Therapeutic Advances in Respiratory Disease
Review
Analysis of exhaled breath condensate in
respiratory medicine: methodological aspects
and potential clinical applications
Therapeutic Advances in
Respiratory Disease
(2007) 1(1) 5–23
DOI: 10.1177/
1753465807082373
©SAGE Publications 2007
Los Angeles, London,
New Delhi and Singapore
Paolo Montuschi
Abstract: Analysis of exhaled breath condensate (EBC) is a noninvasive method for studying
the composition of airway lining fluid and has the potential for assessing lung inflammation.
EBC is mainly formed by water vapor, but also contains aerosol particles in which several
biomolecules including leukotrienes, 8-isoprostane, prostaglandins, hydrogen peroxide,
nitric oxide-derived products, and hydrogen ions, have been detected in healthy subjects.
Inflammatory mediators in EBC are detected in healthy subjects and some of them are
elevated in patients with different lung diseases. Analysis of EBC is completely noninvasive,
is particularly suitable for longitudinal studies, and is potentially useful for assessing the
response to pharmacological therapy. Identification of selective profiles of biomarkers of lung
diseases might also have a diagnostic value. However, EBC analysis currently has important
limitations. The lack of standardized procedures for EBC analysis and validation of some
analytical techniques makes it difficult comparison of results from different laboratories.
Analysis of EBC is currently more useful for relative measures than for quantitative
assessment of inflammatory mediators. Reference analytical techniques are required to
provide definitive evidence for the presence of some inflammatory mediators in EBC and
for their accurate quantitative assessment in this biological fluid. Several methodological
issues need to be addressed before EBC analysis can be considered for clinical applications.
However, further research in this area is warranted due to the relative lack of noninvasive
methods for assessing lung inflammation.
Keywords: exhaled breath condensate; airway inflammation; non-invasive assessment;
therapeutic monitoring; asthma; chronic obstructive pulmonary disease; eicosanoids
Introduction
Inflammation has an important pathophysiological role in respiratory diseases such as
asthma and chronic obstructive pulmonary
disease (COPD). [Sabroe et al. 2007; Wenzel
et al. 2006; Fixman et al. 2007; Barnes 2000;
O’Donnell et al. 2006]. The assessment of lung
inflammation is relevant for the management
of chronic respiratory diseases as it may indicate that pharmacological therapy is required
before onset of symptoms and decrease in
lung function. Moreover, monitoring of airway
inflammation might be useful in the followup of patients with respiratory diseases, and
for guiding pharmacological therapy. Quantification of inflammation in the lungs is currently based on invasive methods including the
analysis of bronchoalveolar lavage (BAL) fluid,
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bronchoscopy, and bronchial biopsies [Adelroth,
1998], semi-invasive methods such as sputum
induction [Berlyne et al. 2000; Hargreave, 2007],
and the measurement of inflammatory biomarkers in plasma and urine which are likely to
reflect systemic rather than lung inflammation.
Exhaled breath consists of a gaseous phase that
contains volatile compounds (e.g. nitric oxide,
carbon monoxide, and hydrocarbons) and a liquid phase, termed exhaled breath condensate
(EBC), that contains aerosol particles in which
several nonvolatile compounds have been identified [Montuschi, 2005a; Mutlu et al. 2001;
Kharitonov and Barnes, 2001; Hunt, 2002;
Kharitonov and Barnes, 2006; Effros et al. 2005;
Horvarth et al. 2005; Cepelak et al. 2007;
Montuschi, 2002; Montuschi and Barnes, 2002].
Measurement of exhaled nitric oxide (NO) is a
Paolo Montuschi
Department of
Pharmacology, Faculty
of Medicine, Catholic
University of the Sacred
Heart, Rome, Italy.
[email protected]
5
Therapeutic Advances in Respiratory Disease
well accepted, standardized, validated and widely
used method for assessing airway inflammation in patients with asthma who are not being
treated with inhaled glucocorticoids, can be used
to monitor the response to these drugs, and
to assess compliance and can predict asthma
exacerbation [Anonymous, 1999; Anonymous,
2005]. Moreover, with the use of exhaled NO
measurements, maintenance doses of inhaled glucocorticoids may be significantly reduced without
compromising asthma control [Smith et al.
2005]. However, the clinical utility of exhaled
NO measurement is currently limited to patients
with asthma, as its role in the management of
other respiratory diseases is not yet known.
Recently, attention has focused on EBC as
a noninvasive method for studying the composition of airway lining fluid [Montuschi, 2002;
Montuschi and Barnes, 2002a; Effros et al.
2005b]. Using urea, which is a freely diffusible
molecule, as a marker, it has been demonstrated
that a measurable fraction (1 in 24 parts) of
the EBC in healthy subjects is derived from
aerosolized airway lining fluid [Dwyer, 2001].
EBC analysis of inflammatory biomarkers is a
noninvasive method which has the potential to
be useful for monitoring airway inflammation in
patients with respiratory diseases, including children [Montuschi, 2002; Montuschi and Barnes,
2002a]. As it is completely noninvasive, EBC
also is suitable for longitudinal studies and for
monitor the response to pharmacological therapy.
Furthermore, different biomarkers might reflect
the different aspects of lung inflammation or
oxidative stress, which is an important component of inflammation. Identification of selective
profiles of biomolecules in different inflammatory
airway diseases might be relevant for differential
diagnosis in respiratory medicine. Collection of
EBC samples is simple, inexpensive, and safe.
However, unlike exhaled NO measurement, EBC
technique does not provide real-time results as
EBC samples need to be assayed for different biomolecules. Several methodological issues,
including standardization of EBC technique and
validation of analytical methods, need to be
addressed before this approach can be considered
for applications in the clinical setting.
This review describes the methodology of
the EBC technique, summarizes the current
knowledge on the biomarkers which have
been identified in EBC, discusses the advantages, the limitations, and the potential clinical
6
applications of EBC analysis, and provides
suggestions for further research in this area.
Experimental setup
The collection of EBC sample is simple and
easy to perform. Home-made and commercially manufactured condensers are available.
Home-made equipment generally consist of a
mouthpiece with a one-way valve connected to
a collecting system which is placed in either ice
or liquid nitrogen to cool the breath [Montuschi,
2005b]. The collecting system consists of a double wall of glass, the inner wall of which is
cooled by ice (Figure 1A) [Montuschi et al.
1999]. Alternatively, jacketed cooling pipes or
tubes in buckets have been used [Mutlu et al.
2001]. Generally, subjects are asked to breath
tidally, with a noseclip on, for 15 min through a
mouthpiece connected to the condenser. Exhaled
air enters and leaves the condensing chamber
through one-way valves at the inlet and outlet
while the chamber is kept closed. If the collecting system consists of two glass containers,
EBC is collected between the two glass surfaces
at the bottom of the outer glass container in a
liquid form [Montuschi et al. 2005b]. Generally, 1.0–2.5 mL of EBC is collected in 15 min
based on respiratory parameters (minute ventilation, respiratory rate, tidal volume), material of
condenser surfaces, temperature, and turbulence
of airflow.
Commercially manufactured condensers are also
available [Montuschi et al. 2000; Montuschi and
Barnes, 2002b]. The EcoScreen® (Jaeger Tonnies,
Hoechberg, Germany) is an electric refrigerated system which consists of a mouthpiece with
a one-way valve and a collecting system connected to a power supply by an extendable arm
(Figure 1B). Subjects breath through the mouthpiece that is connected to a valve block in which
inspiratory and expiratory air are separated. The
valve block is connected to the collecting system,
consisting of a lamellar condenser, and a sample
collection vial. The collecting system is inserted
into a cooling cuff maintained at a cold temperature by a refrigerator. The actual temperature
inside the cooling cuff and, more importantly,
into the cooling system, is not known. According to the information provided by the manufacturers, the temperature inside the cooling
cuff is −10◦ C. During expiration, the air flowing through the lamellar condenser condenses
on the inner surface of the lamellar condenser
and drops into the collecting vial. Despite it’s
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Review
Figure 1. Representation of EBC collecting systems. (A) A homemade EBC collecting system consisting of two
glass containers which form of a double wall of glass for which the inner side of the glass is cooled by ice.
