Molecular Speciated Isotope Dilution Mass Spectrometric Methods for

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Molecular Speciated Isotope Dilution Mass Spectrometric Methods for Accurate, Reproducible and Direct Quantification of Reduced, Oxidized and Total Glutathione in Biological Samples Timothy Fahrenholz,† Mesay Mulugeta Wolle,*,† H. M. “Skip” Kingston,† Scott Faber,‡ John C. Kern, II,§ Matt Pamuku,∥ Logan Miller,† Hemasudha Chatragadda,† and Andreas Kogelnik⊥ †

Department of Chemistry and Biochemistry, Duquesne University, 600 Forbes Avenue, Pittsburgh, Pennsylvania 15282, United States ‡ Department of Medicine, The Children’s Institute, 1405 Shady Avenue, Pittsburgh, Pennsylvania 15217, United States § Department of Mathematics and Computer Science, Duquesne University, 600 Forbes Avenue, Pittsburgh, Pennsylvania 15282, United States ∥ Applied Isotope Technologies, 2403 Sidney Street, Suite 280, Pittsburgh, Pennsylvania 15203, United States ⊥ Open Medicine Institute, 2500 Hospital Drive, Building 2, Mountain View, California 94040, United States S Supporting Information *

ABSTRACT: Novel protocols were developed to accurately quantify reduced (GSH), oxidized (GSSG) and total (tGSH) glutathione in biological samples using molecular speciated isotope dilution mass spectrometry (SIDMS). For GSH and GSSG measurement, the sample was spiked with isotopically enriched analogues of the analytes (310GSH and 616GSSG), along with N-ethylmaleimide (NEM), and treated with acetonitrile to solubilize the endogenous analytes via protein precipitation and equilibrate them with the spikes. The supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the analytes were quantified with simultaneous tracking and correction for auto-oxidation of GSH to GSSG. For tGSH assay, a 310GSH-spiked sample was treated with dithiothreitol (DTT) to convert disulfide-bonded glutathione to GSH. After removing the protein, the supernatant was analyzed by LC-MS/MS and the analyte was quantified by single-spiking isotope dilution mass spectrometry (IDMS). The mathematical relationships in IDMS and SIDMS quantifications are based on isotopic ratios and do not involve calibration curves. The protocols were validated using spike recovery tests and by analyzing synthetic standard solutions. Red blood cell (RBC) and saliva samples obtained from healthy subjects, and whole blood samples collected and shipped from a remote location were analyzed. The concentrations of tGSH in the RBC and whole blood samples were 2 orders of magnitude higher than those found in saliva. The fractions of GSSG were 0.2−2.2% (RBC and blood) and 15−47% (saliva) of the free glutathione (GSH + 2xGSSG) in the corresponding samples. Up to 3% GSH was auto-oxidized to GSSG during sample workup; the highest oxidations (>1%) were in the saliva samples.

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lutathione, the most abundant low molecular mass amino-thiol in the mammalian body, is not only the major antioxidant that maintains a tight redox status control within cells but is also implicated in a multitude of physiological processes such as DNA syntheses, amino acid transport, xenobiotic metabolism and enzymatic activities. 1,2 The tripeptide is virtually produced in all organs, especially in the liver, and is present in every tissue.2 Under normal conditions, glutathione is found almost exclusively in its reduced form (GSH) in the body,3 except in some parts of the body where the oxidized form (glutathione disulfide, GSSG) may be found in relatively high amounts due to the abundance of reactive oxygen species.4 Pathophysiologic conditions causing oxidative stress result in an increase in the level of GSSG, and a lower than normal GSH/GSSG ratio is used as an important indicator of oxidative stress and disease risk.3,5 © 2014 American Chemical Society

Blood is widely used for assessment of glutathione in humans, as it has been confirmed to reflect the status of the biomolecule in less accessible tissues and organs.6 The main portion of glutathione in blood circulation is located in the red blood cells (RBC) and it is used as a valid measure of the erythrocytic ability to overcome oxidative-induced conditions.7 Alterations in GSH/GSSG ratios have been identified in blood or RBC from patients with diabetes,8 autism spectrum disorders,9 acquired immunodeficiency syndrome (AIDS) infection,10 respiratory distress syndrome11 and liver diseases.12 Saliva, a less invasive bodily fluid, can be potentially used for Received: October 21, 2014 Accepted: December 18, 2014 Published: December 18, 2014 1232

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physiological fluids43 and yeast.44 Although the use of internal standards may help to correct for signal suppression and instrument drift, it cannot be used for accurate quantification of analyte species by correcting for their in situ transformation. As discussed later in this paper, true isotope-dilution-based measurement is a direct quantification avoiding the use of relative external calibration curves and response factors. The present study aimed to develop molecular SIDMS methods for accurate, reproducible and direct quantification of GSH, GSSG and tGSH in bodily fluids and cells. For GSH and GSSG assay, samples were spiked with known and proper amounts of the isotopically enriched analogues of the target analytes, and the endogenous and spiked species were equilibrated through solubilization and protein removal. The sample was analyzed by LC-MS/MS utilizing hydrophilic interaction liquid chromatographic (HILIC) separation, and the concentrations of the analytes were determined by the mathematical relationships in SIDMS based on isotopic ratios derived from the peak areas of the product ions of the analytes. A single-spiking IDMS protocol was optimized for tGSH quantitation. The protocols were validated using spike recovery tests and by analyzing synthetic standard solutions. RBC and saliva samples obtained from healthy subjects and whole blood samples collected and shipped from a remote location were analyzed. To the best of the authors’ knowledge, this study is original to demonstrate the use of SIDMS in biomolecular analyses.

