Maternal smoking affects lung function and airway
inflammation in young children with multiple-trigger
Satu Kalliola, MD,aAnna S. Pelkonen, MD, PhD,aL. Pekka Malmberg, MD, PhD,aSeppo Sarna, PhD,b
Mauri H€ am€ al€ ainen, PhD,c,dIlkka Mononen, MD, PhD,c,dand Mika J. M€ akel€ a, MD, PhDa
Helsinki and Turku, Finland
Background: Exposure to tobacco smoke is a well-known risk
factor for childhood asthma and reduced lung function, but the
effect on airway inflammation in preschool-aged children is
Objective: To examine the effect of parental smoking on lung
function and fractional concentration of exhaled nitric oxide
(FENO) in relation to both parental reports and children’s urine
cotinine concentrations in preschool-aged children with
Methods: A total of 105 3- to 7-year-old children with multiple-
trigger wheeze and lung function abnormalities were recruited.
Lung function was assessed by impulse oscillometry, and FENO
measurements were performed. Exposure to tobacco smoke was
determined by parental reports and measurement of children’s
urinary cotinine concentrations.
Results: Forty-three percent of the children were exposed to
environmental tobacco smoke according to parental reports.
The FENO level was significantly higher in children with a
smoking mother (n 5 27) than in children with a nonsmoking
mother (23.4 vs 12.5 ppb, P 5.006). The FENO level expressed as
z score and the cotinine level correlated significantly (P 5 .03).
Respiratory resistance at 5 Hz was higher in children exposed to
maternal smoking than in others (0.99 vs 0.88 kPas/L, P 5.005).
Urinary cotinine concentrations reflected well parental reports
on their daily smoking and increased relative to the number
of cigarettes smoked in the family (P <.01). Atopy was found in
75% of the children, but it was not associated with the FENO
value (P 5 .65).
Conclusion: Maternal smoking was associated with increased
FENO value and poorer lung function in steroid-naive preschool
children with multiple-trigger wheeze. Larger controlled trials
are needed to generalize the results. (J Allergy Clin Immunol
Key words: Pediatric asthma, environmental tobacco smoke,
exhaled nitric oxide, cotinine, lung function
Asthma is a chronic inflammatory airway disease involving
complex interactions of cells and mediators. Modulation of
inflammatory processes occurs not only as a result of endogenous
asthmatic events but also from inhalation of exogenous environ-
mental oxidants1such as environmental tobacco smoke (ETS),
which is a well-known etiological factor in childhood asthma.2-5
Parental smoking has been associated with increased asthma
incidence and poorer lung function in asthmatic children.5,6Ex-
posure to tobacco smoke products during pregnancy has been
experience more exacerbations as well as wheezing episodes,
lower respiratory tract infections, and otitis media if they have
been exposed to tobacco smoke.2,5,6
The fractional concentration of exhaled nitric oxide (FENO) is
Some airway irritants such as ETS can also interfere with the re-
sults of the FENO measurement.10Active smoking and decreased
FENO values have been detected in adults.11,12Acute exposure to
cigarette smoke also produces a decrease in FENO levels.11,12
In healthy children, no association between ETS and FENO
levels has been found.13-15In asthmatic children, nevertheless,
parental smoking and increased FENO values,17buta recent report
showed a negative effect of ETS on FENO values in school-aged
children.16Two studies found no association between ETS and
inflammation in preschool-aged children is unclear.
The exposure to tobacco smoke in children is most often
parental reports can be inaccurate as a measure of children’s
actual inhalation of smoke. Instead, cotinine, a major nicotine
metabolite, canbeusedasa measure ofnicotine intakeinsecond-
hand smoking. Cotinine level determination in serum, saliva,
urine, or hair has been used to monitor exposure to tobacco
The definition of early wheezing symptoms as episodic (viral)
and multiple-trigger wheeze phenotypes is recommended.21Epi-
sodic wheezers wheeze periodically and are well between the ep-
isodes, but multiple-trigger wheezers also wheeze between the
episodes in response to other than viral infection triggers.21,22
Multiple-trigger wheezers suffer from symptoms caused by irri-
tants such as ETS. There are, however, little data to support that
atic studies for this association are warranted.21We hypothesized
that parental smoking reduces lung function and increases
Fromathe Department ofAllergy,Helsinki University CentralHospital;btheDepartment
of Public Health, University of Helsinki;cthe Joint Clinical Biochemistry Laboratory
Department of Clinical Chemistry, University of Turku, Turku.