EBC is collected between the two glass surfaces. (B) A commercially available condenser (EcoScreen). EBC is
collected in the collecting vial as indicated by the arrow. (C) A portable EBC collecting system (RTube) which
consists of a disposable polypropilene collecting system with an exhalation valve that also serves as
syringe-style plunger to pull fluid off the condenser wall. An aluminium cooling sleeve is placed over
the disposable polypropilene tube. (Modified from Montuschi, P., Nature Review Drug Discovery, vol. 1, Indirect
monitoring of lung inflammation, Macmillan Magazines, 2002, pp. 238–242, and from Montuschi, P. Analysis of
exhaled breath condensate: methodological issues. In Montuschi, P. (editor) New perspectives in monitoring
lung inflammation: analysis of exhaled breath condensate, CRC Press, 2005, pp. 11–30. With permission).
wide use, there is currently no evidence of
advantages of the EcoScreen condenser over
homemade devices, except for the possible
immediate freezing of the samples. This can be
important for chemically unstable compounds
including leukotrienes (LTs), particularly LTE4
and cysteinyl-LTs in general. However, as the
temperature inside the cooling cuff is probably higher than −10◦ C throughout the test,
and/or other possible technical problems that
may arise, EBC samples are usually collected
in a liquid or in a mixed liquid/frozen form.
Inconsistencies in collection of samples (liquid,
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frozen, partially frozen) may affect the concentrations of chemically unstable compounds
in EBC and explain part of the variability in
their concentrations reported by different studies.
Anyway, even if samples are collected frozen,
when measuring more than one compound, samples must be thawed at the time of collection
to make the required aliquots. This could be
avoided by replacing the single collecting vial
with some smaller, separate collecting vials. When
considering a large-scale application of EBC
technique, the high cost of this commercial condenser should also be considered.
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Therapeutic Advances in Respiratory Disease
The EcoScreen® II (Jaeger Tonnies, Hoechberg,
Germany) has recently been manufactured.
This condenser allows to measure respiratory
parameters during the collection of EBC. Alternatively, the EcoScreen can be connected to a
pneumotachograph and a computer for online
recording of respiratory parameters [Montuschi
et al. 2003a]. Another advantage of the EcoScreen II is that EBC derived from the airways
or the alveoli can be collected at the same time
into two separate collecting systems consisting
of plastic bags inserted into a refrigerating system. This device might be useful for studying
the origin of biomarkers in the lung compartments (airways vs alveoli). Compared with the
material of the collecting system surface (plastics vs Teflon-coated metal) might also turned
out to be more appropriate for some compounds
(e.g. lipids). However, no published studies are
available with this condenser.
The RTube® (Respiratory Research, Inc.,
Charlottesville, VA) is another commercially
available condenser which has the advantage
of being portable [Hunt, 2002]. This device
consists of a disposable polypropylene collecting system with an exhalation valve that also
serves as syringe-style plunger to pull fluid off
the condenser wall (Figure 1C) [Hunt, 2002].
The refrigerating system consists of an aluminium
cooling sleeve which is placed over the disposable
polypropilene tube (Figure 1C). The temperature of the cooling sleeve can be chosen. The
device prevents salivary contamination, and no
detectable amylase concentrations in EBC have
been reported [Hunt, 2002]. The RTube has
been originally manufactured for measuring pH
in EBC samples, but it can be used for measuring
other compounds [Hunt, 2002]. As measurement
of pH in EBC requires deaeration for removal of
carbon dioxide, a separate device, the pHTube®
(Respiratory Research, Inc., Charlottesville, VA)
can be used for this purpose [Hunt, 2002].
The RTube is portable, which makes it possible to collect EBC samples at home, which
is particularly suitable for longitudinal studies
or when collection of several samples a day is
required; the EBC sample in a polypropylene
tube can be stored in a freezer at home; a sufficient volume of EBC for pH measurement can
be collected in as little as 1 min [Hunt, 2002].
However, the RTube has some limitations as EBC
samples need to be brought to the laboratory
for biochemical assays, and storage conditions in
freezer at home (−20◦ C) are different from those
8
required by some chemically unstable mediators
which need to be stored immediately at −80◦ C.
One study reported that compared with RTube,
collection of exhaled breath by EcoScreen allows
larger volumes to be collected and detects protein
and lipid mediators with greater sensitivity, which
might be due to the differences in the collection temperature [Soyer et al. 2006], but further
studies to address this issue are required.
Careful sterilization of the EBC equipment is
required to avoid cross-contamination, although
standardized procedures for sterilization of condensers are not available. It is reasonable to leave
the EBC equipment in an antibacterial solution such as 1% aqueous solution of sodium
hypochlorite for at least 1 h. After sterilization,
the EBC collecting system needs to be washed
thoroughly with water to remove the antiseptic
solution and dried. Some collecting systems such
as the lamellar condenser in the EcoScreen are
coated with Teflon® (E.I. du Pont de Nemours &
Company, Inc., Washington, DE) to avoid adhesions of biomolecules to the collecting system
surfaces and the consequent artifactual decrease
in their concentrations in EBC. At present, the
effect of different collecting system materials on
the detection of different biomolecules in EBC
is largely unknown. The best collecting system
material is likely to be different depending on the
physicochemical properties of the biomolecule
to be detected. A condenser with borosilicate
glass coating has been shown to be superior to
silicone, aluminium, polypropylene, and Teflon
for detection of albumin in EBC [Rosias et al.
2006]. The effect of antiseptic solutions on the
EBC collecting system materials are not currently
known. Whether the antiseptic solutions interact
and damage the material of the collecting systems
needs to be clarified. Technical improvements
in the design of new condensers would allow
to collect simultaneously several frozen aliquots
making it possible to measure different markers
without thawing the whole sample. The possibility of using selective sensors to make on-line
measurements of hydrogen peroxide [Gajdocsi
et al. 2003] and possibly other specific inflammatory mediators in the breath is currently under
investigation.
Markers of inflammation in EBC
Several biomolecules have been detected in EBC
of healthy subjects and of patients with different inflammatory lung diseases (Table 1 and
Figure 2). In most studies, markers in EBC were
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Review
Table 1. Biomolecules in EBC.
Biomolecule
Isoprostanes
8-Isoprostane (15-F2t -IsoP)
Leukotrienes
LTB4
LTD4
LTE4
Cys-LTs (LTC4 /LTD4 /LTE4 )
Prostanoids
PGE2
PGF2α
PGD2
TxB2
Hydrogen ions
Hydrogen peroxide
Nitrogen reactive species
Nitrite
Nitrate
S-Nitrosothiols
3-Nitrotyrosine
Adenosine
Glutathione
Aldehydes
TBARS
DNA
Electrolytes (sodium, potassium, calcium,
magnesium, chloride)
Keratin
Cytokines
IL-1β
IL-2
IL-4
IL-5
IL-6
IL-8
IL-10
Analytical method
GC/MS [Carpenter et al. 1998], RIA [Montuschi et al. 2003a; Montuschi et al. 2003b;
Mondino et al. 2004; Baraldi et al. 2003a; Montuschi et al. 2006; Montuschi et al.
2005b], EIA [Montuschi et al. 1999; Montuschi et al. 2000; Montuschi and Barnes,
2002b; Antczak et al. 2001; Shahid et al. 2005; Montuschi et al. 2002; Baraldi et al.
