Sleep Medicine Reviews 27 (2016) 1e8

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Sleep Medicine Reviews

journal homepage: www.elsevier.com/locate/smrv

CLINICAL REVIEW

Exhaled breath analysis, a simple tool to study the pathophysiology of

obstructive sleep apnoea
Andras Bikov a, *, James H. Hull b, Laszlo Kunos a
a
b

Department of Pulmonology, Semmelweis University, Budapest, Hungary

Department of Respiratory Medicine, Royal Brompton and Hareeld NHS Foundation Trust, London, UK

a r t i c l e i n f o

s u m m a r y

Article history:
Received 27 April 2015
Received in revised form
30 July 2015
Accepted 30 July 2015
Available online 5 August 2015

Accelerated airway inammation may play a crucial role in the pathophysiology of obstructive sleep
apnoea (OSA); however this phenomenon has been investigated only in a limited number of studies. The
analysis of exhaled breath represents a promising, non-invasive tool to evaluate airway inammation in
this context. The knowledge on exhaled biomarkers in OSA has been growing with an emerging number
of methodological studies which help to interpret exhaled breath data. This article not only summarises
the results of studies on exhaled breath condensate (EBC) biomarkers, exhaled volatile compounds and
exhaled monoxides in OSA, but also aims to critically review methodological limitations and provide
some guideline for further research.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Breath test
Exhaled breath condensate
Exhaled nitric oxide
Obstructive sleep apnoea

Introduction
Obstructive sleep apnoea (OSA) is a common disorder which is
characterised by intermittent and repetitive collapse of the upper
airways and subsequent failure in oxygenation. Repeated mechanical trauma due to upper airway occlusion, accelerated systemic inammation and intermittent nocturnal hypoxia may all
lead to heightened airway inammation and oxidative stress [1].
Currently, there is no clear evidence if airway inammation is only
a consequence of the aforementioned phenomena, or has any
pathogenic effect. In chronic obstructive pulmonary disease (COPD)
persistent airway inammation likely acts to drive and maintain
systemic inammation and thus subsequent comorbidities [2].
Likewise, it is possible that sustained airway inammation in OSA
also contributes to the complex pathophysiology of this disorder
and it is therefore desirable to further evaluate the nature of this
relationship in detail.
Unfortunately, the measurement of inammation and oxidative
stress in the airways is not straightforward. Systemic samples such
as blood or urine do not accurately represent processes in the
airway compartment and thus some means of sampling and
assessing the airway environment is necessary. Direct sampling of
* Corresponding author. Department of Pulmonology, Semmelweis University,
1/C Dios arok, Budapest, H-1125, Hungary. Tel.: 36 206663433; fax: 36
12142498.
E-mail address: andras.bikov@gmail.com (A. Bikov).

airway samples using bronchoscopy (i.e., bronchial biopsy or

bronchoalveolar lavage) is potentially harmful and may indeed
induce inammation itself [3]. Sputum induction is a promising,
semi-invasive method to sample the airways and several studies
have now demonstrated elevated markers of inammation and
oxidative stress in induced sputum samples from individuals with
OSA [4e7]. However, the process of sputum induction has limitations, including the fact that samples can only be reliably obtained
in t and cooperative subjects and results can often be confounded
by salivary/oral contamination. Moreover, the process in itself, may
precipitate low-grade airway inammation and can induce bronchoconstriction in some patients; therefore a delayed time (>24 h)
may be needed before repeated sampling [8].
In contrast to these techniques, exhaled breath analysis offers a
promising alternative method for sampling the airway compartment. The technique is a totally harmless and non-invasive way to
evaluate the airways and sampling can easily be performed in short
time intervals, including in acute illness and in paediatric populations. Moreover, breath can be collected before and after sleep to
study sleep-related changes, pertinent in OSA [9e14]. Moreover,
Amann et al. developed a sampling system to analyse exhaled
volatile organic compounds (VOCs) even during sleep [15,16].
The measurement of exhaled biomarker can be divided into
three separate groups by their clinical and physical properties.
Firstly, exhaled monoxides (i.e., nitric oxide and carbon monoxide)
for which there are well validated measurement techniques, are

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A. Bikov et al. / Sleep Medicine Reviews 27 (2016) 1e8

of EBC in the context of OSA. More importantly, new methodological studies on exhaled breath analysis are also available now,
thus facilitating the interpretation of some of the historical work in
this eld. The aim of this article is to provide a state of the art review of the place of exhaled breath measurement in OSA and its
potential to investigate the pathology of this disease.

