Rapid Method for Glutathione Quantitation Using High-Performance

Article pubs.acs.org/JAFC

Rapid Method for Glutathione Quantitation Using High-Performance Liquid Chromatography with Coulometric Electrochemical Detection Banu Bayram,† Gerald Rimbach,*,† Jan Frank,§ and Tuba Esatbeyoglu† †

Institute of Human Nutrition and Food Science, Christian-Albrechts-University, Hermann Rodewald Strasse 6, 24098 Kiel, Germany Institute of Biological Chemistry and Nutrition, Division Biofunctionality and Safety of Food, University of Hohenheim, Garbenstrasse 28, 70599 Stuttgart, Germany

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ABSTRACT: A rapid, sensitive, and direct method (without derivatization) was developed for the detection of reduced glutathione (GSH) in cultured hepatocytes (HepG2 cells) using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The method was validated according to the guidelines of the U.S. Food and Drug Administration in terms of linearity, lower limit of quantitation (LOQ), lower limit of detection (LOD), precision, accuracy, recovery, and stabilities of GSH standards and quality control samples. The total analysis time was 5 min, and the retention time of GSH was 1.78 min. Separation was carried out isocratically using 50 mM sodium phosphate (pH 3.0) as a mobile phase with a fused-core column. The detector response was linear between 0.01 and 80 μmol/L, and the regression coefficient (R2) was >0.99. The LOD for GSH was 15 fmol, and the intra- and interday recoveries ranged between 100.7 and 104.6%. This method also enabled the rapid detection (in 4 min) of other compounds involved in GSH metabolism such as uric acid, ascorbic acid, and glutathione disulfite. The optimized and validated HPLC-ECD method was successfully applied for the determination of GSH levels in HepG2 cells treated with buthionine sulfoximine (BSO), an inhibitor, and α-lipoic acid (α-LA), an inducer of GSH synthesis. As expected, the amount of GSH concentration-dependently decreased with BSO and increased with α-LA treatments in HepG2 cells. This method could also be useful for the quantitation of GSH, uric acid, ascorbic acid, and glutathione disulfide in other biological matrices such as tissue homogenates and blood. KEYWORDS: glutathione, electrochemical detection, hepatocytes, method validation, buthionine sulfoximine

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INTRODUCTION Glutathione (γ-L-glutamyl-L-cysteinglycine, GSH) is a tripeptide nonprotein thiol antioxidant molecule composed of cysteine, glutamic acid, and glycine. The thiol group of the cysteine residue is the biologically active site of GSH.1,2 GSH has a number of important roles in human metabolism including scavenging of free radicals, detoxifying xenobiotics, maintaining cell homeostasis, protecting against oxidative stress, controlling gene expression, detoxifying heavy metals, and in-signal transduction.2−6 GSH is also involved in regenerating antioxidants involved in the antioxidant network system, such as vitamin E and ascorbic acid.2 GSH is present in all organs with particularly high levels in the liver.1 Severe oxidative stress leads to oxidation of GSH and the formation of glutathione disulfide (GSSG).7 The identification and quantitation of GSH are of great interest due to its critical protective functions in the body. Furthermore, it may be a useful biomarker for oxidative stress, a common occurrence in many disease states. Various chemiluminescence,8 spectrofluorometric,9 and spectrophotometric7 methods are available for the determination of GSH in biological samples. In addition, chromatographic methods with improved specificity and sensitivity have been developed using high-performance liquid chromatography (HPLC) coupled to ultraviolet,10 fluorescence,11 and coulometric electrochemical detections12 as well as mass spectroscopy13 and capillary electrophoresis.14,15 Most of these methods require either precolumn or postcolumn derivatization of GSH with fluorogenic (monobromobimane, o-phthalaldehyde), © XXXX American Chemical Society