2003b; Kostikas et al. 2003; Biernacki et al. 2003; Carpagnano et al. 2003a;
Zanconato et al. 2004]
LC/MS/MS [Montuschi et al. 2004a; Montuschi et al. 2005a], GC/MS [Cap et al. 2004],
EIA [Montuschi and Barnes, 2002b; Montuschi et al. 2003c; Antczak et al. 2001;
Mondino et al. 2004; Biernacki et al. 2003; Montuschi et al. 2005b; Hanazawa et al.
2000; Csoma et al. 2002]
GC/MS [Cap et al. 2004]
GC/MS [Cap et al. 2004], EIA [Mondino et al. 2004; Montuschi et al. 2006; Shibata
et al. 2006]
EIA [Antczak et al. 2001; Baraldi et al. 2003b; Hanazawa et al. 2000; Carraro et al.
2005; Csoma et al. 2002]
GC/MS [Carpenter et al. 1998], RIA [Montuschi et al. 2003b; Montuschi et al. 2005b],
EIA [Montuschi and Barnes, 2002b]
EIA [Montuschi and Barnes, 2002b]
EIA [Montuschi and Barnes, 2002b]
RIA [Vass et al. 2003; Mondino et al. 2004; Huszar et al. 2005], EIA [Montuschi and
Barnes, 2002b]
pH meter or pH microelectrode [Hunt et al. 2000; Vaughan et al. 2003; Kostikas et al.
2002; Larstad et al. 2003; Shimizu et al. 2007; Nicolau et al. 2006; Walsh et al.
2006; Dupont et al. 2006; Prieto et al. 2007; Paget-Brown et al. 2005; Kullmann
et al. 2006]
Spectrophotometry [Horvath et al. 1998; Dekhuijzen et al. 1996], fluorometric assay
[Schleiss et al. 2000; Jobsis et al. 1997], chemiluminescence [Zappacosta et al.
2001]
Spectrophotometry [Cunningham et al. 2000; Corradi et al. 2001], fluorometric assay
[Balint et al. 2001]
Fluorometric assay [Balint et al. 2001]
Spectrophotometry [Corradi et al. 2001], fluorometric assay [Kharitonov et al. 2002]
GC/MS [Larstad et al. 2005; Celio et al. 2006], LC/MS [Goen et al. 2005; Baraldi et al.
2006], HPLC [Celio et al. 2006], EIA [Hanazawa et al. 2000]
HPLC [Vass et al. 2003]
HPLC [Corradi et al. 2003b]
LC/MS [Corradi et al. 2003a; Corradi et al. 2003b], HPLC [Larstad et al. 2002]
Spectrofluorimetry [Nowak et al. 1999]
PCR [Gessner et al. 2004; Carpagnano et al. 2005]
Ion-selective electrodes [Effros et al. 2002], ion chromatography [Effros et al. 2003]
Proteomics [Gianazza et al. 2003], ELISA [Jackson et al. 2007]
EIA [Scheideler et al. 1993], multiplex bead array [Sack et al. 2006]
Flow cytometry [Robroeks et al. 2006]
Protein array [Matsunaga et al. 2006], flow cytometry [Robroeks et al. 2006], ELISA
[Shahid et al. 2002]
ELISA [Profita et al. 2006]
Multiplex bead array [Sack et al. 2006], EIA [Bucchioni et al. 2003; Carpagnano et al.
2003b], ELISA [Rozy et al. 2006]
Protein array [Matsunaga et al. 2006], multiplex bead array [Sack et al. 2006], ELISA
[Cunningham et al. 2000]
Flow cytometry [Robroeks et al. 2006], multiplex bead array [Sack et al. 2006]
(continued)
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Therapeutic Advances in Respiratory Disease
Table 1. Continued.
Biomolecule
Analytical method
Cytokines (continued)
IL-12p70
IL-17
Interferon-γ
IGF-1
Interferon-γ -inducible protein 10
MIP-1α, MIP-1β
PAI-1
RANTES
TGF-β
TNF-α
Multiplex bead array [Sack et al. 2006]
Protein array [Matsunaga et al. 2006]
EIA [Shahid et al. 2002], flow cytometry [Robroeks et al. 2006]
ELISA [Rozy et al. 2006]
Protein array [Matsunaga et al. 2006]
Protein array [Matsunaga et al. 2006]
ELISA [Rozy et al. 2006]
Protein array [Matsunaga et al. 2006]
Protein array [Matsunaga et al. 2006]
RIA [Scheideler et al. 1993], protein array [Matsunaga et al. 2006], ELISA [Rozy et al.
2006], multiplex bead array [Sack et al. 2006]
Abbreviations: GC/MS, gas chromatography/mass spectrometry; EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay; HPLC,
high performance liquid chromatography; IGF-1, insulin-like growth factor-1; IL, interleukin; LC/MS, liquid chromatography/mass spectrometry;
LT, leukotriene; MIP-1, macrophage inflammatory protein-1; PAI-1, plasminogen activator inhibitor-1; PG, prostaglandin; RIA, radioimmunoassay;
TBARS, thiobarbituric acid reactive substances; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α; Tx: thromboxane.
L-arginine
NADPH NOS
O2 oxidase
citrulline
RS-NO
NO
thiols
NO3−
tyrosine
NO2−
cytokines
electrolytes
−
O2
SOD
ONOO−
H2O2
ROS
3-NT
H+
NO2
ROS
5-LO
LTB4
COX
Tx
synthase
5-HPETE
LTA
synthase
LTA4
hydrolase
LTA4
γ -GTP
LTD4
adenosine
8-IP
arachidonic
acid
5-HETE
aldehydes, TBARS
GSH
NO2−
MPO
polyunsaturated fattyacids
PGG2 /PGH2
hydrolysis
TxA2
TxB2
isomerases
LTC4
synthase
PGE2
PGF2α
PGD2
LTC4
dipeptidase
LTE4
Figure 2. Biomarkers of inflammation and/or oxidative stress which have been detected in EBC in healthy
subjects and/or in patients with airway inflammatory diseases.
Abbreviations: COX, cyclo-oxygenase; GSH, glutathione; -GTP, -glutamyl-transpeptidase; 5-HETE,
5-hydroeicosatetraenoic acid; H2 O2 , hydrogen peroxide; 5-HPETE, 5-hydroperoxyeicosatetraenoic acid; 8-IP,
8-isoprostane; 5-LO, 5-lipoxygenase; LT, leukotriene; MPO, myeloperoxidase; NADPH oxidase, reduced
nicotinamide adenine dinucleotide phosphate oxidase; NO, nitric oxide; NOS, nitric oxide synthase; NO−
2 , nitrite;
−
−
NO−
3 , nitrate; 3-NT, 3-nitrotyrosine; O2 , superoxide anion; ONOO , peroxynitrite; PG, prostaglandin; ROS,
reactive oxygen species; RS-NO, RS-nitrosothiols; SOD, superoxide dismutase; TBARS, thiobarbituric acid
reactive substances; Tx, thromboxane.
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measured with immunoassays, which need to be
validated with reference analytical methods such
as mass spectrometry or high performance liquid chromatography (HPLC). These techniques
also will accurately quantify the concentrations
of the different markers in EBC. The presence
of LTB4 [Montuschi et al. 2004a; Montuschi
et al. 2005a], 8-isoprostane [Carpenter et al.
1998] PGE2 [Carpenter et al. 1998], aldehydes
[Corradi et al. 2003a] and free 3-nitrotyrosine in
EBC has been confirmed by mass spectrometry
[Goen et al. 2005; Larstad et al. 2005]. LTB4
[Montuschi et al. 2004a, 2005b] and aldehydes
[Corradi et al. 2003] in EBC have been measured by liquid chromatography/mass spectrometry (LC/MS), whereas 8-isoprostane and PGE2
in EBC have been measured by gas chromatography/mass spectrometry (GC/MS) [Carpenter
et al. 1998]. Free 3-nitrotyrosine has been measured by both LC/MS [Goen et al. 2005] and
GC/MS [Larstad et al. 2005]. Adenosine [Vass
et al. 2003], reduced glutathione [Corradi et al.
