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Research Article
Whole-Body Cryotherapy Decreases the Levels of Inflammatory,
Oxidative Stress, and Atherosclerosis Plaque Markers in
Male Patients with Active-Phase Ankylosing Spondylitis in the
Absence of Classical Cardiovascular Risk Factors
Agata Stanek ,
1
Armand Cholewka,
2
Tomasz Wielkoszyński ,
3
Ewa Romuk ,
3
and Aleksander Sieroń
1
1
School of Medicine with the Division of Dentistry in Zabrze, Department of Internal Medicine, Angiology and Physical Medicine,
Medical University of Silesia, Batorego Street 15, 41-902 Bytom, Poland
2
Department of Medical Physics, Chełkowski Institute of Physics, University of Silesia, 4 Uniwersytecka St., 40-007 Katowice, Poland
3
School of Medicine with the Division of Dentistry in Zabrze, Department of Biochemistry, Medical University of Silesia, Jordana 19
St., 41-808 Zabrze, Poland
Correspondence should be addressed to Agata Stanek; astanek@tlen.pl
Received 26 August 2017; Accepted 19 October 2017; Published 1 February 2018
Academic Editor: Adrian Doroszko
Copyright © 2018 Agata Stanek et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Objective. The aim of the study was to estimate the impact of whole-body cryotherapy (WBC) on cardiovascular risk factors in
patients with ankylosing spondylitis (AS). Material and Methods. We investigated the effect of WBC with subsequent
kinesiotherapy on markers of inflammation, oxidative stress, lipid profile, and atherosclerosis plaque in male AS patients (WBC
group). To assess the disease activity, the BASDAI and BASFI were also calculated. The results from the WBC group were
compared with results from the kinesiotherapy (KT) group. Results. The results showed that in the WBC group, the plasma
hsCRP level decreased without change to the IL-6 level. The ICAM-1 level showed a decreasing tendency. The CER
concentration, as well as the BASDAI and BASFI, decreased in both groups, but the index changes of disease activity were
higher in the WBC than KT patients. Additionally, in the WBC group, we observed a decrease in oxidative stress markers,
changes in the activity of some antioxidant enzymes and nonenzymatic antioxidant parameters. In both groups, the total
cholesterol and LDL cholesterol, triglycerides, sCD40L, PAPP-A, and PLGF levels decreased, but the parameter changes were
higher in the WBC group. Conclusion. WBC appears to be a useful method of atherosclerosis prevention in AS patients.
1. Introduction
Patients with ankylosing spondylitis (AS) have a higher risk
of cardiovascular morbidity and mortality in comparison to
the general population, which may be connected with the
disease’s activity, the functional and mobility limitations,
structural damage, and inflammation [1, 2]. Even AS patients
without concomitant classical cardiovascular risk factors
yet, but in an active phase of the disease, are characterized
by increased levels of oxidative stress, inflammatory states,
higher serum concentrations of soluble CD40 ligand
(sCD40L), and increased carotid intima-media thickness
(IMT) in comparison to the general population. These
factors may accelerate atherosclerosis in this group of
patients [3, 4].
Fortunately, over the last several years, a revolution in the
treatment of AS has taken place through the introduction of
biological and disease-modifying antirheumatic drugs
(DMARDs). Despite these advances, exercise and physio-
therapy still play a very important role [5, 6].
A relatively new physiotherapeutic method used in the
rheumatic disease treatment is whole-body cryotherapy
Hindawi
Mediators of Inflammation
Volume 2018, Article ID 8592532, 11 pages
https://doi.org/10.1155/2018/8592532
(WBC), which is based on the therapeutic exposure of the
entire human body to very low temperatures (below −100
°
C)
for 120–180 seconds [7].
Recent studies have confirmed the anti-inflammatory,
antianalgesic, and antioxidant effects of extremely low tem-
peratures in athletes [8]. WBC procedures also have had a
beneficial influence on lipid profiles in healthy subjects [9]
and in obese people [10].
In addition, noticeably positive effects on the mental
state [11] and antioxidant status of patients with multiple
sclerosis [12] and seropositive rheumatoid arthritis [13] have
been observed when low temperatures were applied to the
entire body.
Little is still known about the role of WBC in the manage-
ment of AS patients. So far, the studies have shown that WBC
procedures in AS patients do not influence ejection fraction,
late ventricular potentials, nor QT dispersion. However, they
do have a beneficial effect on the adaptive processes of the
vegetative nervous system in patients without a significant
pathology in the circulatory system [14].
It has also been proved that in AS patients, WBC proce-
dures with subsequent kinesiotherapy may improve BAS-
DAI (Bath Ankylosing Spondylitis Diseases Activity Index)
and BASFI (Bath Ankylosing Spondylitis Functional Index)
and some spinal mobility parameters and help to decrease
pain [15, 16].
In our preliminary study [17], we showed that WBC may
also have a beneficial influence on some specificinflamma-
tory parameters in AS patients.
In light of the above findings, the primary aim of the
study was to assess the influence of WBC on cardiovascular
risk factors in AS patients with active phase and without
any concomitant classical cardiovascular risk factors.
2. Materials and Methods
2.1. Participants. The study protocol had been reviewed and
approved by the Bioethical Committee of the Medical
University of Silesia in Katowice (permission number: NN-
6501-93/I/07), and all analyzed patients were informed
about the trial and provided written consent for inclusion
in the study. All clinical investigations were conducted
according to the principles expressed in the Declaration of
Helsinki (1964).
The study involved a total of 32 nonsmoking male
patients with ankylosing spondylitis who were divided ran-
domly by a physician into two groups with an allocation ratio
1 : 1. The first group consisted of 16 AS patients exposed to
whole-body cryotherapy procedures with subsequent kine-
siotherapy (WBC group, mean age 46.63 ±1.5 years). The
second group consisted of 16 AS patients exposed only to
kinesiotherapy procedures (KT group, mean age 45.94 ±1.24
years). There was no significant difference in the mean age,
BMI, carotid IMT, BASDAI, BASFI, and comorbiding disor-
ders and distribution of classical cardiovascular risk factors
between these groups.
Computer-generated random numbers were sealed in
sequentially numbered envelopes, and the group allocation
was independent of the time and person delivering the
treatment. The physician (main coordinator) who allocated
the patients to groups had 32 envelopes, each containing a
piece of paper marked with either group WBC or KT. The
physician selected and opened each envelope in the presence
of a physiotherapist to see the symbol and would then direct
the subject to the corresponding group.
