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Clinical Nutrition (2008) 27, 579e586

available at www.sciencedirect.com

http://intl.elsevierhealth.com/journals/clnu

ORIGINAL ARTICLE

Oxidized vitamin E and glutathione as markers of clinical status in asthma*


Lisa G. Wood a,b,*, Manohar L. Garg c, Robert J. Blake c, Jodie L. Simpson a,b, Peter G. Gibson a,b
a Department of Respiratory and Sleep Medicine, Level 3, Hunter Medical Research Institute, John Hunter Hospital, Locked Bag 1, Hunter Region Mail Centre, Newcastle, 2310 NSW, Australia b School of Medicine and Public Health, University of Newcastle, 2308 NSW, Australia c School of Biomedical Sciences, University of Newcastle, 2308 NSW, Australia

Received 2 May 2007; accepted 5 December 2007

KEYWORDS
a-Tocopherol; a-Tocopherol quinone; Glutathione; Glutathione disulde; Antioxidants; Asthma

Summary Background & aims: Antioxidant status is disturbed in asthma. Measurement of both oxidized and reduced forms of antioxidants provides important information regarding the oxidant/antioxidant balance. The aim of this study was to investigate the clinical relevance of key antioxidants (a-tocopherol and glutathione) in asthma, by measuring the oxidized and reduced forms, in the airways (induced sputum) and systemically (peripheral blood). Methods: This cross-sectional study examines stable asthmatics (n Z 44) and healthy controls (n Z 31) recruited through John Hunter Hospital, NSW, Australia. We collected peripheral blood and induced sputum during hypertonic saline challenge. a-tocopherol and a-tocopherol quinone were measured by HPLC. Total glutathione and glutathione disulde were determined by a colorimetric assay. Results: Plasma a-tocopherol was low in asthma versus controls. Subjects with asthma had higher levels of whole blood a-tocopherol quinone and %a-tocopherol quinone than controls and %a-tocopherol quinone correlated with asthma control (p Z 0.009). Sputum supernatant levels of total, reduced and oxidized glutathione were elevated in asthma versus controls. Oxidized glutathione in sputum supernatant negatively correlated with FEV 1 /FVC% (p Z 0.029).

* Conference presentation: Parts of this work have been presented at the Thoracic Society of Australia & New Zealand Annual Conference, Sydney, 2004, and the Nutrition Society of Australia Annual Scientic Meeting, Melbourne, November 2005. * Corresponding author. Department of Respiratory and Sleep Medicine, Level 3, Hunter Medical Research Institute, John Hunter Hospital, Locked Bag 1, Hunter Region Mail Centre, Newcastle, 2310 NSW, Australia. Tel.: 61 2 49885677; fax: 61 2 49855850. E-mail addresses: lisa.wood@newcastle.edu.au (L.G. Wood), manohar.garg@newcastle.edu.au (M.L. Garg), robert.blake@hnehealth. nsw.gov.au (R.J. Blake), jodie.simpson@hnehealth.nsw.gov.au (J.L. Simpson), peter.gibson@hnehealth.nsw.gov.au (P.G. Gibson).

0261-5614/$ - see front matter 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2007.12.002

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Conclusions: In asthma, both systemic and airway antioxidant defences are disturbed. Oxidized forms of a-tocopherol and glutathione are associated with clinical asthma outcomes, and should be further investigated as a tool for monitoring asthma. 2007 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Introduction
Antioxidants are crucial to host antioxidant defence against oxidative stress.1 In asthma there is enhanced oxidative stress, demonstrated by elevated levels of various biomarkers including 8-isoprostane,2 malondialdehyde3e7 and breath ethane.8 Asthma is also characterised by disturbed antioxidant defences, including deciencies in vitamin C,4,5,9e11 vitamin E,4,5,11,12 b-carotene,4,13 lycopene,13 a-carotene,13 lutein13 and b-cryptoxanthin.13 However, interpretation and assessment of antioxidant defences in asthma has been limited by two key issues. Firstly, when measuring antioxidant levels, it is important to differentiate between the reduced and oxidized forms, as these molecules can only act as antioxidants in the reduced state. Secondly, investigation of oxidative stress in airway disease should incorporate analysis of airway lining uid. Most analysis of antioxidant defences in asthma has been carried out in peripheral blood. However, blood markers may not accurately represent conditions at the airway surface,11 the site of oxidative damage. Thus, reliable tools need to be developed for directly assessing antioxidant airway defences. We have sought to address these issues in this study. We have examined two antioxidants that exist in a relatively stable, quantiable form in both the reduced and oxidized states, and we have assessed these in the airway and in circulation. An important dietary antioxidant is vitamin E. The term vitamin E describes a group of eight structurally related molecules, including four tocopherols and four tocotrienols. The most abundant form of vitamin E, both in the diet and in biological systems is a-tocopherol. This exogenous antioxidant is oxidized to form a-tocopherol quinone. Both a-tocopherol and a-tocopherol quinone can be measured using HPLC. While altered a-tocopherol levels have been observed in asthma,4,5,11,12 to our knowledge, atocopherol quinone levels have not been reported in asthma to date. Comparison of the relative amounts of oxidized and reduced a-tocopherol in asthma is important in understanding the role of a-tocopherol in protecting against oxidative stress and affecting clinical asthma outcomes. Another key antioxidant is reduced glutathione. This endogenous antioxidant plays a prominent role in the respiratory tract due its ability to scavenge free radicals, as well as act as a cosubstrate in the glutathione peroxidase-catalysed reduction of H2O2 and lipid hydroperoxides.14 Both the reduced form (GSHr) and the oxidized form, glutathione disulde (GSSG), are quantiable using a colorimetric assay. While altered levels of (GSHt) have been reported in asthma in BAL15 and sputum,16 these studies did not elucidate what form of the antioxidant was driving this increase. Another study found increased GSSG levels in asthma,11 but did not report GSHt and GSHr remained unchanged. Another small study found no changes

