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Volatile organic compound (VOC) profiles emitted in trace concentrations from bacteria or cells has gained increasing importance over the decades. Analysis of VOCs in the headspace does not interfere with in vitro systems and, therefore, offers new options for non-invasive monitoring of cultures. Currently there is not any available standardized in vitro sampling system which considers effects of dilution and contamination onto ppbV to pptV VOC concentrations during. In this study a new in vitro system for online and offline headspace measurement of biological cultures was designed. The system was built from inert materials, equipped with universal sampling ports and easily adjustable volume options. Standard VOC mixtures in the system were analyzed by means of proton-transfer-reaction time-of-flight mass spectrometry and needle-trap-microextraction coupled with gas chromatography/mass spectrometry with a variance of 5%–14% and 10%–15%, respectively. In a proof of concept setup volatile emissions over cell cultures and pure media were assessed. The newly developed system enabled reliable and reproducible headspace analyses of in vitro cultures. As parallel application of different analytical methods is possible and confounding factors could be minimized, this set-up represents an important step towards standardization of headspace analysis over biological cultures.

Volatile molecules in exhaled breath represent potential biomarkers in the setting of infectious diseases, particularly those affecting the respiratory tract. In particular, Pseudomonas aeruginosa is a critically important respiratory pathogen in specific subsets of the population, such as those with cystic fibrosis (CF). Infections caused by P. aeruginosa can be particularly problematic when co-infection with respiratory syncytial virus (RSV) occurs, as this is correlated with the establishment of chronic P. aeruginosa infection. In the present study, we evaluate the volatile metabolites produced by P. aeruginosa (PAO1)-infected, RSV-infected, co-infected, or uninfected CF bronchial epithelial (CFBE) cells, in vitro. We identified a volatile metabolic signature that could discriminate between P. aeruginosa-infected and non-P. aeruginosa-infected CFBE with an area under the receiver operating characteristic curve (AUROC) of 0.850, using the machine learning algorithm random forest (RF). Although we could not discriminate between RSV-infected and non-RSV-infected CFBE (AUROC = 0.431), we note that sample classification probabilities for RSV-infected cell, generated using RF, were between those of uninfected CFBE and P. aeruginosa-infected CFBE, suggesting that RSV infection may result in a volatile metabolic profile that shares attributes with both of these groups. To more precisely elucidate the biological origins of the volatile metabolites that were discriminatory between P. aeruginosa-infected and non-P. aeruginosa-infected CFBE, we measured the volatile metabolites produced by P. aeruginosa grown in the absence of CFBE. Our findings suggest that the discriminatory metabolites produced likely result from the interaction of P. aeruginosa with the CFBE cells, rather than the metabolism of media components by the bacterium. Taken together, our findings support the notion that P. aeruginosa interacting with CFBE yields a particular volatile metabolic signature. Such a signature may have clinical utility in the monitoring of individuals with CF.

Hepatic mitochondrial function (HMF) assessed by the ¹³C-ketoisocaproate breath test (¹³C-KICA BT) has been previously shown to be significantly associated with the severity of biopsy proven non-alcoholic fatty liver disease (NAFLD). However, it is uncertain whether any perturbation in HMF relates specifically to severity of liver disease or factors associated with metabolic syndrome within (NAFLD). Our aim was to investigate whether there was any change in HMF assessed by ¹³C-KICA BT in patients with NAFLD compared to control subjects, and to assess the factors that are independently associated with HMF. Methods. 77 patients with NAFLD and 11 healthy control subjects were studied. HMF was assessed using ¹³C-KICA BT and expressed as cumulative % ¹³C-dose recovered on breath over 1 h (cPDR over 1 h). Liver fat and fibrosis was assessed by transient elastography. Multivariable linear regression modelling was undertaken to test the independence of associations with HMF. Results. HMF (cPDR over 1 h) was lower in NAFLD compared to controls [13.4% (4.8) v. 21.0% (6.3); p < 0.0001)]. In NAFLD, HMF was lower in patients with diabetes versus no diabetes [12.7% (3.4) v. 14.3% (6.1); p = 0.003)]. Regression modelling showed age (β = −0.08; p = 0.01), waist circumference (β = −0.08; p = 0.01), hip circumference (β = −0.04; p = 0.01), aspartate aminotransferase (AST) (β = −0.05; p = 0.01) and diabetes status (β = −1.81; p = 0.01) were independently associated with HMF (R² = 41.5%; p < 0.0001). Conclusions. In patients with NAFLD (compared to healthy subjects), there was a reduction in HMF assessed by the ¹³C-KICA BT. Furthermore, in patients with NAFLD, HMF is independent and inversely associated with age, waist and hip circumference, AST and diabetes status.

