Increased Levels of Oxidative Stress Markers, Soluble
CD40 Ligand, and Carotid Intima-Media Thickness Reflect
Acceleration of Atherosclerosis in Male Patients with Ankylosing
Spondylitis in Active Phase and without the Classical
Cardiovascular Risk Factors
and Aleksander Sieroń
School of Medicine with the Division of Dentistry in Zabrze, Department of Internal Medicine, Angiology and Physical Medicine,
Medical University of Silesia, Batorego St., 15, 41-902 Bytom, Poland
Department of Medical Physics, Chelkowski Institute of Physics, University of Silesia, 4 Uniwersytecka St., 40-007 Katowice, Poland
School of Medicine with the Division of Dentistry in Zabrze, Department of Biochemistry, Medical University of Silesia,
Jordana 19 St., 41-808 Zabrze, Poland
School of Health Sciences in Katowice, Department of Physical Medicine, Chair of Physiotherapy, Medical University of Silesia,
Medyków St., 12, 40-752 Katowice, Poland
Correspondence should be addressed to Agata Stanek; firstname.lastname@example.org
Received 15 June 2017; Accepted 12 July 2017; Published 14 August 2017
Academic Editor: Adrian Doroszko
Copyright © 2017 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 primary aim of the study was to assess levels of oxidative stress markers, soluble CD40 ligand (sCD40L), serum
pregnancy-associated plasma protein-A (PAPP-A), and placental growth factor (PlGF) as well as carotid intima-media thickness
(IMT) in patients with ankylosing spondylitis (AS) with active phase without concomitant classical cardiovascular risk factors.
Material and methods. The observational study involved 96 male subjects: 48 AS patients and 48 healthy ones, who did not
diﬀer signiﬁcantly regarding age, BMI, comorbid disorders, and distribution of classical cardiovascular risk factors. In both
groups, we estimated levels of oxidative stress markers, lipid proﬁle, and inﬂammation parameters as well as sCD40L, serum
PAPP-A, and PlGF. In addition, we estimated carotid IMT in each subject. Results. The study showed that markers of oxidative
stress, lipid proﬁle, and inﬂammation, as well as sCD40L, PlGF, and IMT, were signiﬁcantly higher in the AS group compared
to the healthy group. Conclusion. Our results demonstrate that ankylosing spondylitis may be associated with increased risk
Ankylosing spondylitis (AS) is a chronic inﬂammatory
arthritis aﬀecting primarily the axial skeleton and sacroiliac
joints [1, 2].
Several epidemiological studies conﬁrmed the high risk
of cardiovascular morbidity and mortality in AS patients,
which is associated with an increase of risk for atherosclerosis
independent of traditional risk factors that may be connected
with the disease activity, functional and mobility limitations,
structural damage, and inﬂammation [3, 4].
Inﬂammation, on the one hand, has an important role in
diﬀerent stages of atherogenesis, and, on the other, attenuates
established cardiovascular risk factors [5, 6].
During inﬂammation and phagocytosis reactions, reac-
tive oxygen species (ROS) may be released into the extracel-
lular space and injure the surrounding tissue and thereby
result in the production of acute-phase proteins .
Oxidative Medicine and Cellular Longevity
Volume 2017, Article ID 9712536, 8 pages
Moreover, according to the theory of oxidative stress,
atherosclerosis results from the oxidative modiﬁcation of
low-density lipoproteins (LDL) in the arterial wall by ROS
. Modiﬁed ox-LDL is generally accepted to be an impor-
tant elicitor of promitotic, proinﬂammatory, and atherogenic
eﬀects in vascular cells . What is more, ox-LDL may inter-
act with diﬀerent molecules and form proatherogenic com-
plexes (e.g., ox-LDL/CRP and ox-LDL/β2-glycoprotein 1)
that may not only perpetuate vascular inﬂammation but also
trigger autoimmune responses, accelerating the development
of atherosclerosis [10, 11].
