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Abstract

Background

Platelets are involved in the pathogenesis of aspirin-induced asthma (AIA). AIA patients suffer from an active disease despite avoidance of aspirin, and it has been suggested that administration of aspirin to these patients increases the generation of immediate oxygen products of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), in their platelets. 12-HPETE further activates the 5-lipoxygenase of leukotriene B4-producing inflammatory macrophages precipitating an acute asthmatic attack. Glutathione peroxidase (GPX) has the antioxidant capacity to reduce 12-HPETE, and thus modulate the arachidonic acid metabolic cascade. There is evidence that selenium (Se) nutrition can influence asthma but Se status in AIA patients has not received much attention.

Methods

We measured Se concentrations and GPX activities in platelets and plasma from 13 patients with AIA. Age- and sex-matched healthy individuals served as the control group.

Results

Patients with AIA had significantly higher median platelet Se concentration (102 ng/mg platelet protein) when compared with controls (49 ng/mg platelet protein, P = 0.003). Plasma Se concentrations in patients with AIA and controls were not significantly different (P = 0.59). Median platelet GPX activity was significantly higher in patients with AIA (102.7 mU/mg platelet protein) than in controls (66 mU/mg protein) (P = 0.05). The patient and control groups when combined showed weak, but significant correlation between platelet Se concentration and platelet GPX activity (r = 0.44; P = 0.03).

Conclusion

It is proposed that the higher platelet Se concentration observed in AIA patients contributed to the higher platelet GPX activity seen in these patients. Such an enhanced antioxidant defence system might represent an adaptive response to protect against increasing free radical production by inflammatory cells in AIA and help decelerate ongoing respiratory hypersensitivity.

Introduction

Asthma is a multifactorial disease process with genetic, allergic, environmental, infectious and nutritional components. It is generally accepted that inflammation of the airways is fundamental to the pathophysiology of asthma.

Aspirin and inhibitors of the cyclo-oxygenase pathway precipitate acute bronchospasm in about 10% of adult asthmatics, but the precise mechanism remains poorly understood. Patients with aspirin-induced asthma (AIA) usually suffer from active disease, despite avoidance of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs).

Platelets are involved in the pathogenesis of asthma, including AIA, and Taylor et al.¹ described abnormal release of oxygen-free radicals by platelets from AIA patients. The authors suggested the possible existence of an intrinsic-specific platelet abnormality, and that increased production of oxygen-free radicals might distinguish aspirin-tolerant from aspirin-intolerant subjects. Plaza et al.² showed that incubation of platelets with aspirin increased the production of oxygen-free radicals in aspirin-intolerant subjects. They demonstrated that platelets from AIA patients produced higher quantities of arachidonic acid metabolite at baseline conditions. Maclouf et al.³ demonstrated that 12-hydroperoxyeicosatetraenoic acid (12-HPETE), an immediate oxygenation product of arachidonic acid in platelets, stimulated increased generation of leukotriene B₄ (LTB₄) in mixed platelet–leukocyte suspensions obtained from patients with AIA. They suggested that the administration of aspirin to these patients leads to increased generation of 12-HPETE in their platelets because of an impaired balance of cyclo-oxygenase and 12-lipoxygenase or through inhibition of peroxidase activity in platelets. 12-HPETE could then activate a 5-lipoxygenase of LTB₄-producing circulating leukocytes and pulmonary macrophages, enhancing the production of LTB₄, which then precipitates bronchospasm. Reduction of 12-HPETE has the potential to modulate this metabolic cascade. Glutathione peroxidase (GPX), a selenium (Se)-dependent antioxidant enzyme, has such modulating capacity and it can reduce semi-stable hydroperoxides to less-reactive alcohols.^4–6 A significant positive correlation has been shown between the baseline GPX activity and percentage change in activity after Se supplementation.⁷ Therefore, cellular GPX activity is likely to be altered as a result of a change in Se status, and thus affect normal cellular antioxidant capacity.⁷