Breath analysis has great potential for becoming an important clinical diagnosis method due to its friendly and non-invasive way of sample collection. Hundreds of endogenous trace gases (volatile organic compounds (VOCs)) are present in breath, representing different metabolic processes of the body. They are not only characteristic for a person, their age, sex, habit etc, but also specific to different kinds of diseases. VOCs, related to diseases could serve as biomarkers for clinical diagnostics and disease monitoring. However, due to the large amount of water contained in breath, an identification of specific VOCs is a real challenge. In this work we present a technique of water suppression from breath samples, that enables us to identify several trace gases in breath, e.g., methane, isoprene, acetone, aldehyde, carbon monoxide, etc, using Fourier-transform infrared spectroscopy. In the current state, the technique reduces the water concentration by a factor of 2500. Sample preparation and data acquisition take about 25 min, which is clinically relevant. In this article we demonstrate the working principle of the water reduction technique. Further, with specific examples we demonstrate that water elimination from breath samples does not hamper the concentration of trace gases in breath. Preliminary experiments with real breath also indicate that the concentrations of methane, acetone and isoprene remain the same during the sample preparation.

A reliable method for the determination of carbonyl compounds in exhaled breath based on on-sorbent derivatization coupled with thermal desorption and gas chromatography-tandem mass spectrometry is described. The analytical performances were optimized for a mixture of C2-C9 aldehydes and C3-C9 ketones, particularly interesting for clinical applications, by using an internal standard and applying a 2³ full factorial design. A volume of sample (250 ml) was loaded at 50 ml min⁻¹ into a Tenax GR sorbent tube containing 130 nmol of O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride. All compounds showed a limit of detection lower than 200 pptv. The yield of the derivatization procedure was normalized by adding to the sample a known amount of ⁶D-acetone as an internal standard. This allowed halving the relative standard deviation to 10% and 15% for the mono-and di-carbonyl compounds, respectively, thus improving reliability. The optimized method was applied to the determination of carbonyl compounds in 12 breath samples collected from four patients suffering from heart failure during hospitalization.

The presence of certain volatile organic compounds (VOCs) in the breath of patients with gastric cancer has been reported by a number of research groups; however, the source of these compounds remains controversial. Comparison of VOCs emitted from gastric cancer tissue to those emitted from non-cancerous tissue would help in understanding which of the VOCs are associated with gastric cancer and provide a deeper knowledge on their generation. Gas chromatography with mass spectrometric detection (GC-MS) coupled with head-space needle trap extraction (HS-NTE) as the pre-concentration technique, was used to identify and quantify VOCs released by gastric cancer and non-cancerous tissue samples collected from 41 patients during surgery. Excluding contaminants, a total of 32 VOCs were liberated by the tissue samples. The emission of four of them (carbon disulfide, pyridine, 3-methyl-2-butanone and 2-pentanone) was significantly higher from cancer tissue, whereas three compounds (isoprene, γ-butyrolactone and dimethyl sulfide) were in greater concentration from the non-cancerous tissues (Wilcoxon signed-rank test, p < 0.05). Furthermore, the levels of three VOCs (2-methyl-1-propene, 2-propenenitrile and pyrrole) were correlated with the occurrence of H. pylori; and four compounds (acetonitrile, pyridine, toluene and 3-methylpyridine) were associated with tobacco smoking. Ex vivo analysis of VOCs emitted by human tissue samples provides a unique opportunity to identify chemical patterns associated with a cancerous state and can be considered as a complementary source of information on volatile biomarkers found in breath, blood or urine.

