# Homocysteine May Involve in the Pathogenesis of  Behcet's Disease by Inducing Inflammation

Authors: Selda Pelin Kartal Durmazlar; Ahmet Akgul; Fatma Eskioglu
Journal: Mediators of Inflammation
 (2008)

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## Abstract

Objective. Our aim was to evaluate the significance of homocysteine (Hcy) in Behcet's disease (BD) and the association of elevated Hcy levels associated with the indices of inflammation in BD. Methods. Untreated 70 patients with BD and 33 healthy individuals were included into the study. Hcy, tumor necrosis alpha (TNF-α), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) were evaluated with respect to activity and specific individual clinical manifestations of the disease. Results. Hcy levels were found significantly elevated in active BD when compared to inactive BD and healthy controls. Hcy levels were found to have high correlation with the number of active clinical manifestations increased. A significant positive correlation was found between serum Hcy and TNF-α levels, CRP, and ESR. Hcy was found to be the best predictor of TNF-α among other parameters. Conclusion. Hcy may involve in the pathogenesis of BD by inducing inflammation.


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## Body

## 1. Introduction

In 1969, McCully first proposed homocysteine (Hcy) to
be a cause of cardiovascular disease due to the observation that patients with
homocystinuria, a metabolic disease which results in high Hcy levels, also
displayed atherosclerotic plaques at autopsies [1]. High levels of Hcy were associated
with endothelial damage and oxidative stress [2, 3]. Currently, there is a
classification commonly used for the degree of hyperhomocysteinemia as follows:
normal (5–14 μmol/L), mild
(15–30 μmol/L), moderate (31–100 μmol/L), and
severe (>100 μmol/L) [4]. As many epidemiologic studies supported, mild
hyperhomocysteinemia was found risk factor for both venous and arterial
thrombosis [5–8]. Hcy is an
intermediary sulphydryl-containing amino acid formed during the conversion of methionine to
cysteine. Its sulphydryl group can cause direct endothelial cytotoxicity,
inhibition of glutathione peroxidase and nitric oxide, interference with
clotting factor, and oxidation of LDL [9]. Additionally, high levels of Hcy
cause lipid peroxidation, impaired vasomotor regulation, prothrombic surface,
and, therefore, atherothrombogenesis [10].Behcet's
disease (BD), initially described by Behcet in 1937 [11], is a chronic relapsing
vasculitis, involving both arteries and veins of various sizes. Thrombotic
complications have been reported in approximately 10–40% of BD
patients [12]. Endothelial dysfunction due to inflammation is considered to be
an important factor of thrombosis in BD. Homocysteine is reported to enhance
endothelial leukocyte interaction [13]. Some studies have shown that
hyperhomocysteinemia might be assumed to be an independent and correctable risk
factor for thrombosis in BD [14–19]. Moreover,
the association between Hcy levels and endothelial dysfunction has been shown
in patients with BD [20]. However, endothelial injury itself cannot clearly
account the hypercoagulable status of BD because other vasculitis syndromes do
not increase the risk of thrombosis [21]. Additionally, existence of several
contradictory results with respect to serum Hcy levels leads the role of
homocysteine in BD unclear [22]. As vitamin B6, vitamin B12, and folic acid can
decrease homocysteine levels [23], treatment with these vitamins can be
considered to reduce the risk of venous and arterial thrombosis. Therefore, it
is important to find a clear picture of the role of Hcy, which is thought to
induce proinflammatory cytokines [24] during the course of BD.The
present study was conducted to evaluate serum Hcy as a serological marker for
the assessment of the activity of BD with respect to specific individual clinical manifestations.
Serum levels of Hcy were compared with the values of C-reactive protein (CRP),
an acute phase reactant, the erythrocyte sedimentation rate (ESR), and tumor
necrosis alpha (TNF-α) which is an important proinflammatory cytokine. The
results were controlled with the healthy individuals.In
patients with BD, the question of whether hyperhomocysteinemia depends on the
inflammation has not been answered yet. Therefore, our aim was to evaluate
whether elevated Hcy levels, an independent risk factor for both venous and
arterial thrombosis, associated
with proinflammatory process mediated by cytokines in patients with BD.


