See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/282945166

The whole body cryostimulation modifies irisin concentration and reduces

inflammation in middle aged, obese men

Article in Cryobiology · October 2015

DOI: 10.1016/j.cryobiol.2015.10.143

CITATIONS READS

59 380

7 authors, including:

Katarzyna Dzik Tomasz Grzywacz

Gdansk University of Physical Education and Sport Instytut Sportu
19 PUBLICATIONS 0 CITATIONS 24 PUBLICATIONS 0 CITATIONS

SEE PROFILE SEE PROFILE

Sylwester Kujach Damian Jozef Flis

Gdansk University of Physical Education and Sport Medical University of Gdansk
37 PUBLICATIONS 0 CITATIONS 50 PUBLICATIONS 0 CITATIONS

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Ewa Ziemann on 16 April 2018.

The user has requested enhancement of the downloaded file.

 Cryobiology 71 (2015) 398e404

Contents lists available at ScienceDirect

Cryobiology
journal homepage: www.elsevier.com/locate/ycryo

The whole body cryostimulation modifies irisin concentration and

reduces inflammation in middle aged, obese men
Katarzyna Dulian a, Radosław Laskowski b, Tomasz Grzywacz c, Sylwester Kujach b,
Damian J. Flis d, Mirosław Smaruj e, Ewa Ziemann f, *
a
Gdansk University of Physical Education and Sport, Department of Physiotherapy, Kazimierza Gorskiego 1, 80-336 Gdansk, Poland
b
Gdansk University of Physical Education and Sport, Department of Physiology, Kazimierza Gorskiego 1, 80-336 Gdansk, Poland
c
Institute of Sport, Department of Physiology, Trylogii 2/16, 01-982 Warsaw, Poland
d
Medical University of Gdansk, Department of Bioenergetics and Physiology of Exercise, Marii Skłodowskiej-Curie 3A, 80-210 Gdansk, Poland
e
Gdansk University of Physical Education and Sport, Department of the Theory of Sport, Kazimierza Gorskiego 1, 80-336 Gdansk, Poland
f
Gdansk University of Physical Education and Sport, Department of Physiology and Pharmacology, Kazimierza Gorskiego 1, 80-336 Gdansk, Poland

a r t i c l e i n f o a b s t r a c t

Article history: The anti-inflammatory effect induced by exposure to low temperature might trigger the endocrine
Received 27 April 2015 function of muscle and fat tissue. Thus, the aim of this study was to investigate the influence of the whole
Received in revised form body cryostimulation (CRY) on irisin, a myokine which activates oxygen consumption in fat cells as well
15 August 2015
as thermogenesis. In addition, the relationship between hepcidin (Hpc) e hormone regulating iron
Accepted 11 October 2015
Available online 22 October 2015
metabolism, and inflammation was studied.
A group of middle aged men (n ¼ 12, 38 ± 9 years old, BMI > 30 kg m2) participated in the study.
Subjects were exposed to a series of 10 sessions in a cryogenic chamber (once a day at 9:30 am, for 3 min,
Keywords:
Cold
at temperature 110  C). Blood samples were collected before the first cryostimulation and after
Hepcidin completing the last one. Prior to treatment body composition and fitness level were determined. The
Iron applied protocol of cryostimulation lead to rise the blood irisin in obese non-active men (338.8 ± 42.2 vs
Cardiorespiratory fitness level 407.6 ± 118.5 ng mL1), whereas has no effect in obese active men (371.5 ± 30.0 vs 343.3 ± 47.6 ng mL1).
Fat tissue Values recorded 24 h after the last cryo-session correlated significantly with the fat tissue, yet inversely
Irisin with the skeletal muscle mass. Therefore, we concluded the subcutaneous fat tissue to be the main
source of irisin in response to cold exposures. The applied cold treatment reduced the high sensitivity C-
reactive protein (hsCRP) and Hpc concentration confirming its anti-inflammatory effect.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction hepcidin (Hpc), which is a disulfide-rich peptide produced by he-

patocytes and an iron-regulating hormone [35]. Hepcidin synthesis
Obesity is a serious health problem in developed countries, and is also stimulated by high blood iron concentration [12]. An
the prevalence of obesity has increased dramatically for several increased level of blood hepcidin inhibits iron transport from the
decades [43]. Wide range of co-morbidities such as: insulin resis- duodenum, thus limiting iron absorption [33]. The rise of hepcidin
tance, metabolic syndrome, type 2 diabetes, hypertension, chronic concentration found in a chronic state of inflammation can be
kidney disease, cardiovascular disease, heart failure, cancer, and treated as a non-specific host defense strategy, aimed at limiting
dementia are associated to obesity through the low-grade inflam- the availability of iron for microorganisms [32]. Conversely, in
mation [26]. This stage is characterized by an enhancement of the overweight and obese people with a low-grade systemic inflam-
pro-inflammatory cytokines' concentrations, particularly through mation, a positive association was found between the C-reactive
their release from adipose cells and macrophages [41]. The elevated protein and ferritin [4]. Unexpectedly, obese subjects exhibited low
pro-inflammatory cytokines might stimulate the synthesis of iron concentrations, even anemia, in response to elevated hepcidin
concentrations [1].
There are few strategies decreasing chronic low-grade inflam-
mation in obese people. Since low grade inflammation can be
* Corresponding author.
associated with physical inactivity in healthy subjects [38], physical
E-mail address: [email protected] (E. Ziemann).

