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Scandinavian Journal of Clinical and Laboratory
Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Winter-swimming as a building-up body resistance
factor inducing adaptive changes in the oxidant/
antioxidant status
Anna Lubkowska, Barbara Dołęgowska, Zbigniew Szyguła, Iwona
Bryczkowska, Małgorzata Stańczyk-Dunaj, Daria Sałata & Marta Budkowska
To cite this article: Anna Lubkowska, Barbara Dołęgowska, Zbigniew Szyguła, Iwona
Bryczkowska, Małgorzata Stańczyk-Dunaj, Daria Sałata & Marta Budkowska (2013) Winter-
swimming as a building-up body resistance factor inducing adaptive changes in the oxidant/
antioxidant status, Scandinavian Journal of Clinical and Laboratory Investigation, 73:4, 315-325,
DOI: 10.3109/00365513.2013.773594
To link to this article: http://dx.doi.org/10.3109/00365513.2013.773594
Published online: 20 Mar 2013.
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Citing articles: 4 View citing articles
Correspondence: Anna Lubkowska, PhD, Laboratory of Physical Medicine, Faculty of Health Sciences, Pomeranian Medical University in Szczecin, Poland
ul. Grudziadzka 31, 70-130 Szczecin, Poland. E-mail:annalubkowska@gmail.com
(Rece ived 27 October 2012 ; accep ted 3 February 2013 )
ORIGINAL ARTICLE
Winter-swimming as a building-up body resistance factor
inducing adaptive changes in the oxidant/antioxidant status
ANNA LUBKOWSKA
1,2 , BARBARA DO Ł E˛ GOWSKA
3 , ZBIGNIEW SZYGU Ł A
4 ,
IWONA BRYCZKOWSKA
2 , MA Ł GORZATA STA N
´ CZYK-DUNAJ
5 ,
DARIA SA Ł ATA
3 & MARTA BUDKOWSKA
3
1 Laboratory of Physical Medicine, Faculty of Health Sciences, Pomeranian Medical University in Szczecin,
2 Department of Physiology, Faculty of Biology, Szczecin University,
3 Department of Laboratory Diagnostics and
Molecular Medicine, Pomeranian Medical University in Szczecin,
4 Institute of Human Physiology, University School
of Physical Education, Krakow, and
5 Department of Medical Chemistry , Pomeranian Medical University in
Szczecin , Szczecin , Poland
Abstract
The aim of our research was to examine whether winter-swimming for fi ve consecutive months results in adaptational
changes improving tolerance to stress induced by exposure to cryogenic temperatures during whole-body cryostimulation
(WBC). The research involved 15 healthy men, with normal bodyweight, who had never been subjected to either WBC or
cold water immersion. During the experiment, the participants were twice subjected to WBC (3 min/ ⫺ 130 ° C), namely
before the winter-swimming season and after the season. Blood was taken seven times: In the morning before each cryos-
timulation, 30 min after each cryostimulation and the next morning. Additionally, control blood was collected in the mid-
dle of the winter season, in February. Our analysis concerned changes in hematological parameters as well as in reduced
glutathione and oxidized glutathione, total oxidant status, total antioxidant status and in components of the antioxidant
system: Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase and 8-Iso-
prostanes as a sensitive indicator of oxidative stress. We found signifi cant changes in hemoglobin concentration, the number
of red blood cells, the hematocrit index and mean corpuscular volume of red blood cell and the percentage of monocytes
and granulocytes after the winter swimming season. The response to cryogenic temperatures was milder after fi ve months
of winter-swimming. The obtained results may indicate positive adaptive changes in the antioxidant system of healthy
winter-swimmers. These changes seem to increase the readiness of the human body to stress factors.
Key Words: Cold exposure , cold-stress response , oxidative stress , adaptation , antioxidant effects , cr yotherapy
Introduction
Critical water temperature, i.e. the lowest tempera-
ture at which a naked resting human is able to main-
tain stable body temperature without increased
metabolism, is 32 – 35 ° C. The corresponding critical
air temperature is 22 – 27 ° C. However, these values
are not constant and may be lower in people that
have better adapted to cold [1,2]. Immersion in cold
water during winter-swimming season (4 ° C), accom-
panied by hydrostatic pressure, is undoubtedly a
stress factor for the human body that induces sig-
nifi cant heat and metabolic losses and results in a
disturbance of thermal homeostasis. This leads to
immediate and also long-term physiological and
biochemical reactions, including both hormonal and
metabolic reactions, and the response of the cardio-
vascular system, documented in experiments on ani-
mals and people [3 – 5]. Cold water immersion
decreases skin, subcutaneous, muscle and rectal
temperature [6]. Stimulation of the sympathetic
nervous system and adrenal glands, the observed
increase in the concentration of catecholamines
(mainly norepinephrine, even by four times) and to
a lesser extent in epinephrine, stimulates thermo-
genesis, control vasoconstriction, and together with
cortisol are involved in energy metabolism [7,8].
Scandinavian Journal of Clinical & Laboratory Investigation, 2013; 73: 315–325
ISSN 0036-5513 print/ISSN 1502-7686 on line © 2013 Informa Healthcare
DOI : 10. 3109 /003 65513.2013.773594
316 A. Lubkowska et al.
Additionally, in cold water immersion, the metabolic
rate is affected by the increased concentration of
hormones, such as corticotropin (adrenocortico-
tropic hormone, ACTH), thyrotropin (thyroid-
stimulating hormone, TSH), and vasopressin [9].
The metabolic rate in a cold environment is also
increased by the mechanism of shivering thermo-
genesis, initiated to enhance the endogenous pro-
duction of heat through increased residual tension
of skeletal muscles and more intense shivers – fre-
quent, low-amplitude muscle contractions (shivers).
The work of muscles in cold water immersion, and
also their shivering, increases the blood supply in
body surfaces and lowers their insulating function,
thus heat loss is even greater than before [10].
It is assumed that repeated immersions in cold-
water (used to increase resistance) induce a physio-
logical change of an adaptive character, associated
with a complex cascade of molecular events, which
involve the sympathetic nervous system pathways,
ranging from the release of neurotransmitters to
regulation of gene expression. Winter swimmers
have been observed to have improved thermogen-
esis, especially an increase of non-shivering ther-
mogenesis in adipose tissue, metabolic adaptation
and adaptation of circulatory responses, where the
insulative adaptation indicates an enhancement of
the cutaneous vasoconstrictor response [3,11].
