Martin Thomas Gaugg

Effects of CPAP therapy withdrawal on exhaled breath pattern in obstructive sleep apnoea

Background Obstructive sleep apnoea (OSA) is highly prevalent and associated with cardiovascular and metabolic changes. OSA is usually diagnosed by polysomnography which is time-consuming and provides little information on the patients phenotype thus limiting a personalised treatment approach. Exhaled breath contains information on metabolism which can be analysed by mass spectrometry within minutes. The objective of this study was to identify a breath profile in OSA recurrence by use of secondary-electrospray-ionization-mass spectrometry (SESI-MS). Methods Patients with OSA effectively treated with CPAP were randomised to either withdraw treatment (subtherapeutic CPAP) or continue therapeutic CPAP for 2 weeks. Exhaled breath analysis by untargeted SESI-MS was performed at baseline and 2 weeks after randomisation. The primary outcome was the change in exhaled molecular breath pattern. Results 30 patients with OSA were randomised and 26 completed the trial according to the protocol. CPAP withdrawal led to a recurrence of OSA (mean difference in change of oxygen desaturation index between groups +30.3/h; 95% CI 19.8/h,40.7/h, p<0.001) which was accompanied by a significant change in 62 exhaled features (16 metabolites identified). The panel of discriminating mass-spectral features allowed differentiation between treated and untreated OSA with a sensitivity of 92.9% and a specificity of 84.6%. Conclusion Exhaled breath analysis by SESI-MS allows rapid and accurate detection of OSA recurrence. The technique has the potential to characterise an individuals metabolic response to OSA and thus makes a comprehensible phenotyping of OSA possible. Trial registration number NCT02050425 (registered at ClinicalTrials.gov).

Identification of 2-alkenals, 4-hydroxy-2-alkenals, and 4-hydroxy-2,6-alkadienals in exhaled breath condensate by UHPLC-HRMS and in breath by real-time HRMS.

In recent years, breath analysis in real time has become a noninvasive alternative for the diagnosis of diseases and for molecular fingerprinting of exhaled breath. However, the techniques used lack the capabilities for proper identification of the compounds found in the exhalome. Here, we report the use of UHPLC-HRMS as a tool for the identification of several aldehydes (2-alkenals, 4-hydroxy-2-alkenals, and 4-hydroxy-2,6-alkadienals), biomarkers of lipid peroxidation, in exhaled breath condensate of three healthy subjects (N = 3). Some of the aldehydes studied have never been identified before. Their robust identification is based on retention times, on the generation of fragmentation trees from tandem mass spectra, and on the comparison of these parameters with standards. We also show that the identified compounds can be analyzed and confirmed by MS/MS in breath in real time and, therefore, they could be used as biomarkers for the rapid and noninvasive diagnosis of related diseases.

Expanding metabolite coverage of real-time breath analysis by coupling a universal secondary electrospray ionization source and high resolution mass spectrometry--a pilot study on tobacco smokers.

Online breath analysis is an attractive approach to track exhaled compounds without sample preparation. Current commercially available real-time breath analysis platforms require the purchase of a full mass spectrometer. Here we present an ion source compatible with virtually any preexisting atmospheric pressure ionization mass spectrometer that allows real-time analysis of breath. We illustrate the capabilities of such technological development by upgrading an orbitrap mass spectrometer. As a result, we detected compounds in exhaled breath between 70 and 900 Da, with a mass accuracy of typically  <1 ppm; resolutions between m/Δm 22,000 and 70,000 and fragmentation capabilities. The setup was tested in a pilot study, comparing the breath of smokers (n  =  9) and non-smokers (n  =  10). Exogenous compounds associated to smoking, as well as endogenous metabolites suggesting increased oxidative stress in smokers, were detected and in some cases identified unambiguously. Most of these compounds correlated significantly with smoking frequency and allowed accurate discrimination of smokers and non-smokers.

Translating secondary electrospray ionization-high-resolution mass spectrometry to the clinical environment

While there has been progress in making use of breath tests to guide clinical decision making, the full potential of exhaled breath analysis still remains to be exploited. Here we summarize some of the reasons why this is the case, what we have done so far to overcome some of the existing obstacles, and our vision of how we think breath analysis will play a more prominent role in the coming years. In particular, we envision that real-time high-resolution mass spectrometry will provide valuable information in biomarker discovery studies. However, this can only be achieved by a coordinated effort, using standardized equipment and methods in multi-center studies to eventually deliver tangible advances in the field of breath analysis in a clinical setting. Concrete aspects such as sample integrity, compound identification, quantification and standardization are discussed. Novel secondary electrospray ionization developments with the aim of facilitating inter-groups comparisons and biomarker validation studies are also presented.

On-line Breath Metabolomics in Respiratory Diseases using Secondary Electrospray Ionization-Mass Spectrometry

Every second we are exhaling hundreds of endogenous and exogenous compounds that originate from blood and lung tissue. Obtaining metabolic information via exhaled breath analysis has been an emerging topic since the 1970s. Secondary electrospray ionization-mass spectrometry is a relatively new technique to detect these metabolites on-line in a highly sensitive and specific fashion. Using this technique, several respiratory diseases, including chronic obstructive pulmonary disease, obstructive sleep apnea, idiopathic pulmonary fibrosis, asthma, and lung cancer have been investigated over the past years. Several new potential biomarkers for these diseases were identified and new metabolic insights into their pathophysiology could be obtained.