http://dx.doi.org/10.1016/j.cryobiol.2015.10.143
0011-2240/© 2015 Elsevier Inc. All rights reserved.
 K. Dulian et al. / Cryobiology 71 (2015) 398e404 399

exercise is one of the mechanisms that may be stimulate the anti- not to change any aspect of their daily habits, such as diet, and to
inflammatory response and protection against chronic medical avoid any form of exercise.
disorders induced by this stage [36]. However, in some cases
physical activity was almost completely ineffective in reducing 2.2. Body composition assessment
systemic inflammation [25]. Thus, there is a need to look for
additional methods supporting the anti-inflammatory strategies. Body mass (BM) and body composition were estimated using a
Last papers revealed that the whole-body cryostimulation (CRY) is multi-frequency impedance plethysmograph body composition
considered as the an alternative and complementary method to analyser (In Body 720, Biospace Analyzer, Korea). This analyser
both above mentioned strategies for reducing inflammation accurately measured the amount of body water and body compo-
[16,29,45]. Nonetheless, still physical fitness level might modify the sition, including fat mass, free fat mass, skeletal muscle mass and
effect of CRY [45]. soft lean mass. Additionally, the visceral fat area was determined
Data describing the influence of coldness exposure on irisin is and expressed in cm2. The precision of the repeated measurements
limited [24] with the information about the impact of the whole was expressed as the coefficient of variation, which was 0.6% for fat
body cryostimulation on this protein completely lacking. Irisin is a mass percentage on average [42].
recently discovered myokine, secreted into the circulation
following a proteolytic cleavage from its cellular form, fibronectin- 2.3. Cardiorespiratory fitness measurement
type III domain-containing 5 (FNDC5) in response to exercise [6]. Its
release is induced during exercise in mice and humans, causing an To determine the VO2max, the participants performed a graded
increase in energy expenditure in mice with no changes in move- cycle ergometry test on an electromagnetically braked, cycle
ment or food intake. In addition, irisin activates oxygen consump- ergometer (ER 900 Jaeger, Germany/Viasys Health Care). The par-
tion in white fat cells, and thermogenesis [22]. Consequently, irisin ticipants were allowed a 5-min warm up period at an intensity of
is considered to be beneficial in the treatment of obesity, diabetes, 1.5 W kg1, with a pedalling cadence of 60 rpm. Immediately
and a wide range of pathological conditions characterized by an following the warm-up, the participants began VO2max testing by
imbalance between energy demand and expenditure [22,40]. cycling at increasingly workloads by 25 W min1 until the partic-
Recent papers report that irisin negatively correlates with the BMI, ipant reached the point of volitional exhaustion [20,45]. The re-
waist-hip ratio, and fat mass in men as well as that circulating irisin covery was passive, with the participant in a seated position.
is lower in non-diabetic overweight and obese men [17,30,39]. Lee Breath-by-breath pulmonary gas exchange was measured (Oxy-
and co-workers noted that cold treatment is one of the factors con-Pro, Jaeger -Viasys Health Care, Germany) throughout the
stimulating irisin secretion. Water-infused thermoblankets (from VO2max test; the O2 and CO2 analysers were calibrated prior to each
27  C cooled to 18  C and further lowered by 2  C every 3 min until test using standard gases of known concentrations in accordance
12  C temperature) was found to have stimulated irisin secretion with manufacturer guidelines.
[24]. Hence, irisin seems to be an important link between cold
exposure treatment and obesity. 2.4. Whole-body cryostimulation
Overall, basing on the current information about changes
stimulated by cryotherapy and its efficiency in reducing low grade All of the participants were subjected to a series of coldness
systemic inflammation in obese men, the aim of our study was to exposures (once per day at 9:30 a.m.), including 10 sessions in a
assess the impact of CRY on irisin concentration among obese men cryogenic chamber at the Pomeranian Rheumatologic Centre in
at various fitness level. The second purpose of our investigation was Sopot, Poland, which were conducted by highly qualified medical
to assess the influence of the whole body cryostimulation on staff. Each cryostimulation session lasted 3 min at a temperature
hepcidin concentration and iron status. of 110  C. Entry into the cryo-chamber was preceded by a 20e30 s
adaptation period in the vestibule at a temperature of 60  C. The
2. Materials and methods subjects were dressed in shorts, socks, gloves, and a hat covering
their auricles. The examined individuals did not participate in any
2.1. Subjects other treatment after whole-body cryostimulation to avoid
obscuring the interpretation of the cryogenic effect. Each exposure
Twelve obese men participated in the experiment (38.4 ± 8.2 was preceded by a light breakfast between 07 and 07:30 a.m. ac-
years of age, height of 179.0 ± 6.0 cm). The participants were cat- cording to the instructions given to the subjects [45].
egorised as obese based on their BMI according to the current
guidelines (BMI >30 kg m2) and visceral fat area above 100 cm2 2.5. Blood analysis and collection
[44]. Additionally, the group was divided into two subgroups ac-
cording to the participants' cardiorespiratory fitness, which was Blood samples were taken from the antecubital vein into the
measured in terms of the maximal oxygen consumption (VO2max). vacutainer tubes with EDTAK 2, before, 30 min and 24 h after the
Based on the classification proposed by Astrand [3], subjects with first and last cryo-session. Immediately following the blood
VO2max above 35 mL kg1 min1 were assigned to the high fitness collection one portion of the sample was transferred to centrifuge
level (HFL) group (weight of 93.8 ± 11.5 kg), whereas those with the tubes containing aprotinin (catalog no RK-APRO) from Phoenix
lower VO2 max were enrolled in the low fitness level (LFL) group Pharmaceuticals Inc. The final concentration of aprotinin was 0.6
(weight of 116.5 ± 24.8 kg). All the subjects underwent a medical Trypsin Inhibitor Unit/1 mL of blood. The samples were centrifuged
check-up before being submitted to cold exposure. They were also at 2000 g for 10 min at 4  C. The separated plasma samples were
medication-free. The participants had never previously been sub- frozen and kept at 70  C until later analysis.
jected to any form of cryotherapy. Written, informed consent was Plasma interleukin IL-6 and high sensitivity C-reactive protein
obtained from all subjects. All procedures were approved by the (hsCRP) levels were determined by enzyme immunoassay methods
Bioethical Committee of the Regional Medical Society in Gdansk using commercial kits (R&D Systems, USA, catalog no. HS600B,
NKEBN/245/2009. One week prior to the start of the experiment, DCRP00 respectively). The average intra-assay CV was 8.0% for IL-6
body composition and aerobic capacity were determined for each and hsCRP.
participant. During the experiment, all participants were instructed Quantification of plasma irisin was based on a competitive
400 K. Dulian et al. / Cryobiology 71 (2015) 398e404