Male patients who successfully enrolled in the study had
adefinite diagnosis of AS, did not suffer from any other dis-
eases, had no associated pathologies, and had an attending
physician who did not apply disease-modifying antirheu-
matic drugs (DMARDs), biologic agents, or steroids. The
AS patients were treated with doses of nonsteroidal anti-
inflammatory drugs (NSAIDs), which were not altered
within one month before the beginning of the study and dur-
ing it. All the patients included in the trial fulfilled the mod-
ified New York Criteria for definite diagnosis of AS, which
serves as the basis for the ASAS/EULAR recommendations
[18]. The final selection for the study included only HLA
B27-positive patients, who exhibited II and III radiographic
grades of sacroiliac joint disease and attended a consulting
unit in a health resort in the period of subsidence of acute
clinical symptoms, in order to qualify for sanatorium treat-
ment (physiotherapy). The demographic data of the subjects
is shown in Table 1.
The patients from both groups were asked to abstain
from alcohol, drugs and any immunomodulators, immunos-
timulators, hormones, vitamins, minerals, or other sub-
stances with antioxidant properties for 4 weeks before the
study. All the patients were also asked to refrain from the
consumption of caffeine 12 hours prior to laboratory analy-
ses. The diet of the patients was not modified.
Before the study, each patient was examined by a physi-
cian to exclude any coexisting diseases as well as any contra-
indications for WBC procedures. Prior to the study, a resting
electrocardiogram was performed on all the patients, and
before each session of cryotherapy, the blood pressure was
measured for each patient.
2.2. Whole-Body Cryotherapy and Kinesiotherapy Procedures.
Depending on the group, the AS patients were exposed either
to a cycle of WBC procedures lasting 3 minutes a day with a
subsequent 60-minute session of kinesiotherapy or to a 60-
minute session of kinesiotherapy only, for 10 consecutive
days excluding the weekend.
The WBC procedures were performed in a cryochamber
with cold retention and cooled by synthetic liquid air (pro-
duced by Metrum Cryoflex, Poland), which consists of two
compartments: the antechamber and the proper chamber,
which were connected by a door. In the trial, the tempera-
ture in the antechamber was −60
°
C, whereas in the proper
chamber, it reached −120
°
C. After a 30-second adaptation
process in the antechamber, the patients were exposed to
cryogenic temperatures in the proper chamber for 3
minutes. During the WBC procedure, all the patients were
dressed in swimsuits, cotton socks and gloves, and
wooden shoes and their mouths and noses were protected
by surgical masks and their ears by ear protectors. All
jewelry, glasses, and contact lenses were removed before
entry into the chamber. During the WBC procedure, the
2 Mediators of Inflammation
patients were walking round the chamber without touching
each other.
Immediately after leaving the cryogenic chamber and
changing into track suits and trainers, the AS patients under-
went kinesiotherapy lasting one hour. The program of kine-
siotherapy was the same for all the patients in both groups.
Kinesiotherapy procedures included range-of-motion exer-
cises of the spine and major joints (including the ankle, knee,
hip, wrist, elbow, and shoulder). Chest expansion and breath-
ing exercises were also included. Apart from range-of-
motion exercise, the AS patients received strengthening exer-
cises of the muscles of the major joints (including the ankle,
knee, hip, wrist, elbow, shoulder, thoracolumbar spine, and
cervical spine) as well as aerobic exercise (including cycling
and fast walking). All the exercises were carried out under
the supervision of physical therapists.
All the patients completed the study and no complications
or side effects related to the WBC procedures were observed.
2.3. Blood Sample Collection. Blood samples of all the subjects
were collected in the morning before the first meal. Samples
of whole blood (5 ml) were drawn from the basilic vein of
each subject and then collected into tubes containing ethyl-
enediaminetetraacetic acid (Sarstedt, S-Monovette with
1.6 mg/ml EDTA-K
3
) and into tubes with a clot activator
(Sarstedt, S-Monovette). The blood samples were centrifuged
(10 min, 900gat 4
°
C), and then the plasma and serum were
immediately separated and stored at the temperature of
−75
°
C, until biochemical analyses could be performed. In
turn, the red blood cells retained from the removal of EDTA
plasma were rinsed with isotonic salt solution and then 10%
of the hemolysates were prepared for further analyses. The
hemoglobin concentration in the hemolysates was deter-
mined by the standard cyanmethemoglobin method. The
inter- and intra-assay coefficients of variations (CV) were
1.1% and 2.4%, respectively.
2.4. Biochemical Analyses
2.4.1. Determination of Inflammatory-State Parameters.
High-sensitivity C-reactive protein (hs-CRP) concentration
in the serum was determined by the latex immunoturbidi-
metric method (BioSystems, Spain) and expressed in mg/l.
The inter- and intra-assay coefficients of variations (CV)
were 2.3% and 5.5%, respectively.
The serum ceruloplasmin (CER) oxidase activity was
measured using the p-phenylenediamine kinetic method by
Richterich [19] and expressed in mg/dl after a calibration
with pure ceruloplasmin isolated from a healthy donor serum
pool. The inter- and intra-assay coefficients of variations
(CV) were 3.1% and 6.1%, respectively.
The plasma interleukin 6 (IL-6) and soluble intercellular
adhesion molecule-1 (sICAM-1) concentrations were deter-
mined using the ELISA method from R&D Systems (USA).
The concentrations of IL-6 and sICAM-1 were expressed in
pg/ml and ng/ml. The inter- and intra-assay coefficients of
variations (CV) were 5.1% and 8.8%, respectively, for IL-6
and 4.8% and 9.1%, respectively, for sICAM-1.
2.4.2. Oxidative Stress Marker Analyses
(1) Determination of Lipid Peroxidation Products, Total
Oxidative Status, and Oxidative Stress Index. The intensity
of lipid peroxidation in the plasma and the erythrocytes
was measured spectrofluorimetrically as thiobarbituric acid-
reactive substances (TBARS) according to Ohkawa et al.
[20]. The TBARS concentrations were expressed as malon-
dialdehyde (MDA) equivalents in μmol/l in plasma or in
nmol/gHb in erythrocytes. The inter- and intra-assay coeffi-
cients of variations (CV) were 2.1% and 8.3%, respectively.
The serum concentrations of oxidized low-density lipo-
protein (ox-LDL) and antibodies to ox-LDL (ab-ox-LDL)
were measured with the use of ELISA kits (Biomedica,
Poland). The ox-LDL and the ab-ox-LDL concentrations
were expressed in ng/ml and mU/ml, respectively. The inter-
and intra-assay coefficients of variations (CV) for ox-LDL
were 5.8% and 9.4%, respectively, and −4.1% and 8.7% for
ab-ox-LDL, respectively.
The serum total oxidant status (TOS) was determined
with the method described by Erel [21] and expressed in
μmol/l. The inter- and intra-assay coefficients of variations
(CV) were 2.2% and 6.4%, respectively.
The oxidative stress index (OSI), an indicator of the
degree of oxidative stress, was expressed as the ratio of total
oxidant status (TOS) to total antioxidant capacity (FRAP)
in arbitrary units [22].
