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Aspirin provocation increases 8-iso-PGE2 in exhaled breath condensate of aspirin-hypersensitive asthmatics
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Prostaglandins and Other Lipid Mediators
Aspirin provocation increases 8-iso-PGE2 in exhaled breath condensate of aspirin-hypersensitive asthmatics Lucyna Mastalerz a , Rafał Januszek a , Marek Kaszuba a , Krzysztof Wójcik a , Natalia Celejewska-Wójcik a , Anna Gielicz a , Hanna Plutecka a , Krzysztof Ole´s b , ˛ b , Marek Sanak a,∗ Paweł Strek a b
Department of Medicine, Jagiellonian University School of Medicine, Skawi´ nska 8, 31-066 Kraków, Poland ´niadeckich 2, 31-531 Kraków, Poland Department of Otolaryngology, Jagiellonian University School of Medicine, S
a r t i c l e
i n f o
Article history: Received 24 March 2015 Received in revised form 3 July 2015 Accepted 8 July 2015 Available online xxx Keywords: Aspirin exacerbated respiratory disease Aspirin challenge Exhaled breath condensate Isoprostanes Cysteinyl leukotrienes
a b s t r a c t Background: Isoprostanes are bioactive compounds formed by non-enzymatic oxidation of polyunsaturated fatty acids, mostly arachidonic, and markers of free radical generation during inflammation. In aspirin exacerbated respiratory disease (AERD), asthmatic symptoms are precipitated by ingestion of non-steroid anti-inflammatory drugs capable for pharmacologic inhibition of cyclooxygenase-1 isoenzyme. We investigated whether aspirin-provoked bronchoconstriction is accompanied by changes of isoprostanes in exhaled breath condensate (EBC). Methods: EBC was collected from 28 AERD subjects and 25 aspirin-tolerant asthmatics before and after inhalatory aspirin challenge. Concentrations of 8-iso-PGF2␣ , 8-iso-PGE2 , and prostaglandin E2 were measured using gas chromatography/mass spectrometry. Leukotriene E4 was measured by immunoassay in urine samples collected before and after the challenge. Results: Before the challenge, exhaled 8-iso-PGF2␣ , 8-iso-PGE2 , and PGE2 levels did not differ between the study groups. 8-iso-PGE2 level increased in AERD group only (p = 0.014) as a result of the aspirin challenge. Urinary LTE4 was elevated in AERD, both in baseline and post-challenge samples. Post-challenge airways 8-iso-PGE2 correlated positively with urinary LTE4 level (p = 0.046), whereas it correlated negatively with the provocative dose of aspirin (p = 0.027). Conclusion: A significant increase of exhaled 8-iso-PGE2 after inhalatory challenge with aspirin was selective and not present for the other isoprostane measured. This is a novel finding in AERD, suggesting that inhibition of cyclooxygenase may elicit 8-iso-PGE2 production in a specific mechanism, contributing to bronchoconstriction and systemic overproduction of cysteinyl leukotrienes. © 2015 Elsevier Inc. All rights reserved.
1. Introduction An analysis of exhaled breath condensate (EBC) is a simple and noninvasive technique for monitoring airway inflammation. Lipid mediators derived from arachidonic acid, prostanoids and leukotrienes, can be measured in EBC and used as biomarkers in diseases such as asthma [1]. Isoprostanes are formed mainly during non-enzymatic peroxidation of arachidonic acid (AA), independently of enzymatic cyclooxygenase (COX) activity. As a result of free radical attack, various isomers of different prostaglandins, leukotrienes, and epoxyeicosatrienoic acids are generated [2]. Among them, there is 8-iso-PGF2␣ , also called 8-epi-PGF2␣ , which has been validated
∗ Corresponding author. Fax: +48 12 430 52 03. E-mail address: [email protected] (M. Sanak).
as a biomarker of oxidative stress [3]. In contrast with prostanoids synthesized in a trans-conformation by stereoselective enzymes, isoprostanes have cis-juxtaposition of the alkene residues at the cyclopentane ring. Because isoprostanes are stereoisomers of prostaglandins, they share the same chemical formulas. 8-isoPGF2a was measured in biological fluids including plasma, urine, cerebrospinal fluid, or bronchial lavage [4,5]. Studies on 8-iso-PGE2 , a cis-isomer of the most abundant prostaglandin E2 (PGE2 ), are not numerous because no convenient assay method is available. To date 8-iso-PGE2 was detected in human biological samples and found to cause renal vasoconstriction and platelet activation [3]. Janssen et al. analyzed the effects of 8-iso-PGE2 on the lungs [6]. 8-isoPGE2 constricted pulmonary vessels and bronchia. It was suggested that 8-iso-PGE2 could be a ligand for thromboxane TP receptor, however, an affinity for PGE2 receptors EP was also postulated [3]. Asthma is a clinical entity encompassing many phenotypes, with bronchial inflammation and airway hyperreactivity as common
http://dx.doi.org/10.1016/j.prostaglandins.2015.07.001 1098-8823/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: L. Mastalerz, et al., Aspirin provocation increases 8-iso-PGE2 in exhaled breath condensate of aspirinhypersensitive asthmatics, Prostaglandins Other Lipid Mediat (2015), http://dx.doi.org/10.1016/j.prostaglandins.2015.07.001
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2 Table 1 Clinical characteristics of the study subjects.
Age, years Gender, males/females (%) Duration of asthma, years Skin prick tests, positive/negative (%) Total blood eosinophil count, /mm3 Total serum IgE, IU/mL FEV1 , % of predicted value Asthma severity, NAEPP-EPR3 Asthma control test score (ACT) ICS, yes/no (%) ICS dose, g fluticasone equivalent
AERD (n = 28)
ATA (n = 25)
p Value
46.14 ± 14.01 12/16 (42.9) 10 [5–15] 12/16 (42.9) 349 [149.25–574] 55 [25.45–120.5] 99.1 [84.44–100.0] 4 [3–4] 24 [22.8–25] 25/3 (89.3) 500 [362.5–1000]
43.8 ± 11.48 13/12 (52) 3 [1–13] 15/10 (60) 239 [128–479.75] 59 [22.9–276] 98 [85.92–103] 4 [3–4] 25 [23–25] 21/4 (84) 500 [250–1000]
N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
Values are expressed as counts, arithmetic mean ± SD, and median [lower–upper quartile]. AERD–aspirin-exacerbated respiratory disease, ATA–aspirin-tolerant asthma, ICS–inhaled corticosteroids, N.S.–not significant.
