Breath-based biomarker discovery for asthma with the VOC Atlas

This article is based on a poster originally authored by H. Davies, M. Kerr, M. Ball, H. Chou, W. Arulvasan, J. Greenwood, P. Gordon, O. Birch, R. Mohney, M. Allsworth, B. Boyle, and A. Khider.

Breath comprises a complex mixture of volatile organic compounds (VOCs) that originate from local biological processes or arise systemically. In the context of asthma, inflammatory and immunological mechanisms can alter VOC profiles, underscoring the potential of breath analysis for non-invasive biomarker discovery.

A significant challenge in breathomics is establishing a connection between VOCs and the biological pathways they represent. Many VOCs reflect processes that are less well characterized than metabolites from established matrices such as plasma or tissue.

Without robust reference data, it is difficult to differentiate VOCs that signify pathophysiological relevance from those resulting from normal biological or behavioral variability.

The Breath Biopsy VOC Atlas^® serves as a curated reference dataset of VOCs detected across a representative population, facilitating contextualization against baseline biological variability.

This aids in biological interpretation and the provisional classification of candidate biomarkers:

• Pathophysiology-Associated Biomarkers (PABs): VOCs present in the Atlas cohort but altered under pathophysiological conditions, potentially reflecting broadly responsive processes such as inflammation or metabolic stress.
• Pathophysiology-Specific Biomarkers (PSBs): VOCs not detected in the Atlas cohort, provisionally associated with distinct pathophysiological mechanisms.

The study presented here classifies asthma-associated volatile organic compounds (VOCs) as Pathophysiology-Associated or -Specific Biomarkers by comparing literature-reported compounds with a representative baseline from the Breath Biopsy VOC Atlas^®. This approach enhances biological interpretation and informs the development of breath-based diagnostics.

Literature review

A systematic search identified 39 studies reporting asthma-associated breath VOCs utilizing chemically resolved methods.

The studies exhibited variability in collection approaches (Tedlar^® bags, ReCIVA^® Breath Sampler, direct sampling), analytical platforms (GC-MS, GC×GC-MS, SIFT-MS, SESI-MS), and patient demographics (children, adults, elderly; mild to severe asthma).

Approximately 200 unique candidate VOCs were extracted for comparison (see Figure 2a).

VOC Atlas comparison

The Breath Biopsy VOC Atlas^® served as a reference dataset comprising breath VOCs detected across a representative population (demographics outlined in Table 1), including healthy individuals and those affected by disease without specific disease enrichment. Samples were collected using the ReCIVA^® Breath Sampler (as shown in Figure 1) and analyzed by TD-GC-MS.

Defining VOC presence ("on-breath")

A VOC was considered “on-breath” if it met the following criteria:

• Significantly elevated above paired system background (p < 0.05, paired t-test)
• Detected in more than 10 samples across the Atlas cohort
• Fold difference > 2

Biomarker classification

Candidate VOCs were classified as follows:

• Pathophysiology-Associated Biomarkers (PABs): Detected in the Atlas cohort but reported as altered in asthma.
• Pathophysiology-Specific Biomarkers (PSBs): Not detected in the Atlas cohort, provisionally interpreted as specific to an aspect of asthma-related pathophysiology.

Figure 1. Schematic of the ReCIVA^® Breath Sampler. The device controls the inhalation of clean air and targeted collection of exhaled breath onto sorbent tubes, using CO₂ and pressure sensors to monitor the breath cycle and ensure sample integrity. Image Credit: Owlstone Medical Ltd

Table 1. Age and sex distribution of participants in the Breath Biopsy VOC Atlas^® reference dataset (n = 94), used to define representative baseline breath VOC profiles for biomarker classification. Source: Owlstone Medical Ltd

Results

Asthma VOCs from literature

A systematic search conducted in accordance with PRISMA guidelines (as shown in Figure 2a) identified 315 papers related to asthma and breath VOC analysis.

After abstract screening, 39 publications were selected for detailed review, ultimately resulting in 21 studies included for compound identification.

Studies employing electronic nose technology, exhaled breath condensate, or lacking healthy comparator groups were excluded.

The included studies drew from large-scale clinical cohorts such as ADEM, ALLIANCE, EMBER, U-BIOPRED, MICROMAP, and TASMA, featuring diverse patient populations and methodologies:

• Patient focus: 10 studies examined asthmatic children, one investigated elderly patients, and 13 concentrated on adults.
• Disease severity: Five studies addressed severe asthma exclusively, while two focused on mild-to-moderate cases.
• Collection and analysis: Six studies utilized filtered air, 16 employed Tedlar^® bags, and four included internal reference standards for GC-MS analysis.

In all, 225 unique VOCs were identified as potentially discriminatory between asthmatic and non-asthmatic individuals. Of these, 47 VOCs appeared in multiple publications, enhancing confidence in their relevance to asthma.

