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Acute effect of a single whole-body cryostimulation on
prooxidant–antioxidant balance in blood of healthy, young men
Anna Lubkowska
a,
, Monika Chudecka
b
, Andrzej Klimek
c
, Zbigniew Szygu"a
d
, Barbara Fra˛czek
e
a
Department of Physiology, Faculty of Natural Sciences of Szczecin University, al. Piasto
´w 40b, 71-065 Szczecin, Poland
b
Department of Anthropology, Faculty of Natural Sciences of Szczecin University, al. Piasto
´w 40b, 71-065 Szczecin, Poland
c
Institute of Human Physiology, University School of Physical Education, P.O. Box 62 (AWF), 31-571 Krakow, Poland
d
Department of Sports Medicine, University School of Physical Education, P.O. Box 62 (AWF), 31-571 Krakow, Poland
e
Department of Human Nutrition, University School of Physical Education, P.O. Box 62 (AWF), 31-571 Krakow, Poland
article info
Article history:
Received 18 January 2008
Accepted 27 August 2008
Keywords:
Cryostimulation
Plasma oxidative capacity
Plasma antioxidative capacity
abstract
1. We have examined the prooxidative–antioxidative reaction to extremely low temperatures
(130 1C) during a one-time cryostimulation in 15 young, clinically healthy individuals.
2. The total lipid peroxides as the total oxidative status (TOS) and the total antioxidative status (TAS)
were measured in blood plasma collected in the morning of the day of cryostimulation, 30 min after
the cryostimulation, and on the following morning.
3. The level of stress expressed by total oxidative status in plasma, resulting from exposure to
extremely low temperatures, was statistically significantly lowered 30 min after leaving the
cryochamber than prior to the exposure. The next day, the TOS level still remained lower than the
initial values. The TAS level decreased after leaving the cryochamber and remained elevated
the following day.
&2008 Elsevier Ltd. All rights reserved.
1. Introduction
The whole-body cryotherapy (or cryostimulation) has found its
application in treating many diseases. In professional sports, its
usage has been beneficial in improving biological regeneration
and recovery from post-exercise muscle injury (Banfi et al., 2008;
Zimmer, 2003). Local or systemic use of extremely low tempera-
ture speeds up the healing process of impaired tissues, weakens
inflammable reaction, lowers muscle spasticity, and also has
analgesic properties (Yaumauchi et al., 1981;Bauer and Skrzek,
1999;Hubbard et al., 2004;Nadler et al., 2004;Krasuski and
Dederko, 2005).
Cooling tissue causes a decrease in the efficiency of cellular
respiration, releases enzymes from impaired cells, and inhibits the
breakdown of high-energy compounds (ATP, CP) and glycogen
(Zimmer, 2003). Cryostimulation causes muscle trembling and a
reduction in metabolism by about 50%, which decreases oxygen
demand. After 4 min of exposure to extremely low temperature
(from 100 t o 130 1C), the body experiences considerable
haemangiectasia (angio-osteodystrophy) and an increase in blood
supply to internal organs, which leads to an increase in muscle
oxygen concentration (Bauer and Skrzek, 1999;Zagrobelny and
Zimmer, 1999). A subsequent effect of exposing the body to
cryogenic temperature is a fall in lactate and histamine concen-
tration levels and an increase in bradykinin and angiotensin
concentrations, which, in effect, cause a considerable pain
reduction (Griffin and Reddin, 1981). Cryostimulation has also
been noted to increase the secretion of adrenotropic hormones
(ACTH), cortisol, adrenaline, norepinephrine, and testosterone in
plasma. Stimulation of adrenocorticotropin has been known to
reduce inflammation processes (Leppa
¨luoto et al., 2008). Initially,
cryotherapy was predominantly used on protracted inflammable
states accompanying rheumatic diseases. Research on the influ-
ence of cryostimulation on the production of reactive oxygen
species (ROS), lipid peroxidation and the antioxidative response of
the body, focused mostly on the treatment of people with
rheumatoid arthritis (Yaumauchi et al., 1981;Janiszewski, 1998;
Ksi˛ez
˙opolska-Pietrzak et al., 1999;Metzger et al., 2000). The
cryotherapeutic treatment of these individuals was accompanied
by kinesitherapy as an integral component of this form of
rehabilitation, and in the case of athletes, cryostimulation was
accompanied by physical training (Swenson et al., 1996;Woz
´niak
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Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/jtherbio
Journal of Thermal Biology
0306-4565/$ - see front matter &2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jtherbio.2008.08.003
Corresponding author. Tel.: +48 91 444 2754.