Background. The appetite-regulating effects of non-digestible carbohydrates (NDC) have in part previously been attributed to their effects on intestinal transit rates as well as microbial production of short chain fatty acids (SCFA). Increased colonic production of the SCFA propionate has been shown to reduce energy intake and stimulate gut hormone secretion acutely in humans. Objective. We investigated the effect of the propiogenic NDC, L-rhamnose, on gastrointestinal transit times using a combined ¹³CO₂/H₂ breath test. We hypothesised that L-rhamnose would increase plasma propionate leading to a reduction in appetite, independent of changes in gastrointestinal transit times. Design. We used a dual ¹³C-octanoic acid/lactose ¹³C-ureide breath test combined with breath H₂ to measure intestinal transit times following the consumption of 25 g d⁻¹ L-rhamnose, compared with inulin and cellulose, in 10 healthy humans in a randomised cross-over design pilot study. Gastric emptying (GE) and oro-caecal transit times (OCTTs) were derived from the breath ¹³C data and compared with breath H₂. Plasma SCFA and peptide YY (PYY) were also measured alongside subjective measures of appetite. Results. L-rhamnose significantly slowed GE rates (by 19.5 min) but there was no difference in OCTT between treatments. However, breath H₂ indicated fermentation of L-rhamnose before it reached the caecum. OCTT was highly correlated with breath H₂ for inulin but not for L-rhamnose or cellulose. L-rhamnose consumption significantly increased plasma propionate and PYY but did not significantly reduce subjective appetite measures. Conclusions. The NDCs tested had a minimal effect on intestinal transit time. Our data suggest that L-rhamnose is partially fermented in the small intestine and that breath H₂ reflects the site of gastrointestinal fermentation and is only a reliable marker of OCTT for certain NDCs (e.g. inulin). Future studies should focus on investigating the appetite-suppressing potential of L-rhamnose and verifying the findings in a larger cohort.

Introduction. Tobacco smoke is the leading cause of chronic obstructive pulmonary disease (COPD). Smoking cessation can change the natural history of COPD, as we know from the GOLD guidelines. Little is known about the short-term clinical and functional effects of smoking cessation treatment combined with anti-muscarinic bronchodilators. Objective. To determine whether quitting smoking, obtained by smoking cessation treatment combined with the use of a new long-acting muscarinic antagonist bronchodilator (LAMA), can improve lung function tests and respiratory symptoms more than the use of LAMA alone. Methods. We evaluated, in a retrospective analysis, the functional and clinical data, collected in one year, of 120 patients who were current smokers affected by mild COPD and who quit smoking using smoking cessation treatment combined with glycopirronium. We compared them with a group of 80 patients with mild COPD undergoing the same treatment but who did not quit smoking. All patients underwent functional and clinical tests at baseline and at a third-month check. Measurements and main results. The two groups were homogeneous in terms of demographic data without significant differences. All patients used varenicline for smoking cessation. They all performed the following tests: a spirometry with detection of resistances, the 6 min walking test, haemogasanalysis, the exhaled CO test, the COPD assessment test (CAT) and finally the modified Medical Research Council test (mMRC). A significant improvement in the functional tests at the third-month check was found in both groups—quitters and non-quitters. However, a notable increase in the examined parameters was registered in the group of patients who quit smoking, in particular, we observed a significant increase at the third-month check of the parameter forced expiratory volume in 1 s (FEV1) of more than 200 ml with p < 0.001. A comparison between quitters and non-quitters revealed a major benefit derived from smoking cessation in terms of functional changes and symptom relief. In particular, not only FEV1 but also forced expiratory flow at 25%–75% of vital capacity (FEF 25–75) (p < 0.01) and CAT (p < 0.001) were found to be significantly improved in patients who quit than in patients who did not at the check time point. Conclusions. Smoking cessation treatment obtained by varenicline was confirmed as a crucial therapeutic option, especially when combined with bronchodilator in mild COPD. Patients who quit smoking could already benefit from both treatments in the short term, improving lung function and respiratory symptoms and therefore improving their quality of life.