In addition, high titers of antibodies of ox-LDL have been
reported in patients with myocardial infarction , athero-
slerosis , and rheumatoid disease .
It has been demonstrated that the CD40L concentration
is increased in patients with occlusive carotid artery disease
and may be also a predictor of cardiovascular events [14, 15].
On the other hand, it has been also reported that the pla-
cental growth factor (PlGF) plays an important role in ath-
erosclerosis by stimulating the angiogenesis and atherogenic
migration of monocytes/macrophages into the arterial wall
. It seems to be more eﬀective during the early phase of
atherogenesis, because anti-PlGF antibody treatment signiﬁ-
cantly inhibits early lesions but is ineﬀective during the more
advanced stages of plaque development .
The other marker of atherogenesis is serum pregnancy-
associated plasma protein-A (PAPP-A). It has been shown
that its circulating concentrations are higher in patients with
acute coronary syndrome than in patients with chronic stable
angina and in healthy subjects. Furthermore, increased
serum PAPP-A concentration is associated with the presence
and extent of stable coronary heart disease as well as predic-
tive of future ischemic cardiac events, and the need for percu-
taneous coronary intervention or coronary artery bypass
graft surgery [18, 19].
On the other hand, a few papers postulate that oxidative
stress might be involved both in AS disease onset and pro-
Taking this data into account, the primary aim of the
study was to assess levels of oxidative stress markers,
sCD40L, PAPP-A, and PlGF as well as carotid IMT in AS
patients with active phase without concomitant classical
cardiovascular risk factors.
2. Materials and Methods
2.1. Participants. The research protocol has been reviewed
and approved by the Bioethical Committee of the Medical
University of Silesia in Katowice (Permission number
KNW/022/KB/103/16), and all the subjects we analyzed gave
their informed, written consent for inclusion in the observa-
tional study. It was carried out in accordance with the Decla-
ration of Helsinki (1964). The study involved 96 male
subjects: 48 patients with ankylosing spondylitis (AS group,
mean age 46.06 ±1.44 years) and 48 healthy subjects (control
group, mean age 46.63 ±1.50 years), who did not diﬀer sig-
niﬁcantly regarding age, BMI, comorbiding disorders, and
distribution of classical cardiovascular risk factors. All the
patients included in the observational study fulﬁlled the
modiﬁed New York Criteria for deﬁnite diagnosis of AS,
which served as the basis for the ASAS/EULAR recommen-
dations . Enrollment in the study was performed in the
AS group of male patients, with deﬁnite diagnosis of AS
who did not suﬀer from any other diseases, had no associated
pathologies, and whose attending physician did not apply
disease-modifying antirheumatic drugs (DMARDs), biologic
agents, or steroids. The AS patients were treated with
NSAIDs, which doses were not altered within one month
before the beginning of the study. All the patients with AS
were HLAB27 positive, and they exhibited III and IV radio-
graphic grades of sacroiliac joint disease. The BASDAI was
5.35 ±1.64 and the BASFI was 5.13 ±2.17. The AS patients
did not suﬀer from any other diseases. Similarly, the healthy
subjects had no acute or chronic diseases, nor did they use
any medication. The demographic data of the subjects is
shown in Table 1.
The subjects from both groups were asked to abstain from
alcohol, drugs, and any immunomodulators, immunostimu-
lators, hormones, vitamins, minerals, or other substances
with antioxidant properties for 4 weeks before the study. All
the subjects were also asked to refrain from the consumption
of caﬀeine 12 hours prior to laboratory analysis.
2.2. 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 a basilic vein of each
subject and then collected into tubes containing ethylenedi-
aminetetraacetic acid (Sarstedt, S-monovette with 1.6 mg/
catalogue number 04.1931) and into tubes with
a clot activator (Sarstedt, S-monovette, catalogue number
Table 1: Demographic data of the study subjects.
