I need to summarize these articles attached belowWith focusing on the relation between lipid profile tests and ankylosing spondylitis disease
And HLA-B27 gene with changes in lipid profile
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1473
EXTENDED REPORT
Increased disease activity is associated with a
deteriorated lipid profile in patients with ankylosing
spondylitis
V P van Halm, J C van Denderen, M J L Peters, J W R Twisk, M van der Paardt, I E van
der Horst-Bruinsma, R J van de Stadt, M H M T de Koning, B A C Dijkmans,
M T Nurmohamed
……………………………………………………………………………………………………………….
Ann Rheum Dis 2006;65:1473–1477. doi: 10.1136/ard.2005.050443
See end of article for
authors’ affiliations
…………………..
Correspondence to:
M T Nurmohamed,
Department of Internal
Medicine, VU University
Medical Centre, PO Box
7057, 1007 MB
Amsterdam, The
Netherlands; mt.
nurmohamed@vumc.nl
Accepted 23 April 2006
Published Online First
27 April 2006
…………………..
A
Background: Cardiovascular mortality is increased in patients with ankylosing spondylitis. A possible
explanation might be a more prevalent atherogenic lipid profile in patients with ankylosing spondylitis
than in the general population. It has been postulated that inflammation deteriorates the lipid profile,
thereby increasing cardiovascular risk.
Objective: To explore the association between disease activity and lipid profile in patients with ankylosing
spondylitis.
Methods: Disease activity parameters for ankylosing spondylitis and lipid levels (total cholesterol, highdensity lipoprotein cholesterol (HDLc) and triglycerides) were measured in 45 patients with ankylosing
spondylitis for 6 months after starting treatment with leflunomide or placebo. Findings in this treatment
group were compared with those in 10 patients with ankylosing spondylitis treated with etanercept. A
specialised regression model, adjusting for repeated measurements, age and sex, was used to assess the
influence of the disease activity variables on the lipid levels.
Results: Multilevel regression analyses showed significant associations between disease activity
parameters and lipid levels—for instance, an increase of 30 mm at the end of the first hour in erythrocyte
sedimentation rate was associated with a decrease of about 6% in total cholesterol level and a decrease of
about 11% in HDLc levels. Similar significant associations were found between other disease activity
parameters and lipid levels.
Conclusion: Increase in disease activity was associated with decreases in lipid levels. The decrease in HDLc
levels tended to be almost twice as large as the decrease in total cholesterol levels, resulting in a more
atherogenic lipid profile. Hence, effective treatment of disease activity in patients with ankylosing
spondylitis may lower the cardiovascular risk by improving the lipid profile.
nkylosing spondylitis is a chronic inflammatory disease
that affects predominantly men, starts in young
adulthood and results in immobility of the spine and
sacroiliac joints. Although the number of studies investigating mortality in ankylosing spondylitis is limited, many of
these show an increased total mortality in patients compared
with the general population.1–4 Furthermore, this increased
mortality seems to be predominantly caused by cardiovascular disease (CVD), with a twofold increased cardiovascular
standard mortality.5
A possible explanation for this increased cardiovascular
risk is a higher prevalence of conventional cardiovascular risk
factors, such as a more atherogenic lipid profile.6 An
atherogenic lipid profile is characterised by a reduced level
of high-density lipoprotein cholesterol (HDLc) and increased
levels of total cholesterol, low-density lipoprotein cholesterol
and triglycerides. An important prognostic indicator for
(future) CVD is the atherogenic index, which is the ratio of
total cholesterol to HDLc.7–10
Whether or not an atherogenic lipid profile is present in
patients with ankylosing spondylitis is presently unclear.11–13
Moreover, there is growing evidence that inflammation is
associated with deterioration of the lipid profile,14 15 but so far
data for patients with ankylosing spondylitis are lacking.
Hence, we hypothesise that disease activity in inflammatory
diseases, such as ankylosing spondylitis, worsens the lipid
profile, thereby increasing the risk for (future) CVD.
Consequently, lowering the disease activity in patients with
ankylosing spondylitis may have a beneficial effect on the
lipid profile.
Two recent studies,16 17 investigating the safety and efficacy
of treatment with leflunomide and etanercept in patients
with ankylosing spondylitis, gave us the opportunity to assess
the relationship between disease activity and lipid profile in a
total of 55 patients with ankylosing spondylitis, treated with
leflunomide, etanercept or placebo.
METHODS
Patients
All patients included in the study, aged 18–70 years, fulfilled
the 1984 modified New York criteria for ankylosing spondylitis.18 The first population consisted of 45 consecutive
patients with ankylosing spondylitis participating in a
monocentre, randomised, double-blind, placebo-controlled
phase II trial, in which the safety and efficacy of leflunomide
were assessed.17 Thirty patients were treated with the active
compound at a daily dose of 20 mg and 15 patients received
placebo, for a duration of 24 weeks. During this period, other
pharmacological treatments were maintained stable.
Abbreviations: BASDAI, Bath Ankylosing Spondylitis Disease Activity
Index; CRP, C reactive protein; CVD, cardiovascular disease; ESR,
erythrocyte sedimentation rate; HDLc, high-density lipoprotein
cholesterol
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1474
van Halm, van Denderen, Peters, et al
Table 1 Baseline characteristics of participants
Demographic features
Age (years)
Sex (male:female)
Disease activity parameters
BASG
BASFI
BASDAI
ESR (mm/h)
CRP (mg/l)
Lipids
TC (mmol/l)
HDLc (mmol/l)
Atherogenic index
Triglycerides (mmol/l)
Leflunomide or
placebo (n = 45)
Etanercept (n = 10)
42 (11)
32:13
41 (10)
9:1
6.4 (1.8)
5.3 (1.8)
5.5 (1.4)
15 (10–31)*
13 (4–40)*
6.7 (1.6)
4.4 (1.9)
4.9 (1.2)
37 (13–49)*
11 (4.4–29.4)*
4.9 (1.2)
1.1 (0.9–1.4)*
4.3 (3.1–5.5)*
1.2 (0.8–1.8)*
4.4 (0.8)
1.3 (0.3)
3.7 (1.1)
1.1 (0.7–1.6)*
BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath
Ankylosing Spondylitis Functional Index; BASG, Bath Ankylosing
Spondylitis Global; CRP, C reactive protein; ESR, erythrocyte
sedimentation rate; HDLc, high-density lipoprotein cholesterol; TC, total
cholesterol.
*Values are mean (SD) or median (interquartile range), as applicable.
The findings of the aforementioned patients with ankylosing spondylitis were compared with those of a second
population consisting of 10 patients with ankylosing spondylitis who were treated for 6 months with etanercept
(25 mg twice weekly).18
Disease activity parameters
The Bath Ankylosing Spondylitis Global score,19 the Bath
Ankylosing Spondylitis Functional Index20 and the Bath
Ankylosing Spondylitis Disease Activity Index (BASDAI),21 all
with a scale from 0 to 10, were determined by an experienced
rheumatologist at baseline and weeks 4, 12 and 24.
Furthermore, the erythrocyte sedimentation rate (ESR) and
C reactive protein (CRP) levels were measured at baseline
and weeks 2, 4, 6, 9, 12, 16, 20 and 24. ESR was determined
with local measurement techniques and expressed in
millimetres per hour (in men ,20 mm/h and in women
,30 mm/h). CRP was measured using a turbidimetric assay
Table 2
supplied by Biomedical
Netherlands) (,10 mg/l).
Diagnostics
30 mm/h ESR
30 mg/l CRP
1-point BASG
1-point BASFI
1-point BASDAI
Lipids
TC
HDLc
Atherogenic index
Triglycerides
TC
HDLc
Atherogenic index
Triglycerides
TC
HDLc
Atherogenic index
Triglycerides
TC
HDLc
Atherogenic index
Triglycerides
TC
HDLc
Atherogenic index
Triglycerides
Statistics
Measuring lipid levels and disease activity parameters in a
particular population at several time points, with variable
time intervals, causes an important statistical problem—that
is, ‘‘repeated measurements within subjects’’. To adjust for
repeated measurements within subjects, a multilevel linear
regression analysis was used. This regression technique
allows for both starting levels and progression over time to
differ between patients. It calculates the regression coefficients of the progression over time of the various lipid levels,
adjusted for repeated measurements within subjects and
variable time intervals.22
Multilevel regression analysis is a longitudinal linear
regression analysis. It combines many cross-sectional linear
regression models into one model of one variable over time.
In this study, we measured the lipid levels over time and
investigated the influence of the various other variables, such
as disease activity parameters, on these lipid levels. Multilevel
regression analysis quantifies this influence, or rather the
association between the two variables, and tests it for
statistical significance. Furthermore, the observed association
between the two variables—that is, lipid levels and disease
activity parameters—was also corrected for age and sex.
One advantage of combining the cross-sectional data of the
various time points into one association is that this increases
Absolute
change (mmol/l)
20.29
20.13
0.29
20.15
20.18
20.06
0.10
20.13
0.01
20.01
0.03
20.05
0.02
20.02
0.08
20.06
20.01
20.00
0.01
20.07
Relative
decrease (%)
p Value
26.0
211.4
6.8
212.6
23.8
25.7
2.4
210.9
0.1
20.7
0.6
24.4
0.4
21.7
2.0
24.5
20.3
20.4
0.2
25.8
0.001*
0.01*
0.09
0.04*
0.001*
0.001*
0.16
0.001*
0.79
0.44
0.47
0.01*
0.62
0.25
0.19
0.01*
0.66
0.71
0.87
0.01*
Atherogenic index, ratio of total cholesterol (TC) to high-density lipoprotein cholesterol (HDLc); BASDAI, Bath
Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index; BASG, Bath
Ankylosing Spondylitis Global Score; CRP, C reactive protein; ESR, erythrocyte sedimentation rate.
Influence of disease activity parameters on lipid levels, calculated using multilevel regression analyses correcting for
age, sex and repeated measurements within subjects.
*Indicates significant associations.
www.annrheumdis.com
The
Lipids
Blood samples for lipid measurements were taken after an
overnight fast at baseline and weeks 2, 4, 6, 9, 12, 16, 20 and
24. Serum total cholesterol (,5.0 mmol/l) and triglycerides
(,2.2 mmol/l) were analysed by an enzymatic method using
the appropriate assays supplied by Roche Diagnostics
(Almere, The Netherlands) on a Hitachi 911 analyser
(Roche), according to the instructions of the manufacturer.
Polyethylene glycol-modified enzymes were used for assessing the HDLc levels (in men .0.9 mmol/l and in women
.1.1 mmol/l). The atherogenic index was calculated as the
total cholesterol level divided by the HDLc level.
Influence of disease activity on the lipid levels
Increase in disease
activity parameter
(Apeldoorn,
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Disease activity deteriorates lipid profile in ankylosing spondylitis
the statistical power. Another important advantage of this
method is that it holds into account and corrects for the
possibility that one or a few patients with aberrant values
distort the association between lipid levels and disease
activity—that is, the above-mentioned repeated measurements within subjects. If one or a few patients have, for
example, an extreme level of a certain lipid, it will not distort
the relationship this lipid has with disease activity, because
the multilevel regression analysis will look only at this
relationship and correct for the aberrant starting points.
As the primary goal of this investigation was to study the
relationship between disease activity and lipid levels, it was
not relevant whether the antirheumatic treatment altered
disease activity; hence the data of placebo-treated and
leflunomide-treated patients were put together. The findings
were compared with those of a group of 10 patients with
ankylosing spondylitis treated with etanercept, for whom
similar analyses were conducted.
As the HDLc levels, the atherogenic index and triglyceride
levels were not normally distributed, data were analysed with
the natural logarithms of these values. For clarity, the
regression coefficients for these lipids were retransformed
to geometric means. The multilevel analyses were carried out
with the statistical program MLwiN.23 A p value of (0.05 was
considered significant.