(2) Determination of Activity of Antioxidant Enzymes. The
plasma and erythrocytes superoxide dismutase (SOD -
E.C.1.15.1.1) activity was determined by the Oyanagui
method [23]. Enzymatic activity was expressed in nitrite unit
(NU) in each mg of hemoglobin (Hb) or ml of blood plasma.
One nitrite unit (1 NU) means a 50% inhibition of nitrite ion
production by SOD in this method. SOD isoenzymes (SOD-
Table 1: Demographic data of the study subjects.
Characteristic
WBC
group
(n=16)
Kinesiotherapy
group
(n=16)
Pvalue
Age (years), mean (SD) 46.63 ±1.5 45.94 ±1.24 0.114
Sex (M/F) 16/0 16/0 —
BMI (kg/m
2
), mean (SD) 24.24 ±4.4 23.76 ±6.8 0.880
BASDAI 5.43 ±1.61 5.28 ±1.71 0.720
BASFI 5.20 ±2.29 5.01 ±2.06 1.00
Carotid IMT (mm) 1.1 ±0.13 1.0 ±0.14 0.925
Smoking (yes/no) 0/16 0/16 —
Medication
NSAID (yes/no) 16/0 16/0 —
DMARD (yes/no) 0/16 0/16 —
Biological agents
(yes/no) 0/16 0/16 —
SD: standard deviation; BMI: body mass index; BASDAI: the Bath
Ankylosing Spondylitis Diseases Activity Index; BASFI: the Bath
Ankylosing Spondylitis Functional Index; IMT: intima-media thickness;
NSAID: nonsteroidal anti-inflammatory drug; DMARD: disease-modifying
antirheumatic drug.
3Mediators of Inflammation
Mn and SOD-ZnCu) were measured using potassium cya-
nide as the inhibitor of the SOD-ZnCu isoenzyme. The inter-
and intra-assay coefficients of variations (CV) were 2.8% and
5.4%, respectively.
The catalase (CAT - E.C.1.11.1.6.) activity in erythrocytes
was measured by the Aebi [24] kinetic method and expressed
in IU/mgHb. The inter- and intra-assay coefficients of varia-
tions (CV) were 2.6% and 6.1%, respectively.
The erythrocyte glutathione peroxidase (GPx - E.C.1.11.
1.9.) activity was assayed by Paglia and Valentine’s kinetic
method [25], with t-butyl peroxide as a substrate and
expressed as micromoles of NADPH oxidized per minute
and normalized to one gram of hemoglobin (IU/gHb). The
inter- and intra-assay coefficients of variations (CV) were
3.4% and 7.5%, respectively.
The activity of glutathione reductase in erythrocytes
(GR - E.C.1.6.4.2) was assayed by Richterich’s kinetic method
[19], expressed as micromoles of NADPH utilized per min-
ute and normalized to one gram of hemoglobin (IU/gHb).
The inter- and intra-assay coefficients of variations (CV)
were 2.1% and 5.8%, respectively.
(3) Determination of Nonenzymatic Antioxidant Status. The
total antioxidant capacity of plasma was measured as the
ferric-reducing ability of plasma (FRAP) according to Benzie
and Strain [26] and calibrated using Trolox and expressed in
(μmol/l). The inter- and intra-assay coefficients of variations
(CV) were 1.1% and 3.8%, respectively.
The serum concentration of protein sulfhydryl (PSH)
was determined by Koster’s method [27], using dithionitro-
benzoic acid (DTNB) and expressed in (μmol/l). The inter-
and intra-assay coefficients of variations (CV) were 2.6%
and 5.4%, respectively.
The serum concentration of uric acid (UA) was deter-
mined by a uricase-peroxidase method [28] on the Cobas
Integra 400 plus analyzer and expressed as (mg/dl). The
inter- and intra-assay coefficients of variations (CV) were
1.4% and 4.4%, respectively.
2.4.3. Determination of Lipid Profile. The total, HDL, and
LDL cholesterol (T-Chol, HDL-Chol, and LDL-Chol, resp.)
and triglyceride (TG) concentrations in serum were esti-
mated using routine techniques (Cobas Integra 400 plus ana-
lyzer, Roche Diagnostics, Mannheim, Germany). The
concentrations were expressed in (mg/dl). The inter- and
intra-assay coefficients of variations (CV) were 2.8% and
5.4%, respectively, for T-Chol; 3.2% and 5.4%, respectively,
for HDL-Chol; 2.6% and 6.5%, respectively, for LDL-Chol;
and 2.5% and 7.6%, respectively, for TG. The triglyceride/
HDL cholesterol (TG/HDL) ratio was calculated.
2.4.4. Determination of Atherosclerosis Plaque Instability
Markers and Atherosclerosis Plaque Markers. Serum
pregnancy-associated plasma protein-A (PAPP-A), soluble
CD40 ligand (sCD40L), and placental growth factor (PLGF)
concentrations were assayed by ELISA methods with DRG
Instruments GmbH (Germany). The PAPP-A and sCD40L
concentrations were expressed in ng/ml and the PLGF con-
centration in pg/ml. The inter- and intra-assay coefficients
of variations (CV) were 6.8% and 10.2%, respectively, for
PAPPA-A; 5.1% and 9.4%, respectively, for sCD40L; and
6.2% and 12.1%, respectively, for PLGF.
2.5. Assay of Activity of Ankylosing Spondylitis. The activity of
ankylosing spondylitis was measured by the Bath Ankylosing
Spondylitis Diseases Activity Index (BASDAI) and the Bath
Ankylosing Spondylitis Functional Index (BASFI).
The BASDAI has six questions related to fatigue, back
pain, peripheral pain, peripheral swelling, local tenderness,
and morning stiffness (degree and length). Other than the
issues relating to morning stiffness, all questions were scored
from 0 (none) to 10 (very severe) using a visual analogue
scale (VAS). The sum was calculated as the mean of two
morning stiffness issues and the four remaining issues [29].
The BASFI is the mean score of ten questions address-
ing functional limitations and the level of physical activity
at home and work, assessed on VAS scales (0 = easy,
10 = impossible) [30].
2.6. Assay of Intima-Media Thickness. A high-resolution
Doppler ultrasonography was performed with a Logic-5
device with a high-frequency (11 MHz, 15 MHz) linear
probe. The sonographer was an angiologist who was unaware
of subject’s clinical state. The measurement of intima-media
thickness (IMT) was performed in the right and left common
carotid arteries, and the average of the 2 measurements was
calculated. The IMT was expressed in mm.
