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Rheumatology 2001; 40: 687-690
© 2001 British Society for Rheumatology

Original Papers

Hyperhomocysteinaemia in Behçet's disease

K. Aksu, N. Turgan1, F. Oksel, G. Keser, D. Özmen1, G. Kitapçioglu2, G. Gümüsdis, O. Bayindir1 and E. Doganavsargil

Division of Rheumatology, Department of Internal Medicine,
1 Department of Clinical Biochemistry and
2 Department of Public Health, Ege University School of Medicine, Bornova, Izmir, Turkey


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. Arterial and venous thrombosis are among the clinical features of Behçet's disease (BD), the pathogenesis of which is not completely understood. In this study, we investigated whether hyperhomocysteinaemia, being a well known risk factor for thrombosis, is also a contributive risk factor for the arterial and venous thrombosis of BD.

Methods. Eighty-four patients fulfilling the criteria of the International Study Group for Behçet's Disease (54 males, 30 females, mean age 36±9 yr) were enrolled. All the patients were carefully screened for a history of venous thrombosis and were separated into two groups with respect to thrombosis history. Thirty-six healthy individuals (23 males, 13 females), matched for age and sex with the BD group, were included as a negative control group. Patients were excluded if they had any condition that might affect plasma homocysteine concentration. As methotrexate (MTX) causes hyperhomocysteinaemia, we also included 29 rheumatoid arthritis patients (five males, 24 females) receiving MTX weekly. Fasting plasma homocysteine concentrations were measured by high-performance liquid chromatography. The data were analysed with the {chi}2 test and Student's t-test.

Results. The highest homocysteine concentrations were found in the MTX group (17.5±5.3 µmol/l). Mean plasma homocysteine concentrations in BD patients were significantly higher than in the healthy controls (11.5±5.3 vs 8.8±3.1 µmol/l, P<0.001). Among BD patients with a history of thrombosis, 20 of 31 (64%) had hyperhomocysteinaemia, and this was significantly higher than in those without thrombosis (9%). On the other hand, there was no significant difference between patients with non-thrombotic BD and healthy controls (P>0.05). In patients with thrombosis, we found no correlation between the duration of the post-thrombotic period and homocysteine concentration. Among all the variables investigated, only hyperhomocysteinaemia was found to be related to thrombosis.

Conclusion. Hyperhomocysteinaemia may be assumed to be an independent risk factor for venous thrombosis in BD. Unlike the factor V Leiden mutation, hyperhomocysteinaemia is a correctable risk factor. This finding might lead to new avenues in the prophylaxis of thrombosis in BD.

KEY WORDS: Behçet's disease, Homocysteine, Hyperhomocysteinaemia.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
Behçet's disease (BD) is a chronic relapsing systemic vasculitis, involving both arteries and veins of various sizes. Mucocutaneous, ocular, genital, articular, vascular, central nervous system and gastrointestinal involvements may occur during the course of BD. The vascular involvement is generally in the form of thrombophlebitis and deep-vein thrombosis, and arterial obstruction and aneurysms may also be seen to a lesser extent [1]. In previous studies investigating the causation of thrombosis in BD, the factor V Leiden mutation was the only specific molecular abnormality that was detected [2].

Homocysteine, derived from methionine, is a sulphur-containing essential amino acid. Hyperhomocysteinaemia may occur in the course of diabetes mellitus, hyperlipidaemia, end-stage renal failure, psoriasis and inflammatory bowel disease [3–7]. It may also occur due to vitamin B12 or folate deficiency [8] or secondary to treatment with some drugs, such as methotrexate (MTX) [9]. It is not only an independent risk factor for atherosclerosis [10] but is also a causative risk factor for vascular disease and thrombosis [11, 12]. Hyperhomocysteinaemia, whatever its cause, may be prevented by supplementation with vitamin B12, vitamin B6 and/or folate. Thus, it is a treatable risk factor for thrombosis and atherosclerosis.

