Seasonal Changes in Endotoxin Exposure and Its Relationship to Exhaled Nitric Oxide and Exhaled Breath Condensate pH Levels in Atopic and Healthy Children
Liu C-H (2013) Seasonal Changes in Endotoxin Exposure and Its Relationship to Exhaled Nitric Oxide and Exhaled
Breath Condensate pH Levels in Atopic and Healthy Children. PLoS ONE 8(6): e66785. doi:10.1371/journal.pone.0066785
Seasonal Changes in Endotoxin Exposure and Its Relationship to Exhaled Nitric Oxide and Exhaled Breath Condensate pH Levels in Atopic and Healthy Children
Gwo-Hwa Wan 0
Dah-Chin Yan 0
Tao-Hsin Tung 0
Chin-Sheng Tang 0
Chiu-Hsin Liu 0
Aimin Chen, University of Cincinnati, United States of America
0 1 Department of Respiratory Therapy, College of Medicine, Chang Gung University , Tao-Yuan, Taiwan , 2 Division of Taipei Pediatrics, Department of Pediatrics, Chang Gung Children's Hospital, Chang Gung Memorial Hospital , Taipei, Taiwan , 3 Department of Medicine, College of Medicine, Chang Gung University , Tao-Yuan, Taiwan , 4 Department of Medical Research and Education, Cheng-Hsin General Hospital , Taipei, Taiwan , 5 Department of Public Health, College of Medicine, Fu Jen Catholic University , New Taipei City, Taiwan , 6 Department of Respiratory Therapy, Chang Gung Memorial Hospital , Taipei , Taiwan
Endotoxin, a component of the cell walls of gram-negative bacteria, is a contaminant in organic dusts (house dust) and aerosols. In humans, small amounts of endotoxin may cause a local inflammatory response. Exhaled nitric oxide (eNO) levels, an inflammation indicator, are associated with the pH values of exhaled breath condensate (EBC). This study evaluated seasonal changes on indoor endotoxin concentrations in homes and the relationships between endotoxin exposure and eNO/EBC pH levels for healthy children and children with allergy-related respiratory diseases. In total, 34 children with allergy-related respiratory diseases and 24 healthy children were enrolled. Indoor air quality measurements and dust sample analysis for endotoxin were conducted once each season inside 58 surveyed homes. The eNO, EBC pH levels, and pulmonary function of the children were also determined. The highest endotoxin concentrations were on kitchen floors of homes of children with allergy-related respiratory diseases and healthy children, and on bedroom floors of homes of asthmatic children and healthy children. Seasonal changes existed in endotoxin concentrations in dust samples from homes of children with allergic rhinitis, with or without asthma, and in EBC pH values among healthy children and those with allergyrelated respiratory diseases. Strong relationships existed between endotoxin exposure and EBC pH values in children with allergic rhinitis.
Funding: This study was supported by grants NSC95-2314-B-182-056 and NSC96-2314-B-182-017 from the National Science Council of Taiwan. Also, the authors
would like to thank the Chang Gung Memorial Hospital, Taiwan, for financially supporting this research under grants CMRPD150091. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
The prevalence of allergy-related respiratory diseases has
gradually increased over the last two decades, especially among
children who live in highly urbanized areas. Major risk factors for
such diseases include modernization and considerable time spent
indoors . The development of allergy-related respiratory
diseases involves the result of interactions between different
genetic and environmental factors.
Endotoxin, a component of cell walls of gram-negative bacteria,
is ubiquitous in indoor environments. In its pure form, endotoxin
is a lipopolysaccharide (LPS). Endotoxin has strong
pro-inflammatory properties that can induce airway inflammation and
cytokine upregulation in humans . Acute endotoxin exposure
may induce blood and lung inflammatory responses that involve
neutrophils and macrophages, which can result in fever, shaking
chills, and severe asthma .
Responses to endotoxin inhalation typically differ among
individuals based on genetic factors or degree of tolerance .
Studies have indicated that endotoxin exposure in infancy has a
protective role against asthma development and allergen
sensitization [5,6]. However, endotoxin exposure during childhood and
later in life likely has detrimental effects on both healthy
individuals and those with asthma and respiratory diseases .