enzyme immunoassay and the assay kits were purchased from group. Interestingly, statistical differences were observed in per-
Phoenix Pharmaceuticals Inc (catalog no EK 067-16). Details on the centage of skeletal muscle mass between HFL and LFL groups. Also,
ELISA assay have been described elsewhere [18]. The dilution of the absolute fat mass and percentage of fat tissue values exceeded
sample was applied 1:5. The intra-assay coefficients of variability the recommended ranges for subject age. Moreover an elevated
(CVs) and inter-assay CVs reported by the manufacturer were 4%e amount of visceral fat area (VFA) was observed. HFL group was
6% and 8%e10%, respectively. characterised by significantly lower fat content than LFL group
The changes in hepcidin concentration and iron status have (120.9 ± 13.3 vs 181.5 ± 30.0; p < 0.05).
been determined in the blood samples collected at the baseline and The average values of irisin recorded in the whole group at the
24 h after last (10 th) cold exposure session had ended. The serum baseline was equal 352.4 ± 40.0 ng mL1. Any statistical correla-
hepcidin levels were determined using a DRG Elisa kit. The tions were noted between body composition and irisin concen-
analytical sensitivity of the hepcidin kit was calculated by sub- tration before the exposure to CRY. Taking into account
tracting 2 standard deviations from the mean of 20 replicate ana- cardiorespiratory level, HFL group was characterized by 8.8%
lyses of the Zero Standard (SO) and was established to be elevated irisin concentration at baseline but this was not significant
0.9 ng,ml1 kit. All data were corrected according to the changes in difference to LFL group.
hematocrit. Obtained data confirmed our initial assumption that obese men
Serum ferritin was measured by (SYSMEX XE 2100, Architect ci experienced the low-grade systemic inflammation. Although the
8200, and Test 1 SDL). Iron was measured on the Roche Modular values of hsCRP were still in reference range, the average concen-
System (Roche Diagnostics, Switzerland). tration of hsCRP in HFL group amounted to 2.2 ± 0.6 and in LFL
4.4 ± 1.6 mg L1 (p ¼ 0.003) (Fig. 2). All positive correlations be-
2.6. Statistical calculations tween fat expressed in kg and percentage, VFA and hsCRP were
statistically significant (r ¼ 0.88, r ¼ 0.83, r ¼ 0.77; p < 0.05
Statistical calculations were performed using STATISTICA 10.0.
Results are expressed as the mean, standard deviation (x ± SD). The
normality of data was tested using the ShapiroeWilks W-test. The
level of significance was set at 0.05 for all analyses. In order
determining the differences in of physical performance and body
composition between active and non-active groups U-Mann
Whitney test was applied. The differences between obtained data
before and after the cryotherapy intervention were analysed using
repeated measures of variance (ANOVA) for parametric variables
and U-Mann Whitney test for nonparametric ones. Associations
among measured parameters were analysed using Pearson's linear
regression (coefficient, r).

3. Results

3.1. Baseline data

All participants completed the study, with no adverse events

being reported. The basic anthropometric and physiological char-
acteristics of the subjects are summarised in Table 1. The charac-
teristic is presented according to the cardiorespiratory fitness level.
All participants were categorised as obese, based on the average
BMI of the HFL (30.1 ± 4.0 kg m2) and LFL groups
(35.7 ± 4.5 kg m2). Nonetheless the free fat mass (FFM kg) and
skeletal muscle mass (SMM kg) in HFL group did not differ to LFL

Table 1
Anthropometric and physiological characteristics of participants.

