Exhaled pH, exhaled nitric oxide, and induced
sputum cellularity in obese patients with obstructive
sleep apnea syndrome
GIOVANNA E. CARPAGNANO, ANTONIO SPANEVELLO, ROBERTO SABATO,
ANNARITA DEPALO, VIVIANA TURCHIARELLI, and MARIA PIA FOSCHINO BARBARO
Airway inflammation plays an important role in obstructive sleep apnea syndrome
as well as in obesity. Increasingly, researchers are studying airway inflammation
noninvasively and are studying the new markers of airways inflammation.The aim of
this study was to measure pH in the exhaled breath condensate (EBC), the exhaled
nitric oxide (NO), and the inflammatory cell profile in the induced sputum of obese
patients with and without obstructive sleep apnea syndrome (OSAS). The pH in EBC,
the exhaled NO, and the induced sputum cells were measured in 30 obese patients
with OSAS (OOs), in 20 obese patients without OSAS (ONOs), and in 10 healthy
patients (HPs). Levels of pH in EBC were lower in OOs and in ONOs than in HPs.
Furthermore, the concentrations of exhaled NO and the percentages of neutrophils
in the induced sputum were greater in OOs and in ONOs than in HPs. No significant
differences were found between OO and ONO for other measurements of airway
inflammation. This study shows the presence of airway’s inflammation in obese
patients with and without OSAS and indicates that the “exhaled acidopnea” as well
as exhaled NO and sputum neutrophils are good tools to measure airway inflam-
mation in these subjects. (Translational Research 2008;151:45-50)
Abbreviations: AHI ? apnea/hypopnea index; BMI ? body mass index; EBC ? exhaled breath
condensate; FEV1? forced expiratory volume in 1 s; FVC ? forced vital capacity; HP ? healthy
patient; NO ? nitric oxide; ONO ? obese patient without OSAS; OO ? obese patient with
OSAS; OSAS ? obstructive sleep apnea syndrome; TST SaO2? 90% ? percentage of total sleep
time with oxyhemoglobin saturation ? 90%.
to significant hypoxemia and snoring.1These repetitive
bstructive sleep apnea syndrome (OSAS) is
characterized by repetitive episodes of upper
airway obstruction during sleep, which leads
cycles of hypoxia-reoxygenation and the snoring-re-
lated mechanical trauma lead to inflammation that is
transmitted to the entire respiratory system (from the
nose to the bronchi) and comes close to the systemic
Obesity is the most common predisposing factor for
OSAS. Obesity seems to be involved in the generation
of airway inflammation through the production and
secretion of inflammatory factors mediated by fat
Several studies described the presence of inflamma-
tion in the nose, the uvula, the soft palate,4,5and the
plasma6,7of participants with OSAS, which confirm the
role of the inflammation in the pathogenesis and self-
maintenance of sleep-related breathing disorders. How-
ever, few reports are available on bronchial inflamma-
tion in patients with OSAS. Olopade was the first to
From the Institute of Respiratory Disease, Department of Medical
and Occupational Sciences, University of Foggia, Foggia, Italy, and
the Fondazione Salvatore Maugeri, Care and Research Institute,
Cassano delle Murge, Foggia, Italy.
Submitted for publication February 26, 2007; revision submitted
September 13, 2007; accepted for publication September 14, 2007.
Reprint requests: Giovanna Elisiana Carpagnano, MD, Institute of
Respiratory Diseases, University of Foggia, Ospedale D’Avanzo, Via
degli Aviatori 1, 71100 Foggia, Italy; e-mail: ge.carpagnano@
1931-5244/$ – see front matter
© 2008 Mosby, Inc. All rights reserved.
report the increase of pentane and nitric oxide in the
exhaled air of patients with OSAS after sleep.1,4,8-10
Recently, we characterized airway inflammation in pa-
tients with OSAS by the greater concentrations of in-
terleukin-6 in exhaled breath condensate (EBC)11and
the greater percentage of neutrophils in induced spu-
In consideration of the key role that airway inflam-
mation plays in the pathogenesis of several respiratory
disorders and therefore of OSAS, recent research ad-
dresses the noninvasive study of airways and the new
markers of airway inflammation. The pH measurement
occurs between the noninvasive markers of airway in-
flammation and is the most readily available method to
study the contribution of airway acid stress in respira-
tory diseases.12Normative data and the reproducibility
of pH measurements in EBC are available from Paget-
Brown et al.12In particular, a low exhaled pH, desig-
nated as “acidopnea,” has been described in asthma,
cystic fibrosis, and chronic obstructive pulmonary dis-
ease.13To the best of our knowledge, no study has
measured the exhaled pH in patients with OSAS.
