Respiratory Medicine (2006) 100, 34–38
Methodological improvements for measuring
eicosanoids and cytokines in exhaled breath
Ellen Tufvesson?, Leif Bjermer
Department of Respiratory Medicine and Allergology, University Hospital, 221 85 Lund, Sweden
Received 7 April 2005; accepted 11 April 2005
Background: Exhaled breath condensate (EBC) is simple to collect and as such a
non-invasive method that has attracted substantial interest in the last few years.
However, several methodological concerns have been raised and it has been difficult
to reproduce results between different centres. Because of low concentrations of
inflammatory markers, potential loss in the sampling system may have great
influence. The aim of the present study was to se if evaporation and plastic coating
could facilitate detection.
Methodology: Through methodological improvements, we have now made it
possible to measure EBC concentrations of eicosanoids and cytokines in our system.
Due to absorbance of both fatty acid derivates and proteins to several plastics, the
first step is coating of all surfaces with bovine serum albumin and Tween 20. Since
several assays are sensitive to these factors, the methodology has to be standardised
to avoid false results. Secondly, larger amounts of EBC have to be vacuum-dried, and
thereafter resolved in the respective assay buffers. The EBCs have to be
concentrated 5–10 times, depending on samples and assay sensitivity.
Results: Due to these improvements we can measure, for example, cysteinyl-
leukotrienes, leukotriene B4, prostaglandin E and 8-isoprostane. High sensitivity
assays have also made it possible to measure cytokines, for example, interleukin
(IL)-1b, IL-8 and IL-13.
Summary: We are aware of different results from other labs. However, it seems
essential to coat and to concentrate the samples in order to achieve reliable and
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Exhaled breath condensate (EBC) is a novel
technique of sampling, suitable for assessment of
airway inflammation. The collection of EBC has
been proposed as a simple non-invasive method
that has attracted substantial interest in the last
few years. However, several methodological con-
cerns have been raised1,2and it has been difficult
to reproduce results between different centres. It
is a general impression from several research
groups that EBC is not a reliable technique today,
which was discussed in the ERS Research Seminar:
What is new in Clinical Pathobiology of the Lung?
Invasive Versus Non-invasive Methodologies, 15–16
November 2003, Pavia, Italy.
A number of publications have shown measure-
ments of some eicosanoids and cytokines. These
studies have shown increased levels of leukotriene
B4(LTB4) and cysteinyl-leukotrienes (Cys-LT) in EBC
in asthma3–7and in rhinitis.8Similarly, increased
levels of prostaglandin E2(PGE2) in EBC have been
shown in asthma,6,9as well as 8-isoprostanes in
asthma3,5,7,10and COPD.11Chromatography has
been used in some cases, for measurements,8,12
or validation thereof,10,11supplementary to EIA and
ELISA. Additionally, a few cytokines have also been
detected in EBC, such as interleukin (IL)-1b, IL-4,
IL-6, IL-8,13–18IFN-g19and TNF-a.20
Detectable amounts of these inflammatory mar-
kers have not been found in all samples in several
publications. Because of low concentrations of
inflammatory molecules in EBC, potential loss in
the sampling system may have great influence. The
aim of the present study was to see if concentration
of samples and coating of all plastic surfaces could
Nineteen non-smoking patients with symptomatic
mild asthma (mean age 35 range 19–53) were
investigated. Seventeen of the subjects where
atopic and all had concomitant asthma. Fourteen
of these had perennial rhinitis, (sensitised to house
dust mite and/or cat allergen). Fifteen were
treated with inhaled corticosteroids in low to
medium dose and four used only short-acting
beta-2 agonist on demand. As controls, 12 non-
smoking, non-atopic age-matched healthy subjects
Due to absorbance of both fatty acid derivates
(eicosanoids) and proteins to several plastics (e.g.
the normally low-absorbing polypropylene), the
first step was coating of all plastic surfaces with
1% bovine serum albumin and 0.01% Tween 20 for
30min. This includes the sample containers at-
tached to the condenser tube, as well as all
EBC was collected using a breath condenser
(EcoScreen; Jaeger, Wurzburg, Germany). Subjects
were asked to rinse their mouth with water. While
wearing a nose clip, they breathed tidally for
15min through a mouthpiece and a two-way non-
rebreathing valve, which also served as a saliva
trap. The condensate, 2–3ml per sample, was held
on ice while transferred to sampling tubes, and
immediately stored at ?801C, until analysis.
