Background
There is a need for disease biomarkers that reflect the activity of the underlying pathogenetic pathways that characterise lung disease. These could help diagnose and monitor lung disorders besides providing information on the efficacy of treatment.
In the last decades breath analysis, and particularly the measurement of exhaled nitric oxide (NO), has received a lot of interest because its measurement is simple and its breath levels reflects airway inflammation [
1]. Volatile organic compounds (VOCs) have also been shown to be elevated in inflammatory diseases [
2‐
5], and also to reflect the activity of specific metabolic pathways. For example, acetone is linked to dextrose metabolism and lipolysis [
5,
6], whereas exhaled isoprene correlates with cholesterol biosynthesis [
7], and exhaled levels of sulphur-containing compounds are elevated in liver failure [
5,
8] and allograft rejection [
9]. Different VOC profiles have been identified in several diseases, such as lung cancer [
10‐
13], asthma [
14] and COPD [
15] compared to controls, and the measurement of VOCs has been suggested as a tool for early detection and monitoring of disease.
Contrary to the measurement of exhaled NO, which has been carefully standardised, the parameters potentially affecting VOCs levels in the breath have received little notice [
16,
17]. The lack of standardization of the previously published methods and the poor knowledge of the variables that may affect VOCs have hindered the use of these gases in research. As a result, even though back in 1971 Pauling et. al. [
18] detected more than 200 VOCs in the human breath, to date, breath analysis is still an underused research tool with no current clinical application.
In view of the potential usefulness of VOCs as markers of lung disease we developed a simple method for their measurement using Proton Transfer Reaction Mass Spectrometry (PTR-MS) and crucially, we standardised the breath collection and studied the effect of different breath parameters such as exhalation flow and breath hold on the levels of the measured gases.
Ethanol and acetone were chosen as test gases for standardization because of their ease of measurement and low concentrations in the environment.
Discussion
We standardised breath sample collection and developed a new method for VOC analysis. Because exhalation flow rate and breath hold may affect the expired levels of ethanol and acetone, we suggest that controlling breathing parameters is required to reduce errors and improve the reproducibility of VOC measurements.
Contrary to the measurement of exhaled NO which has been extensively investigated and standardised as described in joint ERS/ATS guidelines [
24], only two preliminary studies have so far investigated the breathing parameters potentially affecting the levels of VOCs in the exhaled breath [
16,
17]. Notably, none of the so far published clinical studies controlled or investigated the effect of breathing parameters on VOC levels. In the current manuscript, we used a previously developed device for exhaled breath collection which allowed us to analyse separately the effect of different breathing manoeuvres.
Ethanol breath levels were significantly decreased at higher exhalation flow rates. As the central airway axial diffusion is an important factor determining flow dependency [
25] this may indicate that ethanol has a significant axial diffusion and the central airways contribute significantly to total ethanol breath levels. Three subjects with low baseline ethanol levels showed no exhalation flow dependency, suggesting that higher gas levels may be more sensitive flow rate reduction. The flow rate of 5 L/min may be a more suitable standard as it is more comfortable. Interestingly, contrary to ethanol, acetone breath concentrations were not exhalation flow rate dependent indicating a poor contribution of the central airways to the total acetone concentrations in the breath. Alternatively, the lack of exhalation flow dependency may result from back diffusion into the tissues allowing the gases to be washed away by the blood stream before axial diffusion can occur. The lack of exhalation flow dependency of acetone shown in a previous study [
16] may be related to the use of much higher exhalation flow rates (15 l/min) which may have cancelled the effect of this variable. The significant effect produced by breath hold on the levels of acetone supports the theory that this gas may have an elevated airway uptake as opposed to ethanol which was not significantly affected by breath hold and therefore may have a higher central airway production/diffusion ration as suggested by its significant flow dependency and lack of reuptake.
Dead space air is mostly a mixture of nasal and ambient air. It is reassuring that the inclusion of dead space air in the breath analysis did not affect the final VOC levels as this suggests low upper airways and nasal VOC concentrations as well as low environmental levels of the measured gases. Even though dead space air did not affect the level of the gasses measured, we advise discarding its collection to reduce the possibility of sporadically elevated environmental levels of acetone and ethanol.
The breathing parameters studied may affect exhaled gases differently depending on their biological and physical properties, therefore, it is crucial to underline that other gases not measured in the current study, with different biophysical properties may be affected. Therefore, we suggest that exhaled breath for VOC analysis should always be collected in a standardised manner as described in the current manuscript.
The presence of water vapour in the exhaled breath presents a technical challenge as it interferes with the measurement of other gases with molecular weights close to that of water. In order to reduce this error, we have limited our analysis to gases with molecular weight dissimilar from water. This approach together with a controlled collection of the exhaled breath has provided an excellent inter and intra- session reproducibility.
Because VOCs are present in the exhaled breath at very low concentrations, another potential error may derive from contamination with environmental air. Some authors have minimised this problem by concentrating the exhaled breath [
26‐
28] or passing it through a scrubber [
29,
30]. Notably, the lack of a significant correlation between exhaled breath VOCs and the concentration of the same gases in concurrent ambient samples may indicate that environmental contamination was not relevant in our study. We controlled environmental contamination by reducing air leaks in the tubing system and carefully sealing the reservoir where the exhaled breath was collected.. Furthermore, exhaling against a resistance producing a mouth pressure of at least 5 cm H
2O may have reduced contamination of the exhaled breath with nasal and environmental air by closing the soft palate as previously described [
20].
Other factors which potentially influence the levels of VOCs in the breath are diet [
31‐
34] and alcohol consumption [
35]. As expected, alcohol rapidly and significantly increased the levels of exhaled ethanol which gradually decreased and returned to baseline levels 3.5 hours after wine consumption. This supports the hypothesis that breath ethanol levels reflect a metabolic process and not alcohol vapours coming from the stomach immediately after drinking wine.
Previous reports have shown that the diet may affect the levels of ethanol [
36,
37]. In our study there was a tendency for higher levels of breath ethanol 30 minutes after the ingestion of food however this was not statistically significant. Interestingly, there was a trend for increased ethanol levels following breakfast rather than lunch even though none of them was statistically significant. This may be related to the higher content of carbohydrates in the former which may have been metabolised to form ethanol.
Competing interests
I confirm that I have no competing interests. I did not receive any fees or funding or have stocks or shares in organizations that may benefit from the publishing of this paper. I am not applying for patents related to the content of this manuscript. I do not have any non-financial competing interests.
Authors’ contributions
AB and KP equally contributed to the manuscript. They both participated in developing the concept and design of the study. In addition, they collected and analysed the data and edited the final draft of the manuscript. RLS built the VOC analyser used in this study, he also participated in the design of the study and writing and editing of the final draft. IH, SAK and PJB participated in the development of the concept, study design, writing and editing of the final draft. PP had a major role designing the study, he also participated in patients recruitment, helped data analysis and supervised the progress of the study, in addition, he wrote the first draft of the manuscript and re edited it following all the other authors input. All authors read and approved the manuscript.