Development of a reliable method based on ultra-performance liquid chromatography coupled to tandem mass spectrometry to measure thiol-associated oxidative stress in whole blood samples

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Highlights

  • Validation of an UPLC–MS/MS analytical method to determine the levels of glutathione, cysteine, homocysteine, and their respective oxidized compounds in whole blood samples.

  • High selectivity and sensitivity method without reaction steps.

  • Suitability of the method in whole blood samples obtained from a consolidated experimental model of hypoxia-reoxygenation in newborn piglets.

  • The method has potential in the application to human newborn clinical assays.

Abstract

The aminothiols are biological compounds with numerous vital functions. One of the most relevant is their role as antioxidant systems. The reduced to oxidized ratios are extremely useful indicators of oxidative stress and cellular redox status. We have validated an ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS) method to determine the levels of glutathione, cysteine, homocysteine, and their respective oxidized compounds in whole blood samples. Results showed excellent linearity for all the analytes with correlation coefficients between 0.990 and 0.997, suitable precision with intra-day coefficient of variation ≤20%, and satisfactory accuracy with recoveries between 75 and 130%. The limits of detection in whole blood samples were 1.16 nmol L−1 for glutathione, 115.8 nmol L−1 for oxidized glutathione, 9.3 nmol L−1 for homocystine, 92.6 nmol L−1 for homocysteine, 347 nmol L−1 for cystine and 0.23 nmol L−1 for cysteine.

The suitability of the method was ascertained in whole blood samples (n = 80) from a consolidated experimental model of hypoxia-reoxygenation in newborn piglets.

Introduction

The aminothiols are biologically active compounds. They are implied in numerous vital functions such as detoxification and regulation of cellular metabolism and enzymatic activity. Primary biological aminothiols are cysteine, homocysteine, cysteinylglycine and glutathione [1]. Glutathione (GSH) is the most abundant non-enzymatic cellular antioxidant, expressed ubiquitously, that has an important function in detoxification (xenobiotics, carcinogens, free radicals, peroxides), and regulation of the immune function. Low concentrations of GSH have been implicated in numerous pathological conditions (diabetes, alcoholic liver disease, AIDS, Parkinson disease or acute pancreatitis [2]) and GSH concentration is broadly used as useful indicator of disease risk in humans. Previous results suggest that whole blood GSH may serve as an accurate indicator of GSH status in human subjects [3]. GSH is present as an intracellular component in blood at concentrations of ∼1 mmol L−1, with the amounts in plasma representing <1% of total. Oxidized glutathione (GSSG) exists as a small fraction of total GSH under normal conditions, but it increases under conditions of oxidative stress. Therefore, the developed methods to determine GSH should be able to determine small amounts of GSSG as well [4]. Since, artifactual oxidation to GSSG can occur during sample storage and treatment, derivatization with an alkylating agent, such as N-ethylmaleimide (NEM) is required immediately after sampling [5].

Nevertheless, another important aspect to take into account is that the aminothiols are metabolically related among them. Actually, the synthesis and degradation of GSH is controlled by reactions of the γ-glutamyl cycle [4]. Cysteine and γ-glutamylcysteine are precursors to GSH, and cysteinyl-glycine is a breakdown product of GSH. In addition to this, homocysteine may be catabolized to cysteine or remethylated to methionine, connecting therefore, GSH synthesis to methylation reactions and epigenetics (see Fig. 1) [6]. It has been speculated that disturbances of the aminothiols concentrations can also correspond to metabolic disorders, and their simultaneous measurement may provide interesting information. Levels of the aminothiols and their oxidized pairs (redox couples) could be regulating the redox environment of cellular organelles such as nucleus or endoplasmic reticulum, which have a reduced and oxidized environment respectively (referred as redox compartmentalization) [7]. In fact, the overall distribution of protein thiols and disulfides in different cellular pools were studied in a previous work in order to define the global redox status of the cells [8].

Pathophysiologycally, it is known that an increase of homocysteine levels in plasma is a risk factor for cardiac infarction and diabetes [9]. On the other hand, redox couples are measured to evaluate the oxidative stress damage [10], [11]. For instance, in a previous study the results showed lower ratios in preeclampsia than normotensive pregnancy, indicating a shift in favor of the oxidized form of those thiols [10]. Moreover, newborn infants undergoing perinatal asphyxia have a significant reduction in the GSH/GSSG ratio especially when resuscitated with high oxygen concentrations, and GSSG concentrations in blood significantly correlate with damage to myocardium and kidney [12], [13]. Therefore, plasma aminothiols and their respective oxidized pairs concentrations are required to be measured for routine clinical diagnosis and translational research about oxidative stress.