## 2. Material and Methods

### 2.1. Subjects

70 patients (40 males, 30 females; mean age of 33 ± 7.79) fulfilling the international study group criteria
[25] for the diagnosis of BD and 33 healthy control subjects (17 males, 16
females; mean age of 30 ± 6.68) were included in this study. Patients with BD
were selected from patients who were being followed up by the departments of
dermatology and cardiovascular surgery, Behcet’s Disease Unit or referred from
other departments. Subjects were newly diagnosed or patients who were not
taking any medicine regarding BD for some reason. The control subjects were
age- and sex-matched healthy hospital staff. Complete blood count, serum
glucose, urea, creatinine, total cholesterol, triglyceride, uric acid, alanine
aminotransferase, aspartate aminotransferease, alkaline phosphatase, vitamin B12, and folic acid levels were evaluated in all of the subjects before being
included into the study. Patients who had other type of illness such as
autoimmune disease that could affect the cytokine levels or patients taking any
medication affecting the immune system or patients with folic acid or vitamin
B12 deficiencies, diabetes mellitus, hyperlipidemia, chronic hepatitis, renal
failure, severe psoriasis, pernicious anemia, and chronic alcoholism which
might affect Hcy levels were excluded from the study. Patients taking vitamin
supplements in the previous 6 months were also excluded from the study. The
study was approved by the local ethics institute. Informed consent was obtained
from all subjects. Pathergy test was performed and the number of active
manifestations recorded for each patient.


### 2.2. Determination of Disease Activity

Since
accepted specific clinical activity scoring system and laboratory screening
profile for BD has not been performed so far, the activity of patients was
considered to have an active disease in case of the existence of two or more
symptoms with worsening of clinical symptoms and lack of wellbeing at the time
of the study. Behcet’s disease
activity index (BDAI) was also performed on our patients according to
the method presented by Bhakta et al. [26] and described elaborately
by Lawton et al. [27]. An
overall disease activity scores derived from adding the scores according to the
evaluation of the duration of each clinical features (from 0 to 4) and patients
and clinicians impressions of the disease activity. As it was agreed that
inclusion of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)
measurements would not add significantly to overall measurement of disease
activity [27] and were not included as criteria in BDAI [26, 27],
ESR and CRP were not used in the determination of the activation of the
disease. The patients who had no symptoms regarding BD in four weeks period or
less than two symptoms with a healing process and an overall wellbeing status
were grouped in inactive BD.


### 2.3. Vascular Examination

All patients and healthy volunteers were examined carefully
for vascular involvement. Either venous or arterial system involvement was
defined as present when confirmed by Doppler ultrasonography, radioisotope
venography, magnetic resonance angiography, conventional angiography, or computerized
tomography.


### 2.4. Serum Homocysteine Analysis

Serum homocysteine levels in the study groups were determined by fluorescence
polarization immunoassay (FPIA) technique (Axis Biochemicals, ASA, Oslo,
Norway). FPIA was run on an IMx analyzer (Abbott, Ill, USA). The 95% confidence interval of plasma Hcy
level suggested by the manufacturer for healthy individuals is 4.45–12.42μmol/L and
serum Hcy level is expected to be up to 10% higher than ethylenediamine
tetraacetate (EDTA) plasma. Five
cc blood samples were drawn using a 25 gauge needle from a peripheral
vein, avoiding haemolysis in the morning hours after an overnight fasting and
30 minutes of supine rest and collected into 10 mL empty evacuated tubes without
EDTA, heparin, or clot activators. Samples were centrifuged at 1000×g for 10
minutes. The serum was separated in aliquots and immediately frozen and stored
at −80°C within 60 minutes until use.


### 2.5. Detection of Tumor Necrosis Alpha by Enzyme-Linked Immunosorbant Assay

The TNF-α level was measured with an enzyme-linked immunosorbant assay (ELISA) kit
(Bender Medsystems, Vienna, Austria, Lot # 223/208-D1)
with a detectable level set at 14 pg/mL. Five cc serum was separated by
centrifugation at 1000×g for 10 minutes and stored at −70°C until use.


### 2.6. C-Reactive Protein, Erythrocyte Sedimentation Rate Analyses

CRP was measured by a highly sensitive turbidimetric method using conventional
biochemical automatic analyzer. The upper limit of the normal range was 5 mg/L.
The ESR was determined by the classic Westergren method.


### 2.7. Statistical Analysis

Results were analyzed using SPSS for Windows VER 11.5 (SPSS
Inc., Chicago, Ill, USA). Each variant was evaluated by one sample
Kolmogorov-Smirnov test for compatibility
with normal distribution. As the data did not fit normal distribution, the
nonparametric Kruskall-Wallis test for intergroups comparison and Mann-WhitneyU test or Bonferroni corrected Mann-Whitney U tests were used for between-group
comparisons as indicated. The relationship between variables was evaluated by
Spearman correlation and regression analysis. Levels of significance set at
0.05 but in Bonferroni corrected test the significance threshold of 0.05
yielded to the value of 0.0167. Data are presented as median values and their
individual ranges (in parenthesis).