E-mail address: annalubkowska@tlen.pl (A. Lubkowska).
Journal of Thermal Biology 33 (2008) 464 –467
Author's personal copy
et al., 2002, 2007b;Krasuski and Dederko, 2005). In both cases, it
is difficult to interpret clearly the influence of low temperature
due to an increase in lipid peroxidation that is a result of an
increased ROS production during physical effort, as confirmed by
numerous studies (Alessio, 1993;Urso and Clarkson, 2003;
Bloomer et al., 2005, 2006;Metin et al., 2003).
An exposure to an acute cold temperature represents an
obvious stress, which could lead to various physiological and
metabolic reactions in the organism. Among others, prooxidan-
t–antioxidant processes play an important role in the develop-
ment of several various pathologies that could also trigger
adaptation changes to protect tissues against disturbances in the
pro–antioxidant balance (Dugue
´et al., 2005;Wozniak et al.,
2007b). Limited studies on the effect of whole-body cryostimula-
tion on total oxidative status (TOS) and total antioxidative status
(TAS) in the plasma of healthy men not involved in physical
exercise exist. Thus, the aim of this study was to assess
pro–antioxidant status in healthy, young men after one session
of whole-body cryostimulation.
2. Materials and methods
Fifteen healthy, 721 year old men, with a normal body weight
(body mass index BMIo28) and never previously exposed to any
form of cryotherapy, were recruited for this study.
The characteristics of the examined group are presented in
Table 1. All participants were asked to sign a written consent. The
project of the study was approved by the Bioethical Committee of
the Regional Medical Society in Cracow. Prior to engaging in the
experiment, all participants underwent a physical examination
to exclude any contraindications against cryostimulation. The
individuals were exposed to a one-time session of extremely low
temperature (130 1C) in a cryogenic chamber at the Ma"opolska
Center of Cryotherapy in Cracow, Poland, in groups of 4 persons
each. The session lasted 3 min.
Each participant’s entry to the cryochamber was preceded by a
30 s adaptation in the vestibule at a temperature of 60 1C. During
the procedure, the participants wore shorts, socks, wooden clogs,
gloves and a hat covering the auricles against frostbite. Blood
samples were obtained from an antecubital forearm vein after a
10 min rest in a sitting position, using vacutainer system tubes
(Sarstedt, Germany). During the day of cryostimulation, blood
specimens were collected after an overnight fasting, in the
morning between 8.00 and 8.30 am (sample A), 30 min after
cryostimulation, e.g. 9.00–9.30 am (sample B), and the next
morning, between 8.00 and 8:30 am (sample C). We examined the
entire blood morphology and carried out the smear analysis. After
centrifugation, the serum and plasma were divided into aliquots
and immediately deep-frozen at 70 1C. The total lipid peroxides
as the total oxidative status (TOS, PerOx) and the total antioxi-
dative status (TAS, ImAnOx) were measured with the photometric
test method, Immunodiagnostik AG, Bensheim-Germany. The
sensitivity of the assay for TOS was 7
m
mol/L, and the intra-
and inter-assay variability were p3.1% and p5.1%, respectively.
Detection limit for the TAS kit was 130
m
mol/L, and the intra- and
inter-assay variability were p2.3% and p2.43%, respectively.
2.1. Statistics
Statistical analysis was performed using the Statistica 6
package. Data were checked for normal distribution using the
Shapiro–Wilk test. Since in some cases the data distribution was
not normal, the Friedman’s ANOVA for repeated measurements
was applied to examine the overall changes. Thereafter, the
Wilcoxon signed-rank test for paired non-parametric data was
used to determine variations from the initial levels during the
experiment, as recommended for this type of data (Cohen, 1988).
Values of po0.05 were considered statistically significant. In
addition, correlation coefficients between variables were calcu-
lated using a Spearman analysis.
3. Results
The morphological parameters and haematologic coefficients
were observed to be within clinical and laboratory norms in all
the examined individuals (Table 1). The results of responses
to one-time whole-body cryostimulation are presented in Figs. 1
and 2.
