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Received 17 August 2001; accepted 3 September 2001

ABSTRACT: Glutathione (GSH) and glutathione disulfide (GSSG) levels in cells constitute a thiol redox system. They can be used asan indicator of oxidative stress of the cell. In this study, a capillary zone electrophoresis (CZE) method is described that enablesquantitation of GSH and GSSG from cellular extracts. The CZE buffer used was 20 mM ammonium acetate containing 5% (v/v) aceticacid at pH 3.1 in conjunction with a polybrene coated capillary operated in reverse polarity mode. Effects of different acids used toprepare cell samples were investigated on CZE performance. The acids include meta phosphoric acid (MPA), trichloroacetic acid(TCA), phosphoric acid (PA) and sulfosalicylic acid (SSA) and are used to stabilize GSH and GSSG before performing CZE analysis.The method features a limit of detection of 4 �M and a limit of quantitation of 12 �M for both GSSG and GSH and recoveries of 94%for GSH and 100% for GSSG. Quantitative analysis of GSSG and GSH in HaCaT cell extracts (5% SSA, w/v) was performed withthis method and changes in the ratio of GSH to GSSG in N-ethylmaleimide treated cell sample was observed by comparing withcontrol cell samples. Copyright � 2002 John Wiley & Sons, Ltd.

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Glutathione (GSH), along with ascorbate and vitamin E,function as antioxidants to prevent and limit oxidativedamage in cell metabolism. The oxidative conversion ofGSH to glutathione disulfide (GSSG) is widely recog-nized as a reliable index of oxidative stress. Therefore,great interest exists in the simultaneous determination ofGSH and GSSG in various biological tissue, organs, andcells (Halliwell and Gutteridge, 1984; Kaplowitz et al.,1985). GSH is also considered an important endogenousantioxidant that may play a role in ameliorating orpreventing the actinic damage that can lead to cutaneousdisorders such as skin cancer, hyperpigmentation andpremature aging (Connor and Wheeler, 1987). Thetransient depletion of GSH and oxidation to GSSG andrapid recoveries of cutaneous GSH levels would becompatible with a role of GSH as an endogenousphotoprotective agent in the skin. This led us to developan analytical method that can be used to monitor the

changes in the ratio of GSH to GSSG and furthermore toquantify GSH and GSSG in cell extracts.

Analytical methods utilized to measure GSH/GSSGratios include enzymatic assay (Tietze, 1969), and, pre-dominantly, HPLC in conjunction with UV (Jayatillekeand Shaw, 1993; Paroni et al., 1995; Yoshida, 1996),fluorescence spectroscopy (Gotti et al., 1994; Haj-Yehiaand Benet, 1995; Yang et al., 1995) or electrochemicaldetection (Kleinman and Richie, 1995; Remiao et al.,2000). More recently Loughlin et al. reported the use ofon-line HPLC-tandem mass spectrometry to determinereduced and oxidized GSH and glutathione conjugates inhepatocytes (Loughlin et al., 2001). An alternative chro-matographic approach has been to employ capillary zoneelectrophoresis (CZE) for such types of analyses (Daveyet al., 1997; Muscari et al., 1998). However, while CZEaffords potentially greater sensitivity and separationefficiency, these previously described approaches possesssome limitations. Hence, in this work we demonstratenovel CZE conditions for the simultaneous analysis andquantitation of GSH and GSSG from cell extracts.

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�������# Metaphosphoric acid (MPA), disodium EDTA,ammonium acetate, acetic acid, Tris-(hydroxymethyl)amino-

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 16: 224–228 (2002)DOI: 10.1002/bmc.129

*Correspondence to: S. Naylor, Guggenheim C009B, Mayo Clinic,Rochester, MN 55905, USA.Email: naylor.stephen@mayo.edu

Abbreviations used: GSH, glutathione; GSSG, glutathione disulfide;MPA, meta phosphoric acid; PA, phosphoric acid; SSA, sulfasalicylicacid; TCA, trichloroacetic acid.