and chromophoric (N-ethylmaleimide or iodoacetic acid) agents or reduction with reducing agents (tributylphosphine, tris(2-carboxyethyl)phosphine),16 which is time-consuming. Among the chromatographic techniques, coulometric electrochemical detection facilitates the resolution and accurate identification of trace amounts of electroactive compounds, including redox-reactive compounds such as thiols and disulfides.17 Thus, HPLC coupled to electrochemical detection (HPLC-ECD) is a valuable tool for analyzing GSH with high specificity and sensitivity without the need of prior derivatization.12,18 In addition, run time is an important aspect of any chromatographic method. The currently available methods for GSH analysis are often longer than 3 min.12,19,20 In this study, we aimed to develop a rapid and sensitive HPLC-ECD method for the analysis of GSH in HepG2 cells and to validate it according to FDA guidelines for bioanalytical method validation.21 The measurement of GSH levels is crucial for determining the influence of treatments on the GSH− GSSG redox system. We therefore tested the application of this method by measuring changes in GSH levels in HepG2 cells treated with buthionine sulfoximine (BSO), an inhibitor, and αlipoic acid (α-LA), an inducer of GSH synthesis. Received: September 13, 2013 Revised: December 10, 2013 Accepted: December 15, 2013

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dx.doi.org/10.1021/jf403857h | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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limit of quantitation (LOQ), limit of detection (LOD), accuracy, precision, recovery, stock solution stability, short-term stability, freeze−thaw stability, and benchtop stability. All of the analyses were performed three times. Linearity, Lower LOD, and Lower LOQ. The linear range of detection was determined using serial dilutions of the stock solution. Calibration curves were obtained by plotting the sum of the peak heights of GSH versus its concentration for eight different concentrations. Standard solutions corresponding to each point in the calibration curve were injected in triplicate and regression parameters calculated. The LOQ was defined as the lowest concentration that could be determined with a deviation from the actual concentration and a coefficient of variation of precision of 98%. Water was of Milli-Q grade, purified by a Milli-Q UV purification system (Millipore, Bedford, MA, USA). Preparation of Standard Solutions. A stock solution of GSH standard was prepared in 1% metaphosphoric acid at a concentration of 10 mM, and working standards were prepared daily from this stock solution by dilution in 1% metaphosphoric acid. It is recommended to prepare the standards daily as an effect of temperature and storage time was observed for GSH in our stability studies. Cell Culture Conditions. HepG2 cells were obtained from Applied Cell Culture (IAZ, München, Germany). Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), and penicillin/ streptomycin were purchased from PAA (Coelbe, Germany). HepG2 cells were cultured in DMEM containing 10% FCS and 1% penicillin/ streptomycin in a temperature- and humidity-controlled incubator (95% air, 5% CO2) at 37 °C. Cells were grown in T75 flasks (Sarstedt, Nümbrecht, Germany) at a density of 8 × 106 cells in DMEM and subcultured twice a week when at 80% confluence. Confluent cells were harvested by trypsination. Extraction of GSH from HepG2 Cells. Confluent cells were seeded at a density of 1.2 × 106 cells per well in 12-well plates (Corning Inc., NY, USA) and cultivated for 24 h. Cells were washed with cold PBS and then incubated with 500 μL of trypsin/EDTA per well for 10 min. Cells were suspended in 1 mL of DMEM and pelleted by centrifugation at 2000g for 5 min at 4 °C. Cell pellets were suspended in 300 μL of PBS and frozen at −80 °C. The cell suspension was then thawed and mixed with 600 μL of PBS. An equal volume of 1% metaphosphoric acid (900 μL) was added before homogenization with a Qiagen TissueLyser (Hilden, Germany) for 2 min. The cell suspension was then centrifuged at 13000g for 10 min at 4 °C, and the supernatants were transferred into HPLC vials for GSH analysis. GSH content was expressed as micromoles per liter. Cytotoxicity Test for BSO and α-LA. The cytotoxicity of BSO and α-LA was determined using the neutral red (NR) assay.22 Viable cells incorporate NR in lysosomes, which can then be photometrically measured at 540 nm. Cell viability was expressed as a percentage of solvent-only exposed control cells. HepG2 cells were incubated with BSO (prepared in distilled water) or with α-LA (prepared in DMSO and the final concentration of DMSO was 800 mV), which can cause detrimental effects to the detection system over time. Therefore, an amperometric boron-doped diamond electrode rather than a coulometric graphite electrode would be preferable if GSSG is analyzed with this method.23 Uric acid

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RESULTS AND DISCUSSION We developed and validated a method for the direct measurement of GSH in HepG2 cells without the requirement of prior derivatization. Cell extracts were acidified with metaphosphoric acid to facilitate protein precipitation and minimize oxidation of GSH. This newly developed and validated method enables the detection of GSH with a retention time of only 1.78 min (Figure 1) and a total run time of 5 min. Currently, we know of no other method for GSH analysis that has a shorter total run time. In addition, the C