1475
6
A
4
TC (mmol/l)
Atherogenic index
Triglycerides (mmol/l)
HDLc (mmol/l)
2
0
Weeks
B
7
4
BASG
BASFI
BASDAI
RESULTS
Patients
The first group of 45 patients with ankylosing spondylitis (13
women and 32 men), with a mean age of 42 (standard deviation
(SD) 11; range 21–66) years, was followed for 24 weeks. Nine
patients had hypertension; eight of them used antihypertensive
agents. Two patients were treated for hypercholesterolaemia.
Non-steroidal anti-inflammatory drugs were used by 39
patients and prednisone was given to one.
The findings of 10 patients with ankylosing spondylitis
(1 woman and 9 men), with a mean age of 41 (SD 10; range
28–60) years, starting treatment with etanercept, were
compared with those of the first population. One patient
was treated for type 2 diabetes mellitus, one for hypertension
and one for hypercholesterolaemia. Nine patients used nonsteroidal anti-inflammatory drugs.
All pharmacological treatments remained unchanged during the entire observation period. Table 1 shows the baseline
characteristics, including demographic and clinical data.
Lipid levels and disease activity parameters
Figure 1 shows the lipid levels and the disease activity
parameters in patients with ankylosing spondylitis, treated
with placebo or leflunomide (n = 45). We found no significant changes in these variables during the 24-week
observation period. The multilevel regression analyses yielded
several significant associations between lipid level progression over time and disease activity parameters; the height of
the disease activity had a significant influence on the height
of the lipid levels. Higher ESR and CRP levels were
significantly (p,0.001) associated with lower total cholesterol levels, with regression coefficients of 20.01 and 20.01,
respectively. The ln-triglyceride levels also had an inverse
relationship with ESR and CRP, with regression coefficients
of 20.005 and 20.004, respectively. A similar relationship
was observed between ln-HDLc and ESR and CRP levels
(regression coefficients of 20.004 and 20.002, respectively).
Moreover, the disease activity parameters tended to have a
linear relationship with the atherogenic index (p = 0.09).
The relationship between disease activity parameters and
lipid levels, or the influence of disease activity on the lipid
levels, is shown in table 2 as absolute values and percentages.
The influence of ESR and CRP on HDLc levels was almost
1
Weeks
C
40
20
CRP (mg/l)
ESR (mm/h)
0
0
2
4
6
9
12
16
20
24
Weeks
Figure 1 (A–C) Mean lipid levels and disease activity variables during
the 24 weeks of placebo treatment or lefunomide treatment.
Antherogenic index, TC/HDLc; BASDAI, Bath Ankylosing Spondylitis
Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional
Index; BASG, Bath Ankylosing Spondylitis Global; CRP, C reactive
protein; ESR, erythrocyte sedimentation rate; HDLc, high-density
lipoprotein cholesterol; TC, total cholesterol.
twice as large as the effect on total cholesterol levels,
resulting in a higher atherogenic index (p,0.001).
The multilevel analyses of patients treated with etanercept
showed that an increased ESR, CRP, Bath Ankylosing
Spondylitis Global score, Bath Ankylosing Spondylitis
Functional Index and BASDAI significantly (p(0.02)
decreased the total cholesterol levels (regression coefficients
of 20.01, 20.02, 20.07, 20.12 and 20.13, respectively).
Higher ESR and CRP levels were associated with lower HDLc
and total cholesterol levels, albeit that the decrease in HDLc
was (again) twice as large as the decrease in total cholesterol
levels, resulting in a higher atherogenic index. Moreover,
increase in other disease activity parameters was also
associated with an increase in the atherogenic index; this
association did not reach significance (p.0.08).
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1476
van Halm, van Denderen, Peters, et al
…………………
DISCUSSION
Our investigation on patients with ankylosing spondylitis
shows that higher disease activity (parameters) is associated
with lower lipid levels and vice versa. This was observed for
total cholesterol, HDLc and triglyceride levels, which were
significantly associated with disease activity parameters as
the ESR, CRP and BASDAI. Moreover, an increase in disease
activity was associated with a decrease in total cholesterol
levels and a more pronounced decrease in HDLc levels,
subsequently resulting in a more atherogenic lipid profile.
The magnitude in which disease activity influences the
lipid levels was limited, which questions its clinical relevance.
However, although the observed influence is small, it should
be noted that ankylosing spondylitis is a chronic inflammatory disease, which means that this small (detrimental)
influence has a potential clinically relevant effect on the
cardiovascular risk for many years. The importance of small
differences in lipid levels over a prolonged period is best
illustrated by several studies in which cardiovascular risk
reduction was found by lowering lipid levels slightly but over
a prolonged period. Firstly, the follow-up of the Framingham
cohort showed that a lower HDLc level of just 0.25 mmol/l
was associated with a 50% higher risk for future vascular
events.24 Secondly, a landmark study on fibrates showed a
22% reduction in the risk for CVD in the group receiving the
active compound versus placebo. The group receiving the
active compound showed only small changes in lipid levels—
that is, 4% decrease in total cholesterol, 6% increase in HDLc
and 31% decrease in triglyceride levels.25 Although these
figures cannot be directly extrapolated to this study, they do
indicate the clinical relevance of small changes in lipid levels.
Growing evidence suggests that inflammation has an
important role in the pathogenesis of CVD, particularly in
atherosclerosis.26 In addition to a postulated direct effect of
inflammation on endothelial cells, mounting evidence
suggests that inflammation can also increase the cardiovascular risk by deterioration of the lipid profile, which is
supported by the findings of a study showing a decrease in
HDLc and apolipoprotein A I levels and an increase in
triglyceride and apolipoprotein B levels during an acutephase response.27 Other investigators found an association
between an increase in lipids as oxidised low-density
lipoprotein cholesterol and proinflammatory cytokines as
CRP, interleukin 6 and tumour necrosis factor a.28 The
findings of the present study confirm these effects of
inflammation on the various lipid concentrations.
High disease activity is characterised by increased cytokine
expression and this could directly lead to altered lipid levels
through effects on the liver or adipose tissue. Moreover, there
may also be an indirect way through various intermediate
factors—for example, metabolic or dietary factors. Patients
with ankylosing spondylitis with high disease activity might
be in a metabolic state comparable to those with rheumatoid
cachexia.29 Decreasing disease activity would improve their
general well-being and physical function, with subsequent
changes in lipid levels. The results of the present study
support this hypothesis, as lower disease activity (parameters) was associated with a more favourable lipid profile.
This investigation is in line with the accumulating evidence
of the intriguing interactions between dyslipidaemia, atherosclerosis and inflammation, showing a worsening of the lipid
profile during increased disease activity. Obviously, more
prospective investigations with cardiovascular end points are
needed to further unravel these relationships.
ACKNOWLEDGEMENTS
We thank Professor Dr YS Smulders and Dr D van Schaardenburg for
their advice and critical reading of the manuscript.
www.annrheumdis.com
Authors’ affiliations
V P van Halm, M J L Peters, M van der Paardt, I E van der HorstBruinsma, B A C Dijkmans, M T Nurmohamed, Department of
Rheumatology, VU University Medical Center, Amsterdam, The
Netherlands
V P van Halm, J C van Denderen, M van der Paardt, R J van de Stadt,
M H M T de Koning, B A C Dijkmans, M T Nurmohamed, Department of
Rheumatology, Jan van Breemen Institute Amsterdam, Amsterdam
J W R Twisk, Department of Clinical Epidemiology and Biostatistics, VU
University Medical Centre
Competing interests: None.
Ethical approval: The local ethics committee approved the study.
REFERENCES
1 Lehtinen K. Mortality and causes of death in 198 patients admitted to hospital
with ankylosing spondylitis. Ann Rheum Dis 1993;52:174–6.
2 Radford EP, Doll R, Smith PG. Mortality among patients with ankylosing
spondylitis not given X-ray therapy. N Engl J Med 1977;11:572–6.
3 Kaprove RE, Little AH, Graham DC. Ankylosing spondylitis: survival in men
with and without radiotherapy. Arthritis Rheum 1980;23:57–61.
4 Smith PG, Doll R. Mortality among patients with ankylosing spondylitis after a
single treatment course with X-rays. BMJ 1982;13:449–60.
5 Peters MJ, van der Horst-Bruinsma IE, Dijkmans BA, Nurmohamed MT.
Cardiovascular risk profile of patients with spondylarthropathies, particularly
ankylosing spondylitis and psoriatic arthritis. Semin Arthritis Rheum
2004;34:585–92.
6 Divecha H, Sattar N, Rumley A, Cherry L, Lowe GD, Sturrock R.
Cardiovascular risk parameters in men with ankylosing spondylitis in
comparison with non-inflammatory control subjects: relevance of systemic
inflammation. Clin Sci (London) 2005;109:171–6.
7 Kwiterovich PO. Detection and treatment of elevated blood lipids and other
risk factors for coronary artery disease in youth. Ann N Y Acad Sci
1995;748:313–30.
8 Rader DJ. High density lipoproteins and atherosclerosis. Am J Cardiol
2002;90(Suppl):i62–70.
9 Lazarevic MB, Vitic J, Mladenovic V, Myones BL, Skosey JL, Swedler WI.
Dyslipoproteinemia in the course of active rheumatoid arthritis. Semin Arthritis
Rheum 1992;22:172–8.
10 Sharett AR, Ballantyne CM, Coady SA, Heiss G, Sorlie PD, Catellier D, et al.
Coronary heart disease prediction from lipoprotein cholesterol levels,
triglycerides, lipoprotein(a), apolipoproteins A-1 and B, and HDL density
subfractions: the atherosclerosis risk in communities (ARIC) study. Circulation
2001;104:1108–13.
11 Masi AT, Aldag JC, Mohan PC, Murugan TSR. Determinants of significantly
lower serum total cholesterol levels in ankylosing spondylitis patients than age, gender-, and medical service-matched control patients: results of multivariate
analysis. Arthritis Rheum 1999;42(Suppl):S300.
12 Jones SM, Harris CPD, Lloyd J, Stirling CA, Reckless JPD, Mc Hugh NJ.
Lipoproteins and their subfractions in psoriatic arthritis: identification of an
atherogenic profile with active joint disease. Ann Rheum Dis
2000;59(11):904–9.
13 Masi AT, Aldag JC, Mohan PC, Murugan TSR. Significantly lower serum
triglyceride levels in ankylosing spondylitis patients than age-, gender-, and
medical service-matched controls: results of multivariate analysis. Arthritis
Rheum 2000;43(Suppl):S104.
14 Cabana VG, Siegel JN, Sabesin SM. Effects of the acute phase response on
the concentration and density distribution of plasma lipids and
apolipoproteins. J Lipid Res 1989;30:39–49.
15 Hulthe J, Fagerberg B. Circulating oxidized LDL is associated with subclinical
atherosclerosis development and inflammatory cytokines (AIR study).
Arterioscler Thromb Vasc Biol 2002;22:1162–7.
16 van Denderen JC, van der Paardt M, Nurmohamed MT, De Ryck YM,
Dijkmans BA, van der Horst-Bruinsma IE. Double-blind, randomised, placebocontrolled study of leflunomide in the treatment of active ankylosing
spondylitis. Ann Rheum Dis 2005;64:1761–4.
17 Calin A, Dijkmans BA, Emery P, Hakala M, Kalden J, Leirisalo-Repo M, et al.
Outcomes of a multicentre randomised clinical trial of etanercept to treat
ankylosing spondylitis. Ann Rheum Dis 2004;63:1594–600.
18 van der Linden S, Valkenburg HA, Cats A. Evaluation of the diagnostic
criteria for ankylosing spondylitis; a proposal for the modification of the New
York criteria. Arthritis Rheum 1984;27:361–8.
19 Jones SD, Steiner A, Garrett SL, Calin A. The Bath Ankylosing Spondylitis
Patient Global Score (BAS-G). Br J Rheumatol 1996;35:66–71.
