The understanding that there is a connection between breath and diseases can date back to over two thousand years ago, when Hippocrates described fetor oris and fetor hepaticus in his report on breath aroma (1). Since then, research into volatile organic compounds (VOCs) exhaled in breath and their involvement in disease physiology has led to exponential progress in the space. VOCs can originate from various parts of the body, be transported through the bloodstream, and exchanged into the air in the lungs. Since these VOCs can stem from metabolic processes occurring in the body, they have potential as non-invasive biomarkers for assessing an individual’s health status (Figure 1).
Figure 1: VOCs can be used as non-invasive biomarkers for gauging an individual’s health status.
The rapid advancement of breath sampling and analysis technology over the last few decades has allowed breath tests to be successfully deployed in clinical settings. This includes fractional exhaled nitric oxide (FENO) for asthma, the 13C-Urea test for H. pylori infection in the stomach, and hydrogen-methane testing for diagnosing small intestinal bacterial overgrowth (SIBO) or carbohydrate malabsorption conditions such as lactose intolerance (2–4). To accelerate the translation of biomarker discoveries into clinical applications and enable the development of tests for a greater range of diseases, conducting studies with animal models is essential.
The need for standardized methodologies
Several challenges remain in the process of VOC biomarker discovery, validation, and clinical translation in breath research. One significant difficulty is the standardization of sampling and analysis to ensure that compounds in human exhaled breath are distinguished from background contamination. Additionally, due to the numerous external and internal sources of variability, such as diet, environment, and individual metabolic differences, clinical trials aiming to distinguish VOC markers between disease and control groups will require many participants to ensure sufficient statistical power to account for these covariates. This requirement not only increases the cost and duration of clinical studies, but also adds complexity to data analysis, as factors such as sample storage time may introduce additional variability.
In vivo studies for biomarker validation
Mouse models are highly advantageous and essential for expediting the identification and validation of breath biomarkers for clinical use for several reasons. Firstly, mouse models are reared in a laboratory under strictly controlled environments, maximizing standardization and minimizing influences such as variations in background and dietary intake. Secondly, the use of inbred mouse strains reduces inter-individual variability, addressing a challenge that is difficult to overcome in human breath studies. Thirdly, the microbiome produces a significant number of VOCs detectable in breath, but controlling the microbiome composition in mouse models is relatively straightforward, making them ideal for biomarker validation.
Recently, a novel pre-clinical method has been developed to collect mouse breath samples in the laboratory and characterize breath VOCs with high accuracy. By modifying the gold-standard respiratory mechanics equipment (flexiVent®) with specific air filters, breath samples can be collected from intubated healthy mice onto industry-standard sorbent tubes for TD-GS-MS analysis with minimal background influence (Figure 2). Using this approach, comparisons between breath and blank samples can effectively distinguish baseline on-breath VOCs in healthy mice from background compounds. This level of differentiation cannot be easily achieved using other sampling methods, such as collecting breath from unrestrained mice directly from a ventilated cage or a nose-only inhalation tube (5,6). Moreover, employing the same air filters, sorbent tubes, and analytical approach in human breath analysis allows for direct comparison of breath VOCs between humans and mice. This comparison revealed 49 VOC-shared compounds, laying the foundation for translating pre-clinical findings and biomarkers into human clinical studies (7).
Figure 2 – A schematic detailing an approach to collecting mouse breath, and how this can be applied for a) an EVOC probe approach for drug discovery and b) using germ-free mice to identify specific VOCs produced by strains of gut bacteria (8).
Speeding up translational research using mouse models
The exhaled breath of mouse models presents a valuable tool for advancing drug development. In recent years, the use of exogenous VOC (EVOC) probes to target specific enzymatic activity has emerged as a novel screening approach for cancer (Figure 2 A). Additionally, the administration of the EVOC probe limonene has demonstrated success in distinguishing liver cirrhosis in a recent clinical study (9). EVOC probes operate on principles similar to the clinically established 13C-labelled urea breath test for H. pylori infection. Upon administration, tumor-associated enzymes in the microenvironment cleave the EVOC probe, releasing isotope-labeled compounds detectable in exhaled breath as reporters.
A recent study demonstrated the potential of EVOC probes in a mouse model (10). A mixture of probes was tested on mice implanted with cancer cells and healthy control mice, leading to the identification of an enzyme target, β-GlcNAc. Elevated levels of the reporter VOC, 13CD5-ethanol, were detected in both in vivo (mouse breath) and ex vivo (tissue headspace) settings. The study further revealed that a commercially available tumor-activated prodrug targeting β-GlcNAc significantly reduced cancer activity in mice transplanted with triple-negative breast cancer (TNBC). This study underscores the power of mouse models in breath research to identify enzyme targets linked to specific diseases, offering a path to accelerate drug discovery.