Figure 2 b illustrates the chemical classification of VOCs, with alkanes, alkene alcohols, cyclic hydrocarbons, aldehydes, ketones, and carboxylic acids representing the predominant chemical classes.

Figure 2. a) PRISMA-style flow diagram summarizing the literature screening process for asthma-related breath VOC studies. An initial search identified 315 abstracts; after abstract screening and full-text review, 21 studies were included for compound identification. b) Distribution of chemical classes among volatile organic compounds (VOCs) identified from asthma-related breath studies. Major classes included alkanes, alcohols, alkene, cyclic hydrocarbons, carboxylic acid and ketones. Minor chemical classes (38 classes in total), accounting for less than 4 % individually, were grouped as ‘minor classes’. Image Credit: Owlstone Medical Ltd

Asthma VOCs from literature

A total of 225 volatile organic compounds (VOCs) have been identified in the literature, of which 46 were also present in the Breath Biopsy VOC Atlas^®.

• 36 VOCs were detected in the representative Atlas cohort and classified as Pathophysiology-Associated Biomarkers (PABs)
• 10 VOCs were not identified in the Atlas and were provisionally classified as Pathophysiology-Specific Biomarkers (PSBs)

Representative examples are illustrated in Figure 3:

• Potential pathophysiology-specific biomarkers, such as nonane and octane, which have been previously associated with lipid peroxidation¹
• Potential pathophysiology-associated biomarkers, including acetone and furfural, which have been linked to fatty acid metabolism and dietary intake^2,3

Beyond presence/absence classification, the VOC Atlas provides quantitative concentration data (in ppb), facilitating the interpretation of observed changes within the context of baseline biological variability.

Figure 3. Representative boxplots showing breath and blank concentrations for candidate VOCs. Nonane and octane are potential disease-specific biomarkers, while acetone and furfural are potential disease-associated biomarkers. Concentrations are plotted as log concentration in breath (ppb). Boxes represent the interquartile range (IQR), whiskers extend to 1.5× IQR, and outliers are shown individually. (n = 94 healthy volunteers). Image Credit: Owlstone Medical Ltd

Conclusions

The Breath Biopsy VOC Atlas^® serves as a representative reference dataset, enabling the contextualization of breath VOCs relative to baseline biological variability.

A comparison of literature-reported asthma biomarkers with Atlas data has led to the provisional classification of 46 VOCs as either Pathophysiology-Associated Biomarkers (PABs) (n = 36) or Pathophysiology-Specific Biomarkers (PSBs) (n = 10).

This classification framework enhances the interpretation of VOCs concerning their potential biological origins:

• PSBs, such as nonane and octane, may indicate processes like lipid peroxidation associated with asthma pathophysiology
• PABs, including acetone and furfural, likely reflect modifiable or systemic factors such as metabolism and diet

However, significant methodological challenges persist. The Atlas was developed using standardized breath collection (ReCIVA^® device) and TD-GC-MS analysis, whereas the literature studies reviewed often employed varied methodologies, such as Tedlar^® bag sampling.

Consequently, it is essential to acknowledge that variability in "on-breath" VOC detection may partially result from differences in sampling and analytical techniques, rather than a true biological absence or presence.

Ongoing efforts to standardize breathomics methodologies are anticipated to diminish these discrepancies over time. As the Breath Biopsy VOC Atlas^® continues to expand, both in participant numbers and across various disease contexts, the capacity to interpret VOC changes within their biological framework will be further strengthened.

References and further reading

1. Ibrahim, W., et al. (2021). Breathomics for the clinician: the use of volatile organic compounds in respiratory diseases. Thorax, 76(5), pp.514–521. https://doi.org/10.1136/thoraxjnl-2020-215667.
2. Wisenave Arulvasan, et al. (2024). High-quality identification of volatile organic compounds (VOCs) originating from breath. Metabolomics, 20(5). https://doi.org/10.1007/s11306-024-02163-6.
3. Ratcliffe, N., et al. (2020). A mechanistic study and review of volatile products from peroxidation of unsaturated fatty acids: an aid to understanding the origins of volatile organic compounds from the human body. Journal of Breath Research, 14(3), p.034001. https://doi.org/10.1088/1752-7163/ab7f9d.
4. Anderson, J.C. (2015). Measuring breath acetone for monitoring fat loss: Review. Obesity, 23(12), pp.2327–2334. https://doi.org/10.1002/oby.21242.
5. Wishart, D.S., et al. (2021). HMDB 5.0: the Human Metabolome Database for 2022. Nucleic Acids Research, 50(D1), pp.D622–D631. https://doi.org/10.1093/nar/gkab1062.

Acknowledgments

Produced from material originally authored by H. Davies, M. Kerr, M. Ball, H. Chou, W. Arulvasan, J. Greenwood, P. Gordon, O. Birch, R. Mohney, M. Allsworth, B. Boyle, and A. Khider from Owlstone Medical Ltd.

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.

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Last Updated: Sep 16, 2025