Increase in inflammatory mediator concentrations in exhaled breath condensate after allergen inhalation

Article Outline

• Abstract
• Methods
  • Subjects
  • Allergen inhalation test
  • Histamine inhalation test
  • Collection of EBC samples
  • Quantification of EBC markers
    • CysLT quantification
    • PGD2 quantification
    • Histamine quantification
    • Tyrosine quantification
  • Urinary LTE4 and 9α, 11β-prostaglandin F2 quantification
  • EDN quantification
  • pH measurement
  • Exhaled nitric oxide measurement
  • Statistical analysis
• Results
  • Clinical characteristics of patients
  • Reproducibility of measuring CysLT concentration in EBC
  • Changes in CysLT concentrations in EBC and urinary LTE4 concentrations after allergen inhalation
  • Changes in PGD2 concentrations in EBC and urinary 9α, 11β-PGF2 concentrations after allergen inhalation
  • Changes in histamine concentrations in EBC
  • Concentrations of other biomarkers
  • Relationship between CysLT concentration in EBC and clinical parameters
• Discussion
• Acknowledgment
• Table E1. 
• References
• Copyright

Background

Although a number of studies have been carried out to examine the baseline concentrations of inflammatory mediators in asthmatic patients, the clinical utility of exhaled breath condensate (EBC) in allergen-induced bronchoconstriction has not yet been clarified.

Objective

We examined whether the release of inflammatory mediators can be detected in EBC after allergen-induced bronchoconstriction in asthmatic patients.

Methods

We quantified mast cell–associated mediators in EBC and their corresponding urinary metabolites before and after allergen inhalation.

Results

Early asthmatic responses (EARs) caused significant increases in the concentrations of cysteinyl leukotrienes (CysLTs; median, 10.4 vs 99.0 pg/mL; P < .0001) and prostaglandin D₂ (PGD₂; median, 2.26 vs 8.72 pg/mL; P = .0077), but not that of histamine, from baseline concentrations. Significant increases in the concentrations of urinary leukotriene E₄ and 9α, 11β-prostaglandin F₂ were detected in patients with EARs. However, the percentage increases in the concentrations of CysLTs and PGD₂ in EBC did not correlate with those of their corresponding urinary metabolites. The increases in concentrations of CysLTs and PGD₂ in EBC in patients with EARs correlated with each other and correlated with the extent of decrease in FEV₁. An insignificant difference in tyrosine concentration before and after the inhalation test demonstrated that errors caused by dilution of inflammatory mediators are negligibly small in EBC collected over a short period.

Conclusion

In patients with allergen-induced EARs, pulmonary generation of mast cell–associated mediators can be evaluated by quantifying CysLTs and PGD₂ in EBC, suggesting that the quantification of EBC mediators might be useful in monitoring acute asthmatic airway inflammation.

Key words: Early asthmatic responses, exhaled breath condensate, cysteinyl leukotrienes, prostaglandin D₂, histamine, inflammatory mediators, mast cells

Abbreviations used: BAL, Bronchoalveolar lavage, CysLT, Cysteinyl leukotriene, EAR, Early asthmatic response, EBC, Exhaled breath condensate, EIA, Enzyme immunoassay, LTE4, Leukotriene E₄, PGD₂, Prostaglandin D₂, PGD₂-MOX, PGD₂-methoxime, 9α, 11β-PGF₂, 9α, 11β-Prostaglandin F₂

In patients with atopic asthma, inhalation of a specific allergen can cause bronchoconstriction within minutes. The binding of an allergen to allergen-specific IgE on mast cells and basophils leads to degranulation and release of various inflammatory mediators, which in turn evoke the clinical symptoms. Allergen-induced changes in the concentrations of inflammatory mediators, such as histamine, cysteinyl leukotrienes (CysLTs), and prostaglandin D₂ (PGD₂), have been examined in various biologic samples, such as plasma,¹ bronchoalveolar lavage (BAL) fluid,² nasal washing fluid,³ induced sputum,⁴ and urine.⁵ There has been considerable uncertainty regarding the appearance of histamine in blood after allergen challenge because of difficulties in measuring histamine concentrations in human plasma.⁶ The results also suggest that the release of mediators is localized in the lungs, and the metabolism of released histamine is very rapid.⁷ BAL is an invasive procedure that is not suitable for routine clinical practice, and it is difficult to repeat at frequent intervals. Collection of induced sputum is relatively invasive because the technique involves inhalation of hypertonic saline, which induces an inflammatory response. Although urinary leukotriene E₄ (LTE₄) has been regarded as an indicator of systemic CysLT biosynthesis, the precise origin of LTC₄ production has not been established. On the other hand, collection of exhaled breath condensate (EBC) is a simple and noninvasive method of obtaining samples from the airways. This type of sample collection can be repeated in short intervals without side effects, and therefore it provides an opportunity to monitor the changes in the concentrations of inflammatory mediators in the airways.

