Exhaled breath condensate (EBC) is a simple, noninvasive technique for monitoring airway inflammation. The measurement of eicosanoids in the expired breath condensate has proven to be a useful noninvasive method for the assessment and monitoring of airway inflammation in inflammatory diseases such as asthma and other pulmonary diseases [
Eicosanoids, including prostaglandins (PGs), thromboxane A2 (TXA2), and leukotrienes (LTs), are lipid mediators involved in the pathogenesis of asthma. TXA2 is rapidly converted to thromboxane B2 (TXB2), a chemically stable metabolite. Thus, thromboxane synthesis in biological tissues has been monitored by measuring TXB2 [
A special regulatory role of eicosanoids was postulated in aspirin-induced asthma (AIA) [
Aspirin-induced changes in the levels of eicosanoids, such as prostaglandins (PGs) and cys-LTs, have been examined in various biologic samples, such as plasma [
The aim of this study was to evaluate the changes in wide eicosanoid spectrum concentrations in EBC during asthmatic response following aspirin inhalation. We hypothesized that the profile of eicosanoids in EBC after local aspirin administrated is markedly different in aspirin-induced asthma patients as compared to asthmatics who tolerate aspirin well. These results were validated by specific analytical techniques, such as gas chromatography/mass spectrometry (GC-MS) or by high-performance liquid chromatography/mass spectrometry (HPLC-MS).
The study population consisted of 21 asthmatic patients sensitive to aspirin (AIA) and 23 asthmatics who tolerated aspirin well (ATA). The diagnosis of asthma was established according to GINA 2008 update. The patients’ characteristics are presented in Table
Clinical characteristics of the patients.
AIA ( | ATA ( | ||
---|---|---|---|
Age (y) | N.S. (0.33) | ||
Female/Male | 10/11 | 15/8 | N.S. (0.36) |
Duration of asthma (y) | N.S. (0.19) | ||
Inhaled steroids yes/no | 18/3 | 22/1 | N.S. (0.34) |
Inhaled steroids ( | N.S. (0.93) | ||
FEV1 baseline (% predicted) placebo/aspirin day | N.S. (0.57) | ||
Total IgE (IU/mL) | N.S. (0.78) | ||
Skin prick test ( | 10/9 | 15/8 | N.S. (0.53) |
Blood eosinophil count | 0.003 |
Values are expressed as mean ± SD, and median (25% and 75% percentiles).
AIA: aspirin-induced asthma.
ATA: aspirin-tolerant asthma.
The diagnosis of aspirin intolerance was confirmed by oral aspirin provocation tests, performed during 36 months preceding the study. All ATA patients occasionally used aspirin without any adverse reactions. The patients had stable asthma and their baseline FEV1 was >70% of the predicted value on the study day. None had experienced an exacerbation or a respiratory tract infection in the 6 weeks preceding the study. Nearly 70% of ATA patients had intermittent asthma, 13% mild persistent asthma, and 17% moderate persistent. In AIA group, 67% of patients had intermittent asthma, 19% mild persistent asthma, and 14% moderate persistent asthma. According to Asthma Control Test 33% of AIA patients and 26% of ATA patients had controlled asthma, and 33% and 48% had partly controlled asthma, 33% and 26% had uncontrolled asthma, respectively. In ATA group, 2 patients were current smokers and 3 exsmokers. There were 7 exsmokers and no current smokers in AIA group. The average level of FEV1 and FEV1/FVC in AIA patients was 89.9% and 73.2%, in ATA patients was 91.5% and 78.5%, respectively.
The subjects were instructed to withhold medications that decrease bronchial responsiveness prior to aspirin challenge. Short-acting
Baseline and following bronchial aspirin challenge, exhaled breath condensate eicosanoids (see Table
Eicosanoids values at baseline and following aspirin challenge in AIA and ATA patients. Results of eicosanoids were recalculated as parts per million (ppm) of palmitic acid (PA).
