Very recently on Twitter, “I have a joke” was trending. The one-liner puns offered by the Twitterati varied from the nonsensical to the mildly excruciating, but a handful of clever offerings earned deserved respect with the coveted likes and retweets numbering into their tens, even hundreds, of thousands. Viewing various contributions inundating my feed, I was half-tempted to offer-up my own ingenious science-related witticism, but I decided to spare my handful of followers the unsolicited groan it would evoke. Besides, the one-liner that came to mind was on a niche area of science that most of my followers likely have only a passing interest in, and the joke was perhaps a little cynical, too.

This blog post is not about Twitter, and certainly not about jokes, but rather the aforementioned niche yet growing field of breath research and the related new reference book, Breathborne Biomarkers and the Human Volatilome, second edition, coedited with Cristina Davis and Joachim Pleil and recently published by Elsevier. Briefly, and rather simplistically, breath research – in the context of the book – is the discipline of searching for chemical compounds in exhaled breath that are indicators (biomarkers) of a specific physiological imbalance (illness) and that can be exploited in a non-invasive, point-of-care breath test, ideally for early-stage diagnostic support. The conceptual charm of this approach to disease screening – and associated high patient compliance – is obvious: there are no painful needles, no messy cups of urine, and samples can be taken on demand, with the promise of immediate results. On top of that, sample material is practically limitless: for context, we exhale an estimated 250 million liters of gas over a lifetime[*], which is quite a lot of sample volume to be throwing away. Before we delve deeper into the subject matter, however, we should take a step back and consider respiration and why our breath has potentially more to offer than might be immediately apparent.

Most of the time we are not consciously aware of our breathing, unless we have a respiratory condition or are specifically focusing on it for one reason or the other (exercise, meditation etc.). Fortunately, the respiratory cycle proceeds subconsciously, so we do not run into problems by forgetting to breathe. In high school biology we learned that the process of respiration has two primary functions, namely to enrich the blood with oxygen (O₂) during inhalation and to remove carbon dioxide (CO₂) waste from the body through exhalation. The ambient air we inhale, which, taken broadly, comprises roughly four parts molecular nitrogen and one part molecular oxygen, is therefore modified fractionally upon exhalation, with a slight reduction in O₂ (from 21 % to about 14 %) and an enrichment of CO₂ (from 0.04 % to 5 %). Together with water vapor (at approximately 6 % volume) and argon from ambient air (just under 1 %), these primary constituents make up more than 99.9 % of exhaled breath.

The remaining fraction of exhaled breath is far more complex, comprising a rich mixture of compounds that are organic in nature and sufficiently volatile to be present in the gas-phase. These VOCs, as they are referred to, are chemically diverse; they are made up mainly of carbon and hydrogen atoms, but many contain oxygen, nitrogen and/or sulfur atoms, among others, all arranged in different structures with varying functional groups. They are also manifold, with recent estimates putting the collective number at almost one-thousand unique compounds in exhaled breath. While the number of different compounds is large, their abundances in breath are low, with concentrations mostly at or below only a few hundred molecules per billion molecules of ‘air’ in any given volume of breath. Further, their biochemical origins are diverse (and often – mostly – unknown), and whereas any one compound can arise from multiple metabolic processes, equally, any one process can generate multiple different compounds. With this knowledge at hand, it can be reasonably hypothesized that perturbations in specific physiological processes can manifest changes in the VOCs generated, qualitatively or quantitatively, or both, and thereby alter the trace gas composition of exhaled breath, which can then be detected. This phenomenon is the premise of exploring and exploiting breathborne biomarkers as indicators of disease.

Interest in exhaled breath as a prospective carrier of disease information has experienced an increasing trend over the past decade or so, but this relatively recent awareness of its potential is predated by several thousand years! In Ancient Greece, the physician Hippocrates of Kos (470-360 BC) – often referred to as the father of modern medicine – described specific odors ‘on the breath’ to indicate a physiological imbalance, such as fetor hepaticus, a foul odor associated with liver dysfunction, or fetor oris, a pungent stench emanating from the mouth, termed halitosis. Odor similarly played a role, in part, in the more recent interest in breath as a diagnostically relevant biological medium, whereby early research leaned on the anecdotal observations of the apparent ability of dogs to ‘smell cancer’. If our canine companions are alerting on a disease using their sense of smell, then it seems reasonable to assume that they must be picking up a unique odor – or, more likely, odor signature – that does not manifest in healthy individuals (canine olfaction is covered extensively in the feature-length Chapter 34 of our new book Breathborne Biomarkers and the Human Volatilome). At this stage it is pertinent to assert that volatile biomarkers are not restricted to exhaled breath, but rather the entire human volatilome, which represents volatile emanations through diverse excretion routes, notably skin (Chapter 25) and urine (Chapter 24). Returning to breath, it is equally worthy of mention here that in addition to the gas-phase, the aerosol fraction of exhaled breath (liquid droplets and particles), which contains semi-volatile compounds and macromolecules such as proteins, is similarly a rich source of potential biomarkers (Chapters 7 and 8), and might equally play a role in the diagnostic capabilities of dogs and beyond.