The aim of this study was to evaluate the changes in inflammatory mediator concentrations in EBC during the development of early asthmatic responses (EARs) after allergen inhalation. We also measured the corresponding urinary metabolites of CysLTs and PGD₂ to examine how the concentrations of inflammatory mediators in EBC reflect those of their corresponding urinary metabolites.

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Methods 

Subjects 

Thirty-one patients with atopic asthma and mild clinical symptoms were enrolled in this study. They had positive results on a skin test and had positive results for the IgE antibody to Dermatophagoides pteronyssinus. They were recruited from among the outpatients of the Pulmonology-Allergy Department at the Sagamihara National Hospital. Patients without respiratory tract infections in the preceding 4 weeks or without exacerbation of asthma for at least 3 months were recruited for the study. The diagnosis of asthma was based on clinical history and examination, pulmonary function parameters, and response to β-agonists in accordance with international guidelines.⁸ All the patients were in stable condition, which was maintained by treatment with low constant doses of inhaled corticosteroids. Patients did not receive leukotriene receptor antagonists, systemic corticosteroids, or allergen immunotherapy. They were confirmed not to have aspirin-intolerant asthma. Permission to conduct this study was obtained from the Ethics Committee of Sagamihara National Hospital, and all the patients provided written informed consent. Sample size was calculated assuming a power of 0.8.

Allergen inhalation test 

At the beginning of the study, urine and EBC samples were collected, and a bronchial provocation test was started at about 9 am. All medications were withheld for at least 24 hours before the start of the study until the end of the study period. The patients inhaled a solution in a DeVilbiss nebulizer (DeVilbiss, Somerset, Pa) operated by air at a flow rate of 5 L/min. The nose was clipped, and aerosols were inhaled through a mouthpiece during tidal breathing. After the measurement of baseline FEV₁, patients inhaled an allergen diluent for 2 minutes. When a change in FEV₁ was not observed, allergen inhalation was performed. The diluted allergen extract (Dermatophagoides pteronyssinus) was inhaled for 2 minutes, and FEV₁ was measured 10 minutes after each inhalation. The starting concentration of the allergen extract for inhalation was determined from the threshold concentration in the skin test, and the inhalation test was stopped when an EAR occurred, which was defined as a decrease in FEV₁ of at least 20% from baseline within 20 minutes after allergen inhalation. Urine and EBC samples were collected immediately after the appearance of a positive reaction. The PD₂₀ caused by an EAR was determined by means of linear interpolation from the relationship between the cumulative dose of inhaled allergen and the percentage change in FEV₁.

Histamine inhalation test 

The histamine inhalation test was performed by using a method described by Chai⁹ with modification. The test was terminated when a decrease in FEV₁ by 20% of the baseline value occurred, and the PC₂₀ value was calculated.

Collection of EBC samples 

EBC samples were collected from the patients by using a condensing chamber (Ecoscreen, Jaeger, Germany) in accordance with American Thoracic Society/European Respiratory Society guidelines.¹⁰ The patients were instructed to breathe out tidally through a mouthpiece connected to the condenser for 15 minutes, and all the patients wore a nose clip to reduce nasal contamination. Approximately 2.0 to 3.0 mL of EBC was collected from the participants of this study, and it was not possible to obtain sufficient amounts of EBC samples for quantifying each mediator in each patient. CysLTs were extracted from EBC samples by using an Empore C18 cartridge (3M, St Paul, Minn) immediately after the collection, as described below, because CysLT concentrations tended to decrease after freezing and thawing. The remaining EBC samples were stored at −80°C until assay. When the amylase concentration was quantified with a commercially available kit (Wako Pure Chemical Industries Ltd, Osaka, Japan), the salivary amylase concentration was 20,246 Caraway units (median; range, 6159-69,727 Caraway units; n = 13) in healthy subjects, and amylase concentrations were less than the detection limit (approximately 60 Caraway units) in all EBC samples.

