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Volume 1 | Issue 1
Eects of Transfer Point Glucan #300 Supplementation on Children exposed to
Passive Smoking - Placebo-driven Double-blind Clinical Trials
Richter Josef1, Král Vlastimil1, Svozil Vladimir2, Rajnohova Dobiasova Lucie1, Pohorska Jitka1, Stiborova
Ivana1, Vetvicka Vaclav*3
1Zdravotní ústav se sídlem v Ústí nad Labem, Czech Republic
2Sanatorium Edel, Zlaté Hory, Czech Republic
3University of Louisville, Department of Pathology, Louisville, KY, USA
*Corresponding author: Dr. Vetvicka Vaclav, University of Louisville, Department of Pathology, 511 S.
Floyd, Louisville, KY 40202, USA, E-mail: vaclav.vetvicka@louisville.edu
Research Article Open Access
Volume 1 | Issue 1
Journal of Nutrition Health
Abstract
Introduction
Lately, there has been increasing interest in the relationship between environmental contaminants and health of the population.
Children are the most sensitive to environmental contaminants, mostly due to their higher ventilation, immature immune system
and, compared to adult population, longer stay outside [1]. e eect of environmental pollution inuences a higher sickness rate,
a higher chance of respiratory infections and the rate of allergic diseases including asthma [1]. It is important to note that con-
taminations of the inside environment, mostly tobacco smoke, are as eective as pollution outside. e eects of passive smoking
on development of allergies demonstrated by elevated levels of IgE at risk children population are well established. e highest
level of eects of pollution is manifested in children suering from asthma and in children with chronic respiratory problems
[2]. Asthmatic children exposed to tobacco smoke have higher IgE levels, which are caused not only by genetic disposition, but
also indirectly by higher penetration of allergens through damaged mucosa [2]. Clinical manifestations of allergies, asthma and
chronic respiratory diseases are further inuence by living close to roads with high trac. Nitrogen oxides and most of all nano-
particles present in exhaust fumes increase clinical problems based on suppression of immunity [3-5]. Monitoring of the eects
of these stress factors by evaluation of levels of salivary cortisol oers important information about the relationship between envi-
ronmental stress and actual the health quality of children. Both aerial and food allergens can serve as triggers of stress reaction [6].
e intensity of stress reaction is inuenced by individual sensitivity. Allergic population, asthmatics and people with respiratory
allergy have higher levels of salivary cortisol [6,7]. Increased response of the hypothalamo-hypophyse-adrenal axis to stress was
found to be higher in healthy individuals with a predisposition to allergic diseases [7]. We believe that β-glucans used in our study
might inuence the individual’s response to the stress [8,9].
In this study, we focused on the eect of β-glucan supplementation of children with chronic respiratory problems. We measured the
levels of cortisol, salivary IgE and cotinine in 56 children and evaluated the eect of 30 day supplementation with 100 mg/day oral
dose of yeast-derived β-glucan. Our results showed strong decrease of cotinine and cortisol levels in saliva of β-glucan-supplemented
children. e increase of total salivary IgE levels in both groups was not statistically signicant. e positive eects of complex cura-
tive treatment using β-glucan were accompanied by increased physical endurance and by signicant reduction of negative clinical
problems of aected children.
Citation: Richter Josef, Král Vlastimil, Svozil Vladimir, Rajnohova Dobiasova Lucie, Pohorska Jitka, et al.
(2014) Eects of Transfer Point Glucan #300 Supplementation on Children exposed to Passive Smoking -
Placebo-driven Double-blind Clinical Trials. J Nutr Health 1: 105
Keywords: Glucan; Children; Saliva; IgE; Cotinine; Smoking
β-Glucan’s role as a biologically active immunomodulator has been well documented for over 50 years. Interest in its immu-
nomodulatory eects was initially raised by experiments that showed that a crude yeast extract stimulated macrophages via acti-
vation of complement system [10]. Subsequent work identied the immunoactive component as β-1,3-D-glucan [11]. Numerous
publications (currently more than 5,000 studies) have shown that β-glucans, either soluble or particulate, exhibit anti-bacterial,
anti-cancerous, anti-infectious and anti-stress activities (for review see [12-14]).