Abbreviations
AHI
ALF
BMI
CO
COPD
CPAP
EBC
ELISA
FENO
H2O2
ICAM
IL
LTB4
MS
NO
OSA
PGE2
TNF
VOC

apnoea/hypopnoea index
airway lining uid
body mass index
carbon monoxide
chronic obstructive pulmonary disease
continuous positive airway pressure
exhaled breath condensate
enzyme linked immunoassay
fractional exhaled nitric oxide
hydrogen peroxide
intercellular adhesion molecule
interleukin
leukotriene B4
mass spectrometry
nitric oxide
obstructive sleep apnoea
prostaglandin E2
tumour necrosis factor
volatile organic compound

Exhaled nitric oxide and carbon monoxide

already in routine clinical use. Secondly, the measurement of water

soluble particles, which are liberated from the airway lining uid
(ALF) in the form of droplets in the exhaled mist and can thus be
analysed with a technique called exhaled breath condensate (EBC).
Finally, thousands of gaseous molecules, found in either nano- or
picomolar concentrations in human breath, can be measured via
direct (i.e., mass-spectrometry) or indirect (i.e., electronic nose)
methods. The key similarities and differences between the techniques are summarised in Table 1.
The data available on exhaled breath analysis in OSA has been
previously summarised in a number of review articles [17e19].
Several of these papers describe exhaled biomarker ndings and
their pathophysiological relevance. However, a recent scoping review, performed with the aim of identifying biomarkers with key
diagnostic potential in various biological matrices, found only a
limited number of exhaled biomarker studies passed strict quality
control criteria [20]. Since the last comprehensive review in 2012
[19], several studies have been now published detailing the utility

The pluripotent role of systemic and airway nitric oxide (NO) in

the pathophysiology of OSA has been summarised previously in a
review by Haight and Djupesland [21]. In this article we therefore
focus only on exhaled NO measurement.
Nitric oxide is the most validated biomarker in exhaled air and
its measurement has become of increasing interest over the last
couple of decades. Fractional exhaled NO (FENO) is now used as a
validated biomarker in the clinical setting of obstructive airway
diseases, especially in asthma. The direct correlation between FENO
and sputum eosinophil level has made FENO a potential marker to
predict worsening of asthma control and steroid responsiveness.
The currently valid guidelines for FENO analysis, issued in 2005 [22],
recommend measurement of FENO at a ow rate of 50 ml/s.
FENO has been measured in numerous studies in OSA, but with
contradictory results. Elevated [5,12,13,23e26] or unaltered
[11,14,27e29] levels were reported when OSA was compared to
health. Because of standardised methodology these studies are
comparable except for the study by Petrosyan et al. where the authors used higher (e.g., use of 250 ml/s as opposed to 50 ml/s) ow
rates [26], and the study by Olopade et al. where expiratory ow
rates were not controlled [11]. In general, FENO values tend to be
higher in individuals with OSA than in healthy individuals (Table 2).
If measured at different expiratory ows it is possible to divide
exhaled NO results into alveolar and bronchial production components [30]. A study investigating alveolar NO showed lower
levels in OSA compared to health with a signicant increase after
continuous positive airway pressure (CPAP) treatment [28]. In
another study, bronchial NO levels were higher and alveolar NO
levels were lower in OSA, but CPAP increased only alveolar NO
levels [24]. In this study, an inverse correlation was found between
alveolar NO and apnoea-hypopnoea index (AHI) [24]. In contrary, a
recent study showed elevated alveolar NO levels in OSA without
any difference in bronchial NO [29]. In addition, a positive relationship was reported between alveolar NO and AHI as well as body

Table 1
Comparison of exhaled monoxide, EBC and exhaled volatile compound measurements.

Level of validation

Exhaled monoxides

Exhaled breath condensate (EBC)

Well-validated, especially exhaled nitric

oxide.

Considerable inter-substance difference in

the level of validation.