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Table 1. Comparison of the Lower Limits of Detection (Given as Total Amount Injected; LOD), Retention Times, Detection Methods, Sample Matrices, and Derivatization Procedures of the Present Method with Other Sensitive Methods for the Quantitation of GSH in Biological Samples Published in the Literaturea LOD

RT (min)

detector

sample

50 fmol 2 fmol 0.2 pmol 10 fmol 0.33 pmol na (LOQ = 0.5 pmol) 4 pmol 15 fmol

3.7 12.3 5.1 4.4 5.23 7.74 3.7 1.8

HPLC-FL HPLC-ECD LC-MS/MS LC-CE HPLC-ECD HPLC-FL LC-MS/MS HPLC-ECD

red blood cells and cultured fibroblasts HL-60 cells mouse liver red blood cells DU145 and A549 cells U373-MG human astrocytoma cell whole blood HepG2 cells

derivatization derivatization none none derivatization none derivatization derivatization none

with OPA

with NEM with ABD-F with NEM

ref 11 12 13 14 18 20 28 this study

a Abbreviations: ABD-F, 4-fluoro-7-aminosulfonylbenzofurazan; CE, capillary electrophoresis; ECD, electrochemical detection; FL, fluorescence; IAA, iodoacetic acid; LC, liquid chromatography; MS, mass spectrometry; NEM, N-ethylmaleimide; OPA, o-phthalaldehyde.

−20, or −80 °C for 24 h. The concentrations after storage were compared with the prestorage values (Table 3). The low-

is a powerful antioxidant and has recently been reported to increase cysteine uptake and GSH synthesis in the brain24 and liver.25 Linearity, Lower LOD, and Lower LOQ. The linearity of the detector response for GSH was evaluated by triplicate injection of standard solutions corresponding to each point on the standard curve on three different days. The detector response was linear between 0.01 and 80 μmol/L with a regression coefficient (R2) >0.99. The LOQ and LOD of GSH were 50 and 15 fmol (given as total amount injected), respectively, which are lower than reported in the literature for most other studies (Table 1). Precision, Accuracy, and Recovery. Precision, accuracy, and recovery were determined by adding low, medium, and high concentrations of the GSH standard to HepG2 cell suspensions (n = 5). Intra- and interday repeatability studies were carried out by analyzing five cell suspensions on three consecutive days. Intraday precision values were 3.75, 1.67, and 0.71%, and interday precision results were 5.27, 6.89, and 9.86% for low, medium, and high GSH concentrations, respectively (Table 2). These precision and accuracy values are within the

Table 3. Stock Solution Stability of GSH Standards at Low, Medium, or High Concentration after Storage at Room Temperature or 4, −20, or −80 °C for 24 h (Short-Term Stability) and at −20 or −80 °C for 4 Months (Long-Term Stability) degradation (%) short-term stability

calcd concn (μmol/L)

2 10 40

2.01 10.36 41.35

2 10 40

2.09 10.40 41.76

recovery (%) Intraday 100.7 103.7 103.5 Interday 104.6 104.2 104.6

accuracy (%)

precision (CV%)