This work was supported by Helsinki University Central Hospital Research Funds, the
Sigrid Juselius Foundation, and Nummela Sanatoriums Stiftelse.
Disclosureofpotentialconflictofinterest: S.Kalliola hasreceivedresearch supportfrom
Helsinki University Central Hospital Research Funds, the Sigrid Juselius Foundation,
and Nummela Sanatoriums Stiftelse. L.P. Malmberg has received lecture fees from
OrionPharma and GlaxoSmithKline. The rest of the authors declare that they have no
relevant conflicts of interest.
Received for publication November 27, 2012; revised January 11, 2013; accepted for
publication January 11, 2013.
Available online February 5, 2013.
Corresponding author: Satu Kalliola, MD, Skin and Allergy Hospital, Helsinki
University Hospital, PO Box 160, Helsinki FIN-00029 HUS, Finland. E-mail: satu.
? 2013 American Academy of Allergy, Asthma & Immunology
ETS: Environmental tobacco smoke
FENO: Fractional concentration of exhaled nitric oxide
IOS: Impulse oscillometry
Rrs5: Respiratory system resistance at 5 Hz
Children referred to the Division of Allergy, Helsinki University Central
Hospital because of suspected asthma were recruited (n 5 105). Children
participated in the trial evaluating the efficacy of the combination of
salmeterol with fluticasone propionate compared with fluticasone propionate
or salmeterol alone in reducing bronchial inflammation in preschool children
with multiple-trigger wheeze.23Study children were aged 3 to 7 years of age
with multiple-trigger wheeze and had a history of wheeze or breathlessness
both during and outside discrete episodes. The median duration of symptoms
was 16 (2-58) months. All children had significant bronchodilator responsive-
ness and/or exercise-induced bronchoconstriction measured by impulse oscil-
lometry (IOS).24-26Patients were excluded if they displayed seasonal
symptoms only, had received systemic or inhaled corticosteroids in the previ-
ous 6 months, or had suffered signs of respiratory tract infection within the
past 2 weeks. All patients had lung function measured by IOS during the first
visit at the study clinic. If the patient fulfilled the inclusion criteria, the patient
was invited to the study clinic to complete the study investigations. The ran-
domization to 3 different treatment groups was done after a run-in phase.
The lung function was assessed again after an 8-week treatment phase. The
trial profile is provided in the article published previously.23The study was
approved by the ethics committee of Helsinki University Central Hospital,
and written informed consent was obtained from the parents.
information and the number of cigarettes smoked in the family per day.
Lung function tests were performed by using IOS, and respiratory system
determined as described previously.24-26Bronchodilator responsiveness was
defined as a decrease of 35% or more in Rrs5.24Typical symptoms and an in-
as evidence of exercise-induced bronchoconstriction.25
FENO level measurements
Measurements of FENO levels were performed with the stationary
chemiluminescence-based analyzer NIOX (Aerocrine AB, Solna, Sweden)
was calibrated according to the manufacturer’s specifications. Children were
seated, without a nose clip, and were asked to fill their lungs completely
with NO-free air, and thereafter to exhale with a mean and instantaneous
flow of 50 6 5 mL/s for at least 6 seconds. Consecutive, acceptable paired
measurements of FENO levels were recorded, and the repeatability and mean
tains a constant rate during exhalation. The FENO values are reported as abso-
lute values and z scores (SD from predicted).28
Children were screened by using skin prick tests for the following
aeroallergens: birch, timothy grass, meadow fescue, mugwort, cladosporium
milk, fish, wheat, shrimp, and peanut.
more against at least 1 of the tested allergens.
Blood samples to assess eosinophil count and eosinophilic cationic protein
were taken during the first or second visit at the study clinic and examined by
Urinary cotinine measurements
Urinary samples (3 3 10 mL) were gathered during the second visit at the
was extracted from urine by using the gas chromatographic method of Feyer-
abend and Russell.20A total of 200 mL of 5-methylcotinine (100 mg/L in
dichloromethane) as internal standard was added and mixed with 200 mg
of urine. Six hundred microliters of sodium hydroxide solution (5 mol/L),
40 mL of antifoam/phenol red mixture (20 mg phenol red/100 mL 5% anti-
foam), and 100 mL of 1,2 dichloroethane were added and the mixture was
centrifuged. The concentrated extract (2 mL) was injected into a Hewlett-
Packard FFAP silica capillary column of the Agilent 7890A gas chromato-
graph equipped with Agilent blos bead nitrogen-phosphorus detector. Helium
was used as a carrier gas, and air and hydrogen were used as detector gases.
