Advances in Experimental Medicine and Biology 840

Neuroscience and Respiration

Mieczyslaw Pokorski Editor

Body
Metabolism
and Exercise
Advances in Experimental Medicine
and Biology

Neuroscience and Respiration

Volume 840

Editorial Board

Irun R. Cohen, The Weizmann Institute of Science, Rehovot, Israel

N. S. Abel Lajtha, Kline Institute for Psychiatric Research, Orangeburg, NY, USA
John D. Lambris, University of Pennsylvania, Philadelphia, PA, USA
Rodolfo Paoletti, University of Milan, Milan, Italy

Subseries Editor

Mieczyslaw Pokorski

For further volumes:

http://www.springer.com/series/13457
Mieczyslaw Pokorski
Editor

Body Metabolism
and Exercise
Editor
Mieczyslaw Pokorski
Institute of Psychology
University of Opole
Poland

ISSN 0065-2598 ISSN 2214-8019 (electronic)

ISBN 978-3-319-10249-8 ISBN 978-3-319-10250-4 (eBook)
DOI 10.1007/978-3-319-10250-4
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Preface

This is a new book series entitled Neuroscience and Respiration, a subseries

of Springers renowned Advances in Experimental Medicine and Biology.
The book volumes present contributions by expert researchers and clinicians
in the field of pulmonary disorders. The chapters provide timely overviews of
contentious issues or recent advances in the diagnosis, classification, and
treatment of the entire range of pulmonary disorders, both acute and chronic.
The texts are thought as a merger of basic and clinical research dealing with
respiratory medicine, neural and chemical regulation of respiration, and the
interactive relationship between respiration and other neurobiological
systems such as cardiovascular function or the mind-to-body connection. In
detail, topics include lung function, hypoxic lung pathologies, epidemiology
of respiratory ailments, sleep-disordered breathing, imaging, and biomarkers.
Other needful areas of interest are acute respiratory infections or chronic
inflammatory conditions of the respiratory tract, exemplified by asthma and
chronic obstructive pulmonary disease (COPD), or those underlain by still
unknown factors, such as sarcoidosis, respiratory allergies, lung cancer, and
autoimmune disorders involving the respiratory system.
The prominent experts will focus their presentations on the leading-edge
therapeutic concepts, methodologies, and innovative treatments. Pharmaco-
therapy is always in the focus of respiratory research. The action and
pharmacology of existing drugs and the development and evaluation of
new agents are the heady area of research. Practical, data-driven options to
manage patients will be considered. The chapters will present new research
regarding older drugs, performed from a modern perspective or from a
different pharmacotherapeutic angle. The introduction of new drugs and
treatment approaches in both adults and children will be discussed. The
problem of drug resistance, its spread, and deleterious consequences will
be dealt with as well.
Lung ventilation is ultimately driven by the brain. However, neuropsy-
chological aspects of respiratory disorders are still mostly a matter of conjec-
ture. After decades of misunderstanding and neglect, emotions have been
rediscovered as a powerful modifier or even the probable cause of various
somatic disorders. Today, the link between stress and respiratory health is
undeniable. Scientists accept a powerful psychological connection that can
directly affect our quality of life and health span. Psychological approaches,

v
vi Preface

by decreasing stress, can play a major role in the development and course of
respiratory disease, and the mind-body techniques can aid in their treatment.
Neuromolecular aspects relating to gene polymorphism and epigenesis,
involving both heritable changes in the nucleotide sequence and functionally
relevant changes to the genome that do not involve a change in the nucleotide
sequence, leading to respiratory disorders will also be tackled. Clinical
advances stemming from basic molecular and biochemical research are but
possible if the research findings are translated into diagnostic tools, thera-
peutic procedures, and education, effectively reaching physicians and
patients. All that cannot be achieved without a multidisciplinary, collabora-
tive, bench-to-bedside approach involving both researchers and clinicians,
which is the essence of the book series Neuroscience and Respiration.
The societal and economic burden of respiratory ailments has been on the
rise worldwide leading to disabilities and shortening of life span. COPD
alone causes more than three million deaths globally each year. Concerted
efforts are required to improve this situation, and part of those efforts are
gaining insights into the underlying mechanisms of disease and staying
abreast with the latest developments in diagnosis and treatment regimens.
It is hoped that the books published in this series will fulfill such a role by
assuming a leading role in the field of respiratory medicine and research and
will become a source of reference and inspiration for future research ideas.
Titles appearing in Neuroscience and Respiration will be assembled in a
novel way in that chapters will first be published online to enhance their
speedy visibility. Once there are enough chapters to form a book, the chapters
will be assembled into complete volumes. At the end, I would like to express
my deep gratitude to Mr. Martijn Roelandse and Ms. Tanja Koppejan from
Springers Life Sciences Department for their genuine interest in making this
scientific endeavor come through and in the expert management of the
production of this novel book series.

Opole, Poland Mieczyslaw Pokorski

Volume 9: Body Metabolism and Exercise

The dynamics of body metabolism are changed in the disease process and
interact with physical activity. The alteration of metabolism and its
consequences raise the need for simple and reliable methods for assessment
of body composition. The chapters aim to investigate various interacting
components converging on metabolic changes in lung and muscle tissues
taking into consideration the drug effects. The effects of exercise and
nutritional status are dealt with to a great extent.

vii
Contents

Body Composition in Heavy Smokers: Comparison

of Segmental Bioelectrical Impedance Analysis
and Dual-Energy X-Ray Absorptiometry . . . . . . . . . . . . . . . . . . . . 1
O. Rom, A.Z. Reznick, Z. Keidar, K. Karkabi,
and D. Aizenbud
Metabolic and Immunological Consequences of Vitamin D
Deficiency in Obese Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
B. Pyrzak, E. Witkowska-Sedek, M. Krajewska,
U. Demkow, and A.M. Kucharska
Markers of Bone Metabolism in Children
with Nephrotic Syndrome Treated with Corticosteroids . . . . . . . . . 21
Magorzata Panczyk-Tomaszewska, Dominika Adamczuk,
Agnieszka Kisiel, Piotr Skrzypczyk, Jerzy Przedlacki,
Elzbieta Gorska, Anna Stelmaszczyk-Emmel,
Urszula Demkow, and Maria Roszkowska-Blaim
Endurance Training and the Risk of Bronchial
Asthma in Female Cross-Country Skiers . . . . . . . . . . . . . . . . . . . . 29
A. Zebrowska, B. Guchowska, D. Jastrzebski,
A. Kochanska-Dziurowicz, A. Stanjek-Cichoracka, and I. Pokora
Effects of Inspiratory Muscle Training on Resistance
to Fatigue of Respiratory Muscles During
Exhaustive Exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
M.O. Segizbaeva, N.N. Timofeev, Zh.A. Donina,
E.N. Kuryanovich, and N.P. Aleksandrova
Nutritional Status in Chronic Obstructive
Pulmonary Disease and Systemic Sclerosis:
Two Systemic Diseases Involving the Respiratory System . . . . . . . . 45
D. Mekal, A. Doboszynska, E. Kadalska, E. Swietlik,
and L. Rudnicka

ix
x Contents

Gradual Versus Continuous Increase of Load

in Ergometric Tests: Are the Results Comparable? . . . . . . . . . . . . 51
A.M. Preisser, M. Velasco Garrido, C. Bittner, E. Hampel,
and V. Harth
Evaluation of Volumetric Changes in Differential
Diagnosis of Brain Atrophy and Active Hydrocephalus . . . . . . . . . 59
E. Szczepek, L. Czerwosz, K. Nowinski,
J. Jurkiewicz, and Z. Czernicki

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Advs Exp. Medicine, Biology - Neuroscience and Respiration (2015) 9: 111
DOI 10.1007/5584_2014_16
# Springer International Publishing Switzerland 2014
Published online: 15 October 2014

Body Composition in Heavy Smokers:

Comparison of Segmental Bioelectrical
Impedance Analysis and Dual-Energy
X-Ray Absorptiometry

O. Rom, A.Z. Reznick, Z. Keidar, K. Karkabi, and D. Aizenbud

Abstract
Smokers tend to have lower body mass index, on one hand, and increased
abdominal obesity, on the other hand. Also, low levels of lean mass
(LM) and bone mineral content (BMC) were found among older smokers
compared with non-smokers. This altered body composition and its
consequences raise the need for simple and reliable methods for assess-
ment of body composition in smokers. This study aimed to compare body
composition assessment by segmental bioelectrical impedance analysis
(sBIA) with the reference method, dual energy X-ray absorptiometry
(DEXA). Body composition was measured by sBIA (Tanita BC-545)
and DEXA (Hologic) in 49 heavy smokers (>15 cigarettes/day, mean
age 43.8
12.0). The comparison included correlations and differences
between measurements obtained using the two methods as well as the
Blande-Altman analysis. Whole-body fat mass (FM) and LM measured by
the two methods were found to be highly correlated (r > 0.9, p < 0.001).
Compared with DEXA, sBIA significantly overestimated whole-body LM
and BMC (1,126 g and 382 g, respectively, p < 0.01). The Bland-Altman
analysis revealed a good agreement for whole-body FM and LM, but a
poor agreement for BMC. The segmental FM percentage and LM were

O. Rom and A.Z. Reznick (*)
Department of Anatomy and Cell Biology, Rappaport
Faculty of Medicine, Technion Israel Institute of
Technology, Efron St., P.O. Box: 9649, Bat Galim, Haifa
31096, Israel
e-mail: reznick@tx.technion.ac.il
Z. Keidar
Rappaport Faculty of Medicine, Technion Israel
Institute of Technology, Haifa, Israel
Department of Nuclear Medicine, Rambam Health Care
Campus, Haifa, Israel
K. Karkabi
Rappaport Faculty of Medicine, Technion Israel
Institute of Technology, Haifa, Israel
Department of Family Medicine, Clalit Health Services,
Haifa, Western Galilee District, Israel
D. Aizenbud
Department of Anatomy and Cell Biology, Rappaport
Faculty of Medicine, Technion Israel Institute of
Technology, Efron St., P.O. Box: 9649, Bat Galim, Haifa
31096, Israel
Department of Orthodontic and Craniofacial Anomalies,
Rambam Health Care Campus, Haifa, Israel

1
2 O. Rom et al.

also highly correlated (r > 0.9, p < 0.001). However, sBIA significantly
overestimated LM of the trunk and legs and underestimated the appendic-
ular FM percentage. Verified by DEXA, sBIA provides reliable measures
of whole-body LM, FM, and trunk FM in heavy smokers. A lesser degree
of agreement was found for BMC, appendicular LM, and FM.

Keywords
Bone mineral content Cigarette smoking Energy X-ray absorptiometry
Fat percentage Lean mass Segmental bioelectrical impedance analysis

1 Introduction of smoking on bone turnover and bone mineral

The effects of cigarette smoking on body weight
and body composition have been studied exten-
sively. A lower body mass index (BMI), on the
one hand, and an increased abdominal adiposity,
on the other hand, are common among heavy
smokers. Compared with non-smokers,
decreased levels of lean mass (LM) and bone
mineral content (BMC) were found among
older smokers.
Large scale population studies reported that
smokers have a lower BMI in comparison to
non-smokers (Clair et al. 2011; Akbartabartoori
et al. 2005; Canoy et al. 2005; Bamia et al. 2004;
Barrett-Connor and Khaw 1989). Although
leaner, smokers were shown to have higher
waist-hip ratios than non-smokers, indicating a
more adverse fat distribution profile of higher
central adiposity (Kim et al. 2012;
Akbartabartoori et al. 2005; Canoy et al. 2005;
Bamia et al. 2004; Barrett-Connor and Khaw
1989). In addition, smoking was previously iden-
tify as a risk factor for sarcopenia, the age related
loss of muscle mass and strength (Lee et al. 2007;
Szulc et al. 2004; Castillo et al. 2003). Szulc
et al. (2004) found that older smokers have a
lower appendicular skeletal muscle mass than
never-smokers. Among the smokers, appendicu-
lar skeletal muscle mass was found to be lower in
subjects who smoked more (number of pack
years >15). Van den Borst et al. (2011) reported
that elderly smokers have significantly lower LM
and BMC when compared with never-smoking
controls. Elgan et al. (2003) studied the influence
density (BMD) in healthy young women. During
a 2-year follow-up period, BMD level decreased
among smokers and smoking was associated
with reduced bone mineral metabolism. BMD
values of females in the post-menopause stage
were also found to be lower among smokers in
comparison with non-smokers (Demirbag
et al. 2006). The adverse effects of smoking on
bone turnover were also described in men. Com-
pared with never-smokers, increased bone
resorption and decreased BMD were found in
male smokers with low body weight (Szulc
et al. 2002). The above studies suggest an altered
phenotype of body composition among smokers
characterized by low body weight, increased
abdominal adiposity, decreased LM, and BMC.
Body composition can be measured by
numerous methods including the skin-fold
method, hydro-densitometry, computerized
tomography (CT), dual energy X-ray absorpti-
ometry (DEXA), and bioelectrical impedance
analysis (BIA). DEXA is a non-invasive and
direct measurement of three components of
body composition: LM, BMC, and fat mass
(FM) (Thibault et al. 2012). This method is
used routinely in clinical practice for the diagno-
sis and follow-up of osteoporosis. In clinical
practice and research, DEXA is considered as
the reference method for the assessment of
body composition. However, its use for the mea-
surement of body composition in clinical prac-
tice is limited due to radiation exposure,
inaccessibility, and high costs (Thibault
et al. 2012). BIA, on the other hand, is the
Body Composition in Heavy Smokers: Comparison of Segmental Bioelectrical. . .
simplest, most reproducible and least expensive
method for measuring body composition
(Thibault et al. 2012; Beeson et al. 2010). It is
based on the capacity of hydrated tissues to con-
duct electrical energy. Total body impedance is
measured allowing the estimation of total body
water and LM which contains body water
(Thibault et al. 2012). Though easy to use and
safe, BIA is criticized for being inaccurate in
comparison with DEXA (Lloret Linares
et al. 2011).
The altered body composition found among
smokers may reflect the metabolic consequences
of smoking in which central obesity, sarcopenia,
osteoporosis, and their associated health risks are
increased. Therefore, the assessment of body
composition in heavy smokers is of great impor-
tance and should receive more attention in an
effort to minimize health risks among smokers.
Ideally, the measurement of body composition in
heavy smokers would be done by DEXA, the
reference method. However, due to its high
costs, low accessibility and risk of radiation
exposure, a simpler, less expensive but reliable
method is required. BIA may serve as an appro-
priate alternative, but its accuracy in the mea-
surement of body composition among smokers
has yet to be compared with DEXA. Therefore,
the aim of the present study was to examine the
reliability and accuracy of segmental BIA (sBIA)
compared with DEXA for the assessment of
whole-body and segmental body composition
among heavy smokers.
2 Methods
Approval for the study was obtained from the
Helsinki Committee of Rambam Health Care
Campus, Haifa, Israel. All participants signed
an informed consent to participate in the study.
2.1 Participants
Forty-nine participants were recruited from the
smoking cessation program of Clalit Health
Services, Haifa, and Western Galilee district,
3
Israel. This program consists of an eight meeting
workshop led by a professional instructor on a
group support basis. The participants were eligi-
ble for the study if they smoked at least
15 cigarettes per day and were in the age range
of 2065. They were advised to use Varenicline
(Champix) as a medical aid to the smoking
cessation process. Thirty-nine (79.6 %) of them
reported using Champix. Exclusion criteria
included cardiovascular and pulmonary diseases,
diabetes, orthopedic conditions, unbalanced thy-
roid disorders, morbid obesity (BMI > 40 kg/
m2), and consumption of more than two alcoholic
drinks per day.
2.2 Body Composition
All measurements were obtained in the morning
after a fast of at least 1.5 h. Height was measured
using a standard wall-mounted measure (Seca
206, Birmingham, UK). Measurements of
body composition by DEXA were conducted at
the Department of Nuclear Medicine, Rambam
Health Care Campus, Haifa, Israel.
Measurements by BIA were performed
1020 min afterwards at the fitness center of
Rambam Health Care Campus.
2.2.1 Assessment of Body Composition
by DEXA
The whole-body and regional body compositions
were estimated in the supine position by DEXA
using a Hologic Explorer device (Hologic,
Bedford, MA). The software provides values for
the masses of lean soft tissue, and fat and bone
mineral for the whole-body and specific regions.
The DEXA device utilizes a constant X-ray
source producing fan beam dual energy radiation
with effective dose equivalents of 5 Sv. The
estimations of FM and LM are based on extrapo-
lation of the ratio of soft tissue attenuation of two
X-ray energies in non-bone containing pixels.
The two X-ray energies are produced by a tung-
sten stationary anode X-ray tube pulsed alter-
nately as 70 kVp (peak) and 140 kVp. The
software performs calculations of the differential
attenuations of the two photon energies and
4 O. Rom et al.
presents data of total mass, FM, LM, BMC, and
BMD. Total LM was calculated as the sum of
LM in the trunk, arms, and legs, assuming that all
non-fat and non-bone tissue is skeletal muscle.
2.2.2 Assessment of Body Composition
by BIA
The BIA measurements of whole-body and seg-
mental body compositions were taken by a
registered dietitian using a BC-545 body compo-
sition monitor (Tanita Corporation, Tokyo,
Japan). The monitor consists of an eight elec-
trode system situated on the metal foot plates
and the handles. The measurement frequency is
50 kHz and the current is 500 A. The signal
flows easily through fluids in the muscle and
other body tissues but meets resistance (imped-
ance) as it passes through body fat. The imped-
ance readings are entered into proprietary
equations not supplied by the manufacturer to
calculate the whole-body and segmental body
compositions including the FM percentage, mus-
cle mass, and body bone mass (bone mineral
level). To equate the terms of body composition
by the two methods, we used the terms LM and
BMC (DEXA) as equivalent to muscle mass and
bone mass (BIA), respectively.
2.3 Smoking Status
Participants were asked to answer a question-
naire regarding their smoking history including
the number of cigarettes smoked per day at dif-
ferent periods of their lives, allowing the calcu-
lation of the number of pack years [pack
years (number of cigarettes smoked per day
 number of years smoked)/20]. To verify the
smoking status of the participants, urine cotinine
was measured by the Cotinine Direct ELISA Kit
(Abnova, Jhongli, Taiwan). Having a longer half-
life than nicotine, cotinine is used preferably as a
reliable marker for smoking status and smoking
cessation studies. Urine samples were taken
before measurements of body composition and
kept at 80  C.
2.4 Statistical Analysis
All data were normally distributed as assessed by
the Kolmogorov-Smirnov test. A paired t-test and
correlation coefficients were used for the compari-
son of whole-body and segmental body
compositions, including FM, LM and whole-body
BMC as measured by sBIA and DEXA. p < 0.01
was considered statistically significant. To further
test the agreement of the two methods, differences
between the methods (bias) and the level of agree-
ment were calculated as the mean difference
between the methods
1.96 SD using the Bland-
Altman analysis and plots (Bland and Altman
1986). Statistical analysis was performed by SPSS
17 software (SPSS Inc., Chicago, IL).
3 Results
3.1 Participants Characteristics
Forty-nine participants (F/M 26/23) of the
mean age of 43.8
12 years were included in
the study. The mean pack years was 31.4
23.3
and the mean urine cotinine was 330
122.6 ng/
ml. For ten participants who reported smoking
cessation for more than 2 weeks, cotinine was
<40 ng/ml. Jhun et al. (2010) previously defined
urinary cotinine levels >100 ng/ml for current
smokers and <40 ng/ml for non-smokers. Demo-
graphic characteristics of the subjects are
presented in Table 1.
3.2 Assessment of Whole-Body
Composition by sBIA vs. DEXA
Highly significant correlations were found
between sBIA and DEXA methods for the
assessment of whole-body FM, FM percentage,
LM, and BMC (Table 2). The corresponding
regression equations are presented in Fig. 1,
showing that the lowest R2 value was found for
the BMC measured by the two methods. The
agreement between the two methods was
assessed from the mean differences using the t-
Body Composition in Heavy Smokers: Comparison of Segmental Bioelectrical. . . 5

Table 1 Participants characteristics

Females (26) Males (23) Combined (49)

Mean
SD Range Mean
SD Range Mean
SD Range
Age (year) 44.7
12.5 664 42.7
11.7 2262 43.8
12.0 2264
Pack years 31.4
23.6 7113 31.3
23.5 490 31.4
23.3 4113
Urine cotinine (ng/ml)a 331.8
103.5 104490 328.2
142.9 45576 330.0
122.6 45576
Height (cm) 164
8 150176 177
7 167194 170
10 150194
Weight (kg) 67.1
12.6 4495 80.4
13.6 60122 73.4
14.6 44122
BMI (kg/m2) 25.0
4.4 1733 25.7
3.4 2036 25.4
4.0 1736
sBIA segmental bioelectrical impedance analysis, BMI body mass index, DEXA dual energy X-ray absorptiometry
a
10 measurements of urine cotinine <40 ng/ml were excluded

Table 2 Assessment of whole-body composition by sBIA vs. DEXA

Measurements sBIA DEXA r Difference (sBIA  DEXA)
FM (%) 27.55
9.57 28.56
8.61 0.95a 1.02
3.06
FM (kg) 20.52
9.03 20.70
7.78 0.97a 0.19
2.30
LM (kg) 50.18
10.96 49.05
11.12 0.94a 1.13
2.09b
BMC (kg) 2.65
0.54 2.26
0.44 0.81a 0.38
0.32b
Values are means
SD
sBIA segmental bioelectrical impedance analysis, DEXA dual energy X-ray absorptiometry, FM fat mass, LM lean
mass, BMC bone mineral content, r correlation coefficient
a
Correlation coefficients are significant at p < 0.001
b
Differences are significant at p < 0.01

test (Table 2) and Bland-Altman analyses as
described earlier in the Methods section (Table 3
and Fig. 2). Compared with DEXA, sBIA was
found to underestimate the whole-body FM
percentage and FM by 1.02 % and 186 g, respec-
tively. However, these differences were insigni-
ficant. In comparison with DEXA, sBIA
significantly overestimated the LM and BMC
by 1,126 g and 382 g, respectively. As shown in
Fig. 2, Bland-Altman plots revealed a good
agreement between the two methods for
measurements of whole-body FM and LM.
However, a poor agreement and a high bias
were found for the BMC measurements.
of FM percentage and LM (Table 4). sBIA sig-
nificantly underestimated the appendicular FM
percentage when compared with DEXA. The
smallest difference between the methods was in
the measurements of the trunk FM percentage
(0.91 %), which was insignificant. In comparison
with DEXA, sBIA significantly underestimated
LM of the right arm and overestimated LM of
both legs. The smallest difference between
methods was found in the measurements of left
arm FM (53 g), which was insignificant.
4 Discussion

Cigarette smoking is a major risk factor of

3.3 Assessment of Segmental Body
Composition by sBIA vs. DEXA
Highly significant correlations were found
between the two methods for the assessment of
segmental FM percentage and LM, but there
were consistent differences in the measurements
numerous pathologies including various cancers,
respiratory and cardiovascular diseases
(Filozof et al. 2004). In addition, previous studies
reported that smoking adversely affects body
composition. Although smokers tend to be leaner
than non-smokers, increased abdominal adipos-
ity was found among smokers (Kim et al. 2012;
6 O. Rom et al.