glutathione assessment, as it contains significant amount of the amino-thiol.13 Among the few studies that explored this biofluid for oxidative stress markers, some reported abnormal levels of salivary glutathione in diabetic14 and periodontopathic patients,15 autistic children16 and patients with head and neck squamous cell carcinoma.17 Over the years, enzymatic, spectrophotometric, fluorometric, electrochemical and separation-based methods have been developed for the measurement of tGSH, GSH and GSSG in samples of various origins.18−20 Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has recently gained a wide use because of its excellent sensitivity and specificity, high sample throughput, easy automation and small sample size requirement.21 However, measurement of the glutathione species has given researchers tremendous difficulty due to the auto-oxidation of GSH to form GSSG artifacts22,23 and the possible reduction of GSSG through enzyme-catalyzed and other processes.24 Such transformation of species can occur during sample collection, preparation, storage and analysis. The challenge becomes even more severe when samples are collected and shipped from distant locations, as analytes and the sample itself become highly vulnerable to degradation and loss during packing, storage and transport. Conventional methods of analyses attempt to preserve the chemical integrity of the analyte species and to avoid their loss in several ways that include freezing the sample during collection and/or processing,20,25,26 centrifuging25−27 and/or deproteinizing25 the sample immediately after collection, masking the thiol group on GSH,24 capturing oxidizing metal ions using chelating agents,28,29 and maintaining the sample pH in the acidic range.18,20 However, these strategies do not guarantee absolute stabilization of the sample and the analytes for several reasons. Oxidation of GSH may accelerate while thawing frozen samples28 especially in tissues containing high amount of γglutamyl transpeptidase.30 GSSG artifacts may also be formed when the pH of acidified samples (during and after deproteinization) is restored to a neutral/alkaline range for subsequent analysis.22,31 Moreover, the efficiency of masking the thiol functionality is affected by the type and concentration of the masking agent,32 the reaction rate, the effect of interfering substances and the sample treatment stage.24 Accurate and reliable analytical measurements require the use of robust and definitive methods that can track and correct for errors originating from the transformation, degradation, partial recovery and loss of analytes. Speciated isotope dilution mass spectrometry (SIDMS, EPA Method 6800)33 is a unique tool that enables solving these analytical problems mathematically. SIDMS entails spiking a known mass of sample with proper amounts of isotopically enriched analogues of the target analytes. After the spikes are equilibrated with the endogenous analytes, sample preparation procedures are applied and the sample is analyzed by a mass spectrometer. The target analytes are quantified, with simultaneous correction for the abovementioned errors, using isotopic ratios and known constants without involving calibration curves. The fundamentals of SIDMS are discussed in patents34,35 and EPA Method 6800.33 So far, the use of this powerful methodology has been demonstrated only in elemental speciation analyses.36−39 In bioanalytical studies, isotope-dilution-based measurements are exclusively made using calibration curves that are constructed on the basis of isotopic response factors where the isotopically enriched spikes are used as internal standards.40 This approach has been used to measure GSH and GSSG in cells,41−43

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MATERIALS AND METHODS Chemicals and Standards. The natural abundant glutathione standards, i.e., L-glutathione reduced, 99%+ peptide purity (C8C2H17N2NO6S, 307GSH) and L-glutathione oxidized, 98% peptide purity (C16C4H32N6O12S2, 612GSSG) were from Sigma-Aldrich. Reduced glutathione (65−70% peptide purity) labeled with two 98% 13 C’s and one 96−98% 15 N (C813C2H17N215NO6S, 310GSH), and oxidized glutathione (65−70% peptide purity) labeled with four 97% 13C’s, (C1613C4H32N6O12S2, 616GSSG) were purchased from Cambridge Isotope Laboratories Inc. All the enrichments of 310GSH and 616GSSG were on the glycine groups of the molecules. Acetonitrile (99.9%), ammonium formate (99%) and disodium salt of ethylenediaminetetracetic acid (EDTA, 95%+) were from Fisher Scientific, and N-ethylmaleimide (NEM, 99%+, Fluka), DL-dithiothreitol (DTT, 99%+, Sigma-Aldrich), formic acid (88%+, Alfa Aesar), ammonium hydroxide solution (28− 30%, BDH) and ultrapure water (18.2 MΩ cm, Branstead NANOpure) were used. Stock solutions of 307GSH, 310GSH, 612GSSG, 616GSSG and NEM were prepared in water. The concentrations of the 310 GSH and 616GSSG solutions were determined by reverse IDMS as described in EPA Method 6800.33 All the solutions were prepared in polypropylene tubes and stored at −20 °C, away from ultraviolet- and sunlight. Aqueous solution of DTT was prepared fresh on the day of every sample preparation for tGSH assay. Materials. Blue-capped trace-metal-free blood drawtubes (6 mL) from Covidien and airtight polypropylene microcentrifuge tubes (1.5 mL) from VWR were used for sample collection and storage. A Sorvall Discovery M150 SE centrifuge with S120AT2 rotor and an SPD1010 SpeedVac concentrator (both from Thermo Scientific) were used. A model 1200 high-performance liquid chromatography (HPLC) system and a model 6460 triple quadrupole mass 1233

