Molecular breath analysis supports altered amino acid metabolism in idiopathic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: Diagnosis of idiopathic pulmonary fibrosis (IPF) is complex and its pathogenesis is poorly understood. Recent findings indicate elevated levels of proline and other amino acids in lung tissue of IPF patients which may also be of diagnostic value. Following these findings, we hypothesized that such altered metabolic profiles would be mirrored in exhaled breath and could therefore be captured non-invasively in real time. METHODS: We aimed to validate these results using real-time exhaled breath analysis by secondary electrospray ionization-mass spectrometry, which can provide a non-invasive, painless and fast insight into the metabolism. Breath analysis was performed in a matched 1:1 case-control study involving 21 patients with IPF and 21 control subjects. RESULTS: We found significantly (P < 0.05) elevated levels of proline, 4-hydroxyproline, alanine, valine, leucine/isoleucine and allysine in breath of IPF patients, whereas pyroglutamic acid and phenylalanine did not show significant differences. This coincides with the amino acid's abundance in pulmonary tissue indicating that our observations reflect progressing fibrosis. In addition, amino acid levels correlated across subjects, further supporting a common underlying pathway. We were able to obtain a cross-validated area under the curve of 0.86, suggesting that these increased amino acid levels in exhaled breath have the potential to be used as biomarkers for IPF. CONCLUSION: We could validate previous findings of elevated lung tissue amino acid levels in IPF and show that online breath analysis might be a practical tool for a rapid screening for IPF.

Real-Time Breath Analysis Reveals Specific Metabolic Signatures of COPD Exacerbations.

BACKGROUND: Exacerbations of COPD are defined by acute worsening of respiratory symptoms leading to a change in therapy. Identifying altered metabolic processes in patients at risk for future exacerbations is desirable for treatment optimization, the development of new therapeutic strategies, and perhaps diagnostic value. We aimed to identify affected pathways using the profiles of volatile organic compounds in exhaled breath from patients with COPD with and without frequent exacerbations (≥ 2 exacerbations within the past 12 months). METHODS: In this matched cohort study, exhaled breath profiles from patients with COPD and frequent exacerbations ("frequent exacerbators") and without frequent exacerbations ("nonfrequent exacerbators") were analyzed during an exacerbation-free interval using real-time secondary electrospray ionization high-resolution mass spectrometry. We analyzed exhaled breath from 26 frequent exacerbators and 26 nonfrequent exacerbators that were matched in terms of age, sex, and smoking history. To obtain new pathophysiological insights, we investigated significantly altered metabolites, which can be assigned to specific pathways. Metabolites were identified by using a Wilcoxon rank-sum test. RESULTS: Metabolite levels from the ω-oxidation pathway, namely ω-hydroxy, ω-oxo, and dicarboxylic acids, were consistently decreased in frequent exacerbators. Additionally, several new nitro-aromatic metabolites, which were significantly increased in frequent exacerbators, were identified. CONCLUSIONS: Real-time breath analysis by secondary electrospray high-resolution mass spectrometry allows molecular profiling of exhaled breath, providing insights about ongoing biochemical processes in patients with COPD at risk for exacerbations. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT02186639; URL: www.clinicaltrials.gov.

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.

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.

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 ß-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.

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.