2.7. Statistical Analyses. Statistical analyses were undertaken
using the statistical package of Statistica 10 Pl software. For
each parameter, the indicators of the descriptive statistics
were determined (mean value and standard deviation
(SD)). The normality of the data distribution was checked
using the Shapiro-Wilk test, while the homogeneity of the
variance was checked by applying Levene’s test. In order
to compare the differences between the groups, an indepen-
dent sample Student t-test was used or alternatively the
Mann–Whitney Utest. In the case of dependent samples,
the Student t-test was used or alternatively the Wilcoxon
test. Correlations between particular parameters were statis-
tically verified by means of Spearman’s nonparametric cor-
relation test. Differences at the significance level of P<0 05
were considered as statistically significant.
3. Results
3.1. Inflammatory-State Parameters, BASDAI, and BASFI. In
the WBC group of AS patients, who underwent a ten-day-
long cycle of WBC procedures with subsequent kinesiother-
apy, it was found that after the completion of the treatment,
the levels of hsCRP and CER decreased significantly. In the
case of hsCRP, the difference prior to post treatment values
in the WBC group was significantly higher in comparison
to those in the KT group patients. Also, in the WBC group,
the level of sICAM-1 showed a decreasing trend. Moreover,
after the completion of the WBC cycle, the level of sICAM-
1 was significantly lower in comparison to the KT group.
But the level of IL-6 did not change significantly in the
4 Mediators of Inflammation
WBC group with subsequent kinesiotherapy after the com-
pletion of treatment.
After the completion of treatment, only the level of CER
decreased significantly from the estimated inflammatory
parameters in AS patients from the KT group who under-
went a cycle of kinesiotherapy only, without being preceded
by WBC procedures. The levels of hsCRP and sICAM-1 did
not change significantly in the KT group. Also, as in the
WBC group, no statistically significant changes in the level
of IL-6 were observed in the KT group.
In turn, the BASDAI and BASFI decreased significantly
in both groups, but in the WBC group with subsequent kine-
siotherapy after the completion of the treatment, the decrease
of these parameters was significantly higher in comparison to
that in the KT group. Moreover, only in the WBC group after
the completion of the treatment, the value of both BASDAI
and BASFI was below 4 (inactive phase of AS disease)
(Table 2).
3.2. Oxidative Stress. We observed that patients in the WBC
group had, after the completion of the treatment, a statisti-
cally significant decrease in erythrocyte levels of MDA,
serum anti-ox-LDL ab, serum TOS, and value of OSI in com-
parison to initial values. What is more, the differences of these
parameters prior to post treatment values in the WBC group
were significantly higher in comparison to the KT group.
The levels of plasma MDA and serum ox-LDL did not change
significantly in the WBC group. In turn, in the KT group, no
significant changes in the levels of plasma and erythrocyte
MDA, serum ox-LDL, serum anti-ox-LDL ab, and serum
TOS and OSI were observed after the completion of the treat-
ment, in comparison to the initial values before the beginning
of the kinesiotherapy cycle (Table 3).
In the WBC group patients, we observed a statistically
significant decrease in erythrocyte activity of GPx after
the completion of a cycle of cryotherapy procedures with
subsequent kinesiotherapy. However, the activity of plasma
and erythrocyte total SOD, plasma SOD-Mn, plasma SOD-
CuZn, erythrocyte CAT, and GR did not change signifi-
cantly in the WBC group after treatment. But in the
WBC group, the activity of plasma SOD-Mn after treat-
ment was significantly higher in comparison to the KT
group. In turn, in the KT group, the activity of erythrocyte
total SOD, GPx, and GR decreased significantly after
Table 2: Levels of inflammatory parameters as well as the value of BASDAI and BASFI (mean value ±standard deviation (SD)) in AS
patients before and after the completion of a cycle of ten whole-body cryotherapy procedures with subsequent kinesiotherapy (WBC
group) or a cycle of ten kinesiotherapy procedures only (KT group), with statistical analyses. (p): plasma; (s): serum; Δ:difference prior to
post treatment.
Parameters WBC group KT group P
hsCRP (s) (mg/l)
Before 13.5 ±16.3 13.9 ±15.2 0.942
After 9.2 ±15.3 13.6 ±16.2 0.438
∗P0.002 0.623
Δ−4.24 ±5.68 −0.27 ±3.25 0.023
CER (s) (mg/dl)
Before 62.83 ±12.61 67.57 ±12.60 0.296
After 51.32 ±10.74 53.51 ±14.26 0.628
∗P0.006 0.003
Δ−11.51 ±16.6 −14.06 ±14.47 0.646
IL-6 (p) (pg/ml)
Before 41.6 ±8.86 41.8 ±10.5 0.957
After 36.6 ±7.89 41.0 ±10.4 0.191
∗P0.121 0.301 0.216
Δ−4.94 ±11.9 −0.74 ±5.71
sICAM-1 (p) (ng/ml)
Before 79.0 ±15.5 84.3 ±21.9 0.432
After 69.2 ±14.2 83.9 ±20.0 0.023
∗P0.088 0.642
Δ−9.84 ±23.0 −0.41 ±16.1 0.191
BASDAI
Before 5.43 ±1.61 5.28 ±1.71 0.720
After 3.29 ±0.91 4.53 ±1.62 <0.05
∗P<0.001 <0.001
Δ−2.14 ±1.23 −0.74 ±0.38 0.001
BASFI
Before 5.20 ±2.29 5.01 ±2.06 1.00
After 3.81 ±2.20 4.35 ±2.23 0.497
∗P<0.001 <0.001
Δ−1.39 ±1.03 −0.66 ±0.39 <0.01
P: statistical significance of differences between both groups of patients; ∗P: statistical significance of differences between values before and after treatment in
particular groups of patients.
5Mediators of Inflammation
treatment in comparison to the WBC group. Additionally,
the activity of plasma SOD-CuZn showed also a decreased
tendency in the KT group. Similarly as in the WBC group
patients, the activity of plasma total SOD and erythrocyte
CAT did not change significantly in the KT group after
treatment (Table 4).
What is more, in the WBC group, the parameters of non-
enzymatic antioxidants, FRAP values, and UA concentration
increased significantly after treatment. The levels of those
parameters were significantly higher in the WBC group in
comparison to the KT group after the completion of the
treatment. The level of PSH did not change significantly in
the WBC group after treatment. In turn, in the KT group,
the FRAP values and PSH level decreased significantly, but
the level of UA did not change significantly after treatment
(Table 5).
3.3. Markers of Lipid Profile, Atherosclerosis Plaque, and
Atherosclerosis Plaque Instability. The levels of T-Chol,
LDL, TG, sCD40L, PLGF, and PAPP-A decreased signifi-
cantly after treatment in both groups, but the differences
prior to post treatment values in the WBC group were signif-
icantly higher in comparison to the KT group, except for T-
Chol. But the TG difference prior to post treatment values
in the WBC group was higher in comparison to the KT
group. The level of HDL-Chol did not change significantly
in both groups. The TG/HDL ratio showed a decreasing ten-
dency in the WBC group in comparison to the KT group
(Table 6).
