Why should we care about the biology behind exhaled VOCs?

An increasing amount of research is going into detecting volatile organic compounds (VOCs) in exhaled breath as a means to detect diseases and personalize treatment for patients. Here, Rianne Fijten, Senior Scientist at Maastro Clinic, Maastricht, shares her perspectives on the field and explores the advantages of identifying the precise biochemical origins of VOCs.

The potential of exhaled breath analysis is huge, with applications in many fields including, but not limited to, the diagnosis and monitoring of disease. However, as with any field of research that is relatively new, a number of crucial challenges still need to be addressed before clinical implementation is possible. These include the lack of standardization, proper statistical methods, and external validation. Yet, there is a much more pressing and fundamental issue to explore: even though thousands of exhaled VOCs have been measured over the years, the understanding of where VOCs originate from in the human body is lacking (Kwak and Preti, 2011; Jia et al., 2019).

Why should we care about where VOCs come from? Isn’t it enough to know that a predictive model based on VOCs works? The simple fact is that these models can be biased, noisy and carry a lot of (statistical) uncertainties, so it is essential to know the biological origin and function of VOCs to have real confidence in a model, especially when it comes to something as important and potentially life-changing as medical diagnostics.

Exhaled breath is prone to variation between individuals and even from a single individual when samples are taken at different times. For example, the food I ate an hour ago and whether or not I brushed my teeth this morning will influence which VOCs can be found in my exhaled breath and at what levels. Knowing the origins of these VOCs helps us to distinguish between those originating from the human body, and those coming from other sources such as diet or pollution. This is an important distinction as it is unclear whether differences in these VOCs are related to environmental variation or reflect real biological changes resulting from disease.

It is absolutely possible that these ‘external’ VOCs are transformed, taken up or released by the liver, fat tissues and other organs, and could therefore possibly provide an indication of overall health status. But with the current level of research, how can we know for sure? Equally, removing VOCs just because they could come from the environment is not the answer. We need to better understand the processes involved.

Regardless of the underlying reason, knowing where VOCs come from is the first step in getting exhaled breath analysis into clinical practice; a step we as a community are determined to take. First, knowing the origins of VOCs will allow us to exclude any that have no biological meaning and only investigate the ones that are produced, transformed, stored or used otherwise inside the human body. Second, looking further into the future, there is a big opportunity for exhaled breath analysis to not just work in the diagnosis domain, but also in disease or treatment monitoring. Since many of the mechanisms underlying disease progression and its response to treatment are known, finding and targeting VOCs that are directly produced as by-products of these mechanisms would encourage widespread acceptance in the medical community.

Only a handful of VOCs have so far been investigated in detail and linked to biological processes in the human body. For instance, dimethylsulfide is exhaled as a result of fetor hepaticus and acetone is excreted via the lungs during ketoacidosis in diabetes (Tangerman et al., 1994; Laffel, 1999). Yet, for most other VOCs, only hypothetical endogenous sources have been proposed yet not confirmed.

In the past few years an increasing amount of research has gone into investigating VOC signatures on a cellular level in the lab using a variety of strategies. Yet these strategies pose a number of new challenges. For instance, comparing cells to cell culture medium (Filipiak et al., 2008; Mochalski et al., 2015; Baranska et al., 2013) may provide insight into overall cell metabolism, but cannot clarify whether VOCs are produced inside the cell or originate from its immediate environment. Another example is the comparison between non-cancerous and cancerous cells (Lavra et al., 2015; Haick et al., 2012; Schallschmidt et al., 2015). Cancer cell lines have changed so much on a molecular level that it is impossible to state that differences between the two are related to specific cancer processes.

Neither of the approaches described above allow us to pinpoint the exact origin of a VOC. If we are to progress, we should seek to challenge cells with an external stimulus such as a carcinogen or drug (Fijten et al., 2017) and subsequently measure cellular changes on multiple levels (DNA, RNA, protein, metabolite) to get a global picture of what happens in the cell and to provide clues to pathways or processes that influence exhaled VOC levels. These newly generated hypotheses can then be further investigated by manipulating key DNA, RNA, protein or (non-volatile) metabolite targets.

You may ask why it isn’t enough to link a VOC to a pathway or process instead of pinpointing the exact gene, protein or metabolite from which a VOC is produced? Linking a VOC to a pathway is the logical first step, but should not be the end. In order to unlock the full potential of exhaled breath analysis, we need to take it into more complex scenarios like disease or treatment monitoring (think lack of response to treatment or monitoring for exacerbations). Since a lot is known about which genes/proteins are the main culprits in disease and treatment responses, pinpointing the exact origin of a VOC would enable use to develop clinically-relevant evidence-based monitoring tools for specific scenarios.

To clarify this concept, let’s look at cancer. If we knew of a VOC produced by proteins that cause resistance to chemotherapy, we could monitor that VOC daily to track the effectiveness of chemotherapy. A lack of response would be picked up quickly and would allow doctors to up the dose or change drugs before it’s too late, thus allowing doctors to use exhaled breath analysis for personalized medicine.

Only through the detailed approaches described above will we be able to say with high certainty where a specific VOC comes from within the human body. Pinpointing the biological origins of VOCs in this way will help to validate existing breathomics results and deliver the kind of reliable and non-invasive clinical diagnostic tests needed to detect more diseases early and save more lives.

In 2018, Rianne was one of the speakers at the inaugural Breath Biopsy conference in Cambridge. Hear more about Rianne in her introductory video from the conference and watch her presentation in full.

References

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Fijten, R.R.R. et al. (2018) Exposure to genotoxic compounds alters in vitro cellular VOC excretion. J. Breath Res., 12. http://dx.doi.org/10.1088/1752-7163/aa9080

Filipiak, W. et al. (2008) Release of volatile organic compounds (VOCs) from the lung cancer cell line CALU-1 in vitro. Cancer Cell Int., 8, 17. http://dx.doi.org/10.1186/1475-2867-8-17

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