代谢物具有足够的稳定性，证明了方法的可靠性。细胞收获时立即用 NEM 衍生化可防止谷胱甘肽的自动氧化，确保 GSH/GSSG 氧化还原比的准确结果，从而可以解释细胞内氧化还原状态。
本文所述的 UPLC-MS/MS 方法提供了一种灵敏、选择性的工具，可快速、同时分析谷胱甘肽途径代谢物及其直接前体和相关化合物。分析性能特征表明该方法适用于不同细胞培养物的应用。因此，通过提供与谷胱甘肽相关的功能代谢读数，该方法可用于研究铁死亡机制和几种病理生理学中氧化应激水平的变化。