Endotoxin is in air and in house dust, and occurs as a
contaminant of organic dusts and environment aerosols .
Inhalation exposure to endotoxin is common in occupational
environments  and in homes [12,13]. Household endotoxin
exposure is a strong risk factor for asthma  and wheezing
[14,15]. Such factors as poverty, number of people in a household,
pets, bedding materials, cleaning methods, frequency of cleaning,
and geographic location influence indoor endotoxin exposure
Endotoxin exposure from floor dust is more critical than that
from airborne particles, as the breathing zone of children is close
to the ground . A clear correlation existed between endotoxin
content in house dust from living room floors and airborne
endotoxin concentrations . Another investigation
demonstrated that the highest and lowest endotoxin concentrations in homes
were on kitchen floors and on bedding, respectively .
Measuring nitric oxide in exhaled breath is a non-invasive
technique for assessing airway inflammation. Studies have shown
that eNO may be a diagnostic factor for asthma  and for
evaluating the anti-inflammatory effects of inhaled corticosteroids
in asthmatic children . Exhaled nitric oxide is also related
to allergic sensitization in childhood asthma and allergic rhinitis
 via the late-phase influx of eosinophils  and nitric oxide
formation after aeroallergen exposure [25,26]. Furthermore, eNO
concentrations are negatively associated with the pH values of
EBC in asthmatics . However, no correlation existed between
eNO and EBC pH in children [28,29]. The EBC pH values in
asthmatic children with or without inhaled corticosteroid
treatment are clearly lower than those in healthy children [30,31].
Environmental factors, particularly endotoxin exposure, have a
significant effect on degree of airway inflammation [32,33].
However, few studies have examined endotoxins effects on eNO
levels and EBC pH values.
To date, no published data exists on seasonal variations of
indoor endotoxin concentration distributions in homes and on the
relationships between endotoxin exposure and eNO/EBC pH
levels in children with allergy-related respiratory diseases and in
healthy children. This study determined seasonal changes in
indoor endotoxin concentrations (on living room floors, bedroom
floors, mattresses, and kitchen floors) in homes of healthy children
and those of children with allergy-related respiratory diseases in
Taipei, Taiwan. Additionally, nitric oxide levels and pH values in
the exhaled breath of healthy children and those with
allergyrelated respiratory diseases were used to evaluate possible
associations with endotoxin exposure in the home.
Materials and Methods
In total, 58 children were recruited: 15 asthmatic children; 9
with allergic rhinitis; 10 with asthma and allergic rhinitis; and 24
healthy children. Age range was 613 years. Children with
allergyrelated respiratory diseases were recruited from the Department of
Pediatrics at Chang Gung Childrens Hospital, Taipei, Taiwan.
All asthmatic children were diagnosed by a pediatrician according
to the guidelines of the Global Initiative for Asthma (GINA) 
Asthma (AS) group
Allergic rhinitis (AR) group AS+AR group
Positive rates of blood allergen-specific IgE test, n (%)
Pulmonary function, % predicted
Endotoxin, EU/mg dust
and the modified National Asthma Education and Prevention
Program (NAEPP) . Although children with allergic rhinitis
did not have hyper-reactive airway responses during methacholine
challenge tests, they met the guidelines in Allergic Rhinitis and its
Impact on Asthma (ARIA) . Healthy children with no history
of allergies or pulmonary disease were recruited from the
Department of Pediatrics at Chang Gung Childrens Hospital
and from the same elementary schools in Taipei City that were
attended by recruited children with allergy-related respiratory
diseases. Study protocol was approved by the Institutional Review
Board of Chang Gung Hospital. Informed written consent was
obtained from each childs parents.
The timing of home visits for indoor air sampling was in the first
two months of each season, and based on then time schedule of
each family. The seasons were defined as follows: spring (February
to April); summer (May to July); autumn (August to October); and
winter (November to January). Environmental measurements of
air temperature, relative humidity (RH), carbon dioxide (CO2),
total volatile organic compounds (TVOCs), and suspended
particulate matter were taken inside the homes of healthy children
and of those with allergy-related respiratory diseases. Indoor
temperature, RH, and CO2 concentration were determined using
a digital psychrometer (TSI, Inc., Shoreview, MN, USA). The
TVOCs levels were determined using a Model PGM 7240
ppbRAEH sampler (RAE Systems, Inc., San Jose, CA, USA). A
portable DUSTcheck monitor (Model 1.108; Grimm
Labortechnik Ltd., Ainring, Germany) was used to measure mass
concentrations of airborne particulate matter.