Only a few studies have confi rmed that winter-
swimming results in oxidative stress, and repeated
immersions in cold water may enhance immune
responses and improve antioxidant protection
[12 – 14]. Previous research has concerned mainly
the evaluation of thermal comfort [15], thermosen-
sitivity and changes in body temperature during
swimming, analgesic response [16], and resting
concentrations of selected hormones [8,17,18],
indicators of the pro-oxidant/antioxidant status
after the winter-swimming season [12], well-being
[19], or whole-body fl uid regulation [20].
It is interesting whether winter-swimming results
in habituation, improving antioxidant parameters,
and if this is the case, whether these changes result
in increased resistance to a strong stressor, i.e. cryo-
genic temperatures acting on the whole body, and
whether it increases resistance to increased oxidative
stress in the body. In order to verify this hypothesis
we carried out research in which we evaluated the
level of selected indicators of the pro-oxidant/anti-
oxidant status in response to a single whole-body
cryostimulation (WBC) applied before and after a
fi ve-month-long winter-swimming season in a group
of healthy men. We examined the changes in concen-
tration of 8-isoprostane (Iso-P) in plasma. Isopros-
tanes are non-classical eicosanoids and are considered
the best available biomarkers of oxidative stress sta-
tus and lipid peroxidation in vivo [21]. With regard
to antioxidant status, our analysis concerned changes
in reduced (GSH) and oxidized (GSSG) glutathione,
total oxidant status (TOS), total antioxidant status
(TAS) and components of the antioxidant system:
superoxide dismutase (SOD, E.C.1.15.1.1), catalase
(CAT, E.C.1.11.1.6), glutathione peroxidase (GPx,
E.C. 1.11.1.9), glutathione reductase (GSSG-R,
E.C.1.8.1.7) and glutathione S-transferase (GST,
E.C.2.5.1.18).
Materials and methods
The research involved 15 healthy men, aged 23 ⫾ 1.47
years, with normal bodyweight (BMI ⫽ 23.8 ⫾ 2.92),
who had never been subjected to either WBC or
cold water immersion. The subjects were a homoge-
neous group with regard to age, level of daily physi-
cal activity. Each participant gave a written consent
before participation in the study, and the Regional
Bioethical Committee issued their formal consent,
according to the Helsinki Declaration. Each partici-
pant was examined by a physician to test for
any contraindications towards cryostimulation and
cold water immersion. The participants were also
subject to anthropometric measurements: Height
and bodyweight, skinfold thickness at the right and
left shoulder. Then the Body Mass Index (BMI) was
determined. The bioimpedance method was used
(Bodystat 1500) to determine the basic body com-
position of the participants, i.e. percentage content
of Lean Body Mass (LBM), water and fat. The char-
acteristics of the examined group are presented in
Table I.
During the experiment the participants swam in
a lake, on average 2 – 3 times a week for a prolonged
period of fi ve months, from November to March.
Each immersion in water took from 2 – 5 min, covered
the whole body, excluding the head. The mean
water temperature ranged from 12 – 15 ° C to 0 – 7 ° C
from the beginning to the end of the experimental
period.
Additionally participants were subjected to cold-
stress factor, as a WBC, twice, before and after the
winter-swimming season (October and April). Each
time, the participants were exposed to a 3-min ses-
sion of extremely low temperature ( ⫺ 130 ° C) in a
two-stage cryogenic chamber, where subjects were
fi rst introduced to the vestibule of the pre-chamber
( ⫺ 60 ° C) and then passed to the main chamber
( ⫺ 130 ° C). Before entering the cryogenic chamber,
participants dried their bodies thoroughly to elimi-
nate the sensation of cold. To protect the upper air-
ways, all participants breathed through a surgical
mask. For protective purposes, all participants wore
gloves, socks, special footwear and head bands to
protect the ears. While in the cryostimulation, the
subjects were advised to move their fi ngers and
legs slightly and to avoid holding their breath. The
cryostimulations took place in the morning between
09:00 and 10:00 h. During the winter-swimming
Adaptation to cold-stress factors 317
season, the participants swam in cold water twice a
week over a period of fi ve months (from November
to March), joining a group of experienced winter-
swimmers. Figure 1 presents the scheme of the
experiment.
Blood was taken seven times: In the morning
before the fi rst cryostimulation (sample T
0 ), 30 min
after the fi rst cryostimulation (sample T
1 ) and in
the next morning (sample T
2 ). The same scheme
was used during the second cryostimulation, after
the winter-swimming season, with the correspond-
ing markings: T
0 ’
, T
1 ’
and T
2 ’
. Additionally, blood
was taken in the middle of the winter season, in
February (T
M ).
Table I. Anthropometric characteristics and hematological indicators of the examined individuals, before
(T
0 ), in the middle (T
M ) and after (T
0 ’
) the winter-swimming season (mean ⫾ SD).