The microbiome produces a significant number of VOCs that can be detected in breath; in the gut, the composition of the microbiome is strongly associated with gastrointestinal conditions such as small intestinal bacterial overgrowth (SIBO), inflammatory bowel disease (IBD), and irritable bowel syndrome (IBS), making breath VOCs highly promising as disease biomarkers. However, linking specific VOCs in human breath to distinct gut microbial species remains a challenge. Mouse models provide a viable solution, as germ-free mice can be transplanted with different gut bacterial species, and their exhaled breath VOCs can be matched to those detected in headspace analysis of the respective bacterial species cultured in vitro (8) (Figure 2 B). This approach connects the dots between VOCs detected in breath and the specific gut bacteria that produce them, providing a deeper understanding of the origins of breath VOCs.
Figure 2 – A schematic detailing an approach to collecting mouse breath, and how this can be applied for a) an EVOC probe approach for drug discovery and b) using germ-free mice to identify specific VOCs produced by strains of gut bacteria (8).
Breath VOCs hold great promise as a tool for future non-invasive, decentralized disease diagnosis and treatment monitoring. The unique advantages of mouse models in breath research, combined with a reliable breath sampling and analysis platform that enables fair comparisons between mouse and human breath compositions, will drive efforts to translate new knowledge into clinical applications. Utilizing mouse models in breath research will bridge the gap between laboratory discoveries and human clinical studies, paving the way for the development of rapid, point-of-care devices for disease diagnosis.
References
- Dweik RA, Amann A. Exhaled breath analysis: the new frontier in medical testing. J Breath Res. 2008 Sep;2(3):030301.
- Patel SK, Pratap CB, Jain AK, Gulati AK, Nath G. Diagnosis of Helicobacter pylori: What should be the gold standard? World J Gastroenterol. 2014 Sep 28;20(36):12847–59.
- Heffler E, Carpagnano GE, Favero E, Guida G, Maniscalco M, Motta A, et al. Fractional Exhaled Nitric Oxide (FENO) in the management of asthma: a position paper of the Italian Respiratory Society (SIP/IRS) and Italian Society of Allergy, Asthma and Clinical Immunology (SIAAIC). Multidiscip Respir Med. 2020 Feb 19;15(1):36.
- Rezaie A, Buresi M, Lembo A, Lin H, McCallum R, Rao S, et al. Hydrogen and Methane-Based Breath Testing in Gastrointestinal Disorders: The North American Consensus. Am J Gastroenterol. 2017 May;112(5):775–84.
- Kistler M, Szymczak W, Fedrigo M, Fiamoncini J, Höllriegl V, Hoeschen C, et al. Effects of diet-matrix on volatile organic compounds in breath in diet-induced obese mice. J Breath Res. 2014 Mar;8(1):016004.
- Hintzen KFH, Smolinska A, Mommers AGR, Bouvy ND, van Schooten FJ, Lubbers T. Non-invasive breath collection in murine models using a newly developed sampling device. J Breath Res. 2022 Feb 14;16(2).
- Taylor A, Blum S, Ball M, Birch O, Chou H, Greenwood J, et al. Development of a new breath collection method for analyzing volatile organic compounds from intubated mouse models. Biology Methods and Protocols. 2024 Nov 14;bpae087.
- Hernandez-Leyva AJ, Berna AZ, Liu Y, Rosen AL, Lint MA, Whiteside SA, et al. The breath volatilome is shaped by the gut microbiota. medRxiv. 2024 Aug 8;2024.08.02.24311413.
- Ferrandino G, Ricciardi F, Murgia A, Banda I, Manhota M, Ahmed Y, et al. Exogenous Volatile Organic Compound (EVOC®) Breath Testing Maximizes Classification Performance for Subjects with Cirrhosis and Reveals Signs of Portal Hypertension. Biomedicines. 2023 Nov;11(11):2957.
- Châtre R, Blochouse E, Eid R, Djago F, Lange J, Tarighi M, et al. Induced-volatolomics for the design of tumour activated therapy. Chem Sci. 14(18):4697–703.
Hsuan (Tina) Chou
Author Bio
Dr. Hsuan (Tina) Chou, senior biomarker scientist, Owlstone Medical
Dr. Hsuan (Tina) Chou is a senior biomarker scientist at Owlstone Medical, where she ensures the successful delivery of customer project results and contributes to manuscript writing. She also supports the biological aspects of internal product development and plays an active role in creating scientific content for the company’s technical sales and marketing efforts. Dr. Chou holds a PhD in Plant Science from the University of Connecticut and has several years of postdoctoral experience working with omics data in the broader biology field at North Carolina State University before joining Owlstone in late 2021.
Filed Under: Biospecimens, Data science, Drug Discovery and Development, Regulatory affairs