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volunteers of 22−64 years old. The subjects refrained from eating and drinking for at least 2 h and rinsed their mouth with water prior to sample collection. Approximately 0.2 g of unstimulated whole saliva that pulls on the floor of the mouth was spit into microcentrifuge tubes designated for tGSH and GSH-GSSG assay and weighed. The tGSH samples were spiked with 0.2 g of a 12.9 nmol/g 310GSH solution, and the GSHGSSG samples with 0.2 g of a solution containing 8.1 nmol/g 310 GSH, 1.6 nmol/g 616GSSG and 32.0 μmol/g NEM (three replicate samples were prepared for each assay per subject). The tubes were gently inverted a few times to ensure mixing, and the GSH-GSSG samples were left to stand for 20 min to let the excess NEM react with the endogenous GSH. The samples were stored at −20 °C until analysis. Analysis of Samples by LC-MS/MS. The frozen spiked samples were thawed to room temperature. For tGSH assay, 0.02 g (RBC or whole blood) or 0.2 g (saliva) of the designated samples were treated with 0.2 g of a 60 mmol/g aqueous DTT solution (the level of tGSH in RBC and whole blood is very high that using 0.02 g of the samples is more than sufficient to quantify the analyte). The samples were vortexed for 10 s, allowed to sit for 30 min and treated with acetonitrile (800 μL) to precipitate out the proteins. For GSH and GSSG assay, 0.2 g of the designated samples (RBC, whole blood or saliva) were treated with 800 μL of acetonitrile, and the mixtures were vortexed for 10 s. All the acetonitrile-treated samples were spun by centrifugation at 5000 rcf (4 °C, 10 min). While the supernatants from the tGSH samples were directly analyzed by LC-MS/MS, those from the GSH-GSSG samples were evaporated in a SpeedVac concentrator at 45 °C for 2.5 h, and the residues were reconstituted in 30 μL of 15 mmol/L ammonium formate (pH 3.8) and 70 μL of acetonitrile, and the resulting solutions were analyzed by LC-MS/MS. The use of higher sample size (0.2 g) along with analyte preconcentration in the GSH-GSSG assay protocol is to help with the detection of the GSSG. The LCMS/MS conditions for tGSH and GSH-GSSG assays are given in the Supporting Information. Sample Preparation and Analysis for Method Validation. A synthetic aqueous standard solution containing 307 GSH and 612GSSG, and whole blood and saliva samples (0.2 g) spiked with known amounts of 307GSH and 612GSSG were prepared for method validation. The samples were spiked with appropriate amounts of 310GSH for tGSH assay, and with 310 GSH, 616GSSG along with NEM for GSH-GSSG assay. The samples were analyzed by LC/MS-MS after applying the necessary sample preparation procedures described in the previous sections. For spike recovery calculations, the concentrations of endogenous tGSH, GSH and GSSG in the original blood and saliva samples (nonspiked with 307GSH and 612 GSSG) were also determined by IDMS (tGSH) and SIDMS (GSH and GSSG). Quality Control. All sample preparation, storage and analyses were carried out in clean rooms equipped with a class-100 high efficiency particulate air filter hood. All measurements were made by mass with 0.0001 g precision. Procedure blanks were prepared along with every sample batch by spiking water with the isotopically enriched analogue(s) of the target analyte(s) and applying all the sample preparation procedures.

spectrometer with a jet stream electrospray ion (ESI) source (all from Agilent Technologies) were used. The column was Atlantis HILIC Silica, (3 μm particle size, 2.1 mm i.d. and 100 mm long) from Waters Corporation. A 0.5 mL polypropylene 96-well plate (Agilent Technologies) with silicone coverings of preslit well caps (Thermo Scientific) was used to keep the samples in the autosampler. All analyses were conducted in positive ionization mode and chromatograms were recorded in multiple reaction monitoring (MRM) scan mode. See the Supporting Information for the optimum LC-MS/MS conditions. Agilent MassHunter software versions B02.01 and B02.00 were used for data acquisition and chromatographic peak integration, respectively. Isotope ratios were calculated based on product ion peak areas. The concentrations of the analytes were calculated by IDMS (tGSH) and SIDMS (GSH and GSSG) using the Glutathione-SPC software from Applied Isotope Technologies Inc. Sample Collection and Preparation for RBC Analysis. Blood samples for RBC analysis were collected from 15 healthy children of 2−9 years of age, who participated in a Duquesne University Institutional Review Board (IRB) approved study at The Children’s Institute of Pittsburgh (IRB 10-36). Blood was drawn from the vein into drawtubes containing anticoagulant (EDTA). The sample was centrifuged at 2000 rcf (4 °C, 15 min) and placed in a nitrogen glovebox to remove the plasma and the white buffy layer. The RBC samples (0.2 g) were transferred into microcentrifuge tubes designated for tGSH and GSH-GSSG assays. The samples designated for tGSH assay were spiked with 0.2 g of a 2.31 μmol/g 310GSH solution, and the GSH-GSSG samples with 0.2 g of a solution containing 1.63 μmol/g 310GSH, 0.013 μmol/g 616GSSG and 32.0 μmol/g NEM (three replicate samples were prepared for each assay per subject). The amounts of the spike solutions were determined to achieve an approximate analyte-to-spike molar ratio of 1:1. An amount of NEM with a minimum 20-fold molar excess of that of GSH was added. The tubes were gently inverted a few times to ensure mixing, and the GSH-GSSG samples were left to stand for 20 min to let the excess NEM react with the endogenous GSH. The samples were stored at −20 °C until analysis. Sample Collection and Preparation for Whole Blood Analysis. Blood samples for whole blood analysis were collected from an adult volunteer in a remote location (Open Medicine Institute, Mountain View, CA, USA). Blood drawtubes were prepared with one set containing 3.0 g of a 1.3 μmol/g 310GSH solution (for tGSH assay), and another set containing 3.0 g of a solution containing 1.3 μmol/g 310GSH, 0.005 μmol/g 616GSSG and 32.0 μmol/g NEM (for GSH and GSSG assay). The tubes were frozen at −20 °C, packed in a cardboard box containing a cold pack and shipped overnight to the sampling site. During sample collection, the frozen solutions in the drawtubes were thawed and approximately 3 mL of blood was drawn from the subject’s vein directly into the tubes. The tubes were gently inverted several times to ensure mixing, frozen at −20 °C and shipped for analysis. The samples arrived at the analytical laboratory in Duquesne University in Pittsburgh, Pennsylvania, USA in 6 days. For comparison, one sample collected for tGSH was shipped frozen in transit and arrived in 1.5 days. All samples were inspected and stored at −20 °C until analysis. Sample Collection and Preparation for Saliva Analysis. Saliva samples were collected from six nonsmoker healthy 1234