features. Inflammation of the airways leads to increased levels of reactive oxygen species and formation of a variety of isoprostanes in asthmatics [7,8]. Aspirin-exacerbated respiratory disease (AERD) is a welldefined phenotype of asthma. It is characterized by a syndromic association of chronic rhinosinusitis with persistent asthma and aspirin hypersensitivity [9]. Prevalence of AERD in the general population is estimated 0.5%, whereas 10–15% of asthmatics have the syndrome. Bronchoconstriction in AERD develops following an ingestion of aspirin or other non-steroidal anti-inflammatory drugs. This asthmatic attack is triggered by a pharmacological inhibition of COX-1 isoenzyme, and is followed by a release of potent pro-inflammatory lipid mediators, including cysteinyl leukotrienes (cys-LTs) [10–12]. Urinary leukotriene E4 (uLTE4 ) is the most reliable biochemical biomarker of AERD and estimates the systemic overproduction of cys-LTs, also elevated in clinically stable asthmatics. We previously reported on urinary metabolites of PGE2 : 13,14–dihydro-15keto-PGE2 and tetranor-PGE-M in AERD, contrasted with aspirin-tolerant asthmatics (ATA), after oral aspirin administration [13]. Despite a similar systemic production of both PGE2 metabolites at baseline, no decline in urinary PGE2 metabolites was found following COX-1 inhibition exclusively in AERD subjects during the bronchoconstriction. However, a similar challenge decreased urinary excretion of PGE2 metabolites in ATA subjects with negative outcome of the challenge. We realized that the analytical method for PGE2 metabolites quantification was insensitive to cis/trans conformation of prostanoids. Therefore, in the current study, we hypothesized that a free radical-mediated mechanism of prostanoid biosynthesis can contribute to the pathogenesis of aspirin hypersensitivity. We developed an assay based on gas-chromatography–mass-spectrometry enabling a separate quantification of PGE2 and 8-iso-PGE2 in biological fluids. In the current study, lysyl-acetylsalicylic acid (L-ASA) was administered by inhalatory challenge to provoke bronchoconstriction in hypersensitive AERD subjects. We analyzed EBC to avoid the background of abundant systemic biosynthesis of PGE2 . However, uLTE4 excretion was measured concurrently to evidence the well known mechanism of bronchoconstriction in AERD. Subjects with AERD or ATA, whose clinical condition was stable, were enrolled to this study. To the best of our knowledge, this is the first study in which 8-iso-PGE2 isoprostane is measured concurrently with PGE2 in asthma. 2. Methods 2.1. Subjects The study subjects comprised 28 AERD patients with a previously diagnosed hypersensitivity to aspirin using the oral challenge
test, and 25 asthmatics who tolerated aspirin well. The diagnosis and evaluation of the disease control complied with Global Initiative for Asthma 2012 update. Severity of asthma was defined using ATS/ERS 2013 recommendations and was evaluated using National Asthma Education and Prevention Program’s Expert Panel Report 3 guidelines [14]. Each subject was assigned the highest level of severity (intermittent, mild, moderate, or severe persistent) according to clinical features. Asthma Control Test was used to assess the disease control [15]. The clinical characteristics of the study patients are summarized in Table 1. None of the study participants experienced an asthma exacerbation or a respiratory tract infection within 6 weeks preceding the study. During the study period all the patients had stable asthma and their baseline FEV1 exceeded 70% the predicted value. The subjects were instructed to withhold any medication decreasing bronchial responsiveness prior to the challenge [16]. None of the patients was treated with systemic corticosteroids or leukotriene modifying drugs. All study participants gave their written informed consent and the study protocol was approved by the Jagiellonian University Ethical Committee.
2.2. Study design A single-blind, placebo-controlled bronchial challenge test with l-ASA (Kardegic, Sanofi Aventis) was carried out during one day in all participants of the study [16]. The test began with 7 inbreathes of saline. FEV1 was monitored at 10 and 20 min after the placebo inhalation. The post-saline FEV1 at 20 min was assumed as the baseline value. Incremental doses of l-ASA obtained by increasing the concentration of l-ASA or by changing number of breaths were inhaled every 30 min and equaled to 0.18, 0.36, 0.90, 2.34, 7.20, 16.2, 39.60, 115.20 mg. FEV1 was measured at 10, 20, and 30 min after each dose. The challenge was interrupted if a bronchospastic reaction occurred (i.e., FEV1 dropped by 20% or more), or the cumulative dose of l-ASA reached 181.98 mg. The cumulative dose of l-ASA causing 20% decrease in FEV1 was the provocation dose (PD20 ). FEV1 and extrabronchial symptoms were recorded before and immediately after the challenge, then every 30 min for the next 6 h. All the AERD subjects had EBC and urine samples collected before the challenge and at the time of the onset of bronchial symptoms. Additional samples of urine were collected 2 and 4 h after the challenge. In ATA group, EBC and urine samples were collected before the challenge and 30 min after the last dose of l-ASA, then additional urine samples were obtained as in AERD group.
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2.3. Lung function measurements Pulmonary function tests were measured using a flowintegrating computerized pneumotachograph (Pneumoscreen, Jaeger GmbH, Hoechberg, Germany). 2.4. Exhaled breath condensate (EBC) collection EBC was collected according to ATS/ERS guidelines using ECO Screen instrument (Jaeger GmbH) equipped with a saliva trap and using a nose clip [17]. During 20 min. of tidal breathing 1–2 mL of the condensate was obtained, then aliquoted on ice and immediately frozen in −70 ◦ C for further analysis. Samples were stored frozen until extraction, which was completed within 3 months after the collection. 2.5. Biochemical assays Prior to the extraction of prostanoids from EBC, chemically identical deuterated standards of PGE2 , 8-iso- PGE2 , 8-iso-PGF2␣ and 11-PGF2␣ (0.5 ng each, Cayman Chemical Co., Ann Arbor MI) were added. Before the measurements, a three step chemical derivatization method was used to block carboxylic, keto and hydroxyl groups of prostanoids by synthesis of penetafluorobenzyl esters, pentafluorobenzyl oximes and trimethylsilyl ethers. In brief, ethyl acetate (pH 3.5) extract of EBC (1 mL) was dried under nitrogen, next dissolved in 30 L of mixture 2,3,4,5,6-pentafluorobenzyl bromide (5% in acetonitrile) with di-isopropylethylamine (1:1 v/v) and incubated for 30 min at room temperature (RT). Next, after evaporation under nitrogen, the material was dissolved in methanol (20 L) and separated by thin-layer chromatography using silica TLC plates (Macherey–Nagel SILGUR-25C, 0.25 mm) (Dueren, Germany) and a mobile phase of ethyl acetate: n-heptane (9:1 v/v). Solvent migration distance was 13 cm. The only difference between PGE2 and 8-iso-PGE2 during preparatory phase was in chromatographic mobility of analytes. PGE2 migration distance was smaller (3.0–3.5 cm) than that of 8-iso-PGE2 (4.6–5.1 cm), whereas, 8-isoPGF2␣ migrated 1.5–2.5 cm. Ethyl acetate extract (1 mL) of scraped silica was dried under nitrogen and subsequently dissolved in 20 L O-(2,3,4,5,6-pentafluorobezyl) hydroxylamine hydrochloride (2% in pyridine) and incubated for 30 min at RT. In the last step of derivatization, dried under nitrogen material was added 20 L of mixture (N,O-bis(trimethylsilyl) trifluoroacetamide) with pyridine (1:1 v/v) for 30 min at RT, dried out and dissolved in 10 L of toluene for GC–MS injection. All reagents for these derivatization steps were from Sigma–Aldrich (St. Louis, MO). Measurements of prostanoids were performed using gas chromatography–mass spectrometry (GC–MS, model Engine 5989B series II Hewlett Packard, Palo Alto, CA). A 30 m long capillary column was used (Rtx-5MS 0.25 ID, Restek, Bellefonte, PA). The instrument was in a negative ion methane chemical ionization mode. Stability of PGE2 spiked to the sample and stored at 4 ◦ C for 5 consecutive days was confirmed by repeated measurements, which coefficient of variation was 2.6%. Details on 8-iso-PGF2␣ measurements and diagnostic ions were published elsewhere [1]. uLTE4 was measured using a commercial competitive LTE4 immunoassay (Cayman Chemical Co.) as published previously [18] with antibody/labeled tracer exhibiting an IC50 (50% B/B0) of 100 pg/mL and a detection limit (80% B/B0) of 25 pg/mL for LTE4 . Concentrations of the measured prostanoids were expressed in picograms per milliliter of EBC. uLTE4 was recalculated to picograms per milligram creatinine.