in GSHt or GSSG.17 Since glutathione is widely recognised as a key antioxidant in the respiratory tract, a comprehensive examination is warranted. In order to examine antioxidant defences in the airways, we have investigated these antioxidants in induced sputum samples. Induced sputum has been used extensively to investigate airway inammation in stable asthma18 and in acute exacerbations of asthma19 and has been shown to be a safe,20 effective and reproducible method.21 Inammatory markers that have been measured in induced sputum in asthma include: IL-8,19,22 IL-2,19 eosinophil cationic protein (ECP),19,22 IL-2,19 neutrophil elastase,22 myeloperoxidase22 and IL-5.22 Induced sputum has also been found to contain biomarkers useful for studying oxidative stress in the lower respiratory tract2 and may provide a useful means of monitoring airway antioxidant defences. This is important as we seek to understand the relevance of antioxidant status to clinical outcomes in asthma. This study therefore aims to investigate the clinical relevance of two key antioxidants (a-tocopherol and glutathione) in asthma, by determining the relative concentrations of the oxidized and reduced forms in the airways (induced sputum) and systemically (peripheral blood).

Materials and methods


Subjects
Adults (over 18 years) with current diagnosis of asthma were recruited from specialist clinics at John Hunter Hospital, Newcastle. Controls were recruited by advertisement and asthma was excluded on the basis of history, normal spirometry and airway responsiveness, together with data review by a respiratory physician. Current smokers were excluded. Atopy was assessed by skin allergy testing. Plasma, whole blood and induced sputum were collected. In cases where the sample volume collected from a subject was insufcient for analysis of all biomarkers, this is indicated [Tables 3 and 4]. All participants gave informed written consent and the study was approved by the Hunter Area Research and University of Newcastle Ethics Committees.

Clinical classication of asthma


Asthma was diagnosed based upon a history of current (past 12 months) episodic respiratory symptoms, a prior doctors diagnosis of asthma (ever), current (past 12 months) use of inhaled asthma therapy and airway hyperresponsiveness to hypertonic saline. Subjects were considered to be unstable, and thus excluded from the study, if their asthma had worsened such that they had needed

Oxidized vitamin E and glutathione to: attend their doctor or hospital, increase the use of their asthma medication (b2-agonist, inhaled or oral steroid), or reduce their activity in the past 4 weeks. Asthma control was measured by a validated questionnaire23 that scores symptoms, activity level, bronchodilator use and lung function during the past week. The asthma control score ranges between 0 and 6 and a higher score indicates worse asthma control.

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Glutathione (GSHt and GSSG) measurements