Parameters T
0 T
M T
0 ’
Age 23.0 ⫾ 1.10 23.0 ⫾ 1.47 23.0 ⫾ 2.07
Height (cm) 181.5 ⫾ 5.08 181.5 ⫾ 5.08 181.5 ⫾ 5.08
Bodyweight (kg) 79.1 ⫾ 11.88 79.0 ⫾ 10.67 80.2 ⫾ 11.48
BMI (kg/m
2 ) 23.8 ⫾ 2.92 23.9 ⫾ 2.03 24.6 ⫾ 3.02
Lean (%) 80.8 ⫾ 2.08 83.0 ⫾ 1.95 84.0 ⫾ 2.65
Fat (%) 14.0 ⫾ 3.69 14.1 ⫾ 4.01 14.2 ⫾ 3.30
Water (%) 59.5 ⫾ 2.12 60.0 ⫾ 0.57 60.5 ⫾ 0.75
Skinfold thickness, total
for arm and forearm(mm)
14.7 ⫾ 3.01 15.0 ⫾ 2.51 15.0 ⫾ 2.51
RBC (10^
12 /L) 5.6 ⫾ 0.71 5.2 ⫾ 0.50 4,9 ⫾ 0.26
*
* T
0
Hb (mmol/L) 10.6 ⫾ 1.11 9.9 ⫾ 1.40
* T
0 8.6 ⫾ 1.36
*
*
* T
0
,T
M
Ht (L/L) 0.51 ⫾ 0.09 0.44 ⫾ 0.05
*
* T
0 0.42 ⫾ 0.03
*
* T
0
MCV (fL) 87.4 ⫾ 6.57 85.5 ⫾ 6.88
*
* T
0 83.8 ⫾ 6.47
*
*
* T
0
,T
M
MCH (fmol) 1.7 ⫾ 0.16 1.7 ⫾ 0.09 1.70 ⫾ 0.06
MCHC (mmol/L) 19.6 ⫾ 1.16 20.1 ⫾ 0.93 19.8 ⫾ 0.78
RDW (%) 12.1 ⫾ 1.06 13.1 ⫾ 1.23
*
* T
0 13.5 ⫾ 1.32
*
* T
0
WBC (10^
9 /L) 6.2 ⫾ 1.15 6.0 ⫾ 1.00 6.5 ⫾ 1.22
LYM (%) 36.9 ⫾ 6.56 36.7 ⫾ 6.75 35.4 ⫾ 3.76
MON (%) 12.9 ⫾ 2.92 9.0 ⫾ 1.71
* T
0 8.6 ⫾ 1.27
*
* T
0
GRA (%) 47.6 ⫾ 6.36 56.6 ⫾ 8.60
*
* T
0 57.9 ⫾ 5.64
*
* T
0
PLT (10^
9 /L) 276.0 ⫾ 79.29 260.9 ⫾ 45.62 269.1 ⫾ 29.98
SBP (mmHg) 129 ⫾ 7.5 127 ⫾ 6.4 137 ⫾ 6.
4
* T0 ,
T
M
DBP (mmHg) 77 ⫾ 6.8 80 ⫾ 6.3 78 ⫾ 6.3
BMI, body mass index; RBC, red blood cells; Hb, hemoglobin; Ht, hematocrit; MCV, mean corpuscular
volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration;
RDW, red blood cell distribution width; WBC, white blood cells; LYM, lymphocytes; MON, monocytes;
GRA, granulocytes; PLT, thrombocytes; SBP, systolic blood pressure; DBP, diastolic blood pressure.
* Statistically signifi cant difference at P ⱕ 0.05;
*
* statistically signifi cant difference at P ⱕ 0.01;
*
*
* statistically
signifi cant difference at P ⱕ 0.001.
Figure 1. The scheme of the experiment. Blood sampling: T
0 , before; T
M, in the middle; T
0 ’
, after the winter-swimming season, in the
morning after overnight fasting. T
1 and T
1 ’
, blood sampling 30 min after the cryostimulation. T
2 and T
2 ’
, blood sampling 24 h after the
cryostimulation.
318 A. Lubkowska et al.
Each time (T
0, T
0 ’
and T
M ) venous blood samples
were obtained after overnight fasting in the morning,
between 08:00 and 09:00 h from an antecubital
forearm vein, after 10-min rest in a sitting position,
using vacutainer system tubes with appropriate anti-
coagulant (Sarstedt, Germany) for biochemical analy-
sis of erythrocytes and plasma (7 mL; EDTA) and to
determine peripheral blood cell parameters (1.2 mL;
anticoagulated with 1 g/L EDTA): number of erythro-
cytes (RBC), hemoglobin concentration (Hb), hemat-
ocrit value (Ht), number of leukocytes (WBC), number
of thrombocytes (PLT). Hematological parameters
were measured by the use of a hematology analyzer
(ABX Micros 60 HORIBA).Intra-assay precision and
accuracy for all hematologic parameters were below
1.5% and inter-assay precision and accuracy for all
hematologic parameters were below 2.0%.
The erythrocytes were separated by centrifuga-
tion (300 rpm, 1500 g , 10 min, 4 ° C), washed three
time with buffered NaCl solution (PBS: 0.01 mol
phosphate buffer 0.14 mol NaCl, pH 7.4) chilled to
4 ° C and fi nally frozen at ⫺ 70 ° C. Plasma was divided
into aliquots and immediately deep-frozen at ⫺ 70 ° C
until the time of analysis, however not longer than
one month.
Total lipid peroxides as the total oxidant status
(TOS) and the total antioxidant status (TAS) were
measured by means of photometric tests (Immuno-
diagnostik AG, Bensheim, Germany). The limits of
detection were 7 μ mol/L for TOS and 130 μ mol/L
for TAS. ELISA kits were used for measuring plasma
levels of 8-isoprostane (Cayman, MI, USA), accord-
ing to the manufacturer ’ s protocol.
Before the analysis, erythrocytes were thawed and
the hemolysate of the washed red blood cells was
diluted with distilled water and chilled to 4 ° C. GSH,
GSSG level, as well as SOD, CAT, GPx and GSSG-R
and GST activities were measured in hemolysate
samples with a BIOXYTECH
®
kit (Oxis Research,
Portland, OR, USA) using a UV/VIS Lambda 40
(Perkin-Elmer, Wellesley, MA, USA) spectropho-
tometer.
The enzyme activity and glutathione concentra-
tion were calculated per 1 g of erythrocyte hemoglo-
bin. In all mentioned cases, hemoglobin levels were
assayed using the Drabkin method [22]. Each sample
was tested in duplicate.
Statistics
The obtained results were statistically analyzed. Data
were checked for normal distribution using the
Shapiro-Wilk test and tested by one-way ANOVA.
The assessment of normality of distribution of con-
tinuous variables (Shapiro-Wilk test) was conducted
and showed non-normal (log-normal) distributions
of parameters. In order to normalize the distribution,
logarithmic transformation was performed of the
continuous variables tested. To assess the differences
between the parameters tested the one-way ANOVA
were used. For the parameters with normal distribu-
tion the results are expressed as the mean value with
standard deviation ( ⫾ SD), in other case the results
are expressed as median. In order to demonstrate
whether the observed correlations were statistically
signifi cant, we used the Spearman ’ s rank correlation
coeffi cient.