20 Calin A, Garrett S, Whitelock H, Kennedy LG, O’Hea J, Mallorie P, et al. A
new approach to defining functional ability in ankylosing spondylitis: the
development of the Bath Ankylosing Spondylitis Functional Index. J Rheumatol
1994;21:2281–5.
21 Garrett S, Jenkinson T, Kennedy LG, Whitelock H, Gaisford P, Calin A. A new
approach to defining disease status in ankylosing spondylitis: the Bath
Ankylosing Spondylitis Disease Activity Index. J Rheumatol
1994;21:2286–91.
22 Twisk JWR. Applied longitudinal data analysis for epidemiology. A practical
guide. Cambridge, UK: Cambridge University Press, 2003.
Downloaded from ard.bmj.com on June 7, 2014 – Published by group.bmj.com
Disease activity deteriorates lipid profile in ankylosing spondylitis
23 Goldstein H, Browne W, Rasbash. Multilevel modelling of medical data. J Stat
Med, 2002;21:3291–315.
24 Sprecher DL, Watkins TR, Behar S, Brown WV, Rubins HB, Schaefer EJ.
Importance of high-density lipoprotein cholesterol and
triglyceride levels in coronary heart disease. Am J Cardiol
2003;91:575–80.
25 Rubins HB, Robins SJ, Collins D, Fye CL, Anderson JW, Elam MB, et al.
Veterans Affairs High-Density Lipoprotein Cholesterol Intervention Trial Study
Group. Gemfibrozil for the secondary prevention of coronary heart disease in
men with low levels of high-density lipoprotein cholesterol. N Engl J Med
1999;341:410–18.
1477
26 Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med
1999;340:115–26.
27 Burger D, Dayer JM. High-density lipoprotein-associated apolipoprotein A-1:
the missing link between infection and chronic inflammation? Autoimmun Rev
2002;1:111–17.
28 Hyka N, Dayer JM, Modoux C, Kohno T, Edwards CK, Roux-Lombard P, et al.
Apolipoprotein A-1 inhibits the production of interleukin-1b and tumour
necrosis factor-a by blocking contact-mediated activation of monocytes by Tlymphocytes. Blood 2001;97:2381–9.
29 Walsmith J, Roubenoff R. Cachexia in rheumatoid arthritis. Int J Cardiol
2002;85:88–99.
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Increased disease activity is associated with a
deteriorated lipid profile in patients with
ankylosing spondylitis
V P van Halm, J C van Denderen, M J L Peters, et al.
Ann Rheum Dis 2006 65: 1473-1477 originally published online April 27,
2006
doi: 10.1136/ard.2005.050443
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Originalien
Z Rheumatol
DOI 10.1007/s00393-016-0092-4
© Springer-Verlag Berlin Heidelberg 2016
Redaktion
U. Müller-Ladner, Bad Nauheim
U. Lange, Bad Nauheim
Introduction
Ankylosing spondylitis (AS) is a prototype of spondyloarthropathies characterized by chronic inflammatory arthritis
with spinal and sacroiliac joint involvement. It may cause structural and functional damage to the skeletal system. In
addition, several systems may be affected,
such as the eyes, skin, gastrointestinal
system, heart and vascular structures [1].
There may be anatomical and functional
changes when both cardiac and vascular involvement are present. Cardiac and
vascular involvement include cardiomyopathy (systolic and/or diastolic dysfunction), valvular diseases (aortic valve insufficiency, regurgitation) and conduction disorders. Furthermore, there may
be an increased risk of atherosclerosis
and coronary artery disease [2, 3].
Atherosclerosis is one of the most important causes of mortality and morbidity
in cardiovascular system (CVS) diseases.
Increased carotid intima–media thickness (CIMT) is a convenient, noninvasive marker that can be used to monitor
atherosclerosis in general, as well as
the progression of cardiovascular diseases [4]. Dyslipidaemia is the most
important risk factor for atherosclerosis. Low-density lipoprotein (LDL)
easily infiltrates the endothelium and
renders the tissue sensitive to oxidative
changes, while high-density lipoprotein
(HDL) has anti-atherogenic properties
and provides a protective effect against
cardiovascular disease [5, 6].
Systemic inflammation is thought to
be involved in lipid profile changes in
A. Kucuk · A. Ugur Uslu · A. Icli · E. Cure · S. Arslan · K. Turkmen · A. Toker · M. Kayrak
Division of Rheumatology, Department of Internal Medicine, Meram School of Medicine, Necmettin
Erbakan University, Konya, Turkey
The LDL/HDL ratio and
atherosclerosis in ankylosing
spondylitis
patients with AS [7]. The presence of systemic inflammation causes the release of
free oxygen radicals via immune-mediated mechanisms, which have been suggested to contribute to the pathogenesis of inflammatory disorders. Systemic
inflammation, endothelial inflammation
and increased oxidative stress are important for the development and progression
of atherosclerosis [8]. Ischemic modified
albumin (IMA) is formed as a result of
changes in the metal ion-binding properties of the N-terminal domain of human
serum albumin. IMA has only recently
been introduced but has rapidly become
a marker of both ischemia and oxidative
stress [9].
Our study is the first to investigate
the relationship between CIMT and the
LDL/HDL ratio and also to assess the
IMA levels. The aim of the study is to
investigate CIMT, the LDL/HDL ratio
and the oxidative stress markers IMA
and total oxidant status (TOS), to determine their relationships among patients
with AS and also to evaluate their use in
atherosclerosis assessment.
Materials and methods
Our study was conducted from June 2013
to June 2014 at the Rheumatology Clinic
of the University Faculty of Medicine.
Sixty AS patients diagnosed using the
Modified New York Criteria and 54 ageand gender-matched controls were included [10]. The Ethics Committee for
Clinical Research of our University Faculty of Medicine approved the present
study. Basic laboratory values and de-
mographics, as well as the Bath AS Disease Activity Index (BASDAI) and the
Bath AS Functional Index (BASFI) were
used to evaluate disease activity and were
recorded for both the patients and the
control group. Participants with any immune deficiency, hypertension, diabetes,
acute and/or chronic infection, coronary
artery disease, chronic obstructive pulmonary disease, a history of malignancy
or suspected malignancy and a history of
smoking were excluded from the study.
Biochemical analyses
Coagulated blood samples were collected
from the patients and the controls following a 12-h fast. After suitable centrifugation, the samples were stored at
–80 ºC until testing. Clinical data and
blood samples were collected over a time
period of 6 months. The serum IMA concentrations were analysed by measuring
the complex composed of dithiothreitol
(DTT) and cobalt chloride (CoCl26H2O)
unbound from albumin by the colorimetric method as described by Bar-Or
et al. [11]. Colour development with
DTT was measured spectrophotometrically at 470 nm and compared with
a serum cobalt blank without DTT. The
results were reported in absorbance units
(ABSU). TOS levels were measured using commercially available kits (Rel Assay, Gaziantep,Turkey). Serum TOS levels were expressed as µmol H2O2 equivalent/l [12]. Total cholesterol (TC), triglycerides (TG) and HDL were measured using a Synchron LX20 system (Beckman
Coulter, Brea, CA, USA) and Beckman
Zeitschrift für Rheumatologie
Originalien
Tab. 1
Demonstrative and laboratory data of the patient and the control groups
AS (n = 60)
Controls (n = 54)
P-value
Carotid intima–media
measurements
Age (years)
41.68 ± 10.98
41.80 ± 9.56
0.953
Gender, m/f (%)
45 (75)/15 (25)
32 (59.3)/22 (40.7)
0.174
BMI (kg/m )
27.09 ± 4.68
28.24 ± 3.77
0.148
Hb (g/dL)
14.38 ± 1.80
14.33 ± 1.39
0.852
WBC (×109/L)
8.12 ± 2.53
6.85 ± 1.35
0.001
ESR (mm/h)
13.31 ± 13.51
6.55 ± 4.87
0.001
CRP (mg/dL)
11.53 ± 22.23
3.33 ± 2.41
0.006
Creatinine (mg/dL)
0.75 ± 0.11
0.82 ± 0.48
0.291
ALT (IU/L)
22.41 ± 11.26
23.61 ± 7.84
0.503
TG (mg/dL)
143.85 ± 66.68
133.81 ± 87.81
0.517
TC (mg/dL)
189.91 ± 37.83
183.25 ± 35.77
0.357
HDL (mg/dL)
43.21 ± 10.04
46.79 ± 12.49
0.114
LDL (mg/dL)
117.92 ± 34.39
109.69 ± 33.58
0.219
LDL/HDL ratio
2.85 ± 1.00
2.47 ± 0.90
0.047
TOS (µmol H2O2
equivalent/L)
212.57 ± 149.44
116.70 ± 122.98
0.005
Statistical analysis
The Statistical Package for Social Sciences
(SPSS) for Windows, version 14.0 (SPSS
Inc, Chicago, IL, USA), was used for our
statistical analyses. Continuous variables
were presented as mean ± standard deviation, while categorical variables were
indicated as a number (n) that was expressed as a percent (%). Variables meeting the parametric assumptions were assessed using the t-test and one-way analysis of variance (ANOVA) in independent groups and Tukey’s HSD test in the
intergroup posthoc evaluation, while categorical variables were assessed by chisquare test. Pearson’s correlation analysis
was carried out to test the correlation of
the data. Any p-values that were less than
0.05 were considered statistically significant.
2
IMA ABSU
0.44 ± 0.17
0.32 ± 0.13
< 0.0001
CIMT (mm)
0.99 ± 0.27
0.76 ± 0.25
< 0.0001
AS ankylosing spondylitis, BMI body mass index, TG triglyceride, TC total cholesterol, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Hb haemoglobin,
WBC white blood cells, ESR erythrocyte sedimentation rate, CRP C-reactive protein, ALT alanine aminotransferase, LDL/HDL ratio low-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio, TOS total oxidative status, IMA ischemic modified albumin, CIMT carotid
intima–media thickness, AS ankylosing spondylitis, m male, f female, BMI body mass index, Hb
hemoglobin, WBC white blood cells, ESR erythrocyte sedimentation rate, CRP C-reactive protein,
ALT alanine aminotransferase, TG triglyceride, TC total cholesterol, HDL high-density lipoprotein,
LDL low-density lipoprotein, LDL/HDL ratio low-density lipoprotein/high-density lipoprotein ratio,
TOS total oxidative status, IMA ischemic modified albumin, ABSU absorbance units, CIMT carotid
intima–media thickness
a
Mean ± SD
Tab. 2
Age, family history, BASFI and BASDAI scores and drug use rates of the patients with AS
AS (n = 60)
Age at diagnosisa, (years)
33.51 ± 9.16
Family history, n (%)
11 (18.3)
BASFIa
2.72 ± 2.16
BASDAIa
4.01 ± 2.14
SLZ, n (%)
24 (40)
ETA, n (%)
8 (13.3)
ADA, n (%)
8 (13.3)
IFX, n (%)
7 (11.7)
AS ankylosing spondylitis, BASFI Bath Ankylosing Spondylitis Functional Index, BASDAI Bath
Ankylosing Spondylitis Disease Activity Index, SLZ sulfasalazine, ADA adalimumab, IFX infliximab,
ETA etanercept
a
Mean ± SD
reagents. HDL levels were assessed using a direct enzymatic method instead of
precipitation. LDL levels were calculated
using the Friedewald formula when TG
levels were lower than 400 mg/dl. If the
Zeitschrift für Rheumatologie
TG levels were higher than 400 mg/dl,
the LDL levels were directly measured.
In all cases, imaging was conducted using
a high-resolution ultrasound machine
(Logiq S6; General Electric, Milwaukee,
WI, USA) with a 12-MHz mechanical
sector transducer. The intima media
thickness of both the right and left
arteria carotis communis (CCA) was
measured at three points on the far walls
in each CCA 2 cm proximal to the CCA
bifurcation. The three locations were
then averaged to obtain the mean IMT
for each side. The average of the twosided measurements was considered as
the patient’s overall mean CIMT.