Preparation for GSHt Sputum was selected from saliva, combined with two volumes of chilled PBS and vortexed. Cell free sputum supernatant was obtained by centrifugation (50 000g, 4  C for 30 min) and stored at 80  C. Plasma, whole blood and selected sputum plugs were also stored at 80  C. Preparation for GSSG Sputum supernatant: Sputum was selected from saliva, combined with 10 mM 1-methyl-2-vinylpyridinium triuoromethanesulfonate (M2VP) and two volumes of chilled PBS and vortexed. Cell free supernatant was obtained by centrifugation (50 000g, 4  C, 30 min) and stored at 80  C. Whole sputum: Sputum was selected from saliva, vortex mixed with 10 mM M2VP, then stored at 80  C. Whole blood and plasma were each mixed with 10 mM M2VP and stored at 80  C. Glutathione assay All glutathione measurements were performed within 30 days of sample collection. On the day of assay, whole blood samples were thawed, 5% metaphosphoric acid was added, the sample was vortex mixed, centrifuged (1000g, 4  C, 10 min) and the supernatant assayed. Whole sputum plugs were thawed, sonicated using a probe sonicator, centrifuged (50 000g, 4  C for 30 min) and the supernatant assayed. GSHt and GSSG concentrations were determined by colorimetric assay (Oxis International Inc., Portland, OR, USA) with standard curves based on dilutions of puried GSSG. GSHr concentrations were calculated using the formula GSHr Z GSHt 2 GSSG. %GSSG was calculated using the formula %GSSG Z (2 GSSG)/GSHt 100. The limit of detection of GSSG was 0.54 mM. This assay was validated for use in sputum supernatant, according to guidelines recently published by the European Respiratory Society, regarding analysis of uid phase mediators in induced sputum.25 Spiking experiments yielded an excellent recovery rate for GSSG in sputum supernatant of 103 (range 97e111)%. Within patient reproducibility was good. Subjects (n Z 5) studied on two occasions, two days apart, showed all values lie within BlandeAltman limits of agreement (mean bias 2SD) of 20.7 to 10.1 mM.

Sputum induction and analysis


Spirometry (KoKo K313100 PD Instrumentation, Louisville, CO, USA) and combined bronchial provocation and sputum induction with hypertonic saline (4.5%) were performed as previously described.24 Sputum induction time was standardized at 15.5 min. A portion of sputum was selected from saliva,24 dispersed with dithiothreitol (DTT), and a total cell count of leucocytes and viability performed. Cytospins were prepared, stained (May-Grunwald Geimsa) and a differential cell count obtained from 400 non-squamous cells.

a-Tocopherol (reduced and oxidized) measurements


Whole blood and plasma were stored at 80  C in tubes coated with 0.01% butylated hydroxytoluene (BHT). Mucus plugs were selected from induced sputum samples and stored at 80  C in BHT coated tubes. The concentrations of a-tocopherol and a-tocopherol quinone in whole blood, plasma and induced sputum were determined using HPLC. Samples were analysed using an extraction method previously described for carotenoid analysis.13 Tocopherol acetate was added as an internal standard. Chromatography was performed on a Hypersil ODS column (100 mm 2.1 mm 5 mm). The mobile phase consisted of acetonitrile:dichloromethane:methanol 0.05% ammonium acetate (85:10:5 v/v) at a ow rate of 0.3 mL/min. Tocopherols and tocopherol quinone were detected at 290 and 260 nm, respectively, using a photodiode array detector. %a-Tocopherol quinone was calculated using the formula %a-tocopherol quinone Z a-tocopherol quinone/(atocopherol a-tocopherol quinone) 100. Recovery of a-tocopherol quinone from spiked samples was excellent for both whole blood (94.1 4.5%) and plasma (80.5 0.6%). Recovery of a-tocopherol was also excellent for whole blood (89.7 4.2%) and plasma (99.4 3.1%). These experiments were performed by measurement of whole blood and plasma samples, with and without the addition of a spike of a-tocopherol quinone and a-tocopherol standard of known concentration, which was added before the extraction process was commenced. In whole blood, inter-assay reproducibility was excellent for a-tocopherol (%CV Z 5.3) and a-tocopherol quinone (%CV Z 4.2), as was intra-assay reproducibility for both a-tocopherol (%CV Z 5.5) and a-tocopherol quinone (%CV Z 7.4). In plasma, inter-assay reproducibility was also excellent for a-tocopherol (%CV Z 3.2) and a-tocopherol quinone (%CV Z 7.8), as was intra-assay reproducibility for both atocopherol (%CV Z 0.7) and a-tocopherol quinone (%CV Z 6.7).

Statistical analysis
Results were analysed using Minitab version 13.32 for Windows (Minitab Inc., State college, PA, USA). Statistical comparisons were performed using the Student t-test for normally distributed data, the ManneWhitney U test for non-parametric data. The mean standard error is reported for normal data, for non-parametric data the median (quartiles 1e3) are reported. Group comparisons were conducted using analysis of variance with ANOVA testing for normally distributed variables and KruskaleWallis testing for non-parametric data. Associations between variables were examined using Pearsons correlation coefcient for normally distributed data and Spearmans rank correlation coefcient for non-parametric data. Signicance was accepted if p < 0.05.

