Targeted eicosanoids lipidomics of exhaled breath condensate in healthy subjects
Introduction
Breath, in several cultures, has been a primordial substance associated with origin of life, as well as life's essential manifestation. Yet in medicine, assessment of chemistry of breath has begun only recently. It came about well over 100 years after the atmospheric air was liquefied [1] and has been based on a similar principle of cooling breath into a liquid state. Thus, in commercial or custom-made devices, during tidal breathing, exhaled air is directed through the one-way inspiratory valve to a cooling trap, resulting in accumulation of a clear liquid. About 99% of it consists of water, but the remaining 1% is of utmost interest [2]. This tiny fraction is composed of both volatile and non-volatile compounds that include several biomarkers. Their origin is not clear. It is assumed that they are present in the liquid lining of the airway surface; they become aerolized and carried up during turbulent airflow.
Exhaled breath condensate (EBC) is of great interest to clinicians. Its collection is a completely non-invasive method of sampling of the respiratory tract that can be repeated several times. Collection devices are portable and can be used in a wide range of settings, including outpatient clinics, intensive care units, workplaces, and at home. The method offers a new insight into pathology of respiratory tract and holds promise for clinical utility. Still, unresolved questions remain, especially sensitivity of the assay techniques (ELISA in most studies) for many biomarkers, contributing to the reported variability [3]. This is particularly true for lipids, present in very low concentrations in EBC. Yet, their role as signaling and inflammatory molecules is being recognized.
Mass spectrometry occupies a leading position in the characterization, identification and quantitation of lipids. Its use has led to emergence of lipidomics, defined as the large-scale study of cellular lipids [4], [5], [6], [7] (i.e. the lipidome). Mass spectrometry was applied sporadically for measurement of single lipid mediators in EBC [8]. Here we present high performance liquid chromatography–tandem mass spectrometry (HPLC–MS2) and gas chromatography–mass spectrometry (GC–MS) techniques focused on the “targeted” lipidomic analysis of multiple derivatives of arachidonic acid.
Section snippets
EBC collection
In a pilot study we compared the results of cys-LTs determination in EBC obtained with two instruments: ECO Screen I and II, both products of Jeager (GmbH Hoechberg, Germany). The measurements were substantially lower with the use of the later than the former device. This was due to adsorption of eicosanoids to large surface of plastic bags present in ECO Screen II, but not I, as already noticed by Tufvesson [9]. Therefore, all further experiments were carried out with ECO Screen I. EBC was
Calculation
Concentration of measured compounds, quantified using a stable isotope dilution method, are presented as medians with the ranges from 25 to 75 centiles. Departure from normal distribution was tested using Shapiro–Wilk statistics. Correlation analyses were performed using Spearman's rank test. Between the groups comparisons were done using non-parametric Mann–Whitney test. Principal component analysis was done with the normally distributed concentrations of eicosanoids and palmitic acid. All
Results
Medians and inter-quartiles ranges of the measured compounds are presented in Table 4.
Statistical analysis of raw data distinguished the metabolites, with normal distribution in EBC samples: PA, LXA4, 11-dehydro-TXB2, 6-keto-PGF1α, and EXC4, while the remaining metabolites distribution suggested a greater inter-individual variation. The most abundant eicosanoids in EBC were tetranor-PGEM, 6-keto-PGF1α, LTB4 and 11-dehydro-TXB2. There was a highly significant positive correlation between PA and
Discussion
The aim of the current study was to develop an analytical approach based on mass spectrometry measurements of eicosanoids of healthy subjects in EBC. Due to the extremely low concentrations in EBC, close to detection threshold of immunoenzymatic assays (∼7.5 pg/mL), and a small sample volume, quantitative analysis of eicosanoids is very limited [9]. Fast non-invasive method of collection and ability to repeat sampling gives EBC an advantage over induced sputum or bronchoalveolar lavage.
Acknowledgments
This work was supported by the grants from EEA Financial Mechanism and from the Foundation for Advancement of Polish Pharmacy and Science.
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