Received Date: April 1, 2014 Accepted Date: May 16, 2014 Published Date: May 20, 2014
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In order to better evaluate the complex eects of sanatorium conditions of glucan-supplemented children, we added not only
evaluation of some immunological parameters, but also clinical evaluation and functional Six Minute Walk Test (6 MWT), which
is valuable test for measuring reaction to physical endurance [15]. Our present study reports signicant benecial eects of addi-
tion of glucan.
e same protocol that was previously described [16-18] was used throughout this study. Briey, a randomized, double-blind, pla-
cebo-controlled trial compared β-glucan #300 and placebo in children. Sixty children from the Sanatorium for respiratory diseases
EDEL Czech Republic were enrolled in the 4-week trial and 56 were used. e reason for exclusion was leaving the Sanatorium
prematurely for non-medical reasons. e clinical trial was conducted at the Sanatorium EDEL (Zlate Hory, Czech Republic) and
the study was approved by the Ethics committees of the Public Health Institute and Sanatorium EDEL. is study was performed
in agreement with Helsinki declaration (revised version 2000.09.01) and was in full compliance with the rules of for clinical testing
in the Czech Republic. Parental consent was given in all cases.
Subjects were randomly assigned to groups which were blinded to intervention. During the intervention period, the subjects
consumed 100 mg/d of β-glucan or placebo. Both glucan and placebo capsules looked identical. Subjects were routinely evaluated
by the medical sta. Four patients were excluded from the study, due to either an abbreviated stay in the sanatorium or due to a
shortened stay based on the parent’s request.
e quality of environment was measured at the place of residence and at the Sanatorium EDEL by testing levels of sulphur diox-
ide, nitrogen oxide and particles of sizes PM 2.5 and PM 10. Overall meteorological conditions were based on countrywide net
of atmosphere monitoring stations. Levels of pollen particles were also measured both at the place of residence and Sanatorium
EDEL using a Burkard 7 Day Recording Volumetric Spore Sampler (Burkard Agronomics TGB). Pollen monitoring stations are
parts of the Czech Pollen Information Service, member of the European Pollen Information Service (www.polleninfo.org).
Glucan
Yeast-derived insoluble Glucan #300 (>85% dry w/w basis) was purchased from Transfer Point (Columbia, SC, USA). is glucan
contains 96% carbohydrates and 2.1% proteins. Neutral sugar analysis conrmed 91.3% glucose and 8% mannose.
Quality of environment
Epidemiological data were obtained using two forms – rst one lled by parents, the second one by medical sta of the Sanato-
rium EDEL. In all tested individuals, we collected samples of saliva using a commercial Salivette device (Sarstead, Orsay, France).
Aer two minutes of chewing, the cotton swab was added into a sterile container and centrifuged at 1,000 g for 15 minutes and
stored at -20 oC. We used identical times (between 8 and 9 AM) for sampling, so the possible inuence of circadian rhythms could
be eliminated. Levels of cotinine were measured in saliva by an ELISA kit Salimetrix according to the manufacturer’s instruction
(Salimetrix Europe, Newmarket, UK). With the suggested laboratory temperature of 20-23 oC, the sensitivity of the method is
around 0.15 ng/ml. In all subject, these measurements were done at day 0 and at day 30. Cortisol levels were also measured by
Salimetrix kits (Salimetrix High Sensitivity Salivary Assay Kit) with sensitivity of 0.0007 μg/L. Total IgE levels were determined
by high anity uoroenzymatic assay using an ImmunoCAP 250 analyzator and kits and equipment Phadia (Uppsala, Sweden)
with sensitivity of 0.005 IU/ml. All participants absolved at the beginning and at the end 6MWT based on suggested development,
including evaluation of additional parameters such frequency before and aer physical stress, oxygen saturation, interruption of
stress due to the tiredness. ese tests were done 2 hrs aer the meal.
Materials and Methods
Protocol
Tests
Statistical analysis
Statistical signicance was evaluated by a pair t-test using a GraphPad Prism 5.04 soware (GraphPad Soware, USA). An aver-
age and standard deviation was evaluated aer determination of composition of standard values (D’Agostino, Pearson). In case of
non-standard composition, we converted the values into logarithms.
