Exhaled Breath Condensate Protein Research Articles

Changes in the proteomics of exhaled breath condensate under the influence of inhaled hydrogen in patients with post-COVID syndrome

Aim. To study the effect of inhalation therapy with an active hydrogen (AH) on the protein composition of exhaled breath condensate (EBC) in patients with post-COVID syndrome (PCS).Material and methods. This randomized controlled parallel prospective study included 60 patients after coronavirus disease 2019 (COVID-19) with PCS during the recovery period and clinical manifestations of chronic fatigue syndrome who received standard therapy according to the protocol for managing patients with chronic fatigue syndrome (CFS). The patients were divided into 2 groups: group 1 (main) — 30 people who received standard therapy and AH inhalations (SUISONIA, Japan) for 10 days, and group 2 (control) — 30 medical workers who received only standard therapy. Patients in both groups were comparable in sex and mean age. All participants in the study were sampled with EBC on days 1 and 10. Samples were subjected to tryptic digestion and high-performance liquid chromatography combined with tandem mass spectrometry analysis using a nanoflow chromatograph (Dionex 3000) in tandem with a high-resolution time-of-flight mass spectrometer (timsTOF Pro).Results. A total of 478 proteins and 1350 peptides were identified using high resolution mass spectrometry. The number of proteins in samples after AH therapy, on average, is 12% more than before treatment. An analysis of the distribution of proteins in different groups of patients showed that only half of these proteins (112) are common for all groups of samples and are detected in EBC before, after, and regardless of hydrogen therapy. In addition to the qualitative difference in the EBC protein compositions in different groups, quantitative changes in the concentration of 36 proteins (mainly structural and protective) were also revealed, which together made it possible to reliably distinguish between subgroups before and after treatment. It is worth noting that among these proteins there are participants of blood coagulation (а-1-antitrypsin), chemokine- and cytokine-mediated inflammation, and a number of signaling pathways (cytoplasmic actin 2), response to oxidative stress (thioredoxin), glycolysis (glyceraldehyde-3- phosphate dehydrogenase), etc.Conclusion. The use of hydrogen therapy can contribute to the switching of a number of physiological processes, which may affect the success of recovery in PCS patients. In particular, the obtained results indicate the activation of aerobic synthesis of adenosine triphosphate in mitochondria by hydrogen therapy, which correlates well with the decrease in the blood lactate level detected by laboratory studies. At the same time, this therapy can inhibit pro-inflammatory activity, negatively affecting the coagulation and signaling pathways of integrins and apoptosis, and, in addition, activate protective pathways, tricarboxylic acid cycle, FAS signaling, and purine metabolism, which may be essential for effective recovery after COVID-19.

A Mycobacterium tuberculosis fingerprint in human breath allows tuberculosis detection

An estimated one-third of tuberculosis (TB) cases go undiagnosed or unreported. Sputum samples, widely used for TB diagnosis, are inefficient at detecting infection in children and paucibacillary patients. Indeed, developing point-of-care biomarker-based diagnostics that are not sputum-based is a major priority for the WHO. Here, in a proof-of-concept study, we tested whether pulmonary TB can be detected by analyzing patient exhaled breath condensate (EBC) samples. We find that the presence of Mycobacterium tuberculosis (Mtb)-specific lipids, lipoarabinomannan lipoglycan, and proteins in EBCs can efficiently differentiate baseline TB patients from controls. We used EBCs to track the longitudinal effects of antibiotic treatment in pediatric TB patients. In addition, Mtb lipoarabinomannan and lipids were structurally distinct in EBCs compared to ex vivo cultured bacteria, revealing specific metabolic and biochemical states of Mtb in the human lung. This provides essential information for the rational development or improvement of diagnostic antibodies, vaccines and therapeutic drugs. Our data collectively indicate that EBC analysis can potentially facilitate clinical diagnosis of TB across patient populations and monitor treatment efficacy. This affordable, rapid and non-invasive approach seems superior to sputum assays and has the potential to be implemented at point-of-care.