Background & Aims. Increased nitric oxide is involved in abnormal hemodynamic parameters and respiratory function of cirrhotic patients. We aimed to quantify partitioning exhaled nitric oxide measurements in exhaled air in liver transplantation (LT) candidates and evaluate their relationships with chronotropic incompetence and aerobic capacity. Methods. We compared exhaled nitric oxide (NO) measurements, heart rate response and peak oxygen uptake during incremental exercise in liver transplantation candidates to those of controls. Results. As opposed to healthy control subjects, LT candidates displayed elevated alveolar NO, blunted chronotropic response and reduced V'O₂ at maximal exercise. In LT candidates, median peak V'O₂ was 18.7 ml kg⁻¹ min⁻¹ (interquartile range (IQR) 16.2; 21.8), corresponding to 65% (IQR 57; 72) of the predicted value. Compared with controls, LT candidates had increased levels of alveolar NO (median (IQR) 2.0 (1.2; 2.2) versus 3.1 (2.3; 4.5), p < 0.001). In controls, no relations were found between alveolar NO and V'O₂ peak or heart rate reserve whereas in cirrhotic patients, negative correlations and negative slopes were found between alveolar NO and V'O₂ peak and heart rate reserve decrease. Conclusions. Increasing alveolar NO could be a specific pathophysiological condition limiting aerobic capacity in LT candidates.

Investigation employing bronchoalveolar lavage supports both increased and decreased iron concentrations in the epithelial lining fluid (ELF) of smokers. Exhaled breath condensate (EBC) is an alternative approach to sampling the ELF. We evaluated for an association between iron homeostasis and both smoking and a diagnosis of chronic obstructive pulmonary disease (COPD) by measuring metal concentrations in EBC samples from non-smoker controls, smoker controls, and individuals diagnosed with COPD. The total number of EBC specimens was 194. EBC iron and zinc concentrations (mean ± standard error) in the total study population were 0.610 ± 0.025 and 40.73 ± 1.79 ppb respectively. In linear regressions, total cigarette smoking in pack years showed a significant (negative) relationship with EBC iron concentration but not with EBC zinc concentration. Iron concentrations in EBC from GOLD stage II, III, and IV patients were all significantly decreased relative to those from non-smoker and smoker controls. In contrast to iron, zinc concentrations in EBC were not significantly different than those from non-smoker and smoker controls. It is concluded that smoking decreases EBC iron concentrations and patients diagnosed with COPD have significantly lower EBC iron concentrations. These results likely reflect an increased burden of cigarette smoke particles in the lower respiratory tract of ever-smokers and patients with COPD and the capacity of components in this particle to complex iron.

Background & aim. Halitosis is defined as an offensive breath odour of whatever source and therefore may affect a person's social interactions. Intra-oral halitosis is a result of bacterial activity. Therefore, probiotics may offer an appropriate and biological solution as a part of the therapy of intra-oral halitosis. The aim of this systematic review was to study the effect of the administration of probiotics alone or as an adjunct to other treatments on the level of halitosis as measured by volatile sulphur compound (VSC) levels, organoleptic scores (ORG) or hydrogen sulphide, methyl mercaptan and dimethyl sulphide levels. In addition, the effect of probiotic usage on oral microbial composition was summarised. Methods. The MEDLINE-PubMed and Embase databases were searched up to September 2017 with language restricted to English. Eligible papers were selected according to pre-set criteria; the data was extracted and analysed descriptively. Results. The search resulted in 1104 original research articles and a final six were selected as being eligible including 129 subjects. These studies used different detection methods and combinations thereof to measure halitosis. Five studies were randomised placebo-controlled clinical trials of which two studies reported a significant reduction in ORG between probiotic and placebo groups, and two studies on the basis of total VSC levels. The two studies reporting a significant improvement in ORG did not find an improvement in total VSC levels. Three studies included a microbiological assessment. In these three studies, the probiotic strain was detected at the end of the treatment period, but no detailed data was reported on the abundance of the strain before and after the treatment period. Conclusions. Probiotics may be beneficial in treating intra-oral halitosis. However, due to limited data and the heterogeneity of the studies, the efficacy of probiotics remains unclear. Studies with more subjects and standardised protocols need to be designed.

