Selenium, Glutathione Peroxidase and Superoxide Dismutase in Maltese Asthmatic Patients: Effect of Glucocorticoid Administration

Selenium, Glutathione Peroxidase and Superoxide Dismutase in Maltese Asthmatic Patients: Effect of Glucocorticoid Administration

Pulmonary Pharmacology & Therapeutics (1998) 11, 301–308 Article No. pu980122 PULMONARY PHARMACOLOGY & THERAPEUTICS Selenium, Glutathione Peroxidase...

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Pulmonary Pharmacology & Therapeutics (1998) 11, 301–308 Article No. pu980122


Selenium, Glutathione Peroxidase and Superoxide Dismutase in Maltese Asthmatic Patients: Effect of Glucocorticoid Administration A. G. Fenech∗, R. Ellul-Micallef Department of Clinical Pharmacology and Therapeutics, University of Malta, Msida, MSD 06, Malta

SUMMARY: Oxidative processes, mediated by free radical chemistry, are recognized to contribute significantly to the inflammatory pathology of bronchial asthma. This study analysed the degree of defence against reactive oxygen species in Maltese, asthmatic patients and in normal individuals, by measuring plasma selenium concentration, erythrocyte glutathione peroxidase (GSH-Px) activity and erythrocyte superoxide dismutase (SOD) activity, in order to determine their antioxidant status. The effect of glucocorticoids on the status of these antioxidants in patients was also investigated. The measurement of antioxidant status was carried out both in mild (n=22) and severe (n=37) asthmatics, as well as in healthy controls (n=49). The same antioxidant profile was then investigated in a group of 16 severe asthmatics following treatment for 4 weeks with inhaled beclomethasone dipropionate (750 lg twice daily), and in a second group of 16 patients suffering from severe asthma, following 2weeks treatment with oral prednisolone (15 mg daily during the first week and 10 mg daily during the second). No statistically significant difference was found in the plasma selenium concentrations and erythrocyte glutathione peroxidase activities between patients and controls. Both mild and severe asthmatics, however, exhibited a statistically significant lower erythrocyte superoxide dismutase activity than normal subjects (mild asthmatics: 62.9 (2.9) SOD 525 U/ml, severe asthmatics: 60.6 (1.9) SOD 525 U/ml, normal: 68.5 (1.1) SOD 525 U/ml, P<0.01). Inhaled beclomethasone dipropionate exerted no effect on this antioxidant profile, while prednisolone caused a significant increase in plasma selenium concentration over pretreatment values (pretreatment: 118.3 (4.4) ng/ ml, post-treatment: 138.1 (4.6) ng/ml, P<0.01). It is thus suggested that asthmatic patients in Malta might be more susceptible to superoxide-induced damage than normal individuals. The reason for the prednisolone-induced augmentation of plasma selenium could not be determined from this study. It is postulated that the drug may decrease the excretion rate of the element, and may thus exert a positive antioxidant effect in individuals of established low selenium status.  1998 Academic Press

KEY WORDS: Asthma, Antioxidants, Glucocorticoids, Enzymes, Malta.

to increased concentrations of the inflammatory mediators prostaglandins, thromboxanes and leukotrienes. A contributing role for ROS in the pathology of asthma has recently been recognized. Oxidative species including O2− ·, ·OH, H2O2 and NO· are released into the airway by activated inflammatory cells such as eosinophils, neutrophils, alveolar macrophages, mast cells and monocytes.2–5 In vitro work has shown that both neutrophils as well as eosinophils obtained from the peripheral blood of atopic asthmatics generate significantly greater amounts of free radicals upon stimulation than do those obtained from healthy subjects.6–8 In asthmatics, the degree of superoxide-anion

