Analyzing breath in mouse models using intubated systems

This study created a method for correctly identifying the volatile organic compounds (VOCs) in the breath of healthy intubated mouse models using GC-MS.

The development of a new breath-collecting method for evaluating volatile organic chemicals in intubated mouse models.

The aim was to use GC-MS to reliably analyze VOCs in the breath of healthy intubated mice and compare the VOCs in mouse breath to those detected in system blank samples and human exhaled breath.

Summary

  • 472 chemicals were discovered in mouse breath.
  • 15.47% of chemicals were detected as 'on-breath', distinguishing them from background contaminants using three quantitative criteria.
  • Mouse and human breath shared 49 similar VOCs.
  • On-breath chemicals shared by humans and mice have been connected to biological functions.

Introduction

Exhaled breath contains volatile organic compounds (VOCs) derived from biological activities in the body, making it a possible biomarker for therapeutic usage.

Breath biomarkers have been identified in a wide range of disease areas,1,2 showing that they have the potential to be widely used in next-generation noninvasive diagnostic and monitoring tools. However, the translation of these biomarkers into breath tests for clinical usage is still limited, owing to a lack of uniform techniques and quality standards in the breath research literature. A reliable pre-clinical breath analysis method for animal models would be beneficial to address these issues.

Establishing a breath analysis approach employing mice in a controlled lab setting as a surrogate for human breath can reduce the unpredictability and problems observed in human studies while also providing a deeper understanding of breath as a sampling medium.

This will speed up identifying and validating breath biomarkers for therapeutic applications.

No previous investigations have directly compared baseline breath VOC patterns in mice and humans using the same analytical approach. Given the number of VOCs detectable in human breath, there may be compounds in mouse breath that could be detected in studies to improve translatability to human biology.

Creating sampling and analytical procedures that can provide an adequate signal-to-noise ratio in mouse breath analysis is essential.

Taylor et al. recently established a method for correctly identifying VOCs in the breath of healthy intubated mice and compared them to system-blank samples and VOCs found in human exhaled breath.3

New pre-clinical platform for breath VOC analysis enables rigorous animal model comparison

Video Credit: Owlstone Medical Ltd

Methods

Mouse breath sampling

This study used male C57BL/6JRj mice to collect breath samples. The mouse breath sampling system was created by modifying a standard flexiVent® FX1 small animal ventilator. The study linked an ambient filter and a flexiVent filter to the ventilator via flexible tubing and a sorbet tube for breath collecting.

A schematic showing the mouse breath sampling system

Figure 1. A schematic showing the mouse breath sampling system used in this study. Image Credit: Owlstone Medical Ltd

Over four days, 15 breath samples were taken from the intubated mice. The mice were ventilated for 45 minutes to collect VOC air, with a thorough lung inflating process performed every two minutes. This resulted in roughly 1.8 L of breath.

A clean backdrop is required for optimum untargeted analysis, which maximizes the signal-to-noise ratio and allows for easier identification of VOCs of interest. Before mouse breath collection, 15 system blank samples were taken.

The approach for collecting system blanks was the same as for sampling mouse breath. To track environmental contamination, ambient blanks were collected at the beginning and conclusion of each day in the room where breath collection was performed.

Human breath sampling

A total of 13 healthy volunteers were recruited, and each subject submitted a single breath sample obtained using the ReCIVA® Breath Sampler, as well as a paired system blank sample collected simultaneously. This distinguishes on-breath chemicals from those in the system background.

Breath analysis

The Breath Biopsy® OMNI® analytical method, which included high-resolution thermal desorption gas chromatography and accurate mass spectrometry (TD-GC-MS), was used to examine mouse and human breath samples.

Data analysis

Three metrics have previously been utilized to distinguish between on-breath and background VOCs, and this study employed them to assess the breath sampling method's performance capabilities. These measures are classified as type 1 (standard deviation), type 2 (paired t-test), and type 3 (ROC-AUC).

Results

Identifying on-breath compounds

A total of 472 molecular characteristics were found in mouse breath. The three metrics were utilized to identify substances on the breath from those in the background air. Using all three metrics, 21 on-breath chemicals were discovered for Type 1, 30 for Type 2, and 56 for Type 3.

The compounds detected by the three metrics significantly overlap, resulting in a range of 15-66 on-breath VOCs discovered in mice, depending on how stringently a definition is applied.

The combined metrics increase the likelihood of detecting potentially informative VOCs that can be considered on-breath and provide greater confidence in the outcomes.

Comparing mouse data to human breath data

To determine the transferability of mouse breath VOCs to human VOCs, the same three metrics were used to count the number of on-breath VOCs in human breath. A total of 49 on-breath VOCs were found in human and mouse breath.

The majority of these chemicals were either related to the gut microbiome or came from plants. Examples include dimethyl sulfone and trimethylamine (TMA). Certain molecules appear to be particular to mouse or human breath; for example, methyl nitrate and 2-butanol appeared to be only present on mice's breath.