The detector temperature was 3308C, and the current was 30 pA. Oven
temperature was programmed from 808C to 1208C at 408C/min, from 1208C
to 2208C at 12 8C /min, and 2208C for 10 minutes. The sensitivity of the assay
was 0.7 mg/L.20
Normal distribution was testedbyusingShapiro-Wilks test.BecauseFENO,
cotinine, and Rrs5 were not normally distributed, nonparametric tests were
used.Parental smokingwas categorizedin 2differentways:binomialvariable
habits at home (no smoking, only father, only mother, and both). Categorical
variables were compared by using Kruskall-Wallis test, Jonckheere-Terpstra
test, or Mann-Whitney U test. Bivariate correlations were made by using
Spearman correlation coefficient. Chi-square test was used to compare 2 cat-
egorical data. Data were analyzed by using SPSS, version 19.0 (SPSS, Inc,
The characteristics of the study population are presented in
Tables I and II. Urine samples from 97 of the 105 children were
available for cotinine analysis, and the FENO level was measured
in 96 children. Therefore, we had complete data for FENO level,
lung function, and urine cotinine levels from 96 children.
According to parental reports, 41 (43%) children were being
exposed to ETS. Smoking was reported by 27 mothers and 28
fathers, and both parents smoked in 14 families.
Cotinine concentrations and parental reports
The median cotinine concentration was less than 0.01 mg/L
(range, 0-17.1 mg/L). Fig 1, A, shows urine cotinine concentra-
tions in relation to exposure to ETS as reported by parents in
the questionnaires. Cotinine concentrations (median, range) in
children with only mother smoking (n 5 13; median, 4.1 mg/L;
range, 0-9.9 mg/L) and with both parents smoking (n 5 14; me-
dian, 4.6 mg/L; range, 0-17.1 mg/L) were significantly higher
than in children with nonsmoking parents (n 5 55; median,
of children to ETS was at a lower level in those families where
only the father smoked (n 5 14; median, cotinine <0.01 mg/L;
range, 0-4.4 mg/L).
J ALLERGY CLIN IMMUNOL
VOLUME 131, NUMBER 3
KALLIOLA ET AL 731
Taken altogether, cotinine levels increased in relation to the
number of cigarettes smoked by either parent (Fig 1, B, P <.01).
The median FENO level was 14.6 (3.5-120.7) ppb, and the FENO
value was significantly higher in children with a smoking mother
(n527) thaninchildren withanonsmokingmother(23.4vs12.5
ppb, P 5.006). The correlation between the FENO z score and the
cotinine level was statistically significant (P 5 .03; Fig 2). Fur-
thermore, the FENO level correlated positively with the number
of cigarettes smoked by the parents (P 5.05), and the FENO level
was higher in children with exposure to smoking only by the
mother (n 5 13; median, 23.3 ppb) or both parents (n 5 14; me-
11.2 ppb; P 5.05; Fig 1, C).
The children underwent IOS for the assessment of lung
function. Rrs5 was higher in children exposed to tobacco smoke
correlation between the cotinine level and Rrs5 did not reach the
significant level. There was no significant correlation between
cotinine levels or reported ETS and respiratory system reactance
at 5 Hz or 10 Hz, bronchodilatation effect, or extent of the
Atopy and ETS
Skin prick test result was positive in 75% of the children.
Cotinine concentrations were not associated with eosinophilia,
eosinophilic cationic protein, or skin prick test positivity. How-
ever, atopy was more pronounced in the children of smoking
P 5.01). The eosinophil count was also higher in children with a
FENO level in this population with high atopy prevalence (P 5
Two important and alarming messages emerge from this study.
First, children with smoking mothers haveclearly increased FENO
levels. Second, despite their young age, these children have
already developed decreased lung function. Our study also high-
lights the significance of dose; in other words, the urine cotinine
concentrations correlated well with the parental reports of their
daily smoking and FENO z score in this population with a high
prevalence of parental smoking (43%).