Cys-LTs, LTB4, 8-isoprostane and PGE concentra-
tions were measured with specific enzyme immu-
noassays (Cayman Chemical, Ann Arbor, MI). The
lower limit of detection for these assays was 13, 6,
5 and 2pg/ml respectively. IL-1b, IL-4, IL-5, IL-8
and IL-13 were measured using ELISA (R&D Systems,
Abingdon, UK). The lower limit of detection for
these assays was 0.1, 0.1, 3, 3.5 and 32pg/ml
respectively. Due to very low levels of these
molecules in EBC, larger amounts of EBC had to
be concentrated 5–10 times, depending on samples
and assay sensitivity. Samples were fully vacuum-
dried in a speed vac centrifuge, and thereafter
resolved in the respective assay buffers.
Data are expressed as means7SEM. The Mann–Whit-
ney non-parametric test for two independent
samples was used for statistical analyses between
Initial experiments showed that coating with the
detergent Tween 20 was crucial for the eicosanoid
analysis, while bovine serum albumin had no
improving effect on these measurements. On the
contrary, bovine serum albumin was the best
improving agent in the cytokine measurements.
Tween 20 also increased the possibility of measur-
ing cytokines, but not to the same extent. Though
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Methodological improvements for measurements in EBC35
IL-1b could be measured in a few samples even
without coating, the concentration was only about
one tenth compared to samples after coating with
bovine serum albumin (data not shown).
There was no effect in these assay systems by
bovine serum albumin itself. No cross-reactivity
was observed in the assays used, neither with
coated controls nor when using 1% bovine serum
albumin as sample. In contrast, Tween 20 affected
several of the eicosanoid assays. Standard series
were prepared in assay buffer containing increasing
concentrations of Tween 20 (0.0005–5%). No influ-
ence on the assay could be observed up to a
concentration of 0.005% Tween 20. We therefore
used 0.01% Tween 20 for coating of all plastic ware
to avoid interference with the assay.
Concentration of samples was necessary to allow
measurements. To check for reliability of this
procedure, different volumes of EBC from different
samples were concentrated and resuspended for
comparison. The original concentrations in the EBC
samples were calculated, and were in agreement
between the different volumes that were used
(data not shown).
It is now in our hands possible to measure several
eicosanoids, e.g. Cys-LTs, LTB4, PGE and 8-isopros-
tane. For visualising these results, we show in Fig. 1
the amount of LTB4in EBC from asthmatic patients
and healthy controls. A significant increase in the
amount of LTB4 in EBC from asthmatic patients
compared to healthy controls (10.372.5 and
3.870.6pg/ml respectively, P ¼ 0:01) could be
Figure 2 shows the amount of 8-isoprostane in
EBC from asthmatic patients and healthy controls.
There was a tendency of an increase in the amount
of 8-isoprostane in EBC from the asthmatic group
(2.170.6pg/mlin the asthmaticgroup and
1.570.2pg/ml in the control group), however, it
was not significant.
Additionally, using this method set-up, we could
also measure Cys-LTs. The level of Cys-LTs was
2–20pg/ml EBC. Similarly, we could measure the
amount of PGE in EBC, where the amount was about
(3–6pg/ml) (data not shown).
