A metabolite of prostaglandin D , 11 β-prostaglandin F ( 11 β-PGF ) , in exhaled breath condensate and serum of asthmatics with airway hyperresponsiveness to distilled water

This study aims at identifying prostaglandin D (PGD ) involvement in osmotic airway hyperresponsiveness of asthmatics. PGD primary plasma metabolite, 11β-PGF , was analyzed in exhaled breath condensate (EBC) in response to ultrasonically nebulized distilled water (UNDW) and in serum in asthmatics with different airway response to the hypoosmotic stimulus. The total group of asthmatics (n=27) had a lower basal level of 11β-PGF (0.38±0.13 pg/ml, mean±SEM) in EBC compared to a group of healthy subjects (0.86±0.31 pg/ml, n=5), which 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 (≥10% FEV drop from baseline, n=14) had a lower concentration of the metabolite (0.28±0.14 pg/ml) as compared to the group without hyperresponsiveness (0.49±0.31 pg/ml, n=10). The 11β-PGF concentration decreased in the 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 . Serum content of 11β-PGF was ranging from 0 to 61 pg/ml in asthmatics (n=17) and from 7.3 to 85.4 pg/ml in healthy subjects (n=8). It was lower in the group with airway hyperresponsiveness to UNDW (8.4±1.7 pg/ml, n=9) than in the group without the hyperresponsiveness (21.0±8.8 pg/ml, n=8). The obtained results do not support the involvement of PGD 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 .


Introduction
Prostaglandin D 2 (PGD 2 ) is produced by mast cells and macrophages increasing in response to allergen exposure.In asthmatics, PGD 2 affects the airways by causing bronchoconstriction, vasodilation, increasing capillary permeability and mucous production 1 .
The role of the prostaglandin in asthmatics with osmotic airway hyperresponsiveness is ill-defined.PGD 2 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.The level of 11β-PGF 2α in EBC was below the announced detection limit (80% B/B 0 ) 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/B 0 vs. 11β-PGF 2α concentration.Obtained values of %B/B 0 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±0.09and 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±0.07pg/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 D 2 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 PGD 2 , apart from 11β-PGF 2α , have been reported 2 which may possess different physiological activities.

Conclusion
The obtained results do not support the involvement of PGD 2 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 PGD 2 .The critical point of this article is the detection limit of the ELISA KIT used.The concentration of metabolite measured in EBC was too much low.I suggest to concentrate the sample by evaporation under vacuum, or use an another KIT with a higher sensitivity.

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The number of healthy subjects from which it was collected EBC is too low.I suggest of collect others EBC samples from healthy subjects.
No competing interests were disclosed.

Competing Interests:
I have read this submission.I believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.The critical point of the article is that the EBC 11β-PGF levels were below the lower detection limit of the ELISA kit used in the study.Thus, the authors can simply not conclude on the results as they might have experienced changes and differences on variations of zero (distilled water)... To resolve this crucial error, I recommend concentrating the sample (i.e.vacuum evaporation), or 2α 1.

2.
resolve this crucial error, I recommend concentrating the sample (i.e.vacuum evaporation), or adding a known concentration of standard to each sample to overcome the detection limit.
The article is too short.Methodology of EBC collection is extremely variable, and to compare results with external findings, EBC methodology needs to be decribed in more detail.
No competing interests were disclosed.

Competing Interests:
I have read this submission.I believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com /doi.org/10.12688/f1000research.8084.1 09 Mar 2016, :307 ( Latest published: 5 ) https://doi.org/10.12688/f1000research.8084.1 /doi.org/10.5256/f1000research.8696.r20065© 2017 Santini G.This is an open access peer review report distributed under the terms of the Creative Commons , which permits unrestricted use, distribution, and reproduction in any medium, provided the original Attribution Licence work is properly cited.Giuseppe SantiniDepartment of Pharmacology, Faculty of Medicine, Catholic University of the Sacred Heart, Rome, Italy https://doi.org/10.5256/f1000research.8696.r17321© 2016 Bikov A. This is an open access peer review report distributed under the terms of the Creative Commons , which permits unrestricted use, distribution, and reproduction in any medium, provided the original Attribution Licence work is properly cited.Andras Bikov Department of Pulmonology, Semmelweis University, Budapest, Hungary I read the article with great interest.Lipid metabolites play a role in the pathophysiology of asthma and their changes during different stimuli are of interest.

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 http
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).Table1) and serum (Table2) samples.The values were produced with different EIA kits on different days.