ORIGINAL ARTICLE

The relationship between plasma homocysteine level and differenttreatment modalities in patients with ankylosing spondylitis

Erhan Capkin • Murat Karkucak • Ayse Akyuz •

Ahmet Alver • Aysegul Kucukali Turkyilmaz •

Elif Zengin

Received: 3 August 2010 / Accepted: 22 May 2011 / Published online: 5 June 2011

� Springer-Verlag 2011

Abstract To determine plasma homocysteine levels in

ankylosing spondylitis (AS) and their correlation with

disease activity measurements. To examine the effects of

different treatment modalities on homocysteine levels. One

hundred eight patients diagnosed with AS according to

New York criteria and 65 healthy individuals matched in

terms of age and gender were enrolled in the study. Patients

were given detailed physical examinations. The Bath AS

Disease Activity Index (BASDAI) was used for disease

activity, the Bath AS Metrology Index (BASMI) for spinal

mobility, the Bath AS Functional Index (BASFI) to

determine functional status and the Bath AS Radiological

Index (BASRI) for radiological analysis. Sedimentation

rate (ESR) and C reactive protein (CRP) levels, acute phase

reactants, were measured. Plasma homocysteine levels,

serum interleukin (IL) -6 and serum tumor necrosis factor-

a (TNF- a) levels were investigated using the enzyme-

linked immunosorbent assay (ELISA) method. Plasma

homocysteine levels in AS patients were statistically sig-

nificantly lower compared with those in the healthy control

group (P = 0.0001). There was no significant difference

among sub-groups established on the basis of medical

treatments and disease activity (BASDAI B4 and [4). No

statistically significant correlation was determined between

homocysteine level and disease activity parameters,

radiological score and functional indices. A significant

negative correlation was, however, established between

age and homocysteine level in the AS group (P \ 0.05,

r = -0.426). Plasma homocysteine was lower in AS

patients compared with the control group. This is not

correlated with disease activation and medical treatment

employed.

Keywords Ankylosing spondylitis � Homocysteine �TNF- a

Introduction

Ankylosing spondylitis (AS) is a systemic, chronic and

inflammatory rheumatismal disease of uncertain etiology

particularly affecting the spine and sacroiliac joints [1].

The disease is more frequent in males and generally begins

in early adulthood. It leads to serious disabilities in at least

1/3 of cases. Symptoms such as pain, fatigue, sleep

impairment and restricted mobility observed throughout

the AS course have a severe impact on daily life [2]. AS

affects other systems as well as the musculoskeletal system

(such as the cardiovascular system) [1, 3].

E. Capkin (&) � M. Karkucak � E. Zengin

Division of Rheumatology, Department of Physical Medicine

and Rehabilitation, Karadeniz Technical University, Medical

School, Farabi Hospital, 61080 Trabzon, Turkey

e-mail: drcapkin@yahoo.com

M. Karkucak

e-mail: muratkarkucak@yahoo.com

E. Zengin

e-mail: elifzengin@yahoo.com

A. Akyuz � A. Alver

Department of Biochemistry, Karadeniz Technical University,

Farabi Hospital, 61080 Trabzon, Turkey

e-mail: ayseakyuz@yahoo.com

A. Alver

e-mail: ahmetalver@yahoo.com

A. K. Turkyilmaz

Physical Medicine and Rehabilitation, Rize Education

and Research Hospital, 53080 Rize, Turkey

e-mail: aysegulyilmaz@yahoo.com

123

Rheumatol Int (2012) 32:2349–2353

DOI 10.1007/s00296-011-1972-1

Homocysteine is a non-essential amino acid containing

sulfur forming with methionine demethylation [4]. Epide-

miological studies have emphasized the correlation between

a high total homocysteine concentration in the blood and

cerebrovascular system diseases, especially the cardiovas-

cular system. A rise in plasma homocysteine level has been

shown to be an independent risk factor for the development

of cardiovascular disease. The literature contains studies

investigating homocysteine levels in systemic rheumatis-

mal diseases, and particularly rheumatoid arthritis [5–10].

Various factors have been suggested in the development of

increased cardiovascular side-effects in rheumatismal dis-

eases, one of which may be homocysteine.

The aim of this study was to determine homocysteine

levels in AS patients and investigate the correlation

between disease activation and medical treatments.