Smoking is on the decline in Finland; according to national
statistics, during the study years 2002 to 2005, 26% of men and
alence rates as 27% of mothers and 28% offathers smoked in our
sample of parents of children with multiple-trigger wheeze. The
importance of ETS as a risk factor for childhood asthma is well
established.1,30ETS is a composition of gases and particles
from burning cigarettes and exhaled mainstream smoke.30It con-
tains about 4000 chemicals including carcinogens and more than
1014oxidants per puff of smoke.1The particle size in ETS is
smaller than in mainstream smoke, which means that it has a
greater penetrability in the airways of children and thus may
evoke airway symptoms. Smoking by the mother seems to be
more harmful than smoking by the father. Similar findings have
dren are often in proximity to their mothers even when mothers
report smoking outside the house and less exposed to paternal
Most of the studies on ETS and children are based on parental
reports of their smoking habits.16,17We used a more exact
TABLE I. Characteristics of study children
No. of subjects
Males, n (%)
Females, n (%)
Age (y), mean (range)
Height (cm), mean (range)
Duration of symptoms (mo), median (range)
ETS, n (%)
Cotinine (mg/L), median (range)
Skin prick test result positive, n (%)
Rrs5 (kPas/L), median (range)
FENO level (ppb), median (range)
Parental atopy (%)
Parental asthma (%)
Animals at home (%)
TABLE II. Characteristics of study children in 2 groups: ETS
exposed and non–ETS exposed
ETS (n 5 41) Non-ETS (n 5 55)
Age (y), mean (range)
Duration of symptoms
(mo), median (range)
Height (cm), mean
Cotinine (mg/L), mean
Skin prick test positivity,
Rrs5 (kPas/L), median
Rrs5, z score, median
FENO level (ppb), median
FENO level, z score,
Parental atopy, n (%)
Parental asthma, n (%)
Animals at home, n (%)
113.9 (98-129)116.2 (99-133).1
3.6 (0-17.1)1.3 (0-13.9).001
35 (85)37 (67).043
0.99 (0.6-2.1)0.84 (0.54-1.74).004
0.57 (21.4 to 4.7) 20.01 (22.1 to 4.8) .04
20 (5-120)12.9 (3.5-93.9).084
2.9 (0.1-6.7)1.9 (20.6 to 6.5) .051
J ALLERGY CLIN IMMUNOL
732 KALLIOLA ET AL
monitoring of ETS bymeasuring urinary cotinine concentrations.
Cotinine has a longer elimination half-time (about 20 h) com-
pared with 2 hours for nicotine and therefore offers a better mea-
are aware, there are only 2 previous studies that have compared
cotinine concentrations with FENO levels in asthmatic children.
Spanier et al6studied 6- to 12-year-old children with doctor-
diagnosed asthma. They found no association between cotinine
levels or reported ETS and FENO levels. De la Riva-Velasco
et al31studied 8- to 18-year-old children with asthma on inhaled
did non–ETS-exposed children. Both the studies had older pa-
tients than in our study, and the children were using inhaled cor-
ticosteroids. The strength of our study is that preschool children
were steroid-naive, preventing the possible masking effect by
medication on the inflammatory response. Our study children
were multiple-trigger wheezers whose symptoms are caused by
many different triggers including exposure to tobacco smoke,
which makes the parental smoking particularly harmful to these
children. In these children, ETS can serve as one of the irritants
that increase airway inflammation and FENO levels. Previous
studies have not examined this effect in this age group.
Different analytical methods for cotinine concentrations have
been postulated to monitor second-hand smoking. Immunoassays
such as radioimmunoassay and ELISA lack the sensitivity and
specificity needed to detect low cotinine levels.32The gas chro-
matographic method used here is highly sensitive and specific
also for ETS monitoring (detection limit, 0.7 mg/L).32The mea-
sured cotinine levels were relatively low as expected in cases of
second-hand smoke exposure, and in 58 (60%) samples, there
were undetectable cotinine concentrations. Still, the significant
correlation between FENO levels expressed as z scores and the
cotinine level could be assessed. The clinical implication of this
finding is unclear. There was also a correlation between the coti-
nine concentrations in the children’s urine and the number of cig-
arettes smoked by parents. This has also been shown in earlier
Measurement of cotinine concentration in a single time spot
can be affected by many different factors.19,33Timing of the
measurement of cotinine concentration and former exposure
to tobacco smoke is important and can have an influence on
the outcome. There may also be some race- and age-related
differences in cotinine metabolism.33Although parental smok-
ing is the principal source of ETS in children, they can be
exposed to ETS in public places or by third-hand smoke.