Exhaled volatile compounds

Validation studies are ongoing or have only

recently been nished. Still no consensus on
the best analytical approach.
No international recommendations are
Number of
Numerous on exhaled nitric oxide, none on One European Respiratory Society/
American Thoracic Society task force article. issued.
guidelines/recommendations exhaled carbon monoxide.
A European Respiratory Society task force
Another document by the European
A European Respiratory Society task force
document is under development.
Respiratory Society is under development
document is under development.
Used still only in research
Used still only in research
Currently used in routine
Exhaled nitric oxide is used in asthma
clinical practice
management.
Exhaled carbon monoxide can be used to
identify smoking avoidance before lung
transplantation
Various factors inuence individual
The effect of some sampling and storageInuencing factors
Very well dened, and include
mediators differently
related factors is general on most of the
Age
substances.
Exhalation ow rate
The inuence of other factors (i.e.,
Breath hold
humidity) depends on the analytical
Atopy
technique.
Smoking
Comorbidities

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A. Bikov et al. / Sleep Medicine Reviews 27 (2016) 1e8

in individuals with OSA which was not related to obesity. In

contrast, no difference was seen in exhaled CO after CPAP therapy
[26].

mass index (BMI) [29]. The same study comparing evening and
morning FENO levels showed a signicant rise in alveolar NO in OSA
patients [29].
Studies investigating the effect of sleep on FENO are inconsistent.
An overnight increase in FENO has been reported in some studies in
OSA [11,12], however in another study this nding applied only in
obese patients with OSA [14]. The study by Olopade et al. reported
an elevation even in healthy volunteers [11]. In contrast, FENO did
not change after night even in OSA in the studies by Przybylowski
et al. [13] and Hua-Huy et al. [29]. No difference was observed
between the morning and afternoon FENO values [31] in children.
FENO was directly related to AHI in some studies [5,23,24],
however other studies have failed to reveal such a relationship [27].
In children there was a tendency for a correlation between FENO
levels and AHI [31]. CPAP treatment reduced the levels of FENO
signicantly in some [12,24,26] but not all studies [28].
Obesity is a risk factor for OSA and may independently alter
airway inammation [32]. Studies evaluating the relationship between FENO and BMI in healthy and asthmatic populations have
shown inconclusive results. Both signicantly negative [33] and
positive [34e37] correlations between FENO and BMI have been
reported in healthy, but not in asthmatic patients [35]. In other
studies, FENO correlated positively with height [38,39] and weight
[38,40]. Signicantly higher FENO levels were reported in obese
non-OSA subjects compared to non-obese non-OSA controls [5,23].
Another study found no difference between obese and non-obese
controls and signicantly higher levels of FENO were reported in
OSA compared only to obese subjects [26]. In contrast, two studies
by the same group demonstrated that FENO levels were higher in
OSA compared only to non-obese healthy volunteers [5,23]. FENO
was higher in overweight children with habitual snoring and in
overweight children with OSA compared to normal and overweight
children without any SDB [31]. Overall in clinical practice FENO level
does not need to be adjusted based on an individual's BMI.
A recent study concluded that FENO has only limited clinical
potential in the screening of sleep disordered breathing (SDB) [41],
however a composite index including BMI, age, carboxyhaemoglobin saturation, FEF50/FIF50, neck circumference and FENO
was predictive for OSA [42]. In children, both OSA and non-OSA
subjects, habitual snoring appears to be associated with higher
FENO levels [31].
Exhaled carbon monoxide (CO) is another, frequently used
marker of airway oxidative stress. CO concentration in exhaled air is
related to heme oxygenase expression and higher levels of exhaled
CO were reported in healthy smokers, and chronic airway diseases
[43]. Only one study examined exhaled CO and found higher levels