The coeffi cient of analytical variation (CV
A ) was
calculated from the mean and standard deviation of
the control blood analyses, which were conducted at
the beginning and end of each winter swimming sea-
son. Desirable analytical performance was assumed
if the imprecision was such that CV
A ⬍ 0.5 ⫻ CV
I
[23 – 25].
For hematological parameters, the coeffi cient of
intra-individual variation (CV
I ) was estimated by the
calculation δ / μ ⫻ 100, where δ denotes the mean of
the individual ’ s results across seven samples and μ
denotes the standard deviation of the seven measure-
ments. As the calculated CV
I includes an analytical
component we applied the Fraser ’ s formula for
each subject to remove the analytical variation (CV
A )
from CV
I using the formula: CV
Ib ⫽ (CV
I
2 ⫺ CV
A
2 )
0,5
[25,26], where CV
Ib denotes the CV
I without ana-
lytical variation. The total intra-individual coeffi cient
of variation (CV
I ) that was applied in RCV (refer-
ence change value) formula was calculated using the
global mean of the CV
Ib . The coeffi cient of variation
between individuals (CV
G ) was calculated according
to the mean and standard deviation of each hemato-
logical parameters obtained between the participants.
The RCV calculation was based on the following
formula: RCV ⫽ 2
0,5 ⫻ Zp(CV
A
2 ⫹ CV
I
2 )
0,5 , where
2
0,5 denotes the probability of bidirectional change
and Zp denotes the standard deviation correspond-
ing to the level of statistical signifi cance in bidirec-
tional change (1.96 ⫽ 95%) [25,27]. Development
of statistical results was performed using STATIS-
TICA PL v 7.1 software (Statsoft, Krakow, Poland).
The accepted level of signifi cance was defi ned as
P ⬍ 0.05.
Results
Anthropometric characteristics of the participants
did not change signifi cantly over the winter season,
neither their weight nor proportions of body compo-
nents. Consequently, BMI remained the same. There
were no changes in the average skinfold thickness in
the arm and forearm. It was found that winter-swim-
ming for fi ve months resulted in signifi cant changes
in the red blood cell system.
Swimming in winter infl uenced the CV
I values
for all hematological parameters. RCVs were always
greater in examined individuals than the values pre-
viously reported in the literature (Table II) [25].
Adaptation to cold-stress factors 319
Seasonal changes for hemoglobin concentration,
red blood cells count and hematocrit index were
more marked in the examined subjects than in the
healthy individuals – values previously reported in
the literature (Table III) [28].
There was a reduction in the number of red
blood cells ( P ⱕ 0.01), although within physiological
ranges. A decrease in hemoglobin, hematocrit index
and MCV ( P ⱕ 0.01) were observed in the middle
of the season, after 3 months of regular swimming,
albeit a decrease in the Hb concentration and MCV
intensifi ed at the end of the season ( P ⱕ 0.001),
reaching the lower limits of physiological standards
for men (83.8 ⫾ 6.47, Hb ⫽ 8.6 ⫾ 1.36 mmol/L).
Other analyzed red cell indicators, i.e. MCH and
MCHC, did not change signifi cantly. There was an
increase in red blood cell distribution width (RDW),
from 12.1% before the season, to 13.5% at the end
of the season.
Within the white blood cells, there was an increase
in the percentage of granulocytes with a simultane-
ous decrease in the percentage of monocytes, not
affecting the total number of white blood cells,
remaining at a level comparable to baseline. There
was also a signifi cantly higher systolic blood pressure
at rest after the season, compared to the level observed
before the season (Table I).
Five months of winter-swimming had a signifi -
cant effect on resting levels of many indicators of the
pro-oxidant/antioxidant status of the participants (T
0
vs. T
M vs. T
0 ’
) (Table IV). Already in the middle of
the season we observed an increased total antioxi-
dant status (TAS) and decreased total oxidant status
(TOS), consequently increasing the TAS:TOS ratio.
At the end of the season, there was a highly signifi -
cant decrease in resting levels of 8-ISOP in plasma
( P ⱕ 0.001) and oxidized glutathione ( P ⱕ 0.001),
which resulted in a reduction in GSH
Total , and the
GSH:GSSG ratio increased two times. Considering
the levels of antioxidant enzymes, an increase in
SOD ( P ⱕ 0.05), CAT ( P ⱕ 0.05 and P ⱕ 0.01) and
RGSSG ( P ⱕ 0.05 and P ⱕ 0.01) activity in the mid-
season was observed, lasting until the end of the sea-
son. There was a signifi cant decrease in the activity
of GPx ( P ⱕ 0.01). There were no changes in GST
activity.
Correlating the level of individual isoprostanes
with antioxidant status indicators, we observed a
positive correlation between isoprostane concentra-
tion and GPx activity ( r ⫽ 0.63, P ⫽ 0.014) before the
Table III. Seasonal variations of some hematological parameters in examined subjects and healthy young
men (control group) in autumn and spring (Means ⫾ SD).
Parameters
Examined group Control group (28)
Autumn Spring % drop Autumn Spring % drop
Hb (mmol/L) 10.6 ⫾ 1.1 8.6 ⫾ 1.4 18.9 9.7 ⫾ 0.1 9.6 ⫾ 0.1 1.0
RBC (10
12 /L) 5.6 ⫾ 0.71 4.9 ⫾ 0.26 12.5 4.9 ⫾ 0.05 5.1 ⫾ 0.04 3.2
Ht (L/L) 0.51 ⫾ 0.09 0.42 ⫾ 0.03 20.0 0.437 ⫾ 0.004 0.452 ⫾ 0.003 3.3
% drop ⫽ [(higher concentration ⫺ lower concentration)/higher concentration] ⫻ 100. Hb, hemoglobin;
RBC, red blood cells; Ht, hematocrit.
Table II. Components of biological and analytical coeffi cients of variation with reference change values (RCV)
for hematological parameters in examined individuals.