To examine deeply the significance of airway inflam-
mation in OSAS, the aim of this study was to measure
pH in the exhaled breath condensate, the exhaled nitric
oxide (NO), and the inflammatory cell profile in the
induced sputum of obese patients with OSAS. To clar-
ify better the responsibility of obesity in the airway
inflammation reported in OSAS, we performed the
same measurements in a group of obese patients with-
MATERIAL AND METHODS
Participants. The study population consisted of 3 groups
of participants, matched for age and gender: 30 obese patients
with OSAS (OOs), 20 obese patients without OSAS (ONOs),
and 10 healthy patients (HPs). All participants were Cauca-
sian, recruited consecutively from the outpatient clinic of the
sleep laboratory of the Respiratory Disease Institute at the
University of Foggia. Written informed consent was obtained
from all participants, and the study was approved by the
Institutional Ethics Committee. A complete physical exami-
nation was performed, which includes neurological, cardio-
pulmonary, and ear, nose, and throat evaluations. The diag-
nosis of OSAS was defined on the basis of an apnea/hypopnea
index (AHI) greater than 10 per hour during the polysomnog-
raphy and on the basis of symptoms of excessive daytime
sleepiness. ONO patients were free of any sleep disorder,
which includes snoring, and no participants reported daytime
sleepiness. HPs were free of sleep disturbances and had an
AHI score of less than 10.
In the groups of OO and ONO, 24 patients had hyperten-
sion controlled by calcium channel antagonists for at least 6
months; 21 patients had hyperlipidemia, 13 patients had hy-
peruricemia, and 3 patients had history of angina pectoris.
No patients had history of cerebrovascular disease. We
considered exclusion criteria for the enrollment in the study
such as endocrinologic diseases; narcolepsy or idiopathic
hypersomnia; neuromuscular disease; psychiatric disorders;
asthma; chronic obstructive pulmonary diseases; respiratory
failure; gastroesophageal reflux; anatomic maxillo mandibu-
lar skeletal abnormalities;, ear, nose, and throat pathologies;
and respiratory and systemic infections. Individuals with al-
cohol abuse or chronic use of any kind of drug were also
excluded. All participants enrolled were nonsmokers. Partic-
ipants enrolled in the current study did not use inhaled, oral,
or nasal steroids; anti-inflammatory drugs; or beta-blockers
for at least 4 weeks before the study. Patients with OSAS
enrolled in the current study were not treated with nocturnal
Study design. During the first day, the participants under-
went anamnesis recording by questionnaire, measurement,
and recording of anthropometric data, bioelectric analysis,
and respiratory function. Polysomnography, exhaled breath
condensate collection after awakening (7 AM), exhaled NO
measurement, and blood collections were performed on the
second day. The participants did not consume anything and
did not perform any physical efforts for 60 min before the
exhaled breath condensate collection.12Finally, on the third
day, the participants underwent sputum induction.
Polysomnography. All participants were evaluated in the
sleep laboratory at the Respiratory Disease Institute at the
University of Foggia for 1 night, and they were monitored
continuously for 8 h using a 19-channel polysomnograph
(Compumedics Limited, Victoria, Australia). Polysomnogra-
phy was performed in all participants after the night of
adaptation in the hospital. Electroencephalographic, elec-
AT A GLANCE COMMENTARY
The airways inflammation plays an important role
in obstructive sleep apnea syndrome (OSAS) as
well as in obesity. An increasing interest is to date
addressed to the non invasive study of airways
inflammation and to the new markers of airways
inflammation. In this study we measured pH in the
exhaled breath condensate (EBC), the exhaled ni-
tric oxide (NO) and the inflammatory cell profile
in the induced sputum of obese subjects with and
The study shows the presence of airways inflam-
mation in obese subjects with and without OSAS
and indicates that the “exhaled acidopnea” as well
as exhaled NO and sputum neutrophils are good
tools to measure airways inflammation in these
subjects and may be proposed in clinical practice.