Variable HFL-group LFL-group P Value

BMI [kg m2] 30.1 (4.0) 35.7 (4.5)* 0.04

FFM [kg] 72.9 (±6.4) 69.8 (±7.0) ns
SMM [kg] 41.7 (±3.8) 39.6 (±4.1) ns
SMM [%] 41.7 (±3.8) 39.6 (±4.1) 0.05
Fat [kg] 20.9 (±6.2) 46.7 (±18.8)* 0.05
Fat [%] 22.0 (±4.6) 38.9 (±6.4)* 0.05
VFA [cm2] 120.9 (±13.3) 181.5 (±30.0)* 0.05
VO2max [mL kg1 min1] 43.2 (±3.4) 26.1 (±1.2)* 0.05

Values are means (±SD); BMI ebody mass index, FFM e free fat mass, SMM e
skeletal muscle mass, % SMM e percentage of skeletal muscle mass, Fat e fat mass,
Fat % e percentage of body fat. Fig. 1. Changes in irisin concentration in response to the first and last session of whole
VFA e visceral fat area, VO2max e maximal oxygen uptake expressed in relatively body cryostimulation (CRY): a. In the whole group of participants (the mean total effect
values HFL(n ¼ 5) high fitness level, LFL(n ¼ 7) low fitness level, * significantly of I CRY vs X CRY intervention is significantly different at p level ¼ 0.04). b. In HFL and
different from HFL group. LFL group (for LFL group the differences were statistically significant p < 0.05, for HFL
ns: non-significant differences. group the changes after 10 sessions of WBC were not differ to the baseline).
 K. Dulian et al. / Cryobiology 71 (2015) 398e404 401

Fig. 2. Alternations in hsCRP in response to the first and last session of whole body cryostimulation in HFL and LFL group (‡ significantly different from baseline (before 1 CRY) HFL
group (p ¼ 0.003), ¶ significantly different: baseline (before 1 CRY) vs 24 h after 1 CRY (HFL p ¼ 0.005; LFL p ¼ 0.001), * significantly different: baseline (before 10 CRY) vs 24 h after
10 CRY (HFL p ¼ 0.004; LFL p ¼ 0,0005).

respectively) among all subjects. In addition, the negative correla- association between irisin and ferritin before CRY was equal r ¼ 
tion between maximal oxygen uptake and hsCRP concentration 0.60 (p < 0.05). Interestingly, among LFL group this relationship was
was noted (r ¼ 0.68; p < 0.05) for the whole group. In HFL group even stronger r ¼ 0.83 (p < 0.05).
the concentration of IL-6 was lower (1.3 ± 0.5 pg mL1) than in LFL
participants (Table 2). The average values of IL-6 in LFL group was 3.2. Effect of 10 sessions of whole-body cryostimulation
69% higher (2.2 ± 0.8 pg mL1) in comparison with those who
characterized by elevated fitness level. Moreover, the group of Although comparison of single measurements did not revealed
obese subjects experienced the relatively high level of hepcidin statistical differences between each values, analysis of means
[19]. Iron and ferritin level among our subjects were in reference calculated at first and last sessions of CRY in all points of blood
range (Table 2). Irisin concentration recorded before CRY correlated collection (Fig. 1a) indicated that the series of 10 cryostimulation
inversely with the ferritin level. For the whole group, the exposures altered irisin concentration among all subjects (the

Table 2
The hepcidin concentration and iron status in response to 10 sessions of whole body cryostimulation.

Variable HFL-group LFL-group Differences P Value

Before first CRY 24 h after last CRY Before first CRY 24 h after last CRY

Serum iron [mg dL1] 105.3 (±5.3) 94.9 (±12.7) 117.7 (±37.5) 104.8 (±21.7) Time ns
Group ns
Time  group ns
Serum ferritin [ng mL1] 104.0 (±16.8) 101.0 (±9.9) 108.7 (±41.4) 105.0 (±41.8) Time ns
Group ns
Time  group ns
Hepcidin [ng ml1] 86.9 (±8.58) 72.2 (±3.08) 103.8 (±42.4) 96.0 (±17.0) Time 0.03a
Group ns
Time  group ns
IL-6 [pg ml1] 1.3 (±0.5) 1.3 (±0.4) 2.2 (±0.8) 2.0 (±1.0) Time ns
Group ns
Time  group ns

Values are means (±SD), ns e no statistical differences for group and time before and after cryostimulation HFL e high fitness level, LFL e low fitness level.
CRY the whole body cryostimulation, 24 h after last 10 session of cryostimulation, differences: time-comparison before and after cryostimulation.
a
Significantly different group-differences between HFL and LFL, time  group e interactions between group and time, ns-non significant differences.
402 K. Dulian et al. / Cryobiology 71 (2015) 398e404