Characteristic AS patients
Sex (M/F) 48/0 48/0 —
Age (years), mean (SD) 46.06 ±1.44 46.63 ±1.50 0.096
), mean (SD) 24.5 ±4.4 23.8 ±5.7 0.674
Smoking (yes/no) 0/48 0/48 —
BASDAI 5.35 ±1.64 ——
BASFI 5.13 ±2.17 ——
HLA B27 antigen (yes/no) 48/0 ——
vascular disease (yes/no) 0/48 0/48 —
Hypertension (yes/no) 0/48 0/48 —
Diabetes mellitus (yes/no) 0/48 0/48 —
Hyperlipidemia (yes/no) 0/48 0/48 —
NSAID (yes/no) 48/0 ——
DMARD (yes/no) 0/48 ——
Biological agents (yes/no) 0/48 ——
SD: standard deviation; BMI: body mass index; BASDAI: the Bath
Ankylosing Spondylitis Disease Activity Index; BASFI: the Bath
Ankylosing Spondylitis Functional Index; HLA B27 antigen: human
leukocyte B27 antigen; NSAID: nonsteroidal anti-inﬂammatory drug;
DMARD: disease-modifying antirheumatic drug.
2 Oxidative Medicine and Cellular Longevity
04.1934). The blood samples were centrifuged (10 min., 900
C), and then the plasma and serum were immediately
separated and stored at the temperature of −70
C, until bio-
chemical analyses were performed. In turn, the red blood
cells retained from removal of EDTA-plasma were rinsed
with isotonic salt solution and then 10% of hemolysates were
prepared for further analyses. Hemoglobin concentration in
hemolysates was determined by standard cyanmethemoglo-
bin method. The inter- and intra-assay coeﬃcients of varia-
tions (CV) were, respectively, 1.1% and 2.4%.
2.3. Biochemical Analyses
2.3.1. Oxidative Stress Marker Analyses
(1) Determination of Activity of Antioxidant Enzymes. The
plasma and erythrocyte superoxide dismutase (SOD—
E.C.184.108.40.206) activity was determined by the Oyanagui
method . 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-
Mn and SOD-ZnCu) were measured using potassium
cyanide as the inhibitor of the SOD-ZnCu isoenzyme. The
inter- and intra-assay coeﬃcients of variations (CV) were,
respectively, 2.8% and 5.4%.
The catalase (CAT—E.C.220.127.116.11.) activity in erythrocytes
was measured by Aebi  kinetic method and expressed in
IU/mgHb. The inter- and intra-assay coeﬃcients of variations
(CV) were, respectively, 2.6% and 6.1%.
The erythrocyte glutathione peroxidase (GPx—E.C.18.104.22.168.)
activity was assayed by Paglia and Valentine’s kinetic method
, 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, respectively, 3.4%
The activity of glutathione reductase in erytrocytes
(GR—E.C.22.214.171.124) was assayed by Richterich’s kinetic
method , expressed as micromoles of NADPH utilized
per minute and normalized to one gram of hemoglobin
(IU/gHb). The inter- and intra-assay coeﬃcients of varia-
tions (CV) were, respectively, 2.1% and 5.8%.
(2) 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  and calibrated with the use of
Trolox and expressed in μmol/l. The inter- and intra-
assay coeﬃcients of variations (CV) were, respectively,
1.1% and 3.8%.
The serum concentration of protein sulfhydryl (PSH)
was determined by Koster et. al’s method  using dithio-
nitrobenzoic acid (DTNB) and expressed in μmol/l. The
inter- and intra-assay coeﬃcients of variations (CV) were,
respectively, 2.6% and 5.4%.
The serum concentration of uric acid (UA) was deter-
mined by a uricase-peroxidase method  on the Cobas
Integra 400 plus analyzer and expressed as mg/dl. The inter-
and intra-assay coeﬃcients of variations (CV) were, respec-
tively, 1.4% and 4.4%.