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Analytical Chemistry Safety Considerations. Material safety data sheets were consulted and essential safety precautions were employed for manipulations of all chemicals and reagents.

sample along with the spike solution is bound with the spiked and the free endogenous GSH to form 309GS-NEM and 306GSNEM, thereby achieving instantaneous analyte−spike equilibration. The NEM also prevents GSH from dimerization as well as from binding with other thiols by masking its sulfhydryl (-SH) group.22,23,49,50 At least 20-fold molar excess NEM than that of the expected GSH was used. Regarding GSSG, since the species exists as free molecule in the biosamples, its equilibration with the spiked analogue did not require other procedure than properly mixing the sample and the spike. The usage of aqueous solutions of the spike(s) and either DTT or NEM helped with equilibration, particularly with the RBC, as the samples are somewhat viscous, and the aqueous solutions added to the samples served to minimize the viscosity and promote efficient analyte−spike mixing. Chromatographic Method. Separation of GSH and GSSG is mostly achieved based on reversed-phase (RP) or ion exchange (IE) chromatographic mechanisms.18 These methods, however, are less convenient because GSH and GSSG are poorly retained on RP columns due to their high polar nature, and the high aqueous content of the eluent (in RP and IE) lowers the sensitivity of the analytical method if the separation is coupled with ESI-MS. HILIC, on the other hand, is suitable for better retention of highly polar solutes and for efficient analyte desolvation/ionization in ESI source as it uses a polar stationary phase with high organic and low aqueous content eluents.51 In HILIC, buffered eluents are commonly prepared from organic salts such as formate or acetate rather than inorganic salts such as phosphate because the latter tend to be insoluble in eluents of higher organic content.52 Iwasaki et al.53 proposed a HILIC method with ESI-MS detection that uses ammonium formate buffer and acetonitrile as eluent for GSH and GSSG determination. In the present study, this method was used after some modifications. A mixture of 10 mM ammonium formate (pH 3.8) and acetonitrile was used for tGSH determination in isocratic mode, and GSH and GSSG were separated in gradient mode using 15 mM ammonium formate (pH 3.8) and acetonitrile. Mostly, buffer concentrations of 5−20 mM are commonly used in HILIC;54 higher concentrations may cause ion suppression in ESI-MS applications. As GSH has an extremely high concentration than that of GSSG in most biosamples including RBC and whole blood, slightly higher buffer concentration (15 mM) was used for GSH and GSSG separation to prevent overlapping of the large GS-NEM peak with the very small GSSG peak that elutes a few minutes later. To further improve the detection of GSSG, the dwell time of the mass spectrometer was lowered to 5 ms after the elution of GS-NEM, i.e., after 6.5 min. Lowering the dwell time for an analyte of small peak width with several individual isotopes being monitored from the peak helps to improve the detection of the target isotopes. At longer dwell times, the analyte peak can finish eluting off before the mass spectrometer sufficiently cycles through all the target isotopes to obtain sufficient number of data points for the isotopes. The optimum chromatographic conditions and chromatograms showing separation of the analytes from a spiked whole blood sample are presented in the Supporting Information. Sharp and nearly symmetrical peaks were obtained for all the analytes, and good resolution was achieved between GS-NEM and GSSG. Analyte Quantification. Two mathematical approaches were used for the quantification of the target analytes. The concentration of tGSH was determined by IDMS using the

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RESULTS AND DISCUSSIONS Reduction of Disulfide Bonds for tGSH Assay. Measurement of tGSH, which represents free glutathione (GSH and GSSG) and protein-bound GSH, typically requires converting the disulfide-bonded glutathione in GSSG and other proteins to free GSH via reduction.18−21 The use of an effective reduction procedure is critical for accuracy in tGSH assay. DTT, a thiol-containing reducing agent, is often used to successfully reduce disulfide linkages.20,21 DTT is a strong reductant and, unlike other disulfide reducing agents such as tris(2-carboxyethyl)phosphine, it is stable in the presence of metal chelators that are normally used to quench oxidizing metal ions such as Fe3+ and Ni2+ in blood.45 The major limitation associated with the use of DTT and other thiolcontaining reductants is the need to subsequently remove or quench the unreacted fraction of the reagent prior to analyzing the sample. However, this requirement holds only if the analyte (GSH) has to be derivatized for detection (e.g., introducing a fluorescent group) as excess of the reductant causes severe interference in the derivatization.20 The use of mass spectrometry avoids the need to remove or quench the unreacted DTT because the analysis does not involve analyte derivatization. In the present study, aqueous solution containing at least 100-fold molar excess of DTT than that of the expected tGSH was used. The reductant solution was prepared fresh on the day of the sample preparation as previous studies reported that the effectiveness of DTT solution decreases with time.46 Protein Removal. Proteins are the most abundant components of biological specimens and their presence in injected samples may attenuate the performance of the analytical method and shorten the lifetime of the instrumentation. Hence, it is essential to remove the proteins before analyzing the sample. The common procedures for protein removal involve acidifying the sample (with trichloroacetic, perchloric, metaphosphoric or sulfosalicylic acid), or treating it with organic solvents such as acetonitrile or methanol.18,20 Acids cause oxyhemoglobin precipitation and H2O2 formation, thereby leading to oxidation of GSH.22,47 Although the oxidation of GSH can be effectively tracked and corrected for by using the SIDMS approach, studies also reported that some acids leave substantial amount of protein in the sample,48 which leads to the use of high volume of the acid that may not be suitable for subsequent analysis of the sample by a molecular mass spectrometer. In the present study, acetonitrile was used because studies demonstrated its efficient use to remove proteins for glutathione assay29 and its friendliness with mass spectrometers. At least a 1:3 sample-to-acetonitrile ratio was found to be optimum as noticed from the reproducibility of the LC-MS/MS results for several samples and from the stability of the analytical column for several months. Analyte−Spike Equilibration. Isotope-dilution-based measurements require equilibration between the endogenous and spiked species, which refers to existence of an analyte and its spiked analogue in chemically indistinguishable forms, before applying the sample preparation and analysis steps.33 In the present study, analyte−spike equilibration in tGSH assay was achieved by converting all the glutathione species to GSH using DTT. In GSH and GSSG assay, the NEM added into the 1235