(A) Trimethylamine (TMA) is an example of an identified on-breath compound in both humans and mice that is significantly different from the background signal. (B) Dimethyl sulfone is on-breath in both humans and mice (Note log axis). (C) Methyl nitrate and (D) 2-butanol are on-breath compounds in mice, but not in humans

Figure 2. (A) Trimethylamine (TMA) is an example of an identified on-breath compound in both humans and mice that is significantly different from the background signal. (B) Dimethyl sulfone is on-breath in both humans and mice (Note log axis). (C) Methyl nitrate and (D) 2-butanol are on-breath compounds in mice, but not in humans. Image Credit: Owlstone Medical Ltd

Discussion

Using animal models for breath biomarker identification enables a more controlled study, minimizing variability due to individual differences, food, and other environmental factors.

The lower probability of false biomarker discovery speeds up discovering promising biomarkers for validation in clinical trials.

Existing literature has established the feasibility of mouse models for breath VOC study, however the number of studies is limited.

The results presented in this study show that three exogenous VOCs commonly linked with environmental sources, xylene, toluene, and styrene, were not identified as on-breath compounds.

This shows that the breath collection method and analytical strategy employed in this investigation allowed for the exclusion of these ambient chemicals.

This study also investigated the possible translatability of the on-breath VOCs detected in mice to those found in human breath. Humans had more VOCs classed as on-breath across all metric kinds than mice.

This is unsurprising given the disparity in relative body size, lung volume, and environmental exposure between mice and humans.

Despite the differences, 49 on-breath VOCs were discovered to be shared by humans and mice, with some of these molecules coming from the microbiome. This is significant because these volatile chemicals are candidates for scientific study into the complex interactions between the microbiota and disease.

The presence of certain frequent on-breath VOCs in both mice and humans suggests that disease-relevant volatile chemicals could be investigated in animal models for human clinical investigations. This strategy provides a promising pathway for converting data from pre-clinical mouse models to clinical human investigations.

Acknowledgments

Produced from materials originally authored by Alastair Taylor, Sylvia Blum, Madeleine Ball, Owen Birch, Hsuan Chou, Julia Greenwood, Shane Swann, Lara Pocock, Max Allsworth, Billy Boyle, and Kerstin Geillinger-Kaestle in Biology Methods and Protocols.

References

  1. Ratiu, I.A., et al. (2020). Volatile Organic Compounds in Exhaled Breath as Fingerprints of Lung Cancer, Asthma and COPD. Journal of Clinical Medicine, 10(1), p.32. https://doi.org/10.3390/jcm10010032.
  2. Kathleen Van Malderen, et al. (2020). Volatomics in inflammatory bowel disease and irritable bowel syndrome. EBioMedicine, 54, pp.102725–102725. https://doi.org/10.1016/j.ebiom.2020.102725.
  3. Taylor, A. et al. (2024). Development of a new breath collection method for analyzing volatile organic compounds from intubated mouse models, Biology Methods and Protocols, 9(1). https://doi.org/10.1093/biomethods/bpae087.

About Owlstone Medical Ltd

Owlstone Medical is developing a breathalyzer with a focus on non-invasive diagnostics for cancer, inflammatory disease and infectious disease, the company aims to save 100,000 lives and $1.5 B in healthcare costs.

The company’s Breath Biopsy® platform has introduced a new diagnostic modality making it possible to discover novel non-invasive biomarkers in breath using a platform with the potential to transition to point-of-care. The award winning ReCIVA Breath Sampler ensures reliable collection of breath samples.

Breath Biopsy is supporting research into early detection and precision medicine with applications in cancer and a wide range of other medical conditions. Highly sensitive and selective, these tests allow for early diagnosis when treatments are more effective and more lives can be saved.


Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Last updated: Jan 9, 2025 at 10:22 AM

Citations

Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Owlstone Medical Ltd. (2025, January 09). Analyzing breath in mouse models using intubated systems. News-Medical. Retrieved on January 09, 2025 from https://www.news-medical.net/whitepaper/20250109/Analyzing-breath-in-mouse-models-using-intubated-systems.aspx.

  • MLA

    Owlstone Medical Ltd. "Analyzing breath in mouse models using intubated systems". News-Medical. 09 January 2025. <https://www.news-medical.net/whitepaper/20250109/Analyzing-breath-in-mouse-models-using-intubated-systems.aspx>.

  • Chicago

    Owlstone Medical Ltd. "Analyzing breath in mouse models using intubated systems". News-Medical. https://www.news-medical.net/whitepaper/20250109/Analyzing-breath-in-mouse-models-using-intubated-systems.aspx. (accessed January 09, 2025).

  • Harvard

    Owlstone Medical Ltd. 2025. Analyzing breath in mouse models using intubated systems. News-Medical, viewed 09 January 2025, https://www.news-medical.net/whitepaper/20250109/Analyzing-breath-in-mouse-models-using-intubated-systems.aspx.

Other White Papers by this Supplier

While we only use edited and approved content for Azthena answers, it may on occasions provide incorrect responses. Please confirm any data provided with the related suppliers or authors. We do not provide medical advice, if you search for medical information you must always consult a medical professional before acting on any information provided.

Your questions, but not your email details will be shared with OpenAI and retained for 30 days in accordance with their privacy principles.

Please do not ask questions that use sensitive or confidential information.

Read the full Terms & Conditions.