On-line Breath Metabolomics with Ambient High-Resolution Mass Spectrometry
Martin Thomas Gaugg
Abstract
Respiratory diseases are among the leading causes of death worldwide and pose a great financial burden on the health care system. During the last decades the medical community has started to recognize that a patient’s individual set of genes, along with environmental factors, are immensely important for the diagnosis and treatment of diseases. This has led to a strong drive towards further developments in personalized and evidencebased medicine. Understanding the underlying metabolic fundamentals of diseases is crucial to provide the appropriate patient care. One of the fastest methods to obtain new insights in this regard is to analyze metabolites in exhaled breath, which offers a non-invasive window into human metabolism, and which can be monitored in real time.
It has been known since the 1970s that human breath is composed not only of the gaseous constituents present in Earth’s atmosphere; rather, it is a complex mixture of various endogenous and exogenous substances. However, these volatile organic compounds (VOCs) are mostly present at extremely low concentrations and in a highly complex matrix. A variety of analytical techniques have emerged to detect these compounds in real time. Secondary electrospray ionization-mass spectrometry (SESI-MS) is a novel technique to achieve this with outstanding sensitivity and specificity. It is able to detect a vast array of analytes and operates at ambient pressure, making it highly modular. Therefore, one can couple it to virtually any type of ambient mass spectrometer, making it possible to exploit the advances in sensitivity and resolution of modern mass spectrometers. Additionally, it is thereby possible to perform unambiguous compound identification by collecting exhaled breath condensate (EBC) and running ultra-high performance liquid chromatography measurements. The work described in this dissertation extends efforts towards the application of SESI-MS to perform noninvasive metabolomics using real-time breath analysis.
An optimized low-flow SESI source that has been developed in our lab was coupled to an Orbitrap mass spectrometer. For the first time, it was thereby possible to explore mass resolutions of up to 105 for real-time breath analysis. Crucially, we showed that some peaks could only be resolved at the highest resolution. The setup was further tested in a pilot study on smoking individuals, which resulted in a perfect discrimination from non-smoking subjects based on their VOC pattern.
Detecting signal levels in real time and performing unambiguous compound identification in EBC allows metabolic interpretations to be made. However, it can be difficult to obtain evidence that the detected compounds are indeed connected via a certain pathway. We therefore expanded the framework to also consider metabolite correlations across people. Using real-time breath data of 146 healthy subjects we were able to identify 3 complete homologous series of fatty acid degradation products that are directly connected via the ω-oxidation pathway. The pronounced observed correlations between the compounds strongly suggest that SESI-MS is able to monitor the entire pathway in exhaled breath.
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease whose cause is still largely unknown. It is characterized by the uncontrollable formation of fibrous connective tissue in the pulmonary interstitium, leading to an irreversible loss of lung function. This results in an average survival of only 2.5–5 years after diagnosis. Building on recent evidence that amino acids related to collagen turnover are elevated in lung tissue of IPF patients, we investigated breath levels of these compounds in patients and healthy controls in a targeted study. Not only could we show that these amino acids are indeed elevated in IPF patients, but also that those with a higher abundance in pulmonary connective tissue show a more pronounced effect. Similar to the ω-oxidation pathway, the compounds also strongly correlated with each other, further strengthening the hypothesis that fibrosis is the underlying process of these observations.
Lipophilic drugs that are administered via inhalation are typically rapidly absorbed. They can therefore be difficult to study in blood samples due to the delayed sampling entailed. The speed of real-time breath analysis offers great opportunities to get an insight into these processes. In a placebocontrolled study involving asthmatics, chronic obstructive pulmonary disease (COPD) patients, and healthy controls, we investigated the metabolic effects of inhaled salbutamol, a common bronchodilator. While it was not possible to detect the drug itself with certainty due to insufficient resolution, pronounced metabolic changes consistent with the known lipolysis stimulating effects of salbutamol were observed.
Untargeted detection is the most challenging approach in metabolomics but also holds the biggest opportunities to obtain entirely new insights into diseases. Exacerbations in COPD are one of the greatest challenges for pulmonologists to date, since their pathophysiology is only poorly understood. We therefore aimed to unravel new associated pathways by comparing COPD patients with and without frequent exacerbations. An entirely new set of metabolites consisting of various nitroaromatic compounds was identified and found to correlate with COPD exacerbations. Additionally, we could show that metabolites from the ω-oxidation pathway are significantly lower in patients with exacerbations and also allow for a good discrimination.
