Tomin Tamara, Schittmayer Matthias, Birner-Gruenberger Ruth
Institute of Chemical Technologies and Analytics, Faculty of Technical Chemistry, Vienna University of Technology-TU Wien, Getreidemarkt 9/164, 1060 Vienna, Austria.
Diagnostic and Research Institute of Pathology, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Stiftingtalstrasse 6, 8010 Graz, Austria.
Metabolites. 2020 Feb 16;10(2):71. doi: 10.3390/metabo10020071.
Determination of the ratio of reduced to oxidized glutathione is of profound clinical interest in assessing the oxidative status of tissues and body fluids. However, this ratio is not yet a routine clinical parameter due to the analytically challenging interconversion of reduced (free) glutathione to oxidized (bound) glutathione. We aimed to facilitate this ratio determination in order to aid its incorporation as a routine clinical parameter. To this end, we developed a simple derivatization route that yields different isotopologues of N-ethylmaleimide alkylated glutathione from reduced and oxidized glutathione (after its chemical reduction) for mass spectrometric analysis. A third isotopologue can be used as isotopic standard for simultaneous absolute quantification. As all isotopologues have similar chromatographic properties, matrix effects arising from different sample origins can only impact method sensitivity but not quantification accuracy. Robustness, simplified data analysis, cost effectiveness by one common standard, and highly improved mass spectrometric sensitivity by conversion of oxidized glutathione to an alkylated glutathione isotopologue are the main advantages of our approach. We present a method fully optimized for blood, plasma, serum, cell, and tissue samples. In addition, we propose production of N-ethylmaleimide customized blood collection tubes to even further facilitate the analysis in a clinical setting.
测定还原型谷胱甘肽与氧化型谷胱甘肽的比例在评估组织和体液的氧化状态方面具有深远的临床意义。然而，由于还原型（游离）谷胱甘肽与氧化型（结合）谷胱甘肽之间的相互转化在分析上具有挑战性，该比例尚未成为常规临床参数。我们旨在促进该比例的测定，以帮助将其纳入常规临床参数。为此，我们开发了一种简单的衍生化方法，该方法可从还原型和氧化型谷胱甘肽（化学还原后）产生不同的N - 乙基马来酰亚胺烷基化谷胱甘肽同位素异构体，用于质谱分析。第三种同位素异构体可作为同时进行绝对定量的同位素标准。由于所有同位素异构体具有相似的色谱性质，不同样品来源产生的基质效应只会影响方法的灵敏度，而不会影响定量准确性。我们方法的主要优点包括稳健性、简化的数据分析、通过一种通用标准实现成本效益以及通过将氧化型谷胱甘肽转化为烷基化谷胱甘肽同位素异构体极大提高质谱灵敏度。我们提出了一种针对血液、血浆、血清、细胞和组织样本进行了充分优化的方法。此外，我们建议生产定制的N - 乙基马来酰亚胺采血管，以进一步方便临床环境中的分析。