The words ‘prospective’ or ‘potential’ are to be found frequently in texts on breath research, as they are in the present text of this blog post, too. Depending on context, this interpretation might act to dampen the apparent prospects of breath analysis as a useful tool, which is unfortunate given that successful tests exist, some of which are widespread (outlined in Chapter 1): examples include exhaled nitric oxide (NO) as a parameter in asthma diagnosis (Chapters 4 and 5) and the breath-ethanol (breathalyzer) test for alcohol intoxication (Chapter 29). However, there are legitimate reasons for using the aforementioned terms: breath research is more challenging and complex than the simplistic concept conveys, and multiple different tests have not been forthcoming, to date. More often than not, new breath tests that show promise lack independent cross-validation, or offer sensitivity and specificity values that fall short of established clinical tests (Chapter 36), rendering them uncompetitive. Further, sets of ‘unique’ putative disease biomarkers transpire to be not unique to a specific disease at all, and environmental confounders and the exposome in general affect exhaled breath and complicate matters. In short, the unique disease biomarker has remained elusive, and current understanding is that intra-individual longitudinal changes in breath composition (and volatilome in general) are more likely to deliver clues on state of health.

Recently, the concept of breath analysis as a medical tool has received increasing attention in the media due to its prospects as a non-invasive and rapid approach for detecting COVID-19. Multiple research groups, analytical instrument manufacturers and start-ups are seemingly ‘jumping on the bandwagon’ of breath research in the hope of developing a quick screening tool for the infection as an attractive alternative to the current tests. While research on COVID-19 is still at its early stages, past studies on other respiratory infections have delivered promising results (Chapter 21), although there are yet many hurdles to overcome, so it remains to be seen how quickly a reliable breath-based test for COVID-19 infection can be established, if at all. Additionally, the current pandemic has raised concerns on the challenges of safely collecting and analyzing breath samples from potentially highly infectious patients (and asymptomatic carriers of the virus), which will have widespread repercussions for breath research in general. Experts across and beyond the field are currently developing protocols for safety measures to address these concerns.

It remains to be seen how the current pandemic will affect progress in breath research, but the concept of being able detect illness and infection via a non-invasive, painless and on-demand test remains an attractive proposition. With every exhalation we are transmitting information about our physiological state into the environment, and ‘catching breath’ for beneficial use is an aspiring goal. Breathborne Biomarkers and the Human Volatilome is a compendium of the state-of-the-art in breath research and represents a comprehensive – and hopefully, highly useful and informative – reference work for novices and experts alike in the field.

If you have read this far, it seems only fair that your (presumed) curiosity on my untweeted tweet be quelled, so I will disclose it here, with a disclaimer that I remain a proponent of breath analysis and the potential it offers, as well as an advocate for due diligence in scientific research; both primary motivators for putting this book together. Anyway, here it is:

“I had a joke about a breath biomarker, but it remains elusive”.

About the book

• Presents recent advances in the field of breath analysis
• Includes an extensive overview of established biomarkers, detection tools, disease targets, specific applications, data analytics, and study design
• Offers a broad treatise of each topic, from basic concepts to a comprehensive review of discoveries, current consensus of understanding, and prospective future developments
• Acts as both a primer for beginners and a reference for seasoned researchers

The book is available on ScienceDirect. Want your own copy? Enter code STC320 at the checkout when you order via the Elsevier store to save up to 30%

About the editor

Jonathan Beauchamp (Twitter handle: @_jbeauchamp)is Manager of the Emissions Analytics and Diagnostics Group at the Fraunhofer Institute for Process Engineering and Packaging IVV in Freising, Germany where his research focus lays in exploring VOCs for different applications, including their potential use as disease-specific biomarkers. He has been involved in breath research for over 15 years, examining breath constituents as early indicators of renal graft rejection and performing pharmacokinetics via online analysis of volatile metabolites in exhaled breath, amongst other applications. He is an active board member of the International Association of Breath Research (IABR) and serves as Associate Editor of Journal of Breath Research, the official journal of IABR.

[*] The value is calculated based on a lifespan of 75 years, with an average tidal volume of 400-500 mL and a breathing rate of 15 breaths per minute.