Materials and methods

One hundred eight patients diagnosed with AS according

to Modified New York Criteria and monitored by the

Karadeniz Technical University Medical Faculty Physical

Medicine and Rehabilitation Department clinic were

enrolled [11]. Patients were given detailed physical

examinations and divided into three sub-groups according

to medical treatment received. Group 1 (n = 47) consisted

of patients administered anti-tumor necrosis factor-a (TNF-

a), Group 2 (n = 37) of patients administered only non-

steroidal anti-inflammatory drugs (NSAID) and group 3

(n = 22) of patients using sulfasalazine (SS) and NSAID.

Patients receiving anti TNF-a treatment consisted of

patients determined with improvement in at least 2 units

from the Bath AS disease activity index (BASDAI) at

3-month evaluation. The control group consisted of 65

healthy adults chosen from patient relatives applying to the

same clinic and matched in terms of age and gender.

Patients with diabetes mellitus, hypertension, congestive

coronary insufficiency or atherosclerotic heart disease, who

had previously suffered myocardial infarct and cerebro-

vascular disease and with chronic systemic diseases such as

peripheral arterial disease and chronic steroid users were

excluded from the study.

Clinical and biochemical assessments

Patients were given detailed physical examinations. Disease

activity was evaluated using BASDI, spinal mobility using

the Bath AS metrology index (BASMI), functional status

using the Bath AS functional index (BASFI) and radio-

logical examination using the Bath AS radiological indices

(BASRI). The validity, reliability and repeatability and

sensitivity to change of the indices used have been

established [12, 13]. Patients with BASDAI values C4 were

regarded as active [14]. Serum and plasma specimens

obtained from blood taken from individuals in the patient

and control groups after at least 12 h of fasting were kept at

-80�C until biochemical measurements were performed.

Serum C reactive protein (CRP) levels were determined by

immunonephelometric measurement (Dade-Behring II).

Plasma homocysteine levels (Axis homocysteine, EIA Kit

FHCY100), serum TNF-a (Biosource, KAP1751) and

interleukin (IL) -6 (Biosource, KAP1261) levels were

measured using ELISA.

Statistical analysis

Compatibility with normal distribution of data obtained by

measurement was investigated using the Kolmogorov–

Smirnov test. Student’s t test was used in comparing two-

way distributed measurement data and the Mann–Whitney

U test for data not normally distributed. In three-way

comparisons, ANOVA (post-hoc Tukey) was used since

this was compatible with normal distribution. The chi-

square test was used in the comparison of qualitative data.

Pearson correlation analysis was used in the correlation of

normally distributed parameters in the analysis of homo-

cysteine levels and disease, and Spearman correlation

analysis for those not normally distributed. P \ 0.05 was

regarded as significant.

Informed consent forms were received from patient and

healthy controls. Ethical Committee approval was obtained

prior to the study.

Results

One hundred seventy-three individuals (108 AS patients

and 65 controls) were enrolled. Mean age of the AS

patients was 36.4 ± 11.2 years, and that of the controls

38.2 ± 13.0 (P = 0.330). Eighty-eight of the AS patients

were men, and 20 women; 49 of the control group were

men and 16 women. There was no statistical difference

between the groups in terms of gender distribution (P = 0.

222). Patients’ symptom duration was 8.4 ± 5.6 years, and

length of first diagnosis 4.2 ± 4.4 years. Patients’ chest

expansion was 3.6 ± 1.6 cm and Schober’s test

12.9 ± 1.6 cm. AS patients’ clinical characteristics and the

parameters used in disease evaluation are shown in

Table 1.

When AS patients and the control group were compared

in terms of plasma homocysteine levels, these were statis-

tically significantly lower in the former (P = 0.0001).

Patients were divided into three sub-groups on the basis of

medical treatment. No statistically significant differences in

ESR, CRP, IL-6 or homocysteine levels were determined

2350 Rheumatol Int (2012) 32:2349–2353

123

among these sub-groups (P = 0.486, P = 0.258, P = 0.342

and P = 0.575, respectively). There was, however, a sig-

nificant difference in TNF-a values (P = 0.037). We clas-

sified disease activation into BASDAI \4 (n = 50) and

BASDAI C4 (n = 49). When groups were evaluated in

terms of ESR, CRP, TNF-a, IL-6 and homocysteine levels,

there was a difference in ESR and CRP but no difference in

terms of other parameters (P = 0. 0001, P = 0. 008, P = 0.