2003], and malondialdehyde (MDA) [Larstad
et al. 2002] in EBC have been detected
by HPLC. In patients with asthma exacerbations, pH values in EBC are more than two
log orders lower than normal and normalize with glucocorticoids [Hunt et al. 2000].
Measurement of pH in EBC is very reproducible, relatively easy to perform, and provides almost real-time results [Vaughan et al.
2003]. This technique could prove to be useful for diagnosis and for monitoring therapeutic response [Vaughan et al. 2003]. pH values
in EBC from patients with other lung diseases
have been reported [Kostikas et al. 2002], but
the biological significance of these data needs
to be clarified.
There is a high interindividual variability in the
total amount of proteins of concentrations in
EBC in healthy subjects (from undetectable to
1.4 mg) [Scheideler et al. 1993]. At present,
the reasons for this variability are unknown.
Establishing the protein concentrations in EBC
under standardized conditions is a priority in this
reasearch area. One study has shown that protein concentrations in EBC in 20 healthy subjects
averaged 2.3 mg/dl [Effros et al. 2002].
Using immunoassays, cytokines have been
detected in EBC in healthy subjects and in
patients with different inflammatory airway diseases [Scheideler et al. 1993; Cunningham
et al. 2000; Shahid et al. 2002]. However,
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immunoassays for cytokines in EBC still need to
be validated by reference analytical techniques.
Moreover, concentrations of cytokines reported
in most studies are close to the detection limit
of the assay. At these concentrations, analytical
data are less reliable. However, due to its biological importance, the issue of the presence of
cytokines in EBC and their accurate quantitative measurement in this biological fluid deserves
further investigation.
Comparisons between absolute concentrations
of EBC markers that have been reported by
different studies are currently difficult due to
differences in the EBC collection procedures,
condensers, and sample storage and handling;
differences in the analytical techniques used;
incomplete identification of factors that affect
EBC analysis; differences in clinical characteristics of study groups (diagnostic criteria, disease
severity, pharmacological therapy); interindividual biological variability. The use of reference
indicators in EBC (e.g. electrolytes or conductivity) has been proposed to take into account
changes in respiratory solute concentrations
which can result from variations in the dilution
of the respiratory droplets [Effros et al. 2002,
2003].
Methodological issues
Standardization of the EBC method and validation of the analytical techniques for measuring
each inflammatory biomarker are required for
comparing data from different laboratories and
assessing the clinical utility of EBC analysis.
Methodological issues that need to be addressed
include flow dependence, time dependence, and
influence of respiratory patterns; origin(s) of
markers in EBC; organ specificity of EBC; possible nasal, saliva, and sputum contamination;
identification of reference indicators in EBC to
ascertain to what extent EBC reflects the composition of airway lining fluid and to adjust
for possible changes in the dilution of respiratory droplets; influence of temperature, humidity,
and collecting-system materials; reproducibility
studies (between days variability, intrasubject
diurnal variability); comparisons of collection
devices; storage issues; possible need for sample pretreatment; for most biomolecules in EBC,
the demonstration of their presence in EBC and
their accurate quantitative measurement by reference analytical techniques (e.g. MS, HPLC);
the validation of sensitive, specific, and reproducible immunoassays to be used routinely.
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Therapeutic Advances in Respiratory Disease
Flow dependence and influence of
respiratory patterns
One study in 15 healthy adults has shown
that hydrogen peroxide concentrations in EBC
depend on expiratory flow rate indicating that
exhaled hydrogen peroxide levels are flow dependent [Schleiss et al. 2000]. In six healthy adults,
MDA concentrations in EBC collected at three
different flow rates were similar [Corradi et al.
2003] indicating that MDA concentrations are
not flow dependent. In four healthy children,
MDA and glutathione concentrations in EBC
samples collected at different flow rates were similar [Corradi et al. 2003b]. To study the influence
of ventilation patterns on 8-isoprostane, a marker
of lipid peroxidation, and PGE2 concentrations
in EBC, we asked 15 healthy adults to breath
at expiratory minute ventilation of 10, 20, and
30 L/min for 10, 10, and 5 min, respectively
[Montuschi et al. 2003a]. There was no difference in mean 8-isoprostane and PGE2 concentrations in EBC collected at different expiratory
minute ventilations indicating that the concentrations of these eicosanoids in EBC are not influenced by this respiratory parameter [Montuschi
et al. 2003a]. The lack of correlation between
8-isoprostane concentrations in EBC and minute
ventilation in mechanically ventilated patients
supports our data [Carpenter et al. 1998]. Likewise, LTB4 and LTE4 concentrations in EBC
were similar in five healthy subjects who breathed
at 14 and 28 breaths/min for 15 min, maintaining
the same tidal volume [Montuschi and Barnes,
2003b]. Other authors have demonstrated that
nitrite and protein concentrations in EBC are
independent of respiratory pattern [McCafferty
et al. 2004] and that ethanol concentrations
in EBC samples collected after tidal breathing
and deep inspiration and expiration are similar
[Rickmann et al. 2001]. These findings indicate
that different markers in EBC behave differently regarding flow dependence and ventilation
patterns. For this reason, each marker should be
studied individually.
For several markers, there is no information
on the influence of airflow and/or ventilation
patterns on their concentrations in EBC and
additional studies are required. Moreover, the
influence of ventilation patterns on markers
in EBC has been mainly studied in healthy
subjects and these findings cannot be directly
extrapolated to patients with inflammatory lung
diseases. Assessing the flow dependence of
different biomarkers in EBC is important for
12
the standardization of this technique as EBC
sampling will be performed either at a constant
flow rate for a fixed time, or at a constant exhaled
volume for a variable time.
8-Isoprostane concentrations in EBC do not
depend on the duration of EBC collection
[Carpenter et al. 1998]. Conflicting results have
been reported for MDA as in one study MDA
concentrations in samples collected at 10 and
20 min were similar [Corradi et al. 2003a],
whereas other authors showed that MDA levels
decreased with prolonged sampling time [Larstad
et al. 2003] in analogy to hydrogen peroxide
concentrations in EBC [Svensson et al. 2003]
Sampling time is likely to be relevant to EBC
concentrations of unstable compounds such as
cysteinyl-LTs or hydrogen peroxide. Additional
studies are warranted to establish the effect of
sampling time on different biomolecules in EBC
and to establish the ideal sampling time for each
of them.
The volume of EBC collected depends on breath
test duration, total exhaled volume, and ventilation rate. In healthy subjects, the mean volumes
of EBC collected in a 15-min test were almost
two-fold higher at 28 breaths/min compared with
those at 14 breaths/min [Montuschi et al. 2002b].
In healthy subjects, EBC volume is correlated
with expiratory minute ventilation, although the
condenser efficiency decreases at the higher expiratory minute ventilation values [McCafferty
et al. 2004]. The mean volume of EBC collected
during 10 min of tidal breathing was about 58%
of that collected during 20 min of tidal breathing [Corradi et al. 2003]. In healthy subjects,
the volume of EBC collected is increased when
subjects inhale through their noses and exhale
through their mouths without wearing a noseclip
probably due to an increase in minute ventilation
when sample collection is performed without a
noseclip [Vass et al. 2003].
Origin(s) of markers in EBC
Three aspects related to the origin of markers in EBC need to be considered: their cellular source; the compartment of the respiratory
system in which they are produced (airways vs
alveolar region); the extent to which EBC reflects
the composition of airway lining fluid and lung
inflammation.