Exhaled breath condensate

The analysis of EBC is dependent on the notion that during tidal
breathing, small droplets are released from the ALF and mix with
water originating from the alveolar space. These droplets are
released by the turbulent airow and/or the burst during airway
opening, suggesting that the formation of EBC may be very variable
due to heterogeneity of airow limitation, especially in obstructive
airway disorders [44]. If the exhaled breath is led through a cooled
chamber, mist can be condensed and molecules of the ALF are
trapped and form exhaled breath condensate [45] (Fig. 1). This
condensate uid can be analysed immediately or stored until
measurement. A great number of articles have now reviewed and
critiqued the methodology for EBC assessment. These are summarised in a European Respiratory Society/American Thoracic Society task force document in 2005 [45], and studies on recent
methodological advances are discussed in topical reviews
[44,46e49], however four specic methodological considerations
are worthy of highlighting in the context of EBC measurement in
OSA.
Firstly, as exhaled breath is led through the oral cavity, one may
argue that EBC does not represent the lower airways exclusively.
This is an important issue in the context of OSA, as heightened oral
inammation has also been reported in this disorder [50,51]. This
potential limitation acknowledged, when performed well, the
salivary contamination in EBC is minimal, as evidenced by a very
low concentration of amylase detectable in EBC, regardless of the
condensing equipment [52,53]. However, oral cavity contamination
may still be a very important bias in the case of EBC pH measurements, due principally to two reasons. Firstly, the main volatile base
determining EBC pH, the ammonia is produced largely in the mouth
[54]. Secondly, acidic gastric droplets may also mix with expiratory
mist resulting in a lower EBC pH [55].
Secondly and a key consideration often overlooked in EBC
measurement, is the impact of dilution factor. Because the ratio of
alveolar/airway water content is variable and unpredictable, a
dilution indicator is warranted to estimate the airway concentration of a molecule from its EBC levels. This indicator must be reliably measurable in EBC with a known concentration in the airways.
Urea levels [56] and total ion content [57] of EBC are potential
markers for dilution, the former because urea freely diffuses
through the bronchial barrier, the later because ALF is isotonic.

Table 2
The results with exhaled nitric oxide in adult patients with OSA.
Authors, reference number

OSA

Control

P Value

FENO (ppb)

FENO (ppb)

Agusti et al., 1999 [27]

Przybyowski et al., 2006 [13]
Foresi et al., 2007 [28]
Depalo et al., 2008 [5]

24
66
34
18

22.2
22.4
21.8
23.1

Carpagnano et al., 2008 [23]

30

31.6 1.6

Culla et al., 2010 [25]

Fortuna et al., 2011 [24]
Chua et al., 2013 [12]
JalilMirmohammadi et al., 2014 [14]

39
30
75
31 obese
16 non-obese
71

23.1 (19.8e28.3)
27.2 18
19.0 7.7
19.8/9.0e44.0/
20.3/5.0e45.0/
17.2 11.5

7
53
29
10
15
10
10
24
30
29
7

19.7 16.7
15.3 8.1
25.1 17.8
7.2 0.6
17.9 2.1
4.8 0.7
27.1 1.8
11.0 (8.7e14.8)
16.7 8
6.9 3.7
11.8/1.0e30.0/

Hua-Huy et al., 2015 [29]

14.7
13.2
11.1
2.1

24

non-obese
obese
non-obese
obese

16.7 14.2

NS
<0.05
NS
<0.001
NS
<0.001
NS
<0.001
<0.05
<0.001
NS
NS
NS

Data are expressed as mean SD, median (95% CI) and median/10e90 percentiles/. FENO-fractional exhaled nitric oxide, ppb-particles per billion, NS-not signicant (p > 0.05),
OSA-obstructive sleep apnoea.

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a [64e66], IL-8 and intercellular adhesion molecule (ICAM) concentrations [6] were higher in OSA. However, the elevation in IL-8
and ICAM may have arisen secondarily to obesity, as a similar
EBC cytokine prole is reported in non-OSA obese subjects [6].
Regardless, the AHI directly correlated with TNF-a [66] and ICAM
[6] and negatively with IL-10 [66] and CPAP signicantly increased
IL-10 [64] and reduced TNF-a [64,68] levels.
Overall, therefore it seems that OSA is characterised by a proinammatory prole, however none of the studies took into account such potential confounding factors as dilution or matrix
effect.
Markers of oxidative stress in EBC

Fig. 1. Schematic diagram of EBC formation. During EBC collection, the patient inhales
air through a two way valve which leads exhaled breath through a cooling device
where the condensate uid is formed. The condensate uid is then sampled and stored
until analysis.