Fig. 1 Correlations between sBIA and DEXA reference line are presented. sBIA segmental bioelectrical
measurements of whole-body composition. (A) FM per- impedance analysis, DEXA dual energy X-ray absorpti-
centage, (B) FM, (C) LM, and (D) BMC by sBIA plotted ometry, FM fat mass, LM lean mass
against DEXA. Corresponding regression equation and

Table 3 Bland-Altman analysis of whole-body composition measured by sBIA and DEXA

Measurements Bias (sBIA  DEXA) 95 % Confidence interval 95 % Limits of agreements
FM (%) 1.02 1.90 0.14 7.02 4.98
FM (kg) 0.19 0.85 0.47 4.70 4.32
LM (kg) 1.13 0.53 1.73 2.96 5.21
BMC (kg) 0.38 0.29 0.48 0.25 1.01
sBIA segmental bioelectrical impedance analysis, DEXA dual energy X-ray absorptiometry, FM fat mass, LM lean
mass, BMC bone mineral content
Body Composition in Heavy Smokers: Comparison of Segmental Bioelectrical. . .
Fig. 2 Bland-Altman analysis for measurements of
whole-body composition between sBIA and DEXA.
(A) FM percentage, (B) FM, (C) LM, and (D) BMC
between sBIA and DEXA. Bias is marked by a solid
line. Limits of agreement were calculated as mean
Akbartabartoori et al. 2005; Canoy et al. 2005;
Bamia et al. 2004; Barrett-Connor and Khaw
1989). Compared with non-smokers, smokers
were also found to have lower levels of LM,
BMC, and BMD (Van den Borst et al. 2011;
Lee et al. 2007; Demirbag et al. 2006; Szulc
et al. 2002, 2004; Castillo et al. 2003; Elgan
et al. 2003). The adverse body composition
among smokers, in which central obesity is
7
difference
1.96 SD and are marked by broken lines.
sBIA segmental bioelectrical impedance analysis, DEXA
dual energy X-ray absorptiometry, FM fat mass, LM
lean mass, BMC bone mineral content
increased and levels of LM and BMC are
decreased, may promote various pathologies
such as cardiovascular diseases, sarcopenia, and
osteoporosis (Akbartabartoori et al. 2005; Szulc
et al. 2002, 2004). To reduce health risks among
smokers, body composition should be examined
more frequently. DEXA is the reference method
for the assessment of body composition, but its
use it limited due to high costs and the risk of
8 O. Rom et al.
Table 4 Assessment of segmental body composition by sBIA vs. DEXA
Measurements Region sBIA
FM (%) Left arm 26.58
10.65
Right arm 25.21
10.35
Left leg 29.24
11.02
Right leg 29.14
11.19
Trunk 26.70
9.52
LM (kg) Left arm 2.77
0.83
Right arm 2.78
0.83
Left leg 8.25
1.85
Right leg 8.40
1.93
Trunk 27.90
5.38
Values are means
SD
sBIA segmental bioelectrical impedance analysis, DEXA dual energy X-ray absorptiometry, FM fat mass, LM lean
mass, r correlation coefficient
a
Correlation coefficients are significant at p < 0.001
b
Differences are significant at p < 0.01
radiation exposure (Thibault et al. 2012). BIA
may provide an inexpensive and simple method
for the assessment of body composition in
smokers (Thibault et al. 2012; Beeson
et al. 2010). However, its accuracy in measuring
whole-body and segmental body composition
among smokers has not previously been com-
pared with DEXA.
This study aimed to examine the reliability
and accuracy of sBIA in the assessment of
whole-body and segmental body composition in
heavy smokers by comparing it to DEXA as a
reference method. The whole-body and segmen-
tal body compositions measured by the two
methods were found to be highly correlated and
no significant differences were demonstrated
between these methods for measurements of
whole-body FM, trunk FM, and LM of left arm.
The Bland-Altman analysis indicated a good
method agreement for measurements of the
whole-body FM and LM, but a poor agreement
for BMC. Significant differences between the
methods were found for the measurement of
whole-body LM, BMC, LM of legs, right arm,
trunk, and overall appendicular FM percentage.
The first step of the comparison between the
two methods in this study included the assess-
ment of correlations coefficients for the various
measurements of body composition (Tables 2, 4,
and Fig. 1). High and significant correlations
were found for all measurements of both whole-
DEXA r Difference (sBIA  DEXA)
30.45
12.76 0.925a 3.87
5.02b
28.35
11.58 0.924a 3.14
4.44b
31.16
10.38 0.893a 1.93
4.00b
31.42
10.41 0.919a 2.28
4.43b
27.61
8.74 0.912a 0.91
3.91
2.82
1.05 0.958a 0.05
0.35
3.23
1.17 0.965a 0.45
0.43b
7.63
2.03 0.959a 0.63
0.58b
7.64
1.97 0.954a 0.76
0.59b
24.45
4.90 0.930a 3.45
1.98b
body and segmental body compositions. The
lowest correlation coefficient was found for the
measurement of BMC (r 0.81). However, cor-
relation coefficient measures only the strength of
a relation between two variables, not the agree-
ment between them. In the present study, we
were interested to examine the agreement
between sBIA and DEXA for the assessment of
body composition among heavy smokers, i.e., to
what degree the measurements of body composi-
tion by sBIA are likely to differ from DEXA in
smokers. To conclude that sBIA can replace
DEXA for the assessment of body composition
among smokers, the differences in the
measurements by the two methods should not
impair the clinical interpretation of the results.
To examine the agreement between the two
methods, the Bland-Altman analysis and plots
(Bland and Altman 1986) were used and the
differences between the methods (sBIA-DEXA)
were plotted against their mean. The agreement
was assessed by calculating the bias as the mean
difference between measurements and by deter-
mining limits of agreements between
1.96 SD
of the differences (Table 3 and Fig. 2). If the
difference between the two methods is found
within the limits of the agreement and has no
clinical importance, it can be concluded that
there is a good agreement and the two methods
can be used interchangeably, i.e., sBIA can
replace DEXA in heavy smokers.
Body Composition in Heavy Smokers: Comparison of Segmental Bioelectrical. . .
The mean differences between the two
methods for measurements of whole-body FM
and FM percentage were insignificant (0.186 g
and 1.02 %, respectively). However, significant
differences were found for the whole-body LM
and BMC (1.13 kg and 0.38 kg, respectively,
p < 0.01). These differences were within the
limits of agreement (Table 3 and Fig. 2). To
further assess the agreement between the two
methods, the clinical significance of these
differences should be evaluated. In the present
study, the mean whole-body LM, as determined
by DEXA and sBIA, were 50.18 kg and 49.05 kg,
respectively. The difference between the
methods of 1.13 kg is only 2.2 % of the whole-
body LM as measured by DEXA. However, the
mean BMC, as determined by DEXA and sBIA,
were 2.65 kg and 2.26 kg, respectively. The
difference between the methods of 0.38 kg is
14.4 % of BMC as measured by DEXA (Table 2).
Thus, regarding LM, although the difference
between the methods was found to be statistically
significant, we believe that the overestimation of
LM by sBIA has little clinical significance.
However, the significant overestimation of
BMC by sBIA when compared with DEXA
may be of greater clinical importance. Therefore,
it can be concluded that the measurements of
whole-body FM, FM percentage, and LM
revealed a good agreement between the two
methods, but a poor agreement for the measure-
ment of BMC.
Regarding the segmental body composition,
differences between the two methods for
measurements of appendicular FM percentage
were all significant. The greatest difference was
found for the measurement of FM percentage of
left arm (3.87 %, p < 0.01). The smallest differ-
ence was found for the measurement of trunk FM
percentage (0.91 %) and was insignificant. The
differences between the two methods for
measurements of appendicular LM were all sig-
nificant except for the left arm (53 g). The
greatest difference in the LM measurements
was found for the trunk LM (3.45 kg,
p < 0.01). Thus, sBIA may be used efficiently
for the measurement of the trunk FM percentage.
However, it appears that compared with DEXA,
9
sBIA measurement of the segmental body com-
position has a poor reliability.
Previous studies compared BIA and DEXA for
the assessment of body composition in various
populations and found similar results. Beeson
et al. (2010) compared BIA with DEXA in diabetic
patients and found that FM, FM percentage, and
free fat mass (FFM) were highly correlated
(r 0.96, 0.91, and 0.95, respectively). Also, the
Bland-Altman analysis showed a general agree-
ment between the two methods. It was concluded
that BIA may provide valid measures of FM, per-
centage of FM, and FFM, and could be used as
practical tool for the assessment of body composi-
tion in diabetics. In addition, Furstenberg and Dav-
enport (2011) compared BIA and DEXA for the
assessment of whole-body and segmental body
composition in hemodialysis patients. It was
found that the whole-body FM and LM measured
by the two methods were highly correlated
(r 0.92 and 0.93, respectively) with a good
agreement using the Bland-Altman analysis (bias
of sBIA-DEXA for FM and LM 1 g and 157 g,
respectively). Similar to our findings, correlation
coefficients for the segmental LM were lower and
biases were greater. Also, BMC measured by the
two methods had weaker correlations (r 0.77)
and sBIA was found to overestimate BMC by
530 g. Furstenberg and Davenport (2011)
concluded that BIA may be used for the measure-
ment of the whole-body FM and LM in hemodial-
ysis patients, but not for the BMC assessment.
The BMC measurement by sBIA is derived
from LM using an algorithm based on a general
healthy population and is not measured directly
as by DEXA (Furstenberg and Davenport 2011).
As mentioned earlier, smokers tend to have lower
levels of BMC and BMD (Van den Borst
et al. 2011; Demirbag et al. 2006; Elgan
et al. 2003; Szulc et al. 2002). This may explain
the overestimation by sBIA in the measurement
of BMC among heavy smokers. Thus, DEXA
appears to be more reliable than sBIA in measur-
ing BMC in smokers.
The strength of the present study is that both
whole-body and segmental body composition
were studied, including the three main
components of body composition: FM, LM and
10
BMC. In addition, the smoking status of the
participants was not determined by self-report
alone, but was objectively verified by the mea-
surement of urine cotinine. The comparison
between sBIA and DEXA was made by extensive
statistical elaboration. Nonetheless, the study has
several limitations. The study examined specific
machines and models of sBIA and DEXA. Dif-
ferent machines may give different results. Sec-
ondly, assessments of body composition by both
sBIA and DEXA were limited to only one mea-
surement. Longitudinal assessments in which
several measurements are taken may also give
different findings. Thirdly, the study had a rela-
tive small study group of 49 participants.
In conclusion, the altered body composition
previously found among smokers and its health
risks raise the need for a simple method of body
composition assessment in smokers. Compared
with DEXA, the reference method for the assess-
ment of body composition, sBIA was found to
provide reliable measures of whole-body LM,
FM, and trunk FM in heavy smokers. Less agree-
ment was found for appendicular LM and FM
and for BMC. To verify our findings, future
studies are needed in which the comparison of
sBIA and DEXA will be repeated in additional
machines and in larger sample sizes.
Acknowledgments This study was supported by grants
from the Rappaport Institute, the Krol Foundation of
Barnegat N.J., the Myers-JDC-Brookdale Institute of Ger-
ontology and Human Development, and ESHEL the
association for planning and development of services for
the aged in Israel.
Conflicts of Interest The authors declare no conflicts of
interest in relation to this article.
References
Akbartabartoori M, Lean ME, Hankey CR (2005)
Relationships between cigarette smoking, body size
and body shape. Int J Obes Relat Metab Disord 29
(2):236243
Bamia C, Trichopoulou A, Lenas D, Trichopoulos D
(2004) Tobacco smoking in relation to body fat mass
and distribution in a general population sample. Int J
Obes Relat Metab Disord 28(8):10911096
O. Rom et al.
Barrett-Connor E, Khaw KT (1989) Cigarette smoking
and increased central adiposity. Ann Intern Med 111
(10):783787
Beeson WL, Batech M, Schultz E, Salto L, Firek A,
Deleon M, Balcazar H, Cordero-Macintyre Z (2010)
Comparison of body composition by bioelectrical
impedance analysis and dual-energy X-ray absorpti-
ometry in Hispanic diabetics. Int J Body Compos Res
8(2):4550
Bland JM, Altman DG (1986) Statistical methods for
assessing agreement between two methods of clinical
measurement. Lancet 1(8476):307310
Canoy D, Wareham N, Luben R, Welch A, Bingham S,
Day N, Khaw KT (2005) Cigarette smoking and fat
distribution in 21,828 British men and women: a
population-based study. Obes Res 13(8):14661475
Castillo EM, Goodman-Gruen D, Kritz-Silverstein D,
Morton DJ, Wingard DL, Barrett-Connor E (2003)
Sarcopenia in elderly men and women: the Rancho
Bernardo study. Am J Prev Med 25:226231
Clair C, Chiolero A, Faeh D, Cornuz J, Marques-Vidal P,
Paccaud F, Mooser V, Waeber G, Vollenweider P
(2011) Dose-dependent positive association between
cigarette smoking, abdominal obesity and body fat:
cross-sectional data from a population-based survey.
BMC Public Health 11:23
Demirbag D, Ozdemir F, Ture M (2006) Effects of coffee
consumption and smoking habit on bone mineral den-
sity. Rheumatol Int 26(6):530535
Elgan C, Samsioe G, Dykes AK (2003) Influence of
smoking and oral contraceptives on bone mineral den-
sity and bone remodeling in young women: a 2-year
study. Contraception 67(6):439447
Filozof C, Fernandez Pinilla MC, Fernandez-Cruz A
(2004) Smoking cessation and weight gain. Obes
Rev 5(2):95103
Furstenberg A, Davenport A (2011) Comparison of mul-
tifrequency bioelectrical impedance analysis and dual-
energy X-ray absorptiometry assessments in outpa-
tient hemodialysis patients. Am J Kidney Dis 57
(1):123129
Jhun HJ, Seo HG, Lee DH, Sung MW, Kang YD, Syn HC,
Jun JK (2010) Self-reported smoking and urinary
cotinine levels among pregnant women in Korea and
factors associated with smoking during pregnancy. J
Korean Med Sci 25(5):752757
Kim JH, Shim KW, Yoon YS, Lee SY, Kim SS, Oh SW
(2012) Cigarette smoking increases abdominal and
visceral obesity but not overall fatness: an observa-
tional study. PLoS One 7(9):e45815
Lee JS, Auyeung TW, Kwok T, Lau EM, Leung PC, Woo
J (2007) Associated factors and health impact of
sarcopenia in older Chinese men and women: a
cross-sectional study. Gerontology 53:404410
Lloret Linares C, Ciangura C, Bouillot JL, Coupaye M,
Decleves X, Poitou C, Basdevant A, Oppert JM (2011)
Validity of leg-to-leg bioelectrical impedance analysis
to estimate body fat in obesity. Obes Surg 21
(7):917923
Body Composition in Heavy Smokers: Comparison of Segmental Bioelectrical. . . 11

Szulc P, Garnero P, Claustrat B, Marchand F, Duboeuf F,
Delmas PD (2002) Increased bone resorption in mod-
erate smokers with low body weight: the Minos study.
J Clin Endocrinol Metab 87(2):666674
Szulc P, Duboeuf F, Marchand F, Delmas PD (2004)
Hormonal and lifestyle determinants of appendicular
skeletal muscle mass in men: the MINOS study. Am J
Clin Nutr 80:496503
Thibault R, Genton L, Pichard C (2012) Body composition:
why, when and for who? Clin Nutr 31(4):435447
Van den Borst B, Koster A, Yu B, Gosker HR,
Meibohm B, Bauer DC, Kritchevsky SB, Liu Y,
Newman AB, Harris TB, Schols AM (2011) Is
age-related decline in lean mass and physical function
accelerated by obstructive lung disease or smoking?
Thorax 66(11):961969
Advs Exp. Medicine, Biology - Neuroscience and Respiration (2015) 9: 1319
DOI 10.1007/5584_2014_81
# Springer International Publishing Switzerland 2014
Published online: 15 October 2014

Metabolic and Immunological

Consequences of Vitamin D Deficiency
in Obese Children

B. Pyrzak, E. Witkowska-Sedek, M. Krajewska, U. Demkow,

and A.M. Kucharska

Abstract
Numerous studies highlighted the link between vitamin D deficiency and
cardiovascular, autoimmune, metabolic diseases, and obesity. However, a
clear role of vitamin D in these disorders is still unknown. Vitamin D
deficiency in children can be a potential risk factor for developing diseases
at a later age. Early prevention and vitamin D supplementation should
become a public health priority. This review highlights the clinical
implications of vitamin D deficiency in adults and children with obesity.

Keywords
Cardiovascular diseases Children Obesity Metabolic syndrome
Vitamin D deficiency

1 Introduction is inversely associated with the 25(OH)D serum

Obesity is a major, increasingly prevalent health
problem affecting modern societies. Vitamin D
deficiency is highly prevalent in patients with
obesity and is strongly connected with its
consequences (Vimeswaran et al. 2013;
Earthman et al. 2012). Body mass index (BMI)
B. Pyrzak (*), E. Witkowska-Sedek, M. Krajewska,
and A.M. Kucharska
Department of Pediatrics and Endocrinology, Medical
University of Warsaw, 24 Marszalkowska St., 00-576
Warsaw, Poland
e-mail: beata.pyrzak@wum.edu.pl
U. Demkow
Department of Laboratory Diagnostics and Clinical
Immunology of the Developmental Age, Medical
University of Warsaw, Warsaw, Poland
level in response to vitamin D supplementation.
The relationship between obesity and 1,25
(OH)2D, the active form of vitamin D, is less
clear and this is due probably to the dynamic
nature of the production and regulation of the
active hormone.
In children, vitamin D has a major biological
action in mineral homeostasis and regulation of
bone remodeling. Moreover, severity and
prolonged duration of vitamin D deficiency can
potentially lead to cardiovascular and metabolic
diseases in adolescence or in early adulthood. A
major form of vitamin D in the circulation is 25
(OH)D and the measurement of its serum level
can be used to evaluate vitamin D status. Vitamin
D deficiency has been defined as 25(OH)D of
less than 20 ng/ml (<50 nmol/l). In healthy