Parameters
Examined individuals Healthy population (25)
CV
A (%) CV
I (%) CV
G (%) II RCV
95% (%) CV
I (%) CV
G (%) RCV
95% (%)
RBC (10
12 /L) 1.0 8.7 8.7 0.99 24.1 3.0 6.1 9.9
Hb (mmol/L) 1.0 13.0 12.5 0.96 34.8 2.8 6.8 8.7
Ht (L/L) 0.7 10.7 17.9 1.67 49.7 2.9 6.4 8.7
MCV (fL) 0.4 7.7 2.4 0.32 7.0 1.1 4.2 4.0
MCH (fmol) 0.8 5.6 4.0 0.71 11.0 1.3 5.2 5.0
MCHC (mmol/L) 0.6 3.6 5.3 1.44 14.8 1.6 2.8 5.3
RDW (%) 1.5 9.8 6.3 0.63 17.7 3.7 5.7 10.8
WBC (10
9 /L) 1.2 18.5 10.5 0.57 29.9 10.8 17.4 33.8
LYM (%) 2.0 13.8 11.4 0.83 32.8 10.8 24.1 32.2
MON (%) 2.1 18.6 21.3 1.14 59.3 10.1 25.0 31.2
GRA (%) 0.8 10.7 12.0 1.12 33.3 17.3 23.1 49.9
PLT (10
9 /L) 3.0 19.3 19.6 1.01 55.0 10.0 21.9 28.2
CV
A , analytical coeffi cient of variation calculated from blood control ( n ⫽ 20); CV
I , intra-individual coeffi cient
of variation; CV
G , between-subject coeffi cient of variation; II, individuality index (II ⫽ CV
I /CV
G ); RCV, reference
change value [%]. RBC, red blood cells; Hb, hemoglobin; Ht, hematocrit; MCV, mean corpuscular volume;
MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red blood
cell distribution width; WBC, white blood cells; LYM, lymphocytes; MON, monocytes; GRA, granulocytes; PLT,
thrombocytes.
320 A. Lubkowska et al.
Table IV. Changes in pro-oxidant/antioxidant mediators level in serum following cryostimulation before (T
0 , T
1 , T
2 ), in the middle of the winter-swimming session (T
M ) and after the fi ve-month-long
winter-swimming season (T
0
’
,T
1
’
, T
2
’
).
Parameters
Cryostimulation before the
winter-swimming season
The middle of the
season
Cryostimulation after the 5-month-long
winter-swimming season
T
0 T
1 T
2 T
M T
0 ’
T
1 ’
T
2 ’
TAS ( μ mol/L) 184.30 ⫾ 15.79 175.20 ⫾ 16.94
* T
0 192.30 ⫾ 14.83
* T
0 190.00 ⫾ 12.06
* T0 192.70 ⫾ 12.32
*
*
* T
0 183.70 ⫾ 9.57
* T
0 ’
190.90 ⫾ 9.40
TOS ( μ mol/L) 136.00 ⫾ 22.42 141.10 ⫾ 21.46
*
* T
0 127.60 ⫾ 15.41 129.10 ⫾ 12.93
* T
0 128.70 ⫾ 17.33
* T
0 133.20 ⫾ 15.2
*
* T
0 ’
127.60 ⫾ 22.96
TAS:TOS 1.39 ⫾ 0.28 1.26 ⫾ 0.20
* T
0 1.52 ⫾ 0.21
* T
0 1.48 ⫾ 0.20
* T
0 1.52 ⫾ 0.24
*
* T
0 1.39 ⫾ 0.17
*
* T
0 ’
1.55 ⫾ 0.35
8-Isoprostane (pg/mL) 591.28 ⫾ 62.27 824.74 ⫾ 77.55
*
* T
0 480.58 ⫾ 78.16
*
* T
0 491.27 ⫾ 67.71
*
* T
0 242.25 ⫾ 90.37
*
*
* T
0 214.28 ⫾ 96.90 223.21 ⫾ 77.26
GSH
Total ( μ mol/g Hb) 1.14 ⫾ 0.28 1.01 ⫾ 0.25 1.06 ⫾ 0.18 1.02 ⫾ 0.20 0.96 ⫾ 0.24
* T0 0.90 ⫾ 0.15 0.93 ⫾ 0.21
GSH
Reduced ( μ mol/g Hb) 1.01 ⫾ 0.28 0.81 ⫾ 0.21 0.84 ⫾ 0.18 0.91 ⫾ 0.19 0.90 ⫾ 0.23 0.82 ⫾ 0.16 0.87 ⫾ 0.26
GSSG ( μ mol/g Hb) 0.13 ⫾ 0.03 0.19 ⫾ 0.08
* T
0 0.21 ⫾ 0.08
*
* T
0 0.11 ⫾ 0.05
*
* T
0 ’
0.05 ⫾ 0.01
*
*
* T
0 0.07 ⫾ 0.02
* T
0 ’
0.08 ⫾ 0.02
* T
0 ’
GSH:GSSG 7.12 ⫾ 3.25 3.54 ⫾ 1.73
*
*
* T
0 3.49 ⫾ 2.21
*
*
* T
0 11.21 ⫾ 14.27
* T
0
*
* T
0 ’
16.10 ⫾ 5.41
*
*
* T
0 11.19 ⫾ 6.07
*
* T
0 ’
9.24 ⫾ 3.52
*
* T
0 ’
CAT (U/g Hb) 1.47 ⫾ 0.26 1.47 ⫾ 0.19 1.52 ⫾ 0.23 1.63 ⫾ 0.21
* T0 1.75 ⫾ 0.14
*
* T
0 1.75 ⫾ 0.12 1.75 ⫾ 0.14
SOD (U/mgHb) 2.18 ⫾ 0.83 1.90 ⫾ 0.54
*
* T
0 2.99 ⫾ 0.42
*
* T
0 2.54 ⫾ 0.52
* T0 2.58 ⫾ 0.55
* T0 2.35 ⫾ 0.49 2.48 ⫾ 0.61
SOD:CAT 1.51 ⫾ 0.60 1.32 ⫾ 0.48 1.98 ⫾ 0.33
* T0 1.58 ⫾ 0.36 1.48 ⫾ 0.35 1.34 ⫾ 0.28 1.42 ⫾ 0.36
GST [U/g Hb] 1.65 ⫾ 0.63 1.31 ⫾ 0.5 4.54 ⫾ 1.56
*
*
* T
0 1.04 ⫾ 0.40 1.88 ⫾ 0.81 1.44 ⫾ 0.51 1.79 ⫾ 0.58
GPx [U/g Hb] 3.84 ⫾ 1.66 3.85 ⫾ 0.87 4.76 ⫾ 0.85
* T0 2.33 ⫾ 0.56
*
* T
0 2.28 ⫾ 0.78
*
* T
0 2.24 ⫾ 0.65 2.42 ⫾ 0.82
GPx:CAT 2.70 ⫾ 1.34 2.69 ⫾ 0.86 3.18 ⫾ 0.73
* T
0 1.45 ⫾ 0.37
*
* T
0 1.29 ⫾ 0.39
*
* T
0
,
*
* T
0 ’
1.28 ⫾ 0.36 1.38 ⫾ 0.46
GSSG-R [U/g Hb] 0.26 ⫾ 0.07 0.25 ⫾ 0.10 0.26 ⫾ 0.07 0.34 ⫾ 0.09
* T
0 0.38 ⫾ 0.08
*
* T
0 0.38 ⫾ 0.08 0.39 ⫾ 0.09
TAS, total antioxidant status; TOS, total oxidant status; GSH, glutathione; GSSG, oxidized glutathione; CAT, catalase; SOD, superoxide dismutase; GST, glutathione S-transferase; GPx, glutathione
peroxidase; GSSG-R, glutathione reductase.