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Table 2 Induced sputum inammatory cell counts Asthma Total cell count (106/mL) % Neutrophils % Eosinophils % Macrophages % Lymphocytes % Columnar epithelial cells % Squamous cells 1.8 (1.4e3.9) 25.5 1.5* 56.8 0.5 2.0 (15.7e44.9) (0.4e5.4) (33.0e76.1) (0.0e1.1) (0.8e5.2) Healthy controls 1.7 (1.0e2.9) 24.3 0.0 66.3 0.94 0.75 (12.1e47.4) (0.0e1.0) (41.9e82.8) (0.1e2.0) (0.3e2.8)

Results
Stable asthmatics (n Z 44) and healthy controls (n Z 31) were recruited. Characteristics of these subjects are described in Table 1. There were no signicant differences in age between the two groups. As expected, asthmatics had reduced lung function and increased sputum eosinophils compared to controls [Tables 1 and 2]. The majority (90%) of subjects with asthma were regularly using inhaled corticosteroids. Concentrations of a-tocopherol and a-tocopherol quinone in whole blood, plasma and whole sputum are described in Table 3. Plasma a-tocopherol levels were low in asthma compared to controls [Table 3]. Similarly, whole blood levels of a-tocopherol tended to be low in asthma versus controls [Table 3, Fig. 1]. Whole blood levels of a-tocopherol quinone and %a-tocopherol quinone were elevated in asthma versus controls [Table 3, Fig. 1]. %aTocopherol quinone in whole blood correlated with asthma control (r Z 0.804, p Z 0.009) [Fig. 2]. Sputum concentrations of a-tocopherol quinone correlated with sputum supernatant concentrations of GSSG (r Z 0.608, p Z 0.047). There was no relationship between a-tocopherol and sputum cell counts. Concentrations of GSHt, GSHr and GSSG in whole blood, whole sputum and sputum supernatant are described in Table 4. Plasma levels of GSHt in asthmatics [0.4 (0.2e1.1) mM] and controls [0.7 (0.4e1.0) mM] were very low, with GSHr and GSSG levels undetectable. In whole blood, glutathione exists predominantly as GSHr [Table 4]. The proportion of GSSG was high in sputum (whole and supernatant) compared to whole blood. Levels of GSHt, GSHr and GSSG in sputum supernatant were elevated in asthma versus controls [Table 4, Fig. 3]. Sputum supernatant GSSG was negatively correlated with FEV1/FVC% (r Z 0.316, p Z 0.029) [Fig. 4]. Correlations also existed between sputum supernatant levels of: GSHt versus total cell count (r Z 0.363, p Z 0.017); GSHt versus

3.2 (0.6e11.4)

6.1 (1.1e17.5)

Data presented as median (quartiles 1e3). *p Z 0.005 versus healthy controls.

%columnar epithelial cells (r Z 0.424, p Z 0.002) and GSSG versus %columnar epithelial cells (r Z 0.403, p Z 0.005).

Discussion
This is the rst study to comprehensively review the status of a-tocopherol in both the reduced and oxidized forms in asthma. It also provides a comparison of this dietary antioxidant, to glutathione, an important endogenous antioxidant which we also examine in the reduced and oxidized states. The data show differences in the antioxidant status of asthmatics compared to controls, with reduced plasma and whole blood levels of a-tocopherol, increased whole blood levels of a-tocopherol quinone and increased sputum supernatant levels of GSHt, GSHr and GSSG in asthma. Importantly, the study also extends previous studies of oxidative stress in asthma, by relating levels of oxidized antioxidants (sputum GSSG and whole blood %a-tocopherol quinone) to clinical outcomes (lung function and asthma control). This highlights the potential of these compounds to be used as markers of disease status in asthma. a-Tocopherol, a key dietary antioxidant, is believed to be the most important lipophilic antioxidant in the lung,26 with a signicant role being the protection of lipids that constitute the surfactant lining the lungs, which is essential for normal lung function.26 In our study we found that concentrations of sputum a-tocopherol were similar to peripheral blood levels. This allocation of a-tocopherol to the lung is evidence that a-tocopherol plays an important role in the lung lining uid. This important role has previously been demonstrated as a-tocopherol concentrations in respiratory tract lining uid (RTLF) increase in response to extreme oxidant burdening of the lungs, probably due to mobilization from other tissues.27 The high levels of a-tocopherol in sputum also suggest the presence of active a-tocopherolsecreting mechanisms that maintain RTLF a-tocopherol levels. Indeed, a-tocopherol is known to be secreted by alveolar type II cells.28 Our study showed low a-tocopherol levels in the plasma and whole blood of asthmatics compared to healthy controls, agreeing with previous reports of disturbed vitamin E status in asthma.4,5,11,12,29 This deciency, however, was not reected in sputum. This may be further evidence of the movement of a-tocopherol into the airways as

Table 1 controls

Characteristics of stable asthmatics and healthy Asthma Healthy controls 31 41.4 2.5 17/14 101.4 2.3 104.1 1.9 80.5 1.3 NA 15 (58%) NA NA

N Age (years)a Sex (M/F) %Predicted FEV1a %Predicted FVCa %FEV1/FVCa PD15 (mLs)c,d Atopy n (%) Asthma control scorea Inhaled corticosteroid use (mg/day)b

44 47.4 2.6 16/28 82.7 3.4* 98.1 2.6 68.8 1.8* 0.65 (0.35) 41 (93%) 1.4 0.1 1000 (500e1600)

*p < 0.001 versus healthy controls. a Data are normally distributed and presented as mean SEM. b Data presented as median (quartiles 1e3). c PD15 is provocation dose resulting in a 15% drop in baseline FEV1. Values are geometric mean (log SD). d n Z 26.