Air samplers were placed in the center of each living room for 8
consecutive hours. Sampling height was 1.01.2 meters above the
floor, a childs breathing zone. The frequency of air sampling was
once per season.
House Dust Collection and Endotoxin Analysis
The timing of house dust sampling was the same as that of
indoor air sampling. Dust samples were collected each season from
four sites in homes: the top surface of a childs mattress; the
bedroom floor next to the mattress; living room floor; and kitchen
floor. A vacuum cleaner fitted with a fresh glass-fiber filter was
used to vacuum a 1 m2 surface area for 2 minutes at each sampling
site. Collected dust samples were sieved through a 425-mm mesh
screen to obtain fine dust.
All endotoxin-free glassware and metals were heat treated at
180uC for 4 hours. A fine dust sample (5 mg) was agitated with
1 mL triethylamine phosphate (TAP) buffer (pH 7.5) for 1 hour to
extract endotoxins. Endotoxin activity in a dust sample was
determined with a chromogenic Limulus amebocyte lysate assay
(Associates of Cape Cod, East Falmouth, MA, USA). Standard
response curves were generated using endotoxin standards in the
range of 0.254.0 EU/mL (correlation coefficient, r = 0.99). A
negative control with pyrogen-free water was used with each assay.
No sample had an activity level below the assays detection limit.
Exhaled Nitric Oxide, EBC pH, and Spirometric
The eNO/spirometric measurement and EBC collection were
performed within 1 week after domestic environmental sampling.
The eNO levels of all children were measured at an expiratory
flow rate of 50 ml/s using a chemiluminescence analyzer (CLD
88 sp; ECO Physics, Du rnten, Switzerland) according to
international standards . A non-invasive cooling device (EcoScreen,
Jaeger Toennies, Hoechberg, Germany) was used to collect the
subjects EBCs. While sitting upright and wearing a nose clip, each
subject breathed normally through a mouthpiece for 15 minutes
for EBC sampling. Approximately 13 mL of EBCs were collected
from each subject and then analyzed. All EBC samples were
deaerated with argon at a flow rate of 350 ml/min to remove
carbon dioxide. Then, EBC pH was determined using a digital pH
meter (UB-5; Denver Instruments, Denver, CO, USA). A
spirometer (MIR Spirolab II, Pinyork, Japan) was used to
determine forced expiratory volume in 1 second (FEV1), forced
vital capacity (FVC), and maximum mid-expiratory flow (MMEF).
The frequency of eNO, EBC pH, and spirometric determinations
for healthy children and children with allergy-related respiratory
diseases was the same as that for indoor air sampling.
Statistical analyses used SPSS version 13.0 (SPSS, Inc.,
Chicago, IL, USA). Figures were graphed with GraphPad Prism
5.0 software (GraphPad Software, Inc., San Diego, CA, USA).
The significance level for all tests was 0.05. The necessary study
sample size was calculated by considering the eNO levels as the
primary outcome . In a Kruskal-Wallis test study, sample sizes
of 15, 9, 10, and 24 were obtained from the asthma group, allergic
rhinitis group, asthma+allergic rhinitis group, and control group
whose means were compared. The total sample of 58 subjects had
an 87% power to detect differences among means versus the
alternative of equal means using an F test with a 0.05 significance
One-way analysis of variance (ANOVA) for normally
distributed data was used to identify group difference in age. The
Kruskal-Wallis test and Mann-Whitney U test for non-normally
distributed data were used to identify group differences in
continuous variables. A chi-squared test was applied to identify
group differences in categorical variables. Seasonal variations in
indoor air indices and endotoxin concentrations in homes, and
levels of eNO and EBC pH in children were assessed by
nonparametric repeated-measures ANOVA for skew distributed data.
The strength of correlation was assessed by the Spearman test for
non-normally distributed data to determine the relationship
between the endotoxin concentrations in dust samples and
eNO/EBC pH levels of all recruited children.