Analyses of exhaled breath condensate cytokines for identification of lung cancer

Abstract Early non-invasive detection of lung cancer is a precondition for enabling better prognosis supported by new innovative therapy regimes. The aim of our study was to evaluate angiogenic and inflammatory proteins in exhaled breath condensate (EBC) as markers for lung cancer. Our report presents a diagnostic study of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and tumor necrosis factor-α (TNF-α) in EBC of 300 individuals, 84 patients with lung cancer, 111 patients with stable chronic obstructive pulmonary disease (COPD), and in 105 healthy controls. Detection of VEGF and bFGF in EBC was applicable to discriminate cancer patients from COPD patients as well as from healthy volunteers. Especially VEGF seems to be suitable to discriminate between non-small cell lung cancer (NSCLC) patients and control groups with highest VEGF values in EBC of patients with progressive NSCLC. The concentration of angiogenic factors correlated with disease progression as well as higher tumor stage. This study supports cytokine analysis in EBC as a suitable noninvasive diagnostic screening method for lung cancer detection and monitoring.

Diagnosis of respiratory diseases using the proteomic analysis of exhaled breath condensate

Investigation of exhaled breath condensate (EBC) is a noninvasive diagnostic method in respiratory diseases. The objective of this study was to compare EBC protein spectrum in healthy volunteers and in patients with chronic obstructive pulmonary disease (COPD), pneumonia and lung cancer (NSCLC), as well as to assess a role of proteomic analysis of EBC for diagnosis and differential diagnosis of these diseases. Methods. We examined 18 patients with COPD, 13 patients with community-acquired pneumonia, 26 patients with lung cancer and 24 healthy non-smoking volunteers. EBC was collected using ECoScreen system (VIASYS Healthcare, Germany) and a standardized method. EBC-samples were lyophilized, hydrolyzed and analyzed by HPLC and tandem mass spectrometry. To identify proteins, we used Mascot (Matrix Science, UK) and IPI-human (version 3.82) databases provided by the European Bioinformatics Institute. Results. Proteomic analysis of EBC identified more than 300 different proteins; most of them were types I and II cytoskeletal keratins. Cytokeratin 5, 6, and 14 concentrations in EBC of NSCLC patients were significantly higher than that in healthy volunteers. Dermcidin, immunoglobulin alpha, kininogen, cytoplasmic actin, serum albumin, and Zn-alpha2-glycoprotein were identified in EBC of healthy volunteers and patients with COPD and pneumonia. High concentration of peroxiredoxin in EBC of COPD patients could be due to severe oxidative stress. High levels of acute-phase and hypoxia proteins (annexins A1 and A2, HSP90B, cystatins M and B, collagen and histones fragments) were detected in EBC of pneumonia patients. Also, β- и α-subunit of hemoglobin, nuclear ubiquitin casein (NUCKS), POTEE, high mobility group protein (HMG-I/HMG-Y) and lactoferrin were identified in EBC of NSCLC patients. Conclusion. We found that EBC in healthy nonsmokers and in patients with COPD, pneumonia and NSCLC had characteristic protein spectrum. Most of the identified proteins could be used for diagnosis of these diseases.

Exhaled breath condensate biomarkers for the early diagnosis of lung cancer using proteomics.