Se has been proposed to regulate inflammatory mediators in asthma and low Se status has been observed in patients with asthma.⁸ A protective role for dietary Se has been proposed⁹ because supplementation of A549-human airways epithelial cell culture with selenite resulted in an increased GPX activity as well as inhibiting the generation of hydrogen peroxide. Other mechanisms, however, have been suggested to explain the protective role of Se in asthma, including the regulation of nuclear factor-kappa B activity possibly by direct oxidation of critical sulfhydryl groups of this transcription factor.¹⁰ Therefore, a diet deficient in Se may lead to exacerbation of asthmatic inflammation, consequently contributing to bronchial hyper-reactivity.

In the UK population, blood Se content has declined approximately by 50% between 1974 and 1991 with intakes only half the reference nutrient intake of 1 μg/kg body weight. Plasma Se responds within one to three weeks to a change of Se status, and the response is influenced by the extent of depletion and form of Se supplementation.^11,12 A priority of Se uptake between organs has been observed in deficiency states and the response varies during repletion of Se with increases in tissue concentrations not reflected by simultaneous increases in plasma values. Therefore, plasma Se may be a poor indicator of overall Se nutritional status.

The dynamics of Se in different cells vary greatly as a result of differences in cell turnover and specific cell functions. Platelets contain high Se concentrations and about half of this Se originates from the irreversible incorporation by bone marrow precursor cells, which satisfies their need for Se even under conditions of Se deficiency.¹³ Platelets show a fairy rapid response to changes in Se intake because of their rapid turnover (8–14 days), and because of this, platelet Se is considered to be the best marker of total body Se content.

The aim of the present study is to compare Se concentration and GPX activity (as a functional index of Se status) in blood platelets and plasma from patients with AIA and normal controls. It was hoped to test the hypothesis that alteration of antioxidant status may play a role in the pathophysiology of AIA. The results are discussed in relation to the role of GPX as part of the body’s cellular defence against free oxygen production and also as a modulator of arachidonic acid metabolism.

Subjects and methods

Subjects

Thirteen otherwise unselected patients with AIA (three males and ten females, aged 23–68 years) attending the Allergy Clinic at the University Hospital of South Manchester were studied. All were currently non-smokers. The diagnosis of aspirin sensitivity was based on the documented firm history of bronchospastic reactions to aspirin. It was not considered ethically justifiable to confirm the diagnosis by provocation. All patients were in a stable clinical condition at the time of testing. None had a history of infection or relevant aspirin exposure for four weeks before blood sampling. The control group was composed of 13 age- and sex-matched apparently healthy subjects.

Platelet isolation

Platelets were obtained by centrifuging 18 mL of blood anticoagulated with 2 mL of sterile acid citrate dextrose [1:9 (v/v)] at 15–25°C, 150 × g for 15 minutes. This was followed by centrifugation of platelet-rich plasma at 15–25°C, 800 × g for 15 minutes. The platelets were then washed thrice in a solution of 12 mmol/L Tris buffer containing 135 mmol/L NaCl, 5 mmol/L glucose, 3 mmol/L Na₂EDTA, pH 7.4 and 4°C. Platelet lysates were obtained by resuspending the platelet pellets in a saturated solution of digitonin in 50 mmol/L Tris buffer containing 2 mmol/L Na₂EDTA and 10 mmol/L amino-n-caproic acid, pH 7.0 at 4°C for 1 hour. Supernatants were stored frozen at − 70°C.