This work presents the performances of silicon micro-preconcentrators chips for breath sampling. The silicon chips were coupled to a handheld battery powered system for breath sampling and direct injection in a laboratory gas chromatography mass spectrometry system through thermal desorption (TD). Performances of micro-preconcentrators were first compared to commercial TD for benzene trapping. Similar chromatographic peaks after gas chromatographic separation were observed while the volume of sample needed was reduced by a factor of 5. Repeatability and day to day variability of the micro-preconcentrators were then studied for a 500 ppb synthetic model mixture injected three times a day four days in a row: 8% and 12% were measured respectively. Micro-preconcentrator to micro-preconcentrator variability was not significant compared to day to day variability. In addition, micro-preconcentrators were tested for breath samples collected in Tedlar^® bags. Three analyses of the same breath sample displayed relative standard deviations values below 16% for eight of the ten most intense peaks. Finally, the performances of micro-preconcentrators for breath sampling on a single expiration were illustrated with the example of volatile tobacco markers tracking. The signals of three smoking markers in breath, benzene, 2,5-dimethylfuran, and toluene were studied. Concentrations of benzene and toluene were found to be 10 to 100 higher in the breath of smokers. 2,5-dimethylfuran was only found in the breath of smokers. The elimination kinetics of the markers were followed as well during 4 h: a fast decrease of the signal of the three markers in breath was observed 20 min after smoking in good agreement with what is described in the literature. Those results demonstrate the efficiency of silicon chips for breath sampling, compared to the state of the art techniques. Thanks to miniaturization and lower sample volumes needed, micro-preconcentrators could be in the future a key technology towards portable breath sampling and analysis.

Rationale: Asthma is often characterised by inflammation, damage and dysfunction of the small airways, but no standardised biomarkers are available. Objectives: Using a novel approach—particles in exhaled air (PExA)—we sought to (a) sample and analyse abundant protein biomarkers: surfactant protein A (SPA) and albumin in adult asthmatic and healthy patients and (b) relate protein concentrations with physiological markers using phenotyping. Methods: 83 adult asthmatics and 21 healthy volunteers were recruited from a discovery cohort in Leicester, UK, and 32 adult asthmatics as replication cohort from Sweden. Markers of airways closure/small airways dysfunction were evaluated using forced vital capacity, impulse oscillometry and multiple breath washout. SPA/albumin from PEx (PExA sample) were analysed using ELISA and corrected for acquired particle mass. Topological data analysis (TDA) was applied to small airway physiology and PExA protein data to identify phenotypes. Results: PExA manoeuvres were feasible, including severe asthmatic subjects. TDA identified a clinically important phenotype of asthmatic patients with multiple physiological markers of peripheral airway dysfunction, and significantly lower levels of both SPA and albumin. Conclusion: We report that the PExA method is feasible across the spectrum of asthma severity and could be used to identify small airway disease phenotypes.