773, P = 0. 129, and P = 0.604, respectively) (Table 2).

Correlation analysis revealed no significant correlation

between homocysteine levels and the disease activity

parameters BASDAI, ESR, CRP, TNF-a and IL-6. There

was no significant correlation between the BASMI, BASRI

and BASFI disease indices used in the study. There was

only a significant negative correlation between homocys-

teine and age in the AS group, but none in the control

group (P \ 0.05, r = -0.426) (Table 3).

Discussion

Plasma homocysteine levels were lower in the AS patients

compared to the control group. This was not correlated

with the clinical and laboratory parameters used in the

evaluation of AS patients.

Homocysteine is a non-essential amino acid, and one

that has been particularly investigated in the pathogenesis

of cardiovascular diseases. Blood levels may be influenced

by a number of factors, such as age, gender, cigarette

consumption, body mass index, folate and vitamin B12 [4].

Homocysteine levels have been shown to decrease as

serum folate and B12 levels rise [15]. Since disease mod-

ifying agents (DMARD) used in the treatment of inflam-

matory diseases have a particular effect on methotrexate

(MTX) folate metabolism, it has been reported that they

may cause a decrease in serum folate levels and a rise in

Table 1 Demographic, clinical

and in biochemical variables

data of patients with AS and

controls

BASDAI bath AS disease

activity index, BASMI bath AS

metroloji index, BASFI bath AS

functional index, BASRI bath

AS radiographic index, CRP C

reaktif protein, ESR eritrosit

sedimentation rate

AS (n = 108)

Mean ± Std

Control (n = 65)

Mean ± Std

P

Gender (M/F) 88/20 49/16 0.222

Age (years) 36.4 ± 11.2 38.2 ± 13.0 0.330

ESR (mm/sa) 26.5 ± 18.8 14.3 ± 11.5 0.000

CRP (mg/dl) 1.32 ± 1.08 0.3 ± 0.2 0.000

Homocysteine 18.9 ± 8.7 23.8 ± 5.8 0.000

TNF alpha (pg/ml) 15.4 ± 24.0 8.9 ± 6.1 0.046

IL-6 (pg/ml) 69.0 ± 262.7 35.2 ± 23.1 0.000

BASDAI 3.7 ± 1.6 –

BASRI 6.9 ± 2.6 –

BASMI 4.0 ± 2.1 –

Symptom duration (years) 8.4 ± 5.6 –

First diagnose (years) 4.2 ± 4.4 –

Schober test (cm) 12.9 ± 1.6 –

Chest ekspan (cm) 3.6 ± 1.3 –

Table 2 Comparison laboratory features according to medical treatment received and disease activity index

CRP (mg/dl)

mean ± Std

ESR (mm/sa)

mean ± Std

Homocysteine

(lmol/L)

mean ± Std

TNF-a (pg/ml)

mean ± Std

IL-6 (pg/ml)

mean ± Std

Medical treatment

Group I (n = 47) 1.5 ± 1.1 25.1 ± 17.1 17.9 ± 9.6 23 ± 3.3 50.9 ± 1.5

Group II (n = 37) 1.0 ± 0.8 27.7 ± 16.8 19.6 ± 7.5 9.4 ± 8.3 31.6 ± 3.2

Group III (n = 22) 1.1 ± 1.2 24.0 ± 25.9 21.0 ± 7.4 11.5 ± 1.1 19.6 ± 1.3

P 0.258 0.486 0.575 0.037 0.342

Disease activitiy

BASDAI \4 (n = 50) 1.1 ± 0.8 17.5 ± 14.3 19.2 ± 9.8 16.9 ± 1.7 28.3 ± 2.1

BASDAI C4 (n = 49) 1.5 ± 1.1 31.9 ± 17.7 18.5 ± 7.6 16.1 ± 3.1 24.7 ± 2.5

P 0.008 0.000 0.604 0.773 0.129

ESR eritrosit sedimentation rate, CRP C reaktif protein, BASDAI bath AS disease activity index

Group 1; Anti TNF-a, Group 2; NSAID, Group 3; Sulfasalazin ve NSAII

Rheumatol Int (2012) 32:2349–2353 2351

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homocysteine levels in patients using these drugs [16].