AIA ( | ATA ( | ||||
Baseline | Challenge | Baseline | Challenge | After the challenge | |
PGD2 | 0.54 | ||||
(parts/million of PA) | 3.18 | 3.69 | 4.27 | 3.85 | |
GC/MS | |||||
PGD2 | Unable | ||||
(parts/million of PA) | 3.56 | 3.00 | 2.99 | 3.33 | |
HPLC/MS/MS | |||||
9 | Unable | ||||
(parts/million of PA) | 1.24 | 1.30 | 0.61 | 0.75 | |
GC/MS | |||||
PGF2 | 0.82 | ||||
(parts/million of PA) | 0.97 | 0.86 | 0.64 | 0.49 | |
GC/MS | |||||
6-keto-PGF1 | 0.74 | ||||
(parts/million of PA) | 32.15 | 32.96 | 16.80 | 16.12 | |
GC/MS | |||||
6-keto-PGF1 | 0.11 | ||||
(parts/million of PA) | 32.08 | 32.51 | 15.60 | 18.15 | |
HPLC/MS/MS | |||||
11-dehydro TXB2 | 0.82 | ||||
(parts/million of PA) | 21.67 | 21.40 | 15.10 | 15.55 | |
GC/MS | |||||
11-dehydro TXB2 | 0.66 | ||||
(parts/million of PA) | 19.69 | 21.48 | 14.49 | 14.65 | |
HPLC/MS/MS | |||||
LTC4 | 0.003* | ||||
(parts/million of PA) | 9.22 | 6.61 | 5.40 | 3.24 | |
HPLC/MS/MS | |||||
LTD4 | 0.67 | ||||
(parts/million of PA) | 2.18 | 2.25 | 2.68 | 2.16 | |
HPLC/MS/MS | |||||
LTE4 | 0.03* | ||||
(parts/million of PA) | 5.19 | 6.38 | 4.88 | 6.54 | |
HPLC/MS/MS | |||||
Total cysLTs | 0.33 | ||||
(parts/million of PA) | 19.74 | 15.70 | 14.14 | 12.94 | |
HPLC/MS/MS | |||||
LTB4 | 0.03* | ||||
(parts/million of PA) | 70.28 | 50.49 | 49.72 | 32.29 | |
HPLC/MS/MS | |||||
5 HETE | Unable | ||||
(parts/million of PA) | 9.98 | 6.78 | 4.53 | 4.22 | |
HPLC/MS/MS | |||||
12 HETE | Unable | ||||
(parts/million of PA) | 12.49 | 6.62 | 7.93 | 5.23 | |
HPLC/MS/MS | |||||
15 HETE | 0.23 | ||||
(parts/million of PA) | 15.48 | 12.80 | 6.72 | 6.13 | |
HPLC/MS/MS | |||||
EXC4 | 0.79 | ||||
(parts/million of PA) | 2.09 | 2.59 | 1.41 | 1.61 | |
HPLC/MS/MS | |||||
EXD4 | 0.69 | ||||
(parts/million of PA) | 2.12 | 1.97 | 2.57 | 2.22 | |
HPLC/MS/MS | |||||
EXE4 | 0.95 | ||||
(parts/million of PA) | 7.06 | 3.82 | 3.79 | 3.65 | |
HPLC/MS/MS | |||||
8-iso-PGF2 | 0.09 | ||||
(parts/million of PA) | 0.68 | 0.76 | 0.67 | 0.73 | |
GC/MS |
Median (25% and 75% percentiles).
AIA: aspirin-induced asthma. ATA: aspirin-tolerant asthma. PA: palmitic acid.
*
* AIA versus ATA at baseline.
*baseline versus challenge in AIA.
*baseline versus challenge in ATA.
The single-blind, placebo-controlled bronchial challenge test with aspirin was carried out during one day in all study patients [
The consecutive doses of lysine-aspirin were inhaled every 30 minutes by increasing the concentration of lysine-aspirin and by changing the number of breaths (increasing doses of 0.18, 0.36, 0.90, 2.34, 7.20, 16.2, 39.60, 115.20 mg, at 0.5 hour intervals, up to the cumulative dose of 181.98 mg). FEV1 was measured at 10, 20, and 30 minutes after each dose. The challenge procedure with aspirin was interrupted, if a bronchospastic reaction occurred (FEV1 dropped ≥ 20%), or if the maximum cumulative dose of aspirin was reached. The cumulative dose of aspirin causing a 20% fall in FEV1 was calculated and recorded as PD20 (provocation dose of aspirin). FEV1 and extrabronchial symptoms were recorded at baseline, before the challenge tests, and then every 30 minutes until 6 hours after the last dose of aspirin.
In patients with positive bronchial aspirin challenge (AIA), exhaled breath condensate samples were collected for wide eicosanoid spectrum (see Table
Pulmonary function tests were performed on a flow-integrating computerized pneumotachograph (Pneumoscreen, E. Jaeger, Germany).