13
14
children, vitamin D deficiency can be caused by a
combination of inadequate intake of vitamin
D-containing foods and insufficient exposure to
sunlight. Seasonal variations of vitamin D level
may occur, depending on the geographic latitude
and sun exposure. There are several conditions,
such as obesity and liver or kidney disorders, in
which the risk of developing vitamin D defi-
ciency is increased (Joergensen et al. 2010).
In Poland, a multidisciplinary group
formulated recommendations for vitamin D sup-
plementation in obese children and adolescents:
1,2002,000 IU/day (3050 g) from September adipocyte-specific
to April, depending on the BMI. These
recommendations are based on the studies of
healthy individuals, taking into consideration
both serum 25(OH)D concentrations and vitamin
D intakes.
2 Vitamin D in Adipogenesis
The regulation of adipogenesis is a key
biological process that is required for both lipid
storage and the development of endocrine
adipocytes (Blumberg et al. 2006). The expression
of vitamin D-metabolizing enzymes has been
demonstrated in human adipose tissue (Wamberg
et al. 2013). Plasma 25(OH)D increases by 27 %
after weight loss in obese individuals; concomi-
tantly, 25-hydroxylase CYP2J2 and
1--hydroxylase CYP27B1 decrease by 71 %
and 49 %, respectively, in subcutaneous adipose
tissue of obese subjects. These findings suggest
that adipose tissue, which can be dynamically
altered during obesity and weight loss, has the
ability to metabolize vitamin D locally. Vitamin
D status may regulate human adipose tissue
growth and remodeling (Blumberg et al. 2006).
Adipocyte precursor cells (preadipocytes) are
present throughout life, and obesity may be par-
tially mediated by stimulating the differentiation
of preadipocytes into adipocytes or by fat accu-
mulation in the differentiated adipocytes
(Nimitphong et al. 2012).
Cellular action of 1,25(OH)2D is mediated by
the vitamin D receptor (VDR), a ligand-
B. Pyrzak et al.
dependent transcription regulator molecule
belonging to the superfamily of nuclear
receptors. There are some reports concerning
the modulatory effect of 125(OH)2D on adipo-
cyte differentiation, but the molecular mecha-
nism of 125(OH)2D-induced modulation
remains unclear and further studies are needed
to clarify this problem (Blumberg et al. 2006).
Differentiation of 3T3-L1 and other types of
preadipocytes is a process highly controlled
through sequential induction of transcription
factors that regulate the expression of
markers. During
adipogenesis, a series of cellular events begins
with a rapid expression of CCAAT/enhancer-
binding protein b (C/EBPb), followed by the
expression of C/EBPa, PPARg, and sterolre-
gulatory element-binding protein 1 (SREBP1)
(Christy et al. 1989).
Blumberg et al. (2006) showed that liganded
VDR downregulates both C/EBPa and PPARg
via inhibition of C/EBPb expression and action.
Liganded VDR is a potent inhibitor of
adipogenesis; the unliganded receptor is not
required for adipogenesis, but may play a role
in some aspect of the process. These data suggest
that the intracellular calcitriol plays a key role in
adipocyte formation.
During adipogenesis, there is an increase in
the expression of genes that produce the adipo-
cyte phenotype, including genes for lipoprotein
lipase and adipocyte lipid-binding protein 2; the
latter being a late marker of adipogenesis
(Christy et al. 1989). In the differentiation
phase, expression of genes encoding lipogenic
enzymes, such as fatty acid synthase, is highly
induced and de novo fatty acid synthesis
increases enormously.
Links between 1,25(OH)2D and adipocyte
lipogenesis has also been supported by other
researchers who demonstrate that the hormone
strongly increases mRNA levels of insulin-
induced gene-2 (Insig-2), a factor blocking fatty
acid synthesis in mature 3T3-L1 adipocytes and
inhibits preadipocyte differentiation (Lee
et al. 2005). Ochs-Balcom et al. (2011) in a
recent study have also shown a positive
Metabolic and Immunological Consequences of Vitamin D Deficiency in Obese Children
association between VDR polymorphisms and
the markers of adiposity.
3 Vitamin D in Autoimmune
Diseases
Deficiency of vitamin D is frequently observed in
patients with autoimmune diseases. Children
with diabetes mellitus, both obese and
non-obese, can develop severe late onset
complications at an older age. The potential risk
can be much greater in patients with vitamin D
deficiency. Autoimmune diseases are
characterized by a loss of immune homeostasis
resulting in impaired self-antigen recognition,
followed by destruction of body tissue by
autoreactive immune cells. A combination of
genetic predisposition, and epidemiological and
environmental contributors can lead to the devel-
opment of autoimmune diseases. One important
factor may be the availability of sufficient vita-
min D levels, as various epidemiological studies
suggest an association between vitamin D defi-
ciency and the incidence of autoimmune
diseases, such as type 1 diabetes (T1D), multiple
sclerosis (MS), systemic lupus erythematosus
(SLE), rheumatoid arthritis (RA), inflammatory
bowel disease (IBD), and Graves disease.
In a recent review, Hewison (2012) has pro-
posed four potential mechanisms by which vita-
min D may influence T cell function:
direct, endocrine effects on T cells, mediated
via systemic calcitriol;
direct, intracrine conversion of 25(OH)D to
calcitriol by T cells;
direct, paracrine effects of calcitriol on T cells
following conversion of 25(OH)D to calcitriol
by monocytes or dendritic cells.
indirect effects on antigen presentation to T
cells mediated via localized antigen-
presenting cells (APC) affected by calcitriol.
Antico et al. (2012) reviewed 219 published stud-
ies and concluded that vitamin D seems to play a
beneficial role in the prevention of autoimmu-
nity, but that randomized controlled clinical trials
confirming this observation are still missing.
15
4 Vitamin D Deficiency and
Its Effects on BMI, Lipid
Metabolism, Diabetes
Mellitus, and Cardiovascular
Disorders in Obese Patients
Current research considers a reduced vitamin D
concentration as a potential risk factor for cardio-
vascular disorders (Earthman et al. 2012), meta-
bolic syndrome (Vimeswaran et al. 2013),
hypertension (Larsen et al. 2012; Lind et al.
1988), diabetes (Kayaniyil et al. 2011; Hypponen
et al. 2001), cancer (Garland et al. 2011),
autoimmune and infectious diseases resulting
from decreased immunity (Hewison 2012).
According to Earthman et al. (2012), obesity
could contribute to low serum 25(OH)D via adi-
pose sequestration of vitamin D. However, adi-
pose tissue has the VDR and can synthesize 1,25
(OH)2D, and there is evidence that vitamin D
may regulate adipose tissue mass, differentiation,
and metabolism; and thus, contributes to obesity.
Vimeswaran et al. (2013) investigated the rela-
tionship between body mass index (BMI) and
vitamin D status and inferred causality by using
genetic variants, namely vitamin D-related single
nucleotide polymorphism (SNPs) and allele
scores, as instruments in bidirectional Mendelian
randomization (MR) analyses. This meta-
analysis was based on data from 21 studies of
adult cohorts comprising 42,024 individuals.
Twelve established BMI-related SNPs, such as
melanocortin 4 receptor, transmembrane protein
18, and brain-derived neurotrophic factor, were
selected for the analysis. Each unit (kg/m2)
increase in BMI was associated with 1.15 %
lower concentrations of 25(OH)D. The inverse
association between BMI and 25(OH)D was
stronger in subjects from North America than
those from Europe, and in women compared
with men, while no variation was seen in relation
to age or BMI. The authors also showed that
higher BMI is connected with lower vitamin D
level. Speliotes et al. (2010) expanded the
Genetic Investigation of Anthropometric Traits
(GIANT) consortium genome-wide association
16
studies of BMI, confirmed 14 known obesity
susceptibility loci and identified 18 new loci
associated with BMI. Some loci are located
near key hypothalamic regulators of energy bal-
ance and one of these loci is located near an
incretin receptor GIPR.
Olson et al. (2012) showed that the mean
serum 25(OH)D level is significantly lower in
children with a higher BMI and high body fat
mass. Data from the National Health and Nutri-
tion Examination Survey (NHANES) 20012004
show that obese children are more likely to have
a low level of 25(OH)D (Kumar et al. 2009).
Significantly lower seasonal variations of 25
(OH)D concentration are observed in obese chil-
dren (Olson et al. 2012). The bioavailability of
25(OH)D is decreased due to of its deposition in
adipose tissue. Obese children often have an
indoor lifestyle with reduced sunlight exposure.
Poor dietary habits also contribute to decreased
vitamin D levels, as unhealthy high caloric food
is usually low in minerals and vitamins. Skipping
breakfast, soda, and juice intake also are
associated with decreased vitamin D levels
(Olson et al. 2012). It is crucial for obese children
to develop healthy dietary habits to ensure an
adequate intake of vitamin D and serum 25
(OH)D levels. Moreover a significant increase
in serum 25(OH)D concentration has been
found in obese children after weight loss.
4.1 Lipid Metabolism
Vitamin D plays a role in lipid metabolism in
adipose tissue. 1,25(OH)2D causes a significant
increase in lipoprotein lipase activity in 3T3-L1
adipocytes. Fatty acid synthase, which facilitates
adipocyte lipogenesis, is downregulated by
1,25(OH)2D in 2T3-L1 cells and VDR can
inhibit lipid metabolism. Mice without VDR are
resistant to high-fat diet-induced obesity, due
probably to increased fatty acid -oxidation in
white adipose tissue, increased expression of
uncoupling proteins in brown fat, and overall
energy expenditure (Wong et al. 2009).
The connection of 25(OH)D with the lipid
profile in children shows a strong correlation
B. Pyrzak et al.
between lower HDL-C, high LDL-C, and TAG,
on the one side, and the decreased level of serum
25(OH)D on the other (Alfawaz and Abdel
Megeid 2013). The positive correlation between
serum 25(OH)D and HDL-C is likely caused by
vitamin D, which maintains the level of apolipo-
protein A-1, the main component of HDL. This
observation corresponds well with another study
reporting an inverse correlation between vitamin
D and total cholesterol (Kardas et al. 2013).
4.2 Type 2 Diabetes
Low 25(OH)D levels are frequent in patients
with both type 1 and type 2 diabetes mellitus.
In children with vitamin D deficiency, the risk of
type 1 diabetes increases by approx. 200 %
(Hypponen et al. 2001). Vitamin D deficiency
increases insulin resistance, decreases insulin
production, and is associated with the metabolic
syndrome. Maestro et al. (2002) suggest a poten-
tially beneficial influence of vitamin D on insulin
sensitivity. They have shown that 1,25(OH)2D
treatment increased insulin receptor mRNA
levels and insulin-stimulated glucose transport
in U-937 promonocytic cells. This effect is pos-
sibly achieved through the upregulation of
phosphatidylinositol 3-kinase activity. Higher
levels of 25(OH)D can predict a better -cell
function and a lower glucose level in patients at
risk of type 2 diabetes (Kayaniyil et al. 2011). In
adults at risk of type 2 diabetes, short-term sup-
plementation with cholecalciferol improves
-cell function. Joergensen et al. (2010)
conducted similar research in patients with type
2 diabetes and showed that the baseline level of
25(OH)D below the 10th percentile predicts an
increased risk of all-cause and cardiovascular
mortality in such patients, but does not predict
micro- or macroalbuminuria. Those results are
consistent with other studies conducted in the
general population and in patients with chronic
kidney disease without diabetes.
Kayaniyil et al. (2011) examined a large
group of multiethnic subjects and confirmed
that a low serum 25(OH)D was significantly
associated with a high incidence of the metabolic
Metabolic and Immunological Consequences of Vitamin D Deficiency in Obese Children
syndrome. Multivariate linear regression analy-
sis showed significant adjusted inverse associa-
tion of 25(OH)D with waist circumference,
triglyceride level, fasting insulin, and alanine
transaminase. A cross-sectional study including
13,331 participants from NHANES III found low
vitamin D levels to be associated with all-cause
mortality. Also, childhood obesity is a risk factor
for the metabolic syndrome and type 2 diabetes.
It is important to identify modifiable risk factors
for metabolic disorders to prevent the develop-
ment of chronic diseases. Several studies have
investigated the correlation between serum 25
(OH)D and impaired glucose tolerance, diabetes
mellitus, dyslipidemia, metabolic syndrome, car-
diovascular disease risk, and hypertension. The
results are sometimes contradictory. There is a
negative association between the serum 25(OH)
D concentration and the level of fasting glucose
(Kardas et al. 2013), insulin concentrations, insu-
lin resistance-HOMA-IR (homeostasis model
assessment-insulin resistance), HbA1c, and a
positive association between 25(OH)D and insu-
lin sensitivity-QUICKY (quantitative insulin-
sensitivity check index) (Roth et al. 2011). This
is in agreement with the results of other
investigations, which show that 25(OH)D is pos-
itively associated with insulin sensitivity and
negatively with serum insulin levels, insulin
resistance (HOMA-IR), and a 2-h glucose level
in an oral glucose tolerance test in obese children
(Olson et al. 2012). Moreover, the association
between 25(OH)D and insulin sensitivity or insu-
lin resistance persisted after adjustment for body
mass. Low 25(OH)D concentrations may be
directly related to insulin resistance, irrespective
of body fat mass. Low serum 25(OH)D
concentrations apparently play a role in the path-
ophysiology of impaired glucose tolerance in
children.
The reports are, however, contentious. A
report, examining vitamin D-deficient (<10 ng/
ml) and insufficient (1020 ng/ml) obese chil-
dren, has demonstrated that insulin resistance
did not statistically differ from that in vitamin
D-sufficient group (>20 ng/ml). There was no
correlation between vitamin D level and insulin
resistance in obese children and adolescents
17
either. Insulin resistance was highly affected by
BMI, BMI-SDS, and BMI% but less so by 25
(OH)D concentration. Some investigators dem-
onstrate a negative correlation between 25(OH)D
levels and HbA1c, while others report no corre-
lation (Olson et al. 2012).
4.3 Cardiovascular Disease
Vitamin D deficiency is an important factor
implicated in the development of cardiovascular
diseases. Its pleiotropic effect is achieved
through the activation of VDR. Calcitriol inhibits
proliferation of vascular smooth muscle cells,
expressing vitamin D receptors via an acute
influx of calcium into cells. It has been shown
that vitamin D deficiency increases cardiovascu-
lar disease mortality rate. Vitamin D has cardio-
vascular and renoprotective effects, because it
has been associated with suppression of the
renin-angiotensin-aldosterone system (RAAS),
inhibits vascular calcification and
atherosclerotic-plaque formation, has anti-
inflammatory and immunomodulatory actions.
Calcitriol therapy reduces blood pressure, plasma
renin activity, and angiotensin II levels. Vitamin
D deficiency causes an increase in the serum
parathyroid hormone (PTH), which may contrib-
ute to cardiovascular disease, increasing cardiac
contractility and myocardial calcification. Low
25(OH)D may influence the activity of
macrophages and lymphocytes in the atheroscle-
rotic plaques, thus promoting chronic inflamma-
tion in the artery wall. The studies by Van den
Berghe et al. (2003) showed that vitamin D sup-
plementation reduces the serum level of CRP,
interleukin-6, and tissue matrix metallopro-
teinases. Hypovitaminosis D and secondary
hyperparathyroidism may promote the acute
phase response and may help to explain how
vitamin D deficiency may act as a risk factor
for cardiovascular diseases.
Hypertension is related to disturbed calcium
metabolism. Calcium levels are lower in patients
with hypertension, because they tend to have
lower dietary calcium intake and a higher renal
calcium loss than those in normotensive subjects.
18
There is an inverse association between the
serum 25(OH)D level and diastolic blood pres-
sure. Larsen et al. (2012) conducted a
randomized, placebo-controlled, double-blind
study in 130 hypertensive patients to investigate
the relationship between supplementation of cho-
lecalciferol and blood pressure. The patients
received 3,000 IU (75 g) cholecalciferol per
day for 20 weeks, a dose that was deemed to
effectively increase vitamin D levels in the
blood. In a subgroup of 92 patients with baseline
25(OH)D levels <32 ng/ml, a significant
decrease in 24-h systolic and diastolic blood
pressure was found during cholecalciferol
supplementation.
In an earlier study, Lind et al. (1988) showed
that calcium metabolism plays a key role in
blood pressure regulation. That prospective,
double-blind, placebo-controlled study included
65 subjects with impaired glucose tolerance. The
findings were that alphacalcidol supplementation
(0.75 g daily) over 12 weeks in patients with
blood pressure of 150/90 mmHg or greater
caused a significant reduction in both systolic
and diastolic; the effect correlated with a reduc-
tion of PTH serum levels. Normal intracellular
calcium levels help maintain normal blood pres-
sure. This relation can explain the therapeutic
effects of calcium-channel blockers in patients
with hypertension. Low adenylate cyclase activ-
ity can lead to a decreased calcium re-uptake into
the sarcoplasmic reticulum, an accumulation of
intracellular free calcium, and an increase in
vascular reactivity and blood pressure. The activ-
ity of this enzyme is calcitriol-dependent. An
enhancement of adenylate cyclase activity may
reduce intracellular calcium concentration. In
children, the link between low 25(OH)D levels
and elevated systolic and diastolic blood pressure
has been confirmed (Kardas et al. 2013). How-
ever, another study reported no correlation
between the level of vitamin D and blood pres-
sure in a pediatric population (Olson et al. 2012).
Adiponectin gene expression may be
upregulated by vitamin D. It has been reported
that adipokine synthesis is regulated by 1,25
(OH)2D. A recent study by Kardas et al. (2013)
showed that adiponectin levels are lower in obese
B. Pyrzak et al.
children (aged 1016 years) and negatively
associated with BMI. Moreover, adiponectin is
strongly associated with HOMA index and
fasting glucose. These results suggest that a low
level of adiponectin is crucial in the development
of insulin resistance and diabetes.
Further clinical investigations are warranted
to examine the role of vitamin D in obesity and to
determine the optimal mode of vitamin D supple-
mentation, especially in obese children.
Conflicts of Interest No conflicts of interests were
declared by the authors in relation to this article.
References
Alfawaz HA, Abdel Megeid FY (2013) Vitamin D defi-
ciency in obese children and its relationship to the
components of the metabolic syndrome. World Appl
Sci J 21(3):320328
Antico A, Tampoia M, Tozzoli R, Bizzaro N (2012) Can
supplementation with vitamin D reduce the risk or
modify the course of autoimmune diseases? A system-
atic review of the literature. Autoimmun Rev
12:127136
Blumberg JM, Tzameli I, Astapova I, Lam FS, Flier JS,
Hollenberg AN (2006) Complex role of the vitamin D
receptor and its ligand in adipogenesis in 3T3-L1
cells. J Biol Chem 281:1120511213
Christy RJ, Yang VW, Ntambi JM, Geiman DE,
Landschulz WH, Friedman AD, Nakabeppu Y, Kelly
TJ, Lane MD (1989) Differentiation-induced gene
expression in 3T3-L1 preadipocytes: CCAAT/
enhancer binding protein interacts with and activates
the promoters of two adipocyte-specific genes. Genes
Dev 3:13231335
Earthman CP, Beckman LM, Masodkar K, Sibley SD
(2012) The link between obesity and low circulating
25- hydroxyvitamin D concentrations: considerations
and implications. Int J Obes (Lond) 36:387396
Garland CF, French CB, Baggerly LL, Heaney RP (2011)
Vitamin D supplement doses and serum
25-hydroxyvitamin D in the range associated with
cancer prevention. Anticancer Res 31:607611
Hewison M (2012) An update on vitamin D and human
immunity. Clin Endocrinol 76:315325
Hypponen EE, Laara E, Reunanen A, Jarvelin M-R,
Virtanen SM (2001) Intake of vitamin D and risk of
type 1 diabetes: a birth-cohort study. Lancet
358:15001503
Joergensen C, Gall MA, Schmedes A, Tarnow L, Parving
HH, Rossing P (2010) Vitamin D levels and mortality
in type 2 diabetes. Diabetes Care 33:22382243
Kardas F, Kendirci M, Kurtoglu S (2013)
Cardiometabolic risk factors related to vitamin D and
Metabolic and Immunological Consequences of Vitamin D Deficiency in Obese Children
adiponectin in obese children and adolescents. Int J
Endocrinol 2013:503270. doi:10.1155/2013/503270
Kayaniyil S, Retnakaran R, Harris SB, Vieth R, Knight
JA, Gerstein HC, Perkins BA, Zinman B, Hanley AJ
(2011) Prospective associations of vitamin D with
-cell function and glycemia: the PROspective Metab-
olism and ISlet cell Evaluation (PROMISE) cohort
study. Diabetes 60:29472953
Kumar J, Muntner P, Kaskel FJ, Hailpern SM, Melamed
ML (2009) Prevalence and associations of
25-hydroxyvitamin D deficiency in US children:
NHANES 20012004. Pediatrics 124:e362e370
Larsen T, Mose FH, Bech JN, Hansen AB, Pedersen EB
(2012) Effect of cholecalciferol supplementation dur-
ing winter months in patients with hypertension: a
randomized, placebo-controlled trial. Am J Hypertens
25:12151222
Lee S, Lee DK, Choi E, Lee JW (2005) Identification of a
functional vitamin D response element in the murine
Insig-2 promoter and its potential role in the differen-
tiation of 3T3-L1 preadipocytes. Mol Endocrinol
19:399408
Lind L, Lithell H, Skarfors E, Wide L, Ljunghall S (1988)
Reduction of blood pressure by treatment with
alphacalcidol. A double-blind, placebo-controlled
study in subjects with impaired glucose tolerance.
Acta Med Scand 223:211217
Maestro B, Molero S, Bajo S, Davila N, Calle C (2002)
Transcriptional activation of the human insulin recep-
tor gene by 1,25-dihydroxyvitamin D3. Cell Biochem
Funct 20:227232
Nimitphong H, Holick MF, Fried SK, Lee MJ (2012)
25-hydroxyvitamin D and 1,25-dihydroxyvitamin D
promote the differentiation of human subcutaneous
preadipocytes. PLoS ONE 7:e52171
Ochs-Balcom HM, Chennamaneni R, Millen AE, Shields
PG, Marian C, Trevisan M, Freudenheim JL (2011)
19
Vitamin D receptor gene polymorphisms are
associated with adiposity phenotypes. Am J Clin
Nutr 93:510
Olson ML, Maalouf NM, Oden JD, White PC, Hutchison
MR (2012) Vitamin D deficiency in obese children
and its relationship to glucose homeostasis. J Clin
Endocrinol Metab 97:279285
Roth CL, Elfers C, Kratz M, Hoofnagle AN (2011) Vita-
min D deficiency in obese children and its relationship
to insulin resistance and adipokines. J Obes
2011:495101. doi:10.1155/2011/495101
Speliotes EK, Willer CJ, Berndt SI, Monda KL,
Thorleifsson G, Jackson AU, Lango Allen H,
Lindgren CM, Luan J, Magi R, Randall JC,
Vedantam S, Winkler TW, Qi L et al (2010) Associa-
tion analyses of 249,796 individuals reveal 18 new
loci associated with body mass index. Nat Genet
42:937948
Van der Berghe G, Van Roosbroeck D, Vanhove P,
Wouters PJ, De Pourcq L, Bouillon R (2003) Bone
turnover in prolonged critical illness: effect of vitamin
D. J Clin Endocrinol Metab 88:46234632
Vimeswaran K, Berry D, Lu C, Pilz S, Hiraki L, Cooper J,
Dastani Z, Li R, Houston D, Wood A (2013) Causal
relationship between obesity and vitamin D status:
bidirectional Mendelian randomization analysis of
multiple cohorts. PLoS Med 10:15491676
Wamberg L, Christiansen T, Paulsen SK, Fisker S,
Rask P, Rejnmark L, Richelsen B, Pedersen SB
(2013) Expression of vitamin D-metabolizing enzymes
in human adipose tissue-the effect of obesity and diet-
induced weight loss. Int J Obes (Lond) 37:651657
Wong KE, Szeto FL, Zhang W, Ye H, Kong J, Zhang Z,
Sun XJ, Li YC (2009) Involvement of the vitamin D
receptor in energy metabolism: regulation of
uncoupling proteins. Am J Physiol Endocrinol Metab
296:E820E828
Advs Exp. Medicine, Biology - Neuroscience and Respiration (2015) 9: 2128
DOI 10.1007/5584_2014_87
# Springer International Publishing Switzerland 2014
Published online: 14 October 2014

Markers of Bone Metabolism in Children

with Nephrotic Syndrome Treated with
Corticosteroids

Magorzata Panczyk-Tomaszewska, Dominika Adamczuk,

Agnieszka Kisiel, Piotr Skrzypczyk, Jerzy Przedlacki,
Elzbieta Gorska, Anna Stelmaszczyk-Emmel,
Urszula Demkow, and Maria Roszkowska-Blaim

Abstract
The aim of the study was to assess bone mineral density, bone metabolism
markers, and vitamin D level in children with idiopathic nephrotic
syndrome in the course of 1-year observation. Twenty five children with
nephrotic syndrome aged 517 years were enrolled into the study.
The median number of relapses was 6 (range 122). All patients were
treated with prednisone and vitamin D (800 IU/day). Bone mineral density
of total body (TB-BMD) and lumbar spine (L-BMD), evaluated by dual
energy X-ray absorptiometry (DXA) expressed as Z-score, and serum
calcium, phosphorus, parathormone (iPTH), alkaline phosphatase
(ALP), bone alkaline phosphatase (BAP), osteocalcin (OC), albumin,
creatinine, 25(OH)D3, 1,25(OH)2D3 and urine calcium/creatinine
ratio (uCa/Cr) were evaluated at the enrollment visit and after 1 year of
therapy. After 1 year significant decreases of TB-BMD Z-score
(from
0.24  1.34 to
0.74  1.31, p < 0.05) and 25(OH)D3 serum
level (from 31.7  16.3 to 23.7  9.3; p < 0.05) were observed. No
other appreciable differences were found. At the study onset, negative
correlations were found between L-BMD Z-score and serum ALP, BAP,
and phosphorus and between TB-BMD Z-score and urine uCa/Cr. After
1 year, L-BMD Z-score correlated negatively with serum BAP and OC,
and positively with serum 25(OH)D3. Multivariate analysis showed that

M. Panczyk-Tomaszewska, D. Adamczuk, A. Kisiel,

P. Skrzypczyk, and M. Roszkowska-Blaim
Department of Pediatrics and Nephrology, Medical
University of Warsaw, 24 Marszalkowska St., 00-576
Warsaw, Poland
J. Przedlacki
Department of Nephrology, Dialysis and Internal
Diseases, Medical University of Warsaw,
1a Banacha St., 02-097 Warsaw, Poland
E. Gorska, A. Stelmaszczyk-Emmel, and
U. Demkow (*)
Department of Laboratory Diagnostics and Clinical
Immunology of Developmental Age, Medical University
of Warsaw, 24 Marszalkowska St., 00-576 Warsaw,
Poland
e-mail: urszula.demkow@litewska.edu.pl

21
22 M. Panczyk-Tomaszewska et al.

BAP was the strongest predictor of L-BMD Z-score (beta
0.49;

p < 0.05). We conclude that children with nephrotic syndrome treated
with corticosteroids are at risk of bone mass loss. Serum BAP concentra-
tion seems to be a good indicator of spongy bone metabolism in these
children, who should be supplemented with vitamin D in an adjustable
dose, possibly higher than 800 IU/24 h to prevent osteopenia.

Keywords
Bone alkaline phosphatase Bone density Bone metabolism markers
Children Corticosteroids Nephrotic syndrome

1 Introduction Lettgen et al. 1994). Nephrotic children frequently

The nephrotic syndrome is a glomerular disease
with massive proteinuria exceeding 50 mg/kg/
24 h, hypoalbuminemia, hyperlipidemia, and
edema, usually responding to corticosteroids
(KDIGO 2012). The idiopathic nephrotic
syndrome is the most common form in children,
with the incidence of 27/100,000 children aged
below 16 (ISKDC 1978). Based on the response
to corticosteroids, the disease is categorized as
steroid-sensitive, steroid-dependent, and steroid-
resistant (KDIGO 2012). Long-term application
of corticosteroids in children with nephrotic syn-
drome may lead to bone metabolism disorders,
whose mechanisms are underlain by both
steroid therapy and biochemical changes due to
proteinuria that influences bone turnover and
mineralization (Feber et al. 2012; Gulati
et al. 2003). The onset and subsequent relapses
of nephrotic syndrome, requiring steroid treat-
ment for several months, frequently coincide
with periods of maximal bone mineral accretion;
thus bone mineral density can be impaired in the
adulthood. Corticosteroids reduce bone formation
by inhibition of bone matrix synthesis, induction
of apoptosis of osteoblasts, and by promotion of
osteoclastogenetic-related bone resorption. In
effect, suppression of bone formation, osteo-
porosis, and impaired growth may develop.
Corticosteroids also affect bone indirectly by
reducing intestinal calcium absorption and increas-
ing urinary calcium loss (Mishra et al. 2009;
Canalis et al. 2002, 2007; Hidalgo et al. 2010;
exhibit disturbances of mineral metabolism, such
as deficiency of circulating 1,25(OH)2D3 and
hypocalcemia, leading to secondary hyperparathy-
roidism and decreased bone mineral density
(BMD) (Bak et al. 2006; Leonard et al. 2004).
Bone alkaline phosphatase (BAP) and osteocalcin
(OC) are used as serum markers of bone formation
(Sasaki et al. 2002), but their usefulness in
predicting alterations in bone mineral density is
unknown. Therefore, the aim of the present study
was to assess bone mineral density, bone metabo-
lism markers, and vitamin D level in corticosteroid-
treated children with nephrotic syndrome in the
course of 1-year observation.
2 Methods
The study protocol was approved by the Ethics
Committee of the Medical University of
Warsaw, Poland. Twenty five children (15 boys
and 10 girls) aged 517 years (mean 9  4 years)
treated in the Department of Pediatrics and
Nephrology of the Medical University of
Warsaw, due to idiopathic nephrotic syndrome
were enrolled into the study after informed con-
sent was obtained from their parents. All test
were performed at times of remission. The chil-
dren had normal renal function and all were
treated with prednisone (a mean starting dose
of 0.7  0.5 mg/kg/48 h) and vitamin D
(800 IU/24 h). Table 1 shows clinical charac-
teristics of the examined children.
Markers of Bone Metabolism in Children with Nephrotic Syndrome Treated with. . . 23

Table 1 Demographic and clinical characteristics of 57 years, and 0.21 for >7 years) (Hoppe and
patients Kemper 2010).
Gender (Female/Male) 10/15 Serum calcium, phosphorus, and alkaline phos-
Age (year) 9  4 (517) phatase were determined using a dry chemistry
Age at onset of NS (year) 4  3 (115) method (MicroSlide, VITROS 5600 Integrated
Duration of NS (years) 53 System; Ortho-Clinical Diagnostics, Rochester,
(0.414)
USA). 1,25(OH)2D3 was assessed by RIA test
Number of relapses (n) 6 (122)
(Biosource; Neville, Belgium), 25(OH)D3 by a
Response to corticosteroids
chemiluminescence method (LIAISON 25OH
Steroid sensitive (n) 12 (48 %)
Vitamin D Total Assay; DiaSorin S.p.A.,
Steroid dependent (n) 10 (40 %)
Steroid resistant (n) 3 (12)
Saluggia, Italy), iPTH by ECLIA test
Renal biopsy (n) 13 (52 %) (F. Hoffmann-La Roche AG, Basel, Switzerland),
Minimal change disease (n) 1 and BAP by EIA OSTASE BAP test and OC by
Mesangial proliferation (n) 11 N-MID Osteocalcin ELISA test (Immunodiagnos-
Focal and segmental 1 tic Systems, Scottsdale, USA).
glomerulosclerosis (n) Data were presented as means  SD and
Current medications ranges. Distribution of data was checked with
Prednisone 25 (100 %) the Shapiro-Wilk test. Pre- vs. post-treatment
Vitamin D 25 (100 %) differences were checked with a t-test or
Previous treatment Wilcoxon test for normal and non-normal
Methylprednisolone pulses (n) 10 (40 %) distributed data, respectively. Frequency of cor-
Cyclosporine A (n) 4 (16 %)
ticosteroid usage at baseline and after 1 year was
Cyclophosphamide (n) 9 (36 %)
compared with Fishers exact test. Pearsons
Chlorambucil (n) 2 (8 %)
coefficient of correlation was used for linear
Levamisole (n) 5 (20 %)
correlations and Spearman rank test for
NS nephrotic syndrome
non-normal distributed variables. Multivariate
analysis of variance was performed using a step-
Bone mineral density of total body wise regression. A p value <0.05 was considered
(TB-BMD), excluding the head, and of lumbar statistically significant. Statistical analysis was
spine L1-L4 (L-BMD) was evaluated by dual performed using Statistica 9.0PL software
energy of X-ray absorptiometry (DXA) and was (StatSoft, College Station, USA).
expressed as Z-score (Discovery A; Hologic,
Bedford, USA). The examinations were per-
formed at the time of enrolment and then 3 Results
repeated after 1 year. The following laboratory
parameters were assessed in the serum: calcium Bone mineral density and biochemical
(Ca, reference range: 2.002.75 mmol/L), phos- parameters of bone metabolism at the study
phorus (P, reference range: 0.952.00 mmol/L), onset and after 1-year therapy with vitamin D
intact parathormone (iPTH, reference range: presented in Table 2. TB-BMD Z-score
1065 pg/mL, alkaline phosphatase (ALP, refer- decreased in 18 (72 %), did not change in
ence range: 80280 U/L, bone alkaline phospha- 1 (4 %), and increased in 6 (24 %) children
tase (BAP, reference range: 5.9152.3 g/L with nephrotic syndrome. Conjointly, there was
(Cavalier et al. 2010), osteocalcin (reference: a significant decline in the mean TB-BMD
range 625 ng/mL), 25(OH)D3, (reference Z-score (p < 0.05). L-BMD Z-score decreased
range: 2060 ng/mL (Dobrzanska et al. 2010), in 6 (24 %), did not change in 2 (8 %), and
1,25(OH)2D3 (reference: range 1570 pg/mL), increased in 17 (68 %) patients. The mean
and the urine calcium/creatinine ratio (uCa/Cr, L-BMD Z-score did not change appreciably
reference range: 0.39 for ages 35 years, 0.28 for 1 year of vitamin D therapy.
24 M. Panczyk-Tomaszewska et al.