Lung cancer (LC) is a leading cause of cancer-related morbidity and mortality globally, and exhaled breath testing has been considered as a fast, convenient and non-invasive way to diagnose LC in its early stages. Volatile organic compounds (VOCs), as markers of LC in exhaled breath, have been widely investigated for cancer diagnosis. However, few studies have reported on the interference of benign pulmonary diseases (BPD) in the selection of VOC markers for LC. During this study, 207 samples were analyzed using thermal desorption instrumentation/gas chromatography/mass spectrometry (TD-GCMS) to detect C₆−C₃₀ VOCs, and all samples were divided into four groups: LC group, BPD group, lung disease (LD) group (including LC group and BPD group) and healthy group. To make up for the deficiency of detecting low carbon hydrocarbons (<C₆), 277 samples were analyzed using solid-phase micro-extraction/gas chromatography/mass spectrometry (SPME-GCMS), divided among the four groups. VOC markers were selected by reference to the receiver operating characteristics curve. With the comparisons among the LC group, BPD group and healthy group from TD-GCMS and SPME-GCMS results, we found that exhaled VOCs are capable of discriminating LC group versus healthy group and BPD group versus healthy group with a consistency of 70%–80%. However, no VOCs can be selected with good discrimination capability between the LC group and BPD group, indicating that BPD interferes significantly in VOC marker selection for LC. To discriminate breath samples from the LD group and healthy group, 11 VOCs, including ten selected from TD-GCMS and one from SPME-GCMS, were chosen as markers for LD diagnosis. The sensitivity, specificity and overall accuracy of the diagnostic model established using ten VOCs were 80.8%, 84% and 82.7%, and those of the model established by using one VOC were 75.6%, 78.9% and 76.7%. These results validate that LD patients can be effectively discriminated and diagnosed using exhaled VOC analysis. (Clinical trial registration number: ChiCTR-DCD-15007106.)

We previously showed that thioether levels in the exhaled breath volatiles of volunteers undergoing controlled human malaria infection (CHMI) with P. falciparum increase as infection progresses. In this study, we show that thioethers have diurnal cyclical increasing patterns and their levels are significantly higher in P. falciparum CHMI volunteers compared to those of healthy volunteers. The synchronized cycle and elevation of thioethers were not present in P. vivax-infection, therefore it is likely that the thioethers are associated with unique factors in the pathology of P. falciparum. Moreover, we found that time-of-day of breath collection is important to accurately predict (98%) P. falciparum-infection. Critically, this was achieved when the disease was asymptomatic and parasitemia was below the level detectable by microscopy. Although these findings are encouraging, they show limitations because of the limited and logistically difficult diagnostic window and its utility to P. falciparum malaria only. We looked for new biomarkers in the breath of P. vivax CHMI volunteers and found that a set of terpenes increase significantly over the course of the malaria infection. The accuracy of predicting P. vivax using breath terpenes was up to 91%. Moreover, some of the terpenes were also found in the breath of P. falciparum CHMI volunteers (accuracy up to 93.5%). The results suggest that terpenes might represent better biomarkers than thioethers to predict malaria as they were not subject to malaria pathogens diurnal changes.

The present work characterized the metabolomic profile of tracheal wash (TW) and exhaled breath condensate (EBC) in healthy horses and horses with respiratory disease. Six asthma-affected horses (group A) and six healthy controls (group H) underwent clinical, endoscopic and cytologic examinations of upper airways to confirm the active phase of asthma. TW and EBC samples were collected from each animal and investigated by proton nuclear magnetic resonance (¹H-NMR) metabolomic analysis. A total of ten out of 38 metabolites found in the TW were significantly different between the groups (p < 0.05). Higher concentrations of histamine and oxidant agents, such as glutamate, valine, leucine and isoleucine, as well as lower levels of ascorbate, methylamine, dimethylamine and O-phosphocholine, were found in group A compared to group H. Eight metabolites were found in equine EBC, namely methanol, ethanol, formate, trimethylamine, acetone, acetate, lactate and butanone, previously observed also in human EBC. Despite the fact that this was a pilot study, the results showed that the metabolomic analysis of TW and EBC has the potentiality to serve as a basis for diagnostic tools in horses with asthma.