3.4. Significant Relationships among the Estimated Parameters
in AS Patients Who Underwent WBC Procedures. After treat-
ment, we noticed significant relationships in the WBC group
between changes of serum hsCRP concentration and erythro-
cyte MDA concentration (r=06). Also, a positive correlation
between serum hsCRP change and plasma FRAP activity
change (r=0 6) was observed. Additionally, a negative corre-
lation between serum hsCRP concentration and plasma
SOD-CuZn activity was found (r=−062). In the case of
the analysis of serum oxLDL-ab, we observed a negative cor-
relation with CAT and SOD activities in erythrocytes (rcoef-
ficients: −0.51 and −0.53, resp.). Furthermore, the ratio of
Table 3: Levels of lipid peroxidation parameters, total oxidative status (TOS), and oxidative stress index (OSI) (mean value ±standard
deviation (SD)) in AS patients before and after the completion of a cycle of ten whole-body cryotherapy procedures with subsequent
kinesiotherapy (WBC group) or a cycle of ten kinesiotherapy procedures only (KT group), with statistical analyses. (p): plasma; (s): serum;
(e): erythrocyte lysates; Δ:difference prior to post treatment.
Parameters WBC group KT group P
MDA (p) (μmol/l)
Before 2.54 ±0.52 2.32 ±0.60 0.272
After 2.30 ±0.75 2.41 ±0.83 0.715
∗P0.278 0.959
Δ−0.24 ±0.81 0.09 ±1.04 0.331
MDA (e) (nmol/gHb)
Before 0.17 ±0.04 0.18 ±0.02 0.418
After 0.15 ±0.03 0.18 ±0.04 0.007
∗P0.013 0.642
Δ−0.02 ±0.03 0.00 ±0.04 0.043
ox-LDL (s) (ng/ml)
Before 249 ±77.6 298 ±122 0.191
After 223 ±100 288±133 0.132
∗P0.301 0.84
Δ−25.9 ±123 −9.6 ±149 0.738
Anti-oxLDL ab (s) (mU/ml)
Before 465 ±209 571±426 0.382
After 347 ±139 490±316 0.111
∗P0.013 0.379
Δ−118 ±178 −80.5 ±323 0.687
TOS (s) (μmol/l)
Before 26.54 ±4.45 23.94 ±11.60 0.414
After 12.09 ±2.55 24.41 ±6.24 <0.001
∗P<0.001 0.605
Δ−14.45 ±4.83 0.46 ±9.11 <0.001
OSI (p/s) (arbitrary unit)
Before 24.10 ±15.94 18.87 ±11.30 0.294
After 8.20 ±6.76 23.65 ±15.68 0.002
∗P0.003 0.301
Δ−15.90 ±16.82 4.78 ±13.88 0.001
P: statistical significance of differences between both groups of patients; ∗P: statistical significance of differences between values before and after treatment in
particular groups of patients.
6 Mediators of Inflammation
TG/HDL was positively correlated with the PLGF serum
concentration after WBC procedures (r=0 58). We also
observed a positive correlation between plasma concentra-
tions of sICAM-1 and MDA (r=066) in the WBC group
after treatment. In the case of erythrocyte GPx activity in
AS patients who underwent WBC procedures with subse-
quent kinesiotherapy, a positive correlation with plasma
PSH (r=054) was visible and a negative correlation was
found with plasma MDA concentration. All the correlations
mentioned above were significant (p<005).
4. Discussion
In our study, we observed that, after the completion of the
treatment, the WBC group of AS patients who underwent a
ten-day-long cycle of WBC procedures with subsequent
kinesiotherapy had significantly decreased levels of hsCRP
and CER. The level of sICAM-1 showed a decreasing trend
in the WBC group. But the level of IL-6 did not change
significantly.
The results of the inflammatory parameters in this study
are consistent with our previous preliminary study [17], in
which AS patients who underwent WBC procedures were
observed to have a decrease in CRP, fibrinogen, mucoprotein,
and sICAM levels.
However, in another study [31], the authors have
observed a decrease in TNF-αand an increase in IL-6 in ten-
nis players after a 5-day exposure to WBC twice a day.
Banfiet al. [32] have also confirmed that a decreased level
of sICAM-1 is induced by WBC treatment and is linked to an
anti-inflammatory response. In another paper, Pournot et al.
[33] have found that WBC (−110
°
C) decreased IL-1βand
CRP levels and increased the IL-1ra level after intense exercise.
But the levels of TNF-α, IL-10, and IL-6 remained unchanged.
Similarly, in our study, we did not observe any changes in
serum IL-6 in AS patients who underwent WBC.
Table 4: Activities of antioxidant enzymes (mean value ±standard deviation (SD)) in AS patients before and after the completion of a cycle
of ten whole-body cryotherapy procedures with subsequent kinesiotherapy (WBC group) or a cycle of ten kinesiotherapy procedures only
(KT group), with statistical analyses. (p): plasma; (e): erythrocyte lysates; Δ:difference prior to post treatment.
Parameters WBC group KT group P
Total SOD (p) (NU/ml)
Before 13.4 ±2.13 12.3 ±1.85 0.145
After 12.1 ±1.88 1.7 ±2.49 0.632
∗P0.233 0.301
Δ−1.28 ±3.13 −0.60 ±2.65 0.512
SOD-Mn (p) (NU/ml)
Before 5.37 ±2.75 4.56 ±1.86 0.336
After 6.27 ±0.99 5.02 ±1.64 0.015
∗P0.163 0.642
Δ0.90 ±2.80 0.46 ±2.46 0.642
SOD-CuZn (p) (NU/ml)
Before 8.09 ±2.74 7.80 ±2.21 0.749
After 7.15 ±1.32 7.05 ±3.09 0.902
∗P0.326 0.063
Δ−0.93 ±2.77 −0.75 ±2.72 0.854
Total SOD (e) (NU/mgHb)
Before 85.5 ±17.3 128.0 ±11.2 <0.001
After 90.5 ±11.9 111.0 ±15.6 <0.001
∗P0.438 0.001
Δ5.02 ±17.3 −17.1 ±11.8 <0.001
CAT (e) (IU/mgHb)
Before 385.0 ±70.3 425.0 ±53.6 0.084
After 375.0 ±58.3 412.0 ±58.6 0.088
∗P0.535 0.352
Δ−9.9 ±57.0 −13.0 ±54.0 0.876
GPx (e) (IU/gHb)
Before 31.2 ±4.90 29.9 ±2.84 0.363
After 29.1 ±2.97 20.4 ±5.05 <0.001
∗P0.039 0.001
Δ−2.09 ±3.61 −9.49 ±6.74 0.001
GR (e) (IU/gHb)
Before 1.72 ±0.56 2.07 ±0.52 0.043
After 1.54 ±0.60 1.65 ±0.59 0.078
∗P0.469 0.002
Δ−0.18 ±0.80 −0.42 ±0.41 0.622
P: statistical significance of differences between both groups of patients; ∗P: statistical significance of differences between values before and after treatment in
particular groups of subjects.