Age range of recruited children was 613. Average age differed
significantly between the four groups (p,0.01; Table 1). No
significant differences existed for gender. Children with
allergyrelated respiratory diseases were mainly sensitized by house dust
mite (D. farinae and D. pteronyssinus) allergens, dog and cat danders,
the cockroach allergen, and Candida albicans. No significant
differences in positive rates of blood allergen-specific IgE tests
existed among the asthma group, allergic rhinitis group, and
asthma+allergic rhinitis group. Mean pulmonary function
parameters for children with allergy-related respiratory diseases were
normal. The FEV1/FVC (% predicted) and MMEF (% predicted)
differed significantly among the four groups of children (p,0.01).
Mean severity of allergy-related respiratory diseases was
mildintermittent level. The highest and lowest median eNO levels were
in asthmatic children (36.9 ppb) and healthy children (11.7 ppb),
respectively. The eNO level of asthmatic children was highest
(p,0.01). Additionally, the EBC pH value (6.3) of asthmatic
children was significantly lower than for the other three groups
(children with allergic rhinitis, EBC pH = 7.9; children with
asthma and allergic rhinitis, EBC pH = 7.9; and healthy children,
EBC pH = 7.3; p,0.01).
During the study period, median air temperatures were 26.4
27.8uC in all homes (Table 1). Median relative humidity was 65.0
69.3% and median CO2 concentrations were 517.8583.4 ppm.
The homes of children with asthma had the highest relative
humidity (p = 0.05). No significant differences in concentrations of
TVOCs and suspended particular matter (PM10, PM2.5, and PM1)
in homes existed among the four groups. The median endotoxin
level in the homes of both asthmatic children (489.9 EU/mg dust)
and children with allergic rhinitis (430.2 EU/mg dust) was
significantly lower than that in the homes of healthy children
(651.9 EU/mg dust; p = 0.021).
Groups were then evaluated separately to identify seasonal
trends in indoor air indices such as temperature, humidity, CO2
concentrations, TVOCs concentrations, and PM levels. A seasonal
trend for variations in air temperature was observed in the homes
of asthmatic children, children with allergic rhinitis, children with
asthma and allergic rhinitis, and healthy children (all p,0.01;
Fig. 1A). Apart from seasonal changes in indoor humidity in the
homes of children with asthma and allergic rhinitis (p = 0.011), the
homes of other groups exhibited no seasonal changes of indoor
humidity (Fig. 1B). The seasonal variation in CO2 concentrations
was significant in homes of the asthma group (p,0.01) and the
control group (healthy children) (p,0.01; Fig. 1C). Moreover, a
seasonal trend for variations in TVOCs concentrations only
existed in the homes of children with asthma (p = 0.026; Fig. 1D).
Seasonal variation in the PM10 level was obvious in the homes of
both children with allergic rhinitis (p,0.01) and healthy children
(p,0.01; Fig. 1E). The levels of PM2.5 and PM1 had significant
seasonal changes in the homes of both the allergic rhinitis group
(PM2.5: p = 0.016, Fig. 1F; PM1: p = 0.017, Fig. 1G) and the
control group (PM2.5: p,0.01, Fig. 1F; PM1: p,0.01, Fig. 1G).
A seasonal trend for variations in endotoxin concentrations only
existed in the homes of both children with allergic rhinitis
(337.6 EU/mg dust in spring; 650.4 EU/mg dust in summer;
527.8 EU/mg dust in autumn; and 399.0 EU/mg dust in winter;
p = 0.015) and children with asthma and allergic rhinitis
(446.0 EU/mg dust in spring; 913.5 EU/mg dust in summer;
541.0 EU/mg dust in autumn; and 390.2 EU/mg dust in winter;
p,0.01; Fig. 2).