Third-hand smoke refers to the type of the ETS that appears
after a cigarette has been extinguished and occurs via the inha-
lation of noxious gases and particles from smoked cigarettes or
FIG 1. A, Urinary cotinine concentrations grouped according to the parental reports of their smoking habits.
Cotinine concentrations were significantly higher in children with a smoking mother (median, 4.1 mg/L;
range, 0-9.9 mg/L) or with both parents smoking (median, 4.6 mg/L; range, 0-17.1 mg/L) than in children
with nonsmoking parents (median, <0.01 mg/L; range, 0-4.4 mg/L; P < .001 by Kruskall-Wallis test). Open
dots indicate outliers. B, Median cotinine concentrations increased relative to the number of cigarettes
smoked by either parent (P < .001, R 5 0.363 by Kruskall-Wallis test). The outliers are represented by
open dots. C, FENO levels in relation to parental reports of their daily smoking. FENO levels are medians
(mother 23.3 ppb, both 25.4 ppb, nonsmoking 12.9 ppb, father 11.2 ppb, P 5 .046 by Jonckheere-Terpstra
trend test). Open dots indicate the outliers. D, Rrs5 grouped according to the maternal smoking. The
mean Rrs5 was higher in children exposed to tobacco smoke via the mother than in children with nonsmok-
ing mothers (0.99 vs 0.88 kPas/L, P 5 .005 by Mann-Whitney test). Open dots indicate outliers.
J ALLERGY CLIN IMMUNOL
VOLUME 131, NUMBER 3
KALLIOLA ET AL 733
objects contaminated by absorbed tobacco smoke such as
furniture or clothes.34,35
We found an association between maternal smoking and
increased FENO levels in these preschool-aged children with
multiple-trigger wheeze. Previous studies in children have
reported controversial results.16-18In active adult smokers, FENO
notable concentrations of NO, and acute ETS exposure is thought
to cause a decrease in FENO levels by negative feedback on induc-
ible NO synthase.10Although acute exposure to tobacco smoke
seems to evoke a reduction in FENO levels in adults, there are lim-
ited data of its long-term effects on airway inflammation, espe-
cially in small children. One study on young children found an
association between an increased FENO value and parental-
informed ETS.17Laoudi et al16observed reduced FENO levels in
school-aged children with nontreated allergic asthma and who
had a smoking parent. Perzanowski et al18found an association
between ETS exposure at age 4 years and increased FENO levels
at age 7 years but an inverse association with present ETS expo-
sure. The discrepancies in study results are most probably due to
and some specific age-related differences in inflammatory re-
sponses on the airway epithelium. A recent study showed strong
TH2-type cytokine responses in infants with smoking parents.36
Genetic differences may also explain part of the inconsistencies
seen in the association between FENO levels and ETS.37,38
Our study children were well-characterized multiple-trigger
wheezers with lung function assessed by noninvasive IOS. While
atopy has been claimed to be associated with FENO levels,9,16,26,28
in our study population, no such connection could be detected.
This could be explained by this highly homogeneous study sam-
ple with steroid-naive multiple-trigger wheezers and the FENO
levels were rather high in most of the study patients. Lung func-
tionwaspoorer in children withsmoking mothers, which has also
been shown by other investigators.3-5It is possible that there is
even a causal relationship between ETS and airway symptoms
and reduced lung function.3-5Confounding factors such as paren-
tal atopy and animals at home were examined and no connection
to increased FENO levels or decreased lung function was found.
Still, there can be some unmeasured socioeconomical factors
that impacted the study results.
In the present study, parental smoking, particularly maternal
smoking, was associated with increased FENO values in steroid-
naive preschool-aged children with multiple-trigger wheeze.
Urinary cotinine concentrations reflected well parental reports
about their smoking habits. Children with smoking mothers
also had poorer lung function, in agreement with the detrimental
matory mechanisms evoked by ETS exposure in small children,
but the findings and message of this study are clear—young
children should not be exposed to ETS.
The authors thank specialist nurse Tuija Rito for her skill and care with the
children, the children themselves, and their parents.
Clinical implications: Environmental tobacco smoke modifies
bronchial inflammation and lung function of young children
with mothers who smoke.
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Cotinine (µ µg g/L)
20,0 15,010,0 5,0,0
FENO z score
FIG 2. The children’s urine cotinine concentration correlated with the FENO level expressed as z score
(P 5 .03 by Spearman correlation test).
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