EBC technique does not provide direct information on the inflammatory cells in the
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respiratory system. Identifying the cellular
source(s) of markers in EBC requires invasive
techniques such as bronchoscopy. A correlation
between the concentrations of a biomolecule
in EBC and a cell type can provide indirect information on the cellular source of that
biomolecule. However, this does not necessarily imply that the biomolecule is released by
that cell type as increased biomolecule concentrations in EBC could also reflect cell activation
without increased cell counts. Flow dependence
might indicate whether a certain inflammatory
mediator is mainly derived from the airways or
the alveoli [Schleiss et al. 2000]. If the concentrations of a biomolecule in EBC depend on
expiratory flow rate, this could imply that the
biomolecule is mainly derived from the airways
as flow rate affects the time available for its
accumulation in the airways, and therefore its
EBC concentrations [Schleiss et al. 2000]. In
contrast, the lack of flow dependence would indicate that the biomolecule is mainly derived from
the alveolar region where flow rate has a minor
role [Schleiss et al. 2000]. As it is flow dependent, hydrogen peroxide in EBC should primarily be derived from the airways [Schleiss et al.
2000], whereas 8-isoprostane [Montuschi et al.
2003a; Carpenter et al. 1998], PGE2 [Montuschi
et al. 2003a; Montuschi and Barnes, 2002b],
glutathione [Corradi et al. 2003a], and aldehydes [Corradi et al. 2003] should primarily be
derived from the alveolar region. These studies
were performed in healthy subjects. In patients
with inflammatory lung disease, the increased
numbers and/or activation of selective inflammatory cell types in different compartments of the
respiratory system and the high-current velocity
which can facilitate formation of aerosol particles
containing nonvolatile inflammatory mediators
[Hunt, 2002] might have a different impact on
the concentrations of a specific biomolecule in
EBC compared with healthy subjects.
The origin of nonvolatile inflammatory markers
which are probably contained in aerosol particles (e.g. proteins) in EBC can also be studied by identifying cell-specific markers such as
keratin and total phospholipid, which is found
primarily in surfactant [Jackson et al. 2007].
Both airway and alveolar compartments contribute to the formation of EBC [Jackson et al.
2007]. Using ethanol as a model compound,
it has been shown that the ratio between the
two compartments depends on ventilation, with
a shift towards the alveolar region during forced
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ventilation [Rickmann et al. 2001]. Studies on
the mechanisms of EBC formation are required
to established to what extent EBC reflects the
composition of airway lining fluid.
Whether the concentrations of inflammatory
mediators in EBC reflects lung inflammation or
systemic production of these compounds, particularly under conditions of increased permeability [Carpenter et al. 1998], is still unknown.
Measurement of inflammatory mediators in
patients with systemic inflammatory diseases
(e.g. systemic lupus erythematosus) and comparisons between concentrations of inflammatory mediators in EBC and in biological fluids
(e.g. plasma and/or urine) reflecting systemic
production of these compounds might be useful.
Dilution reference indicators
The use of dilution reference indicators has been
proposed to 1) estimate the concentrations of
nonvolatile inflammatory mediators in respiratory fluid; 2) to normalize for interindividual
variations in droplet formation. Although most
EBC is derived from water vapor, the presence of nonvolatile compounds in EBC indicates that droplets of the respiratory fluids are
collected [Effros et al. 2002]. Calculation of
airway lining fluid solute concentrations from
EBC requires estimation of the dilution factor.
Using electrolyte concentrations in EBC as dilution reference indicator, it has been estimated
that respiratory fluid represents between 0.01
and 2% of EBC volume [Effros et al. 2002].
Due to this high variability in the dilution of
respiratory droplets by water vapor, it has been
argued that increased concentrations of inflammatory mediators in EBC reported in different
lung diseases could reflect increased droplet formation [Effros et al. 2003]. The selective increase
of biomolecules in EBC and the lack of correlation between concentrations of structurally
related compounds in EBC are not consistent
with this hypothesis [Montuschi and Barnes,
2002b]. Measurements of electrolytes, urea, or
conductivity have been proposed as dilution reference indicators in EBC [Effros et al. 2003], but
their role in EBC analysis is debated. Alternatively, EBC data can be expressed as a change in
ratio of one compound to another.
Salivary contamination
Eicosanoids and other inflammatory mediators
are present in saliva [Zakrzewski et al. 1987;
McKinney et al. 2000] which could affect EBC
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Therapeutic Advances in Respiratory Disease
measurements as reported in one study [Gaber
et al. 2006]. However, amylase concentrations
in EBC samples are generally undetectable,
even with very sensitive assays [Effros et al.
2002]. In the small number of EBC samples in
which amylase concentrations were detectable,
they were less than 0.01% of those in saliva
indicating trivial salivary contamination [Effros
et al. 2002]. Given that leukotriene concentrations in saliva are only seven-fold higher than
those in EBC [Montuschi et al., unpublished
data] possible salivary contamination is unlikely
to have any relevant effect on measurement of
leukotrienes in EBC. Concentrations of adenosine and thromboxane (Tx) B2 in EBC samples
through tracheostomy in mechanically ventilated
patients or through the mouth in healthy subjects were similar [Vass et al. 2003]. However,
most of the ammonia in the EBC is derived
from the mouth, probably as ammonium gas,
as indicated by undetectable concentrations of
ammonia in EBC samples from patients with
tracheostomies [Effros et al. 2002]. These data
indicate that the effect of salivary contamination
on the concentrations of biomolecules in EBC
likely depend on their salivary concentrations
and their physicochemical properties (volatile vs
nonvolatile compounds). For this reason, the
issue of potential salivary contamination of the
EBC samples should be considered separately for
each marker.
Validation of analytical methods
Part of the variability of biomarker concentrations in EBC could be due to analytical
methods. In most studies published so far, concentrations of biomolecules in EBC were measured with commercially available immunoassay
kits which have not been generally validated
with reference analytical techniques. Enzyme
immunoassays generally work well in buffer,
but their behavior in EBC is not known.
Matrix effects due to differences in the composition of EBC and immunoassy buffer could
be relevant and might suggest the need for
sample pre-treatment. Validation of immunoassays with reference analytical techniques (e.g.
HPLC or MS) is essential for demonstrating
the presence of several biomarkers in EBC
and for their accurate quantitative assessment.
We have provided evidence for the specificity
of 8-isoprostane and PGE2 measurements in
EBC by radioimmunoassays (RIAs) developed
in our laboratory [Montuschi et al. 2003b] and
for a commercially available assay for LTB4
14
[Montuschi et al. 2003b]. In these studies, RIAs
for 8-isoprostane and PGE2 and EIA for LTB4
were qualitatively validated by reverse phaseHPLC [Montuschi et al. 2003c; 2003b]. Validation of immunoassays with independent assay
methods is a pre-requisite for large-scale use
of immunoassays in EBC analysis. As reference
analytical techniques are time-consuming, very
expensive and unsuitable routinely, validation of
immunoassays might contribute to the development of EBC analysis in respiratory medicine.
Measurement of markers in EBC for assessing
the response to pharmacological therapy
At present, most of the studies on the effect
of anti-inflammatory drugs on markers in EBC
are cross-sectional. Few single-center, generally
uncontrolled, interventional studies are available.
Large randomized controlled trials using a robust
and standardized methodology are required to
established the effect of drugs on inflammatory mediators in EBC in patients with airway
inflammatory diseases.