Carpagnano et al
trooculographic, and chin electromyographic recordings were
obtained with surface electrodes according to standard meth-
ods.14Airflow was monitored by cannula placed at the nose
and at the mouth. Abdominal and ribcage movements were
assessed by respiratory inductive plethysmography. All night
recordings of hemoglobin oxygen saturation were obtained by
finger pulse oxymetry. Snoring sounds, electrocardiography,
and sleep position were also recorded. Apnea was defined as
cessation of airflow that lasted greater than or equal to 10 s,
and hypopnea was defined as discrete reduction (2/3) of
airflow and/or abdominal ribcage movements lasting greater
than or equal to 10 s and associated with a decrease greater
that 4% in oxygen desaturation or arousals. The frequency of
apneas plus hypopneas per hour of time in bed (AHI) and the
percentage of total sleep time with oxyhemoglobin saturation
? 90% (TST SaO2? 90%) were calculated. Sleep record was
scored according to the standardized criteria.15Finally, sleep
propensity has been measured by the Epworth Sleepiness
Pulmonary function testing. Pulmonary function tests
were performed within 1 day of breath condensate measure-
ments. Forced expiratory volume in 1 s (FEV1) and forced
vital capacity (FVC) and the FEV1/FVC ratio were measured
using a spirometer (PK Morgan Ltd., Gillingham, UK). The
best value of 3 maneuvers was expressed as a percentage of
the predicted normal value.
Induced sputum. Sputum was collected and processed by
the method described by Spanevello et al.17All sputum
counts and measurements were performed blindly to the
clinical details. Selected sputum was defined as adequate only
when fewer than 20% squamous cells were present and via-
bility was greater than 50%. Sputum induction was performed
in 55 patients, without observing any significant adverse
effect or a decrease of FEV1greater than 20%.
EBC. EBC was obtained using a condenser, which col-
lected the nongaseous components of the expiratory air non-
invasively (EcoScreen; Jaeger, Wurzburg, Germany). Partic-
ipants breathed tidally through a mouthpiece and through a
2-way nonrebreathing valve, which also served as a saliva
trap. Participants were asked to breathe at a normal frequency
and tidal volume, wearing a nose clip, for a period of 10 min.
If participants felt saliva in their mouth, they were instructed
to swallow it. The condensate (at least 1 mL) was collected as
ice at ?20°C, transferred to Eppendorf tubes, and stored at
Exhaled pH measurement. A stable pH was achieved in
all cases after deaeration/decarbonation of breath condensate
specimens by bubbling with argon (350 mL/min) for 10 min,
as reported previously.18pH was then measured within 5 min
of condensate collection by means of a pH meter (Jenway-
350, Ltd, Gransmore Green, England) with a 0.00 to 14.00 pH
range and a resolution/accuracy of 0.01 ? 0.02 pH.
The reproducibility of the repeated measurements of pH
was confirmed by the Bland and Altman test and by the
Measurement of exhaled NO. A rapid-response chemi-
luminescence NO analyzer was used to quantify NO. Two-
point calibrations were performed daily using 5.2-parts-per-
million calibration gas. Exhaled NO (FENO) was measured
using a previously described restricted breath technique,
which employed expiratory resistance and positive mouth
pressure to close the velum and exclude nasal NO, and a
constant expiratory flow of 45 mL/s.19Participants inhaled to
total lung capacity and exhaled while targeting a constant
pressure of 20 mm Hg. Exhalations proceeded until a clear
NO plateau of at least 3 s was achieved. Repeated exhalations
were performed until 3 plateaus agreed within 5%.18
Statistical analysis. Data were expressed as means ? SD.
A Mann-Whitney U test was used to compare groups. We
used the Pearson coefficient to evaluate the association be-
tween normally distributed continuous variables and the
Spearman rank correlation test to evaluate the association
between nonparametric variables. Significance was defined as
a P value of less than 0.05.
Anthropometric variables, polysomnography vari-
ables, and functional data of obese participants with
and without OSAS are shown in Table I.
Levels of pH in breath condensate and of exhaled
NO. Levels of pH in the exhaled breath condensate
were significantly less in OO patients than in HPs (7.48
? 0.07 vs 7.99 ? 0.03; P ? 0.01).
The reduction of exhaled pH was also observed in
ONO patients (7.68 ? 0.08) with respect to the HP;
however, the difference does not reach the significance
(P ? 0.1). No significant difference for pH in EBC was
demonstrated between OO and ONO (7.48 ? 0.07 vs
7.68 ? 0.08; P ? NS) (Fig 1).