Results
The general clinical characteristics and
demographics of patients with AS are
summarized in . Tab. 1. There was nosignificant difference between AS patients
and the control group regarding their
age, gender and body mass index (BMI;
. Tab. 1). The clinical characteristics and
the medical treatment of AS patients are
displayed in . Tab. 2.
IMA was higher in AS patients
compared to the control group (0.44
± 0.17 ABSU and 0.32 ± 0.13 ABSU, respectively; p < 0.0001). A higher TOS was
seen in AS patients than it was in the con-
Abstract · Zusammenfassung
Z Rheumatol DOI 10.1007/s00393-016-0092-4
© Springer-Verlag Berlin Heidelberg 2016
A. Kucuk · A. Ugur Uslu · A. Icli · E. Cure · S. Arslan · K. Turkmen · A. Toker · M. Kayrak
The LDL/HDL ratio and atherosclerosis in ankylosing spondylitis
Abstract
Objectives. In ankylosing spondylitis (AS)
patients, cardiac and vascular involvement
may manifest as atherosclerosis and coronary
artery disease. Systemic inflammation,
oxidative stress, increased low-density
lipoprotein (LDL) cholesterol and decreased
high-density lipoprotein (HDL) cholesterol
constitute a significant risk for atherosclerosis.
This study investigated the relationship
between carotid intima–media thickness
(CIMT), LDL/HDL ratio, total oxidant status
(TOS; an indicator of oxidative stress) and
ischemic modified albumin (IMA; an ischemic
marker in AS patients).
Patients and methods. Sixty AS patients
were diagnosed using the Modified New
York Criteria; 54 age- and gender-matched
participants were included as controls. CIMT,
LDL/HDL ratio, TOS and IMA were measured
using the most appropriate methods.
Results. IMA was higher in AS patients
compared to controls (p < 0.0001). TOS was
also increased in AS patients (p = 0.005); as
was CIMT (p < 0.0001). The LDL/HDL ratio
was also greater in AS patients compared to
controls (p = 0.047). A positive correlation
was found between CIMT and LDL/HDL ratio
among AS patients.
Conclusion. Elevated CIMT, IMA and TOS levels
suggest an increased risk of atherosclerotic
heart disease in AS patients. The LDL/HDL
ratio was higher in AS patients compared to
controls, and there was a correlation between
LDL/HDL ratio and CIMT, albeit statistically
weak. Therefore, the LDL/HDL ratio is not
a reliable marker to predict atherosclerotic
heart disease in AS patients.
Keywords
Dyslipidemias · Atherosclerosis · Ankylosing
spondylitis · Ischemia-modified albumin ·
Oxidative Stress
Das Verhältnis von LDL zu HDL und Atherosklerose bei ankylosierender Spondylitis
Zusammenfassung
Ziel. Bei Patienten mit Spondylitis ankylosans
(AS) kann sich eine kardiale und vaskuläre
Beteiligung als Atherosklerose und koronare Herzkrankheit (KHK) manifestieren.
Systemische Entzündung, oxidativer Stress,
erhöhtes Low-Density-Lipoprotein(LDL)Cholesterin und vermindertes High-DensityLipoprotein(HDL)-Cholesterin stellen ein
signifikantes Risiko für Atherosklerose dar. In
der vorliegenden Studie wird die Beziehung
zwischen Karotis-Intima-Media-Dicke (CIMT),
LDL-HDL-Quotient, totalem oxidativem
Status (TOS, Indikator für oxidativen Stress)
und ischämiemodifiziertem Albumin (IMA,
Ischämiemarker bei AS-Patienten) untersucht.
trol group (212.57 ± 149.44 µmol H2O2
equivalent/l and 116.70 ± 122.98 µmol
H2O2 equivalent/l, respectively; p =
0.005). CIMT was higher in AS patients
compared to the control group (0.99
± 0.27 mm and 0.76 ± 0.25 mm, respectively; p < 0.0001; . Fig. 1). A higher
LDL/HDL ratio was seen in AS patients
than in the control group (2.85 ± 1.00
and 2.47 ± 0.90, respectively; p = 0.047;
. Fig. 2). A positive correlation was observed between CIMT and age, BMI and
the LDL/HDL ratio among the patients
(. Tab. 3, . Fig. 3).
There was no correlation between
IMA, TOS and BMI, CIMT, or the
LDL/HDL ratio, erythrocyte sedimentation rate, C-reactive protein or the
Patienten und Methoden. Anhand der
Modified New York Criteria erfolgte die
Diagnosestellung bei 60 AS-Patienten;
Kontrollen waren 54 in Alter und Geschlecht
entsprechende Personen. CIMT, LDL/HDLQuotient, TOS und IMA wurden mit den am
besten geeigneten Methoden ermittelt.
Ergebnisse. IMA war bei AS-Patienten höher
als bei den Kontrollen (p < 0,0001). Auch TOS
war bei AS-Patienten erhöht (p = 0,005) sowie
CIMT (p < 0,0001). Der LDL/HDL-Quotient
war bei AS-Patienten ebenfalls größer als
bei den Kontrollen (p = 0,047). Es fand sich
eine positive Korrelation zwischen CIMT und
LDL/HDL-Quotient bei AS-Patienten.
Schlussfolgerung. Erhöhte Werte für CIMT,
IMA und TOS sprechen für ein erhöhtes Risiko
einer KHK bei AS-Patienten. Der LDL/HDLQuotient war bei AS-Patienten höher als
bei den Kontrollen, und es bestand eine –
allerdings schwache – Korrelation zwischen
dem LDL/HDL-Quotienten und CIMT. Folglich
ist der LDL/HDL-Quotient kein reliabler Marker
zur Vorhersage einer KHK bei AS-Patienten.
leukocyte count. No difference was seen
in CIMT, IMA, TOS or the LDL/HDL
ratio across the treatment options (between when the patients received antitumour necrosis factor alpha treatment
and sulfasalazine).
risk for atherosclerosis [14]. CIMT is
a reliable and inexpensive marker that
has become a predictor of atherosclerosis
and can be used to predict cardiovascular
events [4]. Our results showed a higher
IMA, TOS, CIMT and LDL/HDL ratio
in AS patients compared to the healthy
control group. We also found a positive correlation between the CIMT and
LDL/HDL ratios. However, the correlation between the LDL/HDL ratio and
CIMT was statistically weak.
Although the underlying reasons for
increased atherosclerosis in chronic inflammatory disorders are not fully understood, systemic inflammation exerts
important effects on the process. CIMT is
used to assess early-stage atherosclerosis
Discussion
AS is a systemic inflammatory disorder
that may involve multiple systems and
organs, mainly in the cardiovascular system. Systemic inflammation is involved
in the development of atherosclerosis due
to its direct effects on the vascular structures and also its influence on the lipid
profile [7, 13]. Increased LDL and decreased HDL levels pose an important
Schlüsselwörter
Dysliplidämie · Atherosklerose · Spondylitis
ankylosans · Ischämiemodifiziertes Albumin ·
Oxidativer Stress
Zeitschrift für Rheumatologie
Originalien
Fig. 1 8 Carotidintima–media thickness (CIMT)amongankylosingspondylitis (AS)andcontrol groups
Fig. 2 8 Low-density lipoprotein (LDL)/high-density lipoprotein (HDL) cholesterol ratio among ankylosing spondylitis (AS) and control groups
and is known to have a strong association
with coronary artery disease [15]. Gupta
et al. [16] showed an increased prevalence of CIMT in AS patients compared to
their control group. Additionally, Gonzalez-Juanatey et al. [17] demonstrated
higher CIMT values in AS patients than
Zeitschrift für Rheumatologie
in the control group. In another study,
Mathieu et al. [18] reported increased
CIMT in patients with AS compared to
the control group.
AS patients are known to be at an
increased risk for atherosclerosis, which
is influenced by changes in their lipid
profile. During systemic inflammation, structural changes in endothelial
cells and lipid molecules accelerate the
development of atherosclerosis. LDL
transfers cholesterol from the liver to the
peripheral tissues, and immune system
elements are involved in its placement on
the vascular wall. In contrast, HDL carries cholesterol to the liver and prevents
its adhesion to endothelial cells [19].
Any rise in LDL levels and decreases in
HDL levels are important factors in the
diagnosis of atherosclerosis. Divecha
et al. [20] reported lower HDL levels
in AS patients compared to the control
group in their study. Mathieu et al.
[21] found lower HDL levels in patients
with AS than in their control group.
Considering the effects of LDL and HDL
on atherogenesis, the predictive value
of the LDL/HDL ratio regarding the
atherosclerotic changes on the vascular
wall is even higher. Sathiya et al. [22]
observed a greater LDL/HDL ratio in
patients with coronary artery disease
compared to the control group in their
study. Yang et al. [5] demonstrated
a relationship between the LDL/HDL
ratio and CIMT and carotid plaques.
Recently, the ratio of LDL and HDL
has become a representative marker of
regression and progression in coronary
atherosclerosis [14].
The systemic inflammation commonly seen in patients with AS causes
increased oxidative stress and impaired
endothelial functions. Normally, the
antioxidant system and its mechanisms
balance the effects of oxidative radicals.
Any inconsistency between these systems leads to oxidative stress. TOS is
a recently introduced and commonly
used marker that can assess these oxidative stress products [23, 24]. In their
study, Karakoc et al. [25] found higher
TOS levels in AS patients compared to
the control group. Endothelial dysfunction is the first sign in the development
process of atherosclerosis. The endothelial dysfunction resulting from oxidative
stress plays a major role in cardiovascular
disease pathogenesis [26, 27].
Changes in IMA levels are thought to
be associated with acute and/or chronic
hypoxia and oxidative stress. IMA is
formed as a result of decreased metal ion-
Tab. 3
CIMT
Correlation between serum CIMT and age, body mass index and LDL/HDL ratio
Age (years)
BMI
LDL/HDL ratio
R
0.737
0.315
0.395
P
< 0.0001
0.014
0.002
CIMT carotid intima–media thickness, BMI body mass index, LDL/HDL ratio low-density lipoprotein
cholesterol/high-density lipoprotein cholesterol ratio
Fig. 3 8 The relationship between cholesterol ratio and carotid intima-media thickness (CIMT) among
ankylosing spondylitis (AS) patients
binding capacity due to the effects of free
oxygen radicals on albumin [28]. IMA is
a considerablysensitive marker, and it can
be used as an early indicator of cardiac ischemia [9, 28]. Although there have been
no studies on IMA in AS patients, investigations into autoimmune disorders
have reported an increased frequency
of atherosclerotic heart disease and systemic inflammation, such as rheumatoid arthritis (RA) and systemic sclerosis (SSc). Leitemperguer et al. [29]
demonstrated higher IMA values in RA
patients compared to the control group,
while Montagnana et al. [30] reported
increased IMA values in patients with
SSc compared to the controls.
The cause of 20–40 % of the mortality
seen in AS patients is cardiovascular
disease, and cardiovascular mortality is
increased by approximately two- to threefold in these patients compared to the
general population [17, 18]. Atherosclerotic coronary heart diseases account for
an important portion of cardiovascular
mortality. There are several factors in
AS that may lead to atherosclerotic heart
disease, including metabolic syndrome,
medications, changes in lipid profiles
(low HDL levels) and inflammatory processes [31]. The presence of a chronic
inflammatory process, increased oxidative stress and the interaction between
the two contribute to all stages, including the formation and progression of
atherosclerotic plaque and thrombus
formation [32, 33].