Quantification of EBC markers 

After an aliquot of an EBC sample (1 mL) was loaded on an Empore C18 disk cartridge, CysLTs were eluted with 0.5 mL of a 95% methanol solution. The methanol extract was concentrated to 0.05 mL under reduced pressure and then dissolved in 0.2 mL of assay buffer, which was supplied in a commercial enzyme immunoassay (EIA) kit (Cayman Chemical, Ann Arbor, Mich). CysLT concentration was determined by means of EIA.

After 0.05 mL of 1N HCl was added to an EBC sample (1 mL), the sample was applied to a Bond Elute C18 cartridge (100 mg, 1 mL), which had been preconditioned with methanol, followed by distilled water. The column was rinsed with 1 mL of distilled water. PGD₂ was then eluted with 2 mL of ethyl acetate. The ethyl acetate extract was evaporated under a nitrogen stream, and the residue was dissolved in 0.05 mL of assay buffer, which was supplied in the commercial EIA kit. The solution was incubated with methoxylamine hydrochloride and sodium acetate to convert the PGD₂-methoxime derivative (PGD₂-MOX). After the mixture was diluted with 5 volumes of the assay buffer, PGD₂-MOX concentration in the solution was measured with a PGD₂-MOX EIA kit (Cayman Chemical) in accordance with the manufacturer's instructions.

Histamine concentration in EBC was measured with an EIA (Immunotech/Beckman Coulter, Villepinte, France). Because histamine concentrations in most EBC samples were less than the detection limit (>0.06 ng/mL), histamine was quantified after lyophilization. Briefly, an aliquot of an EBC sample (1 mL) was acidified by adding 0.01 mL of 1N HCl, and then the sample was lyophilized. After the residue was dissolved in 0.1 mL of assay buffer, which was supplied in the commercial EIA kit, the solution was subjected to EIA in accordance with the manufacturer's instructions.

After 25 ng of ¹³C₆-tyrosine (l-tyrosine-ring ¹³C₆, ¹³C-99%; Cambridge Isotope Laboratories, Inc, Andover, Mass) was added to an EBC sample (0.1 mL) as an internal standard, the concentration of tyrosine was determined by means of gas chromatography–mass spectrometry in the negative ion chemical ionization mode, as reported previously.¹¹

Urinary LTE₄ and 9α, 11β-prostaglandin F₂ quantification 

Urine samples were collected within at least 1 hour after the test. The samples were collected in polypropylene bottles containing 4-hydroxy-TEMPO, free radical scavenger (Aldrich Chemical Co, Milwaukee, Wis), and aliquots were stored at −35°C until analysis. Urinary LTE₄ was quantified by means of EIA after purification with HPLC. Briefly, after urine (2 mL) was passed through an Empore C18 disk cartridge, LTE₄ was eluted from the cartridge with methanol. The eluate was injected into an HPLC column. HPLC was performed on a NOVA-PAK C18 column (Waters, Milford, Mass) with a solvent mixture of methanol–distilled water–acetic acid (65:35:0.1 vol/vol/vol) containing 0.1% EDTA (pH adjusted to 5.4 with ammonium hydroxide) at a flow rate of 1.0 mL/min at 37°C. The column effluent corresponding to the retention time of authentic LTE₄ (approximately 13.1 minutes) was collected, and the concentration of LTE₄ was subsequently determined by using an EIA kit (Cayman Chemical). As estimated from tritiated radioactivity, the overall recovery of LTE₄, including extraction with an Empore C18 cartridge and purification by means of HPLC, was about 70%, as reported previously.¹² After extraction with an Empore C18 cartridge, urinary 9α, 11β-prostaglandin F₂ (9α, 11β-PGF₂) was quantified with an EIA kit (Cayman Chemical), as reported previously.¹³ The recovery of 9α, 11β-PGF₂ after purification was approximately 60% to 70% when 40 to 200 pg of authentic standard was added to urine. Urinary LTE₄ and 9α, 11β-PGF₂ concentrations were normalized to urinary creatinine concentrations.