High sensitivity assays have also made it possible
to measure cytokines, for example, IL-1b. In 13 out
of 18 (72%) EBC samples from the asthmatic group,
and 5 out of 12 (42%) from the healthy control
group there were detectable levels of IL-1b. EBC
contained about 0.03–0.5pg/ml IL-1b (Fig. 3). In
several samples there were higher levels of IL-1b in
EBC from the asthmatic group compared to the
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Figure 1 The amount of LTB4in healthy controls and
asthmatic patients. LTB4is expressed as pg/ml EBC. Mean
values are shown by horizontal bars.
Figure 2 The amount of 8-isoprostane in healthy controls
and asthmatic patients. 8-isoprostane is expressed as pg/
ml EBC. Mean values are shown by horizontal bars.
Figure 3 The amount of IL-1b in healthy controls and
asthmatic patients. Samples with undetectable values
were set to 0.01pg/ml ( ¼ one tenth of the detection
limit; corresponding to 10?
sample). IL-1b is expressed as pg/ml EBC. As not all
samples had detectable values no mean values are
concentration of the
E. Tufvesson, L. Bjermer36
Moreover, IL-8 and IL-13 could be measured in
various samples, preferably in EBC from patients
with asthma. The concentrations found in EBC were
0.3–1pg/ml of IL-8 and 5–20pg/ml of IL-13 (data
No detectable amounts of IL-4 and IL-5 could be
observed in EBC.
We are aware of different results from other
research groups. However, it seems essential to
coat all plastic ware and to concentrate the
samples in order to achieve reliable and measur-
The use of polypropylene tubes has previously
been discussed21for diminishing adherence of
inflammatory markers, but this is not sufficient.
Using bovine serum albumin and Tween 20 for
coating decreases adherence of inflammatory mo-
lecules to plastic surfaces. Bovine serum albumin is
mainly essential to avoid adherence of other
proteins, such as cytokines, while the detergent
Tween 20 is crucial for preventing adherence of
fatty acid derivates, such as leukotrienes and
prostaglandins. However, several EIA assays, often
used in previous publications for measurements of
these inflammatory markers, are sensitive to Tween
20, and a balance between coating and the assay is
highly significant to avoid false results. The level of
acceptance in the EIA assays used in this study was
0.005% Tween 20, which has been confirmed by
Concentrating the samples is one way of solving
the problem of low concentrations of inflammatory
markers in EBC. Gessner et al.14,15have previously
used a 10 times concentration of samples through
lyophilisation, which resembles vacuum-centrifuga-
tion used in this study. Additionally, today’s high
sensitivity assays have also made it possible to
measure cytokines, for example, IL-1b, which
would otherwise not be detectable.
It would be preferable to use larger volumes of
EBC to increase the possibility of measuring several
inflammatory markers. However, there is a balance
between the collection time for the patient and the
number of assays that could be run. A collection
time of 15min gives about 2–3ml of EBC, and an
extension of this time could of course be con-
ceivable. However, it is a difficult matter with
respect to the patients to increase the time as
much as should be needed for duplicate samples in
the whole battery of assays. Additionally, the effect
of the increased collection time needs to be
evaluated in this regard.
The patients used in this study are mild asth-
matics, and EBC collected from these patients most
likely contain lower levels of inflammatory markers
than from patients with a more severe disease.
Interestingly, we observed higher concentrations of
LTB4and 8-isoprostane in those patients who were
not treated with inhalation steroid during the time
of collection. The amount of EBC used therefore
depends on the type of patients who are studied, as
well as the assay that is used. We recommend a
5–10-fold concentration of EBC for most applica-
Concerning concentrations of inflammatory mo-
lecules in EBC, these are quite divergent through-
out the publications. In this study, we see lower
levels compared to several other studies. We
cannot explain this phenomenon, but several
factors, for example, the variable dilution of
epithelial lining fluid droplets with condensed
water vapour,1are known to affect the outcome
of these results.
Our method is still under investigation, and
possibly, we may diminish adherence of molecules
even more, to further improve the methodology.
We are now one step forward, enabling measure-
ments of several inflammatory markers in EBC.
We thank Cayman Chemical for analysis regarding
the critical concentration of Tween 20 in their EIA
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