In our study, the higher CIMT, IMA
and TOS values in AS patients compared with the control group may suggest an increased risk of atherosclerotic
heart disease in patients with AS. The
LDL/HDL ratio was higher in AS patients than it was in the control group,
and there was a correlation between the
LDL/HDL ratio and CIMT. However, this
correlation was statistically weak. In reality, a normal or increased LDL level
is not a reliable marker for the risk of
atherosclerosis, since LDL is composed
of various subtypes; small, dense LDL
and oxide subfractions, which are two
of these subtypes, are more atherogenic
[34, 35]. Thus, LDL subgroup analysis
is a more reliable marker used to diagnose atherosclerotic heartdisease. Onthe
other hand, development of atherosclerotic heart disease is not all the time prevented by the increment in the HDL [36].
HDL can be easily dysfunctional and even
it can gain a pro-inflammatory feature
by undergoing oxidation and modification by various oxidant materials [37].
HDL may not show an antioxidant effect
as a result of the polymorphism of the
paraoxonase enzyme, which is responsible for the antioxidant effect of HDL [38].
Increased HDL leads to a higher amount
of lipid transport in the liver; thus, it can
show adverse effects [36, 39]. Therefore,
even though the LDL/HDL ratio of a patient with normal LDL and high HDL
levels is low, there can be high risk of
atherosclerotic heart disease. Similarly,
the LDL/HDL ratio will be disproportionately high in a patient with high LDL
and low atherogenic LDL subtypes. Shah
et al. reported that the LDL/HDL ratio
was not associated with CIMT, and it
did not provide any additional information about atherosclerotic heart disease
[40]. In our opinion, the LDL/HDL ratio
is not a reliable or good marker to determine the risk of atherosclerotic heart
disease. However, it may be beneficial to
assess the LDH/HDL ratio together with
other markers to determine the risks of
atherosclerotic heart disease.
Our study has a few limitations. These
include the small number of patients, the
prospective cross-sectional nature of the
study, and the fact that the differences
between the patients with mild and severe disease activity as per the BASDAI
scoring system were not taken into account.
In conclusion, the high levels of CIMT,
IMA and TOS that were observed in AS
patients in the present study suggest an
increased risk of atherosclerotic heart disZeitschrift für Rheumatologie
Originalien
ease. The LDL/HDL ratio was higher
in AS patients than it was the control
group, and the correlation between the
LDL/HDL ratio and CIMT was statistically weak. The LDL/HDL ratio is therefore not a promising or reliable marker to
detect the risk for atherosclerotic heart
disease. However, it can be useful to
evaluate the LDH/HDL ratio along with
other markers to determine the risk for
atherosclerotic heart disease.
Corresponding address
Dr. A. Kucuk
Division of Rheumatology, Department of
Internal Medicine, Meram School of Medicine,
Necmettin Erbakan University
Konya, Turkey
drademk@yahoo.com
Compliance with ethical
guidelines
Conflict of interest. A. Kucuk, A. Ugur Uslu, A. Icli,
E. Cure, S. Arslan, K. Turkmen, A. Toker and M. Kayrak
state that there are no conflicts of interest.
The Ethics Committee for Clinical Research of Necmettin Erbakan University Faculty of Medicine approved
the present study.
References
1. Daikh DI, Chen PP (2014) Advances in managing
ankylosing spondylitis. F1000PrimeRep 6:78
2. Maghraoui A El (2011) Extra-articular manifestations of ankylosing spondylitis: prevalence,
characteristics and therapeutic implications. Eur J
Intern Med 22:554–560
3. Essers I, Stolwijk C, Boonen A et al (2016)
Ankylosing spondylitis and risk of ischaemic heart
disease: apopulation-based cohort study. Ann
Rheum Dis 75:203–209
4. Katakami N, Kaneto H, Shimomura I (2014) Carotid
ultrasonography: A potent tool for better clinical
practice in diagnosis of atherosclerosis in diabetic
patients. J Diabetes Investig 5:3–13
5. Yang C, Sun Z, Li Y et al (2014) The correlation
between serum lipid profile with carotid intimamedia thickness and plaque. Bmc Cardiovasc
14:181
6. Garcia-Gomez C, Bianchi M, Fuente D de la
et al (2014) Inflammation, lipid metabolism
and cardiovascular risk in rheumatoidarthritis:
A qualitative relationship? World J Orthop
5:304–311
7. Halm VP van, Denderen JC van, Peters MJet al
(2006) Increased disease activity is associated
with a deteriorated lipid profile inpatients
with ankylosing spondylitis. Ann Rheum Dis
65:1473–1477
8. Wan AWN, Sakri F, MokhsinAet al (2015) Low serum
high density lipoproteincholesterol concentration
Zeitschrift für Rheumatologie
is an independent predictor for enhanced
inflammationand endothelial activation. PLOS
ONE 10:0116867
9. Zhong Y, Wang N, Xu H et al (2012) Ischemia-modified albumin instable coronary atherosclerotic
heart disease: clinical diagnosis and riskstratification. Coron Artery Dis 23:538–541
10. Linden S van der, Valkenburg HA, Cats A (1984)
Evaluation of diagnostic criteria forankylosing
spondylitis. A proposal for modification of the New
York criteria. Arthritis Rheum 27:361–368
11. Bar-Or D, Lau E, Winkler JV (2000) A novel assay
for cobalt-albumin binding and its potential as a
marker for myocardial ischemia – a preliminary
report. J Emerg Med 19:311–315
12. Erel O (2005) A new automated colorimetric
method for measuring total oxidantstatus. Clin
Biochem 38:1103–1111
13. Gianturco L, Bodini BD, Atzeni Fet al (2015)
Cardiovascular and autoimmune diseases in
females: The role of microvasculature and
dysfunctional endothelium. Atherosclerosis
241:259–263
14. Nimkuntod P, Tongdee P (2015) Plasma lowdensity lipoprotein cholesterol/high-density
lipoprotein cholesterol concentration ratio and
early marker of carotid artery atherosclerosis. J
Med Assoc Thai 98:58–63
15. Limbu YR, Rajbhandari R, Sharma R et al (2015)
Carotid intima-media thickness (CIMT) and
carotid plaques in young Nepalesepatients with
angiographically documented coronary artery
disease. Cardiovasc Diagn Ther 5:1–7
16. Gupta N, Saigal R, Goyal L et al (2014) Carotid
intimamedia thickness as a marker of atherosclerosis in ankylosing spondylitis. Int Jrheumatol
2014:839135
17. Gonzalez-Juanatey C, Vazquez-Rodriguez TR,
Miranda-Filloy JA et al (2009) The high prevalence
ofsubclinical atherosclerosis in patients with
ankylosing spondylitis withoutclinically evident
cardiovascular disease. Medicine (Baltimore)
88:358–365
18. Mathieu S, Joly H, Baron Get al (2008) Trend
towards increased arterial stiffness or intimamedia thickness in ankylosing spondylitis patients
without clinically evident cardiovasculardisease.
Rheumatology (Oxford) 47:1203–1207
19. Takata K, Imaizumi S, Kawachi Eet al (2014) Impact
of cigarette smoking cessation on high-density
lipoprotein functionality. Circ J 78:2955–2962
20. Divecha H, Sattar N, Rumley A et al (2005) Cardiovascular risk parameters in men with ankylosing
spondylitis in comparison with non-inflammatory control subjects: relevance of systemic
inflammation. Clin Sci (Lond) 109:171–176
21. Mathieu S, Gossec L, Dougados M et al (2014)
Cardiovascular profile inankylosing spondylitis: a
systematic review and meta-analysis. Arthritis
Care Res 63:557–563
22. Sathiya R, Velu VK, Niranjan Get al (2014) A
comparative study of serum uric acid levels and
lipid ratios incoronary artery disease patients. Int J
Biomed Sci 10:124–128
23. Karkucak M, Capkin E, Alver A et al (2010) The effect
of anti-TNF agent on oxidation status in patients
with ankylosingspondylitis. ClinRheumatol
29:303–307
24. Stanek A, Cieslar G, Romuk E et al (2010) Decrease
in antioxidant status of plasma and erythrocytes
from patientswith ankylosing spondylitis. Clin
Biochem 43:566–570
25. Karakoc M, Altindag O, Keles H et al (2007)
Serumoxidative-antioxidative status in patients
with ankylosing spondilitis. Rheumatol Int
27:1131–1134
26. Sloten TT van, Henry RM, Dekker JM et al
(2014) Endothelial dysfunction plays a key role in
increasing cardiovascular risk intype 2 diabetes:
the Hoorn study. Hypertension 64:1299–1305
27. JerkicM, LetarteM(2015)Contributionofoxidative
stress to endothelialdysfunction in hereditary
hemorrhagic telangiectasia. Front Genet 6:34
28. Ali OS, Abdelgawad HM, Mohammed MS et al
(2014) Ischemic heart diseases in Egypt: role of
xanthine oxidase system and ischemia-modified
albumin. Heart Vessels 29:629–637
29. Leitemperguer MR, Tatsch E, Kober H et al (2014)
Assessment of ischemia-modified albumin levels
in patients with rheumatoidarthritis. Clin Lab
60:1065–1070
30. Montagnana M, Lippi G, Volpe Aet al (2014)
Evaluation of cardiac laboratory markers in
patients with systemicsclerosis. Clin Biochem
39:913–917
31. Szabo SM, Levy AR, Rao SRet al (2011) Increased
risk of cardiovascular and cerebrovascular diseases
in individuals withankylosing spondylitis: a
population-based study. Arthritis Rheum
63:3294–3304
32. Valente RL, Valente JM, Castro GR de et al
(2013) Subclinical atherosclerosis in ankylosing
spondylitis: is there a role forinflammation? Rev
Bras Reumatol 53:377–381
33. Şerban C, Dragan S (2014) The relationship between inflammatory and oxidative stressbiomarkers, atherosclerosis and rheumatic diseases. Curr
Pharm Des 20:585–600
34. Cure MC, Tufekci A, Cure E et al (2013) Lowdensity lipoprotein subfraction, carotid artery
intima-media thickness, nitric oxide, and tumor
necrosis factor alpha are associated with newly
diagnosed ischemic stroke. Ann Indian Acad
Neurol 16:498–503
35. Toth PP, Patti AM, Nikolic D et al (2016) Bergamot
reduces plasma lipids, atherogenic small dense
LDL, and subclinical atherosclerosis in subjects
with moderate hypercholesterolemia: a 6 months
prospective study. Front Pharmacol 6:299
36. Saleheen D, Scott R, Javad S et al (2015) Association
of HDL cholesterol efflux capacity with incident
coronary heart disease events: a prospective
case-control study. Lancet Diabetes Endocrinol
3:507–513
37. Icli A, Cure E, Cure MC et al (2015) Endocan
levels and subclinical atherosclerosis in patients
with systemic lupus erythematosus. Angiology.
doi:10.1177/0003319715616240
38. Marsillach J, Becker JO, Vaisar T et al (2015)
Paraoxonase-3 is depleted from the high-density
lipoproteins of autoimmune disease patients
with subclinical atherosclerosis. J Proteome Res
14:2046–2054
39. Ronda N, Favari E, Borghi MO et al (2014) Impaired
serum cholesterol efflux capacity in rheumatoid
arthritis and systemic lupus erythematosus. Ann
Rheum Dis 73:609–615
40. Shah AS, Urbina EM, Khoury PR et al (2013) Lipids
and lipoprotein ratios: contribution to carotid
intima media thickness in adolescents and young
adults with type 2 diabetes mellitus. J Clin Lipidol
7:441–445
Impaired Antiatherogenic Functions of High-density
Lipoprotein in Patients with Ankylosing Spondylitis
Christina Gkolfinopoulou, Efstratios Stratikos, Dimitris Theofilatos, Dimitris Kardassis,
Paraskevi V. Voulgari, Alexandros A. Drosos, and Angeliki Chroni
ABSTRACT. Objective. Ankylosing spondylitis (AS) is a chronic inflammatory disease associated with increased
risk of cardiovascular disease (CVD). High-density lipoprotein (HDL) exerts a series of antiatherogenic properties and protects from CVD. We evaluated whether HDL antiatherogenic properties are
impaired in patients with AS.
