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Abstract The aim of this study was to investigate the

influence of homocysteine (hcy) levels on endothelial

function by the method of brachial artery ultrasonography

and their relation with microvascular complications in

type 2 diabetes mellitus (T2DM) patients without

macrovascular disease. Fifty-nine T2DM patients with a

mean age of 53.48.6 years and diabetes duration of

8.16.2 years and 16 healthy controls with a mean age of

4714.5 years were included in the study. Endothelial-

dependent and endothelium-independent flow-mediated

dilatation (FMD) were evaluated via brachial artery ultra-

sonography. Fasting plasma glucose (FPG), glycosylated

haemoglobin (A1c), lipid profile, hcy, B12 and folic acid

levels were measured. Diabetic patients and control group

individuals were compared with regard to the laboratory

values and brachial artery vascular reactivity. Factors

influencing endothelium-dependent FMD were investi-

gated with linear regression analysis. Age, gender, body

mass index, lipid profiles and hcy levels were similar in

both groups (p>0.05). Endothelium-dependent FMD per-

centages were significantly lower in diabetics than in the

control group (7.75.9 vs. 11.77.1%, p0.05). The upper limit of the reference

hcy value was found to be 12.6 mol/l in the control

group. In the diabetic group, hcy levels were high in 33

patients and normal in 26 patients. No difference wasdetected between the patients with high hcy levels and

those with a normal level with regard to endothelium-

dependent and endothelium-independent FMD values

(p>0.05). Mean hcy levels were 161.7 and 13.34.3

mol/l in T2DM patients with microvascular complica-

tion and those with no microvascular complication,

respectively (p

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Introduction

Type 2 diabetes mellitus (T2DM) patients are at increased

risk for atherosclerotic diseases such as coronary heart

disease, cerebrovascular disease and lower extremityocclusive vascular diseases that cause high mortality and

morbidity [1, 2]. Micro- and macrovascular complications

of diabetes have a complex pathogenesis, including dys-

function or damage of vascular endothelium [3, 4].

Several risk factors such as prolonged hyperglycaemia,

hypertension, smoking, hyperlipidaemia, changes in the

fibrinogen levels and decreased fibrinolytic activity may

accelerate the atherosclerotic process [5]. It is known that

hyperhomocysteinaemia can be associated with

atherothrombotic vascular disease such as coronary artery

disease, stroke and peripheral arterial disease [68].

Hyperhomocysteinaemia has been shown to be a stronger

risk factor in T2DM patients than in non-diabetics, and

has been thought to exert a synergistic effect with diabetes

in accelerating the atherosclerosis process [9]. In our

study we examined the influence of homocysteine (hcy)

plasma levels on endothelial functions as detected by

brachial artery ultrasound and the relation of hyperhomo-

cysteinaemia with the microvascular complications of dia-

betes in T2DM patients.

Patients and methods

The study group consisted of 59 T2DM patients (29 male, 30

female) with a mean age of 53.48.6 years and diabetes duration

of 8.16.2; 16 healthy individuals (8 male, 8 female) with a

mean age of 4714.5 years served as the control group. Diabetic

patients were required to have the diagnosis of diabetes for at

least 1 year. Age, gender, diabetes duration and demographic

characteristics of the patients and controls were recorded. A

complete medical history, with an emphasis on the duration of

diabetes, neuropathic symptoms and complications was taken

and physical examinations were performed. The diagnosis of

retinopathy was made by an ophthalmologist after the examina-

tion of the optic disc following the dilatation of pupils. For the

diagnosis of neuropathy, patients were examined by a neurolo-

gist and patellar reflex, Achilles tendon reflex, sensitivity to heatand sensory stimuli were evaluated. The diagnosis was support-

ed by electromyography when necessary. The mean urinary

albumin excretion measured in three consecutive 24-h urine

samples was used to diagnose nephropathy. Diagnosis of

microalbuminuria was based on the detection of an albumin

excretion of 30300 mg/day in a 24-h urine sample, or 30300

g/mg creatinine measured in the spot urine. All of our patients

were on oral anti-diabetic medication. Medications that could

positively affect the endothelial function, such as ACE inhibitors

and statins, were stopped 2 days before test. Patients with hyper-

tension, pregnant women, women in their menstruation period,

patients diagnosed with coronary artery disease, patients with a

history of myocardial infarction and/or cerebrovascular event

and smokers were not included in the study. Subjects in the con-

trol group were non-smokers with normal lipid levels, no other

cardiovascular risk factor and did not take medications. For both

groups, caffeine-containing drinks were not allowed for 24 h

before the scanning and fasting plasma glucose (FPG), glycosy-

lated haemoglobin (A1c), total cholesterol (TC), high-density

lipoprotein (HDL-C), low-density lipoprotein (LDL-C) and

triglyceride (TG) levels were measured. The venous blood sam-

ples obtained for hcy measurements were collected in EDTA-

containing tubes and measurement was performed with fluores-

cent immunoassay method on a Merck Hitachi L-6200A, Waters

470 (BioRad automatic sampling system AS100) device. Blood

samples were not stored and were measured daily. The upper

limit of the reference hcy values was obtained by calculation of

the mean values of the control group and then adding 2SD.