EBC was collected according to ATS/ERS [
Exhaled breath condensate concentration of eicosanoids was measured by gas chromatography/mass spectrometry (GC-MS) and by high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS2) or both; see Table
Summary statistics were expressed as mean (M), standard deviation (SD), median (Me), and 25% and 75% percentiles. General linear model (GLM) including repeated measures analysis of variance, which takes into account the fact that the outcome measurements are repeated over time within subject was used for multiple comparisons. Logarithmic transformation was used when needed as variance stabilizing transformation. Correlation between variables was estimated with the Spearman rank order correlations. A
There was no statistical difference in the clinical characteristics between patients with aspirin-induced asthma (positive aspirin challenge test), and those who tolerated aspirin well (negative aspirin challenge test) except for blood eosinophil count, Table
At baseline (Table
Following aspirin administration, no significant differences in EBC levels of PGD2 measured by HPLC/MS/MS (results were recalculated as ppm of PA) and 9
In cases when 11-dehydro TXB2 resulted as ppm of PA, at baseline (Table
Negative correlation was founded between provocation doses of aspirin and exhaled PGD2 and its 9
At baseline (Table
In both study groups, following aspirin challenge, EBC level of LTC4 decreased significantly (ANOVA,
The dose of inhaled steroid used by study patients and FEV1 values had no effect on magnitude of the response of the cys-LTs and its duration.
Negative correlation was founded between provocation doses of aspirin and exhaled LTC4 (
At baseline (Table
Following aspirin administration, EBC levels of LTB4 decreased significantly only in aspirin-sensitive patients (ANOVA,
At baseline (Table
At baseline (Table
The dose of inhaled steroid used by patients and FEV1 values had no effect on magnitude of the response of the 5- and 15-HETE. At baseline, negative correlation was found between the doses of steroids and EBC levels of 12-HETE only in aspirin-sensitive subjects (
The dose of aspirin, had no effect on the magnitude of response of 5- and 15-HETE.
Eicosanoids’ levels in exhaled breath condensate before and after bronchial aspirin challenge. (a) 5-HETE, (b) 15-HETE, and (c) 12-HETA. AIA: aspirin-induced asthma. ATA: aspirin-tolerant asthma. B: baseline. Ch: challenge.
At baseline and following aspirin challenge, exhaled breath condensate levels of eoxins C4, D4, and E4 did not differ significantly between the AIA and ATA groups (see Table
At baseline and following aspirin challenge no correlation was found between provocation doses of aspirin, inhaled steroid therapy and FEV1 values, and eoxins EBC levels in aspirin-sensitive and aspirin-intolerant patients.
In this study, we used a validated analytic platform [
Eicosanoids values at baseline and following aspirin challenge in AIA and ATA patients. Results of eicosanoids were recalculated as picograms per milliliter (pg/mL).
AIA ( | ATA ( | ||||
Baseline | After the challenge | Baseline | After the challenge | After the challenge | |
PGD2 | 0.35 | ||||
(pg/mL) | 1.01 | 1.21 | 1.64 | 1.53 | |
GC/MS | |||||
PGD2 | 0.05 | ||||
(pg/mL) | 1.39 | 1.09 | 1.48 | 1.45 | |
HPLC | |||||
9 | 0.59 | ||||
(pg/mL) | 0.36 | 0.34 | 0.29 | 0.28 | |
GC/MS | |||||
PGF2 | 0.82 | ||||
(pg/mL) | 0.28 | 0.27 | 0.30 | 0.23 | |
GC/MS | |||||
6-keto-PGF1 | 0.53 | ||||
(pg/mL) | 7.17 | 7.22 | 7.48 | 7.46 | |
GC/MS | |||||
6-keto-PGF1 | 0.60 | ||||
(pg/mL) | 7.26 | 6.96 | 7.40 | 7.65 | |
HPLC/MS/MS | |||||
11-dehydro TXB2 | 0.18 | ||||
(pg/mL) | 5.74 | 6.17 | 6.81 | 6.62 | |
GC/MS | |||||
11-dehydro TXB2 | 0.03* | ||||
(pg/mL) | 5.49 | 6.03 | 6.63 | 6.18 | |
HPLC/MS/MS | |||||
LTC4 | 0.01* | ||||
(pg/mL) | 2.30 | 2.02 | 2.46 | 1.06 | |
HPLC/MS/MS | |||||
LTD4 | 0.16 | ||||