The aim of this study was to investigate whether hyperhomocysteinaemia is a risk factor for the development of thrombosis in BD.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
Patients
One hundred and thirteen BD patients, fulfilling the criteria of the International Study Group for Behçet's Disease [13] and being followed up by Ege University Rheumatology Department between September 1998 and September 1999, were enrolled in this cross-sectional study. The exclusion criteria were vitamin B12/folate deficiency, hyperlipidaemia, diabetes mellitus, psoriasis, chronic hepatitis, chronic alcoholism, renal failure and pregnancy. Liver and renal function tests, fasting serum glucose, protein, lipid, vitamin B12 and folate and plasma homocysteine concentrations were measured and urinalysis was performed in all patients. All medications were stopped 10 days before homocysteine measurement. Fifteen patients with hyperlipidaemia, seven patients having a vitamin B12 concentration lower than the reference range (133–675 pmol/l) and two patients with renal disease were excluded. Five other patients, in whom drug withdrawal would not be ethical, were also excluded from the study. The remaining 84 BD patients (54 males, 30 females, mean age 36±9 yr, range 20–56 yr, disease duration 80±63 months) were included. These 84 patients were divided into two groups on the basis of deep-vein thrombosis in the past. Plasma homocysteine levels in the thrombotic group were compared with those of BD patients without a history of thrombosis. BD patients were also evaluated with respect to current clinical activity. At the time of the clinical assessment, patients were included in the active group if they had at least two of the following clinical findings: mouth ulcers, genital ulceration, active uveitis, recent arthritis, thrombophlebitis and large-vessel involvement.

Thirty-six healthy controls (23 males, 13 females), all matched with the BD patients for age (39±10 yr, range 22–56 yr) and sex, were included as the negative control group. Twenty-nine patients with rheumatoid arthritis (five males, 24 females), each receiving MTX at a weekly dose of 7.5–15 mg without folic acid supplementation, were also included as the positive control group. The MTX group was matched with the other groups for age (39±6 yr, range 23–51 yr) but not for sex. This group was used only to test the reliability of the method. All the participants were instructed not to take any vitamin supplements within 10 days before the measurements.

Measurements of homocysteine, vitamin B12 and folic acid
The blood samples were collected in ethylenediamine tetraacetate (EDTA)-containing tubes after 12 h of fasting; they were kept on ice in order to prevent homocysteine formation and leakage from erythrocytes, and were centrifuged immediately at 2000 g for 10 min at room temperature. Homocysteine was measured by high-performance liquid chromatography (LC10A; Shimadzu, Kyoto, Japan). The measurement was based on fluorometric detection [14]. The system consisted of a pump (LC10AD, Shimadzu), a fluorescence detector (RF10AXL, Shimadzu) and a C18 column (EC 150/4.6 Nucleosil 100-5 µm; Macherey-Nagel, Düren, Germany). Tri-n-butyl-phosphine in n,n-dimethyl formamide was used to reduce homocysteine disulphide. SBDF (7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonic acid) was used as a derivatization agent. Cysteamine hydrochloride was added as an internal standard. The within-day and inter-day reproducibilities of the method were 2.7 and 3.4% respectively.

Total cholesterol, triglycerides, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol were measured with an automatic analyser (Technicon Dax-48; Bayer Diagnostics, Toshiba, Japan). Serum vitamin B12 and folic acid were measured by chemiluminescence immunoassay (Access; Sanofi Diagnostics Pasteur, Marnes La Coquette, France), the reference intervals being 133–675 pmol/l for vitamin B12 and >6.8 nmol/l for folic acid.

Statistical analysis
SPSS for Windows version 8.0 was used to analyse the data. The data were normally distributed. In the statistical evaluation of the data, the independent samples t-test (Student's t-test) was used to compare the means of the two groups, and the {chi}2 test was used to investigate the relationship between two independent variables. Correlation analysis was performed with the Pearson correlation test. P<0.05 was accepted as significant (confidence interval=95%, {alpha}=0.05).


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
The mean plasma homocysteine concentrations in each study group are shown in Table 1Go. Homocysteine concentrations in the whole BD group were lower than in the patients receiving MTX (P<0.001), but higher than in the healthy controls (P<0.001). Plasma homocysteine concentrations of the BD patients in the thrombotic subgroup were significantly higher than those of the non-thrombotic subgroup (P<0.001). Twenty out of 31 (64%) patients in the thrombotic subgroup had hyperhomocysteinaemia and this was significantly higher than in those without thrombosis (9%) (P<0.005). However, the plasma homocysteine concentration in the thrombotic subgroup was not significantly different from that in the MTX group. There was also no significant difference in plasma homocysteine concentrations between the non-thrombotic subgroup and the healthy controls.


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  		TABLE 1. Plasma homocysteine concentrations

 
Twenty out of 25 (80%) patients with hyperhomocysteinaemia had thrombosis. We observed a strong relationship between hyperhomocysteinaemia and a history of thrombosis in the BD patients (P<0.001). Demographic data and vitamin B12 and folate concentrations of all groups are summarized in Table 2Go .


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  		TABLE 2. Demographic data and plasma vitamin B12 and folate concentrations in all groups (mean±S.D.)