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Table 1. Product Ion Isotopic Abundances (Ax and As) Determined by Analyzing Standard Solutions of 307GSH, 310GSH, 306GSNEM, 309GS-NEM, 612GSSG and 616GSSG Using Positive Mode MS/MS abundance m/z 179.1 180.1 181.1 182.1 183.1 184.1 185.1 186.1 187.1 188.1 total

307

GSH (Ax) 0.8004 0.0688 0.0527 0.0133 0.0112 0.0109 0.0106 0.0106 0.0108 0.0106 1.0000

abundance 310

GSH (As)

m/z

0.0115 0.0115 0.0328 0.7946 0.0502 0.0519 0.0130 0.0117 0.0114 0.0114 1.0000

304.1 305.1 306.1 307.1 308.1 309.1 310.1 311.1 312.1 313.1 total

306

abundance

0.8217 0.1186 0.0521 0.0064 0.0009 0.0002 0.0000 0.0000 0.0000 0.0000 1.0000

GS-NEM (As) 0.0000 0.0002 0.0237 0.8225 0.0979 0.0497 0.0051 0.0008 0.0001 0.0000 1.0000

m/z 355.1 356.1 357.1 358.1 359.1 360.1 361.1 362.1 363.1 364.1 total

612

GSSG (Ax)

616

0.7812 0.1155 0.0878 0.0118 0.0033 0.0003 0.0000 0.0000 0.0000 0.0000 1.0000

GSSG (As) 0.0114 0.0012 0.0013 0.0267 0.7787 0.0844 0.0852 0.0078 0.0030 0.0003 1.0000

product ion peaks for 612GSSG and 616GSSG appear at m/z 355 and 359, respectively. If no transformation of species occurs after equilibration, the concentrations of 307GSH and 612GSSG in the original sample will, respectively, be calculated as

peak areas of the product ions obtained from the LC-MS/MS analysis of the 310GSH-spiked samples. The GSH and GSSG concentrations were determined by SIDMS, with simultaneous correction for in situ interconversions between the species, using the product ion peak areas obtained from the LC-MS/MS analysis of the samples double spiked with 310GSH and 616 GSSG along with NEM. The IDMS and SIDMS equations are described in the following sections. Quantification of tGSH by IDMS. Consider a sample containing tGSH at CX μmol/g concentration. Weigh out WX grams of the sample and spike it with WS grams of CS μmol/g 310 GSH followed by addition of DTT. Analysis of the sample in positive mode MS/MS, with a collision energy adjusted to eliminate a pyroglutamate moiety (129 Da) from a molecule of GSH, gives the most abundant product ion peaks for 307GSH and 310GSH at m/z 179 and 182, respectively. The IDMS relationship to calculate the tGSH concentration in the original sample becomes CX =

309

GS-NEM (Ax)

CxGSH =

307 304 CsGSHW sGSH ⎛ A S − R307/304 A S ⎞ ⎜⎜ ⎟⎟ 304 307 Wx ⎝ R307/304 A X − A X ⎠

and CxGSSG =

359 355 CsGSSGW sGSSG ⎛ A S − R359/355 A S ⎞ ⎜⎜ ⎟⎟ 355 359 Wx ⎝ R359/355 A X − A X ⎠

where 304AX and 307AX are the abundances of m/z 304 and 307 product ions determined by analyzing a standard solution of 307 GSH, 304AS and 307AS are the abundances of m/z 304 and 307 product ions determined by analyzing a standard solution of 310GSH, 355AX and 359AX are the abundances of m/z 355 and 359 product ions determined by analyzing a standard solution of 612GSSG, 355AS and 359AS are the abundances of m/z 355 and 359 product ions determined by analyzing a standard solution of 616GSSG, and R307/304 and R359/355 are the ratios of the product ion abundances (m/z 307 to m/z 304 and m/z 359 to m/z 355, respectively) determined by analyzing the spiked sample by LC-MS/MS. Table 1 lists the isotopic abundances of the product ions (AX and AS) for the aqueous standard solutions of 306GS-NEM, 309GS-NEM, 612GSSG and 616GSSG used in the present study. If the endogenous and the spiked GSH and GSSG undergo in situ bidirectional transformation (oxidation and reduction), the above concentrations of the species will change. As can be seen from Figure 1, the possible forms of GSH and GSSG that may exist in the sample after the transformation of the species will be 307GSH, 310GSH, 612GSSG (endogenous GSSG and those formed from oxidation of 307GSH), 616GSSG, 618GSSG (product of 310GSH oxidation) and 615GSSG (forms if 307GSH binds with 310GSH). In addition, the reduction of 616GSSG forms 309GSH (with two 13C’s on its glycine), which may bind with 307GSH and 310GSH to form 614GSSG and 617GSSG, respectively. In positive mode MS/MS, with a collision energy adjusted to eliminate two pyroglutamate moieties (258 Da) from a GSSG molecule, the most abundant product ion peaks for 614GSSG, 615GSSG, 617GSSG and 618GSSG appear at m/z 357, 358, 360 and 361, respectively.