Methods. HDL (apoB-depleted serum) was isolated from 35 patients with AS and 35 age- and
sex-matched controls. We measured the antioxidant capacity of HDL, the ability of HDL to induce
cholesterol efflux, the activity of HDL-associated enzymes paraoxonase-1 (PON1) and myeloperoxidase (MPO), as well as the ability of HDL to induce Akt kinase activation.
Results. HDL from patients with AS had decreased antioxidant capacity and decreased ability to
promote cholesterol efflux from macrophages compared to controls. HDL-associated PON1 activity
was lower and HDL-associated MPO activity higher in patients with AS compared to controls. Higher
MPO activity correlated positively with lower antioxidant capacity of HDL in patients with AS. In
addition, HDL from patients with AS had impaired endothelial Akt kinase activating properties that
were inversely correlated with the MPO/PON1 ratio and positively correlated with the cholesterol
efflux capacity of HDL.
Conclusion. HDL from patients with AS displays impaired antiatherogenic properties. Attenuation
of HDL properties may constitute a link between AS and CVD. (First Release August 1 2015;
J Rheumatol 2015;42:1652–60; doi:10.3899/jrheum.141532)
Key Indexing Terms:
ANKYLOSING SPONDYLITIS
ANTIOXIDANT EFFECT
HIGH-DENSITY LIPOPROTEINS CHOLESTEROL EFFLUX
CARDIOVASCULAR DISEASE
Ankylosing spondylitis (AS) is a chronic inflammatory
disease primarily affecting the skeleton, leading to chronic
pain in axial and peripheral joints and to functional impairments1. In patients with AS, a higher early mortality of
1.6–1.9 times that of the general population has been
reported2,3. Studies of the causes of death among patients
with AS identified cardiovascular disease (CVD) as the
From the National Center for Scientific Research Demokritos, Agia
Paraskevi, Athens; University of Crete Medical School and Institute of
Molecular Biology and Biotechnology, Foundation for Research and
Technology of Hellas, Heraklion, Crete; Rheumatology Clinic, Department
of Internal Medicine, Medical School, University of Ioannina, Ioannina,
Greece.
Funding provided by the General Secretariat of Research and Technology
of Greece Grant Synergasia 09SYN-12-897 (to DK and AC); the Ministry
of Education of Greece Grant Thalis MIS 377286 (to ES, DK, and AC),
and a Hellenic Rheumatology Society grant (to ES, PVV, AAD, and AC).
C. Gkolfinopoulou, MSc; E. Stratikos, PhD, National Center for Scientific
Research Demokritos; D. Theofilatos, MSc; D. Kardassis, PhD, University
of Crete Medical School and Institute of Molecular Biology and
Biotechnology, Foundation for Research and Technology of Hellas;
P.V. Voulgari, MD, PhD; A.A. Drosos, MD, FACR, Rheumatology Clinic,
Department of Internal Medicine, Medical School, University of Ioannina;
A. Chroni, PhD, National Center for Scientific Research Demokritos.
Address correspondence to A. Chroni, Institute of Biosciences and
Applications, National Center for Scientific Research “Demokritos,” Agia
Paraskevi, Athens 15310, Greece. E-mail: achroni@bio.demokritos.gr
Accepted for publication May 29, 2015.
leading cause2,3. Several studies reported that AS was
associated with an increased risk for ischemic heart disease,
congestive heart failure, peripheral vascular disease, arterial
hypertension (HTN), and cerebrovascular disease4,5. Some
causes of heart disease have been proposed to be direct results
of AS, such as aortitis and aortic insufficiency with the
possible necessity of cardiac surgery, conduction disturbances
of the atrioventricular node with a probable subsequent
indication for a pacemaker, and myocardial involvement with
a possible compromise of left ventricular function6. Analyses
of vascular functional and structural variables associated with
atheroma development in patients with AS showed increased
carotid intima-media thickness (IMT) or flow-mediated
dilation, suggesting the presence of increased risk for atherosclerosis7,8. A systematic literature review and metaanalysis
proposed that patients with AS appear to be at a higher risk
of myocardial infarction2. Overall, these studies indicate that
the excess cardiovascular mortality seen in patients with AS
could be related to functional or structural arterial abnormalities. Unraveling the mechanisms, as well as identification of
new biomarkers of increased risk for atherosclerosis, in
patients with AS is important for both prevention and
treatment.
Numerous clinical and epidemiological studies have
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1652
The Journal of Rheumatology 2015; 42:9; doi:10.3899/jrheum.141532
demonstrated an inverse association between high-density
lipoprotein cholesterol (HDL-C) levels and the risk of
CVD9,10. HDL exerts a series of antiatherogenic functions,
such as the ability to promote cholesterol efflux from artery
wall macrophages, antioxidative activity including the ability
to protect low-density lipoprotein (LDL) against oxidation,
antiinflammatory effects, and protective effects on the
vascular endothelium11,12. HDL or its major apolipoprotein,
apolipoprotein A-I (apoA-I), can have direct effects on
numerous cell types that play roles in cardiovascular and
metabolic health including endothelial cells, vascular smooth
muscle cells, leukocytes, platelets, adipocytes, skeletal
muscle myocytes, and pancreatic β cells13. The various
effects of HDL/apoA-I include the modulation of intracellular
calcium, oxygen-derived free radical production, the activity
of numerous kinases and enzymes and expression levels of
various genes13. Previous studies have shown that during the
course of chronic inflammatory rheumatic diseases, such as
rheumatoid arthritis (RA) and systemic lupus erythematosus
(SLE), the antiatherogenic properties of HDL are affected.
Specifically, patients with RA and SLE were found to have
proinflammatory HDL14,15. In addition, the cholesterol efflux
capacity of HDL was impaired in RA patients with high
disease activity, as well as in SLE patients with the disease
under control16,17.
Several proteomic studies have shown that a large number
of plasma proteins can associate with HDL and this can affect
HDL structure and functions18,19. Under conditions of
acute-phase reaction, as well as in chronic inflammation, the
HDL protein composition is altered, resulting in changes in
HDL function20,21. Among the proteins that associate with
HDL and play a role in its atheroprotective functions is
paraoxonase-1 (PON1), an enzyme linked to the
anti-oxidative, antiinflammatory, and endothelial repair–stimulating effects of HDL19,22. Reduced plasma PON1 activity
has been proposed to be a risk factor for major adverse
cardiac events in humans23. Myeloperoxidase (MPO) is
another protein that, like PON1, binds to HDL and is linked
to oxidative stress and atherosclerosis24,25. Site-specific
oxidation of apoA-I on HDL by MPO has been linked to
impairment of cholesterol efflux capacity and acquisition of
proinflammatory functions26,27. Further, epidemiological
studies have shown an association of plasma MPO with CVD
in the general population25.
Given the observed impairment of HDL antiatherogenic
properties in patients with RA or SLE14,15,16,17, it is tempting
to generalize to other chronic inflammatory diseases such as
AS. Similarly to AS, both RA and SLE have been associated
with excess cardiovascular risk28. In contrast to AS, however,
RA and SLE are characterized by the presence of many
autoantibodies that may cause systemic inflammation29,30.
This pathology is absent in patients with AS29,30. It is
therefore possible that HDL atheroprotective properties are
not affected in AS and the increased predisposition to CVD
for patients with AS is not related to HDL dysfunction. To
test this hypothesis, we evaluated the antiatherogenic
functions of HDL in patients with AS compared to controls.
More specifically, HDL from patients with AS were assessed
for their antioxidant properties using a cell-free assay [dichlorofluorescein (DCF) assay]31,32 that has been used to show
the impairment of antioxidant properties of HDL in patients
with RA and patients with SLE14. Further, we measured the
ability of HDL to induce cholesterol efflux from
macrophages, considered one of the major antiatherogenic
functions of HDL33,34. In addition, the activity of
HDL-associated enzymes PON1 and MPO was measured,
because previous studies have shown that the impairment of
atheroprotective properties of HDL may be associated with
changes in PON1 and MPO activity19,26,27,35. Finally, we
examined the ability of HDL to induce Akt kinase activation.
HDL contributes to cardiovascular protection, particularly in
endothelial cells, by inducing intracellular signaling that leads
to the activation of diverse kinase cascades13. One of these
kinases is Akt, an enzyme that is activated upon phosphorylation.
MATERIALS AND METHODS
Human subjects. Study subjects were recruited among patients diagnosed
with AS and/or followed at the outpatient Rheumatology Clinic of the
University Hospital of Ioannina, Greece. Forty patients were evaluated.
Patients diagnosed with AS should fulfill the 1984 modified New York
criteria36. AS disease activity was measured by the Bath Ankylosing
Spondylitis Disease Activity Index (BASDAI)37. A BASDAI score < 4 is
considered inactive disease. Exclusion criteria were hereditary dyslipidemia,
autoimmune conditions not pertinent to AS, active infection at the time of
the assessment, liver or renal disease, malignancy, alcohol abuse, pregnancy,
and lactation. Five patients were excluded — 2 for liver steatosis, 2 for renal
insufficiency, and 1 for alcohol abuse. All patients were under anti-tumor
necrosis factor-α (anti-TNF-α) therapy, while 6 of them were also taking
disease-modifying antirheumatic drugs (DMARD) and 1 also received
steroids. Controls were recruited who matched patients for age and sex and
met the above exclusion criteria. Subjects were defined as having CVD if
they had documented history of CVD events, such as myocardial infarction,
unstable angina, or cerebrovascular accident. In addition, subjects having a
body mass index (BMI) > 30 were classified as obese. The study subjects’
informed consent and approval from the institution’s ethical committee were
obtained.
Serum lipid and apolipoprotein concentrations. Serum was obtained from
blood samples after an overnight fast and stored at –80°C until use. Total
cholesterol, triglycerides, and apoA-I concentrations were determined using
the commercially available reagents Cholesterol LS (Labkit Chemelex SA),
Infinity triglycerides (Thermo), and ApoA1 (Labkit Chemelex SA),
according to the manufacturer’s instructions. HDL-C was determined in
apoB-depleted serum using the Cholesterol LS reagent. LDL cholesterol
(LDL-C) was calculated using the Friedewald equation38.
HDL preparation. The HDL-containing supernatant (apoB-depleted serum)
from subjects’ serum was isolated by the dextran-Mg2+ method as
described39.
DCF assay. The antioxidant capacity of HDL, prepared by the dextran-Mg2+
method, was tested in the presence or absence of oxidized (ox) LDL by the
DCF assay as described31 with some modifications32. DCF-DA (2,7 dichlorofluorescein diacetate; Molecular Probes/Invitrogen) was dissolved in fresh
methanol at 2.0 mg/ml and incubated at room temperature in the dark for 20
min, resulting in the release of DCF. Upon interaction with oxidants, DCF
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Gkolfinopoulou, et al: Dysfunctional HDL in AS
1653
is oxidized to fluorescent DCF. To compare the HDL antioxidant properties
of affected and unaffected subjects, we normalized for differences in HDL-C
concentration. HDL (final concentration 50 μg cholesterol/ml) in the
presence or absence of oxLDL (final concentration 100 μg cholesterol/ml)
was added into a black 96-well plate in a final volume of 100 μl. The plate
was incubated at 37°C on a rotator for 1 h in the dark. At the end of this
incubation period, 10 μl of DCF solution (0.2 mg/ml) was added to each
well, mixed, and incubated for an additional 2 h at 37°C with rotation in the
dark. Fluorescence was measured with a plate reader (Fluo-Star Galaxy,
BMG LabTech) at an excitation wavelength of 465 nm and an emission
wavelength of 535 nm. All assays were performed in duplicate.
Measurement of PON1 activity. PON1 activity in HDL, prepared by the
dextran-Mg2+ method, was determined using paraoxon as substrate32,40.