(3) Determination of Lipid Peroxidation Products and TOS.
The intensity of lipid peroxidation in the plasma and the
erythrocytes was measured spectroﬂuorimetrically as a thio-
barbituric acid-reactive substances (TBARS) according to
Ohkawa et al. . The TBARS concentrations were
expressed as malondialdehyde (MDA) equivalents in μmol/l
in plasma or nmol/gHb in erythrocytes. The inter- and
intra-assay coeﬃcients of variations (CV) were, respectively,
2.1% and 8.3%.
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 (catalogue num-
bers BI-20022 and BI-20032, Biomedica, Poland) according
to the manufacturer’s instructions. 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 for ab-ox-LDL—4.1% and 8.7%, respectively.
The serum total oxidant status (TOS) was determined
with the method described by Erel  and expressed in
μmol/l. The inter- and intra-assay coeﬃcients of variations
(CV) were, respectively, 2.2% and 6.4%.
2.3.2. Determination of Inﬂammatory State Parameters. The
erythrocyte sedimentation rate (ESR) was determined
immediately in whole blood with EDTA by the classical
The high-sensitivity C-reactive protein (hs-CRP) (cata-
logue number EIA 4584) concentration in serum was
determined by latex immunoturbidimetric method (BioSys-
tems, Spain) and expressed in mg/l. The inter- and intra-
assay coeﬃcients of variations (CV) were, respectively,
2.3% and 5.5%.
The serum ceruloplasmin (CER) oxidase activity was
measured with the use of the p-phenylenediamine kinetic
method by Richterich  and expressed in mg/dl after a cal-
ibration with pure ceruloplasmin isolated from a healthy
donor serum pool. The inter- and intra-assay coeﬃcients of
variations (CV) were, respectively, 3.1% and 6.1%.
2.3.3. Determination of Lipid Proﬁle. Total, HDL-, and LDL-
cholesterol (T-Chol, HDL-Chol, and LDL-Chol, resp.) and
triglicerydes (TG) concentrations in serum were estimated
using routine techniques (Cobas Integra 400 plus analyzer,
Roche Diagnostics, Mannheim, Germany). The concentra-
tions were expressed in mg/dl. The inter- and intra-assay
coeﬃcients of variations (CV) were, respectively, 2.8% and
5.4% for T-Chol, 3.2% and 5.4% for HDL-Chol, 2.6% and
6.5% for LDL-Chol, and 2.5% and 7.6% for TG.
The triglycerides/HDL-cholesterol (TG/HDL) and
LDL-cholesterol/HDL-cholesterol (LDL/HDL) ratios were
2.3.4. Determination of PAPP-A, Soluble CD40 Ligand, and
PlGF. Serum pregnancy-associated plasma protein-A
(PAPP-A) (catalogue number EIA-4512), soluble CD40
3Oxidative Medicine and Cellular Longevity
Ligand (sCD40L) (catalogue number EIA4851), and pla-
cental growth factor (PlGF) (catalogue number EIA-4529)
concentrations were assayed by ELISA methods with
DRG Instruments GmbH (Germany) kits. All assays were
performed according to the manufacturer’s instructions.
The PAPP-A and sCD40L concentrations were expressed in
ng/ml, the PlGF concentration—in pg/ml. The inter- and
intra-assay coeﬃcients of variations (CV) were, respectively,
6.8% and 10.2% for PAPPA-A, 5.1% and 9.4% for sCD40L,
and 6.2% and 12.1% for PlGF.
2.4. Assay of Intima-Media Thickness. A high-resolution
Doppler ultrasonography was performed with a Logic-5
device with a high-frequency (11 MHz and 15 MHz) linear
probe. The sonographer was an angiologist who was unaware
of subject’s clinical state. 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.
2.5. Assay of Activity of Ankylosing Spondylitis. The activity of
ankylosing spondylitis was measured by the Bath Ankylosing
Spondylitis Disease 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
item 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 items and the four remaining items .