−0.71 −3.68 −3.45

3.75 1.67 0.71

−4.59 −4.23 −4.59

5.27 6.89 9.86

room temp

4 °C

0.4 4 40

4.85 2.23 0.25

−5.48 0.08 −2.40

long-term stability

−20 °C −80 °C −20 °C −80 °C −7.60 0.66 −3.30

−5.54 −2.24 2.81

27.29 17.01 13.06

1.93 10.97 11.04

concentration GSH stock solution showed the highest level of degradation (−7.60%) when stored at −20 °C for 24 h. No significant differences in 24 h stability were observed between temperatures. During long-term storage for 4 months, GSH was less stable when stored at −20 °C compared to −80 °C, with the highest level of degradation being observed for the low GSH (Table 3). These results indicate that, to optimize stability, long-term storage of GSH stock solutions and standards should be at −80 °C. To assess short-term stability in cell extracts, HepG2 cells were spiked with GSH at two concentrations (low and high) and analyzed after storage at room temperature or 4, −20, or −80 °C for 24 h. The highest degradation was observed in samples with high concentrations of GSH at −20 °C (−3.68%). There were no clear differences in the stability of GSH between samples stored at room temperature or 4, −20, or −80 °C for 24 h (Table 4). To assess long-term stability, the GSH-spiked HepG2 cells were also stored at −20 or −80 °C for 4 months. Significant degradation was seen in the sample spiked with the high concentration of GSH at −20 °C (12.53%). As with the GSH stock solutions, −80 °C is preferable for long-term storage of cell extracts to protect GSH from degradation (0.13 and 1.16% for high and low concentrations, respectively). The effect of repeated freezing and thawing on the stability of GSH-spiked cell extracts was also assessed. GSH degraded by 2.01% and by −1.87% at low and high concentrations, respectively, indicating that GSH withstands freeze−thawing in cell extracts (Table 4). The stability of HepG2 cell extracts during the time in the autosampler was also investigated. The autosampler temper-

Table 2. Intra- and Interday Precision, Accuracy, and Recovery of GSH Added to Cell Suspensions of HepG2 Cells at Low, Medium, or High Concentrations and Determined in Five Replicate Analyses (n = 5) concn (μmol/L)

concn (μmol/L)

limits prescribed by the FDA (≤20% for concentrations near the LOQ and ≤15% for other concentration levels). The intraday and interday recovery values of GSH in cell suspensions ranged from 100.7 to 104.6% (Table 2). Stability. The stability of analytes prior to injection is an important issue that is often neglected during method development. Therefore, in the present study, we determined both the stability of GSH stock solutions and GSH-spiked cell extracts. Aliquots of GSH stock solutions (low, medium, and high concentrations) were stored at room temperature and 4, D



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Table 4. Short-Term (24 h), Long-Term (4 Months), Freeze−Thaw, and Postpreparative Stabilities (Expressed as Percent Degradation) of GSH-Spiked HepG2 Cell Extracts at Low and High Concentrations degradation (%) short-term stability

long-term stability

concn (μmol/L)

room temp

4 °C

−20 °C

−80 °C

−20 °C

−80 °C

2 40

0.97 1.50

−0.32 −0.57

−2.16 −3.68

−2.91 −1.66

9.83 12.53

1.16 0.13

freeze−thaw stability 2.01 −1.87

postpreparative stability 6h

12 h

18 h

0.6 −2.6

0.5 −3.7

0.8 −3.9

Figure 2. (A) Concentrations of GSH (μmol/L) in HepG2 cells treated with buthionine sulfoximine (BSO) or (B) α-lipoic acid (α-LA) at concentrations ranging between 1 and 250 μmol/L. Values are expressed as arithmetic means ± SD from three individual experiments performed in duplicate (n = 6). Comparison of mean values to control was performed by one-way ANOVA with the Dunnett post hoc test, and significant differences are indicated by asterisks: ∗, P < 0.001.

ature was maintained at 4 °C. Cell extracts containing high and low GSH concentrations were found to be stable for 18 h in the autosampler (Table 4). The highest degradation was observed after 18 h (−3.9%) in the high GSH-spiked samples. Application of the Method for the Quantitation of GSH in HepG2 Cells Treated with BSO and α-LA. The measurement of GSH is useful in assessing the effectiveness of treatments that may affect synthesis of GSH. The developed and validated method was used to analyze the content of GSH

in BSO- and α-LA-treated HepG2 cells. The sensitivity of the analytical method is an important issue when GSH is altered in cells by depleting agents, oxidative stress, or physical agents. BSO acts as an inhibitor of GSH synthesis and has been shown to be effective at lowering GSH levels in a number of cell types both in vitro and in vivo.26,27 According to the results of the neutral red test, no toxic effect of either BSO or α-LA on HepG2 cells was detected at concentrations between 1 and 1000 μmol/L. HepG2 cells were concentration-dependently E



