The Turkish Journal of Pediatrics
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The Levels of Asymmetric Dimethylarginine, Homocysteine and Carotid Intima-Media Thickness in Hypercholesterolemic Children
Alev Hasanoğlu1, İlyas Okur1, Ayşe Ceyda Ören1, Gürsel Biberoğlu1, Suna Oktar2, Fatma Tuba Eminoğlu1, Leyla Tümer1
Departments of 1Pediatric Nutrition and Metabolism, and 2Radiology, Gazi University Faculty of Medicine, Ankara,
|The aim of this study was to examine the intima-media thickness (IMT) of
carotid arteries and endothelial function parameters such as plasma asymmetric
dimethylarginine (ADMA) and homocysteine levels in hypercholesterolemic
children and to investigate the relations of these parameters with
hypercholesterolemia. Fifty-seven hypercholesterolemic and 37 healthy children
were included in the study. Hypercholesterolemia was defined as 155 mg/dl and
above for low-density lipoprotein (LDL)-cholesterol. Plasma concentrations of
ADMA and homocysteine were measured and the measurement of carotid IMT
was determined. Both carotid IMT and plasma ADMA levels were significantly
higher in hypercholesterolemic children than healthy children (p<0.01). No
significant difference was determined in homocysteine concentration between
hypercholesterolemic children and the control group (p>0.05). No significant
correlation was observed between lipid profiles and the levels of ADMA and
homocysteine. However, a significant positive correlation was found between
carotid IMT and total and LDL-cholesterol levels and between the levels of
ADMA and LDL-cholesterol. In conclusion, the progressive increase in ADMA
levels and carotid IMT and the positive relationship between carotid IMT and
serum cholesterol levels support that plasma ADMA levels and carotid IMT
can be indicators of early atherosclerosis in hypercholesterolemic children.|
asymmetric dimethylarginine, carotid intima-media thickness,
|Nitric oxide (NO) is an endogenous vasodilator
released from the endothelium. It also inhibits
platelet adherence and aggregation, reduces
adherence of leukocytes to the endothelium
and suppresses proliferation of vascular smooth
muscle cells. Thus, NO is recognized as the
most potent endogenous molecule against
atherosclerosis. Accordingly, impairment
of NO synthesis bioactivity may increase
the risk of vascular disease. Asymmetric
dimethylarginine (ADMA) is an endogenous
inhibitor of NO synthase that has been linked
to endothelial dysfunction and atherosclerosis
in the general population-. ADMA is formed
endogenously by degradation of proteins
containing arginine residues that have
been methylated by S-adenosylmethioninedependent
methyltransferase. ADMA is
increased in the plasma of humans with
hypertension, chronic renal failure, chronic
heart failure, hyperhomocysteinemia, and other
Bode-Böger et al. and Böger et al.,
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. Moreover,
ADMA is a strong and independent predictor of
cardiovascular events and atherosclerosis.
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. Children with
familial hypercholesterolemia are characterized
by an increased IMT when compared with
healthy controls. 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.
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.
|Material and Methods |
|This study was performed on 57
hypercholesterolemic children, whose lowdensity
lipoprotein (LDL)-cholesterol levels
were >155 mg/dl (4 mmol/L). As the control
group, 37 healthy children of similar age were
included. The detailed medical and family
history of all subjects was obtained and a
complete physical examination, including
the evaluation of height, weight and blood
pressure, was performed. The patients were
classified according to the total number of
points calculated based on the Dutch Lipid
Clinical Network Diagnostic criteria for familial
hypercholesterolemia. Patients who were
scored as ≥3 points were classified as having
familial hypercholesterolemia and those who were scored as <3 points were classified as
having non-familial hypercholesterolemia. The
body mass index (BMI) was calculated as
weight (kg) divided by height squared (m2),
and the subjects who were >95th percentile
according to age and sex for Turkish children
were not included in the study due to obesity.
None of the subjects suffered from any of
the other risk factors for atherosclerosis and
hypercholesterolemia, such as hypertension,
diabetes mellitus, and renal, liver and endocrine
diseases, and none was taking medication, such
as lipid-lowering therapy, immunosuppressive
therapy or vitamin supplements. The study was
approved by the local ethics committee, and
all patients and their family provided written
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
Asymmetric dimethylarginine (ADMA),
arginine and homocysteine concentrations of
the plasma samples were determined with
high performance liquid chromatography with
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 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.
|Age, sex, BMI, and laboratory parameters of
the patient groups and control children are
shown in Table I. No significant differences
were observed between the patient groups and
controls in terms of age and BMI (p>0.05).