In the homes of asthmatic children, median endotoxin
concentration on bedroom floors (341.8 EU/mg dust) was clearly
higher than that on living room floors (219.6 EU/mg dust;
p = 0.046; Fig. 3A). In the homes of children with allergic rhinitis,
median endotoxin concentration on kitchen floors (602.8 EU/mg
dust) was significantly higher than that on living room floors
(292.5 EU/mg dust; p,0.01), on bedroom floors (296.1 EU/mg
dust; p,0.01), and on mattresses (376.0 EU/mg dust; p = 0.011;
Fig. 3B). In the homes of children with asthma and allergic rhinitis,
median endotoxin concentration was highest on kitchen floors
(607.9 EU/mg dust) and lowest on bedroom floors (388.7 EU/mg
dust; p,0.01) and on mattresses (388.0 EU/mg dust; p = 0.02;
Fig. 3C). The distributions of endotoxin concentrations (living
room floors, 379.4 EU/mg dust; bedroom floors, 395.3 EU/mg
dust; kitchen floors, 584.2 EU/mg dust; mattresses, 381.3 EU/mg
dust) in the homes of healthy children were the same as those in
the homes of children with allergic rhinitis (Fig. 3D).
Exhaled nitric oxide levels of .20 ppb were found in 86.7% of
asthmatic children, 41.7% of children with allergic rhinitis, 45.0%
of children with asthma and allergic rhinitis, and 22.9% of healthy
children (data not shown). In terms of possible seasonal variations
in eNO levels, no significant trends existed for children with
allergy-related respiratory diseases and for healthy children
(Fig. 4A). Notably, 58.3% of asthmatic children and 26.0% of
healthy children had EBC samples with pH values ,6.5 (data not
shown). The four groups had significant seasonal variations in
EBC pH values. For asthmatic children (5.9 in spring; 6.4 in
summer; 6.6 in autumn; and 6.3 in winter; p = 0.025) (Fig. 4B); for
children with allergic rhinitis (8.0 in spring; 7.6 in summer; 7.7 in
autumn; and 8.1 in winter; p,0.01); for children with asthma and
allergic rhinitis (7.9 in spring; 7.9 in summer; 7.8 in autumn; and
8.1 in winter; p,0.01); and for healthy children (6.4 in spring;
7.3 in summer; 7.4 in autumn; and 7.4 in winter; p,0.01).
No strong correlations existed between endotoxin exposure and
eNO levels for asthmatic children (r = 0.085, p = 0.518; Fig. 5A),
children with allergic rhinitis (r = 0.159, p = 0.353; Fig. 5B), or
children with asthma and allergic rhinitis (r = 0.258, p = 0.107;
Fig. 5C), a negative correlation was found between endotoxin
exposure and eNO levels for healthy children (r = 0.232,
p = 0.023; Fig. 5D). Further, no strong correlations existed
between endotoxin exposure and EBC pH values for asthmatic
children (r = 0.033, p = 0.800; Fig. 6A), children with asthma and
allergic rhinitis (r = 0.274, p = 0.087; Fig. 6C), and healthy
children (r = 0.172, p = 0.094; Fig. 6D). The EBC pH values were
negatively correlated with endotoxin exposure for children with
allergic rhinitis (r = 0.332, p = 0.048; Fig. 6B).
Based on long-term air monitoring results, 33.6% of the samples
had CO2 levels .600 ppm, a threshold set by the Taiwans
Environmental Protection Agency (EPA). These high CO2 levels
were found in homes of healthy children (30.2%) and in homes of
those with allergy-related respiratory diseases (41.7% of asthmatic
children, 27.8% of children with allergic rhinitis, and 35.0% of
children with asthma and allergic rhinitis). This finding
demonstrates that significant percentages of homes of healthy children
and of those with allergy-related respiratory diseases lacked
adequate ventilation. Only 6.7% of the TVOCs samples from
homes of asthmatic children exceeded the indoor air quality (IAQ)
guideline (3 ppm of TVOCs) set by Taiwans EPA. All PM10
samples were under Taiwans EPA IAQ guidelines for 24-hour
mean level (150 mg/m3). Only 0.4% of the PM2.5 samples from
homes of healthy children exceeded the IAQ guideline (100 mg/
m3 of PM2.5) set by Taiwans EPA. In this study, the
concentrations of suspended particulate matter were determined
only during an 8-hour period. Thus, future studies may extend
sampling duration in homes to evaluate variations in suspended
In this study, the highest concentrations of house dust endotoxin
were on kitchen floors in the homes of children with allergy-related
respiratory diseases and of healthy children. These analytical
results are similar to those in previous studies [8,12,38]. Endotoxin
concentrations on kitchen floors in this study (353.5607.9 EU/
mg dust) were markedly higher than those in US studies (80.5
105 EU/mg dust) [8,38]. A likely reason is the different climatic
conditions of the two countries [7,16]. In addition to kitchen
floors, the bedroom floors (296.1395.3 EU/mg dust) and the
surfaces of mattresses (260.4388.0 EU/mg dust) had higher
endotoxin concentrations. Educating families of children,
espeFigure 5. Associations between endotoxin concentrations and exhaled nitric oxide levels in children. (A) asthma group; (B) allergic
rhinitis group; (C) AS+AR group; (D) control group.