Endothelial function was evaluated non-invasively as

endothelium-dependent flow-mediated dilatation (FMD) by

means of brachial artery ultrasonography. The examination was

performed in the morning. Patients who took oral antidiabetics

were in a fasting state and did not receive medication before theexamination; the control group was also asked not to have break-

fast. Before the imaging process, patients were allowed to rest

for 15 min in supine position in a silent room with its tempera-

ture adjusted to 24C. The examination was performed in the

same room under the same conditions each time. Imaging was

obtained with an ATL HDI 5000 (Philips Bothell, Washington)

echocardiography device by using a 510-MHz linear transduc-

er. Records were obtained with simultaneously accompanying

electrocardiography. A sphygmomanometer was used, with its

cuff placed in the proximal region of the right forearm. Right

brachial artery images were obtained by using B mode imaging

23 cm below the cubital fossa, with the artery visualised in the

longitudinal plane. This part of the artery was magnified and the

initial baseline images were taken. Then, the cuff was inflatedabove a pressure of 100 mmHg and the artery was occluded for

5 min. The longitudinal artery image was obtained 1 min before

releasing the cuff. Records were taken while the cuff was

released. Arterial pulse signal was evaluated with a Doppler

instrument in order to measure the hyperaemic velocity.

Endothelium-dependent FMD was calculated by measuring the

diameter of the brachial artery 60 s after the release of the cuff

in the reactive hyperaemia phase. The patient was then asked to

rest for 15 min. During the follow-up, further baseline images

were obtained. Five milligrams of isosorbid dinitrate tablet was

given sublingually and brachial artery response (endothelium-

independent response) was obtained 3 min later. For endotheli-

um-independent (nitrate-dependent) vasodilatation, baseline

diameter was compared to the diameter measured 3 min after theadministration of sublingual nitrate, and the increase in the

diameter in terms of percentage was obtained. In the diabetic

group patients with A1c values greater than 7 were classified as

having poor glycaemic control and those with A1c lower than 7

were classified as having good glycaemic control.

The data were transferred to the computer using Statistical

Package for Social Science (SPSS). The diabetic patients and the

controls were compared with regard to their risk factors and

brachial artery reactivity. The groups were compared with unpaired

Students t-test. Correlation analysis was used to show the relation

between the variables. All measurements were obtained as mean

valueSD. Multiple linear regression analysis was used to evaluate

the variables influencing endothelium-dependent FMD.

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Results

There was no statistical difference between the diabetic

group and the control group with regard to age, gender

and body mass index (Table 1). FPG, TC, TG, LDL andhcy levels were higher in the diabetic group, but the dif-

ference was not statistically significant (p>0.05). Folic

acid levels were also similar in both groups (p>0.05),

whereas B12 levels were higher in the diabetic group

compared to the control one (p

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When we applied regression analysis to the results of

the diabetic group, the main factors influencing endothe-

lium-dependent FMD were FPG, TC, TG and HDL-C lev-

els (p0.05). Diabetes duration and A1c

levels, although not reaching a statistically significant dif-

ference, were close to being significant (p=0.07, p=0.08

respectively).

In the diabetic group, retinopathy was diagnosed in 12

patients, neuropathy was detected in 23 patients and

nephropathy was detected in 28 patients (Table 3). In 30

patients no complications were diagnosed. Hcy levels

were significantly higher in diabetic subjects who had any

microvascular complication than in those with no compli-

cation detected (167.1 vs. 13.34.3; p

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dependent and endothelium-independent FMD values

were similar in diabetic patients with microvascular com-

plications and in those without (n=29, p>0.05). At linear

regression analysis, both in diabetics with microvascular

complication and in those without, hcy levels did not sig-

nificantly influence endothelium-dependent FMD, but in

diabetics with microvascular complication this was close

to being significant (p=0.09).

When diabetic patients were classified as having goodglycaemic control (28 pts) and on those with poor gly-

caemic control (31 pts), by means of HbA1c levels lower

and greater than 7%, a difference was found with respect to

hcy, B12 and folic acid levels between groups (p

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Hofmann et al. measured soluble thrombomodulin lev-

els as a marker of the impairment of endothelial functions

in an early phase of T1DM patients. They observed that

thrombodulin levels were significantly higher in T1DM

patients, especially in those with high hcy levels and this

finding correlated with hcy levels [27]. In our study, hcylevels were increased in T2DM patients with microvascu-

lar complication, but endothelium-dependent and endo-

thelium-independent FMD were similar to that of the con-

trol group. Endothelium-dependent FMD was not corre-

lated with hcy levels either in patients with complication

or in those without. This finding suggested that hcy levels

do not influence endothelial function of T2DM patients

even when microvascular complications occur. In addi-

tion, the influence of factors such as FPG levels and lipid

parameters is more prominent on endothelium-dependent

FMD than that of hcy, B12 and folate levels.

Soinio et al. [28] followed 830 T2DM patients for 7

years, and they detected a higher cardiovascular mortality

and higher ratios of fatal and non-fatal myocardial infarc-

tion in patients with higher baseline hcy levels, in com-

parison to patients with normal or low hcy levels. They

suggested that plasma hcy level is an independent risk fac-

tor for cardiovascular events and claimed that addition of

folate to dietary intake can decrease cardiovascular event

risk [28]. In our study, endothelial functions detected by

FMD method were impaired both in diabetics with

microvascular complication and in those without. This

means that endothelial function can be impaired in the

early stages of diabetes, before microvascular complica-

tions develop. As hcy levels were found to be higher inpatients with microvascular complication, this could sug-

gest that hcy levels may contribute to the development of

the complications rather than initiating them.

In summary, in patients with T2DM endothelial dys-

function seems to start in the early stages of the disease

before microvascular complications occur. Hcy levels do

not have an influence on the endothelial function evaluat-

ed with FMD by brachial artery ultrasonography in T2DM

patients. The influence of classical atherogenic factors

such as FPG, TG, TC and HDL on endothelial functions

seems to be more prominent. In the presence of these risk

factors hcy may have an accelerating effect on the athero-sclerotic process, especially in patients in whom

microvascular complications have developed.

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