Oxidized vitamin E and glutathione


Table 3 Systemic (peripheral blood) and airway (induced sputum) levels of a-tocopherol and a-tocopherol quinone N (asthma/controls) a-Tocopherol (mM) Asthma Blood (whole) Plasma Sputum (whole) 21 (9/12) 22 (9/13) 19 (7/12) 5.0*** (3.4e6.6) 17.0*,** (13.2e18.9) 4.5 (3.0e5.6) Controls 6.5 (4.9e8.5) 29.1 (15.4e43.2) 5.5 (2.8e6.9) a-Tocopherol quinone (mM) Asthma 5.5* (4.8e7.6) 0.6 (0.2e0.9) 0.4 (0.2e0.5) Controls 3.6 (2.3e5.9) 0.7 (0.1e1.4) 0.3 (0.2e0.4) %a-Tocopherol quinone Asthma 53.8* (47.2e64.4) 2.2 (1.4e5.1) 10.8 (5.6e11.8) Controls

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44.6 (21.0e51.9) 1.8 (0.4e4.5) 5.7 (4.0e6.9)

Data presented as median (quartiles 1e3). *p < 0.05 versus controls; **Data are parametric and analysed using Students t-test; ***p Z 0.076 versus controls.

an adaptive response to oxidative stress,27 resulting in a decrease in circulating a-tocopherol levels while airway levels are maintained. The mechanism by which this may be occurring is uncertain and is an important area for future research. To our knowledge, this is the rst report of atocopherol quinone levels in asthma. In whole blood, the concentration and proportion of a-tocopherol quinone was increased in asthma, also reecting the increased oxidant burden. Whole blood a-tocopherol quinone concentrations are high compared to plasma and sputum, possibly reecting the role of a-tocopherol in protecting erythrocyte membranes from oxidative damage. In contrast to our data, another study examining the a-tocopherol quinone: atocopherol ratio in lung lining uid found a much higher degree of vitamin E oxidation, with a ratio >0.35 in bronchoalveolar lavage (BAL) uid of healthy non-smokers.30 However, these data are not directly comparable to our sputum results, due to the different lung compartment sampled by BAL, the extreme dilution of BAL samples and the possible in vitro production of a-tocopherol quinone, as there was no addition of an antioxidant, such as BHT, prior to storage or extraction of samples. Glutathione is an important endogenous antioxidant in the lining uid of the respiratory tract. The sputum GSHt values obtained in this study agree with other published

data,17 demonstrating that glutathione concentrations in the airways are not as high as glutathione concentrations at the alveolar surface.31 However, this does not reduce the utility of sputum glutathione assessment. This study provides further evidence that measurable concentrations of glutathione are present in sputum, which appear to give a good reection of the redox status of the airways. Glutathione has a central role as both an intra- and extracellular antioxidant, involved in directly scavenging free radicals as well as acting as a cosubstrate in the glutathione peroxidase reduction of hydrogen peroxide.32 The GSHt values obtained in this study in induced sputum supernatant are comparable to other recent reports16,17,33 and are equivalent to whole sputum values, which excludes signicant allocation of glutathione to sputum cells. This suggests that glutathione has an important extracellular role in the airway lining uid. This contrasts with whole blood, where the majority of glutathione is contained within erythrocytes, reecting a predominantly intracellular role. Furthermore, in whole blood, GSSG is efciently recycled to the reduced form, allowing a very low proportion of GSSG to be maintained. In sputum, we observed a much higher %GSSG. This suggests that in the airway lining uid, the mechanisms for replenishing the reduced form are not as effective as in whole blood, probably due to the excessive amount of free radicals present in the airways.

Asthma Control

0 50 75

Figure 1 Whole blood concentrations of a-tocopherol, atocopherol quinone and %a-tocopherol quinone in asthma (n Z 21) versus healthy controls (n Z 11) (ap Z 0.076 versus controls; bp < 0.05 versus controls).

Whole blood % -tocopherol quinone

Figure 2 %a-Tocopherol quinone in whole blood versus asthma control score (r Z 0.804, p Z 0.009). Data analysed using Spearmans rank correlation.