 
In the BD group, 10 (12%) patients were accepted as clinically active. There was no correlation between the plasma homocysteine concentration and disease activity (P>0.05). The clinical features of BD patients are shown in Table 3Go .


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  		TABLE 3. Clinical features of BD patients

 
The plasma homocysteine concentrations in males, in both the BD group and the healthy controls, were significantly higher than in females (P<0.001). The 95th percentile of the homocysteine concentration in the healthy control group (13.81 µmol/l) was taken as the cut-off value. Cut-off values were calculated separately for male and female patients, being 13.86 and 10.54 µmol/l respectively. When we re-evaluated male BD patients according to sex-adjusted cut-off values, hyperhomocysteinaemia was shown to be a risk factor for thrombosis in these patients (P<0.001).

No statistically significant relationship was found between the thrombosis history and any of the serum lipid variables (total cholesterol, HDL cholesterol, LDL cholesterol and triglycerides).

Thirty-four (41%) patients in the BD group, four (14%) in the MTX group and 11 (30%) in the control group were smokers. There was no significant difference between smokers and non-smokers with respect to plasma homocysteine concentration in any of the groups (P>0.05). We found no statistically significant difference between groups of patients with and without a thrombosis history and groups who were and were not smokers (Fisher's exact test, P=0.50).


   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
BD, with typical features such as oral ulcers, genital ulcers and uveitis, is a unique systemic vasculitis characterized by venous and/or arterial thrombosis. Although the pathogenesis of thrombosis in BD is not completely understood, it is generally accepted as the endothelial dysfunction caused by vasculitis [15], as evidenced by increased serum thrombomodulin levels [16]. With respect to coagulation abnormalities, deficiencies of protein S/protein C [17, 18], defects of the fibrinolytic system [19, 20] and the presence of the factor V Leiden mutation [2], a molecular abnormality caused by a point mutation (G1691A) in factor V, were shown to be risk factors for thrombosis in BD. Hyperhomocysteinaemia is also a well-known risk factor for the development of thrombosis. The role of hyperhomocysteinaemia as a contributing risk factor for thrombosis in BD has not been investigated previously. We therefore studied plasma homocysteine concentrations in BD patients with and without a history of thrombosis and compared the results of these two groups with each other and with data on positive and negative control groups. The frequency of hyperhomocysteinaemia and the mean plasma homocysteine concentrations were significantly higher in thrombotic BD patients than in non-thrombotic subjects.

The plasma homocysteine level is determined by genetic and nutritional factors. Other than deficiencies of vitamin B6, B12 and folic acid [8], genetic mutations of enzymes involved in homocysteine metabolism, such as methylene tetrahydrofolate reductase and cystathionine-ß-synthase [21], may also cause hyperhomocysteinaemia.

The exact mechanism by which hyperhomocysteinaemia causes vascular disease and/or thrombosis is not known. However, there are two widely accepted hypotheses. According to the first, hyperhomocysteinaemia has deleterious effects on endothelial cells, causing endothelial cell damage, smooth muscle cell proliferation and increased oxidative stress [22]. In vitro studies are in favour of this hypothesis [23]. In vitro endothelial cell cultures showed that homocysteine interfered with vasomotor regulatory and endothelial anticoagulant functions of vascular endothelial cells. Vessels from monkeys with moderate hyperhomocysteinaemia exhibited increased platelet-mediated vasoconstriction, impaired endothelium-dependent vasodilatation [24] and decreased thrombomodulin-dependent activation of proteinC [25].

According to the second hypothesis, homocysteine-mediated vascular disease/thrombosis occurs via interference with coagulation mechanisms. Homocysteine inhibits the expression and activation of thrombomodulin, which is a cofactor for protein C activation [25]. Homocysteine also suppresses the anticoagulant action of antithrombin III [26].

Besides hyperhomocysteinaemia, there are additional risk factors for venous thrombosis, including the factor V Leiden mutation and deficiency of anticoagulant proteins such as protein C, protein S and antithrombin III. However, hyperhomocysteinaemia differs from other risk factors in that it is correctable. High plasma homocysteine concentrations can be lowered with folic acid and vitamin B12 supplementation.

In conclusion, our findings suggest that hyperhomocysteinaemia is an independent risk factor for the development of thrombosis in BD. Because hyperhomocysteinaemia is a correctable risk factor, this finding might lead to new avenues in the prophylaxis of thrombosis in BD.


   Notes
 
Correspondence to: K. Aksu, 80 Sokak No. 27/3, 35040 Bornova, Izmir, Turkey. Back


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 3 May 2000; Accepted 16 January 2001


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