Folate reinforcement is used to reduce side-effect incidence

in RA patients using MTX. There is a possible increased

risk of cardiovascular disease developing in inflammatory

rheumatismal patients. Divecha et al. [17] described the

risk factors correlated with increased cardiovascular dis-

eases in AS and determined a positive correlation between

IL-6 and CRP levels and cardiovascular risk. Another study

reported a rise in cardiovascular diseases in spondyloar-

thritides. It emphasized that this increased risk was linked

to endothelial dysfunction and the development of athero-

sclerosis. The rise in inflammatory mediators, particularly

CRP, and changes in the lipid profile were indicated as

responsible [18].

Increased homocysteine levels are an independent risk

factor for the development of cardiovascular disease [4].

The increased risk of cardiovascular disease in rheumatis-

mal patients is thought to be correlated with homocysteine

level. In a study of homocysteine levels in AS patients, Wei

et al. [10] compared 103 AS patients with 10 healthy

controls. Homocysteine levels were higher than in the

control group. This difference was statistically significant

in the patients receiving DMARD treatment, while an

insignificant rise was determined in AS patients not using

DMARD. No difference in homocysteine levels was

determined between subject with active disease according

to BASDAI criteria (BASDAI [3) and those without. A

significant reduction in homocysteine levels was also

reported in patients receiving daily folate replacement in

another branch of the study. The authors concluded that AS

patients should be given daily folate replacement.

In our study, homocysteine levels were lower compared

with those in the control group. In contrast to Wei et al.,

none of our patients used MTX as a DMARD. The

important difference is that a great many of our patients

consisted of subjects receiving biological treatment.

Although homocysteine levels were not statistically sig-

nificant in the biological treatment group, they were lower

compared to the other drug groups. Although there are very

positive effects on disease activity scores in connection

with these agents that have been quite frequently used in

AS patients of late, the effects on the vascular system are

debatable. According to some authors, they have a positive

effect on cardiac risk, while others report no effect. But

these drugs probably have positive impacts since they

suppress inflammation very efficiently [19]. In our study,

homocysteine levels were higher in the group using NSAID

and SS compared to the biological treatment group. The

fact that homocysteine levels were significantly lower in

AS patients receiving biological treatment suggests that

these drugs may have a positive effect on the cardiovas-

cular system. However, there is a need for wide-ranging

studies to support this hypothesis, investigating homocys-

teine levels and cardiovascular function before and after

biological treatment. We think that our study can be

regarded as preparatory for such work.

In a study investigating factors influencing plasma

homocysteine levels, Aksoy et al. [4] reported that plasma

homocysteine levels were correlated with gender, age, the

consumption of cigarettes, fruit and vegetables, body mass

index, serum folate and vitamin B12. The reason for the

low homocysteine levels in AS patients in our study may

be linked to other parameters that may impact on homo-

cysteine levels.

We classified patients according to their BASDAI

scores, as inactive, \4, or active, C4. There was no sta-

tistically significant difference in homocysteine levels

between groups at analysis on the basis of BASDAI. In

addition, no significant correlation was determined

between homocysteine levels and parameters showing

disease activity (IL-6, CRP and ESR). The effect of

homocysteine on inflammation in AS may be debated in

the light of this finding. We identified no correlations

between BASRI, BASFI and BASMI scores and homo-

cysteine levels in our analyses. In conclusion, no correla-

tion was established between homocysteine activity and

functional status. A correlation was observed between

homocysteine levels and age.

Table 3 Correlations between the changes in biochemical variables and changes in clinical variables in patients with AS

Homocysteine Age BASDAI ESH CRP TNF-a IL-6

Homocysteine 1 -.426* -.131 .028 .036 .094 .009

Age -.426* 1 .008 .008 -.076 -.032 -.014

BASDAI -.131 .008 1 .447* .274* .056 -.024

ESR .028 .008 .447* 1 .642* .018 .031

CRP .036 -.076 .274* .642* 1 .177 .126

TNF-a .094 -.032 .056 .018 .177 1 .671*

IL-6 .009 -.014 -.024 .031 .126 .671* 1

ESR eritrosit sedimentation rate, CRP C reaktif protein, BASDAI bath AS disease activity index

Results are shown as the correlation coefficients calculated using Pearson’s correlation test. *P value 0.05 by Pearson’s correlation test

2352 Rheumatol Int (2012) 32:2349–2353

123

The lack of any analysis of AS patients included in the

study before and after medical treatment may be consid-

ered a limitation. However, we consider that the fact that

around half the AS patients received biological agents is

noteworthy in terms of evaluating the correlation between

biological agent treatments and homocysteine levels.