INTRODUCTION Oxygen atoms in biological environments have the ability to form highly reactive moieties called reactive oxygen species (ROS). These include hydroxyl radicals (·OH), superoxide (O2− ·), peroxy radicals (RO2·), alkoxy radicals (RO·), organic hydroperoxide radicals (R-OO·), nitric oxide (NO·), hydrogen peroxide (H2O2) and organic hydroperoxides (R-OOH).1 ROS have today been recognized to participate actively in the process of inflammation through the stimulation of the metabolism of arachidonic acid, thereby leading ∗ Author for correspondence. 1094–5539/98/040301+08 $30.00/0


 1998 Academic Press


A. G. Fenech and R. Ellul-Micallef

generation from neutrophils has been shown to correlate with the degree of airway hyperresponsiveness, as quantified by the provocative concentration of inhaled methacholine (PC20).9 Oxygen-derived free radicals from stimulated peripheral blood monocytes of asthmatic patients are released in higher amounts and within a shorter time period than from the equivalent cell population of healthy control individuals.10 The main in vivo antioxidant defences are exerted by the superoxide dismutases (SOD), which convert O2− · to H2O2; and the selenium-dependent glutathione peroxidases (GSH-Px), which convert both H2O2 and R-OOH species to the respective alcohol (R-OH) and water. Selenium is of major importance in antioxidation processes since it is a critical component for the proper synthesis and function of GSH-Px. Widely varying physiological concentrations of the element have been reported in different countries, and these have been generally attributed to the concentrations in soil, vegetables and herbivore meat. Non-enzymatic antioxidants such as vitamin E, glutathione (which possesses some antioxidant properties which are independent of GSH-Px) and vitamin C also exert some action in the airways.11,12 Plasma glutathione peroxidase (pl-GSH-Px) as well as extracellular superoxide dismutase (EC-SOD) present in the epithelial lining fluid may play a significant role by providing first line defence against organic hydroperoxides, hydrogen peroxide and superoxide.13 The present study aimed to investigate the antioxidant profile in Maltese bronchial asthmatics by studying peripheral blood for evidence of alterations to antioxidant parameters. The indices evaluated were plasma selenium concentrations, erythrocyte GSH-Px activity and erythrocyte SOD activity. The aims of the study were: (1) The establishment of baseline values for plasma selenium, erythrocyte GSH-Px and erythrocyte SOD in healthy adult Maltese individuals, and to compare them to those obtained from Maltese bronchial asthmatics; (2) To investigate the effects of high dose inhaled beclomethasone dipropionate and low dose short term oral prednisolone treatment on the antioxidant profile of asthmatic patients. This study had the approval of the Ethics Committee of the Faculty of Medicine and Surgery, University of Malta, Malta. MATERIALS AND METHODS The study was carried out in two phases. Phase 1 established baseline values for plasma selenium concentration, erythrocyte GSH-Px activity, and erythrocyte SOD activity in normal subjects, and compared this data to that obtained from mild and severe asthmatic patients. Phase 2 investigated whether high dose

inhaled and low dose oral glucocorticoid treatment in severe asthmatics exerted any effect on this antioxidant profile. Selection of subjects Healthy controls (n=49) were selected from individuals voluntarily attending the Blood Transfusion Unit, St Luke’s Hospital, Malta, in order to donate blood. The subjects were screened for the presence of disease by the physician on duty. It was ensured that they were not on medication, and were not consuming any form of selenium-containing supplements. Patients were recruited from the Out-Patient Asthma Clinic, St Luke’s Hospital, Malta. Selections for the study were made according to the following criteria, in order to exclude any possible influences arising from other disease states and/or dietary supplementation on the antioxidant profile: (1) Past medical history of bronchial asthma for at least 12 months; (2) no history of other chronic inflammatory diseases, cardiovascular disease or malignant disease; (3) no pregnancy at least during the last 12 months; (4) no history of smoking; (5) no intake of any form of selenium-containing supplements; (6) no other medication except that prescribed for asthma; (7) no inhaled or oral glucocorticoid treatment, at time of selection and at least during the previous 6 weeks.