mean total effect value of 1st CRY vs 10th CRY intervention is Such suggestion is supported by the fact that the rise in irisin was
significantly different, p ¼ 0.04) Interestingly, exposure to coldness inversely proportional to the muscle mass.
in HFL group caused a small drop of irisin (371.5 ± 30.0 ng mL1 at There is limited number of works investigating the influence of
baseline vs 343.3 ± 47.6 ng mL1 24 h after last session of CRY). In coldness on irisin concentration. Lee and co-workers observed the
contrast, the tendency in LFL group was opposite. The irisin irisin concentration to be increased in response to graduate cold
increased in LFL group from 338.8 ± 42.2 ng mL1 to water immersions [24]. Also, Costello noticed that compared to
407.6 ± 118.5 ng mL1 (Fig. 1b). The body composition modified the water immersions, the whole body cryostimulation is more effec-
rise of irisin in our group. The increase of irisin concentration tive in lowering skin temperature [8]. Still, even the exposure
recorded directly before, 30 min and 24 h after 10th sessions of to 110  C (extreme coldness) may not have impacted the muscle
cryostimulation positively correlated with body fat percentage of severely enough to induce shivering, especially in obese individuals
fat mass and visceral fat area. Additionally, the significant relations with an excess of fat tissue. Hence, the impact of CRY in the protocol
were observed between percentage of skeletal muscle mass and of 3-min exposure to low temperature used in our study may have
irisin concentration. In measurements, which were performed been insufficient to induce changes in irisin blood level through
before, 30 min and 24 h after 10th session, subjects with higher muscle shivering. The observed rise of irisin stimulated by coldness
skeletal muscle mass had lower irisin concentration (Table 3). may have either been a short-lasting effect or the irisin has been
The whole body cryostimulation reduced low grade inflamma- taken up very quickly. Similar response was observed by Loffer and
tion, thus the hsCRP concentration in both groups significantly co-workers, who recorded the irisin concentration to have
dropped (Fig. 2). The range of decline was similar in HFL and in LFL increased in adults after a short-term acute exercise. The rise was
group: from at the baseline 2.2 ± 0.9 to 0.8 ± 0.4 mg L1 24 h after detectable only immediately after the exercise and significantly
last session of CRY (HFL) and respectively 4.4 ± 1.6 to diminished in 30 min time following the exercise [27]. This may
1.8 ± 0.5 mg L1 (LFL). Thus the interaction between groups was not explain the smallest correlation observed 24 h after the last session
statistically significant. Nonetheless the drop was essential. More- in our study. Nonetheless, the irisin concentration grew only in the
over, hepcidin concentration was also reduced but this decline was group of obese non-active subjects following the series of cryo-
not accompanied by changes in iron and ferritin concentration. The stimulation sessions, while in the group of active obese man we did
exposure to coldness did not adjust IL-6 concentrations signifi- not record this tendency.
cantly (Table 2). However, recorded small alternations in IL-6 Data concerning the influence of exercise on irisin concentration
concentration corresponded with changes in hepcidin (r ¼ 0.61, are also unambiguous. A one year long lifestyle intervention pro-
p < 0.05). gram was associated with an improvement in anthropometric and
metabolic parameters, resulting in higher irisin levels in obese
4. Discussion children [10]. Other data indicated that single sessions of intense
endurance exercise and heavy strength training lead to transient
The study demonstrates that 10 sessions of the whole body increases in irisin concentrations in blood [34]. In contrary, some
cryostimulation caused the rise of irisin concentration in obese data demonstrated that neither endurance training nor resistance
men. The obtained data confirm our initial hypothesis that expo- training increases irisin concentration [14]. In addition, no signifi-
sure to extremely cold environment might induce muscle shivering cant changes in irisin concentration following a 10-week endur-
and consequently irisin release from the muscle. Still, diverse ten- ance training program were noted in a recent research on habitual
dency in irisin concentration among physically active and non- runners vs non-runners, where runners were characterized by a
active subjects following 10 sessions, might suggest that fat tissue significantly lower body weight and total body fat along with
has also contributed to the irisin secretion. Thermodynamically higher aerobic fitness compared to non-runners [15]. Instead, when
cryotherapy works on the principle of an unidirectional heat compared with pre-training levels, a decreasing tendency in the
transfer from high heat to low heat areas and tissue temperature irisin concentration was observed following the training program,
heat loss to the external cooling modality [28]. In the process, depth independently of each subject's fitness level. Additionally, the mean
of subcutaneous fat is a significant factor affecting the magnitude irisin value tended to be lower in runners compared to non-
and rate of intramuscular cooling [31]. Roca-Rivada and co-workers runners, both before and after single bout of exercise [15].
showed that human subcutaneous as well as visceral fat tissue Since obesity is associated with increased inflammation [23],
express and secret FNDC5/irisin [37]. Contrary to the previous the second aim of the present study was to investigate if CRY
observation [17,30,39] in the present study, no correlations were triggers a decline of hsCRP as a marker of inflammation. Previous
observed at the baseline between body composition and/or research revealed that the protocol with the same number of CRY
cardiorespiratory fitness level and circulating irisin levels. sessions induced an anti-inflammatory effect in obese man through
Conversely, our data showed that after 10 sessions of CRY, the rise in attenuation of TNF-a concentration [45]. Baseline values of hsCRP
blood irisin correlated with the fat tissue amount. These data and IL-6 in the present study indicated that our obese subjects had
suggests that the subcutaneous fat tissue, rather than skeletal experienced a low grade inflammation, while the active, yet obese
muscle, is the main source of irisin in response to extreme coldness. men exhibited lower concentrations of the inflammation markers.

Table 3
The correlations between body composition and irisin concentration after all series of the whole body cryostimulation among all participants.

Variable Irisin BC [ng mL1] Irisin 30 min [ng mL1] Irisin 24 h [ng mL1]

Weight [kg] 0.65* 0.66* 0.46

BMI [kg m2] 0.60* 0.62* 0.41
SMM [%] 0.59* 0.65* 0.48
Fat [kg] 0.65* 0.68* 0.48
Fat [%] 0.57 0.63* 0.45
VFA [cm2] 0.49 0.58* 0.43

Values are Person correlation, *values were significant at P < 0.05, BC-before X cryostimulation, 30 min after X cryostimulation, 24 h 24 h after X cryostimulation.
SMM e skeletal muscle mass, Fatefat mass, Fat%-percentage of body fat, VFA e visceral fat area.
 K. Dulian et al. / Cryobiology 71 (2015) 398e404 403