Table 2 Markers of bone mineral density and bone beginning and 5 (20 %) children at the end of
metabolism, and corticosteroid dose in children with the study, which was an inappreciable difference.
nephrotic syndrome at the onset and after 1-year therapy
The dose of corticosteroids significantly declined
Parameter Before After 1 year p in the course of 1-year therapy (Table 2).
TB-BMD
0.2  1.3
0.7  1.3 <0.05 At the study onset, there were negative
Z-score (
3.32.4) (
3.31.9) correlations between the L-BMD Z-score and
L-BMD
0.5  1.6
0.2  1.3 ns
the serum levels of ALP and BAP (r
0.45,
Z-score (
2.9
3.4) (
3.02.6)
r
0.47, respectively; p < 0.05) (Fig. 1a, b),
Calcium 2.5  0.2 2.5  0.3 ns
(mmol/L)
and phosphorus (r
0.45) (p < 0.05). After
(2.32.8) (2.32.8)
1 year of corticosteroid therapy, L-BMD
Phosphorus 1.8  0.3 1.7  0.2 ns
(mmol/L) (1.42.3) (1.32.2)
Z-score correlated negatively with the serum
iPTH (pg/mL) 19.4  7.2 22.9  13.9 ns
OC (r
0.52; p < 0.05) and continued the
(7.640.2) (3.066.8) negatively correlation with BAP (r
0.47;
ALP (U/L) 191.8  59.3 196.7  84.7 ns p < 0.05), but correlated positively with the
(55288) (69433) serum 25OHD3 (r 0.45; p < 0.05; Fig. 1c).
BAP (g/L) 58.3  32.1 67.4  27.1 ns The TB-BMD Z-score correlated negatively
(5.0105.2) (14.5118.0) with the uCa/Cr ratio at the study onset only
OC (ng/mL) 32.4  33.7 34.3  20.3 ns (r
0.42; p < 0.05; Fig. 1d). In the multivari-
(5.7140.9) (16.258.5) ate analysis, BAP turned out the only significant
25(OH)D3 31.7  16.3 23.7  9.3 <0.05 predictor of the L-BMD Z-score (beta
0.49;
(ng/mL) (9.060.2) (10.460.0) p < 0.05).
1,25(OH)2D3 78.7  20.9 72.5  14.6 ns
(pg/mL) (41.9137.4) (49.496.7)
Urine calcium/ 0.15  0.11 0.16  0.15 ns 4 Discussion
creatinine ratio (0.040.48) (0.010.67)
Prednisone (n 23) (n 12) p < 0.05
Bone mineralization is a complex multifactorial
dose (mg/kg/ 0.7  0.5 0.4  0.6
48 h) process, influenced by genetic, hormonal, and
(0.01.8) (0.01.9)
nutritional factors during the entire lifespan. Dif-
Data are means  SD (range)
ferent studies highlighted a deleterious impact
TB-BMD total body bone mineral density, L-BMD lumbar
spine bone mineral density, iPTH intact parathormone, of corticosteroids on bone metabolism. Bio-
ALP alkaline phosphatase, BAP bone alkaline phospha- chemical markers of bone metabolism and densi-
tase, OC osteocalcin, 25(OH)D3 25-hydroxyvitamin D tometry are useful to trace the bone turnover;
(calcidiol), 1,25(OH)2D3 1,25-dihydroxycholecalciferol,
however, the interpretation of the results in chil-
n number of patients, ns nonsignificant
dren is difficult as many factors like age, gender,
pubertal stage, race, nutritional, and health status
Serum calcium, phosphorus, iPTH, ALP, may weigh in (Ambroszkiewicz et al. 2002).
BAP, and OC were inappreciably different at In the group of children examined, a decrease
the beginning and end of the therapy time. of the total body BMD Z-score without signifi-
Serum ALP activity was within the normal limit cant differences in the lumbar spine BMD
in 23 (92 %) and 20 (80 %) patients at the Z-score was found after 1 year of corticosteroid
beginning and end of the study, respectively. therapy. The BMD Z-score in the present
Eight out of the 25 (32 %) children had a serum study appeared a more sensitive parameter
level of 25(OH)D3 below the recommended than that in a study of Zaniew and Jarmolinski
20 ng/mL at both onset and end of therapy. The (2012). However, the group described by those
mean serum level of 25(OH)D3 decreased signifi- authors was more heterogeneous, as it included
cantly (p < 0.05) after a 1-year therapy, while glomerulopathies other than the idiopathic
that of 1,25(OH)2D3 remained unchanged. The nephrotic syndrome, and only 65 % of children
uCa/Cr ratio was elevated in 4 (16 %) at the received vitamin D supplementation. Zaniew and
Markers of Bone Metabolism in Children with Nephrotic Syndrome Treated with. . .
Fig. 1 Examples of correlations between bone density
markers, biochemical parameters and markers of bone
turn-over (a) ALP, (b) BAP, (c) 25(OH)D3, (d) urine Ca/
Creat. ratio. ALP alkaline phosphatase, BAP bone alkaline
phosphatase, 25(OH)D3 25-hydroxycholecalciferol, uCa/
Jarmolinski (2012) found no differences in the
lumbar spine BMD Z-score in the course of cor-
ticosteroid therapy; the finding coinciding with
that of the present study. Gulati et al. (2003)
reported a high prevalence of bone deficit
(38 %) in children with nephrotic syndrome
supplemented with vitamin D. Moreover, in the
prospective studies of Gulati et al. (2005) and
Bak et al. (2006) supplementation with 200 and
400 IU/day of vitamin D did not prevent bone
loss in children with nephrotic syndrome. In our
group of patients, the serum level of the active
metabolite of vitamin D (25(OH)D3) was signifi-
cantly decreased and stayed below the
recommended level in 32 % of children after
1 years therapy. Moreover, the level of vitamin
D did not increase despite supplementation with
800 IU/day. Our results are consistent with an
25
Cr urine calcium/creatinine ratio, TB-BMD bone mineral
density of total body, L-BMD lumbar spine bone mineral
density, Z-score a statistical measurement of a scores
relationship to the mean in a group of scores
observation of Zaniew and Jarmolinski (2012)
who found that as much as 89 % of children
with nephrotic syndrome are vitamin D deficient.
We also showed that even a dose as high as
800 IU/24 h of vitamin D might be insufficient
to optimize vitamin D status and to prevent a
decrease in BMD in pediatric patients with
nephrotic syndrome. We found that the bone
mineral density was related with vitamin D
level; a similar relation is also present in general
population (Fujiyoshi et al. 2013). This observa-
tion is a consequence of the role of vitamin D in
the bone metabolism. However, vitamin D level
correlated significantly only with the lumbar
spine BMD Z-score after 1 years therapy with
corticosteroid.
The present findings make us to submit that
vitamin D dosing ought to be individualized in
26
nephrotic syndrome children on corticosteroid
therapy, based on a regular evaluation of the 25
(OH)D3 serum level. We found a negative corre-
lation between L-BMD and the bone formation
markers ALP, BAP, and OC. Such a constella-
tion of results is typical for the metabolically
overactive bone tissue, called spongy bone. The
TB-BMD, a compact bone marker, correlated
with the urinary Ca/Cr ratio. Kosan et al. (2012)
analyzed the influence of corticosteroid therapy
on bone mineralization in 21 children at the
onset of steroid-sensitive nephrotic syndrome.
The study evaluated BMD and markers of bone
turnover before and after 4 and 12 weeks of
therapy and found that ALP, BAP, and Ca/Cr
ratio increased, whereas BMD significantly
decreased in the course of therapy. Our finding
of a link between BMD and bone turnover
markers somehow differs from those of other
studies. We did not observe a negative correla-
tion of PTH with BMD, reported by others in the
general population (Fujiyoshi et al. 2013; Arabi
et al. 2012; Khaw et al. 1992). We found that
iPTH was within the normal range, which
opposes the notion that low bone mineral density
is unanimously associated with hyperparathy-
roidism. The discrepancy may possibly be
explained by the influence of corticosteroids on
bone metabolism. These drugs induce osteoclas-
togenesis mediated through enhanced signaling
from the osteoblast receptor, an activator of the
nuclear factor kappaB; the process independent
of PTH (Freundlich et al. 2004).
Osteocalcin (OC) is a non-collagenous protein
of bone matrix, and it is a good marker of bone
formation and a sensitive indicator of inhibitory
effects of corticosteroids (Demiaux et al. 1992;
Kotowicz et al. 1990). The studies in adults have
shown that a single oral dose of 2.5 mg predni-
sone exerts an almost immediate effect on the
serum OC level (Saag et al. 2003). Biyikli
et al. (2004) found a negative influence of long-
term corticosteroid therapy on the OC level in
children with nephrotic syndrome, but the
authors failed to evaluate bone mineral density.
In our present group of patients, osteocalcin was
related significantly to bone mineral density.
M. Panczyk-Tomaszewska et al.
This finding is in line with that of Csakvary
et al. (2013) who revealed a positive correlation
between osteocalcin and both TB-BMD and
L-BMD in a group of 84 prepubertal children.
In contrast, in the CARDIA study, bone turnover
biomarkers were not appreciably associated with
osteocalcin or BMD, except from the left arm
BMD, in 319 healthy adults aged 2436
(Fujiyoshi et al. 2013). These inconsistent results
indicate that further clinical evaluation exploring
the link between osteocalcin and the risk of oste-
oporosis is required.
Bone alkaline phosphatase (BAP) is a protein
produced by osteoblasts and is commonly con-
sidered to be the most specific marker of bone
turnover (Eastell and Hannon 2008). In the pres-
ent study, we found a negative correlation
between BAP and BMD, as it also occurs in the
general population (Fujiyoshi et al. 2013; Rogers
et al. 2000; Ross and Knowlton 1998). In our
study group, BAP was the only significant pre-
dictor of BMD in the multivariate analysis,
which suggests that this marker remains a valu-
able tool in children with the nephrotic syndrome
treated with corticosteroids. We suggest that the
assessment of BAP should be included into the
routine evaluation paradigm of the nephrotic
syndrome.
5 Conclusions
Children suffering from the nephrotic syn-
drome treated with corticosteroids are at risk
of bone mass loss.
Serum bone alkaline phosphatase seems a
good indicator of spongy bone metabolism in
this syndrome.
Children should be supplemented with vita-
min D during corticosteroid therapy of the
nephrotic syndrome in an adjustable dose,
possibly higher than 800 IU/day to prevent
osteopenia.
Conflicts of Interest The authors declare no conflict of
interest in relation to this article.
Markers of Bone Metabolism in Children with Nephrotic Syndrome Treated with. . .
References
Ambroszkiewicz J, Gajewska J, Laskowska-Klita T
(2002) Serum osteocalcin and bone alkaline phospha-
tase in relation to age and gender. Med Wieku Rozwoj
6:257265
Arabi A, Baddoura R, El-Rassi R, El-Hajj Fuleihan G
(2012) PTH level but not 25 (OH) vitamin D level
predicts bone loss rates in the elderly. Osteoporos Int
64:971980
Bak M, Serdaroglu E, Guclu R (2006) Prophylactic
calcium and vitamin D treatments in steroid-treated
children with nephrotic syndrome. Pediatr Nephrol
21:350354
Biyikli NK, Emre S, Sirin A, Bilge I (2004) Biochemical
bone markers in nephrotic children. Pediatr Nephrol
19:869873
Canalis E, Pereira RC, Delany AM (2002) Effects of
glucocorticoids on the skeleton. J Pediatr Endocrinol
Metab 15(Suppl 5):13411345
Canalis E, Mazzioti G, Giustina A, Bilezikian JP (2007)
Glucocorticoidinduced osteoporosis: pathophysiol-
ogy and therapy. Osteoporos Int 18:13191328
Cavalier E, Rozet E, Carlisi A, Bekaert AC, Rousselle O,
Hubert P, Chapelle JP, Delanaye P (2010) Analytical
validation of serum bone alkaline phosphatase
(BAP OSTASE) on Liaison. Clin Chem Lab Med
48:6772
Csakvary V, Puskas T, Oroszlan G, Lakatos P, Kalman B,
Kovacs GL, Toldy E (2013) Hormonal and biochemi-
cal parameters correlated with bone densitometric
markers in prepubertal Hungarian children. Bone
54:106112
Demiaux B, Arlot ME, Chapuy MC, Meunier PJ, Delmas
PD (1992) Serum osteocalcin is increased in patients
with osteomalacia: correlations with biochemical and
histomorphometric findings. J Clin Endocrinol Metab
74:11461151
Dobrzanska A, Charzewska J, Chlebna-Soko D,
Chybicka A, Czech-Kowalska J, Helwich E, Imiela
JR, Karczmarewicz E, Ksiazyk JB, Lewinski A, Lorenc
RS, Lukas W, ukaszkiewicz J, Marcinowska-
Suchowierska E, Milanowski A, Milewicz A,
Pudowski P, Pronicka E, Radowicki S, Ryzko J,
Socha J, Szczapa J, Weker H (2010) Prophylaxis of
vitamin D deficiency Polish recommendations 2009.
Pol Merkur Lekarski 28:130133
Eastell R, Hannon RA (2008) Biomarkers of bone health
and osteoporosis risk. Proc Nutr Soc 67:157162
Feber J, Gaboury I, Ni A, Alos N, Arora S, Bell L, Blydt-
Hansen T, Clarson C, Filler G, Hay J, Hebert D,
Lentle B, Matzinger M, Midgley J, Moher D,
Pinsk M, Rauch F, Rodd C, Shenouda N,
Siminoski K, Ward LM, Canadian STOPP Consor-
tium (2012) Skeletal findings in children recently
initiating glucocorticoids for the treatment of
nephrotic syndrome. Osteoporos Int 23:751760
27
Freundlich M, Jofe M, Goodman WG, Salusky IB (2004)
Bone histology in steroid-treated children with
non-azotemic nephrotic syndrome. Pediatr Nephrol
19:400407
Fujiyoshi A, Polgreen LE, Hurley DL, Gross MD,
Sidney S, Jacobs DR Jr (2013) A cross-sectional asso-
ciation between bone mineral density and parathyroid
hormone and other biomarkers in community-
dwelling young adults: the CARDIA study. J Clin
Endocrinol Metab 98:40384046
Gulati S, Godbole M, Singh U, Gulati K, Srivastava A
(2003) Are children with idiopathic nephrotic syn-
drome at risk for metabolic bone disease? Am J Kid-
ney Dis 41:11631169
Gulati S, Sharma RK, Gulati K, Singh U, Srivastava A
(2005) Longitudinal follow-up of bone mineral den-
sity in children with nephrotic syndrome and the role
of calcium and vitamin D supplements. Nephrol Dial
Transplant 20:15981603
Hidalgo AA, Trump DL, Johnson CS (2010) Glucocorti-
coid regulation of the vitamin D receptor. J Steroid
Biochem Mol Biol 121:372375
Hoppe B, Kemper MJ (2010) Diagnostic examination of
the child with urolithiasis or nephrocalcinosis. Pediatr
Nephrol 25:403413
ISKDC A report of the International Study of Kidney
Disease in Children (1978) Nephrotic syndrome in
children: prediction of histopathology from clinical
and laboratory characteristics at time of diagnosis.
Kidney Int 13:159165
KDIGO Clinical Practice Guideline for Glomerulonephri-
tis (2012) Chapter 3: steroid-sensitive nephrotic syn-
drome in children. Kidney Int Supp 2:163171
Khaw KT, Sneyd MJ, Compston J (1992) Bone density
parathyroid hormone and 25-hydroxyvitamin D
concentrations in middle aged women. Br Med J
305:273277
Kosan C, Ayar G, Orbak Z (2012) Effects of steroid
treatment on bone mineral metabolism in children
with glucocorticoid-sensitive nephrotic syndrome.
West Indian Med J 61:627630
Kotowicz MA, Hall S, Hunder GG, Cedel SL, Mann KG,
Riggs BL (1990) Relationship of glucocorticoid dos-
age to serum bone Gla-protein concentration in
patients with rheumatologic disorders. Arthritis
Rheum 33:14871492
Leonard MB, Feldman HI, Shults J, Zemel BS, Foster BJ,
Stallings VA (2004) Long term, high-dose gluco-
corticoids and bone mineral content in childhood
glucocorticoid-sensitive nephrotic syndrome. N Engl
J Med 351:868875
Lettgen B, Jeken C, Reiners C (1994) Influence of steroids
medication on bone mineral density in children with
nephrotic syndrome. Pediatr Nephrol 8:667670
Mishra OP, Meena SK, Singh SK, Prasad R, Mishra RN
(2009) Bone mineral density in children with steroid-
sensitive nephrotic syndrome. Pediatr Nephrol
76:12371239
28
Rogers A, Hannon RA, Eastell R (2000) Biochemical
markers as predictors of rates of bone loss after meno-
pause. J Bone Mineral Res 15:13981404
Ross PD, Knowlton W (1998) Rapid bone loss is
associated with increased levels of biochemical
markers. J Bone Mineral Res 13:297302
Saag KG (2003) Glucocorticoid-induced osteoporosis.
Endocrinol Metab Clin North Am 32:135157
Sasaki N, Kusano E, And oY, Nemoto J, Iimura O,
Ito C, Takeda S, Yano K, Tsuda E, Asano Y (2002)
M. Panczyk-Tomaszewska et al.
Changes in osteoprotegerin and markers of bone
metabolism during glucocorticoid treatment in
patients with chronic glomerulonephritis. Bone
30:653658
Zaniew M, Jarmolinski T (2012) Vitamin D status and
bone density in steroid-treated children with
glomerulopathies: effect of cholecalciferol and cal-
cium supplementation. Adv Med Sci 57:8893
Advs Exp. Medicine, Biology - Neuroscience and Respiration (2015) 9: 2934
DOI 10.1007/5584_2014_21
# Springer International Publishing Switzerland 2014
Published online: 14 October 2014

Endurance Training and the Risk

of Bronchial Asthma in Female
Cross-Country Skiers

A. Zebrowska, B. Guchowska, D. Jastrzebski,

A. Kochanska-Dziurowicz, A. Stanjek-Cichoracka, and I. Pokora

Abstract
Exercise is one of the crucial factors responsible for asthma development
and exacerbation. The purpose of the present study was to assess the risk
of bronchial asthma in female athletes. Spirometric evaluations and phys-
ical exercise test were performed and exhaled nitric oxide (eNO) levels
were measured in 12 female elite cross-country skiers. Serum
concentrations of interleukin 1 beta (IL-1), tumor necrosis factor-alpha
(TNF-), and interleukin 6 (IL-6) were measured in all subjects before
exercise, immediately after it, and after 15 min of recovery. Peak eNO
values were 18.7
4.8 (ppb) and did not confirm the risk of early
bronchial asthma symptoms. A graded exercise test caused significant
increases in TNF- and IL-1 concentration (p < 0.05) after 15 min of
recovery. A significant negative correlation was found between resting
and post-exercise eNO and IL-6 levels (p < 0.01). Our study did not
confirm an increased risk of bronchial asthma or respiratory tract inflam-
matory conditions among female cross-country skiers exposed to physical
exertion.

Keywords
Athletes Exercise training Lung function Nitric oxide Skiers

A. Zebrowska (*), B. Guchowska, and I. Pokora

Department of Physiological and Medical Sciences,
The Jerzy Kukuczka Academy of Physical Education,
72A, Mikoowska St., 40-065 Katowice, Poland
e-mail: olazebrowska@yahoo.com
D. Jastrzebski
Department of Lung Diseases and Tuberculosis, Medical
University of Silesia, Zabrze, Poland
A. Kochanska-Dziurowicz and A. Stanjek-Cichoracka
Department of Isotope Diagnostics and
Radiopharmaceuticals, Medical University of Silesia,
Sosnowiec, Poland
1 Introduction
Asthma is a chronic inflammatory disease of the
lower respiratory tract. It is characterized by
bronchial hyperresponsiveness to various stimuli
inducing reversible airway obstruction and
bronchospasm. Chronic inflammation can be
caused by an allergen (atopic asthma), infection,
physical exertion, and several unknown factors
(non-atopic asthma). According to previous

29
30
research, physical exertion is one of the crucial
factors provoking the development or exacerba-
tion of asthma (Poon et al. 2012; Parsons and
Mastronarde 2009). In some asthmatics, exercise
may be the only symptom-provoking factor or
it may induce bronchospasm. Increased inci-
dence of asthma is often observed among endur-
ance athletes, frequently exposed to unfavorable
weather conditions. Low temperature, low air
humidity in combination with very high exercise
intensity may cause damage to the airways or
mixed neutrophilic-eosinophilic inflammation.
This accounts for a high rate of asthma morbidity
among cross-country skiers (Elers et al. 2011).
The precise mechanism responsible for
initiating of exercise-induced asthma (EIA) is
still not well understood, but it has been
suggested that the cooling of the airway mucosa
may be important (Anderson and Kippelen 2012;
Haahtela et al. 2008). Other factors that may
influence bronchial hyperresponsiveness include
water loss and its potential to change the osmotic
environment of the airways, chronic exposure to
poorly ventilated wax rooms in Nordic sport, and
exposure to high NO2 concentrations in ice
arenas (Rundell et al. 2000). It should be noted
that athletes often report asthma symptoms while
exercising; they seldom have symptoms at
rest (Elers et al. 2011). A lot of evidence has
indicated that baseline and functional spirometry
performed under laboratory conditions has a low
sensitivity to identify EIA, particularly in cold
weather athletes (Miller et al. 2005; Rundell
et al. 2000, 2004). Therefore, in recent years
a number of investigations have focused on
the relationships between training intensity, fre-
quently under extreme weather conditions, and
the risk of early symptoms of bronchial asthma in
athletes (Anderson and Kippelen 2012; Elers
et al. 2011; Grob et al. 2008).
Exhaled nitric oxide (eNO) measurement is a
non-invasive test which can be used for diagnosis
and monitoring of bronchial asthma as well as
seasonal allergic rhinitis and chronic obstructive
pulmonary disease (Lim and Mottram 2008;
De Gouw et al. 2001; Alving et al. 1993).
Thus, eNO measurement along with the evalua-
tion of pro-inflammatory cytokines might help
A. Zebrowska et al.
assess the risk of inflammation in the lower
respiratory tract. Asthma causes imbalance
between pro- and anti-inflammatory cytokines
resulting in a chronic inflammatory condition.
Interleukin 1 beta (IL-1) and tumor necrosis
factor-alpha (TNF-) are the main overexpressed
pro-inflammatory cytokines which attract and
activate neutrophils through chemotaxis (Berry
et al. 2007). They also stimulate phagocytic cells
and NADPH oxidase which, in turn, catalyzes
reactive oxygen species (ROS) generation.
Increased ROS induce interleukin 6 (IL-6)
which stimulates inflammatory processes but
also, in negative feedback manner, inhibits
TNF- and IL-1 secretion. Therefore, IL-6 acts
as both pro- and anti-inflammatory cytokine.
Cytokines and exposure to allergens and oxidants
increase the expression of inducible nitric oxide
synthase (iNOS), leading to NO production
(Chung and Barnes 1999; Robbins et al. 1994).
NO is a highly reactive molecule expressed by
bronchial epithelial cells, blood vessel endothe-
lium, type 2 pneumocytes, and nerve endings.
It has a beneficial effect on the respiratory tract
(Grob et al. 2008). However, in bronchial asthma,
excessive iNOS-derived NO and ROS intensify
the inflammatory process, increase small vessel
permeability as well as mucus secretion, damage
bronchial epithelium cells, and compromise oxy-
gen uptake by type 2 pneumocytes (Lim and
Mottram 2008; Renauld 2001). As cytokines and
NO are among the most active asthma mediators,
in the present study we attempted to establish
whether intensive exercise training in a cold
environment could affect the level of these
biomarkers in the blood. The aim of the study
also was to assess the risk of bronchial asthma in
a group of female cross-country skiers on the
basis of NO concentration in exhaled air.
2 Methods
2.1 Subjects
The study was approved by the Ethics
Committee of The Jerzy Kukuczka Academy of
Physical Education in Katowice, Poland and was
Endurance Training and the Risk of Bronchial Asthma in Female Cross-Country Skiers
Table 1 Somatic characteristic of the subjects
Variables (n 12) Women (n 12)
Height (m) 1.6
0.1
Weight (kg) 56.6
5.4
BMI (kg/m2) 21.4
1.7
PBF (%) 18.2
3.5
BFM (kg) 9.8
1.9
FFM (kg) 46.3
4.2
Values are means
SD
BMI body mass index, PBF percentage of body fat, BFM
body fat mass, FFM fat-free mass
conducted in a group of 12 elite female cross-
country skiers, aged 22.3
2.1 years. The
participants were members of the Polish biathlon
and cross-country skiing teams and had been
training for approximately 7.6
2.1 years.
Somatic characteristics of the subjects are
presented in Table 1.
2.2 Exercise Protocols
Prior to the study, the participants were training
for 6 weeks in cold or hypobaric hypoxic
conditions (2,015 m above sea level). The
investigations were carried out after the prepara-
tory phase of the annual training cycle. A few
days before the examination, the subjects were
asked to abstain from strenuous exercise and
were put on a standardized normocaloric diet.
All subjects were evaluated in the laboratory
(ambient conditions: 21  C, 60 % relative humid-
ity) after an overnight fast. Blood samples were
taken from the cubital vein in the morning at rest
(between 8:00 and 9:00 a.m.), immediately after
exercise and after 15 min of post-exercise recov-
ery. The level of pro-inflammatory cytokines was
measured by Enzyme-Linked Immunosorbent
Assay kit (ELISA) (Quantikine; R&D System,
MN). Exhaled nitric oxide measurement
was performed using HYPAIR eNO apparatus
(MediSoft, Leeds, UK). The obtained eNO
values (ppb) were compared with normal ranges
(Dweik et al. 2011). The following spirometry
parameters were measured: vital capacity (VC),
forced vital capacity (FVC), forced expiratory
volume in 1 s (FEV1), mid-expiratory phase
31
(FEF2575), and maximal voluntary volume
(MVV). Baseline eNO measurements and spi-
rometry were obtained from each athlete
(Lungtest; MES, Cracow, Poland). Each subject
then performed graded treadmill exercise test
(HP Cosmos; Nussdorf-Traunstein, Germany).
Pulmonary ventilation (VE) and oxygen uptake
(VO2) were measured continuously from minute
6 before the test and at different phase of the
exercise using an Oxycon Pro apparatus (Jaeger,
Hoechberg, Germany).
2.3 Statistical Evaluation
Descriptive statistics were performed to analyze
the data. The results were presented as means

SD. Correlations between maximal oxygen
uptake (VO2max), VE, eNO, and, additionally,
between eNO and TNF-, IL-1, and IL-6 were
verified. Repeated measures analysis of variance
was used to determine any significant changes in
post-exercise cytokines compared with resting
levels thereof. Differences were considered sta-
tistically significant at p < 0.05. The Statistica
package ver. 10 (StatSoft Poland, 10.0) was
used for data processing and analyses.
3 Results
The analysis of the somatic indices demonstrated
low body fat mass and body mass index (BMI);
the latter was however within low-normal range.
Half of the study participants had the percent-
age of body fat (PBF) below the normal range
(1724 %). All subjects exhibited a high fat-free
mass index (FFM) (Table 1).
The measurement of eNO was performed
to assess the risk of early bronchial asthma
symptoms. All investigated female athletes
exhibited eNO levels below 25 ppb, which
falls within normal range (Dweik et al. 2011;
Grob et al. 2008) (Table 2). The levels of
pro-inflammatory cytokines TNF-, IL-1, and
IL-6 in blood samples obtained at rest, immedi-
ately after exercise, and after 15 min of recovery
period are presented in Table 3. Resting levels of
32 A. Zebrowska et al.