The BASFI is the mean score of ten questions addressing
functional limitations and the level of physical activity
at home and work, assessed on VAS scales (0 = easy,
10 = impossible) .
2.6. 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 the Levene test. In order
to compare the diﬀerences between the control group and
the AS group, an independent sample Student’st-test was
used or alternatively the Mann–Whitney Utest. Correlations
between particular parameters were statistically veriﬁed by
means of Spearman’s nonparametric correlation test. Dif-
ferences at the signiﬁcant level of p<0 05 were considered
3.1. Oxidative Stress. In AS patients, there was a signiﬁcantly
higher activity of antioxidant enzymes: plasma SOD, along
with erythrocyte SOD, erythrocyte CAT, erythrocyte GPx,
and erythrocyte GR, was observed in comparison to the
healthy subjects. But the plasma activity of SOD-Mn and
SOD-CuZn isoenzymes in both groups did not diﬀer signiﬁ-
cantly. What is more, in AS patients, a signiﬁcantly lower
concentration of the parameters of nonenzymatic antioxi-
dants, FRAP, PSH, and UA, was observed in comparison to
the healthy subjects (Table 2).
Furthermore, a signiﬁcantly higher concentration of lipid
peroxidation products plasma MDA, along with erythrocyte
MDA, ox-LDL, and ab-ox-LDL, was noted in AS patients in
comparison to the control group of the healthy subjects. In
addition, in AS patients, the concentration of TOS was signif-
icantly higher in comparison to the control group (Table 3).
3.2. Lipid Proﬁle, Inﬂammatory Parameters, and Carotid
Intima-Media Thickness. In AS patients, there was a signiﬁ-
cantly higher concentration of the lipid proﬁle parameters:
T-Chol, LDL-Chol, and TG as well as TG/HDL ratio and
LDL/HDL ratio were noted in comparison to the control
group. But only the concentration of HDL-Chol in both
groups did not diﬀer signiﬁcantly. What is more, also the
concentrations of sCD40L and PlGF as well as carotid IMT
Table 2: Parameters of enzymatic antioxidant status (superoxide dismutase (SOD), its isoenzymes: manganese superoxide dismutase
(SOD-Mn) and copper-zinc superoxide dismutase (SOD-CuZn), catalase (CAT), glutathione peroxidase (GPx), and glutathione
reductase (GR) activity) and nonenzymatic antioxidant status (ferric reducing ability of plasma (FRAP), protein sulfhydryl (PSH), and
uric acid (UA) concentration, as well as activity of ceruloplasmin (CER)) in ankylosing spondylitis (AS) patients and healthy subjects.
Parameter AS patients (n=48) Healthy subjects (n=48)p
SOD (p) (NU/ml) 12.67 ±1.98 10.93 ±2.55 <0.001
SOD-Mn (p) (NU/ml) 5.08 ±2.00 4.59 ±1.88 0.223
SOD-CuZn (p) (NU/ml) 7.64 ±2.31 6.79 ±1.99 0.055
SOD (e) (NU/mgHb) 105.85 ±22.60 95.50 ±19.18 0.017
CAT (e) (IU/mgHb) 410.98 ±63.56 352.55 ±77.21 <0.001
GPx (e) (IU/gHb) 27.23 ±6.43 24.49 ±5.00 0.022
GR (e) (IU/gHb) 1.67 ±0.58 1.38 ±0.43 0.007
FRAP (p) (μmol/l) 550.38 ±76.98 642.17 ±105.67 <0.001
PSH (s) (μmol/l) 474.46 ±192.06 559.07 ±215.14 0.045
UA (s) (mg/dl) 4.73 ±1.39 5.84 ±1.53 <0.001
Values are expressed as means ±standard deviations (SD) of the means; p: plasma; s: serum; e: erythrocyte lysates.
