A metabolite of prostaglandin D₂, 11β-prostaglandin F_2α (11β-PGF_2α), in exhaled breath condensate and serum of asthmatics with airway hyperresponsiveness to distilled water

Introduction

Prostaglandin D₂ (PGD₂) is produced by mast cells and macrophages increasing in response to allergen exposure. In asthmatics, PGD₂ affects the airways by causing bronchoconstriction, vasodilation, increasing capillary permeability and mucous production¹. The role of the prostaglandin in asthmatics with osmotic airway hyperresponsiveness is ill-defined. PGD₂ is an unstable compound rapidly metabolized with 11β-PGF_2α being its primary plasma metabolite. The aim of this study was estimation of 11β-PGF_2α in exhaled breath condensate (EBC) in response to ultrasonically nebulized distilled water (UNDW) and serum in asthmatics with different airway response to the hypoosmotic stimulus.

Methods

The study protocol was approved by the Biomedical Ethics Committee of the Far Eastern Scientific Center of Physiology and Pathology of Respiration (permit #91-1 of 12.01.2015). 39 patients with mild to moderate asthma and 13 healthy subjects participated in the study. The patients were recruited from the Center’s in-patient facilities or invited for follow-up checks. EBC was collected from 27 asthmatics and 5 healthy subjects before and after 3 min provocation with UNDW. Aerosol (the particle size range 0.5–10 μm, average particle size 3 μm) was generated by a Thomex L-2 ultrasonic nebulizer (Poland) operated at 4.5 ml/min. The hypoosmolar challenge test consisted of two consecutive inhalations for 3 min each. The first inhalation was with 30 ml of isotonic solution (0.9% NaCl), and the second with the same volume of distilled water. The temperature of the solution was maintained at 37.3°C. During the inhalation, a participant used a nose-clip and was breathing in a calm manner through a mouthpiece connected via a two-way valve to the container with a solution. Lung function testing was conducted with a spirometer Easy on-PC (ndd Medizintechnik AG, Switzerland) before the provocation test (baseline) and at 1 and 5 min of the recovery period. A drop in forced expiratory volume in 1 sec (ΔFEV1) of 10% or more from baseline after inhalation with UNDW was considered as airway hyperresponsiveness to the hypoosmotic stimulus. Patients who experienced airway responsiveness to isotonic solutions (n=3) were excluded from the test with UNDW, and they were not included in any group except the total group of asthmatics. Patients who experienced any airway responsiveness were given 2 doses of a selective β₂-adrenoceptor agonist to prevent bronchoconstriction. EBC was collected during tidal breathing with a condensing device ECoScreen 2 (Erich Jaeger, Germany). Serum was obtained from 17 patients and 8 healthy subjects before the provocation test. The collected samples were stored at -70°C until analysis. The concentration of 11β-PGF_2α was measured by 11β-prostaglandin F_2α EIA kit (No. 516521, Cayman Chemical Company, USA) in EBC after freeze-drying and in serum after purification by solid phase extraction on C18 cartridges (Strata C18-E, 55 μm, 70A, 500 mg/6 ml, Phenomenex, USA) as recommended by the manufacturer [Cayman’s kit booklet. URL: https://www.caymanchem.com/pdfs/516521.pdf]. The EIA kit utilizes 11β-PGF_2α-specific rabbit antiserum, and mouse anti-rabbit monoclonal antibody. Each sample was assayed in duplicate. The optical densities of the samples were used to calculate the concentrations of 11β-PGF_2α using an automated Excel spreadsheet [A. Swart 2012–2015. URL: http://www.rheumatologie-neuss.net/index_files/RheumatologieNeuss13.htm].

Analysis of the data was performed using standard methods of variational statistics. Statistical differences between groups were calculated by Student’s t test or by the nonparametric criteria of Mann-Whitney and Kolmogorov-Smirnov tests in the case of non-Gaussian distribution of variables (Statistica 8.0, StatSoft Inc., Tulsa, OK, USA, 2008).