3
distribution. Normality of data was tested using Shapiro–Wilk test. Rank correlations were calculated using Spearman’s . Comparisons between the groups were done using nonparametric Mann–Whitney test or 2 test. The Wilcoxon signed-rank test was used for paired measurements. A statistical type I error p < 0.05 was considered significant. All calculations were done using STATISTICA software v. 10.0 (StatSoft Inc., Tulsa OK, USA). 3. Results 3.1. Clinical reactions Clinical characteristics of the study participants did not show significant differences between AERD and ATA groups (Table 1). None of the asthmatics developed symptoms after saline inhalations. In the AERD group, bronchial reactions occurred after l-ASA median dose 9.69 [4.2–56 mg]. All of the respiratory symptoms were relieved by short-acting 2 -agonists. None of the ATA patients developed any clinical symptoms following l-ASA inhalation. 3.2. 8-iso-PGE2 in EBC Following a derivatization with a thin-layer chromatography cleanup, 8-iso-PGE2 was clearly separated from PGE2 using gas chromatography. Both deuterated standards of 8-iso-PGE2 and PGE2 and the native compounds extracted from EBC migrated as two separate peaks because of syn/anti enantiomers of pentafluorobenzyl oxime (Fig. 1). The sum of the areas under these peaks was used for calculation of the prostanoid concentration from a proportion of the deuterated internal standard. Baseline EBC levels of 8-iso-PGE2 did not differ significantly between the study groups (p = 0.42). Following the challenge, EBC concentration of 8iso-PGE2 increased significantly only in AERD patients (from 2.27 [1.54–3.9 pg/ml] to 3.4 [2.11-5.16 pg/ml]; p = 0.014; Table 2). In aspirin-tolerant asthmatics, post challenge 8-iso-PGE2 remained at the same level as before aspirin inhalation therefore tended to be lower than in AERD group at the same time (p = 0.06; Fig. 2). A significant negative correlation was found between l-ASA PD20 and 8-iso-PGE2 level in EBC after aspirin challenge in AERD group ( = −0.435; p = 0.027 Fig. 3). 3.3. 8-iso-PGF2˛ in EBC Baseline EBC levels of 8-iso-PGF2␣ tended to be lower in AERD than in ATA (p = 0.065). Following l-ASA challenge, EBC concentration of 8-iso-PGF2 did not change, thus no difference was present between the study groups (p = 0.26) (Fig. 2). No correlation was found between 8-iso-PGF2␣ and other variables measured after the challenge in AERD group. 3.4. PGE2 in EBC Baseline PGE2 in EBC did not differ between the study groups. Following l-ASA challenge, no significant change in PGE2 concentration was observed in AERD or ATA subjects (Fig. 2). Administered dose of l-ASA had no effect on PGE2 concentration. A positive correlation was present between EBC concentration of 8-iso-PGE2 and PGE2 ( = 0.43; p = 0.023) after the challenge, but only in the AERD group. 3.5. Urinary excretion of LTE4
2.6. Statistical analysis Descriptive statistics was calculated as mean and standard deviation or median and interquartile range, depending on data
The median uLTE4 level was higher in all of the pre- or postchallenge urine samples of AERD than in ATA subjects (Table 3). At the onset of bronchial symptoms, AERD subjects demonstrated
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4. Discussion
Fig. 1. Panel A. Mass spectrograms of pseudomolecular negative ions showing paired peaks of PGE2 and 8-iso-PGE2 separation by gas-chromatography after chemical derivatization. Upper spectrogram of representative EBC sample, lower spectrogram of chemically identical internal deuterated (d4) standards added before sample extraction. Tandem peaks of PGE2 (due to pentafluoro-oxime syn/anti variants) are flanking tandem peaks of 8-iso-PGE2 . Numbers at peaks denote chromatography retention time in minutes. Panel B. Mass spectrograms of pseudomolecular negative ions showing a peak of 8-iso-PGF2␣ separation by gaschromatography after chemical derivatization. Upper spectrogram of representative EBC sample, lower spectrogram of chemically identical internal deuterated (d4) standards for 8-iso-PGF2␣ and 9␣,11-PGF2 which has similar retention time and identical ion size (M/z), added before sample extraction.
a decrease in median uLTE4 excretion (p = 0.036), whereas their uLTE4 returned to its baseline level 2 and 4 h after the test. In AERD group post-challenge 8-iso-PGE2 in EBC correlated positively with uLTE4 level measured at the onset of the symptoms ( = 0.42; p = 0.047). No changes or correlations were noted in uLTE4 in the consecutive urine samples of ATA.
In the current study we focused on differences between aspirin sensitive asthmatics and patients with asthma who tolerate aspirin well. The latter served as a control group. We performed GC–MS quantification of two isoprostanes and PGE2 in EBC samples collected from asthmatics before and after inhalatory challenge with l-ASA. We expected that reactive oxygen species released during acute bronchoconstriction in the AERD group might correlate with isoprostanes biosynthesis. Bioactive isoprostanes, synthesized by a non-enzymatic oxidation of polyunsaturated fatty acids, were proposed as biomarkers of chronic inflammatory lung diseases, e.g., COPD [19]. Both 8iso-PGE2 and 8-iso-PGF2␣ are formed in vivo mainly by a free radical-catalyzed peroxidation of arachidonate [2,3]. However, functional importance of isoprostanes in a disease is poorly understood. 8-iso-PGE2 was described in humans as released by human mononuclear cells in response to pro-inflammatory stimulation [5]. 8-iso-PGE2 has a bronchoconstrictory activity [6]. This also suggested that the mediator could participate in asthmatic inflammation. We analyzed EBC as a relevant and noninvasive biological material, suitable to study fast metabolized lipid mediators. For this purpose we developed a highly specific assay in which 8-iso-PGE2 could be clearly distinguished from its PGE2 stereoisomer. Our results showed that prior to l-ASA challenge, concentrations of 8-iso-PGE2 and 8-iso-PGF2␣ in EBC were similar across the study groups. Antczak et al. reported on elevated 8-iso-PGF2␣ concentration in EBC of steroid naïve AERD patients, but their asthma was more severe than in aspirin tolerant group [20]. In contrast, subjects from both of our studied groups had a good control of the disease. Increased levels of 8-iso-PGE2 were observed following the bronchial challenge only in the AERD group and correlated negatively with l-ASA provocative dose. This novel observation suggests that 8-iso-PGE2 may participate in the mechanism of bronchoconstriction provoked by aspirin in hypersensitive asthmatics. We are aware that the increase in 8-iso-PGE2 EBC concentration is modest, but it may reflect pathophysiologically relevant changes in the airways distinguishing AERD and ATA subjects. Since 8-isoPGE2 is capable of binding to EP4 [21] and TP receptors [22], this may actively promote AERD bronchoconstriction. Moreover, 8-isoPGE2 was to induce airway smooth muscle contractions 10–100 folds more potent than 8-iso-PGF2␣ [8]. Constrictive activity of 8iso-PGE2 was experimentally attenuated by antagonists of EP and TP receptors [22] but bronchial spasm was prevented only by the prostanoid TP receptor antagonist [6]. Aspirin can directly stimulate an intracellular oxidative burst in cultured mast cells of mice and enhance 8-iso-PGF2␣ release similarly to IgE mediated activation [23]. However, during l-ASA challenge we did not find any 8-iso-PGF2␣ response. Thus, during inhalatory l-ASA provocation, 8-iso-PGE2 is released without any particular activation of free radical oxidation, and the mechanism of its formation remains obscure. Cyclooxygenase-depended production of isoprostanes was also described in platelets [24] or monocytes [25]. This should be inhibited at the presence of l-ASA,
Table 2 Exhaled breath condensate concentration of prostanoids before and after inhalatory challenge with lysyl-aspirin (l-ASA). Aspirin-exacerbated respiratory disease (AERD, n = 28)
8-iso-PGE2 8-iso-PGF2␣ PGE2
Aspirin-tolerant asthma (ATA, n = 25)
Before
After
p Value
Before
After
p Value
2.27 [1.54–3.9] 0.28 [0.19–0.49] 1.89 [1.36–3.18]
3.4 [2.11–5.16] 0.25 [0.2–1.1] 2.17 [1.16–4.4]
0.014 N.S. N.S.
1.8 [1.13–4.2] 0.54 [0.35–1.65] 2.35 [1.58/3.68]
1.91 [1.17–3.97] 0.68 [0.37–1.06] 2.15 [1.28/3.34]
N.S. N.S. N.S.
Data is presented as median [lower–upper quartile] in pg/mL.