In conclusion, plasma homocysteine levels were lower

in AS patients. This is not correlated with medical treat-

ment or clinical and laboratory parameters. Homocysteine

levels were lower in the group receiving biological treat-

ment. Wide-ranging studies are needed to investigate the

role of homocysteine in the AS clinical process.

Conflict of interest None.

References

1. Van der Linden S (1997) Ankylosing spondylitis. In: Kelley N,

Ruddy S, Haris E, Sledge C (eds) Textbook of rheumatology. WB

Saunders Company, Philadelphia, pp 969–982

2. Danyan M, Guner A, Tuncer S et al (1999) Disability in anky-

losing spondylitis. Disabil Rehabil 21:74–79

3. Braun J, Khan MA, Sieper J (2000) Enthesitis and ankylosis in

spondyloarthropathy: what is the target of the immune response?

Ann Rheum Dis 59:985–994

4. Aksoy S, Geyikli I, Saygılı E (2006) Determinants of plasma

homocysteine levels in healthy people. Turk J Biochem 31(4):

175–181

5. Montalescot G (1996) Homocysteine: the new player in the field

of coronary risk. Heart 76:101–102

6. Graham I, Daly L, Refsum H et al (1997) Plasma homocysteine

as a risk factor for vascular disease. JAMA 277:1775–1781

7. Fallest-Strobl P, Koch D, Stein J et al (1997) Ho-mocysteine: a

new risk factor for atherosclerosis. Am Fam Phys 56:1607–1610

8. Brattstrom L, Hardebp E, Hultberg B (1984) Moderate homo-

cysteinemia a possible risk factor for arteriosclerotic cerebro-

vascular disease. Stroke 15:1012–1016

9. Taylor L, De Frang R, Harris E et al (1991) The association of

elevated plasma homocyst(e)ine with progression of symptomatic

peripheral arterial disease. J Vasc Surg 13:128–136

10. Wei J, Ming-Shiou J, Chen-Tung Y et al (2007) Plasma homo-

cysteine status in patients with ankylosing spondylitis. Clin

Rheumatol 26:739–742

11. Van der Linden S, Valkenburg H, Cats A (1984) Evaluation of

diagnostic criteria for ankylosing spondylitis: a proposal for modi-

fication of the New York criteria. Arthritis Rheum 27:361–368

12. Garrett S, Jenkinson T, Kennedy L et al (1994) A new approach

to defining disease status in ankylosing spondylitis: the bath

ankylosing spondylitis disease activity index. J Rheumatol

21:2286–2291

13. Jenkinson TR, Mallorie PA, Whitelock HC et al (1994) Defining

spinal mobility in ankylosing spondylitis (AS). The bath as

metrology index. J Rheumatol 21:1694–1698

14. Braun J, Pham T, Sieper J et al (2003) ASAS working group.

International ASAS consensus statement for the use of anti-

tumour necrosis factor agents in patients with ankylosing spon-

dylitis. Ann Rheum Dis 62:817–824

15. Jacques P, Bostom A, Wilson P et al (2001) Determinants of

plasma total homocysteine concentrations in the Framingham

offspring cohort. Am J Clin Nutr 73:613–621

16. Hornung N, Ellingsen T, Stengaard-Pedersen K et al (2004)

Folate, homocysteine, and cobalamin status in patients with

rheumatoid arthritis treated with methotrexate, and the effect of

low dose folic acid supplement. J Rheumatol 31(12):2374–2381

17. 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(2):171–176

18. Heeneman S, Daemen MJ (2007) Cardiovascular risks in

spondyloarthritides. Curr Opin Rheumatol 19(4):358–362

19. Szekanecz Z, Kerekes G, Soltesz P (2009) Vascular effects of

biologic agents in RA and spondyloarthropathies. Nat Rev

Rheumatol 5(12):677–684

Rheumatol Int (2012) 32:2349–2353 2353

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