Table 2 Exhaled nitric oxide measurement, lung func- 15-min post-exercise recovery (p < 0.05)
tion indices, and maximal oxygen uptake in female (Table 3). A significant negative correlation
athletes (n 12)
was revealed between eNO (mL/s) and IL-6
Variable levels at rest (r 0.66), and immediately
eNO (ppb) 18.7
4.8 after the exercise test (r 0.62) (p < 0.01).
eNO (mL/s) 45.7
2.4 The skiers demonstrated a high level of aerobic
FVC (%pred) 112.5
6.3
capacity as confirmed by high VO2max and
FEV1 (%pred) 108.6
6.8
VEmax (Table 2). The mean VC was within nor-
PEF (L/s) 7.0
1.0
mal range (4.4
0.3 L and 108 % of predicted)
(FEF2575) (L/s) 4.9
0.7
in all females examined. The mean value of
FEV1/VC (%) 89.6
4.6
MVV (L/min) 136.9
25.4
forced expiratory volume in 1 s (FEV1), peak
VO2max (mL/kg/min) 54.9
3.0
expiratory flow (PEF), forced expiratory flow of
VEmax (L/min) 109.6
15.3 2575 % (FEF2575), and maximal voluntary
Values are means
SD
ventilation (MVV) exceeded the physiological
eNO exhaled nitric oxide, FVC forced vital capacity, range for healthy population (Table 2). No sig-
FEV1 forced expiratory volume in 1 s, PEF peak expira- nificant correlation was found between baseline
tory flow, FEF2575 forced expiratory flow of 2575 %, eNO and spirometric variables or eNO and
FEV1/VC Tiffeneau index, MVV maximal voluntary ven-
tilation, VO2max maximal oxygen uptake, VEmax maximal
VO2max (p > 0.01).
ventilation

Table 3 Cytokine levels at rest, immediately after 4 Discussion

graded maximal exercise (Max) and after a 15-min recov-
ery period (Rec) (n 12) Chronic inflammation of the airways may lead to
Variable Rest Max Rec Ex% adverse changes in the structure of the bronchial
TNF- 2.3
1.0 5.2
4.7* 5.3
2.6* 187.7 walls and cause significant impairment of the
(pg/mL) respiratory system function (Poon et al. 2012).
IL-1 1.1
0.9 0.9
0.3 2.2
1.6* 168.8 Therefore, early diagnosis and proper medical
(pg/mL)
treatment seem to be crucial (Popov 2011).
IL-6 1.1
0.4 1.4
0.4 1.4
0.6 30.7
(pg/mL) Nowadays, the diagnosis of bronchial asthma
Values are means
SD. Ex% percent change from
is based on a detailed interview, spirometry,
baseline to recovery and bronchial hyperresponsiveness examination
*p < 0.05 significant difference between Max vs. Rest (Porsbjerg et al. 2009; Miller et al. 2005). A
and Rec vs. Rest reduction in forced expiratory flow at 2575 %
of the pulmonary volume, with otherwise normal
interleukins IL-1 and IL-6 were comparable spirometry, might indicate early allergic or
(1.1
0.9 vs. 1.1
0.4 pg/ml). These levels inflammatory involvement of the airways. Moni-
were low as also was that of TNF-, which is in toring of asthma activity includes the measure-
accord with other data reported in endurance- ments of PEF, FEV1, and the assessment of
trained subjects (Larsen et al. 2002; Suzuki use of inhaled medications (2-mimetics). How-
et al. 2002). Maximal graded exercise led to a ever, these parameters do not allow sufficient
significant increase in TNF- compared with the estimation and prediction of asthma course in
resting level (p < 0.05). TNF- level remained athletes. Traditional assessment of exercise-
increased after a 15-min recovery period. Post- induced asthma requires the measurement of
exercise IL-6 concentrations were also increased, FEV1 before and after exercise, or a surrogate
but the differences between pre- and post- of exercise such as eucapnic voluntary hyperpnea
exercise levels were not significant. Interestingly of dry air or mannitol dry powder. Laboratory
enough, the level of IL-1 decreased during exer- spirometry testing has been shown to have a low
cise and then increased significantly during a sensitivity to detect exercise-induced asthma,
Endurance Training and the Risk of Bronchial Asthma in Female Cross-Country Skiers
and exercise testing in the field can be challeng-
ing, particularly in athletes from winter sport
disciplines (Anderson and Kippelen 2012;
Rundell et al. 2000).
A growing body of evidence supports the
utility of the concentration of NO in the exhaled
air at a constant exhalation flow as a marker of
inflammation in asthma (Porsbjerg et al. 2009;
Kharitonov et al. 1994, 1995; Alving et al. 1993).
Therefore, our study aimed to evaluate eNO
exchange in female cross-country skiers. The
findings demonstrate that endurance training
positively affected functional capabilities of the
respiratory system. Both spirometry and eNO
data clearly indicate that female biathlon and
cross-country skiing athletes were rather unlikely
to develop inflammatory conditions in the lower
respiratory tract. The literature data show a high
incidence of bronchial asthma among swimmers,
ice-hockey players, and cross-country skiers
(Elers et al. 2011; Haahtela et al. 2008; Miller
et al. 2005). Cross-country skiers seem to be the
most exposed to extreme weather conditions,
which causes strong and repeated hyperventila-
tion. Hyperventilation-induced heat and water
loss promotes an increase in airway osmolarity.
This may lead to inflammation onset. Changes in
eNO during and after exercise raised hopes of
finding a noninvasive, more accurate method for
the evaluation of exercise-induced inflammatory
conditions within the respiratory tract. It has
been found that increased eNO in asthma is due
to an upregulation of one or more of NOS
isoforms and extension of NO-producing cells
in the airways (Lim and Mottram 2008; Robbins
et al. 1994). Much evidence suggests that epithe-
lial cells express inducible NOS in response
to both pro-inflammatory cytokines, including
TNF-, IL-1, and interferon- (IFN-), and
reactive oxygen species (Berry et al. 2007;
Chung and Barnes 1999; Kharitonov
et al. 1995). Since intensive physical exercise is
associated with systemic cytokine increases to
the levels observed during severe infections,
it has been hypothesized that exercise training,
particularly in cold environment, increases NO
production.
eNO values of cross-country female skiers
indicated a low risk of developing bronchial
33
asthma. Nonetheless, normal amounts of NO in
exhaled air do not exclude asthma, and especially
its non-atopic form. As NO is a nonspecific
inflammatory mediator, we also decided to assess
the levels of pro-inflammatory cytokines. Recent
studies have demonstrated that strenuous exer-
cise induces an increase in pro-inflammatory
cytokines secretion leading to increase suscepti-
bility to infections (Suzuki et al. 2002; Pedersen
et al. 1998). However, a decrease in cytokine
production resulting from regular training has
also been documented (Petersen and Pedersen
2005; Larsen et al. 2002). Noticeable adapta-
tion to physical exertion was also observed in
the present study as the female skiers were
characterized by low levels of cytokines after
exercise and recovery period. The main inducers
of acute phase reactions are the proinflammatory
cytokines TNF- and IL-1. The majority of stud-
ies have shown that the circulating level of these
cytokines is not significantly changed following
exercise. Small changes observed in these
cytokines could be mediated by anti-inflammatory
cytokines, such as IL-6 and IL-10, or by cytokine
inhibitors. Increased cytokine secretion is
observed during high intensity physical exercise
inducing micro-damages to muscle fibers. We did
observe a significant increase in TNF- level
immediately and shortly after a high intensity
exercise test (Ex 187.7 %). Plasma IL-1
levels increased significantly after exercise
(Ex 168.8 %) during a 15-min post-exercise
recovery. This might have been caused by an
increase in anti-inflammatory cytokines or high
exercise levels of TNF-, which, according to
numerous studies, stimulates the production of
IL-1. This finding could also confirm a decrease
in the athletes body response to the exercise test.
Negative correlation seen between the level of
IL-6 at rest and immediately after graded maxi-
mal exercise and respective eNO concentrations
might be associated with IL-6 exhibiting both
pro-and anti-inflammatory activities.
There are some limitations to this study.
Firstly, we did not perform repeated measure-
ments of exercise-induced eNO levels. However,
in healthy subjects, exercise increases NO
elimination due to increased ventilation rates.
Secondly, our study was carried out in the
34
laboratory which could have influenced the
inflammatory response in a different way than
there is during strenuous exercise training in the
field. Nevertheless, there is a need for further
research to clarify the relationship between
eNO and the level of cytokines during sports
competitions.
5 Conclusions
In conclusion, the present study failed to
confirm an increased risk of bronchial asthma
or respiratory tract inflammation among female
cross-country skiers. Nevertheless, it should be
emphasized that the evaluation of exhaled nitric
oxide and pro-inflammatory cytokines might still
prove to be a good adjunct tool for diagnosing
bronchial asthma or respiratory tract inflamma-
tion in professional athletes.
Conflicts of Interest The authors declare no conflicts of
interest in relation to this article.
References
Alving K, Weitzberg E, Lundberg JM (1993) Increased
amount of nitric oxide in exhaled air of asthmatics.
Eur Respir J 6(9):13681370
Anderson SD, Kippelen P (2012) Assessment and
prevention of exercise-induced bronchoconstriction.
Br J Sports Med 46:391396
Berry M, Brightling C, Pavord I, Wardlaw A (2007)
TNF-alpha in asthma. Curr Opin Pharmacol
7(3):279282
Chung KF, Barnes PJ (1999) Cytokines in asthma. Thorax
54(9):825857
De Gouw HW, Marshall-Partridge SJ, Van Der Veen H,
Van Den Aardweg JG, Hiemstra PS, Sterk PJ (2001)
Role of nitric oxide in the airway response to exercise
in healthy and asthmatic subjects. J Appl Physiol
90:586592
Dweik RA, Boggs PB, Erzurum SC, Irvin CG, Leigh
MW, Lundberg JO, Olin AC, Plummer AL, Taylor
DR (2011) An official ATS clinical practice guideline:
interpretation of exhaled nitric oxide levels (eNO) for
clinical applications. Am J Respir Crit Care Med
184:602615
Elers J, Pedersen L, Backer V (2011) Asthma in elite
athletes. Expert Rev Respir Med 5(3):343351
Grob NM, Laskowski D, Dweik RA (2008) A technical
report on exhaled nitric oxide measurement: asthma
monitoring in athletes. J Breath Res 2(3):37027.
doi:10.1088/1752-7155/2/3/037027
A. Zebrowska et al.
Haahtela T, Malmberg P, Moreira A (2008) Mechanisms
of asthma in Olympic athletes-practical implications.
Allergy 63(6):685694
Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R,
Shinebourne EA, Barnes PJ (1994) Increased nitric
oxide in exhaled air of asthmatic patients. Lancet
343:133135
Kharitonov SA, Yates D, Barnes PJ (1995) Increased
nitric oxide in exhaled air of normal human subjects
with upper respiratory tract infections. Eur Respir J
8:295297
Larsen AI, Lindal S, Aukrust P, Toft I, Aarsland T,
Dickstein K (2002) Effect of exercise training on
skeletal muscle fiber characteristics in men with
chronic heart failure. Correlation between skeletal
muscle alterations, cytokines and exercise capacity.
Int J Cardiol 83(1):2532
Lim KG, Mottram C (2008) The use of fraction of
exhaled nitric oxide in pulmonary practice. Chest
133:12321242
Miller MG, Weiler JM, Baker R, Collins J, DAlonzo G
(2005) National Athletic Trainers Association posi-
tion statement: management of asthma in athletes.
J Athl Train 40(3):224245
Parsons JP, Mastronarde JG (2009) Exercise-induced
asthma. Curr Opin Pulm Med 15(1):2528
Pedersen BK, Rohde T, Ostrowski K (1998) Recovery of
the immune system after exercise. Acta Physiol Scand
162(3):325332
Petersen AMW, Pedersen BK (2005) The
ant-inflammatory effect of exercise. J Appl Physiol
98(4):11541162
Poon AH, Eidelman DH, Martin JG, Laprise C, Hamid Q
(2012) Pathogenesis of severe asthma. Clin Exp
Allergy 42(5):625637
Popov TA (2011) Human exhaled breath analysis. Ann
Allergy Asthma Immunol 106:451456
Porsbjerg C, Lund TK, Pedersen L, Backer V (2009)
Inflammatory subtypes in asthma are related to airway
hyperresponsiveness to mannitol and exhaled NO.
J Asthma 46(6):606612
Renauld JC (2001) New insights into the role of cytokines
in asthma. J Clin Pathol 54:577589
Robbins RA, Barnes PJ, Springall DR, Warren JB, Kwon
OJ, Buttery LDK, Wilson AJ, Geller DA, Polak JM
(1994) Expression of inducible nitric oxide synthase in
human bronchial epithelial cells. Biochem Biophys
Res Commun 203:209218
Rundell KW, Wilber RL, Szmedra L, Jenkinson DM,
Mayers LB, Im J (2000) Exercise-induced asthma
screening of elite athletes: field versus laboratory exer-
cise challenge. Med Sci Sports Exerc 32(2):309316
Rundell KW, Anderson SD, Spiering BA, Judelson DA
(2004) Field exercise vs. laboratory eucapnic volun-
tary hyperventilation to identify airway hyper-
responsiveness in elite cold weather athletes. Chest
125(3):909915
Suzuki K, Nakaji S, Yamada M, Totsuka M, Sato K,
Sugawara K (2002) Systemic inflammatory response
to exhaustive exercise. Cytokine kinetics. Exerc
Immunol Rev 8:648
Advs Exp. Medicine, Biology - Neuroscience and Respiration (2015) 9: 3543
DOI 10.1007/5584_2014_20
# Springer International Publishing Switzerland 2014
Published online: 24 September 2014

Effects of Inspiratory Muscle Training

on Resistance to Fatigue of Respiratory
Muscles During Exhaustive Exercise

M.O. Segizbaeva, N.N. Timofeev, Zh.A. Donina,

E.N. Kuryanovich, and N.P. Aleksandrova

Abstract
The aim of this study was to assess the effect of inspiratory muscle training
(IMT) on resistance to fatigue of the diaphragm (D), parasternal (PS),
sternocleidomastoid (SCM) and scalene (SC) muscles in healthy humans
during exhaustive exercise. Daily inspiratory muscle strength training was
performed for 3 weeks in 10 male subjects (at a pressure threshold load of 60 %
of maximal inspiratory pressure (MIP) for the first week, 70 % of MIP for the
second week, and 80 % of MIP for the third week). Before and after training,
subjects performed an incremental cycle test to exhaustion. Maximal inspira-
tory pressure and EMG-analysis served as indices of inspiratory muscle
fatigue assessment. The before-to-after exercise decreases in MIP and centroid
frequency (fc) of the EMG (D, PS, SCM, and SC) power spectrum (P < 0.05)
were observed in all subjects before the IMT intervention. Such changes were
absent after the IMT. The study found that in healthy subjects, IMT results in
significant increase in MIP (+18 %), a delay of inspiratory muscle fatigue
during exhaustive exercise, and a significant improvement in maximal work
performance. We conclude that the IMT elicits resistance to the development
of inspiratory muscles fatigue during high-intensity exercise.

Keywords
Diaphragm EMG Inspiratory muscle fatigue Parasternal Scalene
Sternocleidomastoid

M.O. Segizbaeva (*)

Laboratory of Respiration Physiology, I.P. Pavlov
Institute of Physiology, Russian Academy of Science,
6 Makarova nab., Saint Petersburg 199034, Russia
e-mail: marina@infran.ru 1 Introduction
N.N. Timofeev and E.N. Kuryanovich
Department of Natural Sciences, Military Sport Institute, Inspiratory muscle fatigue develops during high-
63, Bolshoy Sampsonievskyi Pr, Saint Petersburg 194353,
Russia intensity exercise in healthy human (Segizbaeva
et al. 2013; Romer and Polkey 2008) and may
Z.A. Donina and N.P. Aleksandrova
I.P. Pavlov Institute of Physiology, Russian Academy limit maximal work performance (Perlovitch
of Science, Saint Petersburg, Russia et al. 2007; Mador and Acevedo 1991),

35
36 M.O. Segizbaeva et al.

especially during loaded breathing (Segizbaeva Table 1 Baseline characteristics of the subjects
and Aleksandrova 2009) or with a backpack load IMT-group Control-group
(Nadiv et al. 2012). Electromyographic (EMG) Anthropometrics
signs of diaphragmatic fatigue develop in normal Age (years) 19.8  0.6 19.4  0.6
subjects hyperventilating above 70 % of maximal (1823) (1822)
voluntary ventilation as well as in chronic Body height (cm) 176.3  2.3 179.1  1.9
(162186) (169183)
obstructive pulmonary disease (COPD) patients
Body weight (kg) 75.3  2.2 78.1  2.4
(Fitting 1991). Numerous studies have
(6586) (6788)
investigated the effect of specific inspiratory BMI (kg.m 2) 24.2  0.5 24.8  0.7
muscle training (IMT) on inspiratory muscle (21.226.5) (21.027.4)
strength and endurance, respiratory function, Pulmonary function
and exercise performance in healthy subjects FVC (l) 5.0  0.2 4.8  0.3
(Illi et al. 2012; Enright and Unnithan 2011; (4.05.6) (4.25.7)
Verges et al. 2007), and COPD (Decramer FVC (%pred.) 94.6  2.7 93.4  3.1
(85106) (81111)
2009; Hill et al. 2006) and chronic heart failure
FEV1 (l) 4.5  0.3 4.6  0.3
(CHF) patients (Suh-Jen Lin et al. 2012). How- (3.15.6) (3.35.7)
ever, results have been mixed due to differences FEV1 (%pred.) 96.7  1.3 97.5  1.4
in training programs, experimental protocols, (80101) (78106)
and subject selection. Controversy and debate FEV1/FVC (%) 94.7  0.3 96.1  0.4
still exist regarding the mode and intensity of (89107) (84111)
training required to result in improvements in PEF (l/s) 8.0  0.6 7.8  0.8
(5.813.0) (5.912.1)
respiratory function and maximal work perfor-
PEF (%pred.) 89.7  4.2 91.9  3.2
mance. It has been established that inspiratory (79123) (79112)
muscle training intensities lower than 40 % of HbO2Sat (%) 98.5  0.3 98.6  0.2
maximal effort do not translate into quantitative (97100) (98100)
functional outcomes (Enright and Unnithan Values are means  SE (range)
2011). Training intensities of more than 50 % BMI body mass index, FVC forced vital capacity, FEV1
of maximal inspiratory pressure positively influ- forced expired volume in 1 s, PEF peak expiratory flow,
HbO2Sat percutaneous O2 saturation of hemoglobin in
ence exercise capacity in healthy human, COPD, arterialized blood
and CHF patients, but their effects on fatigue
resistance of different inspiratory muscles are
still unknown. In the present study we examined study. Each subject was familiarized with the
the benefits of a short program of incremental experimental procedures and protocol and gave
IMT on inspiratory muscle function and fatigue informed consent to participate in the study.
resistance of diaphragm (D), parasternal (PS), Ten healthy, nonsmoking, moderately trained
sternocleidomastoid (SCM), and scalene (SC) male subjects underwent IMT, while the other
muscles during exhaustive exercise in healthy six served as a control group. The baseline
humans. Respiratory function and electromyog- characteristics of the subjects are summarized
raphy (EMG) served as markers of exercise- in Table 1. There were no significant differences
induced inspiratory muscles fatigue. in anthropometric data, lung function para-
meters, and inspiratory muscle capacity between
the groups. Subjects were free of cardio-
2 Methods respiratory diseases. They were involved in
endurance and power sports and performed low-
The study was performed in accordance with the to-moderate intensity physical activity five times
ethical standards of the Helsinki Declaration for a week. They were requested to keep their indi-
Human Experimentation. A local Committee on vidual physical activity constant 2 weeks prior
Human Research approved the protocol of the to, and throughout the course of the study; except
Effects of Inspiratory Muscle Training on Resistance to Fatigue of. . .
for 2 days before the tests during which they
were requested to refrain from physical activity.
Also, they were asked to refrain from drinking
caffeinated beverages on the test day and to have
last meal at least 2 h prior the test.
2.1 Design Overview
All measurements were performed before the
IMT and within 1 week after its completion.
After familiarization with testing procedures,
subjects underwent pulmonary function testing
and performed an incremental exercise test to
voluntary exhaustion. The maximal inspiratory
pressure (MIP) was measured before and after
the exercise test. Ten subjects performed specific
inspiratory muscle training on a daily basis for
3 weeks, while the other 6 subjects served as a
control group.
2.2 Pulmonary Function and
Incremental Exercise Test
Pulmonary function test was performed with an
ergospirometry-computerized device using a
calibrated turbine for volume measurement
(Schiller AG, Baar, Switzerland) in a sitting posi-
tion. The forced expiratory volume in 1 s (FEV1),
forced vital capacity (FVC), and peak expiratory
flow (PEF) were recorded according to the
recommendations of the ATS/ERS Statement
(2002). Exercise test was performed on an elec-
tronically braked cycle ergometer (Schiller AG,
Baar, Switzerland) using a standard (2-min)
incremental cycle exercise protocol. After
5 min of breathing at rest while seated on the
bicycle, subjects started with a 2-min period of
pedaling with a workload 1 W/kg at 6070
cycles/min. Then, workload was automatically
increased by 0.5 W/kg every 2 min until the
subject was no longer able to maintain the pedal-
ing frequency at a constant level (not <60 per
min). The subjects were strongly encouraged to
cycle until exhaustion was reported.
37
2.3 Ventilatory Parameters
Minute ventilation and its components were
measured continuously using an ergospirometry
device (Schiller AG, Baar, Switzerland) during
the incremental exercise test. Concentration of
expired O2 and CO2 was measured breath-
by-breath with the same device by using para-
magnetic (O2) and infrared absorption (CO2)
gas analyzers. These measures and flow signals
were electronically integrated by a computerized
system to yield 10-s averages of minute ventila-
tion (VE), tidal volume (VT), respiratory rate
(RR), oxygen consumption (VO2 ), CO2 produc-
tion (VCO2), gas exchange ratio (RER), end-tidal
CO2 pressure (PETCO2), and end-tidal O2 pressure
(PETO2). Calibrations were performed before each
test. The heart rate was also measured and used to
obtain the O2-pulse. Arterial hemoglobin oxygen
saturation (SaO2), was monitored noninvasively
using a pulse oximeter ONYX 9500 (Nonin
Medical Inc., Plymouth, MN).
2.4 Maximal Inspiratory Pressure
Measurements
The MIP was measured with a portable device
(Powerbreathe International Ltd., Southam,
Warwickshire, UK) in accordance with ATS/
ERS Statement (2002). MIP was recorded at the
mouth during a quasi-state short maximal inspi-
ration against occluded airways (Mullers
maneuver). The maneuver was performed at
residual volume (RV) (Troosters et al. 2005).
Each participant was instructed to expire slowly
and completely as far as possible and then to
perform a maximal inspiratory effort. The subjects
were verbally encouraged by the operators to
achieve a maximal result. To prevent closure of
the glottis and to avoid significant pressure gener-
ation by the muscles of the cheek, a small leak was
present in the equipment. The subjects performed
a minimum of five maneuvers until two maximal
pressure values were obtained which did not
differ by more than 5 %; the higher of the two
was chosen for analysis. The maneuvers were
38
performed in the sitting position before and
immediately after exercise test. For the sake of
convenience, MIP was expressed in positive
values.
2.5 Surface EMG Recording
Surface EMGs of D, PS, SCM, and SC muscles
were obtained with surface electrocardiographic
electrodes 5 mm in diameter (ARBO, TYCO
Healthcare Group LP, Germany). The EMG of
D was recorded by electrodes applied to the skin
on the anterior axillary line at the level of the
eighth intercostal space (Verin et al. 2002).
The EMG of SCM was obtained with electrodes
positioned longitudinally over the middle third
of the muscle on the right side of the neck, while
the PS EMG was recorded with electrodes placed
in the second right intercostal space close to the
sternum. The EMG of SC was obtained from
electrodes placed in the posterior triangle of
the neck (right side) at the level of the cricoid
cartilage. The place was located during sniff
maneuvers through palpation of the neck in the
lower third of a line drawn between the middle
of the mastoid process and the sternal notch.
Within each electrode pair, the inter-electrode
distance was <2 cm. Impedance was decreased
by careful skin shaving and abrasion with alco-
hol. The wires connected to the electrodes were
carefully secured with tape to minimize move-
ment artifacts. All the EMGs were amplified and
continuously recorded on a six-channel recorder
(Biograph, Russia). EMGs were displayed and
visualized simultaneously. All the data were
stored on PC for future analysis. To quantify the
EMG, the signals were filtered (101,000 kHz)
and integrated on a moving time-average basis
with a time constant of 150 ms. The peak ampli-
tude of integrated EMG was measured for each
inspiratory muscle during valid Mullers maneu-
ver before and after exercise test. The peak
integrated EMG activity was measured in arbi-
trary units and then expressed as a percentage of
the value, reached during Mullers maneuver
before exercise test (control). A fast Fourier trans-
form of EMG was used to compute the power
M.O. Segizbaeva et al.
spectral density. The power spectra were
quantified in terms of the centroid frequency
(fc) (Hary et al. 1982). The fc was measured in
Hz and then expressed as a percentage of the
value reached during inspiration before exercise
test, taken for 100 % (control).
2.6 Training Program
The IMT was performed using an electronic
pressure threshold inspiratory-muscle training
device (Powerbreathe KH1, UK). The IMT
group performed 60 dynamic inspiratory
maneuvers once a day (a.m. session) at a pressure
threshold load of 60 % of individual baseline
MIP for the first week, 70 % of MIP for the
second week, and 80 % of MIP for the third
week, seven times per week. This protocol has
never previously been used to study
improvements in inspiratory muscle function.
Subjects in the control (CON) group performed
a sham-training program, which consisted of
30 breaths, once daily (a.m. session) for 3 week
at the minimum resistive setting; this protocol
has been shown to exhibit no changes in inspira-
tory muscle function (Romer et al. 2002).
Subjects were instructed to begin each inspira-
tory effort from residual lung volume and to
continue until total lung capacity. Breathing fre-
quency during maneuver was reduced to prevent
hyperventilation and hypocapnia. After a session
with 30 inspiratory efforts, a 5 min rest period
followed. Compliance to training was assessed
by monitoring the cumulative number of breaths
completed using a pressure sensor. An inspira-
tory effort was registered when the negative pres-
sure generated during inspiration exceed the set
point on the pressure switch. IMT was performed
in the laboratory and operators supervised each
session.
2.7 Data Analysis
Data are presented as means SE. To assess
the development of respiratory muscle fatigue
during exhaustive exercise, absolute values of
Effects of Inspiratory Muscle Training on Resistance to Fatigue of. . .
maximal inspiratory pressures and centroid
frequency (fc) of power spectra before and after
exhaustive exercise were analyzed at baseline
and after IMT. Differences between post-
exercise MIP as well as fc were compared with
data in the pre-exercise (control, taken for
100 %). For statistical analysis a paired t-test
and Wilcoxons matched pairs test were applied.
All statistical analyses were performed using
standard statistical software (Origin 6.1).
P < 0.05 was considered statistically significant.
3 Results
3.1 Subject Characteristics
and Pulmonary Function
There were no significant differences in age,
weight, BMI, and fitness status among the
control and IMT groups at baseline. Moreover,
there were no differences in MIP, and pulmonary
function variables during exercise before IMT
program (Tables 1 and 2). No changes were
observed in any measure of pulmonary function
in the control group after-training. However,
FVC, FEV1, and PEF tended to increase in the
IMT-group by 8.0, 4.5, and 9.2 % respectively.
3.2 Incremental Cycling Test
Respiratory responses were compared between
pre- and post-training incremental cycle test in
IMT and CON groups. The IMT group showed
a significant increase in maximal work perfor-
mance, PWC170, anaerobic threshold, minute
ventilation and its time-volume parameters,
and in VO2 and VCO2 after IMT intervention
(Table 2). The maximal work performance
during the incremental cycle test remained
unchanged in the CON-group after sham-IMT,
no differences were observed in any cardiopul-
monary variables collected at maximal work rate.
MIP was expressed as a percentage of the
maximal values achieved during Mullers maneu-
ver before exercise test. Peak amplitude of EMGs
was expressed as a percentage of the maximal
39
values achieved during Mullers maneuver before
exercise test. NS non-significant. * P < 0.05 in
comparison to control values.
3.3 Inspiratory Muscle Function
and EMG Analysis
MIP and EMG analyses of D, PS, SCM, and SC
muscles served as markers for the assessment of
inspiratory muscle fatigue. The before-to-after
exercise decrease in MIP pressure was found in
a baseline trial (Table 2). An average drop
in MIP was about 8 % (p > 0.05) in both IMT
and CON groups. Post-exercise values of MIP
tended to decrease in all subjects from the IMT
group despite the peak magnitude of integrated
electrical activity of D, PS, SCM, and SC during
post-exercise Mullers maneuver being signifi-
cantly greater than that pre-exercise in all
subjects. As illustrated in Fig. 1A (bottom
panel), post-exercise Mullers maneuver pro-
duced greater-than-control amplitudes of D, PS,
SCM, and SC in order to provide lower values
of MIP. This fact might be evidence of contrac-
tile inspiratory muscle fatigue after incremental
exercise to exhaustion in normal humans.
Frequency spectrum analysis of the EMG can
detect diaphragmatic fatigue reliably, prior to
the time when the diaphragm fails as a pressure
generator (Gross et al. 1979), as well as other
accessory inspiratory muscles do. The before-to-
after exercise decrease in centroid frequency (fc)
of EMG (D, PS, SCM, and SC) power spectrum
(p < 0.05) was observed in all subjects before
the IMT intervention (Fig. 2) and these changes
may reflect the development of inspiratory
muscle fatigue during heavy exercise.
The IMT increased MIP by 18 % (p < 0.05)
(Table 2). The change in MIP was observed in
all subjects from the IMT, but not CON, group.
To overcome the load required by pressure
threshold training, the subjects demonstrated an
increase in major and accessory muscles activity,
represented by D, PS, SCM, and SC (Fig. 3). All
these muscles were involved in training process,
which led to an improvement in their strength.
Moreover, the peak magnitude of integrated
40 M.O. Segizbaeva et al.