Adaptation to cold-stress factors 321
winter season, and after the season there was a
highly signifi cant negative correlation with the GSH:
GSSG ratio ( r ⫽ ⫺ 0.88, P ⫽ 0.004) and GST activity
( r ⫽ ⫺ 0.76, P ⫽ 0.016).
The course of reaction to WBC changed after the
period of winter-swimming, but in only a few cases
(Table V). In response to the fi rst cryostimulation,
before the season, we observed a very signifi cant
increase in 8-ISOP concentration at 30 min after
treatment (T
1 ), and after 24 h it fell below baseline.
After the winter-swimming season, there were no
changes in this stress marker (T
1 and T
2 ).
TAS decreased at 30 min after the fi rst WBC
and then increased signifi cantly after 24 h above the
baseline value ( P ⱕ 0.05), similar to SOD and GST
activity ( P ⱕ 0.01 and P ⱕ 0.001). After a period of
adaptation to cold, the reaction in TAS was similar
although less intense (reduction at 30 min after the
procedure), and 24 h later the values were compa-
rable to the initial ones (T
1 ’
, T
2 ’
). There were no
signifi cant changes in the activity of SOD and GST
in response to WBC. During the second WBC there
was also a slightly less signifi cant increase in the
concentration of the oxidized form of glutathione
(GSSG): Δ (T
0 – T
1 ) ⫽ ⫺ 0.06 ⫾ 0.08 and Δ (T
0 –
T
2 ) ⫽ ⫺ 0.08 ⫾ 0.08 vs. Δ (T
0 ’
– T
1 ’
) ⫽ ⫺ 0.02 ⫾ 0.02
and Δ (T
0 ’
– T
2 ’
) ⫽ ⫺ 0.03 ⫾ 0.02 and therefore we
observed a positive development in the GSH:
GSSG ratio.
Discussion
The use of immersion in cold water and whole-body
cryotherapy is widely used in sport in recovery [29 –
33]. Winter swimming involves taking a dip in ice-
cold natural waters, regularly throughout the winter
season. It is regarded as a readily available and inex-
pensive method to gain resistance to adverse condi-
tions, a natural type of athletic recovery, and as an
adaptive stimulus against respiratory tract infections
and musculoskeletal pains, especially in northern
countries. Epidemiological reports show a decrease
in respiratory tract infections by 40% in regular win-
ter swimmers [34]. However, its effects on health
have been debated.
Being in water with a temperature below 10 ° C
is clearly a threat to the positive heat balance of the
human body and causes systemic backlash in the
form of stress. Stress can be very severe and can
lead to ‘ collapse ’ , or it can be light, but repeated
regularly may cause activation of the body leading
to hardening (a form of hormesis). It is assumed
that if the impact of a stressor (in this case, cold-
stress) is repeated, the body can ‘ get used to ’ the
stimulus and becomes more resistant, although the
mechanisms of this phenomenon are not yet fully
understood. It is important to dose cold gradually
and at appropriate levels, as mismatched hardening
Table V. Trends in response to cryogenic temperatures during cryostimulation before (T
0 – T
1 , T
0 – T
2 , T
1 – T
2 ) and after (T
0 ’
– T
1 ’
, T
0 ’
– T
2 ’
, T
1 ’
– T
2 ’
) swimming season.