According to the values provided by Immunodiagnostik AG, we
found that the level of stress, expressed by means of the total
oxidative stress (TOS ¼PerOx) in plasma was very low ¼138
m
mol/L (85–160
m
mol/L) in the initial samples (A). As a result of
cryostimulation we observed a statistically significant decrease in
TOS, to 131.1
m
mol/L (64–165
m
mol/L) 30 min after leaving the
cryochamber (B). On the day following the cryostimulation, the
TOS level rose slightly, to 132
m
mol/L (87–143
m
mol/L) (C) and was
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Table 1
Physical characteristics, hematological parameters and cortisol levels in the serum
of the examined men (the values are mean7SD, minimum, and maximum)
n Mean value7standard
deviation (SD)
Min Max
Height (cm) 15 180.1577.59 166.00 190.00
Body mass (kg) 74.7076.52 63.00 86.50
BMI (kg/m
2
) 23.0471.65 21.05 25.40
WBC (10
3
/
m
L) 4.3671.31 1.51 5.54
RBC (10
6
/
m
L) 5.1970.10 5.09 5.41
HGB (g/dL) 15.2370.53 14.5 16.2
HCT (%) 45.5571.18 43.9 47.1
MCV (fL) 87.8272.06 85.2 90.4
MCH (pg) 29.3970.83 28.1 31.0
PLT (10
3
/
m
L) 230.73738.53 139.0 287.0
Cortisol A (
m
g/dl) 10.6373.16 5.78 16.1
Cortisol B (
m
g/dl) 10.1072.77 6.26 15.2
BMI—body mass index; WBC—white body cell, RBC—red body cell, HGB—
hemoglobin, HCT—hematocrit, MCV—mean corpuscular volume, MCH—mean
corpuscular hemoglobin, MCHC—mean corpuscular hemoglobin concentration,
PLT—platelets, Cortisol A—serum cortisol values before, and Cortisol B—30 min
after cryostimulation.
Total oxidative status
TOS
Median
25%-75%
Min-Max
A*
40
60
80
100
120
140
160
180
µmol/l
B* C
Fig. 1. Changes in plasma total oxidative status in healthy subjects at rest (A), at
30 min after cryostimulation (B) and in the morning the day after (C). *pp0.05
statistically significant difference B vs. A.
A. Lubkowska et al. / Journal of Thermal Biology 33 (2008) 464–467 465
Author's personal copy
still lower than the initial values, although the difference was
not statistically significant. Similarly, the initial level (A) of the
plasma total antioxidative status (TAS ¼ImAnOx): 193.00
m
mol/L
(165–228
m
mol/L) was low. The TAS level significantly decreased
to 166
m
mol/L (157–203
m
mol/L) 30 min after leaving the cryo-
chamber (B). The following day (C), an increase in TAS level up to
188
m
mol/L (171–226
m
mol/L) was observed, and was statistically
significant in relation to the B measurement the day before. One-
time cryostimulation did not lead to significant changes in serum
cortisol levels (Table 1).
4. Discussion
The reaction of the human body to extremely low temperature
in a cryochamber initially resulted in a vasospasm and then
vasodilation and massive tissue hyperemia (Janiszewski, 1998;
Zagrobelny and Zimmer, 1999;Nadler et al., 2004). As a result of a
reaction catalyzed by xanthine oxidase due to reperfusion, an
increase of ROS, causing damage to both nucleic acids and
proteins as well as lipid peroxidation was observed. Due
to thermogenic trembling, the production of heat can exceed
3–5 times the level of the heat produced in normal metabolic
state (Jackson and Sammut, 2004). Simultaneously, heat is lost
due to convection and the demand for ATP increases. A higher
demand for ATP increases the metabolism of oxygen in the
mitochondria (Ksi˛ez
˙opolska-Pietrzak et al., 1999;Bartosz, 2003).
This leads to the intensification of ROS generation, through a one-
electron reduction of oxygen (Kopprasch et al., 1997). Due to the
cooling and stimulation of metabolism, the mitochondria in a
body exposed to extremely low temperature conditions produce
10 times more anion-radical superoxides (and after its dismuta-
tion, perhydride) (Bartosz, 2003). Additionally, in hepatocyte
peroxisomes an intensification of the
b
-oxidation of fatty acids
and the consequential generation of perhydride occur (Hamel
et al., 2001).
Prior to carrying out the experiment, we anticipated that an
increase in the concentration of lipid peroxidation products would
be seen, which was to increase plasma total oxidative level in the
subsequent blood samples after a single stay session in the
cryogenic chamber. However, our results show a statistically
significant drop in TOS, accompanied by a decrease in TAS in
plasma. Both parameters correlated highly with each other in the
B samples collected 30 min after leaving the cryochamber. It is
likely to be a result of a decreased ROS generation or a significant
participation of non-enzymatic systems in their removal.