Table 2 Rest and maximal incremental exercise data before-to-after IMT

Before After IMT

Variables Rest Max. exercise Rest Max. exercise
Wmax (W) 248.4  8.1 291.7  12.1*
(194292) (246374)
VE (lmin 1) 6.9  0.6 83.7  5.4 8.2  0.8 107.6  5.8*
(4.211.5) (61120) (4.014.1) (77137)
VT (l) 0.5  0.1 2.5  0.3 0.5  0.1 2.8  0.2
(0.40.9) (1.84.4) (0.40.8) (2.03.9)
fb (breathsmin 1) 13.8  3.9 34.6  1.5 14.2  0.9 40.7  5.7
(8.020.7) (29.344.9) (12.622.1) (31.962.7)
VO2 (lmin 1) 0.3  0.0 3.3  0.2 0.3  0.0 3.7  0.2*
(0.20.4) (2.14.4) (0.20.4) (3.04.5)
VO2 (mlkg 1min 1) 3.5  0.2 39.4  2.5 4.1  0.4 48.9  1.3*
(2.54.5) (28.552.5) (2.55.1) (41.354.0)
VCO2 (lmin 1) 0.2  0.0 3.5  0.2 0.2  0.0 4.1  0.2*
(0.20.3) (2.44.8) (0.10.4) (3.35.0)
VCO2 (mlkg 1min 1) 2.9  0.3 45.9  2.0 3.2  0.3 54.9  2.07*
(2.14.4) (35.656.4) (2.54.4) (38.658.4)
RR 0.7  0.0 1.1  0.0 0.8  0.0 1.1  0.0
(0.60.8) (1.11.1) (0.60.8) (1.01.2)
HR (beatsmin 1) 72.1  3.7 178.7  4.8 64.8  2.4 179.4  5.1
(5484) (159198) (5478) (147210)
O2-pulse (mlbeat 1) 3.1  0.3 19.0  1.3 4.5  0.6 25.9  3.1
(2.14.2) (13.025.6) (2.46.2) (17.546.5)
PETCO2 (mmHg) 33.4  4.0 43.1  2.3 32.8  1.1 42.8  1.6
(27.641.4) (27.951.7) (26.238.1) (34.049.2)
PETO2 (mmHg) 102.0  2.0 109.5  2.2 105.6  1.3 113.7  2.5
(95.8113.0) (101.4123.3) (99.8113.6) (104.2131.2)
RWC170 (W) 229.1  14.8 281.6  16.3*
(181253) (199344)
RWC170 (Wkg 1) 3.3  0.1 3.7  0.1*
(2.74.1) (3.04.3)
AT (W) 219.4  11.3 236.8  17.3*
(148273) (160360)
MIP (cmH2O) 128.4  6.5 119.2  6.4 151.1  10.1* 150.0  11.0
(104197) (95194) (111206) (107214)
Values are means SE
Wmax maximal work performance, VE minute ventilation, VT tidal volume, fb breathing frequency, VO2 oxygen
consumption, VCO2 carbon dioxide production, RR respiratory exchange ratio, HR heart rate, O2-pulse oxygen pulse,
PETCO2 end-tidal CO2, PETO2 end-tidal O2, RWC170 work capacity at 170 beats min 1, AT anaerobic threshold
*p < 0.05 compared with baseline

electrical activity of D, PS, SCM, and SC during

post-exercise Mullers maneuver was unchanged
or significantly lower than that pre-exercise in
the IMT-group the after training program. A
shift toward a decrease in fc of power spectra
after exhaustive exercise was significantly
lower after IMT compared with baseline. These
data are presented in Figs. 1B and 2 and point to
an increase in inspiratory muscle resistance to
fatigue after IMT intervention.
4 Discussion
We evaluated the effects of a 3-week
(21 sessions, 60 inspiratory efforts/day, 7 days/
week) incremental pressure threshold IMT pro-
gram on the inspiratory muscle strength and
exercise-induced inspiratory muscle fatigue in
ten trained male subjects. The IMP increased by
18 % in the IMT group, whereas no changes were
Effects of Inspiratory Muscle Training on Resistance to Fatigue of. . .
Fig. 1 Changes in the mean maximal inspiratory
pressure (top rows) and mean peak amplitudes of the
integrated EMG of the diaphragm (D), parasternal (PS),
Fig. 2 Changes in centroid frequency (fc) of the dia-
phragm (D), parasternal (PS), sternocleidomastoid
(SCM), and scalene (SC) muscles after exhaustive
observed in the control subjects after sham train-
ing. It is in agreement with the majority of other
studies (Enright and Unnithan 2011). On the
basis of EMG analysis, we were able to observe
early signs of the fatiguing process in the D, PS,
SCM, and SC muscles after intensive exercise
to exhaustion in IMT and CON groups before
training. One possible reason for the shift in
the centroid frequency of the power spectrum
toward lower frequencies may be a decrease in
the rate of conducting action potential in muscle
fibers (Gross et al. 1979). This is a contractile
fatigue, which may be related to impaired
excitation-contraction coupling, due possibly to
altered Ca2+ exchange (Edwards et al. 1977).
41
sternocleidomastoid (SCM), and scalene (SC) muscles
during Mullers maneuver (bottom rows) before (Panels
A) and after (Panels B) inspiratory muscle training (IMT)
exercise before and after inspiratory muscle training
(IMT). fc was expressed as a percentage of the mean
values achieved before exercise test
Deceleration of conduction is an initial sign of
metabolic changes in the muscle, associated with
an accumulation of lactic acid and a reduction
of intracellular pH. No significant changes in
MIP and a shift in fc toward lower frequencies
were observed before-to-after exhaustive exer-
cise after IMT training in the IMT group,
whereas both MIP and fc decreased in control
subjects after sham training. The improvements
may be associated with a decrease in blood
lactate concentrations during high-intensive
exercise in trained individuals (Brown et al.
2008). The improvement in inspiratory muscle
function may result in an increase of maximal
work performance. Despite an increase in the
42
Fig. 3 Typical pneumotachogram (PTG), mouth
inspiratory pressure (PmI) and EMG recordings of the
diaphragm (D), intercostal parasternal (PS), sternoclei-
domastoid (SCM), and scalene (SC) muscles during inspi-
ratory training efforts
post-training maximal work performance in
the IMT-group, the development of D, PS,
SCM, and SC muscle fatigue was significantly
reduced in this group after exhaustive exercise.
The physiological mechanisms by which inspi-
ratory muscle training improves exercise
performance remain controversial. The inspira-
tory muscles are morphologically and function-
ally skeletal muscles; thus, they should respond
to training in the same way as any locomotor
muscle if an appropriate physiological load
is applied (Kraemer et al. 2002). It is well
known that skeletal muscles have a high capacity
to adapt to increases in loading, including the
respiratory muscles. Numerous studies have
demonstrated that the number of capillaries,
activity of oxidative enzymes, and the number
of mitochondria are all elevated in the activated
M.O. Segizbaeva et al.
muscles after a period of interval and endurance
training (Taylor and Bachman 1999). High-
intensity interval training results in an increase
in aerobic capacities of skeletal muscle as a result
of expansion of skeletal muscle mitochondria,
as assessed by cytochrome c oxidase activity
(Jacobs et al. 2013). It is possible to suppose
that increases in mitochondrial content after
IMT may provide an improvement in the aerobic
capacity of respiratory muscle and contribute to
maximal work performance increase, delaying
the moment of reaching anaerobic threshold dur-
ing exercise. It has been shown that increases in
both strength and endurance of inspiratory
muscles after IMT in COPD patients might be
related to adaptive structural changes
of respiratory muscles. This improvement is
reported to be associated with a 38 % increase
in the proportion of type I fibers and in the size of
type II fibers (21 %) in the external intercostal
muscles. No such changes have been observed
in the control muscle (Ramirez-Sarmiento
et al. 2002).
We conclude that inspiratory muscle training
increases the resistance of respiratory muscles to
the development of fatigue during high-intensity
exercise and by doing so, improves maximal
work performance in healthy subjects.
Conflicts of Interest The authors declare no conflicts of
interest in relation to this article.
References
ATS/ERS (2002) American Thoracic Society/European
Respiratory Society statement on respiratory muscle
testing. Am J Respir Crit Care Med 166:518624
Brown PI, Sharpe GR, Johnson MA (2008) Inspiratory
muscle training reduces blood lactate concentration
during volitional hyperpnoea. Eur J Appl Physiol
104:111117
Decramer M (2009) Response of the respiratory muscles to
rehabilitation in COPD. J Appl Physiol 107:971976
Edwards RH, Hill DK, Jones DA, Merton PA (1977)
Fatigue of long duration in human skeletal muscle
after exercise. J Physiol 272:769778
Enright SJ, Unnithan VB (2011) Effect of inspiratory
muscle training intensities on pulmonary function
and work capacity in people who are healthy:
a randomized controlled trial. Phys Ther 91:894905
Effects of Inspiratory Muscle Training on Resistance to Fatigue of. . .
Fitting JW (1991) Respiratory muscle fatigue limiting
physical exercise? Eur Respir J 4:103108
Gross D, Grassino A, Ross WRD, Macklem PT (1979)
Electromyogram pattern of diaphragmatic fatigue.
J Appl Physiol 46:17
Hary D, Belman MJ, Propst J, Lewis S (1982) A statistical
analysis of the spectral moments used in EMG tests of
endurance. J Appl Physiol 53:779783
Hill K, Jenkins SC, Philippe DL, Cecins N, Shepherd KL,
Green DJ, Hillman DR, Eastwood PR (2006)
High-intensity inspiratory muscle training in COPD.
Eur Respir J 27:11191128
Illi SK, Held U, Frank I, Spengler CM (2012) Effect of
respiratory muscle training on exercise performance
in healthy individuals: a systematic review and
meta-analysis. Sports Med 42:707724
Jacobs RA, Fluck D, Bonne TC, Burgi S, Christensen PM,
Toigo M, Lundby C (2013) Improvements in exercise
performance with high-intensity interval training coin-
cide with an increase in skeletal muscle mitochondrial
content and function. J Appl Physiol 115:785793
Kraemer WJ, Adams K, Cafarelli E, Dudley GA,
Dooly C, Feigenbaum MS, Fleck SJ, Franklin B,
Fry AC, Hoffman JR, Newton RU, Potteiger J,
Stone MH, Ratamess NA, Triplett-McBride T (2002)
American College of Sports Medicine position stand.
Progression models in resistance training for healthy
adults. Med Sci Sports Exerc 34:364380
Mador MJ, Acevedo FA (1991) Effect of respiratory
muscle fatigue on subsequent exercise performance.
J Appl Physiol 70:20592065
Nadiv Y, Vachbroit R, Gefen A, Elad D, Zaretsky U,
Moran D, Halpern P, Ratnovsky A (2012) Evaluation
of fatigue of respiratory and lower limb muscles dur-
ing prolonged aerobic exercise. J Appl Biomech
28:139147
Perlovitch R, Gefen A, Elad D, Ratnovsky A, Kramer
MR, Halpern P (2007) Inspiratory muscles experience
fatigue faster than the calf muscles during treadmill
marching. Respir Physiol Neurobiol 156:6168
Ramirez-Sarmiento A, Orozco-Levi M, Guell R,
Barreiro E, Hernandez N, Mota S, Sangenis M,
43
Broquetas JM, Casan P, Gea J (2002) Inspiratory
muscle training in patients with chronic obstructive
pulmonary disease: structural adaptation and phy-
siologic outcomes. Am J Respir Crit Care Med
166:14911497
Romer LM, Polkey MI (2008) Exercise-induced respira-
tory muscle fatigue: implications for performance.
J Appl Physiol 104:879888
Romer LM, McConnell AK, Jones DA (2002) Effects of
inspiratory muscle training upon recovery time during
high intensity, repetitive sprint activity. Int J Sports
Med 23:353360
Segizbaeva MO, Aleksandrova NP (2009) Effect of oxy-
gen breathing on inspiratory muscle fatigue during
resistive load in cycling men. J Physiol Pharmacol
60(Suppl 5):111116
Segizbaeva MO, Donina ZA, Timofeev NN, Korolyov
YN, Golubev VN, Aleksandrova NP (2013)
EMG-analyses of human inspiratory muscle resistance
to fatigue during exercise. Adv Exp Med Biol
788:197205
Suh-Jen Lin PT, McElfresh J, Hall B, Bloom R, Farrell K
(2012) Inspiratory muscle training in patients with
heart failure: a systematic review. Cardiopulm Phys
Ther J 23:2936
Taylor AW, Bachman L (1999) The effects of endurance
training on muscle fibre types and enzyme activities.
Can J Appl Physiol 24:4153
Troosters T, Gosselink R, Decramer M (2005) Respira-
tory muscle assessment. In: Gosselink R, Stam H (eds)
Lung function testing, European respiratory mono-
graph 31. European Respiratory Society Journals
Ltd, Wakefield/Sheffield, pp 5771
Verges S, Lenherr O, Haner AC, Schulz C, Spengler CM
(2007) Increased fatigue resistance of respiratory
muscles during exercise after respiratory muscle
endurance training. Am J Physiol Regul Integr Comp
Physiol 292:12461253
Verin E, Straus C, Demoule A, Mialon P, Derenne JP,
Similowski T (2002) Validation of improved record-
ing site to measure phrenic conduction from surface
electrodes in humans. J Appl Physiol 92:967974
Advs Exp. Medicine, Biology - Neuroscience and Respiration (2015) 9: 4549
DOI 10.1007/5584_2014_19
# Springer International Publishing Switzerland 2014
Published online: 14 October 2014

Nutritional Status in Chronic Obstructive

Pulmonary Disease and Systemic Sclerosis:
Two Systemic Diseases Involving
the Respiratory System

D. Mekal, A. Doboszynska, E. Kadalska, E. Swietlik,

and L. Rudnicka

Abstract
This study aimed to assess and compare the nutritional status and life
quality of patients with chronic obstructive pulmonary disease (COPD)
and systemic sclerosis (SSc). Thirty patients with stable COPD and
32 patients with SSc were examined. In all patients, the following
parameters were measured: fat mass, fat-free mass, total body water,
FEV1, and blood gases. COPD patients life quality was assessed with
St. Georges Respiratory Questionnaire, and in SSc patients with a Quality
of Life Questionnaire. The results show that among COPD patients 13 %
had normal body weight, 60 % were obese, and 27 % were overweight.
In SSc patients, 59 % had normal body weight, 31 % were overweight,
1 patient was obese, and 2 were underweight. The mean life quality score
in COPD patients was 57.3
16.5, while that in SSc patients was
35.8
18.2. COPD patients had a statistically significant lower life
quality than SSc patients. The mean value of FEV1 was 45.5
12.2 %
pred. in COPD patients, and 86.8
21.2 % pred. in the SSc group. We
conclude that nutritional disorders are more frequent in COPD patients
compared to those with SSc.

Keywords
Chronic obstructive pulmonary disease Electrical bioimpedance
Nutritional status Quality of life Systemic sclerosis

D. Mekal (*) and E. Kadalska

Department of Clinical Nursing, Warsaw Medical
University, 27 Ciolka St., Warsaw 01-445, Poland
e-mail: dposluszna@o2.pl
A. Doboszynska and E. Swietlik
Department of Pulmonary Medicine, Hospital of L. Rudnicka
Pneumology, Warmia-Masuria University, Olsztyn, Department of Dermatology, Medical University of
Poland Warsaw, Warsaw, Poland

45
46
1 Background
Systemic sclerosis (SSc) and chronic obstructive
pulmonary disease (COPD) are chronic diseases
in which nutritional status has an important prog-
nostic role. Pathophysiological changes in both
these diseases differ. However, both SSc and
COPD are accompanied by chronic inflamma-
tion, which may lead to lesions in internal
organs. Undiagnosed, malnutrition has a nega-
tive effect on treatment, and results in a less
favourable outcome for patients with COPD
and SSC. Extrapulmonary manifestations of
COPD include nutritional status disorders, and a
weakening of muscular force. Previous work
divided extrapulmonary consequences of COPD
into three groups: nutritional disorders, skeletal
muscle dysfunction, and other systemic effects
(Agusti et al. 2003). Malnutrition is the most
frequent nutritional disorder in COPD patients,
especially in the advanced stage of the disease.
Moreover, 1015 % of patients with mild or
moderate COPD, and 50 % of patients with
advanced disease, have malnutrition or body
weight loss. Causes of malnutrition in COPD
are multifactorial, these include: tissue hypoxia,
aging, lack of physical activity, accelerated rest-
ing metabolism, chronic inflammatory processes,
effect of certain medicines (corticosteroids), and
a higher level of catabolism over anabolism.
However, the most common form of malnutrition
in COPD patients is protein-energy malnutrition
(PEM), characterized by low protein- and
energy-rich food intake (Odencrants et al. 2009).
Body weight and body mass index (BMI)
do not provide information on body build.
Therefore, a proper assessment of nutritional
status should include measurements of both fat
free mass (FFM) and fat mass (FM). These
measurements can be achieved with bioelectric
bioimpedance (BIA) or dual energy X-ray
absorptiometric (DEXA) techniques. FFM
measurements are used to calculate fat free
mass index (FFMI) using the following formula:
FFM kg
FFMI
height m2
D. Mekal et al.
where FFMI values over or equal to 15 kg/m2
for women, and 16 kg/m2 for men indicate a
depletion in fat free mass (Kuznar-Kaminska
et al. 2008).
The most frequent signs of malnutrition
include decreased body weight and muscle
mass (Celli et al. 2004). Increased mortality in
COPD underweight patients is attributed to loss
of FFM, which increases incidence of exacerba-
tions leading to a decrease in FEV1, and wors-
ening of the quality of life. BMI measurements
are the most popular screening technique in
malnutrition; however, it has limitations. For
instance, it does not provide information
about body build, which may be a cause of undi-
agnosed disorders. Hence, FFM is a more fre-
quently and commonly used method of the
nutritional status assessment. In addition, studies
indicate that FFM is a better predictor of mort-
ality in COPD compared with BMI, indepen-
dently of FM value (Vestbo et al. 2006).
The objective of the present study was to
assess and compare nutritional status, type of
diet, and life quality of patients with COPD
and SSc.
2 Methods
The study was approved by the Ethics Committee
of Warsaw Medical University in Warsaw,
Poland. There were two patient groups examined
in the study:
COPD group: 30 patients, 21 men (70 %) and
9 women (30 %), aged between 54 and 81 years
(median age: 66 years), with stable COPD;
SSc group: 32 patients, 28 women (88 %) and
4 men (12 %), aged between 17 and 82 years
(median age: 54.4 years), diagnosed with SSc.
Among the COPD patients, 96 % smoked
cigarettes, whereas 41 % of SSc patients smoked
in the past. Anthropometrical measurements
were performed, and body composition was
assessed using electrical bioimpedance (BIA) in
all patients. The following parameters were
measured: fat mass (FM), fat-free mass (FFM),
total body water (TBW), and basal metabolic
rate. In addition, body mass index (BMI) and
Nutritional Status in Chronic Obstructive Pulmonary Disease and Systemic. . .
fat-free mass index were also calculated. Patients
were categorized into the following groups based
on their BMI: underweight (<20 kg/m2), normal
weight (20.024.9 kg/m2), overweight
(25.029.9 kg/m ), and obese (30 kg/m2).
2
Spirometry (to measure FEV1) was also per-
formed in all patients. Finally, life quality was
assessed using St. Georges Respiratory Ques-
tionnaire (SGRQ) for COPD patients and a
Systemic Sclerodermia Quality of Life Question-
naire (SScQLI) developed specifically for SSc
patients developed by L. Rudnicka at Medical
University in Warsaw, Poland (personal commu-
nication). In both questionnaires, higher scores
denote more limitations.
Data are presented as means
SD. The
differences between the two groups of patients
were assessed with a nonparametric U Mann-
Whitney test. Spearmans r (rank correlation
coefficient) value was used to assess correlations
between the parameters measured. Statistical sig-
nificance was defined as p < 0.05.
3 Results and Discussion
Among the COPD patients, 13 % (n 4) had
normal body weight, 60 % (n 18) were obese,
27 % (n 8) were overweight, while none were
undernourished. In the SSc group, 59 % (n 19)
had normal body weight, 31 % (n 10) were
overweight, 1 was obese, and 2 were under-
weight. Body composition measurement with
BIA was contraindicated in 4 patients with SSc.
Fig. 1 Percentage of
patients falling into a
given partition of quality of
life score in chronic
obstructive pulmonary
disease (COPD) and
systemic sclerosis (SSc)
patients
47
COPD patients had a mean BMI of 31.3, while
that of SSc patients was 25.3. This difference was
statistically significant (p 0.001).
None of the COPD patients had an FFM defi-
cit; FFMI of female patients was 15 kg/m2, and
that of male patients was 16 kg/m2. In contrast,
2 (7 %) female patients with SSc had an FFM
deficit. The difference between the FFMI values
of COPD patients (mean FFMI 19.6 kg/m2)
and SSc patients (mean FFMI 14.5 kg/m2)
was statistically significant (p 0.001).
In the COPD group, FM was found to be
increased in 77 % of patients, while 16 % had
normal, and 7 % had values below normal. In the
SSc group, 43 % patients had an excess of FM,
25 % had normal values, and 22 % had an FM
deficit. Decreased TBW was found in 60 % of
COPD and 32 % SSc of patients; whereas, nor-
mal TBW values were measured in 23 % of
COPD and 54 % of SSc patients. For TBW,
above normal values were found in 17 % of
COPD and 14 % of SSc patients.
The group mean score of life quality was
57.3
16.5 points (range 26.088.5) in COPD
and 35.8
18.2 points (range 0100) in SSc
patients (p < 0.002). The difference in life qual-
ity also remained appreciable, in favor of the SSc
patients, when the questionnaires scores were
stratified into three increasing categories:
2549, 5075, and >75 points and the percent-
age of patients falling into a given category was
compared between COPD and SSc, although this
difference tended to taper off with advancing
score, i.e., with more severe conditions (Fig. 1).
48 D. Mekal et al.

