Bode-Böger et al.[6] and Böger et al.[7],[8] demonstrated high levels of ADMA in plasma from hypercholesterolemic rabbits. Their later study showed that ADMA was elevated in young subjects with hypercholesterolemia and that elevation of ADMA was associated with impaired endothelium-dependent vasodilation. Intra-arterial infusion of ADMA causes endothelial dysfunction in humans[9]. Moreover, ADMA is a strong and independent predictor of cardiovascular events and atherosclerosis[10].

Another marker of early atherosclerosis is measurement of the carotid intima-media thickness (IMT) via high-resolution B-mode ultrasound. Several studies have shown that increased carotid IMT is a consistent predictor of the risk of future cardiovascular events and can also predict the presence of coronary artery disease[11]. Children with familial hypercholesterolemia are characterized by an increased IMT when compared with healthy controls[12]. Several investigators have looked for an association between various atherogenic risk factors and IMT of the carotid arteries. Most studies have been conducted among middle-aged and older subjects with hypercholesterolemia, while similar studies in children and adolescents are rare.

High serum homocysteine concentration is increasingly recognized as a new risk factor for atherosclerosis and other vascular diseases. The atherogenic effect of homocysteine is related to cytotoxin action on the endothelial cells and their function[13].

The aim of this study was to examine and compare IMT of the carotid arteries and endothelial function parameters such as plasma ADMA and homocysteine levels between hypercholesterolemic children and healthy controls, and to determine the relations between carotid IMT and atherogenic risk factors, such as lipid and plasma ADMA level.

The blood samples were obtained from the antecubital fossa vein in the morning after 12 hours of fasting and were immediately centrifuged at 2500 rpm for 10 minutes (min). Plasma and serum samples were stored at -80°C. The lipid profile (β-quantification) was analyzed on fresh samples. The subjects whose total cholesterol and LDL-cholesterol were >95th percentile according to age and sex were accepted as hypercholesterolemic. Total cholesterol, triglyceride and high-density lipoprotein (HDL) levels were determined by colorimetric-spectrophotometric Aeroset (Abbott) autoanalyzer at 500 nm according to the Trinder reaction. LDL levels were calculated according to the Friedewald formula (Total cholesterol – (HDL + very low-density lipoprotein [VLDL]).

Asymmetric dimethylarginine (ADMA), arginine and homocysteine concentrations of the plasma samples were determined with high performance liquid chromatography with fluorescence detector[16].

The measurement of carotid IMT was performed by the same sonographer (S.O.) using a General Electric® Logic 9 ultrasonography with a linear array probe of 7.5 MHz.

Statistical Analysis

Statistical analyses were performed using the Statistical Package for Social Sciences (version 11.5, SPSS, Inc., Chicago, IL). All values were expressed as mean and standard deviation (SD). The differences between the two groups were tested by Mann-Whitney testing. Linear regression analysis with Pearson’s coefficients was used to assess the strength of association between variables. Multivariate regression analysis was used to identify determinants of IMT of the carotid artery. The strength of these relationships was expressed as the β coefficient and p value. A p<0.05 was considered statistically significant.

Both carotid IMT and plasma ADMA levels were significantly higher in all hypercholesterolemic groups than in normocholesterolemic children (p<0.001; p<0.01). The levels of plasma arginine were statistically higher in all patients and in children with non-familial hypercholesterolemia (p<0.01) than in the controls. No significant difference was determined in homocysteine concentration between hypercholesterolemic children and the control group (p>0.05) (Table I).

In the correlation analysis, no significant correlation was observed between lipid profiles and the levels of arginine, homocysteine and arginine/ADMA ratio. However, a statistically positive correlation was observed between the levels of plasma ADMA and LDL-cholesterol (Table II). A significant positive correlation was found between carotid IMT and total and LDLcholesterol and triglyceride levels (Table III).

Recent studies have shown that ADMA is an agent for endothelial dysfunction and may be an indicator for atherosclerosis[7],[18]-[20]. Several studies have demonstrated that plasma levels of ADMA are increased in conditions associated with atherosclerosis, including the risk factors of age, hypertension, diabetes, insulin resistance, hypercholesterolemia, hypertriglyceridemia, and hyperhomocysteinemia[2],[7],[17],[20]-[24].

However, the study of ADMA in pediatric diseases has just begun. Unlike prospective data that indicate a direct and independent association between ADMA and cardiovascular endpoints in adult patients with different cardiovascular diseases, pediatric studies have been limited by inadequate power and small sample size[25]. Jehlicka et al.[26] showed that baseline levels of ADMA were significantly higher in children with familial hypercholesterolemia than in diabetes mellitus type 1 and healthy children. In our study, we found that ADMA levels were significantly higher in children with hypercholesterolemia when compared with controls. These findings support the fact that plasma ADMA concentration is a novel risk factor for atherosclerosis in hypercholesterolemic children in the future.

Zhu et al.[27] showed that plasma homocysteine concentrations are high in obese children with hypertension and dyslipidemia, and they suggest that homocysteine levels of these patients should be monitored. Szymczak et al.[28] found high homocysteine levels in hypercholesterolemic children with a family history for cardiovascular disease, and they indicated that high homocysteine level is a predictive risk factor for cardiovascular disease in these children. Sierakowska-Fijalek et al.[29] noted that high plasma homocysteine level is a risk factor for atherosclerosis. According to our findings, there was no significant change in homocysteine levels of the hypercholesterolemic children compared to the control group. However, the effect of homocysteine in the atherosclerotic process is proven[30]. We thus suggest the homocysteine levels may be an independent indicator in atherosclerosis in hypercholesterolemic patients.

Several studies have shown that high resolution B-mode ultrasound measurement of the carotid IMT is a feasible, direct and noninvasive method for evaluating and detecting early atherosclerosis and preclinical lesions of the arterial wall. Increased thickness and stiffness of the carotid artery were noted as an early marker of impaired vascular health[2],[10]. Previous studies that were performed in childhood showed significantly higher IMT in children with type 1 diabetes, obesity, hypertension, and familial hypercholesterolemia[31]-[34]. We demonstrated that carotid IMT is significantly higher in children with hypercholesterolemia when compared with controls and that carotid IMT is significantly correlated with total cholesterol, LDL-cholesterol and triglyceride levels.

Table I. Baseline Characteristics of the Study Groups

Table II. Correlation between ADMA, L-Arginine, L-Arginine/ADMA Ratio, and Homocysteine Levels and Serum Lipid Profile

There are a number of reports in the literature that have shown the association between plasma ADMA levels and carotid IMT in adults[3],[35]. Our study is the first to investigate the association between plasma ADMA levels and carotid IMT in children with hypercholesterolemia. Our results showed that plasma ADMA levels and carotid IMT were significantly higher in children with hypercholesterolemia than healthy children, and also that there was a significant positive correlation between carotid IMT and total and LDL- cholesterol levels. However, no significant positive correlation was observed between plasma ADMA and cholesterol levels.

In conclusion, ADMA level and carotid IMT can be the early leading indicators in hypercholesterolemic children with atherosclerotic risks. The combination of ADMA and advanced imaging methods such as carotid IMT may play an important role in the prediction of cardiovascular risk in hypercholesterolemia in childhood.

Table III. Correlation between C-IMT and Laboratory Measurements