182 179 CSWS ⎛ A S − R182/179 A S ⎞ ⎜⎜ ⎟⎟ WX ⎝ R182/179179A X − 182A X ⎠

where; 179AX and 182AX are the abundances of m/z 179 and 182 product ions determined by analyzing an aqueous standard solution of 307GSH, 179AS and 182AS are the abundances of m/z 179 and 182 product ions determined by analyzing an aqueous standard solution of 310GSH, and R182/179 is the ratio of the abundances of m/z 182 to m/z 179 determined by analyzing the spiked sample by LC-MS/MS. Table 1 lists the isotopic abundances of the product ions (AX and AS) for the aqueous standard solutions of 307GSH and 310GSH used in the present study. Quantification of GSH and GSSG by Double-Spiking SIDMS. Consider a sample containing GSH and GSSG at CGSH X and CGSSG μmol/g concentrations, respectively. Weigh out Wx X gram of the sample and spike it with WGSH and WGSSG grams of S S 310 616 GSH GSH and GSSG standards of CS and CGSSG μmol/g S concentrations, respectively, along with NEM. After equilibration, the endogenous and spiked GSH will be converted to NEM-alkylated adducts, i.e., 306GS-NEM and 309GS-NEM, respectively. In positive mode MS/MS, the peaks for the most abundant product ions resulting from the loss of a pyroglutamate moiety (129 Da) from 306GS-NEM and 309GSNEM show up at m/z 304 and 307, respectively. Similarly, with a collision energy adjusted to eliminate two pyroglutamate moieties (258 Da) from a GSSG molecule, the most abundant 1236

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product ion of the NEM adduct of this 309GSH (308GS-NEM) shows up at m/z 306, its contribution has to be corrected to get 306adj P. The 310GSH standard also has smaller isotope abundance at 307 m/z that needs to be corrected to get 307adj P. Additionally, 306GS-NEM has its most abundant MS/MS peak at m/z 304, and peaks with smaller abundances at m/z 306 and 307; the latter should be accounted for while determining 306adjP and 307adjP, respectively. The most abundant product ion peak for 612GSSG shows up at m/z 355 with smaller peaks at m/z 357 and 358. These contributions should be taken into consideration while determining 357P and 358P coming from 614GSSG and 615GSSG, respectively. The most abundant product ion peak for 616GSSG appears at 359 m/z, with smaller peaks at several other m/z values including 357, 358, 360 and 361 that did not come from oxidation of GSH. These contributions need to be taken into consideration when determining 357P, 358P, 360P and 361P, respectively. The SIDMS equations to calculate the concentrations of endogenous GSH and GSSG after correcting for the transformations of the species become:

Figure 1. Schematic presentation of the in situ transformation pathways for GSH and GSSG in a sample spiked with isotopically enriched analogues of the molecules.

Let α represents the fraction of GSH converted to GSSG, and β represents the fraction of GSSG converted to GSH and/ or to other isotopic forms of GSSG. The two fractions can be calculated as 358

α=

β=

360

P + 2(361P)

P+

307adj

P+

358

P+

359

P + 2(361P)

306adj

P + 1/2( 358

359

355 ⎡ C GSSGW GSSG ⎛ 359A − R ⎞ ⎤ S 359/355 A S s ⎜⎜ ⎟⎟β ⎥ − 2⎢ s 355 359 ⎢⎣ Wx ⎝ R359/355 A X − A X ⎠ ⎥⎦

360

1/2(306adjP +

357

304 ⎡ C GSHW GSH ⎛ 307A − R ⎤ ⎞ 1 ⎞⎟⎥ S 307/304 A S ⎛ s ⎜ ⎜⎜ ⎟ CxGSH = ⎢ s ⎟ 304 307 ⎢⎣ Wx ⎝ R307/304 A X − A X ⎠⎝ 1 − α ⎠⎥⎦

357

P+

P+

360

360

P)

357

P+

360

P)

355 ⎡ C GSSGW GSSG ⎛ 359A − R ⎞ ⎛ 1 ⎞⎤ S 359/355 A S s ⎜⎜ ⎟⎟⎜ CxGSSG = ⎢ s ⎟⎥ 355 359 ⎢⎣ Wx ⎝ R359/355 A X − A X ⎠⎝ 1 − β ⎠⎥⎦

361

where P, P, P, P and P are areas under m/z 357, 358, 359, 360 and 361 peaks derived from the LC-MS/MS chromatogram of the spiked sample; and 306adjP and 307adjP are areas under m/z 306 and 307 peaks, respectively, derived from the LC-MS/MS chromatogram of the spiked sample after subtracting out the abundances of the ions coming from the corresponding natural abundant molecules (GSH and GSSG are polyisotopic molecules and their isotopic patterns should be determined experimentally, as shown in Table 1, so that the contributions from the overlapping isotopes can be subtracted). The 310GSH standard used in this study had small amount of 309 GSH that did not come from reduction of 616GSSG. As the