Results
e glucan-consuming group consisted of 29 children (age 7 – 14 years, mean 9.7; the placebo consuming group used 27 children
(age 6 – 16, mean 10.1). Both groups were the same with respect to birth weight or sex (Table 1). e glucan group had 72% breast-
fed children; the placebo group had 89%. e dierences in duration of breast feeding were statistically not signicant and no dif
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ferences were found in average weight or percentage of children with their BMI higher than 95% or lower than 5% suggested by
CDC. e glucan-supplemented group had slightly elevated allergenic stress by domestic animals allergens. Exposure to passive
smoking was indentical in both groups. e percentage of children with 50 m distance between homes and roads with high trac
frequency were 58.5% in glucan group and 64% in placebo group. e number of children with asthma was same in both groups;
chronical obstruction of lungs was higher in placebo group. Clinical ndings of allergy were 37.9% in the glucan group, 14,8% in
placebo group. 6MWT values at the beginning of our study are given in Table 1.
e environmental characteristics of our patients did not substantially dier from data published previously [16-18]. e levels of
sulphur oxide were almost 4x higher at the place of residence than in Sanatorium EDEL, the same is true about levels of nitrogen
oxide. e highest dierences were found in levels of particles, where PM 10 levels at residence reached 50 mg/m3, whereas levels
at the Sanatorium are 13.0 mg/m3. Almost identical dierences were found in case of PM 2.5 (average 40 mg/m3 vs. 10 mg/m3). e
dierence primarily all due to the distance from frequented routes, which at the Sanatorium is over 1 500 m, whereas more than
50% of evaluated children lives less than 50 m from a road with high frequency of automobile trac (Table 1). Another stress on
the respiratory tract is caused by additional contaminants of environment, particularly pollen. e dierences between home and
Sanatorium levels of pollen were again signicant. Table 2 summarized individual contaminants present during March and April.
Cumulation of negative factors aecting respiratory tract is further increased by high exposure of children to passive smoke, which
is common in almost 50% of children. Relatively high indoor pollution is caused by pets, which is in the asthmatic group higher
than 50% (Table 1). As the types of residences are almost identical in both groups (mostly panel housing), both groups had the
same risk on indoor contamination. Levels of salivary cotinine, which is an indicator of exposure to passive smoking, are shown
in Figure 1. Levels found in the glucan group at the beginning (0.85 ng/ml ± 0.26) and at the end of the study (0.235 ng/ml ± 0.07)
are signicantly dierent from the control group (0.436 ng/ml ± 0.22; 0.28 ng/ml ± 0.22, resp.). e cotinine levels decreased aer
30 days of stay at the Sanatorium in both groups, but the glucan-supplemented group showed a much higher rate of decrease.
e possible eects of diet can be negligible, as both groups were getting identical food during the whole stay. e total levels of
cotinine at the glucan group were strongly inuenced by extremely high levels found in three children. ese levels were most
probably caused by demonstrated multiple exposure to tobacco smoke with three strong smokers in the family.
Figure 2 summarizes the ndings of the cortisol levels. At the beginning of our study, the levels found in both groups were statis-
tically identical (0.45 and 0.42 μg/ml). A decrease in cortisol levels aer the 30 day stay was found in both groups, however, the
decrease was signicant only in glucan group.
Total levels of salivary IgE reached 0.30 ± 0.39 IU/ml in glucan group, in placebo group it was 0.21 ± 0.20 IU/ml. In both groups
we observed an increase in salivary IgE levels, but these changes were not statistically signicant (Figure 3). Detailed study showed
that this increase was most probably caused by seasonal exposure to pollen particles. Evaluation of the 6MWT test demonstrated
an increase of physical capacity from 430 to 445 m (p <0.07) in glucan group, but no changes in the placebo group (Table 1).
G1
G2
P1
P2
G1
G2
P1
P2
Figure 1: B Eects of 4-week oral administration of glucan (100 mg/day) on cotinine levels in saliva. GL 1 represents glucan group at day 1; GL 2
at day 30. C1 represents control (placebo) group at day 1, C 2 at day 30. Data show statisticall signicance between groups.
Cotinine saliva
1.5
1.0
0.5
0.0
ng/ml
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Volume 1 | Issue 1
G1
G2
P1
P2
G1
G2
P1
P2
Figure 2: Eects of 4-week oral administration of glucan (100 mg/day) on cortisol levels in saliva. GL 1 represents glucan group at day 1; GL 2 at
day 30. C1 represents control (placebo) group at day 1, C 2 at day 30. Data show statisticall signicance between groups.