EDN quantification 

After addition of 0.2 mL of PBS containing 1% human serum albumin to an aliquot of an EBC sample (1 mL) to prevent adsorption on the tube, the EBC sample was lyophilized. The residue was dissolved in 0.1 mL of assay buffer, which was supplied in the commercial EIA kit, and the solution was subjected to EIA (MBL, Nagoya, Japan).

pH measurement 

pH was measured with a pH sensor probe for microsamples (FUTURA; Beckman Coulter, Inc, Fullerton, Calif) attached to a pH meter (Beckman Coulter, Inc).

Exhaled nitric oxide measurement 

Exhaled nitric oxide was collected and measured in accordance with the recommendations of the American Thoracic Society/European Respiratory Society at a flow rate of 50 mL/s (Sievers Instruments, Boulder, Colo).¹⁴

Statistical analysis 

Data were analyzed with SPSS for Windows, version 12.0 (SPSS, Inc, Chicago, Ill). Data are expressed as medians with ranges or means ± SD. Paired data were compared by using the Wilcoxon signed-rank test for calculation of significance of differences. In all other calculations, an unpaired t test or the Mann-Whitney test was used. When 2 groups were compared, Kruskal-Wallis ANOVA, followed by the Mann-Whitney test with the Bonferroni correction, or the Friedman repeated-measures ANOVA, followed by the Wilcoxon t test with the Bonferroni correction, was carried out. Correlation was evaluated by using the Spearman rank test. Differences were considered significant at a P value of less than .05.

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Results 

Clinical characteristics of patients 

The clinical characteristics of the patients enrolled in this study are shown in Table I. Statistical evaluation revealed no significant differences between the groups in baseline spirometric values, histamine PC₂₀ values, and total serum IgE levels.

Table I. Clinical characteristics of patients

Data are expressed as medians (ranges).

NO, Nitric oxide.

Although the baseline CysLT concentrations might suggest ongoing generation of LTC₄ in the airways, the baseline CysLT concentration did not correlate with the baseline spirometric and histamine PC₂₀ values. The level of serum allergen-specific IgE antibody in the patients with isolated EARs was significantly higher than that in the patients without EARs (P < .001). Allergen inhalation induced isolated EARs in 18 asthmatic patients, with an FEV₁ decrease of 38% (median) from baseline. On the other hand, 13 patients did not have EARs, even after inhalation of the allergen extract at the highest concentration.

Reproducibility of measuring CysLT concentration in EBC 

A control experiment was performed on 8 patients with mild asthma to establish the reproducibility of measuring CysLT concentrations at rest. EBC samples were collected for CysLT measurement from the patients twice, namely at 9 am and 1 pm at an interval of 4 hours. The CysLT concentrations in 2 consecutive samples were 7.4 ± 4.6 and 6.8 ± 2.2 pg/mL, and the ratio of CysLT concentration at 9 am to that after 4 hours was 1.1 ± 0.3. The results suggest that CysLT concentrations in EBC can be measured with good reproducibility and that CysLT concentrations did not change after an interval of 4 hours, during which the patients did undergo bronchial provocation testing.

Changes in CysLT concentrations in EBC and urinary LTE₄ concentrations after allergen inhalation 

Significant differences were not observed in CysLT concentrations between those measured after inhalation of saline and after inhalation of an allergen diluent (median of 8.9 pg/mL [range, 5.1-24.2 pg/mL] for the concentration after saline inhalation vs 9.6 pg/mL [range, 6.8-24.6 pg/mL] for the concentration after inhalation of allergen diluent; n = 8). The baseline CysLT concentration in EBC was not significantly different between the 2 groups (see Table E1 in this article's Online Repository at www.jacionline.org). CysLT concentrations in EBC significantly increased from the baseline concentration after allergen inhalation in the patients with EARs (P < .0001) but not in those without EARs (Fig 1, A). CysLT concentrations increased in all patients with EARs, as determined by calculating the increased percentage of CysLTs (median, 338% [range, 3% to 5390%]).

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• Fig 1. 