Concentrations of exhaled NO were significantly
greater in OO patients than in HP subjects (31.6 ? 1.6
ppb vs 4.8 ? 0.7 ppb; P ? 0.001). The enhancement of
Table I. Anthropometric functional and
polysomnographic variables in the
(n ? 10)
(n ? 30)
(n ? 20)
NADIR (SaO2 Mmin) 93.2 ? 2.0
TST SaO2 ? 90%
Fat weigh (kg)
42 ? 4
24 ? 0.5
36.9 ? 0.3
39 ? 8
33.2 ? 1.1*
43.4 ? 0.5*
45 ? 8
33.8 ? 1.7*
40.1 ? 1.7*
3.6 ? 0.4
2.5 ? 1.6
59.1 ? 4.1†
11.3 ? 1.4†
87.5 ? 1.9
24.9 ? 5.5†
94.8 ? 13.0
5.7 ? 0.8*
5.6 ? 3.8*
91.8 ? 6.4
1.0 ? 0.4
98.5 ? 6.9
100.9 ? 10.5
85.8 ? 9.3
37.5 ? 4.4*
0.8 ? 1.8
102 ? 3.2
104 ? 6.1 120.2 ? 13
95 ? 2.2
17.1 ? 5.0
85.0 ? 1.6
32.8 ? 2.7*
*P ? 0.05 vs HP.
†P ? 0.01 vs HP.
Volume 151, Number 1 Carpagnano et al
exhaled NO was also observed in ONO patients) (27.1
? 1.8; P ? 0.001). No significant difference for ex-
haled NO was demonstrated between OO and ONO
(31.6 ? 1.6 vs 27.1 ? 1.8 ppb; P ? NS) (Fig 2).
Cell counts in the induced sputum. Two ONO and 3
HPs did not produce adequate sputum samples (which
contained at least 500 nonsquamous cells), and the
expectorates were discharged.
Cell count performed on induced sputum showed that
the percentage of neutrophils was greater in the OO
patients than in the HP subjects (63.3 ? 15.4 vs 28.4 ?
12.3; P ? 0.001). Percentages of neutrophils in the
induced sputum of ONO patients were also increased
significantly with respect to HP subjects (45.8 ? 6.7 vs
28.4 ? 12.3; P ? 0.01). A marked difference was
observed in percentage of neutrophils in the induced
sputum between ONO and OO (45.8 ? 6.7 vs 62.4 ?
6.8; P ? 0.05 (Fig 3).
The percentage of macrophages was less in the OO
patients and in the ONO patients compared with
healthy controls (34.1 ? 11.9 and 52.1 ? 7.3 vs 70.9 ?
12.8; P ? 0.01), whereas no significant differences
were observed in the percentage of eosinophils (1.2 ?
1.1 and 1.6 ? 1.4 vs 0.9 ? 0.7), lymphocytes (1.0 ?
0.8 and 1.0 ? 1.1 vs 1.2 ? 0.9), and epithelial cells
(0.9. ? 1.6 and 1.1 ? 1.3 vs 0.9 ? 1.1).
Correlation between exhaled pH or exhaled NO or neutro-
phils in the induced sputum and anthropometric variables,
polysomnographic variables, and metabolic variables in
obese and OSAS patients. To avoid confounding the re-
sults, healthy participants were excluded by correlation.
The Pearson coefficient between exhaled pH or exhaled
NO or neutrophils in the induced sputum and anthro-
pometric variables, polysomnographic variables, and
metabolic variables in obese patients (with and without
OSAS) is shown in Table II.
Levels of exhaled pH were correlated negatively with
body mass index (BMI), neck circumference, AHI,
Fig 1. Exhaled pH in OOs, ONOs, and HPs.
Fig 2. Exhaled NO in OOs, ONOs, and HPs.
Fig 3. Percent of macrophages and neutrophils in induced sputum of
OOs, ONOs, and HPs. N%, neutrophils %; M%, macrophages %.
Table II. Correlation between exhaled pH or
exhaled NO or neutrophils in the induced sputum
and anthropometric variables, polysomnographic
variables, metabolic variables, and risk factors in
obese subjects (with and without OSAS)
TST SaO2 ? 90%
*P ? 0.05.
Carpagnano et al
TST SaO2? 90% (percentage of total sleep time with
oxyhemoglobin saturation ? 90%). Levels of exhaled
NO were correlated positively with neck circumfer-
ence, AHI, and TST ? 90%. Percentages of sputum
neutrophils were correlated positively with neck cir-
cumference, AHI, and TST ? 90%. However, exhaled
pH, exhaled NO, and percentage of neutrophils did
show correlation between each other.
In the current study, we demonstrated that obese
patients with OSAS present lower airway inflammation
as demonstrated by the acidification of their breath
condensate, the increase of exhaled NO, and the greater
percentage of neutrophils in induced sputum compared
with healthy patients. Signs of airways inflammation
have also been observed in obese non-OSAS patients
compared with healthy controls although their airway
inflammation was less marked compared with obese
patients with OSAS. We found a negative correlation
between levels of exhaled pH with BMI, neck circum-
ference, AHI, and TST SaO2? 90% and a positive
correlation between levels of exhaled NO and percent-
age of sputum neutrophils with neck circumference,
AHI, and TST ? 90%. However, inflammatory mark-
ers did not show correlation with each other.