4 Oxidative Medicine and Cellular Longevity
were signiﬁcantly higher in AS patients in comparison to the
healthy group. The concentration of PAPP-A in both groups
did not diﬀer signiﬁcantly. Furthermore, in AS patients, a
signiﬁcantly higher concentration of all the examined
parameters of inﬂammatory state, ESR, hs-CRP, and CER,
was observed in comparison to the healthy subjects (Table 4).
3.3. Signiﬁcant Relationships among the Estimated
Parameters in AS Patients. In the AS group, a high, statisti-
cally signiﬁcant correlation was observed between acute
phase proteins (hs-CRP versus CER; r=0 59,p<0 05), total
SOD activity in erythrocytes and ceruloplasmin (r=0 52,
p<005), and glutathione cycle enzyme activities in eryth-
rocytes (POX versus GR; r=066,p<0 05) as well as
between uric acid concentration and FRAP activity in
plasma (r=065,p<0 05). Mild but still statistically signif-
icant correlations were shown between CRP concentration
and SOD-CuZn and SOD-Mn plasma activities (r=−035,
p<005), erythrocyte MDA concentration and SOD
erythrocyte activity (r=035,p<005), sCD40L and PlGF
concentration (r=0 51,p<005), plasma MDA and T-Chol
(r=030,p<0 05), and TOS and TG/HDL ratio (r=037,
p=0001). Also, a high correlation between BASFI and
BASDAI was observed (r=0 67,p<0 05) in AS patients.
Unfortunately, no statistically signiﬁcant correlations
between AS activity parameters (BASFI and BASDAI) and
oxidative stress parameters as well as carotid IMT and oxida-
tive stress parameters were obtained.
Brieﬂy, in this observational study, we viewed signiﬁcantly
higher oxidative stress parameters, levels of inﬂammatory
state, and lipid proﬁle parameters, sCD40L, and PlGF as well
as values of TG/HDL, LDL/HDL ratio, and carotid IMT in
AS patients with active phase (BASDAI and BASFI),
compared to the healthy subjects.
In the available literature, only a few, unequivocal reports
concerning the prooxidant-antioxidant status in patients
with ankylosing spondylitis have been published.
In the study , in patients with ankylosing spondylitis,
a signiﬁcantly lower plasma total antioxidant status (TAS)
was demonstrated, as well as higher values of the total
oxidant status (TOS) and oxidative stress index (OSI), in
comparison to the control group of healthy volunteers. That
study did not reveal any signiﬁcant correlation between
the values of the above parameters and the activity of the
In another study , no signiﬁcant diﬀerences were
demonstrated to occur in the activity of SOD, nitric oxide
(NO) metabolites, and the concentration of MDA, between
the group of patients with AS in the active form and the
group of patients with inactive process. The activity of
SOD, NO metabolites, and the concentration of MDA also
failed to demonstrate statistically signiﬁcant diﬀerences when
compared to the control group of healthy subjects, whereas
the activity of CAT and the concentration of MDA in
patients with an active form of the disease were signiﬁcantly
higher, in relation to other groups of subjects studied.
In the study , all antioxidant enzyme activities were
lower, but the MDA level was higher in patients with AS
when compared to the control group.
Table 3: Oxidative stress parameters: malondialdehyde (MDA), oxidized low-density lipoprotein (ox-LDL), antibodies to oxidized low-
density lipoprotein (ab-ox-LDL), and total oxidant status (TOS) concentration in ankylosing spondylitis (AS) patients and healthy subjects.
Parameter AS patients (n=48) Healthy subjects (n=48)p
MDA (p) (μmol/l) 2.51 ±0.63 2.25 ±0.47 0.025
MDA (e) (nmol/gHb) 0.17 ±0.03 0.15 ±0.03 <0.001
ox-LDL (s) (ng/ml) 268.48 ±105.83 162.98 ±63.29 <0.001
ab-ox-LDL (s) (mU/ml) 479.82 ±328.39 323.82 ±210.26 0.007
TOS (s) (μmol/l) 26.99 ±10.65 16.50 ±6.87 <0.001
Values are expressed as means ±standard deviations (SD) of the means; p: plasma; s: serum; e: erythrocyte lysates.