Such observations are backed by different research, where the anti- would like to express their thanks to Robert A. Olek for his cor-
inflammatory effects of exercise was shown [36] and higher levels rections and suggestions in preparing the manuscript and to the
of physical activity associated with a lower concentration of serum medical staff of the hospital where cold exposure took place,
hsCRP [2].  ska and Piotr Krella, MD, for their help
especially Aleksandra Kasin
Despite the lack of differences between the groups (active vs in conducting the research, and all of the participants for their
non-active), the applied protocol of CRY significantly decreased engagement in the experiment.
hsCRP blood concentration in all subjects, thus, endorsing its anti-
inflammatory action reported previously [16,45]. Among other, the
References
inflammation might induced to changes in iron metabolism [13],
hence, the effect of CRY on hepcidin was measured. As a result a [1] E. Aigner, A. Feldman, C. Datz, Obesity as an emerging risk factor for iron
significant reduction in hepcidin level was recorded 24 h after the deficiency, Nutrients 6 (2014) 3587e3600.
[2] D. Aronson, M. Sheikh-Ahmad, O. Avizohar, A. Kerner, R. Sella, P. Bartha,
last CRY session. As outlined above, hepcidin acts by blocking the
W. Markiewicz, Y. Levy, G.J. Brook, C-Reactive protein is inversely related to
ferroportin, which is the only know protein able to transport iron physical fitness in middle-aged subjects, Atherosclerosis 176 (2004) 173e179.
from the cells [11]. Rise in blood hepcidin leads to inhibition of [3] I. Astrand, Aerobic work capacity in men and women with special reference to
ferroportin in many cells, including enterocytes in duodenum, he- age, Acta Physiol. Scand. Suppl. 49 (1960) 1e92.
[4] K.J. Ausk, G.N. Ioannou, Is obesity associated with anemia of chronic disease? a
patocytes, macrophages, adipocytes and many other what may population-based study, Obesity (Silver Spring) 16 (2008) 2356e2361.
result in decrease dietary iron absorption and blood iron but in- [5] S. Bekri, P. Gual, R. Anty, N. Luciani, M. Dahman, B. Ramesh, A. Iannelli,
crease iron accumulation in rest of the cells [9]. Such iron redis- A. Staccini-Myx, D. Casanova, I. Ben Amor, M.C. Saint-Paul, P.M. Huet,
J.L. Sadoul, J. Gugenheim, S.K. Srai, A. Tran, Y. Le Marchand-Brustel, Increased
tribution induced by chronic hyper hepcidinemia may result in adipose tissue expression of hepcidin in severe obesity is independent from
inflammation as iron acts as a pro inflammatory molecule [21]. diabetes and NASH, Gastroenterology 131 (2006) 788e796.
Thus, in our opinion, the observed drop in blood hepcidin should be [6] P. Bostrom, J. Wu, M.P. Jedrychowski, A. Korde, L. Ye, J.C. Lo, K.A. Rasbach,
E.A. Bostrom, J.H. Choi, J.Z. Long, S. Kajimura, M.C. Zingaretti, B.F. Vind, H. Tu,
considered as an anti-inflammatory effect of CRY. It is worth noting S. Cinti, K. Hojlund, S.P. Gygi, B.M. Spiegelman, A PGC1-alpha-dependent
that adipocytes in addition to liver, macrophages and pancreatic myokine that drives brown-fat-like development of white fat and thermo-
islets can also be the source of blood hepcidin [5]. In case of the genesis, Nature 481 (2012) 463e468.
[7] H.L. Cheng, C. Bryant, R. Cook, H. O'Connor, K. Rooney, K. Steinbeck, The
obese individuals we observed both Hpc and BMI as well as the relationship between obesity and hypoferraemia in adults: a systematic re-
body fat % and Hpc to be correlated. These observations suggest view, Obes. Rev. Off. J. Int. Assoc. Study Obes. 13 (2012) 150e161.
that a decrease in blood Hpc may result from its declined release [8] J.T. Costello, K. Culligan, J. Selfe, A.E. Donnelly, Muscle, skin and core tem-
perature after -110 degrees c cold air and 8 degrees c water treatment, PLoS
from the adipose tissue after CRY. Lowering of blood Hpc concen-
One 7 (2012) e48190.
tration along with IL-6 decrease supports the thesis that CRY re- [9] I. De Domenico, D.M. Ward, C. Langelier, M.B. Vaughn, E. Nemeth,
duces obesity-related low grade inflammation. Considering that W.I. Sundquist, T. Ganz, G. Musci, J. Kaplan, The molecular mechanism of
iron deficiency is frequent in advanced stages of obesity possibly hepcidin-mediated ferroportin down-regulation, Mol. Biol. Cell 18 (2007)
2569e2578.
due to high Hpc concentration [7], CRY could be a good method to [10] T. Ebert, D. Focke, D. Petroff, U. Wurst, J. Richter, A. Bachmann, U. Lossner,
