Clinical assay of four thiol amino acid redox couples by LC–MS/MS: Utility in thalassemia☆
Introduction
Sulfur amino acid (SAA) metabolism plays an essential role in the regulation of oxidative stress in biological fluids, cells, and tissues [1], [2], [3], [4]. Four closely integrated metabolic pathways – the transmethylation, transsulfuration, glutathione synthesis, and the glutathione catabolic pathways – produce several key metabolites that are essential for physiological redox regulation [1], [5]. Four unique sulfur-containing redox couples, namely, homocysteine (Hcy)/homocystine (HcySS), cysteine (Cys)/Cystine (CysSS), glutathione (GSH)/glutathione disulfide (GSSG) and the cysteinylglycine (CysGly)/cysteinylglycine disulfide (CysGlySS) are produced by the above pathways. On a metabolic level, these SAA metabolites share tightly regulated precursor–product relationships. For example, Hcy serves as an important metabolic precursor for endogenous Cys synthesis by the transsulfuration pathway [5], [6]; Cys is the rate-limiting substrate for GSH formation [7]; and, CysGly is formed during GSH-breakdown and subsequently can be recycled to yield cysteine in cells [8].
Aside from their metabolic linkages, these compounds also interact with each other and with other protein thiol targets chemically through thiol–disulfide exchange reactions that can ultimately influence cellular thiol/disulfide equilibrium. The specificity of their redox interactions are regulated by their individual concentrations and their specific standard redox potentials, which determine the thermodyamic feasibility for reduction/oxidation reactions between thiol redox couples and oxidants. For example, Hcy, which has a low reduction potential of −200 mV, exists predominantly as mixed disulfides and can act as a pro-oxidant [9]. In contrast, GSH has high reduction potential of −264 mV and exists predominantly in its reduced form even in the highly oxidizing extracellular environment. The steady-state redox states of these compounds appear to be discretely regulated. In human plasma, the Cys/CysSS redox couple is maintained at a more oxidized state than the GSH/GSSG redox couple, and upon exposure to oxidants, the oxidation of the Cys/CysSS precedes the oxidation of the GSH/GSSG redox couple [2], [3], [4], [10]. Distinctive redox regulation of these compounds suggests that comprehensive analysis of all four oxidation states may provide more detailed information on systemic oxidative stress than a “snapshot” view of just a single redox couple [4].
Thalassemias are caused by defective synthesis of either α- or β-globin chains. In β-thalassemia, impaired β-globin synthesis causes an accumulation of unpaired α-globin protein [11]. As a consequence of disrupted globin synthesis, β-thalassemia patients suffer from ineffective erythropoesis and anemia, which necessitates lifelong blood transfusions [12]. Frequent blood transfusions cause secondary iron-overload in thalassemia patients, leading to excessive iron depositions in tissues such as the liver and the heart [13]. Increased availability of redox-active iron may cause oxidative damage to tissues and elevate the risks for cardiovascular and endocrine dysfunctions [12], [14], [15].
Iron-chelation therapy aimed at normalizing tissue iron status can lessen the incidence and the severity of secondary clinical complications in thalassemias [15], [16]. However, owing to the lack of proximate sensitive and accessible markers of the consequences of iron-overload, the clinical management of iron-overload using chelation therapy remains imprecise [17], [18]. Because SAA metabolites are sensitive targets of iron-dependent oxidative stress, alterations in SAA redox states in plasma or in erythroctyes may potentially serve as sensitive biomarkers of iron toxicity in thalassemias. While there are numerous analytical methods for quantifying SAA metabolites [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], existing methods have not been validated for measuring Hcy, Cys, GSH and CysGly redox states in thalassemia.