Isoprostanes
F2 -Isoprostanes, a group of 64 compounds isomeric in structure to PGF2α , are currently
considered among the most reliable biomarkers of oxidative stress and lipid peroxidation
[Montuschi et al. 2004b; Montuschi et al. 2007)
Concentrations of 8-isoprostane, the most known
compound belonging to F2 -isoprostanes, in EBC
are increased in adults with stable asthma, reflecting the severity of the disease and the degree of
inflammation [Montuschi et al. 1999]. Despite
treatment with oral or high doses of inhaled glucocorticoids, patients with severe asthma have
the highest concentrations of 8-isoprostane in
EBC indicating that this eicosanoid is relatively
resistant to inhaled glucocorticoids [Montuschi
et al. 1999]. Other authors reported a similar increase in 8-isoprostane in EBC in patients
with stable moderate asthma who were either
steroid-naive or treated with inhaled glucocorticoids, although subgroup analysis for the effects
of these drugs was not presented [Kostikas et al.
2002]. In contrast to patient with aspirin-tolerant
asthma, patients with aspirin-sensitive asthma
who were steroid-naive had higher 8-isoprostane
concentrations in EBC compared with those
who were treated with inhaled glucocorticoids
[Antczak et al. 2001] indicating that the effect
of glucocorticoids on 8-isoprostane might depend
on asthma phenotype and/or degree of inflammation. In analogy to adults with asthma,
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Review
8-isoprostane concentrations in EBC were elevated in children with asthma who were either
steroid-naive or treated with inhaled glucocorticoids compared with those in healthy children
[Mondino et al. 2004; Baraldi et al. 2003a;
Shahid et al. 2005]. There was no difference
in 8-isoprostane levels in EBC between the two
study groups [Mondino et al. 2004; Baraldi et al.
2003a; Shahid et al. 2005]. In healthy subjects,
pretreatment with a high dose of inhaled budesonide (1600 µg/day for 14 days) has no effect on
the increase in 8-isoprostane in EBC induced by
2 h exposure to ozone at 400 ppb [Montuschi
et al. 2002]. Two open-label studies have shown
that inhaled fluticasone at a dose of 100 µg twice
daily for 4 weeks [Mondino et al. 2004] and oral
montelukast, a LT receptor antagonist, at a dose
of 5 mg once a day for 4 weeks have no effect
on 8-isoprostane concentrations in EBC in atopic
children with asthma [Montuschi et al. 2006].
One study reports that oral glucocorticoids
reduce 8-isoprostane concentrations in EBC in
patients with acute asthma [Baraldi et al. 2003b].
Compared with healthy children, 8-isoprostane
concentrations in EBC were elevated in children with acute asthma [Baraldi et al. 2003b].
In these children, oral prednisolone at a dose of
1 mg/kg/day for 5 days reduced, but not normalized, exhaled 8-isoprostane concentrations
[Baraldi et al. 2003b] However, it should be
noted that 8-isoprostane concentrations reported
in this study were close to the detection limit of
the enzyme immunoassay where data are much
less reliable.
Patients with COPD have elevated 8-isoprostane
concentrations in EBC [Montuschi et al. 2000;
Kostikas et al. 2003]. In contrast to healthy subjects in whom smoking increases 8-isoprostane
concentrations in EBC, smoking habit does
not seem to affect 8-isoprostane levels in EBC
in patients with COPD [Montuschi et al.
2000]. A 41% reduction of mean 8-isoprostane
concentrations in EBC has been reported
in patients with COPD exacerbations after
antibiotic treatment [Biernacki et al. 2003].
Ibuprofen, a nonselective cyclo-oxygenase (COX)
inhibitor, and rofecoxib, a COX-2 selective
inhibitor, have no effect on 8-isoprostane concentrations in EBC in patients with COPD
[Montuschi et al. 2005b]. The lack of effect of
ibuprofen and rofecoxib on exhaled 8-isoprostane
concentrations indicates that this compound is
primarily formed independently of the COX
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pathway [Montuschi et al. 2004b; Montuschi
et al. 2007; Morrow et al. 1990].
In patients with obstructive sleep apnea, a
reduction in exhaled 8-isoprostane concentrations after continuous positive airway pressure
therapy has been reported [Carpagnano et al.
2003]. However, these data need to be interpreted cautiously due to the small size effect
(−1.9 pg/mL) and the reported 8-isoprostane
concentrations which were close to detection
limit of the immunoassay.
Leukotrienes
Elevated cysteinyl-LT and LTB4 concentrations
in EBC have been reported in both adults and
children with asthma [Montuschi and Barnes,
2002b; Montuschi et al. 2006; Antxzak et al.
2001; Mondino et al. 2004; Hanazawa et al.
2000; Zanzonato et al. 2004; Carraro et al. 2005;
Shibata et al. 2006; Cap et al. 2004; Csoma
et al. 2002]. In one study, no difference in cysLT concentrations in EBC was observed between
steroid-naive and steroid-treated patients with
aspirin-tolerant asthma, whereas steroid treatment was associated with lower concentrations
of cysteinyl-LTs in patients with aspirin-sensitive
asthma [Antczak et al. 2001]. These findings indicate that cysteinyl-LTs concentrations in EBC
in patients with aspirin-tolerant asthma are relatively resistant to glucocorticoids, although the
observational study design precludes definitive
conclusions. In children with asthma, inhaled fluticasone at a dose of 100 µg twice daily for
4 weeks has a pronounced effect in reducing
exhaled NO concentrations (−53%) over baseline values, but a limited effect on LTE4 concentrations in EBC (−18%) [Mondino et al.
2004]. In contrast, oral montelukast at a dose
of 5 mg once daily for 4 weeks seems to have
a higher effect on LTE4 concentrations (−33%)
in atopic children with asthma than inhaled glucocorticoids, but a lower effect on exhaled NO
concentrations (−27%) [Montuschi et al. 2006].
Although these studies were open-label, uncontolled, and performed in a limited number of
children with asthma, they point out the possibility of using the analysis of EBC as a noninvasive tool for assessing the effects of drugs
for asthma. Although several studies reported
detectable LT concentrations in EBC in patients
with asthma [Montuschi and Barnes, 2002b;
Montuschi et al. 2005b, 2006; Antxzak et al.
2001; Mondino et al. 2004; Hanazawa et al.
2000; Zanzonato et al. 2004; Carraro et al. 2005;
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Therapeutic Advances in Respiratory Disease
Shibata et al. 2006; Cap et al. 2004; Csoma et al.
2002] and other airway inflammatory diseases,
some authors were unable to detect cysteinylLT concentrations in EBC in adults with asthma
[Sandrini et al. 2003]. Due to the lack of a standardized EBC technique, comparisons of results
from different laboratories are currently difficult.
In contrast to a limited effect of inhaled glucocorticoids on LT concentrations in patients
with stable mild intermittent asthma, a reduction
of 60% of median exhaled cysteinyl-LT concentrations in children with asthma exacerbations after oral glucocorticoids has been reported
[Baraldi et al. 2003b]. This discrepancy might
be partly explained by the different route of
administration (inhaled vs oral) and/or the different disease severity (stable vs exacerbated
asthma).
LTB4 , but not LTE4 , concentrations in EBC
are elevated in patients with COPD [Montuschi
et al. 2003d; Kostikas et al. 2005]. Steroidnaïve and steroid-treated patients with stable
COPD have similar LTB4 concentrations in
EBC [Montuschi et al. 2003d]. In patients with
COPD exacerbations, LTB4 concentrations in
EBC were decreased 2 weeks after treatment
with antibiotics and this effect was maintained
after 2 months [Biernacki et al. 2003]. LTB4
concentrations in EBC are also increased in
patients with cystic fibrosis exacerbations and
are reduced after 2 weeks of antibiotic treatment
[Carpagnano et al. 2003b].