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Fig. 2. Eicosanoids changes following inhalatory challenge with lysyl-aspirin. Prostanoids level in exhaled breath condensate before and after bronchial aspirin challenge. Panel A: 8-iso-PGE2 ; panel B: PGE2 ; panel C: 8-iso-PGF2␣ . Panel D - urinary LTE4 (in pg per mg creatinine–decimal logarithmic scale) excretion before and after 2 or 4 h. Aspirin-exacerbated respiratory disease (AERD) subjects contrasted with aspirin-tolerant asthmatics (ATA).
but analysis of EBC in our study did not reveal any evidence for such an inhibition. The cellular source of 8-iso-PGE2 in EBC is not known, but we may suspect that enzymatically produced 8-iso-PGE2, measured in EBC, comes from the cells with expression of COX [24]. 8-iso-PGE2
detected in EBC may originate not only from epithelial cells (Supplementary Fig. S1) dominating in the airways but also from the cells located in alveoli such as pneumocytes and macrophages. The provocative dose of aspirin had an effect on EBC levels of 8-iso-PGE2 following the positive bronchial aspirin challenge. This
Please cite this article in press as: L. Mastalerz, et al., Aspirin provocation increases 8-iso-PGE2 in exhaled breath condensate of aspirinhypersensitive asthmatics, Prostaglandins Other Lipid Mediat (2015), http://dx.doi.org/10.1016/j.prostaglandins.2015.07.001
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Table 3 Urinary excretion of leukotriene E4 before and after inhalatory challenge with lysyl-aspirin (l-ASA).
Before At symptomsa 2 h later 4 h later
Aspirin-exacerbated respiratory disease (AERD, n = 28)
Aspirin-tolerant asthma (ATA, n = 25)
925 [447–2 699]** 534 [157–1 991]* 1 127 [123–2 330]** 698 [79–5 040]*
275 [34.3–534] 177 [22.3–455] 138 [36.8–353] 204 [40.0–411]
p value 0.036 N.S. N.S
p value N.S. N.S. N.S.
Data is presented as median [lower–upper quartile] in pg/mg creatinine, p-value for comparison with the baseline level before the challenge. * p < 0.05. ** p < 0.01 for between the group comparison. a Urine was sampled in aspirin-tolerant group 30 min after the last dose of aspirin.
Fig. 3. Correlation between lysyl-aspirin provocative dose causing FEV1 drop by 20% (l-ASA PD20) in aspirin-exacerbated respiratory disease asthmatics (AERD) during the bronchial challenge and post-challenge 8-iso-PGE2 concentration in exhaled breath condensate.
was a negative correlation and a lower provocation dose of aspirin resulted in a higher level of 8-iso-PGE2 in EBC. Thus, a hypersensitivity mechanism in which misconfigured 8-epi stereochemistry is linked with biosynthesis of PGE2 at presence of cyclooxygenase inhibitor is suggested. The results of our current study demonstrated a similar baseline EBC levels of PGE2 in both study groups. A similar finding was previously reported in urine [13], as well as in EBC [26] and bronchoalveolar lavage fluid (BALF) [27]. In contrast, Higashi et al. observed significantly lower baseline urinary PGE2 concentrations in AERD as compared to ATA [28]. According to the cyclooxygenase theory of AERD, a pharmacologic inhibition of PGE2 biosynthesis incites bronchoconstriction [10–12]. Decreased biosynthesis of PGE2 following non-steroidal anti-inflammatory drug challenge was documented using samples of BALF or induced sputum [27,29]. Our current results or previous studies with oral [30] and bronchial [31] aspirin challenge did not reveal any effect of aspirin on PGE2 measured in EBC. The plausible explanation for this discrepancy may have methodological nature and it can be due to relatively small fraction EBC originating from the conductive airways. Invasively collected BALF or induced sputum contains much more bronchial epithelial lining fluid (ELF), while EBC is obtained noninvasively and composed of ELF not only from the bronchial mucosa but also from a distal part of the respiratory system [32]. These discrepancies would require further studies using EBC collection enabling to separate condensate from the conductive versus diffusive airways. In our current study, we documented cysteinyl leukotrienes contribution to aspirin-triggered bronchoconstriction in AERD by measurements of uLTE4 excretion. This method was previously validated by ourselves and correlated satisfactorily with mass spectrometry measurement of leukotrienes [33]. Interestingly, we noticed correlation between pulmonary biosynthesis of 8-iso-PGE2 and uLTE4 excretion. However, we did not observe post-challenge increase of uLTE4 , described previously after the oral provocation [34,35]. Likewise, in our previous pilot study EBC did not
reflect increase of cys-LTs following aspirin provocation in AERD [36]. It is plausible, that narrowing of airways caused a decrease of non-volatile compounds content in EBC, blunting any changes accompanying aspirin provocation. Therefore, the current observation on a selective 8-iso-PGE2 increase in EBC offers an attractive hypothesis to explain the mechanism of asthmatic attack in the disease. In conclusion, we describe changes in EBC content of 8-isoPGE2 during a positive bronchial provocation test with aspirin. This isoprostane correlated negatively with l-ASA PD20 . No evidence for a free radical-mediated 8-iso-prostanoids was obtained by measurement of 8-iso-PGF2␣ . It may suggest enzymatic production of 8-iso-PGE2 , which might be involved in bronchoconstriction provoked by aspirin in AERD subjects. The cellular source of this prostanoid could be respiratory epithelium but this would require further studies. This remains unexplained, which is the main limitation of this study. Acknowledgements We thank Iwona Lipiarz for technical assistance. This work was supported by the grant UMO-2012/05/B/NZ6/01012 from the Polish Ministry of Science and the research grant from Switzerland through the Swiss Contribution to the enlarged European Union PSPB 072/2010. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prostaglandins. 2015.07.001 References [1] M. Sanak, A. Gielicz, K. Nagraba, M. Kaszuba, J. Kumik, A. Szczeklik, Targeted eicosanoids lipidomics of exhaled breath condensate in healthy subjects, J. Chromatogr. B 878 (2010) 1796–1800, http://dx.doi.org/10.1016/j.jchromb. 2010.05.012 [2] J.A. Lawson, J. Rokach, G.A. FitzGerald, Isoprostanes: formation, analysis and use as indices of lipid peroxidation in vivo, J. Biol. Chem. 274 (24) (1999) 441–444, http://www.ncbi.nlm.nih.gov/pubmed/10455102 (accessed 10.06.15). [3] J.D. Morrow, The isoprostanes-unique products of arachidonate peroxidation: their role as mediators of oxidant stress, Curr. Pharm. Des. 12 (2006) 895–902, http://www.ncbi.nlm.nih.gov/pubmed/16533158 (accessed 30.11.11). [4] J.A. Awad, J.D. Morrow, K. Takahashi, L.J. Roberts, Identification of non-cyclooxygenase-derived prostanoid (F2-isoprostane) metabolites in human urine and plasma, J. Biol. Chem. 268 (1993) 4161–4169, http://www. ncbi.nlm.nih.gov/pubmed/8440704 (accessed 10.06.15). [5] J.D. Morrow, J. Scruggs, Y. Chen, W.E. Zackert, L.J. Roberts, Evidence that the E2-isoprostane, 15-E2t-isoprostane (8-iso-prostaglandin E2) is formed in vivo, J. Lipid Res. 39 (1998) 1589–1593, http://www.ncbi.nlm.nih.gov/ pubmed/9717718 (accessed 30.11.11). [6] L.J. Janssen, M. Premji, S. Netherton, A. Catalli, G. Cox, S. Keshavjee, et al., Excitatory and inhibitory actions of isoprostanes in human and canine airway smooth muscle, J. Pharmacol. Exp. Ther. 295 (2000) 506–511, http://www. ncbi.nlm.nih.gov/pubmed/11046082 (accessed 30.11.11). [7] Y.S. Cho, H.-B. Moon, The role of oxidative stress in the pathogenesis of asthma, Allergy Asthma Immunol. Res. 2 (2010) 183–187, http://dx.doi.org/ 10.4168/aair.2010.2.3.183
