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Apart from the refined management-oriented clinical stratification of chronic obstructive pulmonary disease (COPD), the molecular pathologies behind this highly prevalent disease have remained obscure. The aim of this study was the characterization of patients with COPD, based on the metabolomic profiling of peripheral blood and exhaled breath condensate (EBC) within the context of defined clinical and demographic variables. Mass-spectrometry-based targeted analysis of serum metabolites (mainly amino acids and lipid species), untargeted profiles of serum and EBC of patients with COPD of different clinical characteristics (n = 25) and control individuals (n = 21) were performed. From the combined clinical/demographic and metabolomics data, associations between clinical/demographic and metabolic parameters were searched and a de novo phenotyping for COPD was attempted. Adjoining the clinical parameters, sphingomyelins were the best to differentiate COPD patients from controls. Unsaturated fatty acid-containing lipids, ornithine metabolism and plasma protein composition-associated signals from the untargeted analysis differentiated the Global Initiative for COPD (GOLD) categories. Hierarchical clustering did not reveal a clinical-metabolomic stratification superior to the strata set by the GOLD consensus. We conclude that while metabolomics approaches are good for finding biomarkers and clarifying the mechanism of the disease, there are no distinct co-variate independent clinical-metabolic phenotypes.

E-cigarettes are widely believed to be safer than conventional cigarettes and have been even suggested as aids for smoking cessation. However, while reasonable with some regards, this judgment is not yet supported by adequate biomedical research data. Since bronchial epithelial cells are the immediate target of inhaled toxicants, we hypothesized that exposure to e-cigarettes may affect the metabolome of human bronchial epithelial cells (HBEC) and that the changes are, at least in part, induced by oxidant-driven mechanisms. Therefore, we evaluated the effect of e-cigarette liquid (ECL) on the metabolome of HBEC and examined the potency of antioxidants to protect the cells. We assessed the changes of the intracellular metabolome upon treatment with ECL in comparison of the effect of cigarette smoke condensate (CSC) with mass spectrometry and principal component analysis on air-liquid interface model of normal HBEC. Thereafter, we evaluated the capability of the novel antioxidant tetrapeptide O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) to attenuate the effect of ECL. ECL caused a significant shift in the metabolome that gradually gained its maximum by the 5th hour and receded by the 7th hour. A second alteration followed at the 13th hour. Treatment with CSC caused a significant initial shift already by the 1st hour. ECL, but not CSC, significantly increased the concentrations of arginine, histidine, and xanthine. ECL, in parallel with CSC, increased the content of adenosine diphosphate and decreased that of three lipid species from the phosphatidylcholine family. UPF1 partially counteracted the ECL-induced deviations, UPF1’s maximum effect occurred at the 5th hour. The data support our hypothesis that ECL profoundly alters the metabolome of HBEC in a manner, which is comparable and partially overlapping with the effect of CSC. Hence, our results do not support the concept of harmlessness of e-cigarettes.

Metabolomics approach that reflects most proximately the status of the airway cells has been proven valuable for determining the phenotypes of COPD. We phenotyped patients with stable COPD by integrating metabolomic biomarkers from exhaled breath condensate and from peripheral blood with clinical/demographic variables. Liquid chromatography/mass spectrometry-based profiles of low molecular weight metabolites, expressed as sets of consecutive mass-to-charge (m/z) ratios, with clinical data (lung function, mMRC scores, major concomitant diseases, exacerbation data and COPD severity scores) from patients with stable COPD were subjected to principal component analysis-based hierarchical clustering. The signals that contributed most for the clustering were selected with parse PLS discriminant analysis. Patients of all COPD stages were included (n=25), 67 (42-78) years of age, with 40 (0-84) pack-years of smoking and FEV1 29 (14-81) % predicted (median, range). Patients9 mMRC score was 2 (1-4) and exacerbation rate during the previous year 2 (0-2). Fourteen patients (56%) were ex-smokers. The 35 clinical/demographic variables distributed equally among the metabolomics ones (5,401 m/z values) to result in 4 clinical/metabolomic phenotypes. Of the clinical indices, depression and theophylline use contributed most to the first 2 principal components (PCs). This integrated setting of clinical-demographical and airway-peripheral metabolomic data subdivides of COPD into 4 phenotypes, whereas the metabolomics indices appear to have more powerful in explaining the variability of COPD compared to the role of clinical indices. Supported by the ESF grants No. 7856, 9043 and 9103.

Human bronchial epithelial cells (HBECs) have first-line contact with harmful substances during smoking, and changes in their metabolism most likely represent a defining factor in coping with the stress and development of airway diseases. This study was designed to determine the dynamics of metabolome changes in HBECs treated with cigarette smoke condensate (CSC), and to test whether normal metabolism can be restored by synthetic antioxidants. Principal component analysis, based on untargeted mass spectra, indicated that treatment of CSC-exposed HBECs with O-methyl-L-tyrosinyl-γ-L-glutamyl-L-cysteinylglycine (UPF1) acted faster than did N-acetylcysteine to revert the effect of CSC. The maximum effect of 10 μg/ml CSC itself on HBEC cell line, BEAS-2B, metabolism was seen at 2 hours after treatment, with return to the baseline level by 7 hours. In primary HBECs, the initial maximum effect was seen at 1 hour after CSC exposure. Certain metabolites associated with redox pathways and energy production were affected by CSC. Subsequent restoration of their content by UPF1 supports the hypothetical protective capacity of UPF1 against the oxidative stress and increased energy demand, respectively. Furthermore, UPF1 up-regulated the contents of phospholipid species identified as phosphatidylcholines and phosphatidylethanolamines in the CSC-exposed HBECs, indicating possible suppression of inflammatory processes along with an increase in spermidine as an endogenous cytoprotector. In conclusion, with this dynamic metabolomics study, we characterize the durability of the CSC-induced metabolic changes in BEAS-2B line cells and primary HBECs, and demonstrate the ability of UPF1 to significantly accelerate the recovery of HBECs from CSC insult.