cially those of children with allergy-related respiratory diseases, to
frequently clean bedroom floors and their bedding is essential.
Peak endotoxin concentrations in dust samples were in
summer/autumn and the lowest were found in spring/winter in
the homes of both children with allergy-related respiratory diseases
and healthy children. This shows that the hottest season (May to
October) promoted growth of gram-negative bacteria and
increased endotoxin concentrations. Thus, childrens families
should increase their housekeeping practices during the hottest
season to decrease bacterial growth and endotoxin concentrations
in their homes. Notably, endotoxin concentrations in the homes of
children with allergy-related respiratory diseases, particularly
asthmatic children and children with allergic rhinitis, were clearly
lower than those in the homes of healthy children, demonstrating
that environmental control education for children with
allergyrelated respiratory diseases and their families in clinical health
teaching practice was beneficial for decreasing endotoxin
concentrations in homes.
In this study, approximately 62.5% of children with
allergyrelated respiratory diseases and 22.9% of healthy children had
high levels of eNO (.20 ppb) and about 25.9% of these children
(25.7% in children with allergy-related respiratory diseases and
26.0% of healthy children) had low EBC pH levels (,6.5). This
analytical finding reveals that significant percentages of these
children had airway inflammation problems. Specially, asthmatic
children had higher eNO levels and lower EBC pH values
compared to those of children with allergic rhinitis, children with
asthma and allergic rhinitis, and healthy children. Further,
patterns of seasonal variations in EBC pH values were clearly
different in asthmatic children for those of the other three groups
in this study. However, eNO levels and EBC pH values were not
directly related to endotoxin exposure in asthmatic children. This
analytical result may be confounded by exposure to other air
pollutants  and environmental allergens  in the homes
of asthmatic children.
Endotoxin exposure was associated with decreased eNO levels
among healthy children. Whether the eNO level was affected by
endotoxin exposure in healthy children was not fully elucidated.
Thus, the mechanism of endotoxins effect on eNO level in healthy
children warrants further clarification. Moreover, the EBC pH
values were negatively related to endotoxin exposure for children
with allergic rhinitis. Future studies can identify the endotoxin
dose needed to affect EBC pH values of children with allergic
rhinitis. Additionally, this study did not measure the concentration
distributions of environmental allergens in the homes of healthy
children and children with allergy-related respiratory diseases.
Therefore, future studies should evaluate the combined effect of
endotoxin and environmental allergens on airway inflammation in
humans. The interactions between exhaled breath indices (eNO
and EBC pH), environmental factors, and medication use in
different seasons also warrant further investigation. This study is
limited by its laboratory method used for endotoxin
determination. The limulus amebocyte lysate method can only detect intact
LPS, whereas a newer cytokine induction assay using a monocytic
cell line can detect both intact and small LPSs (,5 kDa), as well as
peptidoglycans and short bacteria DNA fragments .
In conclusion, the highest endotoxin concentrations were on the
kitchen floors of homes of healthy children and children with
allergy-related respiratory diseases. Obvious seasonal changes
existed in the endotoxin concentrations in dust samples from
homes of allergic rhinitis group and asthma+allergic rhinitis group
and in EBC pH values of healthy children and children with
allergy-related respiratory diseases. Strong correlations existed
between endotoxin exposure and eNO/EBC pH for healthy
children and children with allergic rhinitis, respectively.
The authors thank Yueh-Hsiang Chen for her assistance during this
investigation. Ted Knoy is appreciated for his editorial assistance.
Conceived and designed the experiments: GHW. Performed the
experiments: DCY CST. Analyzed the data: THT CHL. Wrote the
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