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Prostaglandins and Other Lipid Mediators
Aspirin provocation increases 8-iso-PGE2 in exhaled breath condensate of aspirin-hypersensitive asthmatics Lucyna Mastalerz a , Rafał Januszek a , Marek Kaszuba a , Krzysztof Wójcik a , Natalia Celejewska-Wójcik a , Anna Gielicz a , Hanna Plutecka a , Krzysztof Ole´s b , ˛ b , Marek Sanak a,∗ Paweł Strek a b
Department of Medicine, Jagiellonian University School of Medicine, Skawi´ nska 8, 31-066 Kraków, Poland ´niadeckich 2, 31-531 Kraków, Poland Department of Otolaryngology, Jagiellonian University School of Medicine, S
a r t i c l e
i n f o
Article history: Received 24 March 2015 Received in revised form 3 July 2015 Accepted 8 July 2015 Available online xxx Keywords: Aspirin exacerbated respiratory disease Aspirin challenge Exhaled breath condensate Isoprostanes Cysteinyl leukotrienes
a b s t r a c t Background: Isoprostanes are bioactive compounds formed by non-enzymatic oxidation of polyunsaturated fatty acids, mostly arachidonic, and markers of free radical generation during inflammation. In aspirin exacerbated respiratory disease (AERD), asthmatic symptoms are precipitated by ingestion of non-steroid anti-inflammatory drugs capable for pharmacologic inhibition of cyclooxygenase-1 isoenzyme. We investigated whether aspirin-provoked bronchoconstriction is accompanied by changes of isoprostanes in exhaled breath condensate (EBC). Methods: EBC was collected from 28 AERD subjects and 25 aspirin-tolerant asthmatics before and after inhalatory aspirin challenge. Concentrations of 8-iso-PGF2␣ , 8-iso-PGE2 , and prostaglandin E2 were measured using gas chromatography/mass spectrometry. Leukotriene E4 was measured by immunoassay in urine samples collected before and after the challenge. Results: Before the challenge, exhaled 8-iso-PGF2␣ , 8-iso-PGE2 , and PGE2 levels did not differ between the study groups. 8-iso-PGE2 level increased in AERD group only (p = 0.014) as a result of the aspirin challenge. Urinary LTE4 was elevated in AERD, both in baseline and post-challenge samples. Post-challenge airways 8-iso-PGE2 correlated positively with urinary LTE4 level (p = 0.046), whereas it correlated negatively with the provocative dose of aspirin (p = 0.027). Conclusion: A significant increase of exhaled 8-iso-PGE2 after inhalatory challenge with aspirin was selective and not present for the other isoprostane measured. This is a novel finding in AERD, suggesting that inhibition of cyclooxygenase may elicit 8-iso-PGE2 production in a specific mechanism, contributing to bronchoconstriction and systemic overproduction of cysteinyl leukotrienes. © 2015 Elsevier Inc. All rights reserved.
1. Introduction An analysis of exhaled breath condensate (EBC) is a simple and noninvasive technique for monitoring airway inflammation. Lipid mediators derived from arachidonic acid, prostanoids and leukotrienes, can be measured in EBC and used as biomarkers in diseases such as asthma [1]. Isoprostanes are formed mainly during non-enzymatic peroxidation of arachidonic acid (AA), independently of enzymatic cyclooxygenase (COX) activity. As a result of free radical attack, various isomers of different prostaglandins, leukotrienes, and epoxyeicosatrienoic acids are generated [2]. Among them, there is 8-iso-PGF2␣ , also called 8-epi-PGF2␣ , which has been validated
∗ Corresponding author. Fax: +48 12 430 52 03. E-mail address: [email protected] (M. Sanak).
as a biomarker of oxidative stress [3]. In contrast with prostanoids synthesized in a trans-conformation by stereoselective enzymes, isoprostanes have cis-juxtaposition of the alkene residues at the cyclopentane ring. Because isoprostanes are stereoisomers of prostaglandins, they share the same chemical formulas. 8-isoPGF2a was measured in biological fluids including plasma, urine, cerebrospinal fluid, or bronchial lavage [4,5]. Studies on 8-iso-PGE2 , a cis-isomer of the most abundant prostaglandin E2 (PGE2 ), are not numerous because no convenient assay method is available. To date 8-iso-PGE2 was detected in human biological samples and found to cause renal vasoconstriction and platelet activation [3]. Janssen et al. analyzed the effects of 8-iso-PGE2 on the lungs [6]. 8-isoPGE2 constricted pulmonary vessels and bronchia. It was suggested that 8-iso-PGE2 could be a ligand for thromboxane TP receptor, however, an affinity for PGE2 receptors EP was also postulated [3]. Asthma is a clinical entity encompassing many phenotypes, with bronchial inflammation and airway hyperreactivity as common
http://dx.doi.org/10.1016/j.prostaglandins.2015.07.001 1098-8823/© 2015 Elsevier Inc. All rights reserved.
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2 Table 1 Clinical characteristics of the study subjects.
Age, years Gender, males/females (%) Duration of asthma, years Skin prick tests, positive/negative (%) Total blood eosinophil count, /mm3 Total serum IgE, IU/mL FEV1 , % of predicted value Asthma severity, NAEPP-EPR3 Asthma control test score (ACT) ICS, yes/no (%) ICS dose, g fluticasone equivalent
AERD (n = 28)
ATA (n = 25)
p Value
46.14 ± 14.01 12/16 (42.9) 10 [5–15] 12/16 (42.9) 349 [149.25–574] 55 [25.45–120.5] 99.1 [84.44–100.0] 4 [3–4] 24 [22.8–25] 25/3 (89.3) 500 [362.5–1000]
43.8 ± 11.48 13/12 (52) 3 [1–13] 15/10 (60) 239 [128–479.75] 59 [22.9–276] 98 [85.92–103] 4 [3–4] 25 [23–25] 21/4 (84) 500 [250–1000]
N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
Values are expressed as counts, arithmetic mean ± SD, and median [lower–upper quartile]. AERD–aspirin-exacerbated respiratory disease, ATA–aspirin-tolerant asthma, ICS–inhaled corticosteroids, N.S.–not significant.