## 2.1. Subjects

70 patients (40 males, 30 females; mean age of 33 ± 7.79) fulfilling the international study group criteria
[25] for the diagnosis of BD and 33 healthy control subjects (17 males, 16
females; mean age of 30 ± 6.68) were included in this study. Patients with BD
were selected from patients who were being followed up by the departments of
dermatology and cardiovascular surgery, Behcet’s Disease Unit or referred from
other departments. Subjects were newly diagnosed or patients who were not
taking any medicine regarding BD for some reason. The control subjects were
age- and sex-matched healthy hospital staff. Complete blood count, serum
glucose, urea, creatinine, total cholesterol, triglyceride, uric acid, alanine
aminotransferase, aspartate aminotransferease, alkaline phosphatase, vitamin B12, and folic acid levels were evaluated in all of the subjects before being
included into the study. Patients who had other type of illness such as
autoimmune disease that could affect the cytokine levels or patients taking any
medication affecting the immune system or patients with folic acid or vitamin
B12 deficiencies, diabetes mellitus, hyperlipidemia, chronic hepatitis, renal
failure, severe psoriasis, pernicious anemia, and chronic alcoholism which
might affect Hcy levels were excluded from the study. Patients taking vitamin
supplements in the previous 6 months were also excluded from the study. The
study was approved by the local ethics institute. Informed consent was obtained
from all subjects. Pathergy test was performed and the number of active
manifestations recorded for each patient.


## 2.2. Determination of Disease Activity

Since
accepted specific clinical activity scoring system and laboratory screening
profile for BD has not been performed so far, the activity of patients was
considered to have an active disease in case of the existence of two or more
symptoms with worsening of clinical symptoms and lack of wellbeing at the time
of the study. Behcet’s disease
activity index (BDAI) was also performed on our patients according to
the method presented by Bhakta et al. [26] and described elaborately
by Lawton et al. [27]. An
overall disease activity scores derived from adding the scores according to the
evaluation of the duration of each clinical features (from 0 to 4) and patients
and clinicians impressions of the disease activity. As it was agreed that
inclusion of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)
measurements would not add significantly to overall measurement of disease
activity [27] and were not included as criteria in BDAI [26, 27],
ESR and CRP were not used in the determination of the activation of the
disease. The patients who had no symptoms regarding BD in four weeks period or
less than two symptoms with a healing process and an overall wellbeing status
were grouped in inactive BD.


## 2.3. Vascular Examination

All patients and healthy volunteers were examined carefully
for vascular involvement. Either venous or arterial system involvement was
defined as present when confirmed by Doppler ultrasonography, radioisotope
venography, magnetic resonance angiography, conventional angiography, or computerized
tomography.


## 2.4. Serum Homocysteine Analysis

Serum homocysteine levels in the study groups were determined by fluorescence
polarization immunoassay (FPIA) technique (Axis Biochemicals, ASA, Oslo,
Norway). FPIA was run on an IMx analyzer (Abbott, Ill, USA). The 95% confidence interval of plasma Hcy
level suggested by the manufacturer for healthy individuals is 4.45–12.42μmol/L and
serum Hcy level is expected to be up to 10% higher than ethylenediamine
tetraacetate (EDTA) plasma. Five
cc blood samples were drawn using a 25 gauge needle from a peripheral
vein, avoiding haemolysis in the morning hours after an overnight fasting and
30 minutes of supine rest and collected into 10 mL empty evacuated tubes without
EDTA, heparin, or clot activators. Samples were centrifuged at 1000×g for 10
minutes. The serum was separated in aliquots and immediately frozen and stored
at −80°C within 60 minutes until use.


## 2.5. Detection of Tumor Necrosis Alpha by Enzyme-Linked Immunosorbant Assay

The TNF-α level was measured with an enzyme-linked immunosorbant assay (ELISA) kit
(Bender Medsystems, Vienna, Austria, Lot # 223/208-D1)
with a detectable level set at 14 pg/mL. Five cc serum was separated by
centrifugation at 1000×g for 10 minutes and stored at −70°C until use.


## 2.6. C-Reactive Protein, Erythrocyte Sedimentation Rate Analyses

CRP was measured by a highly sensitive turbidimetric method using conventional
biochemical automatic analyzer. The upper limit of the normal range was 5 mg/L.
The ESR was determined by the classic Westergren method.