correct iron metabolism in those subjects. S. Kralisch, J. Kratzsch, J. Beige, I. Bast, M. Anders, M. Bluher, M. Stumvoll,
Our data sets the basis for additional research since many M. Fasshauer, Serum levels of the myokine irisin in relation to metabolic and
renal function, Eur. J. Endocrinol. Eur. Fed. Endocr. Soc. (2014), http://
questions remain to be answered concerning: the connection be- dx.doi.org/10.1530/EJE-13-1053.
tween body composition and irisin concentration, the number of [11] H.P. Erickson, Irisin and FNDC5 in retrospect: an exercise hormone or a
cryostimulation sessions to stimulate irisin release and determi- transmembrane receptor? Adipocyte 2 (2013) 289e293.
[12] T. Ganz, Hepcidin and its role in regulating systemic iron metabolism, he-
nate if changes in hepcidin are also fat tissue dependent. The pre- matology/the education program of the American society of hematology, Am.
sent study, thus, only partly addresses the question whether fat Soc. Hematol. Educ. Program 29e35 (2006) 507, http://dx.doi.org/10.1182/
tissue serves as a source of irisin. Discussion and research are hence asheducation-2006.1.29.
[13] L. Gong, F. Yuan, J. Teng, X. Li, S. Zheng, L. Lin, H. Deng, G. Ma, C. Sun, Y. Li,
likely to continue, taking a careful account of the body temperature
Weight loss, inflammatory markers, and improvements of iron status in
changes. In future projects the following limitations of this study overweight and obese children, J. Pediatr. (2014), http://dx.doi.org/10.1016/
should be addressed: a small number of subjects and time through j.jpeds.2013.12.004.
[14] A. Hecksteden, M. Wegmann, A. Steffen, J. Kraushaar, A. Morsch,
which the positive changes are sustained.
S. Ruppenthal, L. Kaestner, T. Meyer, Irisin and exercise training in humans e
results from a randomized controlled training trial, BMC Med. 11 (2013) 235.
Source of funding [15] T. Hew-Butler, K. Landis-Piwowar, G. Byrd, M. Seimer, N. Seigneurie, B. Byrd,
O. Muzik, Plasma irisin in runners and nonrunners: no favorable metabolic
associations in humans, Physiol. Rep. 3 (2015).
This study was supported by grant No 0026/RS3/2015/53 from [16] H.E. Hirvonen, M.K. Mikkelsson, H. Kautiainen, T.H. Pohjolainen, M. Leirisalo-
the Polish Ministry of Science and Higher Education. Repo, Effectiveness of different cryotherapies on pain and disease activity in
active rheumatoid arthritis. A randomised single blinded controlled trial, Clin.
Exp. Rheumatol. 24 (2006) 295e301.
Conflict of interest [17] K. Hojlund, P. Bostrom, Irisin in obesity and type 2 diabetes, J. Diabetes
Complicat. 27 (2013) 303e304.
There are no professional relationships between any of the au- [18] J.Y. Huh, G. Panagiotou, V. Mougios, M. Brinkoetter, M.T. Vamvini,
B.E. Schneider, C.S. Mantzoros, FNDC5 and irisin in humans: I. Predictors of
thors and manufacturers of equipment utilized in this study. No circulating concentrations in serum and plasma and II. mRNA expression and
conflict of interest occurs. circulating concentrations in response to weight loss and exercise, Metab.
All procedures performed in studies involving human partici- Clin. Exp. 61 (2012) 1725e1738.
[19] Y. Kaneko, H. Miyajima, A. Piperno, N. Tomosugi, H. Hayashi, N. Morotomi,
pants were in accordance with the ethical standards of the insti- K. Tsuchida, T. Ikeda, A. Ishikawa, Y. Ota, S. Wakusawa, K. Yoshioka, S. Kono,
tutional and/or national research committee and with the 1964 S. Pelucchi, A. Hattori, Y. Tatsumi, T. Okada, M. Yamagishi, Measurement of
Helsinki declaration and its later amendments or comparable serum hepcidin-25 levels as a potential test for diagnosing hemochromatosis
and related disorders, J. Gastroenterol. 45 (2010) 1163e1171.
ethical standards.
[20] P.T. Katzmarzyk, J. Gagnon, A.S. Leon, J.S. Skinner, J.H. Wilmore, D.C. Rao,
C. Bouchard, Fitness, fatness, and estimated coronary heart disease risk: the
Acknowledgements HERITAGE Family Study, Med. Sci. Sports Exerc. 33 (2001) 585e590.
[21] D.B. Kell, Iron behaving badly: inappropriate iron chelation as a major
contributor to the aetiology of vascular and other progressive inflammatory
This study was supported by grant No 0026/RS3/2015/53 from and degenerative diseases, BMC Med. Genom. 2 (2009) 2.
the Polish Ministry of Science and Higher Education. The authors [22] D.P. Kelly, Medicine. Irisin, light my fire, Science 336 (2012) 42e43.
404 K. Dulian et al. / Cryobiology 71 (2015) 398e404