Prostanoids
PGE2 concentrations in EBC are elevated in
steroid-naive and steroid-treated patients with
stable COPD and correlate with LTB4 concentrations in EBC indicating that the greater the
lung inflammation, the higher the production of
PGE2 which may have anti-inflammatory effects
in the airways [Montuschi et al. 2003d]. PGE2
concentrations in EBC in steroid-naive and
steroid-treated patients with COPD were similar
[Montuschi et al. 2003d]. Nonselective inhibition
of COX by ibuprofen reduces PGE2 concentrations in EBC in patients with COPD, whereas
rofecoxib, a selective COX-2 inhibitor, has no
effect [Montuschi et al. 2005b] with potential
implications for the modulation of airway inflammation in COPD. There was no difference in
PGE2 concentrations in EBC between patients
with asthma and healthy subjects [Mondino et al.
2004; Baraldi et al. 2003] and between patients
with asthma who were either steroid-naïve or
16
treated with inhaled glucocorticoids [Mondino
et al. 2004; Baraldi et al. 2003a].
TxB2 , the stable hydrolysis product of TxA2 ,
a potent bronchoconstrictor, was detected in
some patients with asthma who were steroidnaïve [Montuschi and Barnes, 2002b], whereas it
was not detected in either patients with COPD or
healthy subjects [Montuschi et al. 2003d]. Other
authors reported detectable TxB2 concentrations
in EBC in most of the healthy subjects [Huszar
et al. 2005].
PGD2 and PGF2α , which are bronchoconstrictors, were detected in some patients with
asthma and healthy subjects [Montuschi and
Barnes, 2002b]. In those subjects, in whom these
eicosanoids were detectable there was no difference in their concentrations between the two
groups [Montuschi and Barnes, 2004b].
pH
Measurement of pH is very reproducible and
might prove clinically useful for the diagnosis and monitoring of pharmacological therapy
in inflammatory airway diseases [Hunt, 2002;
Vaughan et al. 2003].
pH of deaerated EBC is over two log orders
lower in adults with acute asthma than in
healthy subjects and normalizes with glucocorticoid therapy [Hunt et al. 2000]. pH values in
EBC in children with acute asthma are lower
than those in children with stable asthma and
normalize with inhaled glucocorticoid therapy
[Brunetti et al. 2006]. Treatment with lansoprazol, a protein pump inhibitor, for two months
increases pH values in EBC in patients with
moderate asthma and gastroesophageal reflux
disease, but not in asthmatic patients without
gastroesophageal reflux disease [Shimizu et al.
2007]. Mean pH values are lower in patients
with COPD than in patients with asthma and
healthy subjects [Kostikas et al. 2002]. One study
reported no difference in EBC pH between children with and without parentally reported symptoms suggestive of asthma and no consistent
association between EBC pH and lung function,
AHR, and airway inflammation [Nicolau et al.
2006]. In patients with COPD, pH values in
EBC are negatively correlated with sputum neutrophils and hydrogen peroxide concentrations
in EBC [Kostikas et al. 2002]. In one study,
there were no differences in pH values between
patients with COPD who were steroid-naïve and
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Review
those who were treated with inhaled glucocorticoids [Kosikas et al. 2002]. EBC pH can be
continuously monitored in mechanically ventilated patients and pH values normalize with
recovery [Walsh et al. 2006]. pH values in EBC
are lower in patients with allograft rejection
after lung transplantation compared with those
without rejection or healthy subjects [Dupont
et al. 2006].
pH values in EBC are unaffected by salivary
or therapeutic artifact, and identical to samples taken directly from the lower airway [Hunt
et al. 2002]. However, one study has shown that
EBC pH values in samples obtained by RTube
are lower than those in samples obtained by
EcoScreen condenser and that the storage for
8 weeks had a significant influence on the pH
of samples analyzed without deaeration [Prieto
et al. 2007]. An extensive normal data reveals
median EBC pH value is 8.0 with interquartile (25–75%) range of 7.8–8.1 [Paget-Brown
et al. 2006]. There were no differences based
on age, sex, or race and the distribution was
skewed, with 6.4% of EBC samples having a pH
range <7.4 (Paget-Brown et al.2006). However,
some methodological aspects including the effect
of ambient CO2 , need for sample deaeration,
and optimal time for measurement need to be
addressed.
One study has shown that condensate CO2
partial pressure influences pH measurements
[Kullmann et al. 2006]. Determination of pH at
a standard CO2 level provides the most reproducible condensate pH values to date [Kullmann
et al. 2006]. Large controlled studies are required
to establish the effect of inhaled glucocorticoids
on pH in EBC in patients with inflammatory
airway diseases including asthma and COPD.
Hydrogen peroxide
Hydrogen peroxide is detected in EBC but is
likely to be less stable than other markers of
oxidant stress such as isoprostanes. Compared
with healthy nonsmokers, exhaled hydrogen peroxide is increased in adults and children with
asthma [Horvath et al. 1998; Jobsis et al. 1997;
Antczak et al. 1997; Emelyanov et al. 2001]
and in patients with COPD [Dekhuijzen et al.
1996; Nowak et al. 1999; De Benedetto et al.
2001], bronchiectasis [Loukides et al. 1998]
and ARDS [Kietzmann et al. 1993]. Hydrogen peroxide concentrations in EBC are further
elevated in patients with COPD exacerbations
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[Dekhuijzen et al. 1996] and in children with
acute asthma [Dohlman et al. 1993], but not
in adults with unstable asthma [Horvath et al.
1998]. Treatment with montelukast at a dose
of 10 mg once daily for 2 weeks does not
affect the levels of hydrogen peroxide in EBC in
patients with mild asthma [Sandrini et al. 2003].
However, one study reported that nasal triancinolone acetonide reduced hydrogen peroxide
concentrations in EBC in patients with allergic
rhinitis with and without asthma, supporting the
one airway disease hypothesis [Sandrini et al.
2003b].
The effect of inhaled glucocorticoids on hydrogen peroxide concentrations in EBC in patients
with COPD has not been established [Ferreira
et al. 2001; van Beurden et al. 2003a]. One
double-blind crossover placebo-controlled study
performed in 20 stable nonsmoking patients with
COPD showed that beclomethasone at a dose of
500 µg twice daily for 2 weeks had no effect
on hydrogen peroxide concentrations in EBC
[Ferreira et al. 2001]. In contrast, another study
reported that glucocorticoids with more peripheral deposition (HFA-beclomethasone dipropionate at a dose of 400 µg twice daily for 4 weeks)
and with more central deposition (fluticasone
dipropionate at a dose of 375 µg twice daily
for 4 weeks) reduced hydrogen peroxide concentrations in EBC in patients with stable moderate COPD [van Beurden et al. 2003a]. In
25 patients with COPD who were admitted to
hospital because of exacerbations due to lower
respiratory tract infections, exhaled hydrogen
peroxide concentrations were not reduced during treatment with intravenous dexamethasone,
inhaled salbutamol/ipratropium and antibiotics
as needed [Kasielski et al. 2001]. In a doubleblind, double-dummy, placebo controlled, parallel group study in 44 patients with stable
COPD, treatment with oral N-acetyl-cysteine,
which can have antioxidant effects, at a dose of
600 mg a day for 12 months reduced hydrogen peroxide concentrations in EBC starting
from 9 months [van Beurden et al. 2003b].
Despite this evidence of reduced oxidant stress, a
multicenter randomized placebo controlled trial
has shown that N-acetyl-cysteine is ineffective
at prevention of deterioration in lung function and prevention of exacerbations in patients
with COPD [Decramer et al. 2005]. Moreover,
hydrogen peroxide concentrations in EBC in
patients with COPD are transiently increased
30 min after nebulization of N-acetyl-cysteine
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Therapeutic Advances in Respiratory Disease
[Rysz et al. 2007]. In children with cystic fibrosis exacerbations, antibiotic treatment reduces
hydrogen peroxide concentrations in EBC [Rysz
et al. 2007].
Elevated levels of hydrogen peroxide in EBC are
reduced, but not normalized, after two months
of antibiotic treatment in patients with active
tuberculosis [Kwiatkowska et al. 2007].