Results

The level of 11β-PGF_2α in EBC was below the announced detection limit (80% B/B₀) of the kit (5.5 pg/ml). For the estimation of the 11β-PGF_2α level in EBC before and after the provocation test and comparison of the changes in the groups of healthy subjects and asthmatics with and without airway hyperresponsiveness to UNDW, calibration curves were built using the 11β-PGF_2α standard in the range of 0–25.6 pg/ml with additional dilutions of the standard down to 0.64 and 0.256 pg/ml and plotting %B/B₀ vs. 11β-PGF_2α concentration. Obtained values of %B/B₀ for the dilutions were different from 100% (Dataset 1, Table 1).

As a result, the calculated content of 11β-PGF_2α in EBC was in the range of 0–3.1 pg/ml (Table 1). The total group of asthmatics had a lower basal level of 11β-PGF_2α (0.38±0.13 pg/ml, mean±SEM) compared to the group of healthy controls (0.86±0.31 pg/ml), which further decreased following the UNDW challenge to 0.30±0.09 and 0.53±0.12, respectively. The group of asthmatics with airway hyperresponsiveness to UNDW was found to have a lower concentration of the metabolite (0.28±0.14 pg/ml) as compared to the group without the hyperresponsiveness (0.49±0.31 pg/ml). The 11β-PGF_2α concentration decreased in both groups after the challenge: 0.20±0.04 and 0.23±0.07 pg/ml in the groups with and without hyperresponsiveness to UNDW, respectively.

The content of 11β-PGF_2α in serum was higher ranging from 0 to 61 pg/ml in the total group of asthmatics and from 7.3 to 85.4 in healthy subjects (Table 1). Once again, it was lower on average in the total group of asthmatics than in the healthy subjects (14.3±4.3 vs. 33.7±12.1 pg/ml), and lower in the group with airway hyperresponsiveness to UNDW (8.4±1.7 pg/ml) than in the group without hyperresponsiveness (21.0±8.8 pg/ml). Due to the high variation of 11β-PGF_2α content in the subjects, all differences were statistically insignificant (p>0.05).

Since prostaglandin D₂ is considered to be a mast cell- and macrophage-specific eicosanoid, the lack of an increase in the concentration of its major metabolite 11β-PGF_2α found in the present study suggests a diminished role of these immune cells in the pathogenesis of the inflammatory reaction in asthma patients with osmotic airway hyperresponsiveness. However, the formation of different metabolites of PGD₂, apart from 11β-PGF_2α, have been reported² which may possess different physiological activities.

Conclusion

The obtained results do not support the involvement of PGD₂ in the pathophysiology of asthma with airway hyperresponsiveness to a hypoosmotic stimulus unless other conversions of the prostaglandin occur in the airway under these conditions with formation of metabolites different from 11β-PGF_2α. It would be interesting to investigate the level of other possible metabolites of PGD₂.

Data availability

F1000Research: Dataset 1. Data for calibration curves, 10.5256/f1000research.8084.d115417³

F1000Research: Dataset 2. 11β-PGF_2α content (pg/ml) in exhaled breath condensate (EBC) of individual subjects before and after provocation with ultrasonically nebulized distilled water and change in forced expiratory volume in 1 sec (% ΔFEV1 from baseline) after the provocation, 10.5256/f1000research.8084.d115418⁴

F1000Research: Dataset 3. 11β-PGF_2α content (pg/ml) in serum of individual subjects before provocation with ultrasonically nebulized distilled water and change in forced expiratory volume in 1 sec (% ΔFEV1 from baseline) after the provocation (if applicable), 10.5256/f1000research.8084.d115419⁵

Consent

The study protocol was approved by the Biomedical Ethics Committee of the Far Eastern Scientific Center of Physiology and Pathology of Respiration (permit #91-1 of 12.01.2015), and the participants gave written informed consent.