## 2.7. Statistical Analysis

Results were analyzed using SPSS for Windows VER 11.5 (SPSS
Inc., Chicago, Ill, USA). Each variant was evaluated by one sample
Kolmogorov-Smirnov test for compatibility
with normal distribution. As the data did not fit normal distribution, the
nonparametric Kruskall-Wallis test for intergroups comparison and Mann-WhitneyU test or Bonferroni corrected Mann-Whitney U tests were used for between-group
comparisons as indicated. The relationship between variables was evaluated by
Spearman correlation and regression analysis. Levels of significance set at
0.05 but in Bonferroni corrected test the significance threshold of 0.05
yielded to the value of 0.0167. Data are presented as median values and their
individual ranges (in parenthesis).


## 3. Results

Detected number of patients suffering from individual active clinical manifestations of
the disease in various degrees was 31 with oral ulceration (44.28%), 23 with genital ulceration
(32.85%), 18 with ocular involvement (25.71%), 37 with vascular involvement (52.85%),
25 with arthritis (35.71%), and 31 with cutaneous lesions like erythema nodosum
(EN) and papulopustular eruption (PPE) (44.28%). Thirty five patients (50%)
were found to have positive pathergy test. According to our evaluation scale, 34
patients were considered to have active disease with a median of 4 symptoms ranging between
2 and 5 and 36 patients to have inactive disease with a median of 1 symptom
ranging between 1 and 2. Consistently, active group had a statistically
significant high BDAI score with a median of 16 ranging between 9 and 25
compared to inactive group with a median of 2 ranging between 1 and 5 (P=.00)
(Table 1). According to vascular examination, venous involvement (deep venous thrombosis
with or without thromboflebitis) was found in 23 patients and either arterial
occlusion or aneurysm was found in 14 patients in active group. 17 patients in
inactive group had short duration of thromboflebitis with a wellbeing status.Table 1
Comparision of serum homocysteine, TNF-α,
CRP, and ESR values and BDAI scores between groups.
Homocysteine (μmol/L)TNF-α (pg/mL)ESR (mm/h)CRP (mg/L)BDAI scoresMedian (range)Group I (active BD) (n=34)20.95 (13.7–39.25)93.5 (30–228)24 (2–95)20.5 (3–400)16 (9–25)Group II (inactive BD) (n=36)13 (6.43–18)19 (15–30)27 (8–49)6.65 (3–34)2 (1–5)Group III (healthy control) (n=33)8 (5–8)0 (0–18)6 (2–18)3.1 (3–10)Significance for groupsI-II-IIIPa=.00*Pa=.00*Pa=.00*Pa=.00*Pc=.00*I-IIPb=.00*Pb=.00*Pb=.001*Pb=.00*I-IIIPb=.00*Pb=.00*Pb=.00*Pb=.00*II-IIIPb=.00*Pb=.00*Pb=.008*Pb=.00*P*a Significantly different by Kruskal-Wallis
test (P<.05).
P*b Significantly different by Bonferroni
corrected Mann-Withney U test  (P<.0167).
P*c Significantly different by Mann-Withney U test
(P<.05).
Data are presented  as
median values and their individual ranges within parentheses.In intergroup
comparison of active BD, inactive BD, and healthy controls according to serum
levels of Hcy, TNF-α, CRP, and ESR significant difference was detected for
each parameter (P=.00 for each parameter) (Table 1). To determine which
group/groups cause the difference, between-group comparisons performed.
According to this, Hcy, TNF-α, CRP, and ESR levels were found elevated in
active BD compared to inactive BD and healthy controls. Hcy, TNF-α, CRP, and
ESR levels of inactive BD were found elevated compared to healthy controls. Hcy
and TNF-α suggested having a strong association of disease activity (Table 1).