Contract/grant sponsor: Blandin Foundation.

Copyright 2002 John Wiley & Sons, Ltd.

ORIGINAL RESEARCH

methane (TRIS), polybrene, ethylene glycol, sodium hydroxide,methanol, sulfosalicylic acid (SSA), phosphoric acid (PA),trichloroacetic acid (TCA), and N-ethylmaleimide (NEM) wereall purchased from Sigma (St Louis, MO). Silica capillary of 75 �mi.d. � 365 �m o.d. was purchased from Polymicron TechnologiesInc. (Phoenix, AZ).

��������� ���������� %��� ������ ���� ���������#All separations were performed on a Beckman P/ACE 5010 system(Palo Alto, CA) equipped with a UV absorbance detector set to200 nm. The sample was pressure injected (0.5 psi) for 10 s andseparated at �10 kV. The capillary length was 69 cm from inlet tothe detection window and 76 cm in total length. The CZEbackground electrolyte used is reported in each of the figures.Data were collected at a rate of 5 Hz and processed with theBeckman P/ACE software version 1.0. The coated CZE capillarywas prepared by rinsing with 1 M NaOH followed by methanol,each for 30 min, then rinsed with polybrene (5% polybrene and 2%ethylene glycol in water) for 5 min and left in the lumen for 5 min.The coating procedure was repeated twice before rinsing withmethanol and CZE separation buffer (20 mM ammonium acetate in5% (v/v) acetic acid, pH 3.1), each for 10 min. Finally, a voltage of�10 kV was applied to condition the capillary. Between eachsample injection, the capillary was rinsed with CZE buffer for1 min to equilibrate the capillary surface. Finally rinsing withmethanol for 0.2 min and then with CZE buffer was used to removecontaminants from the previous analytical run. Samples wereinjected at the cathode and detected at the anode (reverse polarity).The capillary was maintained at 28°C during capillary coating andthroughout all CZE analyses.

������� �������# Samples were prepared from the culturedhuman keratinocyte HaCaT cell line. After growing to confluencyin tissue culture dishes, the cells were treated with 10 �M NEM for60 s or irradiated with UV-B (Meves et al., 2001; Peus et al.,1999). Control cells were not subject to either of these twotreatments. The cell medium was subsequently removed, and thecells were washed twice with ice-cold PBS then with 0.4 mLextraction solvent [either 3% (w/v) MPA containing 1 mM

disodium EDTA, or 5% (w/v) SSA] was added. After 2 minincubation on ice, the cells were harvested, homogenized with apre-cooled Dounce (tight pestle) and centrifuged at 14 000 rpm for10 min at 4°C. The supernatant was transferred into a Milliporemicro-concentrator with Mr cut-off of 5000 (Ultrafree-MCNMWL) and was ultrafiltered by centrifugation at 4800g for90 min (4°C). The filtrate was subsequently used for CZE analysis.

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It has previously reported that CZE can be used for thesimultaneous quantitative analysis of GSH and GSSG(Muscari et al., 1998). They utilized a CZE bufferconsisting of 100 mM boric acid containing 25 mM Tris,at pH 8.2. When we used their conditions with GSH andGSSG standards dissolved in H2O, we effected a baselineseparation differing in migration times by �1 min. The

separation was effected in �5 min, and is shown in Fig.1(A). However, when we attempted to separate GSH andGSSG using conditions employed to analyze cellextracts, we encountered problems. The extractionsolvent used to lyse, extract, and stabilize GSH andGSSG from cells contains 3% (w/v) MPA and 1 mM

Figure 1. (A) Electropherogram of GSSG (15 �M) and GSH(25 �M) in water. CZE buffer contained 100 mM boric acid and25 mM Tris, pH 8.2. Fused silica capillary (75 �m i.d. � 67 cm)was maintained at a constant temperature of 28°C. Sampleswere injected by applying a pressure (0.5 psi) for 10 s and wereseparated by applying a voltage 30 kV across the capillary. UVdetection was set at 200 nm. (B) Electropherogram of GSSGand GSH in 3% MPA containing 1 mM EDTA. The concentra-tions of GSSG and GSH were the same and the CZE conditionsare as in (A).