Alternatively, dilution can be estimated by the measurement of

conductivity in lyophilised [58] or vacuum evaporated [52,59]
condensate samples. Unfortunately, none of the EBC studies, performed to date, in OSA estimated dilution factor, thus potentially
confounding the interpretation of some results. Of note however,
no difference in the rate of dilution was shown between health and
airway diseases, including asthma [59] or COPD [52] and therefore
signicant alterations in mediator content between the OSA and
control groups may still represent pathologically relevant airway
change.
Thirdly, because EBC is a very dilute uid, mediators may be
found in concentrations close to the lower limit of detection of the
currently used analytical techniques. In addition, these methods,
such as enzyme linked immunoassay (ELISA) were not developed
and validated specically for EBC. The use of invalidated media to
determine cytokine levels with ELISA may thus potentially lead to
false positive results due to a matrix effect [60].
Finally, the material from which the collection device is constructed may also signicantly affect the composition of EBC [61,62]
and this effect needs to be tested for each biomarker.
Overall, despite these limitations the technique of EBC when
performed well, with appropriate consideration for oral contamination and dilution effects, offers the potential for a non-invasive
window to the airway environment.
EBC cytokines
A variety of cytokine proles have been evaluated using EBC in
OSA. Elevated levels of the pro-inammatory cytokine interleukin6 (IL-6) were reported in the EBC of both adult [63e66] and paediatric [67] subjects with OSA, with a direct relationship found
between EBC IL-6 and disease severity, determined by the AHI
[63,66] as well as neck circumference [63]. CPAP therapy signicantly reduced EBC IL-6 concentrations in some [64,68], but not all
[69] studies. Upper airway surgery did not appear to reduce EBC IL6 levels, however only nine patients were investigated [69].
Other studies have revealed that the immunosuppressive EBC
IL-10 levels [64,66] were lower, while tumour necrosis factor (TNF)-

8-isoprostane is the stable end-product of lipid peroxidation,

and therefore EBC 8-isoprostane is widely used to evaluate and
potentially characterise oxidative stress in the airways [70].
Elevated levels of 8-isoprostane in EBC have been reported in both
adult [10,26,63e66] and paediatric [67] OSA. Moreover, a signicant correlation between 8-isoprostane and AHI [10,26,63,66] as
well as neck circumference has been reported [10,63]. A signicant
elevation in EBC 8-isoprostane levels was observed in the morning
compared to the evening [10] and CPAP treatment signicantly
reduced 8-isoprostane concentrations in the majority [10,64,68]
but not in all [26] studies.
Despite these results, it is important to interpret the result
carefully, as demonstrated in two subsequent studies of the same
workgroup [10,63]. Indeed despite identical methodologies for
collection and measurement, EBC 8-isoprostane levels in healthy
subjects (6.7 0.2 pg/ml) in the later study [10] were very similar to
the EBC 8-isoprostane concentrations measured in patients with
OSA (7.4 0.7 pg/ml) in the former work [63]. Although no alterations in analytical methods were noted, the considerable variation
in average values within healthy (5.0 0.3 pg/ml vs. 6.7 0.2 pg/
ml) and OSA groups (7.4 0.7 pg/ml vs. 9.5 1.9 pg/ml) between
the two studies are noteworthy [10,63].
Higher levels of hydrogen peroxide (H2O2), another marker of
oxidative stress, have also been found in adults with OSA using EBC
[26]. A direct relationship between EBC H2O2 and AHI is reported
[26], however CPAP treatment did not change EBC H2O2 levels [26].
In children, EBC H2O2 levels were higher only in moderate to severe
OSA subjects with no difference between mild OSA and healthy
subjects [71]. This was related to AHI and the percentage of sleep
time with oxygen saturation of haemoglobin <95% [71]. Of note, it
was demonstrated that EBC H2O2 levels are signicantly related to
the breathing pattern [72], which was not controlled in the previous studies [26,71]. In addition, as obesity signicantly reduces EBC
H2O2 concentrations, this effect has to be taken into consideration
[26].
Uric acid is a major antioxidant within the human body, and
elevated levels of this molecule are thus potentially indicative for
accelerated oxidative stress [73]. Higher levels of EBC uric acid have
been reported in children with OSA. This elevation was not
apparent in serum concentrations, indicating that uric acid excess
was arising from the airway compartment [74]. Having said this, it
is important to note, that the volume of EBC was lower in OSA
suggesting that higher EBC uric acid levels in this group were
related potentially to a reduced dilution by alveolar water and not
necessarily the true uric acid concentration [74].
Markers of nitrosative stress in EBC
Together with oxidative stress, excessive nitrosative stress has
also been described in chronic inammatory airway diseases [75].
Higher EBC nitrate levels were reported in OSA without any