[23] P.G. Kopelman, Obesity as a medical problem, Nature 404 (2000) 635e643. [34] H. Nygaard, G. Slettalokken, G. Vegge, I. Hollan, J.E. Whist, T. Strand,
[24] P. Lee, J.D. Linderman, S. Smith, R.J. Brychta, J. Wang, C. Idelson, R.M. Perron, B.R. Ronnestad, S. Ellefsen, Irisin in blood increases transiently after single
C.D. Werner, G.Q. Phan, U.S. Kammula, E. Kebebew, K. Pacak, K.Y. Chen, sessions of intense endurance exercise and heavy strength training, PLoS One
F.S. Celi, Irisin and FGF21 are cold-induced endocrine activators of brown fat 10 (2015) e0121367.
function in humans, Cell Metab. 19 (2014) 302e309. [35] C.H. Park, E.V. Valore, A.J. Waring, T. Ganz, Hepcidin, a urinary antimicrobial
[25] C.A. Libardi, G.V. De Souza, C.R. Cavaglieri, V.A. Madruga, M.P. Chacon-Mikahil, peptide synthesized in the liver, J. Biol. Chem. 276 (2001) 7806e7810.
Effect of resistance, endurance, and concurrent training on TNF-alpha, IL-6, [36] A.M. Petersen, B.K. Pedersen, The anti-inflammatory effect of exercise, J. Appl.
and CRP, Med. Sci. Sports Exerc. 44 (2012) 50e56. Physiol. 98 (2005) 1154e1162.
[26] D. Lloyd-Jones, R. Adams, M. Carnethon, G. De Simone, T.B. Ferguson, K. Flegal, [37] A. Roca-Rivada, C. Castelao, L.L. Senin, M.O. Landrove, J. Baltar, A. Belen Cru-
E. Ford, K. Furie, A. Go, K. Greenlund, N. Haase, S. Hailpern, M. Ho, V. Howard, jeiras, L.M. Seoane, F.F. Casanueva, M. Pardo, FNDC5/irisin is not only a
B. Kissela, S. Kittner, D. Lackland, L. Lisabeth, A. Marelli, M. McDermott, myokine but also an adipokine, PLoS One 8 (2013) e60563.
J. Meigs, D. Mozaffarian, G. Nichol, C. O'Donnell, V. Roger, W. Rosamond, [38] A. Rull, J. Camps, C. Alonso-Villaverde, J. Joven, Insulin resistance, inflamma-
R. Sacco, P. Sorlie, R. Stafford, J. Steinberger, T. Thom, S. Wasserthiel-Smoller, tion, and obesity: role of monocyte chemoattractant protein-1 (or CCL2) in the
N. Wong, J. Wylie-Rosett, Y. Hong, Heart disease and stroke statisticse2009 regulation of metabolism, Mediat. Inflamm. 2010 (2010).
update: a report from the American Heart Association Statistics Committee [39] F. Sanchis-Gomar, R. Alis, H. Pareja-Galeano, E. Sola, V.M. Victor, M. Rocha,
and Stroke Statistics Subcommittee, Circulation 119 (2009) e21e181. A. Hernandez-Mijares, M. Romagnoli, Circulating irisin levels are not corre-
[27] D. Loffler, U. Muller, K. Scheuermann, D. Friebe, J. Gesing, J. Bielitz, S. Erbs, lated with BMI, age, and other biological parameters in obese and diabetic
K. Landgraf, I. Viola Wagner, W. Kiess, A. Korner, Serum irisin levels are patients, Endocrine (2014), http://dx.doi.org/10.1007/s12020-014-0170-9.
regulated by acute strenuous exercise, J. Clin. Endocrinol. Metab. (2015) [40] F. Sanchis-Gomar, G. Lippi, S. Mayero, C. Perez-Quilis, J.L. Garcia-Gimenez,
jc20142932, http://dx.doi.org/10.1210/jc.2014-2932. Irisin: a new potential hormonal target for the treatment of obesity and type 2
[28] M.A. Merrick, L.S. Jutte, M.E. Smith, Cold modalities with different thermo- diabetes, J. Diabetes 4 (2012) 196.
dynamic properties produce different surface and intramuscular tempera- [41] H. Tilg, A.R. Moschen, Adipocytokines: mediators linking adipose tissue,
tures, J. Athl. Train. 38 (2003) 28e33. inflammation and immunity, Nature reviews, Immunology 6 (2006) 772e783.
[29] E. Miller, Cryostimulation as an antioxidative factor in sclerosis multiplex, Pol. [42] E. Volgyi, F.A. Tylavsky, A. Lyytikainen, H. Suominen, M. Alen, S. Cheng,
Merkur. Lek. Organ Pol. Tow. Lek. 31 (2011) 186e189. Assessing body composition with DXA and bioimpedance: effects of obesity,
[30] J.M. Moreno-Navarrete, F. Ortega, M. Serrano, E. Guerra, G. Pardo, physical activity, and age, Obesity (Silver Spring) 16 (2008) 700e705.
F. Tinahones, W. Ricart, J.M. Fernandez-Real, Irisin is expressed and produced [43] A. von Ruesten, A. Steffen, A. Floegel, A.D. van der, G. Masala, A. Tjonneland,
by human muscle and adipose tissue in association with obesity and insulin J. Halkjaer, D. Palli, N.J. Wareham, R.J. Loos, T.I. Sorensen, H. Boeing, Trend in
resistance, J. Clin. Endocrinol. Metab. 98 (2013) E769eE778. obesity prevalence in European adult cohort populations during follow-up
[31] W.J. Myrer, K.A. Myrer, G.J. Measom, G.W. Fellingham, S.L. Evers, Muscle since 1996 and their predictions to 2015, PLoS One 6 (2011) e27455.
temperature is affected by overlying adipose when cryotherapy is adminis- [44] WHO, Obesity: preventing and managing the global epidemic. Report of a
tered, J. Athl. Train. 36 (2001) 32e36. WHO consultation, World Health Organ. Tech. Rep. Ser. 894 (i-xii) (2000)
[32] E. Nemeth, S. Rivera, V. Gabayan, C. Keller, S. Taudorf, B.K. Pedersen, T. Ganz, 1e253.
IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the [45] E. Ziemann, R.A. Olek, T. Grzywacz, J. Antosiewicz, S. Kujach, M. Luszczyk,
iron regulatory hormone hepcidin, J. Clin. Investig. 113 (2004) 1271e1276. M. Smaruj, E. Sledziewska, R. Laskowski, Whole-body cryostimulation as an
[33] G. Nicolas, L. Viatte, M. Bennoun, C. Beaumont, A. Kahn, S. Vaulont, Hepcidin, a effective method of reducing low-grade inflammation in obese men, J. Physiol.
new iron regulatory peptide, Blood Cells Mol. Dis. 29 (2002) 327e335. Sci. JPS 63 (2013) 333e343.

View publication stats