Validation of a liquid chromatography tandem mass spectrometry method to measure oxidized and reduced forms of glutathione in whole blood and verification in a mouse model as an indicator of oxidative stress
Introduction
Increasing evidence suggests that oxidative stress is linked to various human diseases such as chronic lung diseases, cardiovascular diseases, neurodegenerative disorders, diabetes, rheumatoid arthritis, and malignancy, as well as the general aging process [1], [2], [3], [4], [5], [6]. However, this association still needs to be confirmed, and has likely been underestimated because of recognized shortcomings of the methods for assessing oxidative stress in vivo in humans [3], [7]. Glutathione (γ-glutamyl-l-cysteinylglycine) is an important antioxidant that plays a key role in the cellular resistance to oxidative damage [8]. It is present mainly in its reduced form (GSH), however, under oxidative conditions two GSH molecules are oxidized to form glutathione disulfide (GSSG). A low GSH level, high GSSG level, and/or a low GSH/GSSG ratio are interpreted as increased oxidative stress [9]. As functional tissue is less accessible, whole blood is a noninvasive alternative to use for GSH and GSSG measurement, which has been confirmed to reflect the status of other tissues [3], [10]. Therefore, the levels of GSH and GSSG in blood have been considered an index of whole body glutathione status and have been used in clinical diagnosis and investigations of many diseases [3].
The authors of many previous studies have measured whole blood GSH and GSSG, comparing concentrations obtained in healthy individuals with those measured in persons affected by various diseases, and reporting a relationship between the disease and low GSH/GSSG ratios [2], [11], [12]. However, various methods have been used for the determination of glutathione, which has induced large differences in the reported values of GSH and GSSG between investigations, even for control values in healthy people [1], [13]. As the main cause of this wide variation is considered to be artifactual GSH oxidation during sample processing and consequently marked overestimation of GSSG level, particular attention should be paid to sample processing [3], [9]. To prevent artifactual GSH oxidation, methods derivatizing GSH with alkylating agents such as N-ethylmaleimide (NEM) [9], iodoacetic acid [14], 5-iodoacetamidfluorescein [15], phthalimide [16], and dithionitrobenzoate [17] have been devised. Among these agents, NEM has been most widely used, and pretreatment of samples with NEM immediately after blood collection has been recommended as the most reliable method [9]. A wide variety of methods including spectrophotometry, fluorometry, high pressure liquid chromatography (HPLC), and HPLC coupled to tandem mass spectrometry (HPLCMS/MS) have been used for GSH and GSSG measurement [18]. Among these methods, HPLCMS/MS offers the most sensitive and selective glutathione measurement [19], [20].
In this study, we developed a new liquid chromatography tandem mass spectrometry method for determination of whole blood GSH and GSSG, modifying a recently proposed method by Moore et al. [21]. In addition, we validated the relevance of the GSH/GSSG ratio as a marker of oxidative damage in a well-documented animal model of chronic oxidative stress.
Section snippets
Mouse model development
A previously well-established mouse model of chronic iron overload was employed for this experiment [22]. This mouse model consists of 89 weeks old (2530 g) male inbred BALB/C mice (OrientBio, Seoul, Korea), which have genetic and phenotypic uniformity. Iron loading was achieved by intraperitoneal (IP) injections of iron dextran (CosmoFer for injection, Pharmbio Korea Co., Ltd., Seoul, Korea) over a defined experimental period. The current investigation acquired institutional approval from the
Chromatography
Typical chromatograms of GSH-NEM and GSSG produced by LCMS/MS are shown in Fig. 1. GSH-NEM and GSSG were eluted from the column at 3.1 and 3.8 min, respectively. The type and concentration of electronic modifiers in the mobile phase can have a noticeable effect on the retention, selectivity, and the peak symmetry of solutes separated on Hyercarb columns. As mobile phase additives, either 0.1% formic acid, 0.1% diethylamine, or 0.1% TFA was evaluated. Optimal symmetric peak shape was achieved
Discussion
Despite considerable interest in GSH and GSSG ratio as an index of oxidative stress, there has been no standardized protocol for sample preparation and analysis for the quantification of these molecules in biological samples [3], [8], [9]. Consequently, various research groups have reported rather different levels of GSH and, particularly, GSSG, even in the blood of healthy individuals used as controls [3], [9]. The major artifact that can occur during GSH and GSSG quantification is about 515%
Conclusions
We have developed and validated an accurate and reliable method for quantification of whole blood GSH and GSSG using LCMS/MS. Artifactual GSH auto-oxidation was prevented through derivatization with NEM immediately after drawing whole blood. The precipitation of proteins using 10% SSA containing isotopically labeled internal standard also minimized the preanalytical and analytical variations between samples. Our data showed sufficiently precise and accurate measurements of both GSH and GSSG.
Conflict of interest
All authors declare no conflict of interest.
Acknowledgment
This research was supported by a faculty research grant of Yonsei University College of Medicine (6-2014-0013).
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