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difference between obese and non-obese healthy controls [26]. A

signicant positive relationship between EBC nitrate and AHI has
also been reported [26]. When EBC nitrite and nitrate were
measured together, no difference was shown in OSA [71]. CPAP
treatment did not change EBC nitrate levels [26,69]. However, EBC
nitrotyrosine signicantly decreased following CPAP [68]. The
somewhat conicting data on airway nitrosative stress in OSA urge
to conduct more studies to have a better picture on true processes.
EBC acidity
The pH of the EBC uid potentially provides an insight into the
acidity of the airway tract, however because gastric droplets may
also mix with EBC, EBC pH may be lower in the case of gastrooesophageal reux [55]. Numerous physiological and methodological factors were identied which may affect EBC pH [76]. Data
on the effect of obesity on EBC pH are inconsistent. In healthy
subjects, no difference was shown [23,26,77], while in asthmatic
subjects low EBC pH was associated with high BMI [78].
In OSA, reduced EBC pH was reported by various [23,26,65], but
not all [79] studies. Obesity may have biased the results, as in one
study no difference was found between non-OSA and OSA obese
patients [23]. EBC pH was inversely related to AHI and neck
circumference in one [23], but not in the other study [79]. CPAP
signicantly increased EBC pH in one [26], but not in the other
studies [69,79]. Upper airway surgery did not change EBC pH values
[69]. Overall therefore the ndings are mixed but the positive
studies potentially highlight the possibility that an acid environment in the airways of subjects with OSA is present. However, it is
not clear if it truly represents an accelerated airway inammation
and oxidative stress or a proximal gastro-oesophageal reux often
seen in obese patients.
Lipid mediators in EBC
Leukotrienes are products of lipoxigenase enzyme and play an
important role in chronic airway diseases [80]. It seems that obesity
does not have a pronounced effect on airway leukotrienes, as it did
not affect EBC leukotrine B4 (LTB4) [81] or cysteinyl leukotriene
levels [77] in asthma. Higher levels of EBC LTB4 were reported in
OSA without any difference between obese and non-obese subjects
without OSA [26]. Similarly, in children, the levels of EBC LTB4 were
higher than in control subjects [82]. A direct relationship between
EBC LTB4 and AHI was noted [26]. CPAP treatment did not change
EBC LTB4 levels [26]. Cysteinyl leukotrienes were higher in EBC in
children with OSA [82]. No difference in EBC prostaglandin E2
(PGE2), another lipid mediator was found in children with OSA
compared to health [82].
Other markers
EBC leptin was higher in OSA especially in obese patients [4]. No
difference in EBC erythropoietin levels were shown between COPD,
health and OSA [83].
Exhaled volatile organic compounds
Exhaled breath contains thousands of VOCs which are related to
airway and systemic metabolism as well as oxidative stress [84].
VOCs can be detected and quantied in breath using sophisticated
methods, such as mass-spectrometry (MS) coupled with a separation technique, such as gas-chromatography (GCeMS), selected-ion
ow-tube (SIFT-MS) or proton-transfer-reaction system (PTR-MS),
etc [85]. These techniques use electron (i.e., MS) or chemical (i.e.,
PTR-MS) ionisation to reliably identify VOCs. However, these