Briefly, the assays were performed in a final volume of 250 µl containing 5
µl of HDL, 5.61 mmol/l paraoxon (paraoxon-ethyl, Sigma Aldrich), 2
mmol/l CaCl2, and 100 mmol/l Tris–HCl, pH 8.0. The rate of p-nitrophenol
formed by the hydrolysis of paraoxon was measured by monitoring the
increase in absorbance at 405 nm for 15 min at room temperature in a
microplate spectrophotometer. PON1 activity was expressed as units per liter
of HDL samples. One unit is the activity that catalyzes the formation of 1
µmol p-nitrophenol per minute.
Measurement of MPO activity. The activity of MPO in HDL, prepared by
the dextran-Mg2+ method, was determined as described16 using the
InnoZyme MPO activity assay kit (EMD Chemicals), according to the
manufacturer’s instructions. Briefly, 80 µl of HDL were added to a 96-well
plate with an immobilized polyclonal antibody specific for human MPO.
Activity of captured MPO was measured using a detection reagent
containing tetramethyl benzidine (TMB) and hydrogen peroxide. Following
color development, the reaction was stopped with sulphuric acid and the
absorbance of the oxidized TMB detected at 450 nm.
Cellular cholesterol efflux assay. The cholesterol efflux capacity of HDL
was quantified as described34,41. To begin, J774 mouse macrophages plated
in 48-well plates were labeled with 0.2 ml of labeling medium [0.25 µCi/ml
4(14C)cholesterol in Dulbecco modified Eagle’s medium DMEM (high
glucose) supplemented with 0.2% (w/v) BSA]. Following 24 h of labeling
and washing, cells were equilibrated for 24 h with 0.3 mM
8-(4-chlorophenylthio)- cyclic adenosine monophosphate (cAMP) in 0.2 ml
of DMEM (high glucose) supplemented with 0.2% (w/v) BSA. Subsequently, efflux media containing 2% v/v HDL, prepared by the dextran-Mg2+
method, in DMEM (high glucose) were added for 4 h. At the end of the
incubation, the supernatants were collected and the cells were lysed in 200
μl of lysis buffer (PBS containing 1% (v/v) Triton X-100) for 30 min at room
temperature by gentle shaking. The radioactivity in 50 μl of the supernatant
and 100 μl of cell lysate was determined by liquid scintillation counting. The
percentage of secreted (14C)cholesterol was calculated by dividing the
medium-derived counts by the sum of the total counts present in the culture
medium and the cell lysate. All assays were performed in duplicate. To
correct for plate-to-plate and day-to-day variations, HDL samples from the
same 3 control subjects were included on each plate.
Akt kinase activation assay. The human umbilical vein endothelial
cell–derived line EA.hy926 (300,000 cells/well in 6-well plates) was
cultured in DMEM (10% fetal bovine serum, 2% pen-strep) to 80%
confluence. Cells were starved for 4 h and then treated in the presence or
absence of HDL, prepared by the dextran-Mg2+ method, at a final concentration of 40 µg apoA-I/ml for 20 min42. Western blot assays were performed
using antibodies for phospho-Akt (Ser473) or total Akt (Cell Signaling).
Image quantitation was performed using the ChemiDoc XRS+ Gel Imaging
System (Bio-Rad) and the Image Lab software (Bio-Rad).
Statistical analysis. Statistical analyses were performed using GraphPad
Prism 5 (GraphPad Software) and IBM SPSS Statistics version 22 (IBM
Corp.) software. All data presented are mean ± SD, unless otherwise
indicated. Patient and control groups were compared using Student’s t test
for continuous variables and the chi-square test of association for categorical
variables. Where indicated, p was adjusted for sex, smoking, HTN, obesity,
diabetes mellitus, CVD, and statin use as covariates using general linear
model univariate analysis. Analysis of the distribution of the data by the
D’Agostino and Pearson omnibus K2 normality test showed that, except for
the values obtained from the DCF assay, all other data do not follow a normal
distribution. Therefore, correlations between variables were evaluated using
the Spearman’s correlation coefficient for nonparametric data. P values
< 0.05 were considered significant.
RESULTS
Study group characteristics. Thirty-five patients with AS
were compared to 35 age- and sex-matched controls for alterations in their HDL atheroprotective properties. On average,
patients had longstanding disease with low disease activity
(BASDAI < 4; Table 1). All patients received treatment with
anti-TNF-α drugs (infliximab), 6 also received DMARD
(methotrexate), and 1 also received methylprednisolone.
There was no statistical difference for C-reactive protein
values between patients with AS and controls. The demographic, lifestyle, and clinical characteristics of patients and
controls are summarized in Table 1. BMI and percentage of
smokers were similar in patients and controls. There were
more subjects using statins among patients, but this did not
reach statistical significance. Further, there was no significant
difference for the presence of CVD, HTN, diabetes, and
obesity between patients with AS and controls. Serum total
cholesterol, HDL-C, LDL-C, and apoA-I concentrations did
not differ statistically between patients with AS and controls.
Serum triglyceride levels were lower in patients with AS
compared to controls. Similar lipid profiles between patients
with AS with inactive disease and controls have been
reported43. Disease-related treatment followed by improvement of disease activity has been shown to result in the
improvement of lipid profile of patients with AS44,45,46.
Antioxidant capacity of HDL. HDL samples were assessed
for their antioxidant properties using the DCF assay31,32. In
the absence of oxLDL, HDL (used at equal HDL-C concentration) from both patients and controls produced similar
fluorescence signal levels, indicating comparable oxidation
status. However, in the presence of oxLDL we saw a marked
increase in fluorescence signal when using HDL from
patients with AS compared to HDL from the control group,
indicating that the capacity of HDL to suppress the oxidative
potential of oxLDL is reduced in patients with AS (p
< 0.0001; Figure 1). Similar results were obtained when the
values for patients with AS and controls who were under
statin treatment were excluded (p < 0.0001).
HDL-mediated cellular cholesterol efflux. Measurement of
cholesterol efflux from macrophages using 2% apoB-depleted
serum showed that HDL from patients with AS had decreased
capacity to promote cholesterol efflux compared to HDL
from controls (p < 0.0001; Figure 2A). The cholesterol efflux
capacity of HDL in patients with AS remained decreased
compared with controls (p < 0.005), even when cholesterol
efflux values were normalized for HDL-C levels (Figure 2B).
The cholesterol efflux capacity of HDL in patients with AS
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1654
The Journal of Rheumatology 2015; 42:9; doi:10.3899/jrheum.141532
Table 1. Clinical characteristics, lipids, and lipoproteins of patients with AS and controls. Values are mean ± SD
unless otherwise indicated.
Age, yrs
Men, n (%)
Disease duration, yrs
BMI, kg/m2
CRP, mg/dl
BASDAI (0–10)
Anti-TNF-α use, n (%)
DMARD use, n (%)
Steroid use, n (%)
Current smokers, n (%)
CVD, n (%)
HTN, n (%)
DM, n (%)
Obesity, n (%)
Statin use, n (%)
Total cholesterol, mg/dl
HDL-C, mg/dl
LDL-C, mg/dl
Triglycerides, mg/dl
apoA-I, mg/dl
Patients, n = 35
Controls, n = 35
p*
46.1 ± 13.3
30 (86)
19.0 ± 11.1
24.9 ± 4.8
4.9 ± 3.2
3.2 ± 0.7
35 (100)
6 (17)
1 (3)
13 (37)
1 (3)
8 (23)
1 (3)
5 (14)
9 (26)
170.1 ± 38.9
49.9 ± 17.2
105.4 ± 40.2
89.1 ± 45.6
144.1 ± 49.8
45.3 ± 12.6
26 (74)
—
24.7 ± 2.3
3.7 ± 3.0
—
—
—
—
12 (34)
1 (3)
3 (9)
0
1 (3)
3(9)
182.5 ± 49.6
49.3 ± 13.7
110.3 ± 48.8
114.8 ± 54.1
165.7 ± 42.0
0.840
0.232
p (adjusted)**
0.806
0.110
0.803
1.000
0.101
0.314
0.088
0.057
0.249
0.855
0.649
0.035
0.054
0.232
0.627
0.491
0.005***
0.202
*p calculated by Student’s t test for continuous variables and chi-square test of association for categorical variables.
**p calculated by general linear model univariate analysis including sex, smoking, HTN, obesity, DM, CVD, and
statin use as covariates. *** Adjustment for each covariant independently of the others resulted in p values between
0.016 and 0.047. AS: ankylosing spondylitis; BMI: body mass index; CRP: C-reactive protein; BASDAI: Bath
Ankylosing Spondylitis Disease Activity Index; anti-TNF-α: anti-tumor necrosis factor-α; DMARD:
disease-modifying antirheumatic drugs; CVD: cardiovascular disease; DM: diabetes mellitus; HTN: hypertension;
HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; apoA-I: apolipoprotein A-I.
Figure 1. HDL antioxidative capacity in patients with AS and controls. The
fluorescence intensity resulting from oxidation of DCFH by test HDL
(50 μg cholesterol/ml), prepared by the dextran-Mg2+ method, in the
presence or absence of oxLDL (100 μg cholesterol/ml) was measured in a
spectrofluorometer as described. ***p < 0.0001. AS: ankylosing spondylitis;
C: controls; P: patients; AU: arbitrary units; HDL: high-density lipoprotein;
DCF: dichlorofluorescein; oxLDL: oxidized low-density lipoprotein.
was also lower compared to controls when the values for
patients with AS and controls using statins were excluded
(p < 0.0001 and p < 0.05 without or with normalization for
HDL-C levels, respectively).
PON1 and MPO activities of HDL. Measurement of PON1
and MPO activity using equal HDL volume for each sample
showed that the HDL-associated PON1 activity was significantly lower (p < 0.05) and the HDL-associated MPO activity
significantly higher (p < 0.05) in patients with AS compared
to controls (Figure 3A, C). Normalization of PON1 and MPO
activities for HDL-C levels showed that HDL-associated
PON1 activity was also significantly lower (p < 0.05) and
MPO activity significantly higher (p < 0.05) in patients with
AS compared to controls (Figure 3B, D). Similar results were
obtained when the values for patients with AS and controls
who were under statin treatment were excluded (p < 0.05 for
PON1 activity with or without normalization for HDL-C
levels; p < 0.05 for MPO activity without normalization for
HDL-C levels, and p < 0.005 for MPO activity with normalization for HDL-C levels).
When all measurements were adjusted for equal HDL-C
levels, the relationship between HDL-associated PON1 or
MPO activity and the antioxidant or cholesterol efflux
capacity of HDL from patients with AS was examined by
Spearman’s rank correlation. While there was no correlation
between PON1 activity and antioxidant or cholesterol efflux
capacity, neither between MPO activity and cholesterol efflux
capacity, the analysis showed that higher MPO activity correlated positively with worse antioxidant function of HDL from
patients with AS (r 0.359, p = 0.020).
Personal non-commercial use only. The Journal of Rheumatology Copyright © 2015. All rights reserved.
Gkolfinopoulou, et al: Dysfunctional HDL in AS
1655
Figure 2. HDL-mediated cholesterol efflux in patients with AS and controls. The capacity of HDL (2% v/v),
prepared by the dextran-Mg2+ method, to promote total cholesterol efflux from J774 mouse macrophages treated
with chlorophenylthio-cAMP, was measured as described. All assays were performed in duplicate. A. Values are
expressed as % cholesterol efflux of total cell cholesterol. B. Values are expressed as % cholesterol efflux of total
cell cholesterol divided by HDL-C concentration in mg/dl. **p < 0.005; ***p < 0.0001. AS: ankylosing
spondylitis; C: controls; P: patients; HDL: high-density lipoprotein; HDL-C: HDL cholesterol; cAMP: cyclic
adenosine monophosphate.