Fig. 2 Correlation
between body mass index
and fat free mass index
(FFMI) in chronic
obstructive pulmonary
disease (COPD; solid linear
regression line: r 0.62,
p < 0.05) and systemic
sclerosis (SSc; dotted linear
regression line: r 0.59,
p < 0.05) patients

Physical fitness domain scores were lower in
COPD patients (65.5
19.2), as measured with
SGRQ; while in SSc patients, the lowest scores
were due to anxiety related to the prognosis and
the course of the disease. The mean values of
FEV1 were 45.5
12.2 % pred. in COPD and
86.8
21.2 % pred. in SSc patients (p < 0.001).
COPD patients showed positive correlations
between FEV1 and the following parameters:
grip strength (r 0.64, p < 0.05), FFM (%)
(r 0.48, p < 0.005), and FFM (r 0.51,
p < 0.05); whereas negative correlations were
noted between FEV1 and FM (%) (r 0.56,
p < 0.05) and life quality (total score)
(r 0.44, p < 0.05). FEV1 correlated posi-
tively with and basal metabolic rate in both
COPD and SSc patients (r 0.65, p < 0.05).
Finally, a strong positive correlation was noted
between BMI and FFMI in both patient groups
(Fig. 2).
Based on the questionnaire items concerning
changes in body weight from the time of initial
diagnosis to the time of survey, it was estimated
that body weight appreciably increased in 58 %
of COPD patients. In contrast, body weight
increased in 26 %, did not change in 45 %, and
decreased in 29 % of SSc patients. When COPD
patients were asked about the diet habits, 40 %
declared a high-caloric and 40 % a low-caloric
diet. In contrast, 83 % of SSc patients declared a
low caloric diet. Thus, it appears that the type of
disease exerts an effect on the caloric content
of diet.
4 Conclusions
Nutritional disorders were more frequent in
COPD patients compared with those suffering
from systemic sclerosis. Obesity affected 60 %
of COPD patients, with 77 % of them having fat
mass over normal values. In contrast, in the sys-
temic sclerosis group, 57 % of patients had nor-
mal body weight, with only 43 % having fat mass
above normal. Finally, life quality of COPD
patients is significantly worse than that of sys-
temic sclerosis patients.
Conflicts of Interest The authors declare no conflicts of
interest in relation to this article.
References
Agusti A, Noguera A, Sauleda J, Sala E, Pons J, Busquets
X (2003) Systemic effects of chronic obstructive pul-
monary disease. Eur Respir J 21:347360
Celli BR, MacNee W, Agusti A, ATS/ERS Task Force
(2004) Standards for the diagnosis and treatment of
patients with COPD: a summary of the ATS/ERS
position paper. Eur Respir J 23:932946
Nutritional Status in Chronic Obstructive Pulmonary Disease and Systemic. . . 49

Kuznar-Kaminska B, Batura-Gabriel BB, Kaminski J Vestbo J, Prescott E, Almdal T, Dahl M, Nordestgaard

(2008) Analysis of nutritional status disorders in
patients with chronic obstructive pulmonary disease.
Pneumonol Alergol Pol 76:327333
Odencrants S, Ehnfors M, Ehrenberg A (2009) Nutritional
status and body composition among persons with
chronic obstructive pulmonary disease. J Nursing
Health Chron Illness 1:6070
BG, Andersen T, Sorensen TI, Lange P (2006) Body
mass, fat free body mass and prognosis in patients
with chronic obstructive pulmonary disease from a
random population sample. Am J Respir Crit Care
Med 173:7983
Advs Exp. Medicine, Biology - Neuroscience and Respiration (2015) 9: 5158
DOI 10.1007/5584_2014_15
# Springer International Publishing Switzerland 2014
Published online: 26 September 2014

Gradual Versus Continuous Increase

of Load in Ergometric Tests:
Are the Results Comparable?

A.M. Preisser, M. Velasco Garrido, C. Bittner,

E. Hampel, and V. Harth

Abstract
Standard exercise testing (ET) comprises progressive exercise provocation
with cardiovascular monitoring. Exercise tolerance is estimated by work-
load. Cardiopulmonary exercise testing (CPX) is a non-invasive measure-
ment of ventilatory gas exchange which provides more accurate
quantifications of cardiorespiratory fitness (CRF). Workload is usually
increased stepwise in ET and continuously (ramp) in CPX. Our aim was
to examine the comparability of the results. Thirty two healthy volunteers
(17 females/15 males, age 26.8
6.1 years, BMI 24.5
3.0) underwent
exercise testing on a bicycle ergometer up to maximum physical exhaus-
tion; under ramp protocol (CPX) and 27 days later with a stepwise increase
of workload (ET). We compared the physical work capacity under both
methods at maximum workload, at heart rate of 150 and 170 beats/min
(PWC150 and PWC170), and the exercise duration. We found that there
were no statistically significant differences in the maximum heart rate
(CPX: 177.1
11.7/min vs. ET: 178.5
11.2/min) or maximal workload
(CPX: 219.8
50.6 vs. ET: 209.4
42.5). PWC150 and PWC150/kg were
higher with CPX than those with ET (156.6
51 vs. 146.4
42.3,
p < 0.001 and 2.1
0.5 vs. 1.9
0.4, respectively, p < 0.001). Exercise
duration was almost equal (12.1 vs. 11.3 min). We conclude that overall
physical performance was higher with CPX. Since the results are similar,
we recommend the CPX: wattage and other parameters in performance
assessment are to be determined directly, interpolations are obsolete.

A.M. Preisser (*), M. Velasco Garrido, C. Bittner, and

V. Harth
Institute for Occupational and Maritime Medicine,
University Medical Center Hamburg-Eppendorf,
Seewartenstrasse 10, 20459 Hamburg, Germany
e-mail: a.preisser@uke.de
E. Hampel
WiDi-Kontor, Hamburg, Germany

51
52
Keywords
Cardiopulmonary exercise testing Cycle ergometer Oxygen uptake
Physical work capacity Ramp protocol Standard exercise test
1 Introduction
Standard exercise testing (ET) with stepwise
progressive exercise provocation on the bicycle
ergometer in combination with serial ECGs,
hemodynamics, oxygen saturation, and subjec-
tive symptoms is a well-established technology
to diagnose and predict cardio-vascular diseases
with ECG alterations, exercise intolerance, and
dyspnea (ATS/ACCP 2003). To test physical
fitness, ET is also common, and in some coun-
tries ET is used to determine the suitability
for heavy physical activities (DGUV 2010).
Exercise tolerance must be estimated indirectly
from workload; it is estimated from the workload
achieved at a heart rate of 150 beats/min or
170 beats/min, and reported as physical work
capacity (PWC150 and PWC170).
Cardiopulmonary exercise testing (CPX) is a
non-invasive measurement of ventilatory gas
exchange during exercise test which provides
more accurate quantifications of cardiorespiratory
fitness (CRF) and in depth analyses. CPX can be
performed on a bicycle ergometer, but also on the
treadmill or with other types of load. In the medi-
cal field the bicycle ergometer is preferred.
The bicycle ergometer (mechanical tread
dynamometer) was invented by Elisee Bouny in
1896 (Prinz 1993). First experiments with addi-
tional analysis of gas metabolism date back to
Atwater and Benedict (1903). In Germany, CPX
has been studied from the early 1950s; it was
developed and optimized for sport and cardiovas-
cular research mainly by Hollmann et al. (2006)
in Cologne. At about the same time, Wasserman
et al. (1967, 2002, 2004) also have been engaged
in this technology in the U.S. At the beginning,
main obstacles were the application of the instru-
ments on a moving and sweating body, and the
correct measurement (e.g., blood pressure and
heart rate) under these circumstances. The main
A.M. Preisser et al.
objective, however, was the measurement of
oxygen uptake, inter alia, to determine the meta-
bolic parameters for the energy turnover. On a
bicycle ergometer, the wattage is given, and in
ET only the direct performance at the bicycle
crank is taken into account. Beside the wattage
on a bicycle, ergometer VO2max is increasingly
being used as a measure of physical fitness, as
well as in other forms of exercise testing such as
treadmill or step-tests (Forman et al. 2010).
CPX on a bicycle ergometer allows the deter-
mination of both workload and oxygen uptake.
CPX uses digital and computerized methods to
measure and document the parameters and to
analyze the results. The investigation method is
somewhat more complex and the technical
equipment for modern CPX is more expensive
than for ET.
CPX usually works with a continuous (ramp)
increase of the demanded physical activity on the
ergometer, while ET operates traditionally with a
stepwise increase of physical activity. The latter
is explained by the fact that in the past only
mechanically braked bicycle ergometers were
available. Both approaches are used today to
assess physical fitness and cardiac performance,
for example, in aptitude tests for firefighters or
divers as well as to determine the right time for a
heart transplant or a lung resection. In our study
we addressed the question of whether the results
of these methods are comparable.
2 Methods
The study protocol was approved by an institu-
tional Ethics Committee and the study was
performed in accordance with the Declaration of
Helsinki for Human Research. The participants
were informed about the experimental character
of the exercise tests and took part voluntarily.
Gradual Versus Continuous Increase of Load in Ergometric Tests: Are. . .
We included healthy individuals consecu-
tively seen at our outpatient department under-
going an aptitude health examination for diving.
A total of 32 subjects (17 female/15 male, age
26.8
6.1, 75.1
12.8 kg, 174.8
8.5 cm,
BMI 24.5
3.0) participated in the study. Men
and woman did not differ significantly regarding
their age (27.7
6.6 vs. 26.1
5.7) and body
mass index (BMI), but differed in height and
weight (Table 1).
The test was a precondition to participate in a
university scientific diving course. The aptitude
examination included medical history, blood
tests (blood count, erythrocyte sedimentation,
blood glucose, creatinine, gamma-GT, and
ALT), urinalysis, physical examination, spirom-
etry, body plethysmography, fitness test, visual
test, and audiometry. The volunteers did not take
any cardiac or respiratory effective medications
and had no history of cardiac or lung disease.
For the fitness test, participants had to step on
a bicycle ergometer up to the maximum physical
exhaustion. The volunteers underwent two
fitness tests, first under the terms of the CPX
and 27 days later under the standard ET
conditions. All exercise tests were carried out
on a calibrated, electronically braked cycle
ergometer which maintains a steady work rate
at variable speeds (Ergoselect 200P, Ergoline,
Bitz, Germany), supplemented with continuous
recording of the ECG and oscillometric blood
pressure measurement every 3 min. Participants
were instructed to maintain a pedaling rate of
6070 rotations per min. CPX was performed
with a comprehensive cardiopulmonary breath-
Table 1 Demographic data and predicted values
All (n 32)
Age 26.8
6.1
Height (cm) 174.8
8.5
Weight (kg) 75.1
12.8
BMI (kg/m2) 24.5
3.0
Days between CPX and ET 2.5
2.2
Reference values (means)
Max. heart rate predicted (bpm) 190.6
Wattage predicted (W) 208.3
VO2 max-predicted (ml/min) 2638.4
Values are means
SD
53
by-breath and intra-breath testing system
(Oxycon Pro, CareFusion, Hochberg, Germany).
The continuous CPX started with a warm-up
period of 2 min with an external workload of
25 W followed by an increase of 1215 W/min,
performed in very small steps of 4 or 5 W every
20 s. The stepwise increase of workload in the
standard exercise test included also 2 min
warming up with 25, 50, or 75 W followed by a
gradual rise with 25 W every 2 min, according to
the recommendations for occupational health
checks of the German Statutory Accident Insur-
ance (DGUV 2010). Stress duration of 812 min
should be achieved. Both tests were terminated
primary by physical exhaustion; in this respect
the maximum heart rate was not relevant, but
was recorded.
The measurements included exercise dura-
tion, initial and maximum workload, PWC150,
PWC170; the latter three related to kg bodyweight
(kgBW). CPX also allowed a continuous record-
ing of oxygen uptake (VO2), carbon dioxide
output (VCO2), and respiratory exchange rate
(RER). Furthermore, pulmonary ventilation
with respiratory frequency, tidal volume, and
flow-volume curve was recorded. The anaerobic
threshold (AT) was non-invasively determined
by a combination of V-slope and ventilatory
equivalent for oxygen (VE/VO2).
As the reference values for maximum
heart rate, we used the formula 220 minus age,
for maximum load the formulas Wmax
kgBW*310 % for every age decade over
30 years (men), and Wmax kgBW*2.510 %
for every age decade over 30 years (women)
Male (n 15) Female (n 17)
27.7
6.6 26.1
5.7
181.6
6.5 168.7
4.4
82.8
10.4 68.2
10.8
25.1
2.7 23.9
3.2
3.5
1.9 2.8
2.4
191.3 189.9
267.9 155.7
3289.0 2064.4
54
(Reiterer 1975). For maximum VO2 we used the
reference values of Wasserman et al. (2004) and
Hansen et al. (1984). Differences in individual
parameters and comparisons with the reference
values were analyzed using a paired t-test.
P < 0.05 was defined as statically significant.
3 Results
3.1 Comparison of ET and CPX
All participants achieved a comparable maximum
heart rate under both test conditions (CPX:
177.1
11.7/min; ET: 178.5
11.2/min). None
of them suffered immoderately about excessive
dyspnea or reported chest pain. With CPX, the
output was about 10 W higher than with ET (219.8

50.6 vs. 209.4
42.5, p < 0.001) as well as
the PWC150 and the PWC150/kg were higher
(156.6
51 vs. 146.4
42.3, p < 0.001 and
2.1
0.5 vs. 1.9
0.4, respectively,
p < 0.001). Four participants showed the same
performance with both methods. Twenty subjects
reached a higher wattage (520 W) by CPX, eight
were between 5 and 55 W worse off than under
ET conditions. According to PWC150, a similar
distribution was observed: five subjects achieved
Fig. 1 Maximum wattage (a) and physical work
capacity at a heart rate of 150 beats/min (PWC150)
(b) under continuous increase (CPX) (ordinate) and step-
wise increase (ET) (abscissa) of load on bicycle
A.M. Preisser et al.
better results under ET conditions; 4 subjects
performed equally in both settings. Figures 1a, b
show a comparison of the maximum wattage and
PWC150, respectively, achieved under CPX (ordi-
nate) and ET (abscissa). Here it becomes clear that
the slight difference in performance is due to a
better capacity. Values for men and women are
displayed separately in Fig. 2a, b; showing that a
higher wattage is achieved with CPX by men.
The PWC170 and the PWC170/kg was determined
only in 28 cases, because four subjects did not
reach the heart rate of 170 bpm. In these subjects,
PWC170 also differed about 10 W (195.5
53.3
vs. 179.8
48.3, p < 0.005 and 2.6
0.5 vs.
2.4
0.5, p < 0.005).
The exercise duration was almost equal for
both test protocols (12.1 vs. 11.3 min) and
corresponded to the recommended time (Meyer
et al. 2013; Balady et al. 2010) (Table 2).
3.2 Comparison by Gender
and with Reference Values
The results of the ET and the CPX with gas
measurement are presented in Table 2. We found
no significant difference in the maximum heart
rate between the males and females in both tests
ergometer; each symbol represents one subject; the solid
line is a linear regression trend and dashed line is the
identity line as visual reference
Gradual Versus Continuous Increase of Load in Ergometric Tests: Are. . . 55

Fig. 2 Maximum wattage (a) and physical work capac- load on a bicycle ergometer. Each symbol represents one
ity at a heart rate of 150 beats/min (PWC150) (b) for men subject; the solid line is a linear regression trend and dashed
(rhombs) and women (triangles) under continuous increase line is the identity line as a visual reference. Men achieved
(CPX) (ordinate) and stepwise increase (ET) (abscissa) of a better performance with CPX

Table 2 Results of cardiopulmonary exercise tests with standard stepwise (ET) and continuous (CPX) increase
of physical workload
All (n 32) Male (n 15) Female (n 17)
ET
Max. heart rate (bpm) 178.5
11.2 177.3
11.0 179.6
11.5
Max. wattage (W) 209.4
42.5 240.0
35.1 182.4
27.6
Max. Watt/kg of body weight (W/kg) 2.8
0.5 2.9
0.5 2.7
0.4
PWC150 (W) 164.4
42.3 173.3
39.5 122.6
28.7
PWC150/kg (W/kg) 1.9
0.4 2.1
0.5 1.8
0.4
PWC170 (*n 28) (W) 179.8
48.3 210.4
48.2 156.9
34.5
PWC170/kg (*n 28) (W/kg) 2.4
0.5 2.5
0.5 2.4
0.5
Start wattage (W) 71.1
25.5 86.7
24.8 57.4
17.1
Exercise duration (min) 12.1
2.6 12.9
2.4 11.4
2.7
Difference max. wattag/pred. wattage 0.8
0.4 0.6
0.4 0.9
0.4
CPX
Max. heart rate (bpm) 177.1
11.7 176.6
10.6 177.6
12.9
Max. wattage (W) 219.8
50.6 259.0
39.5 185.1
29.5
Max. Watt/kg of body weight (W/kg) 2.9
0.5 3.1
0.5 2.7
0.5
PWC150 (W) 156.6
51.0 194.0
42.8 123.5
31.2
PWC150/kg (W/kg) 2.1
0.5 2.4
0.5 1.9
0.4
PWC170 (an 28) (W) 195.5
53.3 232.3
45.4 163.7
36.9
PWC170/kg (an 28) (W/kg) 2.6
0.5 2.8
0.5 2.5
0.5
Exercise duration (min) 11.3
2.0 11.8
2.0 10.9
2.0
Max VO2 (ml/min) 2810.4
737.5 3348.4
625.4 2335.7
448.1
Max VO2/kg (ml/min/kg) 37.5
7.4 40.7
7.3 34.6
6.5
Difference max. VO2/pred. VO2 +172.0
498.7 +59.4
574.5 +271.4
413.1
Difference max. wattage/pred. wattage +11.4
39.9 8.9
43.5 +29.4
26.5
VO2 at AT (% pred. VO2 max) 72.1
23.6 69.4
24.7 74.6
23.1
Heart rate at AT (bpm) 139.1
21.5 135.1
21.3 142.9
21.7
Difference max. wattage ET/CPX (W) 10.4
18.1 19.0
16.7 2.8
16.1
Values are means
SD
a
4 subjects did not reach the heart rate of 170 bpm
56
(ET: 177.3
11.0 vs. 179.6
11.5; CPX:
176.6
10.6 vs. 177.6
12.9, respectively),
but there were significant gender differences
in the maximum wattage (ET and CPX) and
the maximum O2 uptake (CPX) with mostly
higher performance values for men. The
mean values for body mass-related wattage
(W/kgBW) was also slightly higher in men
(ET: 2.9
0.5 vs. 2.7
0.4; CPX: 3.1
0.5
vs. 2.7
0.5, p < 0.01). Accordingly, PWC150
and PWC170 were also higher in men. Likewise,
females achieved lower body mass-related O2
uptake in CPX (VO2/kgBW) (40.7
7.3 vs.
34.6
6.5).
Overall, the participants showed no signifi-
cant differences in the predicted values for
maximal heart rate. However, women achieved
a significantly better wattage compared with the
predicted values (185.1
29.5 vs. 155.7 W
pred., p < 0.001). This phenomenon was also
seen for VO2, although differences compared
with the reference values were less pronounced
(2335.7
448.1 vs. 2064.4 ml/min pred.,
p < 0.05). Men showed only a slightly better
performance of oxygen uptake than predicted
(3348.4
625.4 vs. 3289.0 ml/min pred.).
However, the wattage here was below the
selected reference value (240.0
35.1 vs.
267.9 W pred.).
3.3 Oxygen Uptake at the Anaerobic
Threshold
For the entire group, oxygen uptake at the
anaerobic threshold was with 72.1
23.6 of
the predicted VO2 max. within the normal
range. In a separate analysis of men and
women, oxygen uptake of 69.4
24.7 and
74.6
23.1, respectively, the endurance per-
formance was within the normal range for both
sexes and there were no significant differences.
However, there was a difference in heart rate at
the anaerobic threshold (males 135.1
21.3 vs.
females 142.9
21.7).
A.M. Preisser et al.
4 Discussion
Comparing the two types of exercise testing,
the measured physiological values differed
only slightly. Higher maximum wattages were
achieved more frequently with the CPX, espe-
cially by men. This is explainable, since this
peak power must be held for a shorter time than
under ET conditions. Also, the PWC values were
slightly, although significantly, higher in CPX.
The wattage in CPX is determined directly,
interpolation is obsolete. With the VO2 value,
the CPX allows an additional performance
assessment.
The exercise testing and maximum output are
usually determined by the maximum heart rate
achieved, adjusted to predicted values. PWC also
uses this construct. There is little intraindividual
variation in the maximum heart rate reached in
both settings. Our study shows, however, that the
achieved heart rates vary widely, not only
between the sexes, but also between individuals.
Thus, we think that the predicted values of the
age-related heart rate are to be reviewed. The
target values of oxygen uptake, however, are
regularly checked and updated. The actual values
are in good agreement with the reference values
according to Hansen et al. (1984) and
Wasserman et al. (2004). This is also true when
the reference values of Koch et al. (2009) are
taken, which when compared with those of
Hansen et al. (1984), are slightly higher for the
age group of 2025 years.
The performance of men and women differed
significantly, not only in the achieved maximum
output, but also in the pulse rate (shown in the
PWC) and, more slightly, in relation to body
weight. Thus, this finding confirms previous
results (Reiterer 1975). In aptitude tests, this is
often taken into account and a lower performance
of women is required, even if it must be assumed
that the same activities are demanded of them in
diving or in the workplace; being, for instance, a
firefighter. It is still unclear whether the same
energy metabolism and oxygen consumption
Gradual Versus Continuous Increase of Load in Ergometric Tests: Are. . .
Fig. 3 Comparison of maximal required wattage/kg
and PWC150/kg body weight, separately for men and
women, both with linear regression trend lines. The max-
imum wattage increases in men stronger, especially at
high outputs, compared with PWC150
are required by both sexes for the same per-
formance. For this purpose, gender studies on
the oxygen uptake in certain activities which
require aptitude tests would be necessary.
Wultsch et al. (2012) showed gender-specific
differences in oxygen uptake in workers with
heavy workload. In their sample, however,
only a 20-min representative work period was
measured without quantifying the heaviness of
the work accurately.
When comparing the sexes in terms of the
relation of maximum wattage and PWC150, we
found major differences. With reference to body
weight, the differences between men and women
were leveled out by calculation. However, com-
paring the maximum wattage with the perfor-
mance at the pulse rate of 150, the progression
shows different interdependencies in men and
women (Fig. 3). For both sexes, measuring
performance by PWC150 did not allow drawing
conclusions about the actual performance of an
individual. A direct comparison of the achieved
values of men and women was not possible
as based on the PWC150/kg. This difference
57
between the sexes is not yet taken into account
in the recommendations for an aptitude test.
A methodological weakness of our study
could be that the sequence of the tests was not
randomized. The CPX was always performed at
the initial examination and the ET followed in
the second examination. This could have led to
a higher motivation in the first test, and be a
cause for a better performance during the CPX.
However, women showed an overall better per-
formance compared with the gender-specific
predicted values. It is striking that their perfor-
mance was about 13 % higher than the predicted
values for maximal oxygen consumption
(Wasserman et al. 2004; Hansen et al. 1984)
and even 20 % higher than the reference values
for wattage (Reiterer 1975).
The predicted values for maximum oxygen
uptake are well validated for the age group of
2535 years and they are also in line with the
current set points by Koch et al. (2005).
The relatively low predicted values for the
wattage, which are regularly used in Germany,
relate to measurements in the 1970s and need to
be reviewed.
In conclusion, both loading protocols (CPX
and ET) allow qualified statements, in the con-
text of both aptitude test and diagnostic exami-
nation of heart and lung diseases. The choice of a
method may, therefore, be based on the technical
and financial capabilities. Basically, we would
recommend the ramp load increase, since no
interpolations are required, so the devices show
clear and legible results. In addition, other
parameters are not disturbed by an abrupt
increase in load and the compliance of subjects
is better even in the cardiac or pulmonary
practice.
The gradual load increase is attribuable to
the former limited options with mechanically
braked ergometers. We recommend replacing
the gradual by continuous increase in load. This
examination type is better in sports medicine
or cardiology diagnostics, especially when a
steady-state of low load is not aimed at.
The acceptance of the CPX-based aptitude test
was higher in our study population. The target
58
values of the wattage on a bicycle ergometer
need to be reviewed, particularly those for
women. Because of the observed different heart
rate at the anaerobic threshold we want to stimu-
late a discussion on the significance of PWC150
and PWC170.
Acknowledgements We thank Sabine Boler and
Christina Jeske for their technical assistance.
Conflicts of Interest The authors declare to have no
conflicts of interest in relation to this article.
References
ATS/ACCP (2003) Statement on cardiopulmonary
exercise testing. Am J Respir Crit Care Med 167
(2):211277
Atwater WO, Benedict FG (1903) Experiments on the
metabolism of matter and energy in the human body,
19001902 Bulletin No 136. US Department of Agricul-
ture, Office of Experiment Stations, Washington, DC
Balady GJ, Arena R, Sietsema K, Myers J, Coke L,
Fletcher GF, Forman D, Franklin B, Guazzi M,
Gulati M, Keteyian SJ, Lavie CJ, Macko R,
Mancini D, Milani RV (2010) Clinicians Guide to
cardiopulmonary exercise testing in adults: a scientific
statement from the American Heart Association.
Circulation 122(2):191225
Deutsche Gesetzliche Unfallversicherung (DGUV)
(2010) Berufsgenossenschaftliche Grundsatze fur
arbeitsmedizinische Vorsorgeuntersuchungen.
5. Gentner, Aufl. Stuttgart
Forman DE, Myers J, Lavie CJ, Guazzi M, Celli B, Arena
R (2010) Cardiopulmonary exercise testing: relevant
but underused. Postgrad Med 122(6):6886
Hansen JE, Sue DY, Wasserman K (1984) Predicted
values for clinical exercise testing. Am Rev Respir
Dis 129(2):S49S55
A.M. Preisser et al.
Hollmann W, Struder HK, Predel HG, Tagarakis
CVM (2006) Spiroergometrie. Schattauer,
Stuttgart/New York
Koch A, Ivers M, Gehrt A, Schnoor P, Rump A,
Rieckert H (2005) Cerebral autoregulation is tempo-
rarily disturbed in the early recovery phase after
dynamic resistance exercise. Clin Auton Res. 15
(2):8391
Koch B, Schaper C, Ittermann T, Spielhagen T, Dorr M,
Volzke H, Opitz CF, Ewert R, Glaser S (2009) Refer-
ence values for cardiopulmonary exercise testing in
healthy volunteers: the SHIP study. Eur Respir J 33
(2):389397
Meyer FJ, Borst MM, Buschmann HC, Ewert R,
Friedmann-Bette B, Ochmann U, Petermann W,
Preisser AM, Rohde D, Ruhle KH, Sorichter S,
Stahler G, Westhoff M, Worth H (2013) Belastung-
suntersuchungen in der Pneumologie. Empfehlungen
der Deutschen Gesellschaft fur Pneumologie und
Beatmungsmedizin e.V. (Exercise testing in respira-
tory medicine). Pneumologie 67(1):1634
Prinz JP (1993) Elisee Bouny. Der Erfinder des
Fahrradergometers. In: Tittel K, Arndt K-H, Hollmann
W (eds) Sportmedizin. Gestern Heute Morgen.
Bericht vom Jubilaumssymposium des Deutschen
Sportarztebundes, Oberhof, 2527. September 1992.
Barth, Leipzig. ISBN 3-335-00346-2
Reiterer W (1975) Methodik eines rektangularen-
triangularen Belastungstestes. Herz-Kreislauf
7:457462
Wasserman K (2002) Exercise gas exchange, breath-
by-breath. Am J Respir Crit Care Med 165
(3):325326
Wasserman K, Van Kessel AL, Burton GG (1967) Inter-
action of physiological mechanisms during exercise.
J Appl Physiol 22(1):7185
Wasserman K, Hansen JE, Sue DY, Stringer WW, Whipp
BJ (2004) Principles of exercise testing and interpre-
tation. Lippincott Raven, Baltimore
Wultsch G, Rinnerhofer S, Tschakert G, Hofmann P
(2012) Governmental regulations for early retirement
by means of energy expenditure cut offs. Scand J
Work Environ Health 38(4):370379
Advs Exp. Medicine, Biology - Neuroscience and Respiration (2015) 9: 5967
DOI 10.1007/5584_2014_85
# Springer International Publishing Switzerland 2014
Published online: 14 October 2014

Evaluation of Volumetric Changes

in Differential Diagnosis of Brain
Atrophy and Active Hydrocephalus

E. Szczepek, L. Czerwosz, K. Nowinski, J. Jurkiewicz, and

Z. Czernicki

Abstract
Despite a variety of diagnostic methods, differentiation of symptoms of
normal pressure hydrocephalus from those of atrophic processes of the
brain is still a difficult task. In the present study an attempt of non-invasive
differential diagnosis of normal pressure hydrocephalus (NPH) and brain
atrophy (BA) was presented using volumetric analysis of CT images of
the head by means of VisNow proprietary software. The analysis was
based on the number of voxels converted to the amount of cerebrospinal
fluid (CSF) in the subarachnoid space, skull base casters, and the ventric-
ular system. The results demonstrate that the mean volumes of CSF in
these compartments in patients with NPH differed significantly from those
in BA. Similarly, the mean volumes of CSF in the subarachnoid space and
skull base casters in patients with BA differed significantly from those in
NPH. Volumetric assessment presented in the paper by application of
VisNow software seems useful in the evaluation of NPH and brain BA.