Δ (T
0 – T
1 ) Δ (T
0 – T
2 ) Δ (T
1 – T
2 ) Δ (T
0 ’
– T
1 ’
) Δ (T
0 ’
– T
2 ’
) Δ (T
1 ’
– T
2 ’
)
TAS 9.07 ⫾ 24.69 ⫺ 8.00 ⫾ 17.40
* Δ (T0 ’ – T2 ’ ) ⫺ 17.07 ⫾ 23.55 9.00 ⫾ 6.05 1.78 ⫾ 9.07
* Δ (T0 – T2) ⫺ 7.21 ⫾ 9.76
TOS ⫺ 5.14 ⫾ 3.65 8.35 ⫾ 18.77 13.50 ⫾ 19.09 ⫺ 4.50 ⫾ 3.39 1.14 ⫾ 17.19 5.64 ⫾ 17.55
TAS:TOS 0.13 ⫾ 0.18 ⫺ 0.13 ⫾ 0.18 ⫺ 0.26 ⫾ 0.12 0.12 ⫾ 0.09 ⫺ 0.02 ⫾ 0.24 ⫺ 0.15 ⫾ 0.27
GSH
Total 0.13 ⫾ 0.35 0.07 ⫾ 0.34 ⫺ 0.05 ⫾ 0.20 0.05 ⫾ 0.33 0.00 ⫾ 0.25 ⫺ 0.05 ⫾ 0.37
GSH
Reduced 0.19 ⫾ 0.37 0.16 ⫾ 0.39 ⫺ 0.03 ⫾ 0.21 0.08 ⫾ 0.32 0.03 ⫾ 0.24 ⫺ 0.04 ⫾ 0.36
GSSG ⫺ 0.06 ⫾ 0.08 ⫺ 0.08 ⫾ 0.08
*
* Δ (T0
′ – T2
′ ) ⫺ 0.02 ⫾ 0.07 ⫺ 0.02 ⫾ 0.02 ⫺ 0.03 ⫾ 0.02
*
* Δ (T0 – T2) 0.00 ⫾ 0.01
GSH:GSSG 3.57 ⫾ 4.30 3.62 ⫾ 4.15
* Δ (T0
′ – T2
′ ) 0.05 ⫾ 2.94 4.90 ⫾ 5.77 6.85 ⫾ 4.19
* Δ (T0 – T2) 1.94 ⫾ 7.34
CAT ⫺ 0.06 ⫾ 0.27 ⫺ 0.05 ⫾ 0.29 ⫺ 0.04 ⫾ 0.16 0.00 ⫾ 0.20 0.00 ⫾ 0.20 0.00 ⫾ 0.18
SOD 0.27 ⫾ 1.30 ⫺ 0.80 ⫾ 0.83
*
* Δ (T0
′ -T2
′ ) ⫺ 1.08 ⫾ 0.79
*
* Δ (T1
′ – T2
′ ) 0.23 ⫾ 0.40 0.10 ⫾ 0.54
*
* Δ (T0 – T2) ⫺ 0.13 ⫾ 0.31
*
* Δ (T1 – T2)
SOD:CAT 0.18 ⫾ 0.98 ⫺ 0.47 ⫾ 0.73 ⫺ 0.66 ⫾ 0.41
*
* Δ (T1
′ – T2
′ ) 0.13 ⫾ 0.30 0.06 ⫾ 0.37 ⫺ 0.07 ⫾ 0.23
*
* Δ (T1 – T2)
GPx ⫺ 0.05 ⫾ 1.93 ⫺ 0.91 ⫾ 1.86 ⫺ 0.90 ⫾ 1.15 0.04 ⫾ 0.40 ⫺ 0.13 ⫾ 0.60 ⫺ 0.17 ⫾ 0.67
GPx:KAT 0.01 ⫾ 1.61 ⫺ 0.47 ⫾ 1.70 ⫺ 0.48 ⫾ 0.70 0.00 ⫾ 0.21 ⫺ 0.08 ⫾ 0.34 ⫺ 0.09 ⫾ 0.45
GSSG-R 0.00 ⫾ 0.09 0.00 ⫾ 0.09 0.00 ⫾ 0.08 0.00 ⫾ 0.10 0.00 ⫾ 0.08 0.00 ⫾ 0.04
TAS, total antioxidant status; TOS, total oxidant status; GSH, glutathione; GSSG, oxidized glutathione; CAT, catalase; SOD, superoxide dismutase; GST, glutathione S-transferase; GPx, glutathione
peroxidase; GSSG-R, glutathione reductase.
* Statistically signifi cant difference at P ⱕ 0.05;
*
* statistically signifi cant difference at P ⱕ 0.01;
*
*
* statistically signifi cant difference at P ⱕ 0.001.
322 A. Lubkowska et al.
WBC and winter swimming) lead to habituation,
especially concerning thermal sensation and com-
fort. Additionally, Lepp ä luoto et al. [43] showed that
sustained cold-induced stimulation of norepineph-
rine was remarkably similar between WBC and win-
ter swimming.
The present study shows that the single exposi-
tion to cryogenic temperatures during WBC after the
season of winter-swimming caused much smaller
changes in prooxidant/antioxidant status than WBC
without adaptation. Winter-swimming signifi cantly
lowered the resting plasma levels of 8-ISOP, which
was observed already in the middle of the season,
and WBC after the season did not cause a signifi cant
increase in their concentrations, as was the case
before the season. Isoprostanes are not only the indi-
cators of increased free radical reactions – they also
play a role of mediators of oxidant damage both in
physiological and pathophysiological processes. Our
previous studies showed that 8-ISOPs are a sensitive
indicator of oxidative stress in exposure to cold,
which has also been confi rmed in this study [42].
According to Roberts and Morrow [21], there are a
number of attributes confi rming the importance of
measurement of isoprostanes as a reliable indicator
of oxidative stress in vivo . Their formation is modu-
lated by antioxidant status and their levels are not
affected by the lipid content of the diet. They are
stable compounds, specifi c products of lipid peroxi-
dation.
Particularly signifi cant changes in our study were
WBC-induced changes in the glutathione system
and glutathione-dependent enzymes. The changes
in GSH level are one of the earliest signs of oxidative
stress, and blood and tissue levels of GSH are sensi-
tive to oxidative stress. We observed a signifi cantly
reduced resting level of glutathione disulfi de (in
favor of the GSH fraction in total GSH), and also
the nature of the concentration changes in response
to WBC after the season of winter-swimming indi-
cates the benefi cial adaptive changes in the body.
Erythrocytes contain glutathione as an antioxidant,
mainly to prevent against the oxidation of hemog-
lobin. Intracellular glutathione is rapidly oxidized to
GSSG in response to an increase in free radicals
(e.g. the presence of H2O2 and hydroperoxides
enzymatically with glutathione peroxidase) but is
rapidly reduced back to GSH if the oxidative stress
is not severe and the antioxidant system is effi cient
enough. If the stress exceeds the capacity of the cell
to reduce GSSG to GSH, the increase in GSSG in
the body may be used as a marker of oxidative stress
[44]. A smaller WBC-induced increase in GSSG
after a period of adaptation (winter-swimming)
indicates favorable changes in the antioxidant sys-
tem, which was further confi rmed by an increase in
the resting activity of GSSG-R in this period, a fl a-
voprotein whose function is to maintain the correct
concentration of GSH in cells thanks to the ability
conditions can cause irreversible changes and dam-
age in the body [35].
In our study, the winter swimmers swam on aver-
age 2 – 3 times a week for a period of fi ve months,
from November to March. Each immersion in water
took from 2 – 5 min, covered the whole body, exclud-
ing the head. In this study we attempted to identify
the extent to which winter-swimming induced
adaptive changes in the body, in relation to the
pro-oxidant/antioxidant status. Additionally, it was
suspected that adaptation to cold stimuli and the
improvement in body hardening could be related to
an increase in the protection against oxidative stress
which occurs during exposure to cold. Literature
data confi rm that regular winter swimmers have an
increased resting number of white blood cells, mono-
cytes and plasma interleukin-6 [36,37].