We explored whether the proteomic analysis of exhaled breath condensate (EBC) may provide biomarkers for noninvasive screening for the early detection of lung cancer (LC). EBC was collected from 192 individuals [49 control (C), 49 risk factor-smoking (S), 46 chronic obstructive pulmonary disease (COPD) and 48 LC]. With the use of liquid chromatography and tandem mass spectrometry, 348 different proteins with a different pattern among the four groups were identified in EBC samples. Significantly more proteins were identified in the EBC from LC compared with other groups (C: 12.4 ± 1.3; S: 15.3 ± 1; COPD: 14 ± 1.6; LC: 24.2 ± 3.6; P = 0.0001). Furthermore, the average number of proteins identified per sample was significantly higher in LC patients, and receiver operating characteristic curve (ROC) analysis showed an area under the curve of 0.8, indicating diagnostic value. Proteins frequently detected in EBC, such as dermcidin and hornerin, along with others much less frequently detected, such as hemoglobin and histones, were identified. Cytokeratins (KRTs) were the most abundant proteins in EBC samples, and levels of KRT6A, KRT6B, and KRT6C isoforms were significantly higher in samples from LC patients (P = 0.0031, 0.0011, and 0.0009, respectively). Moreover, the amount of most KRTs in EBC samples from LC patients showed a significant positive correlation with tumor size. Finally, we used a random forest algorithm to generate a robust model using EBC protein data for the diagnosis of patients with LC where the area under the ROC curve obtained indicated a good classification (82%). Thus this study demonstrates that the proteomic analysis of EBC samples is an appropriated approach to develop biomarkers for the diagnosis of lung cancer.

Early diagnosis of lung cancer based on proteome analysis of exhaled breath condensate

A comparative study of the exhaled-breath-condensate (EBC) proteome that was obtained for four donor groups was carried out using ion cyclotron resonance mass spectrometry with electrospray ionization. The groups included subjects with diagnosed lung cancer, chronic obstructive pulmonary disease, community-acquired pneumonia, and healthy nonsmoking control subjects. More than 300 proteins were identified, while 19 of them were found in the EBC samples of the donors who were diagnosed with lung cancer in the early stages and are potentially significant in the development of a diagnostic lung-cancer biomarker panel. It was shown that the EBC protein profiles of different donor groups can be distinguished. It may be possible to highlight a specific protein group that is typical for certain conditions/diseases of the respiratory system. Thus, the EBC analysis could be a promising non-invasive method for early diagnosis of lung cancer.

Russian

It has been performed complex examination of 150 school-aged children with bronchial asthma, which included a study of exhaled breath condensate and a determination of deletions in the genes of glutathione-S-transferase (GSTM1 and GSTT1). A greater activity of bronchial inflammation manifested by increased activity of oxidative stress with the accumulation of products of oxidative modification of proteins in exhaled breath condensate has been noticed during asthma exacerbation in children with deletion polymorphism of genes GSTT1 and GSTM1 compared with patients without those genetic changes.

Exhaled Breath Condensate for Proteomic Biomarker Discovery

Exhaled breath condensate (EBC) has been established as a potential source of respiratory biomarkers. Compared to the numerous small molecules identified, the protein content of EBC has remained relatively unstudied due to the methodological and technical difficulties surrounding EBC analysis. In this review, we discuss the proteins identified in EBC, by mass spectrometry, focusing on the significance of those proteins identified. We will also review the limitations surrounding mass spectral EBC protein analysis emphasizing recommendations to enhance EBC protein identifications by mass spectrometry. Finally, we will provide insight into the future directions of the EBC proteomics field.

Pattern of cytokines and chemokines in exhaled breath condensate is related to the characteristics of mechanical ventilation

Ventilator-associated lung injury (VALI) is an inflammatory response of the lung caused by mechanical ventilation (MV) and is related to tidal volumes (TV), positive end-expiratory pressure (PEEP) and peak pressures (Ppeak) [1]. In experimental settings, VALI is characterized by a local inflammatory response measured in tissue or lavate. It is difficult to obtain this material in the critically ill [2]. Exhaled breath condensate (EBC) is obtained in patients on MV using an easy non-invasive technique. In this pilot study we examined the relation between levels of inflammatory proteins in EBC of patients on MV and characteristics of MV.