(pg/mL) | 0.69 | 0.65 | 1.34 | 0.95 | |
HPLC/MS/MS | |||||
LTE4 | 0.04* | ||||
(pg/mL) | 1.83 | 2.33 | 1.93 | 2.45 | |
HPLC/MS/MS | |||||
Total cysLTs | 0.24 | ||||
(pg/mL) | 5.97 | 6.01 | 7.24 | 4.90 | |
HPLC/MS/MS | |||||
LTB4 | 0.02* | ||||
(pg/mL) | 29.66 | 16.99 | 27.73 | 15.35 | |
HPLC/MS/MS | |||||
5 HETE | 0.79 | ||||
(pg/mL) | 2.94 | 3.41 | 2.29 | 1.67 | |
HPLC/MS/MS | |||||
12 HETE | 0.02* | ||||
(pg/mL) | 4.05 | 2.82 | 3.10 | 1.93 | |
HPLC/MS/MS | |||||
15 HETE | 0.30 | ||||
(pg/mL) | 5.96 | 4.77 | 2.95 | 2.39 | |
HPLC/MS/MS | |||||
EXC4 | 0.74 | ||||
(pg/mL) | 0.79 | 0.59 | 0.80 | 0.61 | |
HPLC/MS/MS | |||||
EXD4 | 0.909 | ||||
(pg/mL) | 0.73 | 0.62 | 1.06 | 0.74 | |
HPLC/MS/MS | |||||
EXE4 | 0.07 | ||||
(pg/mL) | 2.15 | 1.05 | 1.59 | 1.35 | |
HPLC/MS/MS | |||||
8-iso-PGF2 | 0.92 | ||||
(pg/mL) | 0.20 | 0.21 | 0.35 | 0.34 | |
GC/MS |
Median (25% and 75% percentiles).
AIA: aspirin-induced asthma. ATA: aspirin-tolerant asthma.
*
* AIA versus ATA at baseline.
*baseline versus challenge in AIA.
*baseline versus challenge in ATA.
The aim of this study was to compare a wide profile of eicosanoids released to the epithelial surface of the asthmatic lung in subjects with and without aspirin hypersensitivity at baseline and following bronchial aspirin challenge, most of them on chronic inhaled steroid therapy. We demonstrated for the first time exhaled eicosanoids following local administration of aspirin in aspirin-induced asthma patients.
Comparing subjects with AIA and ATA no significant differences were observed in EBC levels of cyclooxygenase pathway prostanoid products (PGD2 and its metabolite 9
Our results revealed a significant upregulation of some arachidonate lipoxygenation products in asthmatic subjects with aspirin hypersensitivity, as manifested by high baseline levels of 5-, 15-HETE in EBC. This data is consistent with the latest observations [
No elevation of baseline 12-HETE in EBC comparing AIA and ATA subjects was observed. Whereas, after bronchial aspirin administration, there was a statistically significant decrease in 12-HETE concentration noted only in ATA subjects. On that basis, we assume blood platelets, the main source of 12-LO, may possibly play some role in pathogenesis of aspirin hypersensitivity. However, 12-LO was originally cloned from respiratory epithelia, where 15-LO activity was also found [
Our study did not demonstrated baseline local overproduction of cys-LTs in the airways in AIA and is consistent with an earlier study [
As was reported in childhood asthma [
The high-sensitivity eicosanoid profiling of lipoxygenation products (5HETE, 15HETE) in EBC makes it possible to detect alterations in asthma, especially in its distinct phenotype characterized by hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs. Cysteinyl-leukotriene levels in EBC after aspirin challenge in stable asthma patients, not steroid naive, most probably cannot be used as a reliable and sensitive index for aspirin hypersensitivity. In stable AIA patients on chronic inhaled steroid therapy of global (urinary) rather than in local (breath condensate) production of postchallenge cys-LTs is of greater and more sensitive value for aspirin hypersensitivity. We believe that quantitate cell analysis and measurements of released eicosanoides in induced-sputum will be more applicable for that purpose.
Aspirin-intolerant asthma
Aspirin-tolerant asthma
Exhaled breath condensate
Gas chromatography-mass spectrometry
High-performance liquid chromatography-tandem mass spectrometry
Cyclooxygenase
Prostaglandin
PGD2 semistable metabolite
Prostacyclin metabolite
Thromboxane A2 metabolite
Prostaglandin receptor 2
Lipoxygenase
Hydroxyeicosatetraenoic acid
Leukotriene
Cysteinyl leukotrienes
Isoprostane F2
Nonsteroidal anti-inflammatory drugs.
This work was supported by the Grant K/PBW/0000552 from the Polish Ministry of Science.