While the previously published studies also show
regular winter-swimmers to have an increase in the
baseline concentration of reduced glutathione and a
decrease in the concentration of oxidized glutathi-
one in the erythrocyte, higher activities of erythro-
cytic catalase glutathione peroxidase and superoxide
dismutase [12,38], there are still no reports in lit-
erature which would describe any change in response
to stress factors (in this case cryogenic tempera-
tures) after a period of adaptation to the cold, that
would testify to improved resistance to stress factors
and could explain the widely recognized mechanism
of improved resistance by the cold hardening pro-
cess. We have managed to show just this in our
experimental scheme. We observed an improvement
in reaction to cold-induced stress after a period of
adaptation, with prolonged immersion in water at a
temperature in the range of 4 – 15 ° C used as a hard-
ening factor (cold-wet) to the reaction to whole-
body exposure to cryogenic air at ⫺ 130 ° C in a
cryogenic chamber.
Our previous studies showed that a single WBC
is a stress to the body that disrupts the balance of
the body ’ s pro-oxidant-antioxidant balance [39].
Subsequent studies confi rmed that the repetition of
these treatments in a series of 10, 15, or 20, affects
the lipid profi le [40], the level of infl ammation mark-
ers [41] and increases the antioxidant capacity of the
system [42]. For fi nancial reasons, WBC is usually
limited to 10 up to 20 daily treatments, often as
complement to primary therapy or in sports injuries,
which means an average of 4 weeks of exposure.
However, it should be noted that in most cases ben-
efi cial changes typically occur after 20 sessions and
its stability depends on the duration of the WBC
treatment. Therefore, it seems reasonable to seek
long-term methods that are less expensive than WBC.
Although it could be expected that exposure to dry
cold in a cryochamber would be better tolerated by
the body and less noticeable than immersion in cold
water, research conducted by Smolander et al. [15]
showed that regular short-term cold stimuli (both
Adaptation to cold-stress factors 323
to convert GSSG into GSH with NADPH as a co-
enzyme.
The increased GSSG after the fi rst WBC, before
the season of winter-swimming, resulted in a lower
activity of GST at 30 min after WBC and increased
GST activity after 24 h, which can be explained by
the necessity of removing the oxidized glutathione
outside the cell, i.e. glutathione produced during the
reduction reaction of hydrogen peroxide with GSH
as a proton donor. Glutathione S-transferase inacti-
vates endogenous unsaturated aldehydes, epoxides
and peroxides, or reactive products of oxidative stress
[45]. Reduced proportions of oxidized glutathione in
the total amount of glutathione after the period of
hardening is very benefi cial for the body, because
glutathione, apart from scavenging ROS and regen-
erating other antioxidants, is also involved in restor-
ing damaged cell components, mainly proteins and
lipids of cell membranes, and is involved in maintain-
ing a proper redox potential of cells [46] which is
important in the regulation of intracellular metabo-
lism [47] and also in the processes of growth and cell
differentiation and apoptosis [48,49].
From a clinical point of view, it is important to
mention the unfavorable changes in the red blood
cell system that appeared during the mid-winter
swimming season. In the case of some indicators
these changes even intensifi ed during the rest of the
season, which may be a factor detracting the use of
winter bathing. Of particular note is the signifi cant
progressive decrease in the number of red blood
cells, hemoglobin and hematocrit, which was
accompanied by an increase in red blood cell dis-
tribution width. Perhaps prolonged exposure to low
temperatures in winter-swimming could have led to
changes in iron metabolism or increased blood
hemolysis, but this is diffi cult to interpret without
the determination of parameters of iron metabo-
lism. The simultaneous increase in RDW red blood
cell distribution width can suggest the appearance
of a greater number of juvenile red blood cells in
peripheral blood. It should be noted that the lack
of a control group is a weakness in our study;
although changes in hematologic indices in the
study group were analyzed with regard to their ini-
tial level of output, which was a type of control.
However this is not enough to exclude the possibil-
ity of changes resulting from wintertime cold accli-
matization responses.
The biological variation could complicate the
interpretation of our results, performed consecutive
for the same participants during the 5-month period
of the experiment.
In order to make sure whether the changes in
hematological parameters did not result only from
the biological or seasonal variation, we calculated
intra-individual coeffi cient of variation (CV
I ),
between-subject coeffi cient of variation (CVG), ref-
erence change value (RCV) and the percent drop
between autumn and spring in examined subjects
(Tables II and III). The CV
I values and RCV
S were
higher for all red blood cells parameters, monocytes
and PLT than the values for healthy population pre-
viously reported in the literature [25], also higher
% drop was observed in subjects exposed to winter
swimming compare to reference value for seasonal
variation [28]. The obtained results indicate that
prolonged winter swimming contributed to a
decrease in hemoglobin concentration, hematocrit
and erythrocyte counts. Although it was possible to
refer the results for erythrocyte to reference values
in the literature, the biological variability of pro-
oxidant/antioxidant mediators is still not estab-
lished, whereas our previous results do not concern
a suffi ciently large and comparable population that
could serve as reference in terms of age and sex of
subjects.
On the contrary, the observed increase in the rest-
ing activity of superoxide dismutase and catalase, the
main endogenous antioxidant enzymes, after the sea-
son of winter-swimming, should be seen as a benefi -
cial effect for the human body. The results indicate
benefi cial adaptive changes in the antioxidant system
in healthy individuals who decide to use winter-
swimming to increase their resistance. Therefore they
confi rm the hypothesis that regular winter-swimming
may increase the capacity and effi ciency of the anti-
oxidant system. In addition, winter-swimming may
also increase the resistance and improve the body ’ s
defensive reaction to the factors increasing oxidative
stress, thereby increasing tolerance of the human
body to stressors.
Acknowledgements
This paper was supported by the Polish Ministry of
Science and Higher Education (Grant no. N N404
312940).
Declaration of interest : The authors report no
confl ict of interest. The authors alone are responsible
for the content and writing of the article.
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