Copyright 2002 John Wiley & Sons, Ltd. Biomed. Chromatogr. 16: 224–228 (2002)

Oxidized and reduced glutathione analysis ORIGINAL RESEARCH 225

EDTA. Analyzing spiked GSH/GSSG samples inMPA:EDTA resulted initially in compromised resolu-tion, deterioration in signal:noise, and contaminantinterference, and this is highlighted in Fig. 1(B). Overtime with multiple injections, resolution deteriorated andmigration times became highly variable. We encounteredsimilar problems employing the CZE conditions sug-gested by Davey et al. (1997). In both cases we couldpartially alleviate these issues by diluting the MPAextract (25% dilution) with either H2O or CZE buffer.However, this dilution effect significantly compromisedconcentration limits of detection in CZE.

In order to overcome all these problems, as well asminimize analyte losses to the capillary wall, wedeveloped a CZE separation method utilizing a coatedcapillary and a CZE buffer composed of 20 mMammonium acetate and 5% (v/v) of acetic acid (pH3.1). The coating procedure is described in detail in theExperimental section, however the inner surface ofcapillary is covered with positively charged polybreneions. Since GSSG and GSH and other ions in the sampleare positively charged at pH 3.2, they are repelled by thecapillary wall. As a result, analyte loss to the largecapillary surface is minimized. As shown in Fig. 2, a low

concentration of spiked GSSG (3.3 �M) was wellseparated from a comparatively higher concentration ofspiked GSH (323 �M) in a cell extract prepared in 3%MPA containing 1 mM EDTA. Other cell componentsand extraction solvents do not appear to interfere withanalyte separation performance. In addition, under suchconditions, one capillary coating allows analysis of 20injections without deterioration in CZE performance.After such a large number of injections, the overall CZEconditions could be recovered simply with capillarywashing and re-coating.

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A variety of methods have been used to prepare differentbiological samples for the determination of GSSG andGSH concentration levels (Floreani et al., 1997).Typically some type of acid extraction is carried out.The function of such acids is two-fold: firstly, the acid(s)is used to deactivate �-glutamyltraspeptidase, the enzymeresponsible for the first step in the degradation of GSHand, secondly, the acid(s) also provides sufficienthydrogen ions to ensure chemical stabilization of GSHand GSSG. We therefore evaluated the effects of variousacids, including TCA, PA and SSA on CZE performance.While the effectiveness of TCA and SSA have been welldocumented in the quantitation of GSH and GSSGderived from biological tissue (Roberts and Francetic,1993), we also wanted to ascertain the effectiveness ofPA and MPA in the same extraction process. GSH(25 �M) and GSSG (15 �M) were spiked into cell lysates,and extracted with individual acids. Samples were thenanalyzed on CZE using run conditions described above.These data are summarized in Fig. 3. While MPAextraction appeared to be most efficient, there were someinconsistencies in reproducible migration times of GSHand GSSG. Based on efficient recovery of GSH andoptimal resolution conditions we decided to use SSA.This solvent has been reported to be excellent forextraction of GSH in blood and a variety of biologicaltissues, including liver, spleen, lung, and heart (Robertsand Francetic, 1993).

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The calibration line was obtained from six standardmixtures, each of them containing 0, 5, 15, 25, 35 50 �M

GSSG and 0, 5 25, 50, 80 and 100 �M GSH in 5% (w/v)SSA and was injected in triplicate. The correlationcoefficients were calculated to be 0.999 for both GSSGand GSH. The limit of detection (LOD) was calculated tobe 4 �M and the limit of quantitation (LOQ) was about12 �M for both GSSG and GSH. The detectability wascalculated based on the definitions provided by ICH (theInternational Conference on Harmonization of Technical

Figure 2. Electropherograms of spiked GSSG (3.73 �M) andGSH (323 �M) in a cell extract prepared in 3% MPA (w/v)containing 1 mM EDTA. CZE conditions are as follows:capillary (75 �m i.d. � 76 cm) coated with polybrene asdescribed in the Experimental section. The CZE buffer contains20 mM ammonium acetate and 5% (w/v) acetic acid, pH 3.1.