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5.9 (0.2e13.8) 76.7 (63.8e79.8) 75.2 (50.0e100.0)
20 a Controls 15 Asthma

L.G. Wood et al.


90 80 70 60

Controls

( M)

10

1.7 (0.3e5.0) 84.2 (33.1e93.7) 76.6 (58.1e87.2)

40 30 20 10

Asthma

%GSSG

GSHt

GSHr

GSSG

%GSSG

24.8 (0.7e42.4) 3.5 (2.6e4.6) 2.6 (1.8e5.1)

Controls

Figure 3 Sputum supernatant concentrations of GSHt, GSHr, GSSG and %GSSG in asthma (n Z 37) versus healthy controls (n Z 20) (ap < 0.005 versus controls; bp < 0.05 versus controls).

Systemic (peripheral blood) and airway (induced sputum) levels of glutathione

Data presented as median (quartiles 1e3). *p < 0.05 versus controls; **p < 0.005 versus controls. GSHt Z total glutathione; GSHr Z reduced glutathione; GSSG Z glutathione disulde.

The elevated levels of GSHt, GSHr and GSSG in asthmatic sputum supernatant compared to controls, reect the increased oxidative burden in asthmatic airways [Fig. 3]. Disturbed glutathione status has been reported previously in asthma, with GSHt15 and GSSG11 being elevated in BAL uid, and GSHt being elevated in sputum.16 However, this study extends previous observations by demonstrating that the elevated GSHt levels in asthma are driven by an increase in both GSHr and GSSG. This suggests glutathione synthesis and/or transport has increased as an adaptive response to the presence of excess oxidants. Increased GSH synthesis may be due to the redox-sensitive transcriptional upregulation of mRNA for g-glutamylcysteine synthetase.34 The increase in GSHr levels in asthma has enabled a constant glutathione redox ratio to be maintained, despite an increased oxidant burden and consequent increase in GSSG concentrations. A compensatory but probably inadequate increase in GSHt has been reported in other conditions of oxidative stress.35,36 Epithelial shedding is a key feature of asthma37 and it has been demonstrated that free radicals generated by activated inammatory cells may contribute to this process.38

834 (705e924) 13.6* (10.4e15.6) 15.3** (10.0e22.4)

GSHt (mM)

908 (698e1033) 10.4 (6.6e12.1) 7.0 (4.7e14.3)

Controls

832 (698e900) 2.2 (0.7e8.8) 4.1* (1.4e6.8)

GSHr (mM)

Asthma

836 (628e995) 2.7 (1.6e3.5) 1.2 (0.0e3.8)

Controls

5.7 (1.0e19.2) 3.5 (2.2e7.5) 5.9** (4.0e8.4)

GSSG (mM)

Asthma

Asthma

100 90 80 70 60 50 40 30 0 2 4 6 8 10 12 14 16 18

N (asthma/control)

Sputum (whole)

Sputum (supernatant)

Blood (whole)

32 (21/11) 16 (10/6) 57 (37/20)

FE V1/FVC%

Sputum GSSG Concentration (uM)

Figure 4 Sputum supernatant concentrations of GSSG versus FEV1/FVC% (r Z 0.316, p Z 0.029) in subjects with asthma and healthy controls. Data analysed using Spearmans rank correlation.

Table 4

(%)

50

Oxidized vitamin E and glutathione The correlations in this study between %columnar epithelial cells and both GSHt and GSSG in induced sputum support the hypothesis that an increased oxidant burden in the airways, which causes an inux of GSHt and increased levels of GSSG, leads to increased shedding of airway epithelial cells thus contributing to the pathophysiology of asthma. Epithelial cell lysis/shedding is also likely to be directly contributing to the glutathione levels observed in induced sputum. No relationship was found between airway and blood antioxidant levels. This is further evidence that active transport mechanisms exist for both a-tocopherol and glutathione, whereby airway antioxidant defences are enhanced or maintained in response to a high oxidant load in the lungs. Schock et al.39 also found no correlation between serum and BAL concentrations of a-tocopherol. This agrees with the previous observation that blood biomarkers do not always accurately describe events in the airways.11 This highlights the need for further studies investigating mechanisms by which airway antioxidant defences may be augmented. It also highlights the need for studies to ascertain the relevance of antioxidant status to clinical asthma outcomes. Importantly, this study found several correlations that extend previous studies of oxidative stress in asthma, by relating levels of oxidized antioxidants to clinical outcomes. The inverse association between GSSG and airway obstruction (FEV1/FVC%) suggests that a high oxidant burden may worsen respiratory status. Similarly, the relationship between whole blood %a-tocopherol quinone and asthma control suggests that oxidative stress may contribute to a worse clinical outcome. These data are important because they suggest that clinical status may be improved using strategies aimed at reducing oxidative stress, such as antioxidant supplementation. In conclusion, it appears that both the antioxidants examined, a-tocopherol and glutathione, are important in the respiratory tract, with both of these antioxidants moving into the airway lining uid as an adaptive response to increased oxidative burden. Antioxidant defences are disturbed in asthma compared to controls. Furthermore, oxidized forms of both of the antioxidants examined, i.e. %a-tocopherol quinone and GSSG, are related to clinical status. This suggests that investigations of antioxidant defence should incorporate measurement of oxidized antioxidants as they may provide important information regarding clinical status. Future work should focus on determining whether using antioxidant supplementation to improve the oxidant-antioxidant balance in asthma affects clinical outcomes.