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treated with 1−250 μmol/L of BSO, and significant GSH depletion was achieved even at a low concentration (5 μmol/L) (Figure 2A, P < 0.001). Conversely, treatment with the GSH inducer α-LA, when used at concentrations of 100 and 250 μmol/L, significantly increased cellular GSH (Figure 2B, P < 0.001). In conclusion, we developed and validated a simple, fast, direct, sensitive, and specific HPLC-ECD method for the determination of GSH in HepG2 cells. This chromatographic method has a number of advantages, including a quick sample preparation, as it requires no derivatization step, a very good sensitivity with markedly lower LOD than for most other methods reported in the literature, and a very short retention time of GSH of only 1.78 min, which results in the fastest analysis time among the published HPLC methods for GSH. This method furthermore allows the elution of GSH as well as three other compounds involved in GSH metabolism (ascorbic acid, uric acid, and GSSG) in 4 min, resulting in a total analysis time of under 5 min. The method may thus be useful for the analysis of these compounds, too. The developed method was successfully applied to analyze GSH in HepG2 cells treated with BSO and α-LA. It needs to be established whether our method can also be applied for the analysis of GSH in other biological samples such as blood and tissues.

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(10) Giustarini, D.; Dalle-Donne, I.; Colombo, R.; Milzani, A.; Rossi, R. An improved HPLC measurement for GSH and GSSG in human blood. Free Radical Biol. Med. 2003, 35, 1365−1372. (11) Cereser, C.; Guichard, J.; Drai, J.; Bannier, E.; Garcia, I.; Boget, S.; Parvaz, P.; Revol, A. Quantification of reduced and total glutathione at the femtomole level by high-performance liquid chromatography with fluorescence detection: application to red blood cells and cultured fibroblasts. J. Chromatogr., B 2001, 752, 123−132. (12) Hiraku, Y.; Murata, M.; Kawanishi, S. Determination of intracellular glutathione and thiols by high performance liquid chromatography with a gold electrode at the femtomole level: comparison with a spectroscopic assay. Biochim. Biophys. Acta 2002, 1570, 47−52. (13) Norris, R. L.; Eaglesham, G. K.; Shaw, G. R.; Smith, M. J.; Chiswell, R. K.; Seawright, A. A.; Moore, M. R. A sensitive and specific assay for glutathione with potential application to glutathione disulfide, using high-performance liquid chromatography−tandem mass spectrometry. J. Chromatogr., B 2001, 762, 17−23. (14) Piccoli, G.; Fiorani, M.; Biagiarelli, B.; Palma, F.; Potenza, L.; Amicucci, A.; Stocchi, V. Simultaneous high-performance capillary electrophoretic determination of reduced and oxidized glutathione in red blood cells in the femtomole range. J. Chromatogr., A 1994, 676, 239−246. (15) Yao, X.; Wang, Y.; Chen, G. Simultaneous determination of aminothiols, ascorbic acid and uric acid in biological samples by capillary electrophoresis with electrochemical detection. Biomed. Chromatogr. 2007, 21, 520−256. (16) Monostori, P.; Wittmann, G.; Karg, E.; Túri, S. Determination of glutathione and glutathione disulfide in biological samples: an indepth review. J. Chromatogr., B 2009, 877, 3331−3346. (17) Iwasaki, Y.; Saito, Y.; Nakano, Y.; Mochizuki, K.; Sakata, O.; Ito, R.; Saito, K.; Nakazawa, H. Chromatographic and mass spectrometric analysis of glutathione in biological samples. J. Chromatogr., B 2009, 877, 3309−3317. (18) Spadaro, A.; Bousquet, E.; Santagati, N. A.; Vittorio, F.; Ronsisvalle, G. Simple analysis of glutathione in human colon carcinoma cells and epidermoid human larynx carcinoma cells by HPLC with electrochemical detection. Chromatographia 2005, 62, 11−15. (19) Khan, A.; Khan, M. I.; Iqbal, Z.; Shah, Y.; Ahmad, L.; Nazir, S.; Watson, D. G.; Khan, J. A.; Nasir, F.; Khan, A. A new HPLC method for the simultaneous determination of ascorbic acid and aminothiols in human plasma and erythrocytes using electrochemical detection. Talanta 2011, 84, 789−801. (20) Steele, M. L.; Ooi, L.; Münch, G. Development of a highperformance liquid chromatography method for the simultaneous quantification of glutathione and related thiols. Anal. Biochem. 2012, 429, 45−52. (21) U.S. FDA. Guidance for Industry: Bioanalytical Method Validation; U.S. Departmentof Health and Human Services, FDA, Center for Drug Evaluation and Research, Rockville, MD, 2001; www. fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/ guidances/ucm070107.pdf. (22) Valacchi, G.; Rimbach, G.; Saliou, C.; Weber, S. U.; Packer, L. Effect of benzoyl peroxide on antioxidant status, NF-κB activity and interleukin-1α gene expression in human keratinocytes. Toxicology 2001, 165, 225−234. (23) Park, H. J.; Mah, E.; Bruno, R. S. Validation of high-performance liquid chromatography-boron-doped diamond detection for assessing hepatic glutathione redox status. Anal. Biochem. 2010, 407, 151−159. (24) Aoyama, K.; Matsumura, N.; Watabe, M.; Wang, F.; KikuchiUtsumi, K.; Nakaki, T. Caffeine and uric acid mediate glutathione synthesis for neuroprotection. Neuroscience 2011, 181, 206−215. (25) Allameh, A.; Maleklou, N.; Zargar, M.; Sanati, M. H. The influence of uric acid treatments on liver glutathione system prevents oxidative damages in experimental autoimmune encephalomyelitis mice. Neurosci. Lett. 2008, 439, 111−115.