Glutathione peroxidase assays

GPX activity in platelet, and in plasma was measured using the coupled assay of Paglia and Valentine¹⁴ as modified by Lavender et al.¹⁵ The volume of test sample was adjusted according to enzyme activity and incubated in a final volume of 1 mL containing 2 μmol glutathione, 1 unit of glutathione reductase and 103 μmol NADPH at pH 7.0 and 37°C. The reaction was initiated by adding 20 μL of 15 mmol/L t-butyl hydroperoxide, and the absorbance of the reaction at 340 nm was monitored for 4 minutes. One milliunit GPX activity was defined as the oxidation of 1 nmol of NADPH per minute and was related to the protein content of platelet lysate (or plasma) as determined by the Lowry method.¹⁶

Selenium measurement

Se was measured in plasma and platelet lysates using graphite furnace atomic absorption spectrometry with Zeeman background correction by a modification of the method described by Welz et al.¹⁷ The analyses were calibrated against Seronorm 103 lyophilized human reference serum (Nycomed, Oslo). Analyses were performed on coded specimens, without information concerning aspirin status or GPX activity.

Results

The median (ranges) Se concentrations and GPX activities are shown in Tables 1 and 2. The patients with AIA had a significantly higher median platelet GPX activity than did the healthy subjects (P = 0.05). The median plasma GPX activity was higher in the patients than the controls, but the difference did not reach significance (P = 0.08). Median platelet concentrations of Se were also higher in the patients than in the controls (P = 0.03). There was no significant correlation between either parameters measured in platelets and plasma.

Table 1

Median (range) glutathione peroxidase activities in platelets and plasma

	Platelets (mU/mg protein)	Plasma (mU/mg protein)
Patients with AIA	102.7* (17.9–250.3)	87.5 (67.7–116.5)
Controls	66.0 (15.6–145.3)	78.3 (23.4–99.3)

AIA, aspirin-induced asthma

*Mann-Whitney U test P = 0.05; P = 0.08

Table 2

Median (range) Selenium concentrations in platelets and plasma

	Platelets (ng/mg protein)	Plasma (μg/L protein)
Patients with AIA	102.0* (49.0–163.0)	96.0 (88.0–130.0)
Controls	49.0 (36.0–101.0)	103.0 (68.0–153.0)

AIA, aspirin-induced asthma

*Mann-Whitney U test P = 0.003; P = 0.59

Se concentration correlated significantly with GPX activity, only in platelets, for the comparison group (Spearman correlation 0.65; P = 0.02) and the two groups combined (Spearman correlation 0.44; P = 0.03).

Discussion

Prior to comparing Se concentrations in patients and controls it seemed appropriate to classify the Se status of the study population as described by Neve et al.¹² Given the wide range of Se intake, these authors proposed a classification of three categories of plasma Se concentration. Concentrations below 50–60 μg/L are designated as 'low', values above 100–120 μg/L are 'high', and those between these two ranges are 'intermediate'. However, this classification does not indicate 'inadequacy' or 'adequacy' of Se status.¹² Our results showed that the study population had plasma Se values that placed half in the 'intermediate' category and half in the 'high' category. These results are consistent with those estimated by Pearson et al.¹⁵ and the values were considered to be as expected for a population originating from the north-west of the UK.

This study is the only one to show that adult patients with AIA have higher platelet Se concentrations than healthy controls. Hasselmark et al.¹⁸ showed that the mean concentration of platelet Se in nine patients with NSAID intolerance tended to be lower than in the control group. In order to explain the differences in platelet GPX activity between AIA patients and controls, Pearson et al.¹⁹ measured plasma Se concentration in these subjects. No significant difference was observed and the authors gave no reason for the lower platelet GPX activity observed in the patient group. Stone et al.²⁰ measured Se concentration and GPX activity in plasma and platelets from 49 unselected asthma patients. Their patients had significantly lower plasma Se concentrations than in the controls. However, there was no difference between platelet Se concentration and GPX activity in the patients and the controls. Ward et al.²¹ showed that blood Se concentrations in treated asthmatic children from the north-east of Scotland were not significantly different from those of a control group, although whole blood GPX was significantly increased. Qujeq et al.²² assessed serum GPX activity and Se concentration in 46 asthmatic patients and 75 age- and sex-matched non-asthmatic subjects and found that the asthmatic patients had significantly lower serum concentrations of Se and GPX than those in controls. Current data indicate that plasma Se is not an accurate index of cellular Se content and consequently, it is not possible to rely on the plasma Se measurements to explain differences in observed cellular GPX activities.