Study phase 1 The selected patients (n=59) were subsequently classified into mild (n=22) or severe (n=37) categories, as defined below. This classification was based on the principles laid out by Jones.14 Mild Asthmatic patients who only suffered from occasional mild attacks, which did not in any way interfere with their lifestyle, and who did not require more than the occasional use of inhaled bronchodilator therapy. Severe Patients who presented with an unstable condition had FEV1 and PEF values of 75% or less than predicted, and complained of frequent attacks of dyspnoea and wheezing, and of being woken up at night with these symptoms. All patients used salbutamol inhaler treatment as

Antioxidant Status in Asthma: Effect of Glucocorticoids

required to relieve their symptoms. Salbutamol has already been shown by Burgess et al,15 not to exert any effect on antioxidant status when administered by inhalation. Atopy was present in 19 patients with mild symptoms and 32 patients with severe symptoms. This was determined from total serum IgE concentrations in excess of 100 IU/ml and a positive allergen skin prick test result.


lamp (Photron Pty Ltd, Victoria, Australia). Withinrun precision, calculated by carrying out 10 repeat determinations of control bovine serum, produced a coefficient of variation (CV) of 2.5%. Between-run precision, calculated using 10 determinations of the same control serum over a period of 1 month, produced a CV of 4.1%. Erythrocyte GSH-Px analysis

Study phase 2 The severe asthmatic patients either required high dose inhaled glucocorticoids or a low dose oral prednisolone rescue course to stabilize their condition. The former (n=16) were prescribed 750 lg beclomethasone dipropionate twice daily, by metered dose inhaler via a large volume spacer, and were seen again after 4 weeks. The latter (n=16) were prescribed oral prednisolone for 3 weeks, starting at 15 mg daily as a single morning dose during the first week, followed by 10 mg during the second week and 5 mg during the third and final week. These patients were seen again 2 weeks after commencing their prednisolone rescue course. During each visit, spirometry was carried out, and 5 ml of venous blood was collected in a sterile blood tube containing ethylene diamine tetraacetic acid (EDTA) as anticoagulant. Five of the 37 severe asthmatics did not turn up for the second visit and were therefore excluded from the study. Spirometry Pulmonary function testing was carried out on patients using a Flowmate electronic spirometer (Jaeger Instruments, Wuerzburg, Germany), and the standard procedures for a forced expiratory manoeuvre were followed. Results were expressed as a percentage of the predicted normal values for individuals of European descent, taking into consideration each person’s gender, age and height. All spirometric measurements, as well as blood sampling, were carried out between 0900 and 1100 h, in order to eliminate the influence of any diurnal variations. Selenium analysis Plasma selenium analysis was carried out by graphite furnace atomic absorption spectroscopy using the method of standard additions. The procedure was based on that published by Jacobson and Lockitch,16 with an optimized temperature program. The atomic absorption instrument employed was a Varian SpectrAA-300, incorporating graphite furnace atomizer, Zeeman background correction, and autosampler (all from Varian Australia Pty Ltd, Victoria, Australia), together with a Photron boosted discharge selenium

Erythrocyte GSH-Px activity was measured using the standard method of Sinet et al.17 This consisted of the classic Paglia and Valentine,18 coupling reaction method, with hydrogen peroxide being replaced by tert-butylhydroperoxide, and measurement carried out at 37°C instead of 20°C. The CV for within-run precision for erythrocyte GSH-Px activity determinations was 3.3%, while the CV for between-run precision was 5.5%. Erythrocyte SOD analysis Erythrocyte SOD activity was measured photometrically using the method described by Nebot et al,19 as developed and commercially produced by Bioxytech S.A., Bonneuil sur Marne, France. A CV of 4.6% was obtained for within-run precision, while the between-run precision gave a CV of 5.8%. All absorbance measurements were carried out using a GBC 918 double-beam UV/VIS spectrophotometer fitted with peltier-effect thermostatted cuvette holders (GBC Scientific Equipment Ltd, Victoria, Australia). Statistical analysis All data sets were subjected to normal probability plots to determine whether the data points were normally distributed. It was subsequently decided to use parametric methods to analyse all data. Data comparison between different groups was carried out using the Student’s t-test. Pre- and post-treatment statistical comparisons in the same patient groups were carried out using the paired-sample t-test. Correlation statistics between variables were performed by calculating the Pearson coefficient. All statistical calculations were performed with the aid of the PC-90 statistical package (BMDP Statistical Software, Cork, Ireland) for IBM compatible computers. RESULTS Study phase 1 Normal subjects The data for normal subjects is presented in Table 1. This group consisted of 30 adult males and 19 agematched females, all of whom were between 18 and