Cortisol saliva
0.6
0.4
0.2
0.0
μg/l
G1
G2
P1
P2
G1
G2
P1
P2
Figure 3: B Eects of 4-week oral administration of glucan (100 mg/day) on IgE levels in saliva. GL 1 represents glucan group at day 1; GL 2 at ay
30. C1 represents control (placebo) group at day 1, C 2 at day 30. Data show statisticall signicance between groups.
lgE
1.5
0.3
0.2
0.0
IU/ml
0.8
0.1
0.4
5
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Table 1: Basic information on tested groups of children
Zlaté HoryBohumínPlant species
17468249AlderAlnus
1710BirchBetula
03BornbeamCarpinus
4911413HaselCorylus
1141306PoplarPopulus
2991184YewTaxus
95243willowSalix
Table 2: e number of pollen grains (1m3 ) in residence children (Bohumín – Moravia) and Sanatorium EDEL
(Zlaté Hory) (March and April 2011)
Placebo groupGlucan group
n= 27n=29Child characteristics
6 - 167 - 14Age (min-max)
10,1 (2,7)9,7 (2,0)Age Mean (SD)
15 (55,6)16 (55,2)Male sex (%)
12 (44,4)13 (44,8)Female sex (%)
3182,73404,6Birth weight (grams)
88,972,4Breastfed (%)
9,777,38Breastfed Month Mean
18,2 (4,4)18,3 (3,5)BMI (SD)
5 (18,5)% 4 (13,8)BMI – for – age percentils >95%
4 (14,8)% 2(6,9)BMI – for – age percentils <5%
8 (29,6)15 (51,7)Pet keeping (%)
12 (44,4)15(51,7)Passive smoking (%)
16 (64)17 (58,6)Living within 50 m of a main street N(%)
9 (36)8 (27,6)Living within 500 m of a main street N(%)
0 (0)4 (4)Living within more m of a main street N(%)
4 (14,8)11 (37,9)Allergy (%)
14 (51,9)16 (55,2)Asthma (%)
9 (33,3)2 (6,9)
COPD (chronic obstructive pulmon. disease )
%
440 (85)430 (38)6 Minute Walk Test I. (SD)
435 (75)445 (57)6 Minute Walk Test II. (SD)
Values are absolute/values (percentages)or means (SD)
Discussion
Currently there is little doubt about the fact that contamination of environment strongly aects the health problems of the whole
population, and children in particular [1]. e overall quality of environment has strong eects on development of immune sys-
tem and on some diseases aecting both mental and physical development [1,3]. Environmental stress is reected in respiratory
tract diseases, development of allergies, increase in occurrence of asthma bronchiale and other health problems [19]. e increase
in prevalence of asthma bronchiale in highly polluted region of the Czech Republic in last decade (2002-2012) achieved more
than 10% and represents a heavy economical strain for the health industry. e only possible way for improvement of the current
state is signicant reduction of all aspects adding to the contamination of the environment. An actual solution can be short term
limination of exposure to environmental contaminants caused by the locality [19]. e eects of this solution were demonstrated
during the Olympic games in Atlanta, were the reduction in automobile trac caused an decrease in overall sickness. e World
Health Organization describes trac-based contaminants as primary causes leading to the developments of allergic diseases. e
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Volume 1 | Issue 1
authors conclude that the distance of the residence to the frequented road represents signicant risk factor [5], where living up
to 20 m from heavy frequented street increased the risk of respiratory problems up to 15 percent [3]. In might be important to
note that these ndings will dier among dierent countries, which can be explained not only by dierent age and general state
of the vehicles, but also by prevalent type of engine. An increase of environmental pollution in the Czech Republic is inuenced
not only by high percentage of diesel engines, but also by an average age of cars, which is greater than 10 years. Taken together, we
consider it necessary to monitor quantity and development of pulmonary functions that are heavily inuenced by distance from
trac. Gauderman and his group found signicant changes of pulmonary functions in individuals living closer than 500 meters
from roads compared to people living further than 1 000 meters [4]. In addition, these damages can oen last an entire lifetime. In
our conditions, children oen spent signicant periods of time outdoors; therefore, it is necessary to also calculate the localization
of the school. Among children tested in our report, the distance between the school and busy street was more than 100 meters.
Exposure to passive smoke represents an important health stress aecting the development and quality of the immune system.
Evaluation of the levels of salivary cotinine represents a non-invasive and highly precise indicator of exposure to tobacco smoke.