Hcy and TNF-α were found to have high correlation with BDAI scores (r=0.836, P=.00
and r=0.924, P=.00, resp.) and the number of active clinical
manifestations increased (r=0.694, P=.00 and r=0.842, P=.00, resp.)
while ESR and CRP also showed a positive correlation with both parameters with
relatively less significance (Table 2).Table 2
The relationship between variables.
HcyESRCRPTNFTotal†BDAISpearman correlation coefficient andP-valuesHcyr= 0.561r= 0.645r= 0.889r= 0.694r=
0.836P=.00P=.00P=.00P=.00P=.00ESRr= 0.561r= 0.421r= 0.538r= 0.304r= 0.408P=.00P=.00P=.00P=.011P=.00CRPr= 0.645r= 0.421r= 0.670r= 0.549r= 0.583P=.00P=.00P=.00P=.00P=.00TNFr= 0.889r= 0.538r= 0.670r= 0.842r= 0.924P=.00P=.00P=.00P=.00P=.00Total†r= 0.694r= 0.304r= 0.549r= 0.842r= 0.842P=.00P=.011P=.00P=.00P=.00BDAIr= 0.836r= 0.408r= 0.583r= 0.924r= 0.842P=.00P=.00P=.00P=.00P=.00Total† number of active clinical manifestations for each patient.The evaluation of association between specific individual clinical manifestations
and serum levels revealed increased Hcy levels in patients with oral ulcer (P=.03),
genital ulcer (P=.005), the presence of positive pathergy test (P=.007),
ocular lesion (P=.00), and vascular lesion of high significance (P=.00)
(Table 3). No association was found
between Hcy levels and EN/PPE and arthritis.Table 3
The significance of the association of Hcy with versus without individual
clinical manifestations and pathergy reaction.
Hcy levels (μmol/L) in patients withHcy levels (μmol/L) in patients withoutManifestationsNo. of PatientsMedian(Range)Median(Range)P-valuesOral ulcer3118.03(8–39.3)14.28(6.4–29)P=.003*Genital ulcer2318.1(11.9–38.1)14.28(6.4–39.3)P=.005*Ocular lesion1821.59(14–39.3)14.15(6.4–38)P=.00*Vascular lesion3719(11.9–39.3)13.75(6.4–26)P=.00*Arthritis/arthralgia2516(6.4–39.3)15(8–38.1)P=.99EN/PPE3116(8.1–38.1)14.88(6.4–39.3)P=.306(+) pathergy3517.2(7.9–38.1)14(6.4–39.3)P=.007*P*<.05 by Mann-Whitney U test (statistically
significant with versus without clinical manifestations).
Data are presented  as median values and their individual ranges within  parentheses.The serum levels of TNF-α were found to have strong association with oral ulcer (P=.00),
genital ulcer (P=.00), the presence of positive pathergy test (P=.00),
ocular lesion (P=.00), and vascular lesion (P=.000) (Table 4). No
association was found between TNF-α levels
and EN/PPE and arthritis.Table 4
The significance of the association of TNF-α with versus without individual
clinical manifestations and pathergy reaction.
TNF-α levels (pg/mL) in patients withTNF-α levels (pg/mL) in patients withoutManifestationsNo. of patientsMedian(Range)Median(Range)P-valuesOral ulcer3170(15–228)20(15–150)P=.00*Genital ulcer2354(17–228)21(15–228)P=.00*Ocular lesion1899(30–228)21(15–180)P=.00*Vascular lesion3784(15–228)21(15–145)P=.00*Arthritis/arthralgia2538(15–228)28(15–228)P=.49EN/PPE3145(15–228)22(15–228)P=.086(+) pathergy3545(15–228)21(15–228)P=.00*P*<.05 by Mann-Whitney U test (statistically
significant with versus without clinical manifestations).
Data are presented  as
median values and their individual ranges within  parentheses.The association of CRP with genital ulcer (P=.001), ocular lesion (P=.001),
vascular lesion (P=.001), EN/PPE (P=.043), and the presence of
positive pathergy test (P=.016) was found. No association was found between CRP levels and ocular lesion and
arthritis (Table 5). ESR showed no significant association with any of the
specific individual clinical manifestations (Table 6).Table 5