features. Inflammation of the airways leads to increased levels of reactive oxygen species and formation of a variety of isoprostanes in asthmatics [7,8]. Aspirin-exacerbated respiratory disease (AERD) is a welldefined phenotype of asthma. It is characterized by a syndromic association of chronic rhinosinusitis with persistent asthma and aspirin hypersensitivity [9]. Prevalence of AERD in the general population is estimated 0.5%, whereas 10–15% of asthmatics have the syndrome. Bronchoconstriction in AERD develops following an ingestion of aspirin or other non-steroidal anti-inflammatory drugs. This asthmatic attack is triggered by a pharmacological inhibition of COX-1 isoenzyme, and is followed by a release of potent pro-inflammatory lipid mediators, including cysteinyl leukotrienes (cys-LTs) [10–12]. Urinary leukotriene E4 (uLTE4 ) is the most reliable biochemical biomarker of AERD and estimates the systemic overproduction of cys-LTs, also elevated in clinically stable asthmatics. We previously reported on urinary metabolites of PGE2 : 13,14–dihydro-15keto-PGE2 and tetranor-PGE-M in AERD, contrasted with aspirin-tolerant asthmatics (ATA), after oral aspirin administration [13]. Despite a similar systemic production of both PGE2 metabolites at baseline, no decline in urinary PGE2 metabolites was found following COX-1 inhibition exclusively in AERD subjects during the bronchoconstriction. However, a similar challenge decreased urinary excretion of PGE2 metabolites in ATA subjects with negative outcome of the challenge. We realized that the analytical method for PGE2 metabolites quantification was insensitive to cis/trans conformation of prostanoids. Therefore, in the current study, we hypothesized that a free radical-mediated mechanism of prostanoid biosynthesis can contribute to the pathogenesis of aspirin hypersensitivity. We developed an assay based on gas-chromatography–mass-spectrometry enabling a separate quantification of PGE2 and 8-iso-PGE2 in biological fluids. In the current study, lysyl-acetylsalicylic acid (L-ASA) was administered by inhalatory challenge to provoke bronchoconstriction in hypersensitive AERD subjects. We analyzed EBC to avoid the background of abundant systemic biosynthesis of PGE2 . However, uLTE4 excretion was measured concurrently to evidence the well known mechanism of bronchoconstriction in AERD. Subjects with AERD or ATA, whose clinical condition was stable, were enrolled to this study. To the best of our knowledge, this is the first study in which 8-iso-PGE2 isoprostane is measured concurrently with PGE2 in asthma. 2. Methods 2.1. Subjects The study subjects comprised 28 AERD patients with a previously diagnosed hypersensitivity to aspirin using the oral challenge
test, and 25 asthmatics who tolerated aspirin well. The diagnosis and evaluation of the disease control complied with Global Initiative for Asthma 2012 update. Severity of asthma was defined using ATS/ERS 2013 recommendations and was evaluated using National Asthma Education and Prevention Program’s Expert Panel Report 3 guidelines [14]. Each subject was assigned the highest level of severity (intermittent, mild, moderate, or severe persistent) according to clinical features. Asthma Control Test was used to assess the disease control [15]. The clinical characteristics of the study patients are summarized in Table 1. None of the study participants experienced an asthma exacerbation or a respiratory tract infection within 6 weeks preceding the study. During the study period all the patients had stable asthma and their baseline FEV1 exceeded 70% the predicted value. The subjects were instructed to withhold any medication decreasing bronchial responsiveness prior to the challenge [16]. None of the patients was treated with systemic corticosteroids or leukotriene modifying drugs. All study participants gave their written informed consent and the study protocol was approved by the Jagiellonian University Ethical Committee.
2.2. Study design A single-blind, placebo-controlled bronchial challenge test with l-ASA (Kardegic, Sanofi Aventis) was carried out during one day in all participants of the study [16]. The test began with 7 inbreathes of saline. FEV1 was monitored at 10 and 20 min after the placebo inhalation. The post-saline FEV1 at 20 min was assumed as the baseline value. Incremental doses of l-ASA obtained by increasing the concentration of l-ASA or by changing number of breaths were inhaled every 30 min and equaled to 0.18, 0.36, 0.90, 2.34, 7.20, 16.2, 39.60, 115.20 mg. FEV1 was measured at 10, 20, and 30 min after each dose. The challenge was interrupted if a bronchospastic reaction occurred (i.e., FEV1 dropped by 20% or more), or the cumulative dose of l-ASA reached 181.98 mg. The cumulative dose of l-ASA causing 20% decrease in FEV1 was the provocation dose (PD20 ). FEV1 and extrabronchial symptoms were recorded before and immediately after the challenge, then every 30 min for the next 6 h. All the AERD subjects had EBC and urine samples collected before the challenge and at the time of the onset of bronchial symptoms. Additional samples of urine were collected 2 and 4 h after the challenge. In ATA group, EBC and urine samples were collected before the challenge and 30 min after the last dose of l-ASA, then additional urine samples were obtained as in AERD group.
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2.3. Lung function measurements Pulmonary function tests were measured using a flowintegrating computerized pneumotachograph (Pneumoscreen, Jaeger GmbH, Hoechberg, Germany). 2.4. Exhaled breath condensate (EBC) collection EBC was collected according to ATS/ERS guidelines using ECO Screen instrument (Jaeger GmbH) equipped with a saliva trap and using a nose clip [17]. During 20 min. of tidal breathing 1–2 mL of the condensate was obtained, then aliquoted on ice and immediately frozen in −70 ◦ C for further analysis. Samples were stored frozen until extraction, which was completed within 3 months after the collection. 2.5. Biochemical assays Prior to the extraction of prostanoids from EBC, chemically identical deuterated standards of PGE2 , 8-iso- PGE2 , 8-iso-PGF2␣ and 11-PGF2␣ (0.5 ng each, Cayman Chemical Co., Ann Arbor MI) were added. Before the measurements, a three step chemical derivatization method was used to block carboxylic, keto and hydroxyl groups of prostanoids by synthesis of penetafluorobenzyl esters, pentafluorobenzyl oximes and trimethylsilyl ethers. In brief, ethyl acetate (pH 3.5) extract of EBC (1 mL) was dried under nitrogen, next dissolved in 30 L of mixture 2,3,4,5,6-pentafluorobenzyl bromide (5% in acetonitrile) with di-isopropylethylamine (1:1 v/v) and incubated for 30 min at room temperature (RT). Next, after evaporation under nitrogen, the material was dissolved in methanol (20 L) and separated by thin-layer chromatography using silica TLC plates (Macherey–Nagel SILGUR-25C, 0.25 mm) (Dueren, Germany) and a mobile phase of ethyl acetate: n-heptane (9:1 v/v). Solvent migration distance was 13 cm. The only difference between PGE2 and 8-iso-PGE2 during preparatory phase was in chromatographic mobility of analytes. PGE2 migration distance was smaller (3.0–3.5 cm) than that of 8-iso-PGE2 (4.6–5.1 cm), whereas, 8-isoPGF2␣ migrated 1.5–2.5 cm. Ethyl acetate extract (1 mL) of scraped silica was dried under nitrogen and subsequently dissolved in 20 L O-(2,3,4,5,6-pentafluorobezyl) hydroxylamine hydrochloride (2% in pyridine) and incubated for 30 min at RT. In the last step of derivatization, dried under nitrogen material was added 20 L of mixture (N,O-bis(trimethylsilyl) trifluoroacetamide) with pyridine (1:1 v/v) for 30 min at RT, dried out and dissolved in 10 L of toluene for GC–MS injection. All reagents for these derivatization steps were from Sigma–Aldrich (St. Louis, MO). Measurements of prostanoids were performed using gas chromatography–mass spectrometry (GC–MS, model Engine 5989B series II Hewlett Packard, Palo Alto, CA). A 30 m long capillary column was used (Rtx-5MS 0.25 ID, Restek, Bellefonte, PA). The instrument was in a negative ion methane chemical ionization mode. Stability of PGE2 spiked to the sample and stored at 4 ◦ C for 5 consecutive days was confirmed by repeated measurements, which coefficient of variation was 2.6%. Details on 8-iso-PGF2␣ measurements and diagnostic ions were published elsewhere [1]. uLTE4 was measured using a commercial competitive LTE4 immunoassay (Cayman Chemical Co.) as published previously [18] with antibody/labeled tracer exhibiting an IC50 (50% B/B0) of 100 pg/mL and a detection limit (80% B/B0) of 25 pg/mL for LTE4 . Concentrations of the measured prostanoids were expressed in picograms per milliliter of EBC. uLTE4 was recalculated to picograms per milligram creatinine.