A. G. Fenech and R. Ellul-Micallef Table 1 Summary of antioxidant data obtained from normal subjects. Antioxidant parameter

Male (n=30)

Female (n=19)

Total (n=49)


Plasma selenium (ng/ml) RBC GSH-Px (U/gHb) RBC SOD (SOD 525 U/ml)

118.0 (6.6) 32.1 (1.1) 67.6 (1.4)

108.8 (6.2) 31.6 (1.3) 69.8 (1.7)

114.4 (4.7) 31.8 (0.9) 68.5 (1.1)


Values are presented as mean (SEM). NS=not significant.

Table 2 Summary of antioxidant defence and pulmonary function parameters, across all subject groups.

Antioxidant parameters Plasma Se (ng/ml) RBC GSH-Px (U/gHb) RBC SOD (SOD 525 U/ml) Pulmonary function parameters FEV1 (% of predicted) FVC (% of predicted) PEF (% of predicted)

Mild (n=22)

Severe (n=37)

Controls (n=49)

116.4 (4.8) 30.5 (2.2) 62.9 (2.9) (0.53–10.67)a

117.3 (3.2) 31.7 (1.7) 60.6 (1.9) (3.7–12.1)b

114.4 (4.7) 31.8 (0.9) 68.5 (1.1)∗∗

97.4 (2.6) 102.1 (2.8) 96.0 (2.9)

61.4 (2.2) 81.1 (3.0) 62.1 (2.0)

— — —

Values are presented as mean (SEM). ∗∗ P<0.01 when compared to mild asthmatics and also when compared to severe asthmatics. a 95% confidence interval for the mean differences (mild vs. controls). b 95% confidence interval for the mean differences (severe vs. controls).

49 years of age. The mean age for males was 31 (11) years while that for females was 30 (9) years (SEM). The Student’s t-test demonstrated no statistically significant gender-dependent differences in any variable measured within normal subjects, at the 0.05 level of significance. No correlation was found between any parameter and age, demonstrating a lack of agedependency, within this age bracket. The degree of erythrocyte GSH-Px activity failed to correlate with plasma selenium concentrations. Patients The data obtained for the patient groups is presented in Table 2. The mild asthmatic group consisted of 22 subjects (17 males and five females), while the severe asthmatics totalled 37 (16 males and 21 females). Both asthmatic groups were age-matched to the controls. The three categories showed no statistical differences for erythrocyte GSH-Px activity (mild asthmatics – 30.5 (2.2) U/gHb, severe asthmatics – 31.7 (1.7) U/ gHb, controls – 31.8 (0.9) U/gHb) or plasma selenium concentrations (mild asthmatics – 116.4 (4.8) ng/ml, severe asthmatics – 117.3 (3.2) ng/ml, controls – 114.4 (4.7) ng/ml). With respect to erythrocyte superoxide dismutase, however, the control group exhibited a significantly higher activity than did either asthmatic group (mild asthmatics – 62.9 (2.9) SOD 525 U/ml, severe asthmatics – 60.6 (1.9) SOD 525 U/ml, controls – 68.5 (1.1) SOD 525 U/ml; P<0.01). The comparison