instruments are expensive and require specialist skills and expertise to obtain reliable results.
Another possible approach is to measure numerous volatile
molecules in combination. It is now recognized that disorders
involve several interrelated processes which cannot be investigated
at the level of single molecules. Electronic noses are composites of a
sensor array and an in built processor. These instruments functionally resemble the biological olfactory receptors, in that they are
not selective for a single ligand and upon activation by an odour the
sensor array gives a signal pattern [86]. The analytical potential of
these devices depends heavily on the material of the sensors [87].
Because electronic noses cannot identify individual substances, but
are able to compare and distinguish complex molecular patterns,
appropriate statistical approaches in electronic nose analysis are
essential [88]. In addition, as numerous physiological and
sampling-related methodological factors might modify exhaled
volatile compound pattern [87], an external validation should be
performed in an independent cohort to ensure results are reliable
and valid [89]. The commensurate measurement with direct VOC
analysis (i.e., with GCeMS) is also encouraged, as it may help to
validate results and to understand the pathophysiological background of observed differences in VOC patterns. For a more detailed
description of electronic nose technology and its use in breath
research please refer to references [85,87,90].
There are a number of important methodological and technical
considerations when evaluating VOCs. Firstly, volatile compounds
are heavily inuenced by previous exogenous exposure (e.g., food,
smoking or medications) which needs to be taken into consideration [91]. Secondly, expiratory ow rate, breath hold and exclusion of anatomical dead space may signicantly affect exhaled
VOC concentration [92,93] and thus needs to be tightly regulated.
This is particularly important for electronic nose analysis, where it
was shown that the aforementioned factors can signicantly
affect the ability of the electronic nose to detect pulmonary malignancy [93]. Finally, sampling material, transportation and
sample storage may all inuence VOC levels [94]. Both individual
VOCs and gaseous mixtures have been investigated in OSA. Unfortunately, the aforementioned factors have been generally
poorly accounted for and controlled in studies with OSA. In
addition, the levels of some exhaled VOCs relate to BMI [95] which
fact should also be taken into consideration, especially in OSA. Not
surprisingly, exhaled volatile compound pattern was different in
obesity [96].
This acknowledged, exhaled levels of pentane, a product of lipid
hydroperoxide decomposition, is increased in the morning
compared to the evening samples in OSA [11]. Unfortunately, OSA
and non-OSA groups were not compared, but both morning
(7.0 1.3 nM vs. 4.7 1.1 nM, mean SEM) and evening
(4.2 0.4 nM vs. 3.1 0.6 nM) values tended to be higher in OSA
than in controls [11]. Of note, pentane is one of the volatile compounds in which levels are inuenced by the expiratory ow rate
[97], but in the only study in OSA, expiratory ow rate was not
controlled [11].
Altered volatile compound mixtures were reported in OSA
compared to health by various workgroups [9,79,96,98]. All of these
studies used the commercially available Cyranose 320 electronic
nose, which is a composite of conducting polymer sensors and is
sensitive for polar compounds. The accuracy of Cyranose 320 to
detect OSA was between 80 and 90% in independent studies
[9,79,96]. Interestingly, this difference was present only in the
morning and not before sleep which was supported by the fact that
the evening and morning breathprints were different in OSA [9]. A
signicant relationship was found between volatile compound
mixtures and AHI, in one [79], but not in the other study [9]. CPAP
therapy signicantly altered exhaled breathprints [79], however

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A. Bikov et al. / Sleep Medicine Reviews 27 (2016) 1e8

other studies have shown that these alterations are different if OSA
is accompanied with comorbidities such as diabetes mellitus,
metabolic syndrome and chronic heart failure [99].

Conicts of interest
The authors report no conict of interest.

Summary and recommendations

Using exhaled breath methodologies, several studies have now
demonstrated an altered airway inammatory prole together
with heightened oxidative stress in both adults and children with
OSA. Importantly several of the differences in mediator levels have
been shown to relate to the severity of OSA and respond to CPAP
therapy. Despite this, there is still progress needed to truly establish
the place and value of exhaled breath analysis in OSA and important considerations include:
1) several important methodological factors must be taken into
consideration when measuring exhaled breath samples. To date
this has not been approached in a robust manner and whilst
different studies are sometimes not comparable in OSA, technical and methodological factors may at least in part explain the
discrepancy in results.
2) obesity as well as other important co-morbid conditions
including metabolic and cardiovascular consequences of OSA
may lead to heightened systemic and subsequent airway
inammation themselves. Moreover, airway inammatory
markers are not necessarily specic for OSA and are elevated in
other chronic airway disorders as well. Future studies should
thus carefully control for these factors when possible and be
cognisant of their impact when interpreting results otherwise.
3) studies measuring numerous mediators showed a concordant
change of several rather than only one biomarker. This suggests
that the whole pattern of airway inammation is likely to be
modied, and the results on individual biomarkers are thus not
specic and unlikely to be rewarding when studied in isolation.

Practice points
1) The analysis of exhaled breath is a non-invasive, harmless still not fully standardised method to investigate the
pathophysiology of OSA.
2) Markers of airway inflammation and oxidative stress are
elevated in exhaled breath of patients with OSA.
3) Obesity and comorbidities which accompany OSA
frequently may alter exhaled biomarkers themselves.

Research agenda
1) With established methodological recommendations of
exhaled breath measurement, studies should be
repeated with controlled circumstances to avoid
methodology-related biases.
2) Large scale studies using powerful bioinformatics are
needed to identify clusters of biomarkers which correlate
with different characteristics of OSA. This may help to
understand the role of airway inflammation in the complex pathophysiology of this disorder.

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