  Changes in CysLT concentrations in EBC (A), LTE₄ concentrations in urine (B), PGD₂ concentrations in EBC (C), and 9α, 11β-PGF₂ concentrations in urine (D) before and after allergen inhalation.

We examined the time course of increase in urinary LTE₄ excretion after bronchial provocation testing in detail. Urinary LTE₄ levels increased from a baseline concentration of 252.4 ± 257 pg/mg of creatinine (mean ± SD) to 7008 ± 4183 pg/mg of creatinine during the first hour and to 2155 ± 2866 pg/mg of creatinine between the 1- and 3-hour collection periods. The findings suggest that about 80.7% ± 22.9% of LTE₄ was excreted by 1 hour after allergen inhalation. Thus we collected urine samples within 1 hour after provocation. The concentrations of LTE₄ significantly increased in urine samples collected within 1 hour after allergen inhalation (P < .0001). The patients without EARs did not show increased urinary LTE₄ concentrations after allergen inhalation (Fig 1, B). After EAR induction, the percentage increases in CysLT concentrations in EBC did not correlate with those in urinary LTE₄ concentrations.

Changes in PGD₂ concentrations in EBC and urinary 9α, 11β-PGF₂ concentrations after allergen inhalation 

PGD₂ concentrations significantly increased in the patients with EARs after allergen inhalation (P = .0039, n = 9) but not in the patients without EARs (n = 7; Fig 1, C). An increased concentration of urinary 9α, 11β-PGF₂ was detected in the patients with EARs (P = .0016, n = 18; Fig 1, D). There was a significant correlation between increased concentrations of CysLTs and of PGD₂ in EBC collected in the patients with EARs after allergen inhalation (r = 0.88, P = .001, n = 9, Fig 2). However, the increase in urinary 9α, 11β-PGF₂ concentrations did not correlate with either the increase in PGD₂ concentrations in EBC or that in LTE₄ concentrations in urine.

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• Fig 2. 

  Correlation between increase in concentration of CysLT (abscissa) and that of PGD₂ (ordinate) in EBC samples from patients with EARs after allergen inhalation.

Changes in histamine concentrations in EBC 

A standard curve was constructed in the concentration range of 0.111 to 11.1 ng/mL in accordance with the manufacturer's instructions. When the concentration of histamine was less than the detection limit in EBC, the data were expressed as a half the concentration of the detection limit, which was 0.006 ng/mL, because EBC was concentrated 10-fold. Although histamine concentrations in EBC increased in 6 of 18 patients with EARs, this increase was not significant (n = 15), despite the finding that CysLT concentrations significantly increased in EBC (P = .0007, n = 15). Then we measured histamine concentrations in EBC samples collected from 8 patients who inhaled a cumulative dose of 0.234 to 19.9 mg of histamine dissolved in 2 to 8 mL of saline. In 2 patients histamine concentrations were less than the detection limit in EBC samples collected after histamine inhalation testing. Histamine concentrations increased from 0.1 to 0.15 ng/mL, from less than 0.1 to 1.12 ng/mL, and from 0.29 to 0.35 ng/mL in 3 patients, respectively. Histamine concentrations decreased in another 3 patients. The concentrations of CysLTs did not change in EBC after bronchoconstriction induced by histamine inhalation (17.1 pg/mL [range, 11.5-19.4 pg/mL] for the baseline concentration vs 15.7 pg/mL [range, 10.2-29.4 pg/mL] for the postinhalation concentration, n = 8).

Concentrations of other biomarkers 

There were no significant differences in pH or tyrosine concentrations between the patients with EARs and those without EARs, and pH and the tyrosine concentrations did not change after allergen inhalation (see Table E1). In addition, there was no significant change in EDN concentrations in both EBC and urine in 6 patients with EARs (EBC: 0.74 ng/mL [range, 0.65-1.12 ng/mL]; urine: 487 ng/mg of creatinine [range, 235-820 ng/mg of creatinine] for baseline concentration vs EBC: 0.75 ng/mL [range, 0.65-0.82 ng/mL]; urine: 512 ng/mg of creatinine [range, 227-761 ng/mg of creatinine] for postinhalation concentration), suggesting that eosinophils might not participate in the generation of CysLTs.