of erythrocyte SOD data across the three subject groups is shown graphically in Fig. 1. Study phase 2 High-dose inhaled BDP treatment Paired-sample t-test analysis in 16 patients revealed no significant changes in any of the measured antioxidant parameters, following 4 weeks of inhaled beclomethasone dipropionate (750 lg twice daily) (Table 3). A clinical amelioration in symptoms in all patients was, however, evident, and this was reflected in statistically significant improvements in pulmonary function values. Low-dose oral prednisolone treatment Plasma selenium levels increased significantly from a mean of 118.3 (4.4) ng/ml to a mean of 138.1 (4.6) ng/ ml (SEM) (paired t-test, P<0.01), following 2 weeks of oral prednisolone treatment (Table 4, Fig. 2). No changes were observed in erythrocyte GSH-Px (preprednisolone – 31.9 (2.7) U/gHb, post-prednisolone – 33.7 (2.9) U/gHb) and SOD (preprednisolone – 59.3 (3.1) SOD 525 U/ml, post-prednisolone – 59.7 (2.7) SOD 525 U/ml) activities following the same treatment period. A clinical amelioration, which was reflected in significantly improved pulmonary function values, was observed in all patients (Table 4).

Antioxidant Status in Asthma: Effect of Glucocorticoids

Erythrocyte superoxide dismutase activity (SOD 525 U/ml)










Fig. 1 Distribution profiles of erythrocyte superoxide dismutase activities in mild asthmatics, severe asthmatics and normal subjects. The horizontal lines denote the means while the error bars denote the SEM. ∗∗ P<0.01.

DISCUSSION In healthy Maltese subjects, selenium was found to be present at a mean concentration of 114.4 (4.7) ng/ ml, with no age- or gender-dependent variations. This is higher than most published data reported from other countries.20–29 Since the element enters the food chain from soil stores, the locally measured plasma concentrations strongly suggest a high concentration of selenium in Maltese soil.

The lack of gender- or age-correlations of the element is in agreement with studies carried out in other countries.30,31 Age-dependent changes have only been reported at the extremes of age, in countries where the general population selenium status tends to be low.32,33 No relationship between selenium concentration and GSH-Px activity was observable within any of our subject groups. This is in contrast to results published by other workgroups.34–36 Our observations may be explained by the fact that whole blood GSHPx activity saturates at whole blood selenium levels exceeding 100 ng/ml.37 Since the plasma concentrations (which are lower than whole blood values) measured locally already exceed this threshold, they are unlikely to linearly correlate with locally measured activities of erythrocyte GSH-Px. The similar plasma concentrations of the element observed in Maltese patients and controls is in agreement with work reported by Flatt et al,28 as well as Hasselmark et al.34 Conversely, Stone et al,30 observed significantly lower plasma (P<0.01) and whole blood (P<0.001) concentrations in asthmatics, while Pearson et al,36 observed normal serum selenium concentrations in aspirin-sensitive asthmatics, but significantly higher concentrations in aspirin-tolerant patients. The mean plasma concentration observed in the Maltese control population is 21–88% higher than those reported in the previously mentioned studies. Any changes in selenium absorption, distribution or excretion which may occur in asthmatics, secondary to the disease, therefore appear unlikely to be observed in a population whose normal baseline concentration is relatively high. This study did not identify any variations in erythrocyte GSH-Px activity across the whole spectrum of patients and controls. The lack of gender- or agedependent variations of glutathione peroxidase is in agreement with previously published material.38,39 Indeed, age-dependent variations in enzyme activity

Table 3 Antioxidant defence parameters in patients suffering from severe bronchial asthma, before and after beclomethasone dipropionate administration (n=16). Before

Antioxidant parameters Plasma Se (ng/ml) 117.8 (5.9) RBC GSH-Px (U/gHb) 31.1 (2.3) RBC SOD (SOD 525 U/ml) 62.2 (2.4) Pulmonary function parameters 65.3 (3.7) FEV1 (% of predicted) PEF (% of predicted) 70.4 (4.1) FVC (% of predicted) 75.8 (3.9) Results are presented as mean (SEM). ∗∗∗ P<0.001.