The significance of the association of CRP with versus without individual
clinical manifestations and pathergy reaction.
CRP levels (mg/L) in patients withCRP levels (mg/L) in patients withoutManifestationsNo. of patientsMedian(Range)Median(Range)P-valuesOral ulcer3112.4(3–400)8.7(3–43)P=.159Genital ulcer2318(3–100)8.4(3–400)P=.001*Ocular lesion1819.25(3–400)8.5(3–68)P=.001*Vascular lesion3720.5(3–400)8.4(3–51)P=.001*Arthritis/arthralgia258.4(3–400)12.9(3–400)P=.201EN/PPE3112.9(3–400)8.9(3–400)P=.043*(+) pathergy3514.8(3–400)8.7(3–400)P=.016*P*<.05 by Mann-Whitney U test (statistically
significant with versus without clinical manifestations).
Data are presented as median values and their individual
ranges within  parentheses.Table 6
The significance of the association of ESR with versus without individual
clinical manifestations and pathergy reaction.
ESR levels (mm/h) in patients withESR levels (mm/h) in patients withoutManifestationsNo. of patientsMedian(Range)Median(Range)P-valuesOral ulcer3121(2–95)12(2–70)P=.113Genital ulcer2322(2–95)14(2–60)P=.121Ocular lesion1822(2–86)14(2–95)P=.291Vascular lesion3720(2–70)14(2–95)P=.642Arthritis/arthralgia2522(3–60)14(2–95)P=.317EN/PPE3124(2–95)12(2–70)P=.070(+) pathergy3522(2–95)14(3–49)P=.173P*<.05
by Mann-Whitney U test (statistically significant with versus without clinical
manifestations).
Data are presented  as
median values and their individual ranges within  parentheses.A significant positive correlation was found between serum Hcy and TNF-α levels
(r=0.89, P=.00), CRP (r=0.645, P=.00) and ESR (r=0.561, P=.00)
(Table 2). Among parameters, Hcy was found to be the best predictor of TNF-α which
was used as a dependent variable (Beta = 6.45, R2=0.88, P=.00) (Table 7).Table 7
Predictors of plasma TNF-α level: regression analysis.
Predictor of TNF-αR2BetaP-valueHcy0.886.45P=.00CRP0.390.559P=.00ESR0.1461.15P=.00Dependent variable: TNF-α.Serum levels of Hcy in active BD patients with vascular involvement were found
significantly increased when compared in patients with active BD without
vascular involvement. In addition, serum levels of Hcy in inactive BD with
vascular involvement were found significantly increased compared to inactive BD
(Table8).Table 8
Comparison of Hcy intragroups of active and inactive patients according to presence of
vascular involvement.
Hcy levels (μmol/L)MedianRangeP-valuesActive BD (n=34)With vascular involvement (n=20)2619–39.25.00*Without vascular involvement (n=14)15.9813.7–26Inactive BD (n=36)With vascular involvement (n=17)14.2811.94–18.00*Without vascular involvement (n=19)10.756.43–16P*<.05 by Mann-Whitney U test. Data are presented
as median values and their individual ranges.Serum levels of active BD patients with vascular involvement, oral ulcer, arthritis/arthralgia,
EN/PPE and the presence of positive pathergy test were found significantly
increased compared to inactive BD patients with the same clinical symptoms
(Table9).Table 9
Comparison of Hcy between groups of active and inactive patients according to the presence of
individual clinical manifestations and positive pathergy.
No. of patientsHcy levels (μmol/L)P-valuesActiveInactiveActiveInactiveVascular involvement201726 (19–39.25)14.28 (11.94–18).00*Oral ulcer26520.85 (13.7–39.25)11.94 (8–14).00*Arthritis/arthralgia141119 (13.7–19)10.83 (6.43–17).00*EN/PPE211019 (13.7–38.1)12.36 (8.1–16).00*Positive pathergy241120.85 (13.7–38.1)14.28 (7.91–17.21).00*P*<.05 by Mann-Whitney U test. Data are presented
as median values and their individual ranges.