Zagrobelny et al. (1993) observed a significant increase in
the concentration of adrenaline, norepinephrine, ACTH, and
beta-endorphins in blood serum 30 min after cryostimulation.
Catecholamines—important regulators of metabolism—may have
some impact on the production of reactive oxygen species.
Whilst only few studies on the influence of cryostimulation on
oxidative and antioxidative processes in the cells of ill and healthy
individuals have been published, it is suggested that repeated
cryochamber sessions may cause adaptive changes, for example
an increase in antioxidative capacity (Janiszewski, 1998). Woz
´niak
et al. (2007b) when compared with the activity of superoxide
dismutase, catalase, and glutathione peroxidase after training
accompanied by cryostimulation. It was found that their activities
were lower when training was preceded by exposure to extremely
low temperature conditions. Additionally, in the next experiment
it was found that low temperature solely caused neither a
labialisation of lysosomal membranes nor significant changes in
the activities of lysosomal hydrolases (Woz
´niak et al., 2007a).
Siems and Brenke (1992) and Siems et al. (1994) observed that
acute cold stimuli (such as winter swimming) induced a decrease
in major plasma antioxidants (i.e. ascorbic acid and uric acid) and
an increase in the concentration of hydroxynonenal in plasma
(a marker of lipid peroxidation).
Dugue
´et al. (2005) compared acute and long-term changes in
plasma antioxidant capacity in women who attended whole-body
cryotherapy regularly. They observed a significant increase in the
value of total peroxyl radical trapping antioxidant capacity of
plasma (TRAP) 2 min after cold stress in the first 4 weeks of their
study. Thirty-five minutes after application of cold stress, the
values of TRAP did not vary from the baseline values. These data
may suggest that cold stress activates antioxidant defense in the
body, especially at the initial stages of an adaptation period.
However, changes in the TRAP values showed significant varia-
tions between subjects. High individual variation of response to
stress caused by cold is confirmed by our own research, in
particular with regard to total oxidative status, which enables
estimation of lipid peroxidation increase.
Cortisol, the concentration of which increases in response to
stressogenic factors, is generally the most frequently investigated
marker in cryostimulation research. Reports on changes in its
concentrations are often contradictory. In this research, we did not
note any changes in cortisol concentration in serum caused by a
one-time session of cryotherapy. Zagrobelny and Zimmer (1999)
reported an increase in the concentration of this hormone in blood
serum after cryotherapy treatment, but Woz
´niak et al. (2007a) did
not report any statistically significant changes in cortisol con-
centrations both after a single stay session in a cryochamber, and
after 6 days of training accompanied by cryostimulation. They
observed, however, an increasing tendency in cortisol levels after
the sixth day of training, and then a decrease after the 10th day of
training in conjunction with cryostimulation. Leppa
¨luoto et al.
(2008) studying the effects of long-term winter swimming and
whole-body cryotherapy, observed that plasma cortisol exhibited
an insignificant increase after the first winter swimming, as did
ACTH. During whole-body cryotherapy, plasma cortisol at 15 min
after exposure was significantly lower in week 4 than in week 1.
After the 11th week of the study, plasma cortisol levels were lower
at 15 and 35min in comparison with the period preceding the
start of the experiment.
Limited literature on the influence of cryogenic temperatures
on antioxidative mechanism and on the generation of free radicals
leaves room for future research. The next step should be to
estimate the influence of cryostimulation series (in arrangements
most often used by patients and athletes) on plasma oxidative–
antioxidative coefficients.
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Total antioxidative status
TAS
Median
25%-75%
Min-Max
150
160
170
180
190
200
210
220
230
240
µmol/l
A* B** C**
Fig. 2. Changes in plasma total antioxidative status in healthy subjects at rest (A),
at 30 min after cryostimulation (B), and in the morning the day after (C). *pp0.05
statistically significant difference B vs. A, **pp0.01 statistically significant
difference B vs. C.
A. Lubkowska et al. / Journal of Thermal Biology 33 (2008) 464–467466
Author's personal copy
In conclusion, one session of whole-body cryostimulation
causes disturbances in the prooxidant–antioxidant balance—the
level of total oxidative status in plasma was statistically
significantly lowered 30 min after leaving cryochamber and
remained low the following day, whereas the level of total
antioxidative status decreased after cold exposure and elevated
the next day.
Acknowledgment
The authors express special thanks to all participants for their
participation in this research project.
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