Breath VOC Profiles are Impacted by Dynamic Variables

Volatile organic compounds (VOCs) are a promising class of compounds to serve as next-generation biomarkers for many physiological processes in the body. A major clinically relevant advantage of VOCs is that they can be non-invasively sampled from exhaled breath. The ultimate goal we envision is the ability to analyze the abundance of VOCs in a breath sample and compare the levels to a reference range to non-invasively diagnose, screen for, or monitor the development of disease.

Many metabolic processes in the body such as inflammation produce VOCs that are detectable in breath, and therefore certain changes in breath VOC composition will be clinically relevant. However, the VOCs in the body can originate from multiple sources (including those from the environment through inhalation, and the diet). This means that eventual VOC abundance in the breath is impacted by many variables that are important to understand before they can be used for clinical interpretation.

Measurements of the dynamic behavior of VOC profiles in the exhaled breath in response to normal variation in human biology are a key starting point to better understand VOCs as potential biomarkers. In this case study, we explore the impact that under-appreciated variables such as breathing patterns and biological rhythms have on breath VOC levels. We will overview the literature that indicates what is known to impact VOC levels in the breath that are unrelated to disease, and other more well–understood variables such as age, BMI, gender, diet, and smoking.

Changes in breathing patterns

Several studies have associated different breathing patterns with the levels of VOCs detectable in exhaled breath (1–7), likely due to ultimately altering gas-exchange kinetics in the lungs. This means that VOCs of exogenous origin are likely to be less impacted by this effect than endogenous VOCs. Certain physicochemical properties such as volatility, solubility, and gas partition coefficients can also impact which VOCs are most impacted by changes in breathing patterns.

Evidence from the effect of forced expiratory volume (FEV) maneuver on the composition of exhaled breath supports this, as compounds such as acetonitrile were less affected than isoprene, which is known to originate endogenously from skeletal muscle and has low aqueous solubility and high vapor pressure (8,9). In comparison, VOCs with high aqueous solubility such as acetone are significantly less impacted by changes in breathing dynamics (Figure 1) (6).

Figure 1 – Image adapted from Miekisch et al., 2022 (10).

In order to control for this effect in breath studies, consistent normal breathing patterns are important to maintain while collecting a breath sample. However, it is a known effect that upon asking participants to breathe normally, they actually begin to hyperventilate, measurable through lowering end-tidal carbon dioxide levels Although the precise impact that this will have on VOC concentrations is not known, the solution for this potential covariable is real-time tracking of participants’ breathing patterns during breath collection, which can then be controlled. Since this paper was published 20 years ago, there have been technological advances in terms of breath collection, including the development of the ReCIVA® Breath Sampler.

The ReCIVA® Breath Sampler has built-in carbon dioxide and pressure sensors that monitor and learn the breath patterns of each subject in real time and only activate the pumps that draw air into the adsorbent tubes to capture the desired breath fraction (i.e. the end-tidal breath). All of this metadata is saved and can be utilized alongside VOC data for accurate interpretation of the results of a study.

Biological rhythms affect VOC profiles

A dizzying number of compounds are produced all over the body constantly, through the multitude of metabolic processes ongoing, so unsurprisingly, the abundance of VOCs is hypothesized to dynamically shift depending on the metabolic state of the body. Levels of hydrogen in the breath are known to be associated with circadian rhythms, with levels of hydrogen high in the morning and decreasing to their lowest by 16:00, before rising again after dinner and staying high throughout the night (12).

Inflammatory diseases such as asthma are also known to be linked to circadian rhythms, with narrowing of the airway and eosinophilic inflammation being at its peak early in the morning – associated with an increase in symptoms and asthma deaths at this time (13–15). Associated with that, it has also been reported that fractional exhaled nitric oxide (FENO) and VOCs in breath in those with asthma also fluctuated throughout the day (16).

This demonstrates that time of day, and other normal cycles are important to consider when designing and interpreting breath analysis data, especially in untargeted early studies. Participants should ideally be sampled at the same time of day regardless of cross sectional or longitudinal study design, to reduce inter and intra variability.

The menstrual cycle has also been associated with changes to the VOC profile of exhaled breath (17). In this study, it was shown that the most significant changes were at ovulation, with ammonia, isoprene, and dimethyl sulfide significantly lower in abundance, and acetone significantly raised in abundance (P-value ≤ 0.005). The effect of the menstrual cycle on VOC profile is often neglected as studies generally use gender as a covariate. When designing breath analysis studies this factor should be added into further consideration, especially in studies with a focus on women’s health.

This highlights the importance of gaining and tracking demographic factors of breath study participants, as well as the time of day that breath samples were collected. Recording demographic data, even if precise demographic matching is not a possible means of statistical analysis, can be conducted to investigate the impact of these potential covariables in your study.

Conclusion

It is important to note that almost all biomarkers currently in use are impacted by other variables that clinicians or researchers need to consider when interpreting the data. Knowledge of what factors can impact different VOC levels in the breath is a crucial step toward the translation of breath VOCs into clinical practice and does not detract from the potential usefulness of breath as a biomarker platform.

For example, blood glucose levels can provide diagnostic information regarding diabetes, but must be taken after a strict fast for 8 to 10 hours to be reliable. One of the other beneficial attributes of breath is that it can also strengthen the use of a breath test. Dynamic longitudinal data of how VOCs change over time can easily be collected, as breath is produced by the body almost constantly as a waste product and a large volume can be collected serially.

This means that it isn’t just one static measure of VOC abundance that can be utilized in clinical practice, the kinetics of how a VOC changes over a set time can be interpreted. This can be combined with a probe-based approach to target specific pathways of interest, such as our LIBRA test using limonene to screen for liver disease.

The Breath Biopsy Collection Station, featuring the ReCIVA® Breath Sampler and CASPER® Portable Air Supply enables reliable, reproducible collection of breath VOCs for a wide range of applications. Using an advanced analysis platform built on GC-Orbitrap™ technology we can support high-resolution targeted and untargeted breath analysis for biomarker discovery.

Our team of experts in breath analysis can perform statistical analyses and provide a detailed report of results to help identify and develop biomarkers of interest for a range of applications. In order to better understand the composition of human breath both in terms of understanding the diversity present in a healthy population, and the differences in different disease states, we have developed the Breath Biopsy VOC Atlas®, a catalog of identified and quantified volatile organic compounds (VOCs) found in exhaled breath.

The Atlas also provides insight and scientific context to identified compounds to enable the confident selection of candidate biomarkers for a variety of diseases. Sign up to gain exclusive access to the Atlas.

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