## 4. Discussion

The main
pathology in BD is an inflammatory process of small arteries and veins and
thrombosis as a result of vasculitis of the vaso vasorum [24]. Hyperhomocysteinemia
has been shown to be a risk factor for thrombosis in BD in some recent studies [14–19]. In our study,
serum levels of Hcy were found to have a positive correlation with BDAI scores
and total number of active clinical manifestations. We also found significantly
elevated levels of serum total Hcy in active BD when compared with inactive and
control groups. Our
results supported that total serum Hcy was a reliable marker for the activity
of disease [17]. Aksu et al. [14] divided their patients with BD into two
groups on the basis of thrombosis and they found hyperhomocysteinemia in
patients with a history of thrombosis. However, Leiba et al. [22] could not
find different serum total Hcy levels between patients with and without a
history of thrombosis. The main limitations of this study were its
retrospective nature and the long lag period between the thrombotic events and
the time of analyses performed. Aksu et al. [14] evaluated their patients with
respect to current clinical activity and 10 patients were accepted as
clinically active, though they could not find association between serum Hcy levels
and disease activity. The small sample size was a limitation of this study.The main factor responsible for the increased frequency of thrombosis in BD is thought
to be endothelial dysfunction caused by vascular inflammation [14, 15]. The
association between Hcy levels and endothelial dysfunction and its correlation
to the degree of endothelial damage has been shown in patients with BD [20]. Hcy generates superoxide and hydrogen peroxide, both
of which have been linked to endothelial damage [15]. Hcy-induced vascular
problems are thought
to be multifactorial, including direct Hcy damage to the endothelium, enhanced
lipid peroxidation and increased platelet aggregation by the effects on the
coagulation system [15, 17].In our study, the evaluation of association between specific individual clinical
manifestations and serum levels revealed increased Hcy levels in patients with
oral ulcer, genital ulcer, the presence of positive pathergy test and
particularly with ocular and vascular lesions of high significance. No association was found between Hcy levels and EN/PPE and
arthritis. Er et al. [15] showed the association of Hcy levels with disease
activity and ocular BD. Other specific individual clinical manifestations were
not assessed in this study. Ateş et al. [16] showed the association of Hcy
levels with vascular involvement in BD. However, they could not find
statistically significant difference between active and inactive diseases and association
with mucocutaneous involvement in BD. On the other hand, Korkmaz et al. [28] reported
no statistically significant association between vascular involvement and
homocysteine levels, though 50% of patients with vascular involvement had
hyperhomocysteinemia. Although the mechanism is unknown, all these findings
indicate that Hcy associates with BD. However, the causes of these different
study outcomes are not clear. The different sample sizes, different disease
durations or study patients under treatment that might affect outcomes might
have contributed. In addition, the question whether hyperhomocysteinemia depends
on the inflammation or not remains to be unanswered.Although the etiopathogenesis of BD has not yet been clarified, different immunological
abnormalities have been reported and increased spontaneous secretion of TNF-α,
Interleukin-6 (IL-6), and Interleukin-8 (IL-8) in monocyte cultures obtained
from BD patients [29]. Necrotizing, neutrophilic (leukocytoclastic)
obliterative perivasculitis, and venous thrombosis with lymphocytic and
monocytic cellular infiltration of the veins, capillaries, and the arteries of
all sizes and endothelial dysfunction are the hallmarks of BD [30]. Histopathological
studies revealed cellular infiltration consisting of lymphocytes, plasmocytes,
monocytes, and peripheral polymorphonuclear leukocytes (PMNs) in varying degrees, depending on the
stage of lesion in BD [30, 31]. Since cytokines are involved in the regulation
of functions of lymphocytes and phagocytes, they are playing important role in
the pathogenesis of the disease [30, 31]. Although any organ system may be
affected, there is high prevalence of venous or arterial thrombosis in BD and
most clinical features are associated with systemic vasculitis. It has been
suggested that vasculo-Behcet disease should be classified as a neutrophilic
vasculitis targeting the vaso
vasorum [32]. Aneurysm formation may be related to degeneration of arterial
wall caused by inflammation of the vaso vasorum. As Hcy has profound effects concerning vascular
lesions and thrombosis, vascular and ocular involvements in BD deserve special
attention. Indeed, we found significantly elevated levels of Hcy with respect
to the presence of vascular involvement in the intragroup comparisons of separately
active and inactive BD patients.  However,
in our study Hcy was found to be able to evaluate the fluctuating activity of
disease in an objective fashion and the number of additional organ systems
affected. This finding may imply that Hcy cannot simply be assumed to be an
independent risk factor for only vascular and ocular vaso-occlusive diseases in
BD [14–19]. That is because most clinical features are thought
to be associated with systemic vasculitis and dermal blood vessels embolized by
thrombus are observed even at the sites of needle-prick reaction [30].
Furthermore, endothelial injury is a characteristic finding in BD and can be
observed even in patients without any clinical vascular involvement [24, 33, 34].
These data provides
insight as to why Hcy is able to detect overall disease activity
with respect to different system involvements and why ascribed significance of
Hcy is not limited only to the individual clinical manifestations of vascular or
vaso-occlusive ocular involvements? Consistently, we found the association of
Hcy not only  with vascular and ocular vaso-occlusive
disease but also with genital ulceration, positive pathergy test, and oral
ulcers in BD patients, though with less significance. Therefore, our results not only support the
suggestion that Hcy is a risk factor for vascular and ocular vaso-occlusive
diseases in BD [15, 16] but also identify Hcy as a risk factor for the
development of most clinical features that are known to be associated with
vasculitis.We found a significant positive correlation between serum Hcy, TNF-α and CRP levels,
and ESR with relatively less significance. Hcy was found to be the best