Total and LDL-cholesterol concentrations
were statistically higher in the patient groups
than in the control group (p<0.001), but no
significant difference was observed between the
patients and controls in terms of triglyceride
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
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).
|Endothelial dysfunction has an important role
in the atherosclerotic process, and the key
mechanism of this loss of function is NO-based.
In many studies, it was reported that there
are defects in the biological activity of NO
in hypercholesterolemia and atherosclerosis.
The first step of the atherosclerotic process
is endothelial dysfunction caused by leukocyte adhesion and platelet aggregation.
Recent studies have shown that ADMA is an
agent for endothelial dysfunction and may be
an indicator for atherosclerosis,-. 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
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. Jehlicka et al. 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. 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. 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.
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. We thus
suggest the homocysteine levels may be an
independent indicator in atherosclerosis in
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,. Previous
studies that were performed in childhood
showed significantly higher IMT in children
with type 1 diabetes, obesity, hypertension,
and familial hypercholesterolemia-. 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
| ||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,. 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
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|
1. Cooke JP, Dzau VJ. Nitric oxide synthase: role in the
genesis of vascular disease. Annu Rev Med 1997; 48:
2. Cooke JP. ADMA: its role in vascular disease. Vasc
Med 2005; 10 (Suppl): S11-17.
3. Furuki K, Adachi H, Matsuoka H, et al. Plasma levels
of asymmetric dimethylarginine (ADMA) are related
to intima-media thickness of the carotid artery: an
epidemiological study. Atherosclerosis 2007; 191: 206-
4. Moncada S, Higgs A. The L-arginine-nitric oxide
pathway. N Engl J Med 1993; 329: 2002-2012.
5. Böger RH. Asymmetric dimethylarginine (ADMA)
and cardiovascular disease: insights from prospective
clinical trials. Vasc Med 2005; 10 (Suppl): S19-25.
6. Bode-Böger SM, Böger RH, Kienke S, Junker W, Frölich JC. Elevated L-arginine/dimethylarginine ratio
contributes to enhanced systemic NO production by
dietary L-arginine in hypercholesterolemic rabbits.
Biochem Biophys Res Commun 1996; 219: 598–603.
7. Böger RH, Bode-Böger SM, Szuba A, et al. Asymmetric
dimethylarginine: a novel risk factor for endothelial
dysfunction. Its role in hypercholesterolemia.
Circulation 1998; 98: 1842–1847.
8. Böger RH, Bode-Böger SM, Tsao PS, Lin PS, Chan
JR, Cooke JP. An endogenous inhibitor of nitric
oxide synthase regulates endothelial adhesiveness for
monocytes. J Am Coll Cardiol 2000; 36: 2287–2295.
9. Zoccali C, Bode-Böger SM, Mallamaci F, et al. Plasma
concentration of asymmetrical dimethylarginine and
mortality in patients with end-stage renal disease: a
prospective study. Lancet 2001; 358: 2113–2117.
10. Geroulakos G, O’Gorman DJ, Kalodiki E, Sheridan DJ,
Nicolaides AN. The carotid intima–media thickness as a
marker of the presence of severe symptomatic coronary
artery disease. Eur Heart J 1994; 15: 781–785.
11. Chambless LE, Heiss G, Folsom AR, et al. Association
of coronary heart disease incidence with carotid
arterial wall thickness and major risk factors: the
Atherosclerosis Risk in Communities (ARIC) study,
1987–1993. Am J Epidemiol 1997; 146: 483–494.
12. Pauciullo P, Iannuzzi A, Sartorio R, et al. Increased
intima-media thickness of the common carotid artery
in hypercholesterolemic children. Arterioscler Thromb
1994; 14: 1075–1079.
13. Pfeiffer CM, Huff DL, Gunter EW. Rapid and accurate
HPLC assay for plasma total homocysteine and cysteine
in a clinical laboratory setting. Clin Chem 1999; 45:
14. World Health Organization. Familial
hypercholesterolemia—report of a second WHO
Consultation. Geneva, Switzerland: World Health
Organization; 1999. (WHO publication no. WHO/
15. Bundak R, Furman A, Gunoz H, Darendeliler F, Bas
F, Neyzi O. Body mass index references for Turkish
children. Acta Paediatr 2006; 95: 194-198.