The clinical significance of these findings is
currently unknown. More controlled studies to
establish the effects of pharmacological therapy
on exhaled hydrogen peroxide in patients with
COPD are required. As with other biomolecules,
a reliable methodology for EBC analysis is
required for planning and interpreting the
results of interventional studies.
Nitrogen reactive species
High levels of nitrite/nitrate have been found in
EBC of adults [Ganas et al. 2001] and children with asthma [Ratnawati et al. 2006]. In
one study, nitrite/nitrate concentrations in EBC
were lower in adults with asthma who were
treated with inhaled glucocorticoids compared
with those in steroid-naïve adults with asthma
[Ganas et al. 2001]. Increase in nitrite concentrations in EBC is more pronounced during asthma exacerbations [Hunt et al. 1995]
and severe asthma [Corradi et al. 2001]. In a
double blind, parallel group, placebo controlled,
randomised trial, inhaled budesonide at a dose
of 100 µg or 400 µg once daily for 3 weeks
caused a rapid decrease in nitrite/nitrate concentrations in EBC which was not dose-dependent
[Kharitonov et al. 2002]. Nitrite/nitrate concentrations returned to baseline values one week
after treatment withdrawal [Kharitonov et al.
2002].
A significant reduction in exhaled S-nitrosothiols,
which are derived from interaction of NO or
reactive nitrogen species with thiol groups, compared with pretreatment levels was only seen at
the end of weeks 1 and 3 of treatment with
high dose budesonide [Kharitonov et al. 2002].
However, whereas reduction in nitrite/nitrate
concentrations in EBC is likely to contribute to
the anti-inflammatory effects of glucocorticoids
in the airways, the reduction in S-nitrosothiols
might have a different biological significance as
S-nitrosothiols have bronchodilator effects and
reduced concentrations of these compounds in
the airways has been causally linked to acute
18
respiratory failure in children [Gaston et al.
1998].
Some studies reported elevated levels of
3-nitrotyrosine in EBC in both adults [Hanazawa
et al. 2000] and children with asthma [Baraldi
et al. 2006] and in patients with cystic fibrosis
[Balint et al. 2001]. In a single-blind placebocontrolled study, inhaled flunisolide at a dose of
800 µg a day for 8 weeks reduced 3-nitrotyrosine
concentrations in EBC in children with mild-tomoderate asthma [Bodini et al. 2006]. However,
a study in which 3-nitrotyrosine was measured
with both GC/MS and HPLC showed that free
3-nitrotyrosine in EBC fails as a marker for
oxidative stress in children with stable cystic
fibrosis and asthma [Celio et al. 2006] although
larger study groups are required to draw definitive conclusions.
Other markers
Using immunoassay kits, cytokines including
interleukin (IL)-1β, [Scheideler et al. 1993; Sack
et al. 2006] tumor necrosis factor-α (TNF-α)
[Scheideler et al. 1993; Sack et al. 2006;
Rozy et al. 2006; Matsunaga et al. 2006], IL-4
[Shahid et al. 2002; Matsunaga et al. 2006;
Robroeks et al. 2006], IL-5 [Profiat et al. 2006],
IL-6 [Sandrini et al. 2003; Sack et al. 2006;
Rozy et al. 2006], IL-8 [Cunningham et al. 2000;
Sack et al. 2006; Matsunage et al. 2006], IL-10
[Sack et al. 2006; Robroeks et al. 2006], IL-12p70
[Sacks et al. 2006] insulin-like growth factor-1
[Rozy et al. 2006], interferon-γ , [Shahid et al.
2002; Robroeks et al. 2006] plasminogen activator inhibitor-1 [Rozy et al. 2006] were detected
in EBC in patients with different lung diseases.
However, concentrations of cytokines are generally close to the detection limit of the immunoassay [Bucchioni et al. 2003; Cunningham et al.
2000; Shahid et al. 2002; Carpagnano et al.
2003b; Robroeks et al. 2006]. At these concentrations the analytical variability is high and the
reliability of data is questionable. As with other
immunoassays matrix effects due to differences in
composition between assay buffer and EBC may
play a role.
Using a chemiluminescence-based membrane
protein array, expression of IL-4, IL-8, IL-17,
TNF-α, RANTES, interferon-γ -inducible protein 10, transforming growth factor-β, and
macrophage inflammatory protein 1α and 1β
in EBC were found elevated in steroid-naïve
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patients with asthma [Matsunage et al. 2006].
However, using flow cytometry, IL-2, IL-4,
interferon-γ , and IL-10 were detected only in 16,
16, 11, and 9%, respectively, of all samples in
patients with asthma and cystic fibrosis at EBC
concentrations which were close to the detection
limit of the assay [Robroeks et al. 2006]. Using a
multiplex fluorescent bead immunoassay (cytometric bead array), IL-1β, IL-6, IL-8, IL-10,
TNF-α, and IL-12p70 have been found elevated
in EBC patients with acute lung injury compared with healthy subjects [Sack et al. 2006].
However, cytokines concentrations in EBC were
in the order of few pg/ml. Protein array systems
are convenient for analyzing several biomolecules
at the same time, but the development of more
sensitive assays and the identification of cytokines
in EBC with reference analytical techniques
still stand.
Keratin concentrations have been detected in
EBC [Jackson et al. 2007; Gianazza et al. 2003]
and found elevated in healthy smokers [Gianazza
et al. 2003].
Some authors reported that DNA can be
detected in EBC [Gessner et al. 2004;
Carpagnano et al. 2005] whereas other authors
failed to detect Psedomonas aeruginosa and
B. Cepacia by PCR in the EBC of patients with
cystic fibrosis [Vogelberg et al. 2003]. Whether
gene expression analysis in EBC will have diagnostic relevance is not known.
Conclusions
Measurement of biomolecules in EBC might
provide insights into the pathophysiology of lung
diseases. Selective profiles of inflammatory mediators in EBC might have a differential diagnostic
value in respiratory medicine. As it is completely noninvasive, analysis of EBC is potentially
useful for assessing lung inflammation, monitoring pharmacological therapy, testing new drugs
for lung diseases, and clarifying the mechanism(s) of action of the existing drugs, providing a more rational pharmacological basis for
their administration. However, EBC analysis is
currently limited to research purposes, due to the
lack of standardization and methodological limitations that make it difficult to compate data
from different laboratories. Future research in
this area should include: the identification of
reference values for the different inflammatory
markers; large longitudinal studies to ascertain if
sequential EBC analysis in the individual patient
http://tar.sagepub.com
reflects the degree of inflammation and/or disease severity; studies of the relationships between
EBC markers and symptoms, pulmonary function, and other indices of airway inflammation
(e.g. exhaled NO and eosinophil counts in sputum); the accurate quantitative assessment of
biomolecules and the identification of profiles
of biomolecules in EBC in different lung diseases; large prospective controlled interventional
studies to establish the usefuleness of EBC analysis for monitoring pharmacological therapy in
lung diseases, as the available studies are mainly
cross-sectional; studies to establish the usefulness of EBC analysis for predicting therapeutic
response and assessment of new drugs; identification of other biomolecules in EBC; studies to
establish the feasibility and significance of gene
expression analysis in EBC; studies on drug disposition (e.g. antibiotics); studies on formation of
EBC, its origin in the respiratory system and its
relationships with airway lining fluid.
Whether and when EBC analysis will be applicable in the clinical setting cannot be anticipated.
Due to the important pathophysiological role of
inflammation in lung diseases such as asthma
and COPD, the relative lack of noninvasive techniques for monitoring airway inflammation and
therapy, and the importance of its potential applications, further research on EBC analysis is
warranted.
Acknowledgments
Supported by Catholic University of the Sacred
Heart, Fondi di Ateneo 2005–2007.
Conflict of interest statement
The authors declare that there is no conflict of
interest.
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