A significant correlation was observed between platelet Se concentration and GPX activity. This phenomenon was not observed in plasma. Such a correlation between Se concentration and GPX activity has been demonstrated in Se-deficient states, in naturally or artificially Se-deficient animals, and following subsequent Se-supplementation.^23–25 Similar responses have been observed in humans.^26,27 In other studies, involving probable Se adequacy, inconsistent results have been reported. On the one hand, some studies^28–30 have failed to show a correlation between Se concentration and GPX activity, whereas others have suggested that a positive correlation does exist.^31–33

It is possible that the observed increase in platelet GPX activity in patients with AIA can potentially provide antioxidant protection against the greater amounts of reactive-oxygen species generated by platelets and may enhance the survival of these cells at the sites of inflammation.

The higher platelet GPX activity observed in patients with AIA may in part be explained by the higher platelet Se concentration observed in these patients. However, other mechanisms should also be considered; for example GPX activity may be up-regulated by inflammation. There are numerous reports of marked induction of the antioxidant enzyme systems copper–zinc–superoxide dismutase (CuZnSOD) and glutathione reductase during hyperoxia in animal models and tissue cultures.^34–37 Increased GPX activity may be an adapter mechanism to enhanced activities of other antioxidant enzymes. An increase in CuZnSOD is accompanied in several systems by an elevated activity of GPX.^35,38–40 Amstad et al.³⁸ studied the effects of the balance between CuZnSOD and GPX on cellular sensitivity to oxidants. They introduced a bovine GPX expression vector into cells that had been transfected with human CuZnSOD. They reported that moderate increases in GPX activity in the double transfectants compensated for the hypersensitivity caused by CuZnSOD over-expression. It is also possible that increased GPX activity might relate to over-expression of the GPX gene. Misso et al.³⁹ showed increased expression of GPX mRNA in eosinophils from AIA patients. Comhair et al.⁴⁰ reported an eight-fold increase in GPX mRNA in bronchial epithelial cells after exposure to reactive-oxygen species and glutathione, a cofactor for GPX. They found that alterations in intracellular and extracellular oxidized and reduced glutathione were temporarily associated with GPX induction, and thus supporting redox mechanisms in gene expression. The effect of steroids on GPX activity needs to be addressed because corticosteroid preparations have been reported to boost blood and plasma GPX activity in treated asthmatics,^41–44 and are likely to have similar effects on cellular GPX. Both gender and age appear to influence GPX activity, although the relative importance of these factors may differ in asthmatic and non-asthmatic populations. Misso et al.⁴⁵ supported the effect of age and sex on GPX activity. Their study included 41 asthmatics and revealed that mean whole blood GPX activity was higher in females and showed a positive correlation with age. Platelet GPX activity might also vary in relation to the severity of asthma. Picado et al.⁴⁶ demonstrated that platelet GPX activity was significantly lower in the most severe asthma patients. The patients in the current study were less-severe asthmatics stabilized on inhaled steroids for at least one-year prior to the study.

In conclusion, the findings of this study of a higher platelet (and plasma) GPX activity observed in our cohort of patients with AIA when compared with controls, support the hypothesis that this might represent an adaptive response to sustained oxidant stress. As plasma GPX is qualitatively different from the cellular enzyme^45,46 this implies that several regulatory mechanisms are involved in combating oxidant stress in asthma. Further studies are required to investigate the relationship between aspirin and concentration of GPX activity and Se concentrations before and after aspirin challenge and thereafter at specified time intervals, to demonstrate whether GPX is implicated in the development of bronchospasm. Experimental animal models may be the only acceptable approach to conduct such studies.

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Selenium status in patients with aspirin-induced asthma

Abstract

Background

Methods

Results

Conclusion

Introduction

Subjects and methods

Subjects

Platelet isolation

Glutathione peroxidase assays

Selenium measurement

Results

Discussion

References