Table 4: Parameters of lipid proﬁle (total cholesterol (T-Chol), low-
density lipoprotein cholesterol (LDL-Chol), high-density lipoprotein
cholesterol (HDL-Chol), triglicerydes (TG) concentration, TG/HDL,
and LDL/HDL ratio), concentration of PAPP-A, soluble CD40
ligand (sCD40L), PlGF, and value of carotid intima-media
thickness (IMT), as well as parameters of inﬂammatory state
(erythrocyte sedimentation rate (ESR) value, high sensitivity C-
reactive protein (hs-CRP), and ceruloplasmin (CER)
concentration) in ankylosing spondylitis (AS) patients and
Parameter AS patients
T-Chol (s) (mg/dl) 217.73 ±35.48 187.09 ±18.57 <0.001
LDL-Chol (s) (mg/dl) 140.49 ±33.64 112.57 ±22.89 <0.001
HDL-Chol (s) (mg/dl) 61.10 ±18.08 57.49 ±15.16 0.291
TG (s) (mg/dl) 190.48 ±47.30 139.74 ±47.66 <0.001
TG/HDL ratio 3.37 ±1.13 2.57 ±1.23 <0.001
LDL/HDL ratio 2.55 ±1.1 2.02 ±0.58 <0.05
PAPP-A (s) (ng/ml) 17.82 ±16.22 14.24 ±4.35 0.281
sCD40L (s) (ng/ml) 8.93 ±3.74 5.54 ±2.37 <0.001
PlGF (s) (pg/ml) 25.8 ±8.99 19.77 ±3.27 <0.001
Carotid IMT (mm) 1.1 ±0.13 0.55 ±0.08 <0.001
ESR 27.13 ±21.55 5.94 ±3.91 <0.01
hs-CRP (s) (mg/l) 14.92 ±15.55 1.58 ±2.00 <0.001
CER (s) (mg/dl) 48.12 ±12.67 38.68 ±4.84 <0.001
Values are expressed as means ±standard deviations (SD) of the means;
5Oxidative Medicine and Cellular Longevity
The next paper  reported that ESR, CRP, and lipid
peroxidation products were higher in patients with AS than
in healthy subjects, but vitamins A, C, E, and β-carotene con-
centrations in plasma, reduced glutathione, and glutathione
peroxidase activity values in erythrocyte were lower in
patients with AS than in healthy subjects. But the authors
estimated only some chosen parameters of prooxidant-
antioxidant status. Furthermore, the level of oxidative stress
was shown to be correlated with the intensity of inﬂamma-
tion in patients with AS .
Contrastingly, there are many papers, which reported
increased cardiovascular risk in AS patients [39–43].
In the current study, a signiﬁcant increase in acute-phase
protein concentration (hs-CRP and CER) was observed in AS
patients compared to the healthy subjects. A signiﬁcant pos-
itive correlation between hs-CRP and CER was shown as well
as a signiﬁcant negative correlation between hs-CRP and
Some researchers found that increased AS disease activity
was associated with decreases in lipid levels and the decrease
in HDL-Chol levels, which tended to be almost twice as large
as the decrease in total cholesterol levels, resulting in a more
atherogenic lipid proﬁle [43, 44].