A recent study proposed that the MPO/PON1 ratio could
be a useful marker for coronary artery disease (CAD) risk
assessment through modulation of HDL properties47. As
shown in Figure 3E, HDL-associated MPO/PON1 ratio is
significantly higher for patients with AS compared to controls
(p < 0.05). Exclusion of values for patients with AS and
controls that were using statins resulted in similar results
(p < 0.05).
Akt kinase-activating properties of HDL. Incubation of
endothelial cells with HDL (used at equal HDL-apoA-I
concentration) from patients with AS resulted in a reduced
ratio of phosphorylated (p) Akt/Akt compared to cells
incubated with HDL from controls (Figure 4A), indicating
that HDL from patients with AS has reduced capacity (p <
0.005) to promote signaling events through Akt activation.
Similar results were obtained after normalization for HDL-C
levels (p < 0.001; Figure 4B) and after exclusion of values
for patients with AS and controls that were using statins (p <
0.001 for values without normalization for HDL-C levels and
p < 0.01 for values after normalization for HDL-C levels).
The pAkt/Akt ratio in endothelial cells after treatment with
HDL from patients with AS showed an inverse correlation
with the HDL-associated MPO/PON1 ratio in patients with
AS (r -0.294, p = 0.046). In addition, the pAkt/Akt ratio was
positively correlated with the cholesterol efflux capacity of
HDL from patients with AS, after normalization for HDL-C
levels (r 0.354, p = 0.020). A positive and stronger correlation
between the pAkt/Akt ratio and the cholesterol efflux
capacity of HDL was also observed for control subjects
(r 0.520, p = 0.0007).
DISCUSSION
Autoimmune rheumatic diseases have been associated with
atherosclerosis and increased risk of cardiovascular morbidity
and mortality48. The impairment of HDL atheroprotective
properties as a mechanism that leads to increased atherosclerotic risk has been studied for the rheumatic diseases RA and
SLE14,15,16,17. Extension of these conclusions to AS should,
however, be done with caution because of significant differences in the pathology among these diseases. Specifically,
RA and SLE are characterized by the presence of many
autoantibodies that may cause systemic inflammation, a
pathology that is absent from AS29,30. Because systemic
inflammation could be the reason behind the impairment of
HDL-mediated atheroprotection in RA and SLE, this issue
has to be addressed separately in AS. Further, earlier studies
reported increased levels of HDL-associated serum amyloid
A and reduced plasma PON1 activity in patients with
active AS, suggesting impaired HDL antiatherogenic
functions45,49,50.
In our current study, we show that HDL atheroprotective
properties, such as cholesterol efflux ability and antioxidant
capacity, are also impaired in patients with AS. Moreover,
the atheroprotective properties of HDL in patients with AS
are impaired despite the fact that all patients with AS had
received disease-related treatment and the disease was clinically under control. This suggests that changes in the
functions of HDL can occur even in the absence of marked
systemic inflammation and remain persistent even in clinically managed disease.
Differences between patients with AS and controls were,
in all cases, unaffected by normalization for HDL-C levels,
confirming that our results reflect differences in HDL composition and functionality. Specifically, we find that despite
normal serum HDL-cholesterol and apoA-I levels, patients
with AS have HDL with (1) reduced capacity to promote
cholesterol efflux from macrophages, (2) impaired antioxidant properties, (3) decreased PON1 activity, (4) increased
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1656
The Journal of Rheumatology 2015; 42:9; doi:10.3899/jrheum.141532
Figure 3. HDL-associated PON1 and MPO activity in patients with AS and controls. HDL-associated
PON1 (A, B) and MPO (C, D) activity was measured using 5 µl and 80 µl of HDL, respectively, prepared
by the dextran-Mg2+ method, as described. A. Values are expressed as HDL-associated PON1 activity in
u/l. B. Values are expressed as HDL-associated PON1 activity in u/l divided by HDL-C concentration in
mg/dl. C. Values are expressed as HDL-associated MPO activity in ng/ml. D. Values are expressed as
HDL-associated MPO activity in ng/ml divided by HDL-C concentration in mg/dl. E. HDL-associated
MPO/PON1 ratio. *p < 0.05. AS: ankylosing spondylitis; C: controls; P: patients; HDL: high-density
lipoprotein; HDL-C: HDL cholesterol; PON1: paraoxonase-1; MPO: myeloperoxidase.
MPO activity, and (5) impaired endothelial Akt kinase
activating properties. The cholesterol efflux capacity of HDL
from macrophages was demonstrated to have a strong inverse
association with carotid IMT and the likelihood of angiographic CAD34 and more recently to be inversely associated
with the incidence of cardiovascular events in a popula-
tion-based cohort33. The proinflammatory/antiinflammatory
properties of HDL were shown to distinguish patients with
CVD or CVD equivalents from control subjects better than
HDL cholesterol51 and were associated with an increased
prevalence of carotid plaques and with a higher IMT in
patients with SLE52. Therefore, the impairment of cholesterol
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Gkolfinopoulou, et al: Dysfunctional HDL in AS
1657
Figure 4. Activation of Akt kinase by HDL from patients with AS and controls. A. The property of HDL (40 μg
apoA-I/ml), prepared by the dextran-Mg2+ method, to activate the Akt kinase in HUVEC-derived line EA.hy926
was measured as described. Cellular pAkt (Ser473) and total Akt levels were measured by immunoblotting (a
representative set of images is shown in the upper panel). Western blots were scanned and quantified by Image
Lab software (lower panel). The normalized levels of pAkt against total Akt in the treated cells are shown as fold
activation relative to the non-treated cells. B. Values are expressed as levels of pAkt against total Akt in the treated
cells with HDL, calculated as fold activation relative to the non-treated cells, divided by the HDL-C amount in
µg for each sample. **p < 0.005; ***p < 0.001. C: controls; P: patients; HDL: high-density lipoprotein; HDL-C:
HDL cholesterol; AS: ankylosing spondylitis; HUVEC: human umbilical vein endothelial cell; apoA-I:
apolipoprotein A-I; pAkt: phosphorylated Akt.
efflux and antioxidant capacity of HLD in patients with AS
may in part underlie the increased CVD-associated mortality
for this patient group.
HDL can turn from antiinflammatory to proinflammatory
during periods of acute or chronic inflammation because of
changes in HDL proteomic and lipidomic composition20,21.
Changes in proteomic composition can lead to decreased
PON1 activity and increased MPO activity19,27. The latter
enzyme also affects HDL cholesterol efflux capacity26,27.
Both PON1 and MPO activities have been linked to cardiovascular risk23,25. In our study, we determined lower
HDL-associated PON1 activity and higher HDL-associated
MPO activity in patients with AS compared to controls. None
of the 2 enzyme activities were correlated with the cholesterol
efflux capacity of HDL from patients with AS, suggesting
that other changes in HDL composition or other factors are
responsible for the reduced cholesterol efflux capacity of
HDL in these patients. Regardless, the HDL-associated MPO
activity was found to associate with the impairment of the
antioxidant capacity of HDL from patients with AS, similarly
to what has been previously shown for patients with RA15.
A previous study suggested that in patients with coronary
artery disease the reduced HDL-associated PON1 activity led
to the reduction of endothelial Akt phosphorylation at Ser473,
inhibition of eNOS-activation, and subsequent loss of the
endothelial antiinflammatory and endothelial repair-stimulating effects of HDL22. Another study demonstrated that
MPO-oxidized HDL results in lower Akt phosphorylation in
endothelial cells, as well as reduced endothelial repair in
mice53. HDL from patients with AS showed reduced capacity
for endothelial Akt phosphorylation at Ser473 compared to
controls. This reduction was not correlated to HDL-associated PON1 or MPO activity when all measurements were
adjusted for equal HDL-C levels. However, it was correlated
to the HDL-associated MPO/PON1 ratio, indicating a
combined effect of changes in HDL composition on
HDL-mediated endothelial Akt phosphorylation and subsequent signaling. This is consistent with a recent study that
proposed the serum MPO/PON1 ratio as a potential indicator
of dysfunctional HDL47.
The pAkt/Akt ratio in endothelial cells after treatment with
HDL showed a positive correlation with the HDL-mediated
cholesterol efflux capacity in controls and a weaker, but
statistically significant, correlation in patients with AS. This
finding may suggest that in endothelial cells, the efflux of
cholesterol can activate signaling pathways and that
impairment of HDL cholesterol efflux capacity also affects
HDL-mediated signaling. Further studies are needed to
elucidate the interactions between HDL-mediated signaling
and cholesterol efflux pathways.
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1658
The Journal of Rheumatology 2015; 42:9; doi:10.3899/jrheum.141532
A limitation of our present study is that we have no
pretreatment data for the subjects. Such data would allow the
examination of the relationship between inflammation or
autoimmunity and HDL atheroprotective functions in patients
with AS and whether treatment improves the HDL atheroprotective functions in these patients. Such analyses will be
approached in future studies.
Overall, this is, to our knowledge, the first study to
evaluate antioxidant, cholesterol efflux, and signaling
capacity of HDL from patients with AS. The attenuation of
HDL properties observed in patients with AS suggests a
molecular link between AS and CVD. The fact that the disease
was substantially controlled by therapy in our patient group
suggests that the impairments in HDL atheroprotective
properties observed in patients with AS are not dependent on
a persistent highly active inflammatory status. It is possible
that specific mediators of chronic immune responses or other
genetic factors can influence HDL functions in a persistent
manner and do not allow easy recovery to normal levels when
the disease is under control. Our results provide novel insight
into the increased cardiovascular risk observed in AS2,3,6,7,8,
suggesting that AS affects HDL composition and function and
this may result in increased atherosclerosis and cardiovascular
risk. Therefore, the functional integrity of HDL may be an
important, still unexplored longterm prognostic marker to
evaluate risk for atherosclerosis and CVD in patients with AS.
REFERENCES
1. Braun J, Sieper J. Ankylosing spondylitis. Lancet 2007;369:1379-90.
2. Mathieu S, Gossec L, Dougados M, Soubrier M. Cardiovascular
profile in ankylosing spondylitis: a systematic review and
meta-analysis. Arthritis Care Res 2011;63:557-63.
3. Papagoras C, Voulgari PV, Drosos AA. Atherosclerosis and
cardiovascular disease in the spondyloarthritides, particularly
ankylosing spondylitis and psoriatic arthritis. Clin Exp Rheumatol
2013;31:612-20.
4. Han C, Robinson DW Jr., Hackett MV, Paramore LC, Fraeman KH,
Bala MV. Cardiovascular disease and risk factors in patients with
rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis.
J Rheumatol 2006;33:2167-72.
5. Szabo SM, Levy AR, Rao SR, Kirbach SE, Lacaille D, Cifaldi M, et
al. Increased risk of cardiovascular and cerebrovascular diseases in
individuals with ankylosing spondylitis: a population-based study.
Arthritis Rheum 2011;63:3294-304.
6. Lautermann D, Braun J. Ankylosing spondylitis—cardiac
manifestations. Clin Exp Rheumatol 2002;20:S11-5.
7. Sari I, Okan T, Akar S, Cece H, Altay C, Secil M, et al. Impaired
endothelial function in patients with ankylosing spondylitis.
Rheumatology 2006;45:283-6.
8. Peters MJ, van Eijk IC, Smulders YM, Serne E, Dijkmans BA, van
der Horst-Bruinsma IE, et al. Signs of accelerated preclinical
atherosclerosis in patients with ankylosing spondylitis. J Rheumatol
2010;37:161-6.
9. Gordon T, Castelli WP, Hjortland MC, Kannel WB, Dawber TR.
High density lipoprotein as a protective factor against coronary
heart disease. The Framingham Study. Am J Med 1977;62:707-14.
10. Di Angelantonio E, Sarwar N, Perry P, Kaptoge S, Ray KK,
Thompson A, et al. Major lipids, apolipoproteins, and risk of
vascular disease. JAMA 2009;302:1993-20...