AUTHOR INFORMATION

Corresponding Author

*(G.R.) E-mail: [email protected]. Phone: +49 431 880 2583. Fax: +49 431 880 2628. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Pastore, A.; Federici, G.; Bertin, E.; Piemonte, F. Analysis of glutathione: implication in redox and detoxification. Clin. Chim. Acta 2003, 333, 19−39. (2) Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M.; Mazur, M.; Telser, J. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell B 2007, 39, 44−84. (3) Bald, E.; Chwatko, G.; Głowacki, R.; Kusmierek, K. Analysis of plasma thiols by high-performance liquid chromatography with ultraviolet detection. J. Chromatogr., A 2004, 1032, 109−115. (4) Potesil, D.; Petrlova, J.; Adam, V.; Vacek, J.; Klejdus, B.; Zehnalek, J.; Trnkova, L.; Havel, L.; Kizek, R. Simultaneous femtomole determination of cysteine, reduced and oxidized glutathione, and phytochelatin in maize (Zea mays L.) kernels using high-performance liquid chromatography with electrochemical detection. J. Chromatogr., A 2005, 1084, 134−144. (5) Petrlova, J.; Mikelova, R.; Stejskal, K.; Kleckerova, A.; Zitka, O.; Petrek, J.; Havel, L.; Zehnalek, J.; Adam, V.; Trnkova, L. Simultaneous determination of eight biologically active thiol compounds using gradient elution-liquid chromatography with Coul-Array detection. J. Sep. Sci. 2006, 29, 1166−1173. (6) Livingstone, C.; Davis, J. Targeting therapeutics against glutathione depletion in diabetes and its complications. Br. J. Diabetes Vasc. Dis. 2007, 7, 258−265. (7) Rossi, R.; Milzani, A.; Dalle-Donne, I.; Giustarini, D.; Lusini, L.; Colombo, R.; Di Simplicio, P. Blood glutathione disulfide: In vivo factor or in vitro artifact? Clin. Chem. 2002, 48, 742−753. (8) Wang, S.; Ma, H.; Li, J.; Chen, X.; Bao, Z.; Sun, S. Direct determination of reduced glutathione in biological fluids by Ce(IV)quinine chemiluminescence. Talanta 2006, 70, 518−521. (9) Pullela, P. K.; Chiku, T.; Carvan, M. J.; Sem, D. S. Fluorescencebased detection of thiols in vitro and in vivo using dithiol probes. Anal. Biochem. 2006, 352, 265−273. F



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(26) Beddowes, E. J.; Faux, S. P.; Chipman, J. K. Chloroform, carbon tetrachloride and glutathione depletion induce secondary genotoxicity in liver cells via oxidative stress. Toxicology 2003, 187, 101−115. (27) Will, O.; Mahler, H. C.; Arrigo, A. P.; Epe, B. Influence of glutathione levels and heat-shock on the steady-state levels of oxidative DNA base modifications in mammalian cells. Carcinogenesis 1999, 20, 333−337. (28) Moore, T.; Le, A.; Niemi, A. K.; Kwan, T.; Cusmano-Ozog, K.; Enns, G. M.; Cowan, T. M. A new LC−MS/MS method for the clinical determination of reducedand oxidized glutathione from whole blood. J. Chromatogr., B 2013, 929, 51−55.

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Rapid Method for Glutathione Quantitation Using High-Performance

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