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distribution. Normality of data was tested using Shapiro–Wilk test. Rank correlations were calculated using Spearman’s . Comparisons between the groups were done using nonparametric Mann–Whitney test or 2 test. The Wilcoxon signed-rank test was used for paired measurements. A statistical type I error p < 0.05 was considered significant. All calculations were done using STATISTICA software v. 10.0 (StatSoft Inc., Tulsa OK, USA). 3. Results 3.1. Clinical reactions Clinical characteristics of the study participants did not show significant differences between AERD and ATA groups (Table 1). None of the asthmatics developed symptoms after saline inhalations. In the AERD group, bronchial reactions occurred after l-ASA median dose 9.69 [4.2–56 mg]. All of the respiratory symptoms were relieved by short-acting 2 -agonists. None of the ATA patients developed any clinical symptoms following l-ASA inhalation. 3.2. 8-iso-PGE2 in EBC Following a derivatization with a thin-layer chromatography cleanup, 8-iso-PGE2 was clearly separated from PGE2 using gas chromatography. Both deuterated standards of 8-iso-PGE2 and PGE2 and the native compounds extracted from EBC migrated as two separate peaks because of syn/anti enantiomers of pentafluorobenzyl oxime (Fig. 1). The sum of the areas under these peaks was used for calculation of the prostanoid concentration from a proportion of the deuterated internal standard. Baseline EBC levels of 8-iso-PGE2 did not differ significantly between the study groups (p = 0.42). Following the challenge, EBC concentration of 8iso-PGE2 increased significantly only in AERD patients (from 2.27 [1.54–3.9 pg/ml] to 3.4 [2.11-5.16 pg/ml]; p = 0.014; Table 2). In aspirin-tolerant asthmatics, post challenge 8-iso-PGE2 remained at the same level as before aspirin inhalation therefore tended to be lower than in AERD group at the same time (p = 0.06; Fig. 2). A significant negative correlation was found between l-ASA PD20 and 8-iso-PGE2 level in EBC after aspirin challenge in AERD group ( = −0.435; p = 0.027 Fig. 3). 3.3. 8-iso-PGF2˛ in EBC Baseline EBC levels of 8-iso-PGF2␣ tended to be lower in AERD than in ATA (p = 0.065). Following l-ASA challenge, EBC concentration of 8-iso-PGF2 did not change, thus no difference was present between the study groups (p = 0.26) (Fig. 2). No correlation was found between 8-iso-PGF2␣ and other variables measured after the challenge in AERD group. 3.4. PGE2 in EBC Baseline PGE2 in EBC did not differ between the study groups. Following l-ASA challenge, no significant change in PGE2 concentration was observed in AERD or ATA subjects (Fig. 2). Administered dose of l-ASA had no effect on PGE2 concentration. A positive correlation was present between EBC concentration of 8-iso-PGE2 and PGE2 ( = 0.43; p = 0.023) after the challenge, but only in the AERD group. 3.5. Urinary excretion of LTE4
2.6. Statistical analysis Descriptive statistics was calculated as mean and standard deviation or median and interquartile range, depending on data
The median uLTE4 level was higher in all of the pre- or postchallenge urine samples of AERD than in ATA subjects (Table 3). At the onset of bronchial symptoms, AERD subjects demonstrated
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4. Discussion
Fig. 1. Panel A. Mass spectrograms of pseudomolecular negative ions showing paired peaks of PGE2 and 8-iso-PGE2 separation by gas-chromatography after chemical derivatization. Upper spectrogram of representative EBC sample, lower spectrogram of chemically identical internal deuterated (d4) standards added before sample extraction. Tandem peaks of PGE2 (due to pentafluoro-oxime syn/anti variants) are flanking tandem peaks of 8-iso-PGE2 . Numbers at peaks denote chromatography retention time in minutes. Panel B. Mass spectrograms of pseudomolecular negative ions showing a peak of 8-iso-PGF2␣ separation by gaschromatography after chemical derivatization. Upper spectrogram of representative EBC sample, lower spectrogram of chemically identical internal deuterated (d4) standards for 8-iso-PGF2␣ and 9␣,11-PGF2 which has similar retention time and identical ion size (M/z), added before sample extraction.
a decrease in median uLTE4 excretion (p = 0.036), whereas their uLTE4 returned to its baseline level 2 and 4 h after the test. In AERD group post-challenge 8-iso-PGE2 in EBC correlated positively with uLTE4 level measured at the onset of the symptoms ( = 0.42; p = 0.047). No changes or correlations were noted in uLTE4 in the consecutive urine samples of ATA.
In the current study we focused on differences between aspirin sensitive asthmatics and patients with asthma who tolerate aspirin well. The latter served as a control group. We performed GC–MS quantification of two isoprostanes and PGE2 in EBC samples collected from asthmatics before and after inhalatory challenge with l-ASA. We expected that reactive oxygen species released during acute bronchoconstriction in the AERD group might correlate with isoprostanes biosynthesis. Bioactive isoprostanes, synthesized by a non-enzymatic oxidation of polyunsaturated fatty acids, were proposed as biomarkers of chronic inflammatory lung diseases, e.g., COPD [19]. Both 8iso-PGE2 and 8-iso-PGF2␣ are formed in vivo mainly by a free radical-catalyzed peroxidation of arachidonate [2,3]. However, functional importance of isoprostanes in a disease is poorly understood. 8-iso-PGE2 was described in humans as released by human mononuclear cells in response to pro-inflammatory stimulation [5]. 8-iso-PGE2 has a bronchoconstrictory activity [6]. This also suggested that the mediator could participate in asthmatic inflammation. We analyzed EBC as a relevant and noninvasive biological material, suitable to study fast metabolized lipid mediators. For this purpose we developed a highly specific assay in which 8-iso-PGE2 could be clearly distinguished from its PGE2 stereoisomer. Our results showed that prior to l-ASA challenge, concentrations of 8-iso-PGE2 and 8-iso-PGF2␣ in EBC were similar across the study groups. Antczak et al. reported on elevated 8-iso-PGF2␣ concentration in EBC of steroid naïve AERD patients, but their asthma was more severe than in aspirin tolerant group [20]. In contrast, subjects from both of our studied groups had a good control of the disease. Increased levels of 8-iso-PGE2 were observed following the bronchial challenge only in the AERD group and correlated negatively with l-ASA provocative dose. This novel observation suggests that 8-iso-PGE2 may participate in the mechanism of bronchoconstriction provoked by aspirin in hypersensitive asthmatics. We are aware that the increase in 8-iso-PGE2 EBC concentration is modest, but it may reflect pathophysiologically relevant changes in the airways distinguishing AERD and ATA subjects. Since 8-isoPGE2 is capable of binding to EP4 [21] and TP receptors [22], this may actively promote AERD bronchoconstriction. Moreover, 8-isoPGE2 was to induce airway smooth muscle contractions 10–100 folds more potent than 8-iso-PGF2␣ [8]. Constrictive activity of 8iso-PGE2 was experimentally attenuated by antagonists of EP and TP receptors [22] but bronchial spasm was prevented only by the prostanoid TP receptor antagonist [6]. Aspirin can directly stimulate an intracellular oxidative burst in cultured mast cells of mice and enhance 8-iso-PGF2␣ release similarly to IgE mediated activation [23]. However, during l-ASA challenge we did not find any 8-iso-PGF2␣ response. Thus, during inhalatory l-ASA provocation, 8-iso-PGE2 is released without any particular activation of free radical oxidation, and the mechanism of its formation remains obscure. Cyclooxygenase-depended production of isoprostanes was also described in platelets [24] or monocytes [25]. This should be inhibited at the presence of l-ASA,
Table 2 Exhaled breath condensate concentration of prostanoids before and after inhalatory challenge with lysyl-aspirin (l-ASA). Aspirin-exacerbated respiratory disease (AERD, n = 28)
8-iso-PGE2 8-iso-PGF2␣ PGE2
Aspirin-tolerant asthma (ATA, n = 25)
Before
After
p Value
Before
After
p Value
2.27 [1.54–3.9] 0.28 [0.19–0.49] 1.89 [1.36–3.18]
3.4 [2.11–5.16] 0.25 [0.2–1.1] 2.17 [1.16–4.4]
0.014 N.S. N.S.
1.8 [1.13–4.2] 0.54 [0.35–1.65] 2.35 [1.58/3.68]
1.91 [1.17–3.97] 0.68 [0.37–1.06] 2.15 [1.28/3.34]
N.S. N.S. N.S.
Data is presented as median [lower–upper quartile] in pg/mL.