A new approach to study exhaled proteins as potential biomarkers for asthma

Asthma is a complex clinical disease characterized by airway inflammation. Recently, various studies reported on the analysis of exhaled breath condensate (EBC) in the search for potential biomarkers for asthma. However, in a complex disease such as asthma, one biomarker might not be enough for early diagnosis or follow-up. The use of proteome analysis may reveal disease-specific proteolytic peptide or protein patterns, and may lead to the identification of novel proteins for the detection of asthma. Liquid chromatography and mass spectrometry were used to separate and detect proteins (proteolytic peptides) present in EBC samples from 30 healthy children and 40 children with asthma in the age group of 6-12 years. Support vector machine analysis resulted in differentiating profiles based on asthma status. These proteolytic peptide patterns were not correlated to some well known (spirometry, exhaled nitric oxide) and more recently described exhaled markers (EBC pH, LTB₄). The more abundant proteins in EBC were identified as cytokeratins, albumin, actin, haemoglobin, lysozyme, dermcidin, and calgranulin B. Although the exact role in the disease development or physiological state of the airways of the proteins described in the presented pattern is not clear at this moment, this is an important step in the search for exhaled biomarkers for asthma. This study shows that EBC contains proteins that are of interest for future non-invasive asthma diagnosis or follow-up.

Non‐invasive biomarker sampling and analysis of the exhaled breath proteome

Exhaled breath condensate (EBC) is a biological fluid that contains trace amounts of secreted pulmonary proteins, and is emerging as a potentially valuable and non-invasively obtained source of disease biomarkers. Proteome analysis of these samples could lead to the identification of prognostic indicators of airway diseases. The objective of this study was to develop a protocol for proteome analysis of EBC samples. In this report, an improved procedure for EBC sample preparation and concentration is presented, together with a method for comparison of the protein profiles between two groups. The presented approach enabled to study the condensed exhaled breath proteome for biomarker analysis, and revealed proteins not previously identified in an EBC proteomics approach. In a comparative pilot study, EBC protein profiles obtained from smokers and non-smokers showed distinct differences and are illustrative for its potential in clinical studies. EBC from smokers contained higher concentrations of the more abundant proteins, such as cytokeratins, compared to non-smokers, and calgranulin B was identified uniquely in EBC samples from smokers.

Assessment of individual lung cancer risk by the proteomic analysis of exhaled breath condensate

Lung cancer is one of the leading causes of cancer-related deaths. Several diagnostic strategies are available but these are frequently ineffective, either because of their cost and organizational difficulty or because of the involvement of high radiations. As recent data from spiral computerized axial tomography have shown limited sensitivity and limited impact on cancer-related fatality, several options have been proposed in order to identify biological fluid-based biomarkers. Evaluating whether proteomic analysis of alveolar fluid obtained in the form of exhaled breath condensate (EBC) can be valuable for detecting and effectively diagnosing lung cancer. Careful review of recently published papers on proteomic EBC analysis, together with experience in the authors' laboratory, allows the discussion of benefits, pitfalls and possible future development of this approach. The rapid advancements of proteomics are expected to validate EBC protein(s) as lung pathology biomarker(s). Accessibility of an early marker of lung cancer will be a great advantage for potentially early treatment by surgical procedures with limited tissue removal, possibly preceding metastasis development.

Human skin keratins are the major proteins in exhaled breath condensate

Exhaled breath condensate (EBC) may be an attractive noninvasive alternative to bronchoscopy, bronchoalveolar lavage and induced sputum for diagnosis and monitoring of pulmonary disease. The aim of the present study was to identify proteins in EBC by mass spectrometry. Protein in EBC was characterised by gel electrophoresis of freeze-dried EBC samples, and individual proteins were identified by mass spectrometry. Saliva, ambient air condensate (AAC) and EBC were collected from normal human volunteers with or without a filter to remove particles from air. In some instances, EBC was condensed by breathing compressed air. Samples were freeze-dried and analysed by SDS-PAGE and peptide mass fingerprinting. Three major bands were seen in EBC and AAC, and were identified by peptide mass fingerprinting. The probability-based Mowse score was significant only for cytokeratin (CK) 1, CK2 and CK10. In the catalogue of human cytokeratins, CK1, CK2, CK9 and CK10 are described in keratinising epidermis. Saliva did not contain keratin and compressed air EBC contained markedly less keratin. Filtration of inspired air did not remove contaminating keratin. In conclusion, skin keratin in exhaled breath condensate derives from ambient air and not from the respiratory tract.