7Mediators of Inflammation
In the present study, we also saw a significant decrease in
the BASDAI and BASFI after the completion of the WBC
treatment in a cryochamber with cold retention. Similar
results were observed in a closed cryochamber of a type called
“Wrocławski”, cooled by liquid nitrogen [15]. In the both
studies, after the completion of a cycle consisting of ten daily
3-minute-long WBC procedures with subsequent kine-
siotherapy (−120
°
C, with a weekend break), the BASDAI
and BASFI decreased below 4. This indicates that the AS dis-
ease entered an inactive phase after the completion of treat-
ment. Our results are also consistent with a study [16], in
which the AS patients underwent 8 daily WBC procedures
(−110
°
C, 3 minutes).
There are not many reports on the impact of WBC on
the prooxidant-antioxidant balance. It has been noticed that
WBC procedures may have a beneficial influence on antiox-
idant status. In the study performed by Dugué et al. [34], a
significant increase has been seen in the TAS value in healthy
men at the end of a cycle of 45 procedures of WBC (−110
°
C,
2 minutes, coolant liquid nitrogen) performed three times a
week. In another study, Miller et al. [12] have noticed an
increase in total antioxidant status, SOD activity, and uric
acid level in the plasma of multiple sclerosis patients who
underwent WBC treatment (−110
°
temperature, daily 10
procedures with weekend break, coolant medium liquid
nitrogen). What is more, WBC was advocated to possibly
enhance antioxidant capacities and, thus, counteract the
exercise-induced reactive oxygen species production [12].
However, in a different study [13], patients with seropos-
itive rheumatoid were observed by the authors to have only a
short-term increase in TRAP during the first treatment ses-
sion of WBC (−110
°
C, three times daily for 7 consecutive
days) and the cold treatment did not cause any significant
oxidative stress or adaptation.
In our study, we observed a significant decrease in oxida-
tive stress, which may also be linked to the decrease in
systemic inflammation in AS patients who underwent WBC
treatment. After treatment, in the WBC group, we observed
positive correlations between plasma concentrations of
sICAM-1 and MDA as well as serum hsCRP and erythrocyte
MDA concentrations. In addition, negative correlations
between serum hsCRP concentration and plasma SOD-
CuZn activity were found.
Furthermore, we observed the similar results in healthy
subjects who underwent WBC procedures performed in a
cryochamber with cold retention [35].
The differences in the results of various studies may be
related to the type of cryochamber being used and the
coolant medium, in addition to the time of exposure to
cryogenic temperatures.
Only a few papers have estimated the impact of WBC on
lipid profile. In rats exposed to WBC for 5 or 10 days, HDL
and LDL cholesterol fraction decreased and total cholesterol
levels in animals subjected to −60
°
C sessions for 10 days
remained unchanged. The authors have also observed an
increase in triglycerides in the blood serum of animals sub-
jected to cryostimulation compared to control. A decrease
in HDL cholesterol in rats after cryostimulation can be
explained by the fact that HDL is the main fraction transport-
ing cholesterol in rats, while in humans, most cholesterol is
found in low-density lipoproteins [36].
In another study [9], the authors have observed reducing
T-Chol, LDL-Chol, and TG and increasing HDL-Chol after
20 sessions of WBC in healthy men, but after 10 sessions of
WBC, only LDL-Chol decreased, while a simultaneous
HDL-Chol increase was observed in healthy men (cryogenic
temperature −130
°
C).
In another study by these authors [14], a significant
decrease in the level of LDL-Chol and TG has been
observed, with a slight increase in high-density lipoprotein
concentration after WBC treatment, including two cryosti-
mulation treatments of 20 daily sessions in the second and
Table 5: Levels of nonenzymatic antioxidants (mean value ±standard deviation (SD)) in AS patients before and after the completion of a
cycle of ten whole-body cryotherapy procedures with subsequent kinesiotherapy (WBC group) or a cycle of ten kinesiotherapy procedures
only (KT group), with statistical analyses. (p): plasma; (s): serum; Δ:difference prior to post treatment.
Parameters WBC group KT group P
FRAP (μmol/l)
Before 587.1 ±58.3 550.0 ±91.3 0.183
After 636.1 ±62.3 499.3 ±74.6 <0.001
∗P0.010 0.001
Δ49.0 ±31.7 −50.8 ±39.4 <0.001
PSH (s) (μmol/l)
Before 402.6 ±91.7 393.2 ±90.0 0.772
After 392.6 ±87.4 364.7 ±28.4 0.239
∗P0.836 0.017
Δ−9.9 ±108.1 −28.5 ±92.6 0.605
UA (s) (mg/dl)
Before 5.40 ±1.39 4.34 ±1.15 0.025
After 6.62 ±2.07 4.61 ±1.25 0.003
∗P0.011 0.196
Δ1.22 ±1.70 0.27 ±0.70 0.052
P: statistical significance of differences between both groups of patients; ∗P: statistical significance of differences between values before and after treatment in
particular groups of patients.
8 Mediators of Inflammation
the last month of intervention, without diet modification in
obese subjects.
In our study, we also observed a significant decrease
in T-Chol, LDL-Chol, and TG. But the HDL-Chol level
did not change after completing WBC procedures in the
AS patients. What is more, in our study, we observed a sig-
nificant decrease in the levels of sCD40, PAPP-A, and
PLGF. Additionally, in the present study, the ratio of TG/
HDL was positively correlated with the PLGF serum con-
centration after WBC procedures. The impact of WBC on
these markers in AS patients has been estimated for the
first time.
A significant decrease in lipid profile, atherosclerotic
plaque and oxidative stress, and inflammatory parameters,
as well as a reduction in the proportion of TG cholesterol
to HDL cholesterol (TG/HDL ratio), seems beneficial enough
to consider WBC treatment as a useful method for athero-
sclerosis prevention in AS patients.
The present study has some limitations. First, the
study did not provide long-term follow-up (at least 3
months), and thus, we do not know how long the benefi-
cial effect of WBC with subsequent kinesiotherapy would
be maintained after the completion of a WBC cycle. Sec-
ond, the cycle of WBC with subsequent kinesiotherapy
Table 6: Levels of lipid profile parameters, atherosclerosis plaque markers, and atherosclerosis plaque instability and values of TG/HDL ratio
(mean value ±standard deviation (SD)) in AS patients before and after the completion of a cycle of ten whole-body cryotherapy procedures
with subsequent kinesiotherapy (WBC group) or a cycle of ten kinesiotherapy procedures only (KT group), with statistical analyses.