The inflammation of airways plays a key role in the
pathogenesis of OSAS; however, the exact mechanism
remains unknown.5The first publications regarding air-
way inflammation focused on upper airway inflammation,
particularly that in the nose, uvula, and soft pal-
ate.4,5,10,21,22Recently, concomitant bronchial inflamma-
tion has been described. A few years ago, Olopade et al
reported the increase of the exhaled pentane and NO
concentrations in participants with moderate–severe
OSAS. Our group has described the high levels of inter-
leukin-6 in the EBC and the percentage of increased
neutrophils in the induced sputum of these patients.2,5,23
The interest in studying associations between inflam-
matory the markers of airways and the measurement of
pH in body fluids has increased. Only recently, an
endogenous airway acidification designated “acidop-
nea,” as assessed by the condensate pH, has been im-
plicated in the pathophysiology of inflammatory airway
diseases and, therefore, has been added to the list as a
useful assessment relevant to their diagnosis and man-
pH measurement in EBC is the most technically
validated of all measurements. The pH of EBC has
been measured already in several inflammatory dis-
eases such as asthma, cystic fibrosis, and COPD, in
which pH levels are high.13,18To the best of our knowl-
edge, this study is the first to measure the exhaled pH in
patients with OSAS. We observed the acidification of
exhaled pH in obese patients with OSAS compared
with healthy controls.
Low exhaled pH as an expression of airway inflam-
mation was expected in obese patients with OSAS. The
stress exerted on the mucosa of the respiratory system
by the snoring, the pressure gradient caused by the
intermittent obstruction, and the activation of the neural
receptors by proinflammatory peptides are recognized
as the main cause of airway inflammation. These mech-
anisms may determine acidopnea in OSAS patients.
As with previous reports, we found greater concen-
trations of exhaled NO and of sputum neutrophils in
obese OSAS, which confirmed the presence of bron-
chial airways inflammation in these patients.5,25
To clarify better the role of the obesity in the gener-
ation of airway inflammation, we measured the exhaled
pH, the exhaled NO, and the inflammatory cells in the
induced sputum of obese patients without OSAS. To
the best of our knowledge, this study is the first to
analyze these inflammatory markers in the obese non-
OSAS patients. We observed a marked reduction of
exhaled pH and an increase of exhaled NO and per-
centage of neutrophils in these patients compared with
healthy patients. The increase of these markers was,
however, less marked between ONO and healthy pa-
tients than it was between OO patients and healthy
patients. The presence of inflammation in the pathogen-
esis of the obese patients was recognized a long time
ago, and our study confirms these findings.14
The increase of inflammatory markers either in
plasma or in the EBC of obese patients has been attrib-
uted to the accumulation of excess fat around the neck,
which could be the source of the inflammatory cells and
mediators.14Several studies demonstrated that adipose
tissue is an important source of cytokines; fat produces
and releases several factors, collectively called adipo-
kines, which are implicated in systemic and vascular
inflammation.23Visceral fat seems to produce several
of these adipokines more actively than does adipose
tissue.23Reduction in fat mass correlates with the de-
crease in the serum levels of many of these factors,
which implies that approaches designed to promote fat
loss should be useful in attenuating the proinflamma-
tory milieu associated with obesity.
The observed correlation among exhaled pH, NO and
neutrophils, and BMI and neck circumferences sup-
ports this theory.
In our study, obese patients with and without OSAS
were very similar with respect to age, BMI, and other
anthropometric characteristics such as neck circumfer-
ence, so it is difficult to establish whether the increased
levels of inflammatory markers are linked to the obesity
or to the sleep apnea. A limit of this study is that we
failed to enroll nonobese OSAS patients who could
Volume 151, Number 1Carpagnano et al
help to study the airway inflammation in obesity, and
that we did not enroll patients with OSAS to CPAP to
evaluate the possible reversibility of the inflammation
described in OSAS patients.
In conclusion, our results indicate that obese patients
with OSAS and obese patients without OSAS present
upper airway inflammation that could be monitored
with the exhaled pH, exhaled NO, and percentage of
neutrophils in the induced sputum.
However, additional studies are required to clarify
better different mechanisms that cause airway’s inflam-
mation in obesity and OSAS.20
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