predictor of TNF-α among other parameters. In other words, Hcy was found to
explain 88% of TNF-α change while CRP and ESR were found to explain 39% and 14%
of TNF-α change, respectively. The significance of Hcy in BD represents either vascular endothelium damaged by Hcy or immune system
stimulation by Hcy, our study may demonstrate the link between Hcy and
inflammation pathways in BD. That is because TNF-α, CRP, and ESR are indices
of inflammation. Although there are conflicting results in the literature with
respect to the reliability of ESR and CRP as markers to detect
the activity of BD and they are currently not involved as criteria in BDAI
scores [27], CRP deserves special attention because it has a strong predictive
power of vascular events [35]. We found both of them were elevated in the
active stage of the disease. However, unlike CRP, ESR showed no significant
association with any of the specific individual clinical manifestations.
Relatively large sample size of our patients with vascular involvement might be
a factor affecting our study outcome in favor of the significance of CRP in BD.Chemotactic and phagocytic activities
of neutrophils in patients with BD have been reported to be high [31] and TNF-α is a major factor
modulating inflammatory responses which is known to be increased in
inflammatory diseases. Recent studies showed increased TNF-α level in BD patients,
especially in the exacerbation period [24, 30]. Synthesis of CRP in the liver
is induced by several proinflammatory cytokines including TNF-α. This marks CRP
as a precise objective index of inflammatory activity and surrogate of
underlying cytokine stimulus and a sensitive marker of underlying systemic
inflammation [35]. It has been demonstrated that coagulation indices including
fibrinogen, von Willebrand factor, and plasminogen activator activity are
increased in BD and Hcy is thought to promote the clotting cascade via several
actions including inactivation of protein C, activation of coagulation factor
V, increased vascular smooth muscle cell proliferation, and inhibition of
thrombomodulin [15]. CRP is thought to have effects into the mechanism linking
inflammation and coagulation [35]. Hcy is thought to induce proinflammatory
cytokines [24, 36–38]. In
vasculitis such as BD, endothelial-leukocyte interaction is an important part
of the inflammation [21] and Hcy has been reported to increase endothelial-leukocyte
interaction [13]. Hcy has been shown in vivo and in vitro to promote inflammatory
process such as the adhesion of neutrophils to endothelial cells as well as the
release of the inflammatory cytokine IL-8 and monocyte chemoattractant
protein-1 (MPC-1) [39]. Hcy was shown to enhance the cytokine-stimulated
expression of endothelial cell adhesion molecules and monocyte and T-cell
adhesion to endothelial cells [39]. Adhesion molecules are characterized not only by their ability
to cause directed migration of leukocytes into inflamed tissue, but they may
also have biological effects on other cell types, such as endothelial cells,
fibroblasts, and smooth muscle cells [40]. Hcy was shown to promote TNF-α
mediated induction of vascular cell adhesion molecule-1 (VCAM-1) in endothelial
cells [41]. With respect to innate inflammatory markers such as TNF-α and CRP,
serum Hcy has been shown to contribute to innate immune response and to be a
determinant of TNF-α in hypertensive patients [36]. In addition, Hcy has been
shown to enhance inflammation markers in murine models [42]. The correlation of
Hcy levels with nitric oxide has been shown in patients with BD [15]. Hcy is
known to increase nitric oxide production in vascular smooth muscle cells and nitric
oxide is known to be released as a response to systemic immunoinflammatory
diseases [15]. A study demonstrated that targeting the TNF pathway
significantly decreased Hcy and CRP levels in patients with psoriatic arthritis
and in this study, CRP showed a positive correlation with Hcy at baseline [43].
CRP has been shown to correlate with Hcy in BD [19]. Hcy has been suggested to
be related to the markers of inflammation and its elevated levels as a result
of immunosuppressive treatment [19]. Indeed, immunosuppressive treatments may
affect serum levels of Hcy and TNF-α [44]. This is the reason why we studied
with the patients who were not taking any medication regarding BD. This definite
selection decreased the number of our study population as a study limitation.
In our study, serum levels of active BD patients with vascular involvement,
oral ulcer, arthritis/arthralgia, EN/PPE and the presence of positive pathergy
test were found significantly increased compared to inactive BD patients with
the same clinical symptoms. Therefore, serum levels of Hcy were
found to explain the wide clinical spectrum of the activity of the specific
individual symptoms.In summary, Hcy
and CRP have effects into the mechanism linking inflammation and coagulation
and Hcy is thought to induce proinflammatory cytokines including TNF-α, which
is known to increase chemotactic and phagocytic activities of neutrophils, endothelial-leukocyte
interaction, and CRP levels. We found a high correlation between serum levels
of Hcy and TNF-α and Hcy was found to explain the wide clinical spectrum of the
activity of the disease and specific individual symptoms. Taking all these
findings into account, a theory might be put forward which is open to further
in vivo and in vitro studies; Hcy-induced inflammation may be responsible for
tissue damage as well as endothelial dysfunction in BD and enhanced Hcy may
account for the differences observed between clinically distinct subgroups.Lowering Hcyby selected vitamin supplements or TNF blockade may be the future
direction of prevention from developing the risk of thrombosis and vasculitis
of different organ systems and thereby activation of BD disease.


## 5. Conclusion

Our data identified Hcy as a risk factor for the
development of not only vascular and ocular involvement but also most clinical
features that are known to be associated with vasculitis in BD. In addition, serum
Hcy showed a strong association of disease activity and correlated with BDAI
scores and the number of active clinical manifestations increased. Therefore, Hcy might be assumed as a reliable
marker for the activity of disease. Hcy was found to be the best predictor of
TNF-α and positively correlated with inflammatory markers. Our findings
suggest that Hcy might contribute to the pathogenesis of BD by inducing
inflammation. Further investigation in clinical and experimental studies is required to
conclusively demonstrate the association between Hcy and inflammatory pathways.



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