16. Chen BM, Xia LW, Zhao RQ. Determination of
N(G),N(G)-dimethylarginine in human plasma by
high-performance liquid chromatography. J Chromatogr
B Biomed Sci Appl 1997; 692: 467-471.
17. Cooke JP. Does ADMA cause endothelial dysfunction?
Arterioscler Thromb Vasc Biol 2004; 20: 2032-2037.
18. Miyazaki H, Matsuoka H, Cooke JP, et al. Endogenous
nitric oxide synthase inhibitor. A novel marker of
atherosclerosis. Circulation 1999; 99: 1141-1146.
19. Mügge A, Hanefeld C, Böger RH. Plasma concentration
of asymmetric dimethylarginine and the risk of coronary
heart disease: rationale and design of the multicenter
CARDIAC study. Atheroscler Suppl 2003; 4: 29-32.
20. Sahinarslan A, Cengel A, Biberoglu G, Hasanoglu
A, Turkoglu S, Timurkaynak T. Plasma asymmetric
dimethylarginine level and extent of lesion at coronary
angiography. Coron Artery Dis 2006; 17: 605-609.
21. Surdacki A, Nowicki M, Sandmann J, et al. Reduced
urinary excretion of nitric oxide metabolites and
increased plasma levels of asymmetric dimethylarginine
in men with essential hypertension. J Cardiovasc
Pharmacol 1999; 33: 652-658.
22. Abbasi F, Asagmi T, Cooke JP, et al. Plasma
concentrations of asymmetric dimethylarginine are
increased in patients with type 2 diabetes mellitus.
Am J Cardiol 2001; 88: 1201-1203.
23. Böger RH, Bode-Böger SM, Szuba A, et al. Asymmetric
dimethylarginine (ADMA): a novel risk factor for
endothelial dysfunction: its role in hypercholesterolemia.
Circulation 1998; 98: 1842-1847.
24. Stuhlinger MC, Oka RK, Graf EE, et al. Endothelial
dysfunction induced by hyperhomocyst(e)inemia: role
of ADMA. Circulation 2003; 108: 933-938.
25. Tain YL, Huang LT. Asymmetric dimethylarginine:
clinical applications in pediatric medicine. J Formos
Med Assoc 2011; 110: 70-77.
26. Jehlicka P, Stozický F, Mayer O Jr, et al. Asymmetric
dimethylarginine and the effect of folate substitution
in children with familial hypercholesterolemia and
diabetes mellitus type 1. Physiol Res 2009; 58: 179-
27. Zhu W, Huang X, Li M, Neubauer H. Elevated plasma
homocysteine in obese school children with early
atherosclerosis. Eur J Pediatr 2006; 165: 326-331.
28. Szymczak E, Chelchowska M, Radomyska B, Laskowska
KT. Homocysteine and some lipid parameters in
hypercholesterolemic children. Med Wieku Rozwoj
2001; 5: 158-164.
29. Sierakowska-Fijalek A, Kaczmarek P, Pokoca L, Smorag
I, Wosik EM, Baj Z. Homocysteine serum levels and
lipid parameters in children with atherosclerosis risk
factors. Pol Merkur Lekarski 2007; 22: 146-149.
30. Scott CH, Sutton MS. Homocysteine: evidence for a
causal relationship with cardiovascular disease. Cardiol
Rev 1999; 7: 101-107.
31. Jarvisalo MJ, Jartti L, Nantö-Salonen K, et al. Increased
aortic intima-media thickness: a marker of preclinical
atherosclerosis in high risk children. Circulation 2001;
32. Sorof JM, Alexandrov AV, Cardwell G, Portman
RJ. Carotid artery intimal-media thickness and left
ventricular hypertrophy in children with elevated blood
pressure. Pediatrics 2003; 111: 61–66.
33. Aggoun Y, Bonnet D, Sidi D, et al. Arterial mechanical
changes in children with familial hypercholesterolemia.
Arterioscler Thromb Vasc Biol 2000; 20: 2070–2075.
34. Reinehr T, Kiess W, de Sousa G, Stoffel-Wagner
B, Wunsch R. Intima media thickness in childhood
obesity. Relations to inflammatory marker, glucose
metabolism and blood pressure. Metab Clin Exp 2006;
35. Vladimirova-Kitova L, Deneva T, Marinov B.
Predictors of the intima-media thickness of carotid
artery in asymptomatic newly detected severe
hypercholesterolemic patients. Clin Physiol Funct
Imaging 2010; 30: 250-259.
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