Because EBC was diluted at various degrees with water vapor, it might be difficult to directly compare inflammatory mediator concentrations between EBC samples. We used tyrosine concentration for correcting EBC dilution errors because tyrosine concentration can easily be measured in a small volume of a sample (0.1 mL of EBC), and there were significant correlations among the concentrations of tyrosine, urea, and total protein in BAL fluid (data not shown). Tyrosine concentrations in EBC remain constant before and after allergen inhalation. When we used tyrosine concentration in EBC for correcting dilution error, the ratio of CysLT concentration to tyrosine concentration in EBC significantly correlated with CysLT concentration in EBC (r = 0.87, P = .0003, n = 18).

Relationship between CysLT concentration in EBC and clinical parameters 

The percentage increase in CysLT concentration in EBC, which was calculated as the ratio of CysLT concentration after allergen inhalation to that at baseline, significantly correlated with RAST score (r = 0.57, P = .032), histamine PC₂₀ value (r = −0.52, p = .048), and the maximum decrease in FEV₁ (r = 0.707, P = .003; Fig 3) in the patients with EARs. There was also a significant correlation between allergen PD₂₀ and either CysLT concentration in EBC (r = −0.55, P = .042) or percentage increase in CysLT concentration in EBC (r = −0.59, p = .033). Similar to CysLT in EBC, the percentage increase in PGD₂ concentration in EBC significantly correlated with the 4 clinical parameters.

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• Fig 3. 

  In the patients with allergen-induced EARs, the decreased percentage in FEV₁ significantly correlated with the increase in CysLT concentration in EBC (A) but not with the increase in urinary LTE₄ concentration (B) after allergen inhalation.

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Discussion 

This is the first study showing that the concentrations of both CysLTs and PGD₂ are pronouncedly increased in EBC after allergen-induced EARs, which confirms that changes of inflammatory mediator concentration have been measured in EBC after allergen inhalation and that EBC provides a means of investigating local inflammatory processes in the airways without the need to undertake invasive procedures, such as bronchoscopy.

The novel emphasis here is that we were able to successfully measure these inflammatory mediators in EBC, a very dilute fluid, after concentration of EBC. The baseline concentration of mediators in EBC was very low, which is similar to previous reports. Therefore we considered that it is indispensable to purify and concentrate EBC for accurate measurement.

There are some issues in question. First, the concentration of CysLT in EBC has been directly quantified by means of EIA without purification or condensation. Because EBC consists mostly of distilled water, the composition of the assay buffer in the well for generating a standard curve might be different from that in the well containing EBC, which probably results in inaccurate quantification.

Second, because the mediator concentration in EBC is significantly low, it falls below the detection limit when EBC is quantified by means of EIA without condensation. These problems have been pointed out only recently.¹⁵, ¹⁶ Thus the usefulness of the quantification of the mediator concentration in EBC for understanding the clinical conditions has not yet been clarified.

We think that one way to address these problems is to examine whether the mediator generation accompanying airway contraction in the allergen inhalation test can indeed be assessed by means of the quantification of mediator concentration in EBC by purifying and concentrating EBC.

All the patients who participated in this study had isolated EARs. LTC₄ is released mostly from mast cells, basophils, and eosinophils, as well as from monocytes/macrophages and bronchial epithelial cells¹⁷ at lower concentrations. PGD₂ is reported to be produced from not only mast cells but also macrophages/monocytes,¹⁸ T cells,¹⁹ fibroblasts,²⁰ and skin Langerhans cells.²¹ Because the increased concentrations of CysLTs significantly correlated with those of PGD₂ in EBC (Fig 2) and it is generally accepted that asthmatic responses to an inhaled allergen result from IgE-mediated mast cell activation, these inflammatory mediators might be generated from mast cells through an IgE antibody–dependent mechanism. For all these, it might be possible that cells other than mast cells are activated either directly by an allergen or indirectly by mediators released from mast cells. Unexpectedly, we could not observe the increase in histamine concentration in EBC after allergen inhalation. It is reasonable to consider that histamine released by allergen stimulation has disappeared rapidly in the airways because we were not able to detect an increase in histamine concentration in EBC, even after inhalation of histamine solution. Little is known about the rate of clearance of histamine in the airways, and it has been reported that airway epithelial cells degrade histamine through 2 enzymes, namely histamine N-methyltransferase and histaminase.²², ²³, ²⁴ These findings indicate that the concentrations of CysLTs and PGD₂ in EBC might be more sensitive indicators of mast cell activation than the concentration of histamine.