95% confidence interval for the mean differences

111.8 (6.8) 30.6 (3.0) 60.3 (3.7)

— —

78.9 (4.0)∗∗∗ 86.8 (4.3)∗∗∗ 92.0 (3.5)∗∗∗

(8.1–19.1) (10.5–22.2) (8.2–24.3)

A. G. Fenech and R. Ellul-Micallef


Table 4 Antioxidant defence parameters in patients suffering from severe bronchial asthma, before and after prednisolone administration (n=16).

Antioxidant parameters Plasma Se (ng/ml) RBC GSH-Px (U/gHb) RBC SOD (SOD 525 U/ml) Pulmonary function parameters FEV1 (% of predicted) PEF (% of predicted) FVC (% of predicted)



95% confidence interval for the mean differences

118.3 (4.4) 31.9 (2.7) 59.3 (3.1)

138.1 (4.6)∗∗ 33.7 (2.9) 59.7 (2.7)

(5.05–34.5) —

61.1 (3.5) 62.8 (3.3) 86.1 (4.3)

73.4 (4.1)∗∗∗ 76.9 (5.8)∗ 97.6 (3.7)∗∗

(6.8–19.0) (2.7–25.7) (4.1–18.2)

Results are presented as mean (SEM). ∗ P<0.05; ∗∗ P<0.01; ∗∗∗ P<0.001.

Plasma selenium (ng/ml)


150 **



Before treatment

After treatment

Fig. 2 Changes in plasma selenium status following oral prednisolone administration. The error bars represent the SEM. ∗∗ P<0.01.

only appear to be evident in the elderly and in neonates.38,40–43 However, the lack of statistical difference between the erythrocyte GSH-Px activity observed in Maltese patients and controls contrasts with the results of studies carried out by other research workers.28,34,44,45 Unlike our reported mean plasma selenium concentration in healthy subjects [114.4 (4.7) ng/ml], mean plasma or serum concentrations in control subject groups participating in these published studies did not exceed 94 ng/ml (Table 2), while the concentrations in patients were either

equivalent or less. Any tendency towards a diseaseinduced decrease in GSH-Px activity in Maltese asthmatics might therefore have been masked by an already selenium-saturated enzyme activity. The results of this study demonstrate a lowered superoxide defence system in the blood of asthmatic patients, including those whose symptomatology appeared to be of a mild, easily controllable nature. This lowered erythrocyte SOD activity might contribute to a situation of increased oxidative stress. The cause underlying these levels cannot be established from this work; a decreased rate of synthesis or increased rate of inactivation of the enzyme may be envisaged. The latter might occur due to the presence of higher amounts of peroxide and superoxide. Such inactivation has indeed been shown to occur in vitro.46 The increase in plasma selenium after short-term low-dose oral prednisolone is in agreement with the results observed by Marano et al following treatment with high-dose prednisolone (40–100 mg daily).47 It may be reasonable to postulate that prednisolone may alter the pharmacokinetics of the element, either as a primary effect or via intermediate processes. Prednisolone is known to possess a small degree of mineralocorticoid effect, and besides retaining sodium it might also decrease the excretion rate of selenium. The failure of high dose beclomethasone dipropionate to exert a similar effect is probably a consequence of low systemic levels of the drug following administration by metered dose inhaler. Inhaled beclomethasone dipropionate is converted to the active metabolite 17-beclomethasone monopropionate by lung esterases. This is in turn rapidly metabolized to inactive products by the liver. This work suggests that Maltese asthmatics have adequate selenium-dependent enzymatic protection against H2O2 and R-OOH reactive species, but concurrently demonstrate a lower superoxide defence than healthy individuals. The plasma selenium-increasing effect of prednisolone is beneficial to individuals of

Antioxidant Status in Asthma: Effect of Glucocorticoids

low selenium status and GSH-Px activity. Such an action augments selenium-dependent antioxidant activity, thereby decreasing the degree of oxidant stress. Indeed, this may be another mechanism by which glucocorticoids exert their antiinflammatory actions.

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Date Received: 12 December. Date Revised: 23 September. Date Accepted: 2 October.