In the current work, we developed a simple and fast sample processing and analysis method for Hcy, Cys, GSH, and CysGly quantification. In this method, analytes are sequentially derivatized with iodoacetamide (IAM) and isopropylchlroformate (IPCF), and the derivatives are chromatographically resolved and detected by electrospray positive ionization–tandem mass spectrometry. Immediate mixing of blood samples with IAM minimizes the potential ex vivo oxidation in blood samples. This step is especially critical for thalassemia samples, where increased iron and hemoglobin availability increase the likelihood of thiol oxidation during plasma and erythroctye isolation from whole blood. The subsequent IPCF derivatization improves the chromatographic resolution of amino acid analytes [29], [30]. For IPCF derivatization, a commercially available EZ-FAAST™ Amino Acid LCMS kit is used. The EZ-FAAST kit includes a proprietary strong cation exchange resin filled solid-phase extraction (SPE) tips and reversed-phase HPLC column that greatly enhances analyte enrichment and improve chromatographic separation of analytes. The SPE tips included in this kit cannot be purchased individually and provides a convenient small volume analyte enrichment solution that cannot be easily replicated with standard SPE products. The combined usage of sequential IAM/IPCF derivation procedure with the proprietary SPE tips and HPLC column provided in the EZ-FAAST kit, efficiently minimized the ex vivo oxidation, improved the enrichment of low abundant oxidized disulfides, and the specificity of detection of the SAA metabolites.
Section snippets
Reagents and analytical standards
The following reagents of highest analytical grade were purchased from Sigma–Aldrich (St. Louis, MO): homocysteine (Hcy), homocystine (HcySS), cysteine (Cys), cystine (CysSS), glutathione (GSH), glutathione disulfide (GSSG), cysteinyl glycine (CysGly), cysteinyl glycine disulfide (CysGlySS), diethylene triamine pentaacetic acid (DTPA), dithiothreitol (DTT), Tris-base and iodoacetamide (IAM). Homoglutathione (hGSH) was purchased from BACHEM America Inc. (Torrance, CA). Stable isotopes,
Optimization of derivatization procedure
In the current method, thiol containing SAA metabolites stabilized with IAM prior to acidification and S-carboxyamidomethylated thiols were further derivatized with IPCF. The optimal condition for S-carboxyamidomethylation was to maintain plasma pH at 8.0 and use 20 mM concentrations of IAM. This is achieved by mixing equal volume of plasma with 20 mM solution of IAM dissolved in 100 mM Tris–HCl, pH 8.0 solution.
The reaction sequence of IPCF derivatization of IAM-modified Cys is shown as an
Conclusion
A LC/MS/MS method was developed and validated for the quantification of major SAA metabolites. The sample processing involves sequential derivatization with IAM and IPCF, using the commercially available EZ-FAAST LCMS amino acid analysis kit. The three main advantages of using the EZ-FAAST kit are (1) the convenience and the efficiency of their proprietary SPE tips for sample enrichment, (2) the improved performance of their proprietary reversed-phase column and (3) the ability to standardize
Acknowledgements
This work was supported by grant support from PO1 AT002620-01 to B.N. Ames and J.H. Suh, NIH/NIDDKD T32 DK078514-06A2 fellowship and S.D. Bechtel Jr. Foundation Award to J.H. Suh and Minority Health Disparity Center Grant P60MD00222 to M.K. Shigenaga.
References (43)
- et al.
J. Nutr.
(2006) - et al.
Free Radic. Biol. Med.
(2001) - et al.
Biol. Chem.
(1975) - et al.
Atherosclerosis
(2000) - et al.
Free Radic. Biol. Med.
(2003) - et al.
Can. J. Cardiol.
(2009) Blood
(2006)- et al.
J. Chromatogr. B
(2001) - et al.
J. Pharm. Biomed. Anal.
(2001) - et al.
J. Chromatogr. B
(2003)
J. Pharm. Biomed. Anal.
Anal. Biochem.
J. Chromatogr. B
J. Chromatogr. B
Clin. Chim. Acta
J. Chromatogr. B
J. Chromatogr. B
Clin. Chim. Acta
Free Radic. Biol. Med.
Free Radic. Biol. Med.
Blood
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This paper is part of the special issue “Analysis of Thiols”, I. Dalle-Donne and R. Rossi (Guest Editors).