Real-Time Monitoring of Tricarboxylic Acid Metabolites in Exhaled Breath

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathway for cellular respiration in aerobic organisms. It provides and collects intermediates for many other interconnecting pathways and acts as a hub connecting metabolism of carbohydrates, fatty acids, and amino acids. Alteration in intracellular levels of its intermediates has been linked with a wide range of illnesses ranging from cancer to cellular necrosis or liver cirrhosis. Therefore, there exists an intrinsic interest in monitoring such metabolites. Our goal in this study was to evaluate whether, at least the most volatile metabolites of the TCA cycle, could be detected in breath in vivo and in real time. We used secondary electrospray ionization coupled with high-resolution mass spectrometry (SESI-HRMS) to conduct this targeted analysis. We enrolled six healthy individuals who provided full exhalations into the SESI-HRMS system at different times during 3 days. For the first time, we observed exhaled compounds that appertain to the TCA cycle: fumaric, succinic, malic, keto-glutaric, oxaloacetic, and aconitic acids. We found high intraindividual variability and a significant overall difference between morning and afternoon levels for malic acid, oxaloacetic acid, and aconitic acid, supporting previous studies suggesting circadian fluctuations of these metabolites in humans. This study provides first evidence that TCA cycle could conveniently be monitored in breath, opening new opportunities to study in vivo this important metabolic pathway.

119 Exhaled breath analysis by real-time mass spectrometry in patients with pulmonary fibrosis

Introduction: Idiopathic pulmonary fibrosis (IPF) is recognized as a distinct clinical disorder. However, despite major endeavors to understand the pathogenesis, the diagnosis of IPF remains elusive [1]. Metabolic profiling of biopsied tissue specimens has been shown promise to gain insights into IPF pathogenesis [2]. Thus we hypothesized that analysis of exhaled metabolites may also provide further insights. Methods: 21 patients with IPF and 21 matched controls were recruited for this exploratory study. Exhaled breath analysis was performed by Secondary Electrospray Ionization-Mass Spectrometry (SESI-MS) [3]. Significant differences in exhaled metabolite levels in IPF were subsequently sought. A two-sample t-test followed by estimation of false discovery rate in multiple comparisons was used for this purpose. Statistical significance was set at p < 0.05. Finally, we attempted to predict IPF vs control in a leaveone-out-cross-validation (LOOCV) using a set of 15 metabolites per sample predicted. Results: Fig. 1A shows an example of six replicate exhalations of one subject for one particular metabolite with the molecular formula C16H23NO5 (m/z 310.1640). Fig.1B shows the same compound exhaled in a selected group of controls (left-hand-side) and IPF patients (right-hand-side). The graph suggests that this particular compound is exhaled in higher concentrations in IPF patients than in controls. Fig. 1C shows the ROC curve obtained as a result of the LOOCV. The obtained area under the curve was 0.8 (0.61-0.91). Conclusions: Analysis of exhaled breath by real time mass spectrometry shows promise to help in the diagnostic process of IPF. Breath analysis complements ongoing efforts to characterize IPF at the metabolic level using more invasive approaches (e.g. biopsy). Ongoing in-depth structural identification of altered breath metabolites is likely to provide insights on IPF pathogenesis. These findings will require further validation in larger and independent cohorts.

Secondary electrospray ionization proceeds via gas-phase chemical ionization

Our main goal was to gain further insights into the mechanism by which gas-phase analytes are ionized by interaction with plumes of electrospray solvents. We exposed target vapors to electrosprays of either water or deuterated water and mass analyzed them. Regardless of the solvent used, the analytes were detected in protonated form. In contrast, when the ionization chamber was humidified with deuterated water, the target vapors were detected in deuterated form. These observations suggest that either there is no interaction between analytes and electrospray charged droplets, or if there is any, a subsequent gas-phase ion–molecule reaction governs the process. Implications in practical examples such as breath analysis are discussed.

Mass-Spectrometric Detection of Omega-Oxidation Products of Aliphatic Fatty Acids in Exhaled Breath

Omega-oxidation is a fatty acid degradation pathway that can occur alternatively to the dominant β-oxidation. The dysregulation of fatty acid oxidation has been related with a variety of diseases, termed fatty acid oxidation disorders. This work shows evidence for real-time detection in exhaled breath of the complete series of saturated linear ω-hydroxyalkanoic acids, ω-oxoalkanoic acids, and alkanedioic acids with carbon chain lengths of 5-15. We present a comprehensive analytical workflow using online and subsequent offline methods: secondary electrospray ionization mass spectrometry of exhaled breath and UHPLC-HRMS/MS experiments using exhaled breath condensate, respectively. By analyzing online breath measurements of 146 healthy individuals, we were able to obtain strong evidence for the correlation of these metabolite families. This enabled us to monitor the full ω-oxidation pathway in human exhaled breath. We could unambiguously identify these compounds, many of which have never been reported in breath so far. This comprehensive study on breath metabolites reinforces the notion of breath as a valuable source of information, which is underexploited in metabolomics.