(p): plasma; (s): serum; (e): erythrocyte lysates; Δ:difference prior to post treatment.
Parameters WBC group KT group P
T-Chol (s) (mg/dl)
Before 221.3 ±39.17 200.33 ±21.33 0.074
After 202.40 ±24.40 190.70 ±22.57 0.51
∗P0.0006 0.04
Δ−18.90 ±20.54 −9.63 ±18.38 0.20
LDL-Chol (s) (mg/dl)
Before 125.2 ±32.6 145.3 ±28.3 0.073
After 93.3 ±36.9 132.4 ±24.7 0.002
∗P<0.001 0.005
Δ−31.9 ±28.6 −12.9 ±15.1 0.027
HDL-Chol (s) (mg/dl)
Before 50.5 ±14.1 58.0 ±18.0 0.198
After 47.0 ±9.0 56.4 ±18.2 0.078
∗P0.079 0.109
Δ−3.5 ±9.1 −1.7 ±10.1 0.590
TG (s) (mg/dl)
Before 185.1 ±18.9 178.6 ±15.9 0.299
After 156.7 ±11.2 165.2 ±20.4 0.158
∗P0.001 0.001
Δ−28.4 ±22.4 −13.4 ±19.7 0.053
TG/HDL ratio
Before 3.95 ±1.18 3.32 ±0.96 0.150
After 3.44 ±0.60 3.18 ±0.99 0.320
∗P0.055 0.250
Δ−0.51 ±0.92 −0.14 ±0.43 0.190
sCD40L(s) (mg/ml)
Before 9.21 ±3.88 7.25 ±2.20 0.180
After 5.01 ±2.55 5.85 ±2.06 0.171
∗P0.0004 0.006
Δ−4.19 ±2.17 −1.4 ±1.78 0.0001
PLGF(s) (pg/ml)
Before 30.17 ±10.23 21.69 ±3.54 0.007
After 19.32 ±5.53 18.31 ±2.91 0.641
∗P0.001 0.004
Δ−10.84 ±7.05 −3.38 ±2.13 0.0001
PAPP-A (s) (ng/ml)
Before 17.74 ±7.78 14.48 ±4.52 0.162
After 11.24 ±3.12 11.79 ±3.72 0.920
∗P0.0004 0.003
Δ−6.51 ±8.40 −2.69 ±3.65 0.008
P: statistical significance of differences between both groups of patients; ∗P: statistical significance of differences between values before and after treatment in
particular groups of patients.
9Mediators of Inflammation
consisted of only ten procedures. A greater number of
procedures (e.g., 20–30) could probably increase the treat-
ment effect. Third, the study should involve a larger num-
ber of AS patients.
5. General Conclusion
Whole-body cryotherapy with subsequent kinesiotherapy
facilitates a decrease in oxidative stress, lipid profile, athero-
sclerosis plaque, and its instability, as well as inflammatory
parameters, and appears to be a useful method of atheroscle-
rosis prevention in AS patients.
Conflicts of Interest
The authors declare that there is no conflict of interests
regarding the publication of this paper.
Acknowledgments
This work was supported by grants from the Medical Uni-
versity of Silesia (KNW-1-045/K/7/K and KNW-640-2-1-
376/17).
References
[1] A. Bremander, I. F. Petersson, S. Bergman, and M. Englund,
“Population-based estimates of common comorbidities and
cardiovascular disease in ankylosing spondylitis,”Arthritis
Care & Research, vol. 63, no. 4, pp. 550–556, 2011.
[2] N. Bodnar, G. Kerekes, I. Seres et al., “Assessment of subclini-
cal vascular disease associated with ankylosing spondylitis,”
The Journal of Rheumatology, vol. 38, no. 4, pp. 723–729, 2011.
[3] A. Stanek, A. Cholewka, T. Wielkoszyński, E. Romuk,
K. Sieroń, and A. Sieroń,“Increased levels of oxidative stress
markers, soluble CD40 Ligand and carotid intima-media thick-
ness reflect acceleration of atherosclerosis in male patients with
ankylosing spondylitis in active phase and without the classical
cardiovascular risk factors,”Oxidative Medicine and Cellular
Longevity, vol. 2017, Article ID 9712536, 8 pages, 2017.
[4] D. van der Heijde, S. Ramiro, R. Landewé et al., “2016 update
of the ASAS-EULAR management recommendations for axial
spondyloarthritis,”Annals of the Rheumatic Diseases, vol. 76,
no. 6, pp. 978–991, 2017.
[5] L. A. Passalent, “Physiotherapy for ankylosing spondylitis: evi-
dence and application,”Current Opinion in Rheumatology,
vol. 23, no. 2, pp. 142–147, 2011.
[6] J. Tyrrell, W. Schmidt, D. H. Williams, and C. H. Redshaw,
“Physical activity in ankylosing spondylitis: evaluation and
analysis of an eHealth tool,”Journal of Innovation in Health
Informatics, vol. 23, no. 2, pp. 510–522, 2016.
[7] X. Guillot, N. Tordi, L. Mourot et al., “Cryotherapy in inflam-
matory rheumatic diseases: a systematic review,”Expert Review
of Clinical Immunology, vol. 10, no. 2, pp. 281–294, 2014.
[8] G. Lombardi, E. Ziemann, and G. Banfi,“Whole-body cryo-
therapy in athletes: from therapy to stimulation. An updated
review of the literature,”Frontiers in Physiolology, vol. 8,
no. 258, pp. 1–16, 2017.
[9] A. Lubkowska, G. Banfi, B. Dolegowska, G. V. d’Eril, J. Łuczak,
and A. Barrasi, “Changes in lipid profile in response to three
different protocols of whole-body cryostimulation treat-
ments,”Cryobiology, vol. 61, no. 1, pp. 22–26, 2010.
[10] A. Lubkowska, W. Dudzińska, I. Bryczkowska, and
B. Dołęgowska, “Body composition, lipid profile, adipokine
concentration, and antioxidant capacity changes during inter-
ventions to treat overweight with exercise programme and
whole-body cryostimulation,”Oxidative Medicine and Cellu-
lar Longevity, vol. 2015, Article ID 803197, 13 pages, 2015.
[11] J. Rymaszewska, D. Ramsey, and S. Chładzińska-Kiejna,
“Whole-body cryotherapy as adjunct treatment of depressive
and anxiety disorders,”Archivum Immunologiae et Therapiae
Experimentalis, vol. 56, no. 1, pp. 63–68, 2008.