However, in the study  in AS patients, the authors did
not observe a signiﬁcant diﬀerence in T-Chol, LDL-Chol,
HDL-Chol, and TG concentration compared to healthy
subjects. But in AS patients, the values of HDL/LDL ratio,
complex intima-media, and TOS concentration as well as
inﬂammatory state parameters were signiﬁcantly higher than
in controls. In this study, a signiﬁcant increase in T-Chol,
LDL-Chol, and TG as well as TG/HDL and LDL/HDL ratio
was observed in comparison to the healthy subjects. But
HDL-Chol concentration did not diﬀer between AS patients
and healthy subjects. In addition, the carotid IMT in AS
patients was also signiﬁcantly higher in comparison to the
A high level of cholesterol, especially in LDL, may acti-
vate thrombocytes and cause the release of substances that
activate phospholipase A
. Then, the accumulated arachi-
donic acid is metabolized to leukotriene by a lipoxygenase
pathway and thromboxane, prostaglandin, and MDA by a
cyclooxygenase pathway. During this metabolism, ROS
may be produced, and under insuﬃcient antioxidant capac-
ity, they may also trigger lipid peroxidation . What is
more, it was shown that the TG/HDL ratio estimates ath-
erogenic small, dense low-density lipoprotein cholesterol
and predicts arterial stiﬀness and hard cardiovascular
events in adults [46, 47]. In our study, we observed a
signiﬁcant positive correlation between TOS and TG/
Additionally, it was also shown that an increase in serum
LDL levels leads to an increase in the adherence of circulating
monocytes to arterial endothelial cells and at the same time
to an increased rate of entry of LDL into the intima . It
is also possible that TG enrichment may alter the physico-
chemical properties of LDL, which is considerably more
susceptible to oxidation .
In this study, we also observed the increased concentra-
tion of lipid peroxidation products in plasma and erythrocyte.
MDA is one the most abundant aldehydes, resulting from
peroxidation of arachidonic, eicosapentaenoic, and docosa-
hexaenoic acid [50, 51]. MDA reacts with lysine residues by
forming Schiﬀbases  and plays a major role in LDL mod-
iﬁcation and their deviation towards macrophages .
In the current study, a signiﬁcantly higher concentration
of MDA in plasma as well as in erythrocytes in AS patients
was observed compared to healthy subjects. A positive corre-
lation between plasma MDA and T-Chol as well as erythro-
cyte MDA and SOD in AS patients was also shown. We did
not observe any correlation between MDA and other esti-
mated parameters. The explanation of this fact may be that
TBARS assay does not measure MDA exclusively, because
it reacts to compounds other than MDA .
However, so far, there are no reports estimating ox-LDL
and ab-ox-LDL concentration in ankylosing spondylitis. In
the present study, a signiﬁcantly higher concentration of
ox-LDL as well as ab ox-LDL in AS patients was observed
compared to healthy subjects.
In the study , it was shown that antibodies to ox-LDL
were correlated signiﬁcantly with ESR and CRP in patients
with early rheumatoid arthritis and suggested that the
occurrence of these antibodies must be related to inﬂamma-
tion. However, in the current study, no correlation was
observed between ox-LDL and ab-ox-LDL and other esti-
In our study, we observed a signiﬁcantly higher levels of
sCD40L and PlGF as well as a positive correlation between
them. Furthermore, in this study, the level of PAPP-A did
not diﬀer between AS patients and healthy subjects. It may
be connected with the fact that PAPP-A is expressed in
unstable but not in stable atherosclerotic plaques .
The study proved that increased oxidative stress, the
levels of sCD40L and PlGF, the disturbance of lipids, and
the inﬂammation process may enhance atherogenesis in
However, the study has some limitations. First, it
involved only 48 AS patients and thus a greater number of
patients should be examined. Second, patients in diﬀerent
stages of AS should be involved in the study.
5. General Conclusion
Our results demonstrate that increased oxidative stress,
higher serum concentrations of PlGF and sCD40L, and
increased IMT may reﬂect the acceleration of atherosclerosis
in male AS patients in active phase and without concomitant
classical cardiovascular risk factors.
Conflicts of Interest
The authors declare that there is no conﬂict of interests
regarding the publication of this paper.
This work was support by a grant from the Medical
University of Silesia (KNW-1-045/K/7/K).
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