Although EBC was diluted at various degrees with water vapor, the concentrations of inflammatory mediators in EBC have been compared between samples without knowing the degree of dilution. The marker used to correct the difference in the degree of dilution has not yet been established. Inflammatory mediators increase the permeability of vascular cells and bronchial epithelial cells, and therefore total protein concentration increases in lavage fluid immediately after endobronchial challenge with an allergen.² It is assumed that tyrosine concentration might increase concurrently with total protein concentration. However, tyrosine concentration did not change in EBC before and after allergen inhalation, and CysLT concentration did not correlate with tyrosine concentration in EBC. Therefore there might be no marked differences in both the generation of aerosol particles from airway lining fluid and dilution of aerosol particles by water vapor during the inhalation test, suggesting that it is possible to compare CysLT concentrations in the short term without correcting dilution errors, even after bronchoconstriction.

To date, there have been no studies evaluating the relationship between an increase in CysLT concentration in EBC and that in urinary LTE₄ concentration. After an intravenous administration of tritiated LTC₄ to human subjects or monkeys, tritiated LTE₄ was found to be a predominant metabolite in their urine, and about 5% of the total radioactivity was recovered as LTE₄; a substantial radioactivity was associated with more polar compounds.²⁵ Thus the concentration of urinary LTE₄ is associated with different metabolic rates in the liver and/or different kinetics of renal elimination of LTE₄ between individuals. Metabolic degradation of CysLTs can also occur in the airways. This might contribute to the lack of correlation between the increase in CysLT concentration in EBC and that in urinary LTE₄ concentration after allergen inhalation. Taken together, we consider that the possible clinical implication of EBC has been successfully demonstrated in the monitoring of acute allergic airway inflammation in this study. On the other hand, basal concentration of CysLT in EBC did not correlate with any data of pulmonary function tests. Thus the pathophysiologic significance of the baseline concentration remains to be elucidated.

In conclusion, our study indicated that in patients with allergen-induced EARs, pulmonary generation of mast cell–associated mediators can be evaluated by quantifying CysLTs and PGD₂ in EBC, suggesting that the quantification of EBC mediators might be useful in the monitoring of acute asthmatic airway inflammation.

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We thank Keiichi Kajiwara and Itsuko Ito for technical assistance.

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Table E1. 

Concentrations of biomarkers in EBC and urine before and after allergen inhalation

	Patients without EARs (n = 13)		Patients with EARs (n = 18)
	Baseline	After allergen inhalation	Baseline	After allergen inhalation
EBC
pH	6.24 (4.93-6.82, n = 13)	6.30 (5.47-6.83, n = 13)	6.33 (4.71-6.85, n = 18)	6.14 (5.26-6.85, n = 18)
CysLTs (pg/mL)	11.7 (5.1-32.3, n = 13)	10.4 (6.5-26.0, n = 13)	13.0 (5.4-151.5, n = 18)	99.0†‡§ (9.2-505.6, n = 18)
Tyrosine (ng/mL)	40.3 (14.0-119.3, n = 13)	44.7 (13.1-93.9, n = 13)	43.7 (18.8-470.0, n = 18)	38.5 (15.4-236.0, n = 18)
PGD2 (pg/mL)	1.67 (0.62-3.05, n = 7)	2.26 (1.22-3.74, n = 7)	0.93 (0.49-2.97, n = 9)	8.72∗‡§ (5.72-14.0, n = 9)
Histamine (ng/mL)	0.09 (0.06-1.40, n = 8)	0.12 (0.06-2.24, n = 8)	0.09 (0.03-2.55, n = 15)	0.11 (0.02-7.64, n = 15)
Urine
LTE4 (pg/mg cre)	164 (88-773, n = 13)	196 (100-818, n = 13)	183 (2-815, n = 18)	1020†‡§ (208-9949, n = 18)
9α,11β-PGF2 (pg/mg cre)	78 (13-293, n = 13)	79 (18-267, n = 13)	56 (9-280, n = 18)	97∗‡§ (5-3875, n = 18)

Data are expressed as medians [range, (n)].

cre, Creatinine.

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