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Fig. 2. Eicosanoids changes following inhalatory challenge with lysyl-aspirin. Prostanoids level in exhaled breath condensate before and after bronchial aspirin challenge. Panel A: 8-iso-PGE2 ; panel B: PGE2 ; panel C: 8-iso-PGF2␣ . Panel D - urinary LTE4 (in pg per mg creatinine–decimal logarithmic scale) excretion before and after 2 or 4 h. Aspirin-exacerbated respiratory disease (AERD) subjects contrasted with aspirin-tolerant asthmatics (ATA).
but analysis of EBC in our study did not reveal any evidence for such an inhibition. The cellular source of 8-iso-PGE2 in EBC is not known, but we may suspect that enzymatically produced 8-iso-PGE2, measured in EBC, comes from the cells with expression of COX [24]. 8-iso-PGE2
detected in EBC may originate not only from epithelial cells (Supplementary Fig. S1) dominating in the airways but also from the cells located in alveoli such as pneumocytes and macrophages. The provocative dose of aspirin had an effect on EBC levels of 8-iso-PGE2 following the positive bronchial aspirin challenge. This
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Table 3 Urinary excretion of leukotriene E4 before and after inhalatory challenge with lysyl-aspirin (l-ASA).
Before At symptomsa 2 h later 4 h later
Aspirin-exacerbated respiratory disease (AERD, n = 28)
Aspirin-tolerant asthma (ATA, n = 25)
925 [447–2 699]** 534 [157–1 991]* 1 127 [123–2 330]** 698 [79–5 040]*
275 [34.3–534] 177 [22.3–455] 138 [36.8–353] 204 [40.0–411]
p value 0.036 N.S. N.S
p value N.S. N.S. N.S.
Data is presented as median [lower–upper quartile] in pg/mg creatinine, p-value for comparison with the baseline level before the challenge. * p < 0.05. ** p < 0.01 for between the group comparison. a Urine was sampled in aspirin-tolerant group 30 min after the last dose of aspirin.
Fig. 3. Correlation between lysyl-aspirin provocative dose causing FEV1 drop by 20% (l-ASA PD20) in aspirin-exacerbated respiratory disease asthmatics (AERD) during the bronchial challenge and post-challenge 8-iso-PGE2 concentration in exhaled breath condensate.
was a negative correlation and a lower provocation dose of aspirin resulted in a higher level of 8-iso-PGE2 in EBC. Thus, a hypersensitivity mechanism in which misconfigured 8-epi stereochemistry is linked with biosynthesis of PGE2 at presence of cyclooxygenase inhibitor is suggested. The results of our current study demonstrated a similar baseline EBC levels of PGE2 in both study groups. A similar finding was previously reported in urine [13], as well as in EBC [26] and bronchoalveolar lavage fluid (BALF) [27]. In contrast, Higashi et al. observed significantly lower baseline urinary PGE2 concentrations in AERD as compared to ATA [28]. According to the cyclooxygenase theory of AERD, a pharmacologic inhibition of PGE2 biosynthesis incites bronchoconstriction [10–12]. Decreased biosynthesis of PGE2 following non-steroidal anti-inflammatory drug challenge was documented using samples of BALF or induced sputum [27,29]. Our current results or previous studies with oral [30] and bronchial [31] aspirin challenge did not reveal any effect of aspirin on PGE2 measured in EBC. The plausible explanation for this discrepancy may have methodological nature and it can be due to relatively small fraction EBC originating from the conductive airways. Invasively collected BALF or induced sputum contains much more bronchial epithelial lining fluid (ELF), while EBC is obtained noninvasively and composed of ELF not only from the bronchial mucosa but also from a distal part of the respiratory system [32]. These discrepancies would require further studies using EBC collection enabling to separate condensate from the conductive versus diffusive airways. In our current study, we documented cysteinyl leukotrienes contribution to aspirin-triggered bronchoconstriction in AERD by measurements of uLTE4 excretion. This method was previously validated by ourselves and correlated satisfactorily with mass spectrometry measurement of leukotrienes [33]. Interestingly, we noticed correlation between pulmonary biosynthesis of 8-iso-PGE2 and uLTE4 excretion. However, we did not observe post-challenge increase of uLTE4 , described previously after the oral provocation [34,35]. Likewise, in our previous pilot study EBC did not
reflect increase of cys-LTs following aspirin provocation in AERD [36]. It is plausible, that narrowing of airways caused a decrease of non-volatile compounds content in EBC, blunting any changes accompanying aspirin provocation. Therefore, the current observation on a selective 8-iso-PGE2 increase in EBC offers an attractive hypothesis to explain the mechanism of asthmatic attack in the disease. In conclusion, we describe changes in EBC content of 8-isoPGE2 during a positive bronchial provocation test with aspirin. This isoprostane correlated negatively with l-ASA PD20 . No evidence for a free radical-mediated 8-iso-prostanoids was obtained by measurement of 8-iso-PGF2␣ . It may suggest enzymatic production of 8-iso-PGE2 , which might be involved in bronchoconstriction provoked by aspirin in AERD subjects. The cellular source of this prostanoid could be respiratory epithelium but this would require further studies. This remains unexplained, which is the main limitation of this study. Acknowledgements We thank Iwona Lipiarz for technical assistance. This work was supported by the grant UMO-2012/05/B/NZ6/01012 from the Polish Ministry of Science and the research grant from Switzerland through the Swiss Contribution to the enlarged European Union PSPB 072/2010. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prostaglandins. 2015.07.001 References [1] M. Sanak, A. Gielicz, K. Nagraba, M. Kaszuba, J. Kumik, A. Szczeklik, Targeted eicosanoids lipidomics of exhaled breath condensate in healthy subjects, J. Chromatogr. B 878 (2010) 1796–1800, http://dx.doi.org/10.1016/j.jchromb. 2010.05.012 [2] J.A. Lawson, J. Rokach, G.A. FitzGerald, Isoprostanes: formation, analysis and use as indices of lipid peroxidation in vivo, J. Biol. Chem. 274 (24) (1999) 441–444, http://www.ncbi.nlm.nih.gov/pubmed/10455102 (accessed 10.06.15). [3] J.D. Morrow, The isoprostanes-unique products of arachidonate peroxidation: their role as mediators of oxidant stress, Curr. Pharm. Des. 12 (2006) 895–902, http://www.ncbi.nlm.nih.gov/pubmed/16533158 (accessed 30.11.11). [4] J.A. Awad, J.D. Morrow, K. Takahashi, L.J. Roberts, Identification of non-cyclooxygenase-derived prostanoid (F2-isoprostane) metabolites in human urine and plasma, J. Biol. Chem. 268 (1993) 4161–4169, http://www. ncbi.nlm.nih.gov/pubmed/8440704 (accessed 10.06.15). [5] J.D. Morrow, J. Scruggs, Y. Chen, W.E. Zackert, L.J. Roberts, Evidence that the E2-isoprostane, 15-E2t-isoprostane (8-iso-prostaglandin E2) is formed in vivo, J. Lipid Res. 39 (1998) 1589–1593, http://www.ncbi.nlm.nih.gov/ pubmed/9717718 (accessed 30.11.11). [6] L.J. Janssen, M. Premji, S. Netherton, A. Catalli, G. Cox, S. Keshavjee, et al., Excitatory and inhibitory actions of isoprostanes in human and canine airway smooth muscle, J. Pharmacol. Exp. Ther. 295 (2000) 506–511, http://www. ncbi.nlm.nih.gov/pubmed/11046082 (accessed 30.11.11). [7] Y.S. Cho, H.-B. Moon, The role of oxidative stress in the pathogenesis of asthma, Allergy Asthma Immunol. Res. 2 (2010) 183–187, http://dx.doi.org/ 10.4168/aair.2010.2.3.183
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Please cite this article in press as: L. Mastalerz, et al., Aspirin provocation increases 8-iso-PGE2 in exhaled breath condensate of aspirinhypersensitive asthmatics, Prostaglandins Other Lipid Mediat (2015), http://dx.doi.org/10.1016/j.prostaglandins.2015.07.001