304 ⎡ C GSHW GSH ⎛ 307A − R ⎞ ⎤ S 307/304 A S s ⎜⎜ ⎟⎟α ⎥ − 1/2⎢ s 304 307 ⎢⎣ Wx ⎝ R307/304 A X − A X ⎠ ⎥⎦

Analysis of RBC, Whole Blood and Saliva Samples. Red Blood Cells. RBC samples collected from 15 healthy children were analyzed as described in the Materials and Methods section. The concentrations of tGSH, GSH and GSSG found in the tested samples are given in Table 2. The measured tGSH concentrations agreed with previously reported values for RBC

Table 2. Concentrations (μmol/g) of tGSH, GSH and GSSG in RBC Samples from Healthy Children (n = 9, 95% CL)

a

subject

gender

age

RBC 1 RBC 2 RBC 3 RBC 4 RBC 5 RBC 6 RBC 7 RBC 8 RBC 9 RBC 10 RBC 11 RBC 12 RBC 13 RBC 14 RBC 15 mean

M M F M M M F M M M M M M M F

6 5 9 5 3 6 2 3 5 6 5 2 5 9 6 5

tGSH 2.12 1.94 1.90 2.12 2.24 1.91 2.05 1.58 2.15 1.99 1.78 2.10 1.86 2.06 2.09 1.99

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.04 0.04 0.04 0.08 0.10 0.05 0.05 0.04 0.01 0.05 0.10 0.07 0.08 0.03 0.86

GSH 1.80 1.72 1.58 1.75 1.80 1.55 1.75 1.42 1.79 1.97 1.38 1.57 1.40 1.92 2.01 1.69

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.04 0.03 0.03 0.01 0.03 0.01 0.02 0.03 0.04 0.25 0.21 0.01 0.02 0.01 0.10 0.83

GSSG

%GSHa

%GSSGb

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

99.66 99.81 98.85 97.77 99.63 99.67 99.33 99.25 99.23 99.81 99.73 98.44 98.87 99.30 99.80 99.28

0.35 0.18 1.15 2.23 0.36 0.34 0.67 0.75 0.76 0.19 0.27 1.56 1.13 0.70 0.20 0.72

0.0031 0.0016 0.0092 0.0200 0.0033 0.0026 0.0059 0.0054 0.0069 0.0019 0.0019 0.0124 0.0080 0.0068 0.0020 0.0061

0.0004 0.0002 0.0005 0.0001 0.0004 0.0002 0.0003 0.0003 0.0003 0.0003 0.0002 0.0001 0.0002 0.0001 0.0002 0.0038

%GSH = GSH × 100/(GSH + 2xGSSG). b%GSSG = (2xGSSG) × 100/(GSH + 2xGSSG). 1237

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Analytical Chemistry

Table 3. Concentrations (nmol/g) of tGSH, GSH and GSSG in Saliva Samples from Healthy Nonsmoking Adults (n = 9, 95% CL)

a

subject

gender

age

S1 S2 S3 S4 S5 S6 mean

M M M F M M

37 27 24 22 64 31 34

tGSH 8.5 3.1 11.3 24.8 13.4 4.3 10.9

± ± ± ± ± ± ±

GSH

0.4 0.3 0.7 1.8 0.1.2 0.4 4.8

0.9 1.2 0.9 8.0 4.4 0.9 2.7

± ± ± ± ± ± ±

0.2 0.1 0.1 0.7 0.4 0.2 1.7

GSSG

%GSHa

%GSSGb

± ± ± ± ± ± ±

52.9 66.7 52.9 53.3 84.6 52.9 60.0

47.1 33.3 47.1 46.7 15.4 47.1 40.0

0.4 0.3 0.4 3.5 0.4 0.4 0.9

0.1 0.1 0.1 0.2 0.1 0.1 0.7

%GSH = GSH × 100/(GSH + 2xGSSG). b%GSSG = (2xGSSG) × 100/(GSH + 2xGSSG).

Table 4. Spike Recoveries of tGSH, GSH and GSSG from Whole Blood and Saliva (n = 9, 95% CI) fluid

thiol

initiala

spikedb

totalc

recovery (%)d

blood (μmol/g)

tGSH GSH GSSG tGSH GSH GSSG

1.91 ± 0.14 1.46 ± 0.07 0.0024 ± 0.0004 6.20 ± 0.92 0.78 ± 0.08 0.34 ± 0.02

2.22 1.63 0.003 7.51 1.25 0.50

4.25 ± 0.05 2.98 ± 0.05 0.0053 ± 0.0011 14.20 ± 1.22 1.97 ± 0.12 0.87 ± 0.04

105.4 93.3 96.7 102.6 95.2 106.0

saliva (nmol/g)

Initially measured concentration of the analyte in the unspiked fluid. bConcentration of the natural abundant analytes spiked into the fluid. cTotal concentration of the analyte measured in the spiked fluid. dRecovery (%) = (total concentration − initial concentration) × 100/spiked concentration. a