[12] E. Miller, M. Mrowiecka, K. Malinowska, K. Zołynski, and
J. Kedziora, “Effects of the whole-body cryotherapy on a total
antioxidative status and activities of some antioxidative
enzymes in blood of patients with multiple sclerosis-
preliminary study,”The Journal of Medical Investigation,
vol. 57, no. 1,2, pp. 168–173, 2010.
[13] H. Hirvonen, H. Kautiainen, E. Moilanen, M. Mikkelsson, and
M. Leirisalo-Repo, “The effect of cryotherapy on total antioxi-
dative capacity in patients with active seropositive rheumatoid
arthritis,”Rheumatology International, vol. 37, no. 9,
pp. 1481–1487, 2017.
[14] L. Jagodziński, A. Stanek, J. Gmyrek, G. Cieślar, A. Sielańczyk,
and A. Sieroń,“Evaluation of whole-body cryotherapy on the
circulatory system in patients with ankylosing spondylitis by
analysis of duration and QT interval dispersion,”Polish Jour-
nal of Physiotherapy, vol. 7, no. 3, pp. 362–369, 2007.
[15] A. Stanek, A. Cholewka, J. Gaduła, Z. Drzazga, A. Sieroń, and
K. Sieroń-Stołtny, “Can whole-body cryotherapy with subse-
quent kinesiotherapy procedures in closed type cryogenic
chamber improve BASDAI, BASFI, some spine mobility
parameters and decrease pain intensity in patients with anky-
losing spondylitis?,”BioMed Research International, vol. 2015,
Article ID 404259, 11 pages, 2015.
[16] M. W. Romanowski, W. Romanowski, P. Keczmer,
M. Majchrzycki, W. Samborski, and A. Straburzynska-Lupa,
“Whole body cryotherapy in rehabilitation of patients with
ankylosing spondylitis. A randomised controlled study,”Phys-
iotherapy, vol. 101, article e1294, Supplement 1, 2015.
[17] A. Stanek, G. Cieślar, K. Strzelczyk et al., “Influence of cryo-
genic temperatures on inflammatory markers in patients with
ankylosing spondylitis,”Polish Journal of Environmental
Study, vol. 19, no. 1, pp. 167–175, 2010.
[18] S. van der Linden, H. A. Valkenburg, and A. Catts, “Evaluation
of diagnostic criteria for ankylosing spondylitis,”Arthritis &
Rheumatology, vol. 27, no. 4, pp. 361–368, 1984.
[19] R. Richterich, Clinical Chemistry: Theory and Practice, Aca-
demic Press, New York, 1969.
[20] H. Ohkawa, N. Ohishi, and K. Yagi, “Assay for lipid peroxides
in animal tissues by thiobarbituric acid reaction,”Analytical
Biochemistry, vol. 95, no. 2, pp. 351–358, 1979.
[21] O. Erel, “A new automated colorimetric method for measuring
total oxidant status,”Clinical Biochemistry, vol. 38, no. 12,
pp. 1103–1111, 2005.
[22] M. Harma, M. Harma, and O. Erel, “Increased oxidative stress
in patients with hydatidiform mole,”Swiss Medical Weekly,
vol. 133, no. 41-42, pp. 563–566, 2003.
[23] Y. Oyanagui, “Reevaluation of assay methods and establish-
ment of kit for superoxide dismutase activity,”Analytical Bio-
chemistry, vol. 142, no. 2, pp. 290–296, 1984.
10 Mediators of Inflammation
[24] H. Aebi, “[13] Catalase in vitro,”Methods in Enzymology,
vol. 105, pp. 121–126, 1984.
[25] D. Paglia and W. Valentine, “Studies on the quantitative and
qualitative characterization of erythrocyte glutathione peroxi-
dase,”The Journal of Laboratory and Clinical Medicine,
vol. 70, no. 1, pp. 158–169, 1967.
[26] I. F. F. Benzie and J. J. Strain, “The ferric reducing ability of
plasma (FRAP) as a measure of “antioxidant power: the FRAP
assay”,”Analytical Biochemistry, vol. 239, no. 1, pp. 70–76,
1996.
[27] J. F. Koster, P. Biemond, and A. J. Swaak, “Intracellular and
extracellular sulphydryl levels in rheumatoid arthritis,”Annals
of the Rheumatic Diseases, vol. 45, no. 1, pp. 44–46, 1986.
[28] Y. Zhao, X. Yang, W. Lu, H. Liao, and F. Liao, “Uricase based
methods for determination of uric acid in serum,”Microchi-
mica Acta, vol. 164, no. 1-2, pp. 1–6, 2009.
[29] S. Garrett, T. Jenkinson, L. G. Kennedy, H. Whitelock,
P. Gaisford, and A. Calin, “A new approach to defining disease
status in ankylosing spondylitis: the Bath Ankylosing Spondy-
litis Disease Activity Index,”Journal of Rheumatology, vol. 21,
no. 12, pp. 2286–2291, 1994.
[30] A. Calin, S. Garrett, H. Whitelock et al., “A new approach to
defining functional ability in ankylosing spondylitis: the devel-
opment of the Bath Ankylosing Spondylitis Functional Index,”
The Journal of Rheumatology, vol. 21, no. 12, pp. 2281–2285,
1984.
[31] E. Ziemann, R. A. Olek, S. Kujach et al., “Five-day whole-body
cryostimulation, blood inflammatory markers, and perfor-
mance in high-ranking professional tennis players,”Journal
of Athletic Training, vol. 47, no. 6, pp. 664–672, 2012.
[32] G. Banfi, M. Melegati, A. Barassi et al., “Effects of whole-body
cryotherapy on serum mediators of inflammation and serum
muscle enzymes in athletes,”Journal of Thermal Biology,
vol. 34, no. 2, pp. 55–59, 2009.
[33] H. Pournot, F. Bieuzen, J. Louis et al., “Time-course of changes
in inflammatory response after whole-body cryotherapy multi
exposures following severe exercise,”PLoS One, vol. 6, no. 7,
article e22748, 2011.
[34] B. Dugué, J. Smolander, T. Westerlund et al., “Acute and long-
term effects of winter swimming and whole-body cryotherapy
on plasma antioxidative capacity in healthy women,”Scandi-
navian Journal of Clinical and Laboratory Investigation,
vol. 65, no. 5, pp. 395–402, 2005.
[35] A. Stanek, K. Sieroń-Stołtny, E. Romuk et al., “Whole-body
cryostimulation as an effective method of reducing oxidative
stress in healthy men,”Advances in Clinical and Experimental
Medicine, vol. 25, no. 6, pp. 1281–1291, 2016.
[36] B. Skrzep-Poloczek, E. Romuk, and E. Birkner, “The effect of
whole-body cryotherapy on lipids parameters in experimental
rat model,”Polish Journal of Balneology, vol. 44, no. 1–4, pp. 7–
13, 2002.
11Mediators of Inflammation
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