585 Institute, NSW, Australia, the John Hunter Hospital Research Committee, NSW, Australia, and the Asthma Foundation of NSW. Assistance with sample collection and analysis was received from Naomi Timmins, Rebecca Oldham, Joanne Smart, Glenda Walker, Kellie Fakes, Noreen Bell and Philippa Talbot from Respiratory and Sleep Medicine, Hunter Medical Research Institute, John Hunter Hospital, Newcastle, NSW, Australia. LGW, MLG and PGG originated and designed the study. LGW and JLS coordinated the study. LGW, MLG, RJB and PGG were responsible for various analytical and clinical measurements. LGW wrote the paper. All authors read and approved the nal manuscript.

References
1. Wood LG, Gibson PG, Garg ML. Biomarkers of lipid peroxidation, airway inammation and asthma. Eur Respir J 2003;21: 177e86. 2. Wood LG, Garg ML, Simpson JL, et al. Induced sputum 8-isoprostane concentrations in inammatory airway diseases. Am J Respir Crit Care Med 2005;171:426e30. 3. Rahman I, Morrison D, Donaldson K, MacNee W. Systemic oxidative stress in asthma, COPD and smokers. Am J Respir Crit Care Med 1996;154:1055e60. 4. Kalayci O, Besler T, Kilinc K, Sekerel BE, Saraclar Y. Serum levels of antioxidant vitamins (alpha tocopherol, beta carotene, and ascorbic acid) in children with bronchial asthma. Turk J Pediatr 2000;42:17e21. 5. Shanmugasundaram KR, Kumar SS, Rajajee S. Excessive free radical generation in the blood of children suffering from asthma. Clin Chim Acta 2001;305:107e14. 6. Ozarus R, Tahan V, Turkmen S, et al. Changes in malondialdehyde levels in bronchoalveolar uid and serum by the treatment of asthma with inhaled steroid and beta2-agonist. Respirology 2000;5(3):289e92. 7. Antczak A, Nowak D, Shariati B, Krol M, Piasecka G, Kurmanowska Z. Increased hydrogen peroxide and thiobarbituric acid-reactive products in expired breath condensate of asthmatic patients. Eur Respir J 1997;10:1235e41. 8. Paredi P, Kharitonov SA, Barnes PJ. Elevation of exhaled ethane concentration in asthma. Am J Respir Crit Care Med 2000;162:1450e4. 9. Aderele WI, Ette SI, Oduwole O, Ikpeme SJ. Plasma vitamin C (ascorbic acid) levels in asthmatic children. Afr J Med Sci 1985;14:115e20. 10. Vural H, Uzun K. Serum and red blood cell antioxidant status in patients with bronchial asthma. Can Respir J 2000;7(6): 476e80. 11. Kelly FJ, Mudway I, Blomberg A, Frew A, Sandstrom T. Altered lung antioxidant status in patients with mild asthma. The Lancet 1999;354:482e3. 12. Mohan IK, Das UN. Oxidant stress, antioxidants, nitric oxide and essential fatty acids in bronchial asthma. Med Sci Res 1997;25:307e9. 13. Wood LG, Garg ML, Blake RJ, Garcia-Caraballo S, Gibson PG. Airway and circulating levels of carotenoids in asthma and healthy controls. J Am Coll Nutr 2005;24: 448e55. 14. Beier J, Beeh KM, Kornmann O, Buhl R. Induced sputum methodology: validity and reproducibility of total glutathione measurement in supernatant of healthy and asthmatic individuals. J Lab Clin Med 2004;144:38e44. 15. Smith LJ, Houston M, Anderson J. Increased levels of glutathione in bronchoalveolar lavage uid from patients with asthma. Am Rev Respir Dis 1993;147:1461e4.

Conict of interest statement


None declared.