E. Szczepek
Department of Neurosurgery, Second Faculty of
Medicine, Medical University of Warsaw, Warsaw,
Poland
Department of Neurosurgery, Medical Research Center,
Polish Academy of Sciences, 5 Pawinskiego St., 02-106
Warsaw, Poland
Bioinformatics Laboratory, Medical Research Center,
Polish Academy of Sciences, 5 Pawinskiego St., 02-106
Warsaw, Poland
L. Czerwosz (*)
Bioinformatics Laboratory, Medical Research Center,
Polish Academy of Sciences, 5 Pawinskiego St., 02-106
Warsaw, Poland
Department of Respiratory Research, Medical Research
Center, Polish Academy of Sciences, 5 Pawinskiego St.,
02-106 Warsaw, Poland
e-mail: czerwosz@imdik.pan.pl
K. Nowinski
Interdisciplinary Center for Mathematical and
Computational Modelling, Warsaw University, Warsaw,
Poland
J. Jurkiewicz
Department of Neurosurgery, Second Faculty of
Medicine, Medical University of Warsaw, Warsaw,
Poland
Z. Czernicki
Department of Neurosurgery, Second Faculty of
Medicine, Medical University of Warsaw, Warsaw,
Poland
Department of Neurosurgery, Medical Research Center,
Polish Academy of Sciences, 5 Pawinskiego St., 02-106
Warsaw, Poland

59
60
Keywords
Cerebral atrophy Cerebrospinal fluid volume Normal pressure
hydrocephalus Volumetric assessment
1 Introduction
The differentiation between symptoms of normal
pressure hydrocephalus (NPH) and brain atrophy
(BA) presents significant difficulties. A diagnostic
method that gives the highest probability of appro-
priate recognition is an infusion test (Juniewicz
et al. 2005). The results of such a test should
always be interpreted in conjunction with other
elements of the diagnostic process: neuropsycho-
logical assessment (Fersten et al. 1997) and brain
imaging (Czernicki et al. 1992). Since the infusive
test represents an invasive method encumbered
with severe complications, attempts have been
made to apply non-invasive procedures such as
neuropsychological testing, evoked potentials,
and morphometry of fluid spaces to differentiate
between both pathological syndromes.
Particularly noteworthy is a non-invasive
diagnostic method of differentiating NPH and
BA the evaluation of CT images (ASOT)
introduced to clinical practice by Czernicki
and Jurkiewicz (1991). Using the ASOT method
Jurkiewicz (1996) developed a mathematical
formula to determine the content and distribution
of cerebrospinal fluid in intracranial fluid spaces.
On this basis Marszaek et al. (1997) developed a
multistage method to identify NPH by applying
morphometric assessment of CT images. Subse-
quently, an index of hydrocephalus (Ih) has been
calculated as a ratio of the number of fluid points
within the ventricles to the total number of fluid
points within the tested layer.
The following equation determines the Ih
index: Ih Kk/Kw  100 %, where Kk is the
number of points (pixels) falling within the cere-
bral ventricles and Kw is the number of points
(pixels) fitted on the surface of visible layer
assigned as fluid.
The methods to differentiate between NPH
and BA have lately been enriched by introducing
E. Szczepek et al.
a non-invasive diagnostic test of NPH consisting
of balance assessment using posturography on
Pro-Med force-plate and of gait analysis
performed by means of a dynographic measure-
ment system (Czerwosz et al. 2008, 2012, 2013;
Szczepek et al. 2008). A new computational
parameter called visual index has been
introduced which indicates a degree of changes
in postural stability related to eyes opening/
closing. This parameter turned out to be an
important indicator in the differentiation of
NPH from BA.
The object of this study was an attempt to
apply a novel non-invasive method of
differentiating NPH from BA by means of the
VisNow software (http://visnow.icm.edu.pl) that
enables a volumetric analysis of the measure-
ment of CT head scans.
2 Methods
The study was approved by the Ethics Committee
of the Medical University of Warsaw in Poland
(permit no. kb/64/2014) and was performed in
accord with the Declaration of Helsinki for
Human Experimentation of the World Medical
Association.
Volumetric analysis of CT imaging has been
performed in 10 patients diagnosed with NPH
and 17 patients with BA (mean age 58.5  7.7
and 71.4  5.1 years, respectively). The qualifi-
cation of patients diagnosed with NPH was based
on the following attributes:
ventricular enlargement on CT of the head,
Evans index >0.3,
none or minor features of cortical atrophy,
neurological symptoms (triad Hakim) at
least two symptoms
intracranial pressure measured by lumbar
puncture 10 cmH2O,
Evaluation of Volumetric Changes in Differential Diagnosis of Brain Atrophy. . .
resorption resistance 11 mmHg/ml/min,
neuropsychological examination.
Patient selected for BA group included:
ventricular enlargement in CT of the head,
Evans index < 0.3,
features of cortical subcortical atrophy,
uncharacteristic neurological symptoms,
intracranial pressure measured by lumbar
puncture < 10 cmH2O,
resistance resorption <11 mmHg/ml/min,
neuropsychological examination.
Neuropsychological examination was based on a
battery of tests developed and performed by
Marszaek et al. (1997).
Volumetric methods are techniques in which
measurements, in hand-selected areas, are automat-
ically aggregated by an algorithm. The methods
allow accurate volume assessment, which is of
key significance in brain structural changes of
NPH and BA. The assessment was carried out
using the proprietary software system VisNow,
developed at Warsaw University in Poland (ICM
2011). The VisNow is an open platform integrating
algorithms and methods of visual data analysis,
which enables to integrate the users own
algorithms of processing of experimental and
computational modeling data with the advanced
visualization methods implemented in the system.
Data can be stored in the DICOM format density
tissue arrays, in a three-dimensional map of
the magnetic resonance signal, or as diffusion
tensor MRI. DICOM data acquired from CT or
MRI scanners are organized as stacks of
two-dimensional slices containing scalar density
or diffusion tensor data. The slices are usually
squares of 256 by 256 or 512 by 512 matrices
covering square slices of body scan. On the other
hand, brain surface is represented by irregular mesh
of points in a three-dimensional space and a list of
triangular patches.
The VisNow system contains an internal data
formats library and a library of modules processing
and visualizing data. Modules are graphically
presented in the network editors as rectangular
blocks connected with lines symbolizing a flow
of data. VisNow modules include:
data input in various formats including dicom
and some vendor defined formats,
61
Fig. 1 Network of VisNow program modules in the
design window of data flow network
data denoising,
interpolation to user defined computational
mesh,
data volume segmentation,
data presentation, e.g., in the form of slices,
contour surfaces, or volume rendering.
A sample of network DICOM volume
denoising, segmentation, and the presentation of
segmented objects as areas within surfaces is
shown in Fig. 1. Modules are presented as rect-
angular blocks, lines indicate the flow of data or
control. The network follows a method of three-
dimensional visualization of CT data stored in
DICOM format. The data is denoised by means
of digital filter, segmented, and then visualized in
the next window as a surface determined by the
points of equal values of some selected signal
components such as points lying on the border
between two tissue densities. The VisNow
system also enables the construction and testing
of a network of modules in a relatively uncom-
plicated manner. Thus, generation of experimen-
tal techniques of processing and presentation of
the data is ensured.
Sequentially processed data produce a number
of virtual 2D and 3D x-ray images reproducing
the scanned brain. Networks of modules
performing subsequent stages of image data
processing and visualization can be easily
prototyped and then converted to stand-alone
applications for end users (Figs. 2 and 3).
62
Fig. 2 CT scan of a head in normal pressure hydro-
cephalus. (a) Frontal, coronal section; (b) Sagittal sec-
tion; (c) Axial section; (d) Volumetric image of space
On basis of head CT imaging in patients with
NPH and BA, the presented method allows
assigning and counting of voxels in the following
areas:
intracranial volume,
brain volume,
subarachnoid space and basal cisterns,
intracranial ventricular system.
A statistical analysis of the number of voxels
was carried out in the subarachnoid space and
skull base casters, and in the volume of intrac-
ranial ventricular system on the basis of CT
imaging of the head in patients classified as
NPH or BA (see algorithms for intracranial
volume estimation in the appendix).
E. Szczepek et al.
with a separated volume of intracranial subarachnoid
space and skull base casters (green) and a volume of
intracranial ventricular system (red)
3 Results
Figure 4 illustrates the mean volume of
cerebrospinal fluid (CSF) contained in the sub-
arachnoid space and skull base casters and in the
intracranial ventricular system estimated on the
basis of CT images of the head in patients classi-
fied as NPH and BA. The average volumes of
CSF in these spaces differed significantly
between the BA and NPH patients (p < 0.001;
t-test). An average volume of CSF in the intra-
cranial cerebral ventricles of the NPH patients
also differed significantly from that of the BA
patients (p < 0.004).
Evaluation of Volumetric Changes in Differential Diagnosis of Brain Atrophy. . . 63

Fig. 3 CT scan of the head in brain atrophy. (a) volume of the intracranial subarachnoid space and skull
Frontal, coronal section; (b) Sagittal section; (c) Axial base casters (green) and a volume of intracranial ventric-
section; (d) Volumetric image of space with a separated ular system (red)

Figure 5 is a two-dimensional chart, where

the X-axis coordinate corresponds to the value
of CFS volume in the intracranial ventricular
system and the Y-axis corresponds to the value
of CSF in the subarachnoid space and skull
base casters in NPH and a BA patients. Each
point represents an individual patient. The
graph clearly contrasts the NPH and BA patients.
A high value of CSF volume is present in the
intracranial ventricular system of NPH, but in the
subarachnoid space and skull base casters of BA.

4 Discussion

The present study determined changes in CSF

Fig. 4 Mean volume of CSF contained in the subarach-
volume in different fluid compartments of the
noid space and skull base casters and in the intracranial brain in normal pressure hydrocephalus and
ventricular system in patients with NPH and BA brain atrophy. The aim was to attempt to
64
differentiate between these two pathological
conditions on the basis of subtle differences in
CSF volume and its distribution.
We employed a new kind of volumetric
assessment of CSF distribution in brain fluid
spaces; a non-invasive method in which measu-
rements are performed in hand-selected areas of
brain CT scans and are then automatically
aggregated by a computerized algorithm of the
VisNow software (ICM 2011). The calculation of
the mean volume of CSF voxels was 79.4 % in
brain ventricles and 20.6 % in the subarachnoid
space in NPH patients, whereas in BA the voxel
volumes were 44.2 % in brain ventricles and
55.8 % in the subarachnoid space and basal
cisterns together. These percentages result from
the conversion of data contained in Table 1
above outlined.
The volumetric method, as opposed to the
classical morphometry, gives a real mean
Fig. 5 Scatter plot of the CSF volume in the subarach-
noid space and skull base casters and in the intracranial
ventricular system in patients with NPH and BA
Table 1 Calculation of CSF volume (cm3) in the subarachnoid space and skull base casters and in the intracranial
ventricular system in patients with NPH and BA
Volume
Subarachnoid space and skull base casters
Ventricular space
Data are means  SD
NPH normal pressure hydrocephalus, BA brain atrophy
E. Szczepek et al.
number of CSF voxels converted to CFS volume
contained in the intracranial fluid system.
The proportions of CFS distributed between the
ventricles and the subarachnoid space we found
in the present study were somewhat different
than those in a study of Marszaek et al. (1997),
which employed a classical morphometric
method which neglected the CSF contained in
the basal cisterns. In contrast, we calculated an
absolute actual average number of voxels in both
subarachnoid space and basal cisterns.
There are many pieces of software described
in the literature for estimating the volume of the
brain or various brain compartments. Ishii
et al. (2013) described software that enables
automatic measuring of the regional CSF space
and comparing the volumes of ventricular
systems, Sylvian fissures and sulci at high con-
vexity in NPH patients, Alzheimers patients,
and healthy volunteers. The analysis was based
on 1.5 and 3 tesla MRI. Voxel-based morpho-
metry (VBM)/volumetry was performed with
their own volumetric software AVSIS program
improved for the study to automatically measure
the CSF volumes. The relative sulci at high
convexity and the midline volume in NPH
patients was the smallest among the three
investigated groups. The volume of ventricular
systems in NPH patients was significantly larger
than those in Alzheimers patients and healthy
volunteers. The authors concluded that the
volume measurement can be used to delineate
the characteristic changes in CSF space and is
useful in the diagnosis of NPH.
Ambarki et al. (2011) examined the CFS
volume in the ventricular and subarachnoid
spaces. The magnetic resonance MRI 3 tesla-T2
and FLAIR (axial) images were analyzed in
hydrocephalus using the commercially available
NPH BA
48.8  13.3 146.8  42.5 <0.001
187.8  37.8 116.2  40.0 <0.004
Evaluation of Volumetric Changes in Differential Diagnosis of Brain Atrophy. . .
QBrain software in a fully automatic way
and were compared with a manual method.
The automatic calculations provided a rapid
assessment of brain volumes of selected fluid
spaces, but there were significant differences
between the mean volumes calculated by manual
and automatic methods. Furthermore, the auto-
matic method did not recognize the cerebral
aqueduct or the fourth ventricle. The QBrain
software is tailored only for the use with MRI,
but not CT, images.
In contrast, the VisNow software used in the
present study is suitable for the analysis of both
MRI and CT images and it enables the manual
inclusion of the fourth ventricle and cerebral
aqueduct into the volume of intracranial CSF
ventricular systems, which provides more
dependable results. The reliability of the VisNow
software in clinical diagnostics has been
confirmed in other recent studies (Kapinski
et al. 2013; Borucki et al. 2011, 2012).
Comparing the VisNow volumetric method
with the morphometric one introduced by
Marszaek et al. (1997), one should note that
both methods allow the assessment of the ratio
between the content of CSF in the ventricular
and subarachnoid spaces. However, the volu-
metric method evaluates this proportion in a
far more accurate way as it shows the actual
number of fluid voxels inside the ventricles,
subarachnoid spaces, and skull base casters.
In contrast, the morphometric method is able
to evaluate just three selected layers, which are
not always representative and thus may lead to a
substantial overestimation of hydrocephalus in
differential diagnosis. The volumetric method
makes use of routine diagnostic CT imaging
and, despite the need of manual indication
of the areas of interest, it performs the voxel
evaluation in the automatic mode, with simul-
taneous visualization of the areas included in
the specified CFS reservoirs. The result is
given directly in calibrated units of volume.
The method enabled to confirm the assumption
put forward by Jurkiewicz (1996) that an appre-
ciable amount of CSF volume is contained in
the brain ventricles in NPH patients.
In conclusion, use of a new volumetric
method of assessment of CT images seems a
65
promising tool in the differential diagnosis of
various pathological central nervous pathologies,
in particular normal pressure hydrocephalus and
brain atrophy.
Acknowledgments The research support was partially
provided by the Biocentrum-Ochota project
POIG.02.03.00-00-003/09.
Conflicts of Interest The authors declare no conflicts of
interest in relation to this article.
Appendix
1. The application developed for intracranial
volume estimation comprises the following
modules:
(a) DICOM reader reading CT scans
available in DICOM formats and
pre-processing them by:
(b) Trilinear interpolation to a regular grid
with 1 mm resolution
(c) Clamping original data range to the 064
Hunsfield unit range increasing the contrast
between essential regions (cortico-spinal
fluid, brain tissue and bone structures)
facilitating volume segmentation.
2. Optional anisotropic denoising module
implementing the following two-stage
algorithm:
Step 1: The original volume v(x,y,z) is Gauss
smoothed with the window width 46
pixels:
um; n; p
vm i, n j, p kei j k
iR
r
jR
r
kR
r 2 2 2
R R R ei j k
2 2 2
iR jR kR
where v is an original image, w an inter-
mediate image, R 3. . .6.
The smoothed volume w(x,y,z) is radically
denoised at the cost of smoothing the
image edges (inter-region boundaries).
Step 2: The resulting image u(x,y,z) is formed
by local weighted averaging with position-
dependent weight calculated from the
smoothed image w:
66
ir
r
jr
r
kr
r
vm i, n j, p ke
um; n; p
ir
r
jr
r
kr
r
e
where u is the resulting image and
the term h(w(m + i, n + j, p + v) 
w(m, n, p))2 provides anisotropic
denoising: the local denoising kernel
i2 j2 k2 2

with CSF.
e r2 hwmi, nj, pvwm;n;p decre-
ases rapidly in the direction perpendicular
to the boundary between two subregions of
the image v with significantly different
values of v and is reduced to an ordinary
Gaussian kernel e-i jr2k in the areas of
2 2 2
uniform density. The denoising radius r is
assumed as 2 or 3 for our CT denoising
problem. While computationally intensive,
the algorithm can be easily parallelized for
multiprocessor systems.
3. The final segmentation and volume calcula-
tion is performed in a module based on the
following semi-interactive algorithm:
Step 1: The user picks set S of starting points in
the area of interest from the chosen slices;
Step 2: For every volume point p, the shortest
path (p p0, p1,. . .,pn) of consecutive points
linking p with some point pn from the starting
set S of points is chosen. The path length
is defined as in 1 (v ( pi)  v ( pi  1))2,
where v( p) is the value (density) in point p.
The formula assures that a path passing
through an area of uniform values is short
while each crossing of a sharp contour
between significantly differing areas
increases radically the path length;
Step 3: The user sets a threshold t for path
length and the algorithm assigns to the
segmented area all points that can be
connected with a point from S by a path
shorter than t.
A variation of this method assigns to the
segmented area all points that can be
connected to the starting set S by a path
passing through voxels p satisfying the
E. Szczepek et al.
i2 j2 k2


hwmi, nj, pvwm;n;p2
r2
i2 j2 k2


hwmi, nj, pvwm;n;p2
r2
condition tmin < v( p) < tmax (for example,
taking tmin 12HU and tmax 17HU, so
that we can segment particular areas filled
References
Ambarki K, Wahlin A, Birgander R, Eklund A, Malm J
(2011) MR imaging of brain volumes: evaluation of a
fully automatic software. Am J Neuroradiol
32:408412
Borucki B, Nowinski K, Chlebiej M, Rutkowski A,
Adamczyk P, Laskowski JM (2011) Automated geo-
metric features evaluation method for normal foot
skeleton model. Int J Comput Assist Radiol Surg
1:110111
Borucki B, Nowinski K, Adamczyk P, Laskowski JM
(2012) Automatic classification of hallux valgus
deformations with the use of automatic evaluation of
geometric descriptors. Int J Comput Assist Radiol
Surg 1:196198
Czernicki Z, Jurkiewicz J (1991) Disorders of intracranial
volume relations pressure. Pol J Neurol Neurosurg
25:671677
Czernicki Z, Walecki J, Jurkiewicz J, Grochowski W,
Tychmanowicz K (1992) Intracranial volume reserve
determination using CT images, numerical analysis
and lumbar infusion tests. An experimental study.
Acta Neurochir 115:4346
Czerwosz L, Szczepek E, Sokoowska B, Jurkiewicz J,
Czernicki Z (2008) Recognition of gait disturbances in
patients with normal pressure hydrocephalus using a
computer dynography system. J Physiol Pharmacol 59
(Suppl 6):201207
Czerwosz L, Szczepek E, Sokoowska B, Jurkiewicz J,
Czernicki Z (2012) Recognition of posture and gait
disturbances in patients with normal pressure hydro-
cephalus using a posturography and computer
dynography systems. InTech, Chapter 12, pp 189214
Czerwosz L, Szczepek E, Sokoowska B, Jurkiewicz J,
Czernicki Z (2013) Posturography in differential diag-
nosis of normal pressure hydrocephalus and brain
atrophy. Adv Exp Med Biol 755:311324
Fersten E, uczywek E, Jurkiewicz J, Dowzenko A
(1997) Evaluation of perceptive activity in persons
with low pressure hydrocephalus treated surgically.
Pol J Neurol Neurosurg 31:7788
Evaluation of Volumetric Changes in Differential Diagnosis of Brain Atrophy. . .
ICM Interdisciplinary Center of Mathematical and
Computational Modeling (2011). http://visnow.icm.
edu.pl. Accessed 12 Nov 2013
Ishii K, Soma T, Shimada K, Oda H, Terashima A,
Kawasaki R (2013) Automatic volumetry of the cere-
brospinal fluid space in idiopathic normal pressure
hydrocephalus. Dement Geriatr Cogn Disord Extra
3:489496
Juniewicz H, Kasprowicz M, Czosnyka M, Czosnyka Z,
Gizewski S, Dzik M, Pickard JD (2005) Analysis of
intracranial pressure during and after the infusion test
in patients with communicating hydrocephalus.
Physiol Meas 26:10391048
Jurkiewicz J (1996) Negative-pressure hydrocephalus.
J Neurosurg 85:364365
67
Kapinski N, Borucki B, Nowinski K (2013) Error
assessment and minimization in 4D motion tracking
for functional orthopaedics diagnostics. Int J Comput
Assist Radiol Surg 8:157159
Marszaek P, Jurkiewicz J, Fersten E, uczywek E,
Czernicki Z, Gielecki J, Bogucki J (1997) Multi-
stage method for the diagnosis of low-pressure hydro-
cephalus. Pol J Neurol Nuerosurg 31:527539
Szczepek E, Czerwosz L, Dabrowski P, Dudzinski K,
Jurkiewicz J, Czernicki Z (2008) Posturography and
computerized gait analysis in the computer
dynography system as non-invasive methods for eval-
uation of normal pressure hydrocephalus progression.
Pol J Neurol Nuerosurg 42:139152
Advs Exp. Medicine, Biology - Neuroscience and Respiration (2015) 9: 69
DOI 10.1007/5584_2014
# Springer International Publishing Switzerland 2014

Index

A M
Athletes, 3034 Metabolic syndrome, 1517

B N
Bone alkaline phosphatase (BAP), 2226 Nephrotic syndrome, 2126
Bone density, 25 Nitric oxide, 3032, 34
Bone metabolism markers, 2126 Normal pressure hydrocephalus (NPH), 6065
Bone mineral content (BMC), 210 Nutritional status, 4548

C O
Cardiopulmonary exercise (CPX) testing, 52, 55 Obesity, 3, 7, 1318, 48
Cardiovascular diseases, 5, 7, 1718 Oxygen uptake, 3032, 52, 53, 56, 57
Cerebral atrophy, 60, 62, 65
Cerebrospinal fluid volume (CSF), 60, 6266
Children, 1318, 2126 P
Chronic obstructive pulmonary disease (COPD), 30, 36, Parasternal muscle, 36, 41
42, 4548 Physical work capacity, 52, 54, 55
Cigarette smoking, 2, 5
Corticosteroids, 2126, 46
Cycle ergometer, 37, 5255, 5758 Q
Quality of life, 46, 47

D
Diaphragm (D), 36, 3842 R
Ramp protocol, 52, 57

E
Electrical bioimpedance, 46 S
Electromyographic (EMG), 36, 3842 Scalene muscle, 36, 41, 42
Energy X-ray absorptiometry, 110 Segmental bioelectrical impedance analysis, 110
Exercise training, 30, 33, 34 Skiers, 2934
Standard exercise test, 52, 53
Sternocleidomastoid muscle (SCM), 36, 3842
F Systemic sclerosis, 4548
Fat percentage, 9

V
I Vitamin D deficiency, 1318, 25
Inspiratory muscle fatigue, 35, 36, 39, 40 Volumetric assessment, 61, 64, 65

L
Lean mass (LM), 210
Lung function, 32, 36

69