By measuring the levels of salivary cotinine we can determine the level of exposure and monitor the end of the exposure [20]. e
total levels of cotinine are aected by the combination of exposure and the size of the interior, as well as the relationship between
the smoker and the passive smoker. e highest levels of cotinine can be found in children exposed to the smoke by mother,
whereas the exposure by father leads to the lower levels (probably by forcing the father to smoke outside by non-smoking mother).
e relationship between the cotinine levels and tobacco smoke from both smoking parents is clear and our results are in agree-
ment with results obtained in children from countries such as the United Kingdom, Wales or Sweden [20]. Approximately 75% of
nicotine is converted to cotinine, most of all by cytochrome P450 2A6 [21]. e half-life of cotinine is 16 hours, its levels are stable
throughout the day and the correlation between the ndings in saliva and blood is extremely high. Metabolism of cotinine can be
inuenced by genetic factors, race, sex, and is usually higher in children exposed to passive smoke than in similarly stressed adults
[21]. Higher levels were found in children with asthma bronchiale [21], which our study conrmed. In addition, we pointed out
the eects of environmental contaminants on increase of cotinine levels. In these cases the cotinine levels are higher than the 3
ng/ml cut-o point (NHANES 1999-2004) distinguishing between passive and active smoking [21]. Information of the eects of
passive smoking on children’s health, particularly on their pulmonary system, is in families with aected children oen overlooked
and signicant public education is necessary. Based on the forms utilized in our study, only 30% of the population has any knowl-
edge about the possible eects of passive smoking on induction of asthmatic or allergic diseases. In addition, the full knowledge of
these interactions is not totally understood even in the professional population. A 1/2 imbalance induced by smoking is not
studied in detail, similarly we do not have enough information about T regulation system in passive smokers.
All this oers one of possible ways as to how to inuence health status of passive smokers. It is well known that β-glucans are
strong immunomodulators with positive eects on immunocompetent cells [22]. An optimal application of β-glucan in individu-
als with heavy physical stress reduced respiratory illnesses, increased phagocytic and NK cell activity [22]. Several experiments
demonstrated that glucan treatment aer stress resulted in the decrease of lactate levels, lower activity of creatinine kinase as well
as additional markers demonstrating stronger endurance during higher stress [23]. Eects of β-glucans on activation of mac-
rophages and neutrophils together with their stimulation of phagocytosis, cytotoxicity and antimicrobial activity via activation
of specic receptors (such as CR3, Dectin-1, lactosyl or TLR2-6) are well established (for review see [24]) and can be utilized in
suppression of stress of our group of children. e reasons why we used a relatively short supplementation are twofold – rst, we
wanted to evaluate the hypothesis that glucan can help even aer short interval, and second the 30 days corresponded to the length
of stay in the Sanatorium.
Dynamics of changes in salivary IgE is dierent based on exposure to passive smoking. Higher original levels were found in chil-
dren exposed to smoking, which corresponds to the previous ndings [2]. When re-evaluated aer the 30 day stay at the Sanatori-
um, we found a small insignicant increase, which was most probably caused by seasonal allergens. is exposure was, compared
to residence, signicantly smaller, due to the low allergen exposure from lawn surrounding the whole Sanatorium campus.
It is necessary to understand that children evaluated in our study are under the inuence of chronic stress of complex etiology
(environment, passive smoking, social and economic inuences) [6]. Taking out from long term stress resulted in pronounced
decrease of cortisol levels, which was most pronounced in glucan-supplemented group. Measuring cortisol during morning hours
had no inuence on levels of salivary cotinine, known for increased levels during the day. It is known that allergic patients react
to stress by increase of cortisol levels [6]. e positive eects of glucan on several types of stress have been already described [8].
Later, the positive eects of glucans on respiratory symptoms in individuals with mood stress [9]. Based on our data, we conclude
that immunostimulatory eects of β-glucan resulted in improvements of medical parameters during and aer the medical stay at
the Sanatorium [16,17,25,26].
Positive eects of complex curative treatment using β-glucan are accompanied by increased physical endurance and by signicant
reduction of negative clinical problems of aected children [27]. ese nding are supported by improvements in 6MWT, where
the data in the glucan-treated group corresponds with the normal child population of the same age group [15].
7
Journal of Nutrition Health
e positive eects of complex curative treatment using β-glucan were accompanied by increased physical endurance and by sig-
nicant reduction of negative clinical problems of aected children. Based on these data, we conclude that natural immunomodu-
lator β-glucan signicantly improves the overall health of children with chronic respiratory problems caused by environmental
stress and/or passive smoking.
Acknowledgement
is study was supported by the Technology Agency of the Czech Republic TACR TA 0202094.
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