In the literature we can find a large-scale study reporting pediatric values of total GSH, GSH, and GSSG in whole blood samples previously hemolyzed [14]. As regards the sample treatment procedures found in literature, most of them consisted of a reduction step in order to determine total free aminothiols, followed by precipitation of proteins with acid or organic solvents, and a derivatization step depending on the analytical technique and detector employed [15], [16], [17].

Among the variety of techniques to measure aminothiol concentrations in plasma samples, liquid chromatography (HPLC) with fluorescence detection is the most widely used [18]. Other methods are based on HPLC with UV-photometric [5] or electrochemical detection [19], gas chromatography coupled to mass spectrometry (MS) [17], ion-exchange chromatography with photometric detection, capillary electrophoresis with fluorimetric [20] or UV-photometric[21] or electrochemical detection [22] and enzyme immunoassay with photometric detection [23]. In general, the results obtained from these methods are considered suitable for GSH and GSSG, since they take into account several pre-analytical and analytical conditions in order to obtain suitable analytical performance [5], [18], [19], [21]. However, the methods based on immunoassays are characterized by high cross reactivity (interferences), and matrix effect [23].

It is important to highlight that although HPLC–MS/MS has several advantages, such as high sensitivity and selectivity, few validated methods to determine aminothiols can be found in literature [24]. Also, few analytes were determined in these works, being GSH, GSSG, and homocysteine the most studied compounds [25], [26], [27].

The aim of this work was to validate a relative simple method to determine simultaneously pairs of reduced/oxidized aminothiols (redox couples), since they constitute a reliable index of oxidative stress to follow up certain circumstances such as birth asphyxia and resuscitation. However, the proposed method, with minor modifications, could be applied in future analysis to determine mixed disulfides which can represent a large fraction in blood. They are very important in the redox regulation since disulfide stress is associated with oxidation of thiols (cysteine, glutathione). In fact, in a previous work from our research group, the sample treatment to measure protein mixed disulfides was optimized [2]. Briefly, it consisted of the following steps: i) sample acidification; ii) recovery and washing of the protein pellet; iii) protein re-solubilization at neutral pH; iv) reduction with dithiothreitol (DTT); iv) derivatization of new formed thiols with N-ethylmaleimide (NEM); and v) final acidification. In this way, all the aminothiols conjugated with NEM were derived from mixed disulfides with proteins.

To our knowledge this is the first validated method to determine redox couple ratios in whole blood samples by means of ultrahigh performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS/MS). Herewith, we aim to apply this method in clinical studies, such as the analysis of blood samples from asphyxiated newborn infants, with hypoxic-ischemic encephalopathy and therapeutic hypothermia in the frame of a multicenter randomized controlled trial.

Section snippets

Sample collection and storage

Biological samples (acidified supernatans) were obtained from Noroc (LyxLD) newborn piglets provided by the group of the Pediatric Research Department of the Oslo University Hospital (Prof. O.D. Saugstad). The Norwegian National Animal Research Authority (NARA) approved the experimental protocol, and the animals were cared for and handled in accordance with the European Guidelines for Use of Experimental Animals certified by FELASA (Federation of European Laboratory Animals Science

Analytical performance data

The reliability of the method described was evaluated by employing standards in H2O (0.1% v/v HCOOH) that contained the analytes in the 1.9–1900 nmol L−1 concentration range, except for GSSG (9.3–9500 nmol L−1), cysteine (9.3–9500 nmol L−1) and GSH (185.5–190000 nmol L−1). As shown in Table 2, the method provided an adequate linearity for all the analytes within the corresponding concentrations ranges, with R2 values between 0.990 and 0.997. As regards precision, the intra-day and inter-day

Conclusions

The present work describes the development of a reliable, simple and sensitive UPLC–MS/MS method for the simultaneous determination of three ratios of reduced/oxidized aminothiols. Of note, the lack of validated liquid chromatographic methods to determine all these compounds simultaneously in whole blood samples adds relevance to this new method. The described UPLC–MS/MS method can be considered a simple alternative to previously published methods, which require aminothiol reduction, and/or

Conflict of interest

The authors declares no conflict of interest.

Acknowledgements

CC-P acknowledges a Carmen & Severo Ochoa Grant (City Government; Valencia); JK acknowledges the ‘Sara Borrell’ grant (CD12/00667) from the Instituto Carlos III (Ministry of Economy and Competitiveness, Spain). AS-I and MV acknowledge the financial support from the Spanish Maternal and Child Health and Development Network SAMIDRD12/0022/12 from the Instituto Carlos III (Spanish Ministry of Economy and Competitiveness) also supported by (FEDER funds from the European Union.). MV acknowledges a

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