Acknowledgements
LGW was supported by a Postdoctoral Fellowship from the National Health and Medical Research Council, Australia. PGG was supported by a Practitioner Fellowship from the National Health and Medical Research Council, Australia. This study was funded by the Hunter Medical Research

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16. Beier J, Beeh KM, Semmler D, Beike N, Buhl R. Increased concentrations of glutathione in induced sputum of patients with mild or moderate allergic asthma. Ann Allergy Asthma Immunol 2004;92:459e63. 17. Dauletbaev N, Rickmann J, Viel K, Buhl R, Wagner T-O-F, Bargon J. Glutathione in induced sputum of healthy individuals and patients with asthma. Thorax 2001;56:13e8. 18. Pin I, Gibson PG, Kolendowicz R, et al. Use of induced sputum cell counts to investigate airway inammation in asthma. Thorax 1992;47:25e9. 19. Pizzichini MM, Pizzichini E, Efthimiadis A, et al. Asthma and natural colds. Inammatory indices in induced sputum: a feasibility study. Am J Respir Crit Care Med 1998;158:1178e84. 20. Wark PAB, Simpson JL, Hensley MJ, Gibson P. Safety of sputum induction with isotonic saline in adults with acute severe asthma. Clin Exp Allergy 2001;31:1745e53. 21. Pizzichini E, Pizzichini MM, Efthimiadis A, et al. Indices of airway inammation in induced sputum: reproducibility and validity of cell and uid-phase measurements. Am J Respir Crit Care Med 1996;154:308e17. 22. Norzila MZ, Fakes K, Henry RL, Simpson J, Gibson PG. Interleukin-8 secretion and neutrophil recruitment accompanies induced sputum eosinophil activation in children with acute asthma. Am J Respir Crit Care Med 2000;161:769e74. 23. Juniper EF, OByrne PM, Guyatt GH, Ferrie PJ, King DR. Development and validation of a questionnaire to measure asthma control. Eur Respir J 1999;14:902e7. 24. Gibson PG, Wlodarczyk J, Hensley M, et al. Epidemiological association of airway inammation with asthma symptoms and airway hyperresponsiveness in childhood. Am J Respir Crit Care Med 1998;158:36e41. 25. Kelly MM, Keatings V, Leigh R, et al. Analysis of uid-phase mediators. Eur Respir J 2002;37(Suppl.):24se39s. 26. Kolleck I, Sinha P, Rustow B. Vitamin E as an antioxidant of the lung. Am J Respir Crit Care Med 2002;166:S62e6. 27. Elsayed NM. Antioxidant mobilisation in response to oxidative stress: a dynamic environmental-nutritional interaction. Nutrition 2001;17:828e34.

L.G. Wood et al.


28. Rustow B, Haupt R, Stevens PA, Kunze D. Type II pneumocytes secrete vitamin E together with surfactant lipids. Am J Physiol e Lung Cell Mol Physiol 1993;265(9):L133e9. 29. Misso NLA, Brooks-Wildhaber J, Ray S, Vally H, Thompson PJ. Plasma concentrations of dietary and nondietary antioxidants are low in severe asthma. Eur Respir J 2005;26: 257e64. 30. Pacht ER, Kaseki H, Mohammed JR, Cornwell DG, Davis WB. Deciency of vitamin E in the alveolar uid of cigarette smokers. J Clin Invest 1986;77:789e96. 31. van der Vliet A, ONeill C, Cross CE, et al. Determination of low-molecular-mass antioxidant concentrations in human respiratory tract lining uids. Am J Physiol e Lung Cell Mol Physiol 1999;276:L289e96. 32. Dickinson DA, Forman HJ. Glutathione in defense and signaling. Ann NY Acad Sci 2002;973:488e504. 33. Beeh KM, Beier J, Koppenhoefer N, Buhl R. Increased glutathione disulde and nitrosothiols in sputum supernatant of patients with stable COPD. Chest 2004;126:1116e22. 34. Rahman I, MacNee W. Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease. Am J Physiol 1999;277:L1067e88. 35. Toth KM, Berger EM, Beehler CJ, Repine JE. Erythrocytes from cigarette smokers contain more glutathione and catalase and protect endothelial cells from hydrogen peroxide than do erythrocytes from nonsmokers. Am Rev Respir Dis 1986;134: 281e4. 36. Borm PJ, Bast A, Wouters EF, Slangen JJ, Swaen GM, Border DE. Red blood cell antioxidant parameters in healthy elderly control subjects versus silicosis patients. Free Radic Res Commun 1987;3:117e27. 37. Barnes PJ. Reactive oxygen species and airway inammation. Free Radic Biol Med 1990;9:235e43. 38. Henricks PAJ, Nijkamp FP. Reactive oxygen species as mediators in asthma. Pulm Pharmacol Ther 2001;14:409e21. 39. Schock BC, Young IS, Brown V, et al. Antioxidants and protein carbonyls in bronchoalveolar lavage uid of children: normal data. Pediatr Res 2001;49:155e61.