obtained from healthy individuals.41 The average GSH and GSSG concentrations found in the present sample accounted for 99.28% and 0.72% of the free glutathione that was calculated as GSH + 2xGSSG (one mole of GSSG is composed of two moles of GSH). The GSSG concentrations found in this study are significantly lower than previously reported erythrocytic GSSG levels in healthy individuals (6.5%− 20%),25,28,55,56 and are in good agreement with the expected physiological range of GSSG in healthy persons, which is less than 1%.57 The average free glutathione in the RBC samples accounted for 85.5% of the mean tGSH. The difference in concentration between tGSH and free glutathione is because the former represents the sum of GSH, GSSG and proteinbound GSH. Whole Blood Sampled and Shipped from a Remote Location. For the testing of blood glutathione in samples collected and shipped from remote sources, an important step forward was previously reported where a blood drawtube containing NEM was used for sample collection.50 When blood is drawn into the tube, the NEM binds with GSH, thereby minimizing the conversion of the analyte to GSSG. In the present method, blood drawtubes containing measured amount(s) of the isotopically enriched analogues of the analyte(s) were shipped to the sampling location. Blood was drawn into the tubes and sent to the analytical laboratory in two different shipping ways as described in the Materials and Methods section. The tGSH concentration measured in the blood sample that was received after 1.5 days was 1.12 ± 0.12 μmol/g and was statistically indistinguishable from the one found in the sample that arrived after 6 days (1.09 ± 0.08 μmol/g). The concentrations of GSH and GSSG measured in the 6 day sample were 1.06 ± 0.02 μmol/g and 0.0015 ± 0.0005 μmol/g, respectively. The GSSG found in the blood, which was equal to 0.28% of the free glutathione, was with in the expected range for a healthy person.57 The fact that, even after 6 days of shipping, the GSH and GSSG quantification was successfully completed suggested that the SIDMS protocol is

effective for accurate quantification of the species in samples shipped from remote locations. Saliva. Saliva samples collected from healthy nonsmoking subjects were analyzed as described in the Materials and Methods section. In most studies, stimulants and/or absorbents are used for saliva sampling. However, reports indicate that stimulants used to enhance saliva generation might cause interference in the assay or alter the level of the analyte(s) in the fluid, and absorbents may collect localized rather than whole saliva depending on the location of the mouth where the materials are placed.58 The use of absorbents is also problematic if the volume of the saliva is small because quantitative recovery of the fluid becomes difficult once it disperses across the material.58 In the present study, unstimulated saliva samples were collected as described in the Materials and Methods section. The measured concentrations of tGSH in the tested samples were within previously reported salivary tGSH ranges55 and 2 orders of magnitude lower than those found in the RBC and whole blood samples analyzed in this study (Table 3). Unlike the case of RBC and blood, the mean GSH and GSSG concentrations in the saliva samples represented 60% and 40% of the free glutathione, respectively. The higher salivary GSSG may be due to the abundance of oxygen in the mouth. Auto-oxidation of GSH. Oxidation of GSH to GSSG that occurred after the analytes and spikes are equilibrated was tracked and corrected for using the mathematical relationships in double-spiking SIDMS. The results show that up to 3% of the GSH in the tested samples was auto-oxidized to GSSG; the highest conversions (>1%) were in the saliva samples. The results indicate that, even with the use of NEM, there might still be auto-oxidation of GSH that should be accounted for to achieve accurate GSH and GSSG assay. The SIDMS procedure complements the use of NEM by determining the extent to which the thiol-masking agent prevented the conversion of GSH to GSSG thereby ensuring true quantification of the analytes. Although the method-induced oxidation of GSH in 1238

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Analytical Chemistry Notes

the RBC and whole blood samples seems to be very low, it may have significant effect on the GSSG concentration as the specimens contain at least 3 orders of magnitude lower GSSG than GSH; oxidation of 1% GSH will increase the GSSG concentration by 10% and more depending on the actual GSH/ GSSG ratio. Method Validation. The IDMS and SIDMS protocols developed in this study were validated based on analysis of a synthetic standard solution and recovery tests from spiked whole blood and saliva samples. The sample preparation and analysis procedures are described in the Materials and Methods section. The measured concentrations of tGSH (2.09 ± 0.30 μmol g−1), GSH (2.27 ± 0.32 μmol g−1) and GSSG (0.0623 ± 0.0028 μmol g−1) in the synthetic standard solution were in statistical agreement with the expected values, i.e., 2.30, 2.00 and 0.0601 μmol g−1, respectively. Table 4 shows the recovery values for tGSH, GSH and GSSG from the spiked whole blood and saliva samples. Spike recoveries of 105% and 103% were obtained for tGSH from the blood and saliva samples, and 93%−106% of the spiked GSH and GSSG were recovered from the two samples.

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CONCLUSIONS

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ASSOCIATED CONTENT

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Heinz Endowments, Richard King Mellon Foundation and Health Resources and Services Administration (HRSA R1CRH20683) for supporting the research, Agilent Technologies Inc. for the instrumentation and phlebotomist Becky Peckar for her contributions.

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Despite the availability of a broad range of methods for GSH, GSSG and tGSH quantitation, there is no standard protocol for such assay because none of the reported methods are capable of tracking and correcting for the in situ transformations, partial recoveries, degradation and loss of the analytes. Taking this into consideration, the present study demonstrated a unique and successful adaptation of the SIDMS methodology for accurate and reproducible measurement of the glutathione species in biological fluids and cells. In the assay, spiking the samples during or immediately after collection enabled tracking and correction for transformation of species that occurred during sample workup and analysis. The present study is original in demonstrating the unique capability of SIDMS for accurate measurement of reactive biomolecular species such as those of glutathione. The demonstrated analytical merits (accuracy and reproducibility) of the method proved the capability of the method to overcome the commonly encountered imprecisions associated with GSH and GSSG measurement, and to solve chain of custody problems that reduce accuracy in clinical assays. The study also demonstrated the robustness of the method to accurately measure the target analytes in samples shipped from remote locations. Previously, results from such analyses were highly questionable because of the potential sample/analyte decomposition that cannot be accounted for by traditional methods of analysis.

* Supporting Information S

Pictorial presentation of the IDMS and SIDMS methods, optimum conditions of the LC-MS/MS method and chromatograms showing detection of tGSH, GSH and GSSG are presented. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*M. M. Wolle. E-mail: [email protected], [email protected]. Fax: +1-412-396-4013. Tel.: +1-412-396-4106. 1239

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