Copyright 2002 John Wiley & Sons, Ltd. Biomed. Chromatogr. 16: 224–228 (2002)

226 ORIGINAL RESEARCH Q. Yang et al.

Requirements for Registration of Pharmaceuticalsfor Human Use) as follows (ICHQ2B, 1996): LOD =3.3*(�/S), LOQ = 10*(�/S), where S was estimated fromthe slope of the calibration line and � was estimated fromthe standard deviation of y intercept of the regressionline. The within-day repeatability were obtained from sixcell extracts spiked to contain 5 �M GSSG and 60 �M

GSH and was calculated to be 10% for both GSSG andGSH. The accuracy of the method was evaluated fromspiked cell extracts with mixtures of GSSG and GSH in5% (w/v) SSA. Each of the samples was injected threetimes. The recoveries were estimated to be 94% for GSHat a concentration of 70 �M and 100% for GSSG at aconcentration of 19 �M.

As shown in Table 1, in the first three cell extractsanalyzed contained GSSG below 1 �M, which was muchlower than the LOD of this method. While, GSHconcentrations ranged from 50 to 59 �M in these samples.We also applied the method to two cell samples (samples4 and 5) in order to evaluate whether treatment withNEM, an agent known to deplete GSH, could cause

changes of GSSG and GSH in HaCaT cell line. As shownin Fig. 4, we observed a dramatic increase in GSSG

Figure 3. Summary of the effects and efficiency of extraction of SSA, TCA, PA and MPAfor GSH (25 �M) and GSSG (15 mM) spiked in cell extracts.

Table 1. Concentrations of GSSG and GSH in cell extracts

Cell extractGSSG, �M

(mean � SD)GSH, �M

(mean � SD)

1 (n = 3) 0.70 � 0.20 58.66 � 0.342 (n = 3) 0.66 � 0.15 55.67 � 2.573 (n = 3) 0.25 � 0.04 50.72 � 3.374 (n = 2) 22.07 � 0.14 189.95 � 2.015 (n = 2) 57.90 � 2.10 118.70 � 9.45

Figure 4. Electropherograms of GSSG and GSH in a HaCaTcell sample treated with NEM followed by extraction with 5%(w/v) SSA and a control cell sample extracted with 5% (w/v)SSA. The CZE conditions are as in Fig. 2: (—) control; (---)treated with NEM.

Copyright 2002 John Wiley & Sons, Ltd. Biomed. Chromatogr. 16: 224–228 (2002)

Oxidized and reduced glutathione analysis ORIGINAL RESEARCH 227

concentration in the NEM treated cell sample and adecrease in GSH. It indicates the effectiveness of themethod for the authentic biological samples. Investiga-tions to examine the conversion of GSH to GSSG in UV-B-treated HaCaT cells with the established method are inprogress.

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We have developed an analytical method that simulta-neously detects and quantifies GSSG and GSH in cellextracts without any further treatment other than filtrationwith a 5 kDa micro-centrifuge. The resolution wassignificantly enhanced by using coated capillary, andthe method allows the detection of GSSG in cell extractfeaturing high ionic strength which enables directinjection of cell extracts containing up to 5% acids. Thedirect sample injection not only ensures stability of bothGSSG and GSH in biological samples but also avoidstime-consuming sample pretreatment before the analysis.The established CE conditions are also potentially usefulfor CE-MS due to use of a volatile CE buffer.

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Q. Yang and S. Naylor would like to acknowledge afinancial support from Blandin Foundation, USA.

".3.".'�.�

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