Content uploaded by Robson Barbosa de Miranda
Author content
All content in this area was uploaded by Robson Barbosa de Miranda on Aug 03, 2019
Content may be subject to copyright.
E-Mail karger@karger.com
Original Paper
A n n N u t r M e t a b 2 0 1 3 ; 6 3 : 2 5 – 3 1
DOI: 10.1159/000350314
L a c k o f A s s o c i a t i o n o f H o m o c y s t e i n e C o n c e n t r a t i o n s
with Oxidative Stress, Alterations in Carotid Intima
Media Thickness and Endothelial Reactivity in
Prepubertal Children
FabíolaIsabelSuanodeSouza a,e VâniaD’Almeida f
FernandoLuizAffonsoFonseca b SoniaHix c RobsonMiranda d
RosanaGomesdeTorresRossi a RenataRibeiro b RoseliOselkaSaccardoSarni a,e
a Pediatric Department, b Division of Biochemistry, Department of Morphology and Physiology, c Biochemistry Course, and
d Angiology and Vascular Surgery, Faculdade de Medicina do ABC, and e Pediatric Department and f Laboratory of Innate
Errors of Metabolism, Universidade Federal de São Paulo, São Paulo , Brazil
groups regarding the lipid profile and fasting glycaemia,
blood pressure or oxidative stress. Overweight and obesity
(body mass index z-score > +1) were more frequent in the
group with Hcy ≥ 7.3 μmol/l when compared to the group
with Hcy ≤ 4.1 μmol/l [13/35 (37.1%) vs. 4/29 (13.8%); p =
0.035]. After adjusting for nutritional status, there were no
differences in the intima media thickness and endothelium-
dependent vasodilation between children with high and low
Hcy levels. Conclusions: Among the cardiovascular risk fac-
tors investigated in the present study, which was based on
prepubertal children, only overweight and obesity were as-
sociated with increased Hcy concentrations ( ≥ 7.3 μmol/l).
Copyright © 2013 S. Karger AG, Basel
Introduction
The association between elevated concentrations of
homocysteine (Hcy) and the development of cardiovas-
cular diseases (CVDs) has been reported in the literature
[1] . Some mechanisms thought to contribute to this as-
Key Words
Homocysteine · Cardiovascular diseases · Child · Oxidative
stress · Endothelium
Abstract
Background and Aims: Our purpose was to assess the pres-
ence of risk factors for cardiovascular diseases (alterations in
the lipid profile, fasting glycaemia, high arterial pressure val-
ues, oxidative stress, increased intima media thickness and
impaired endothelium-dependent vasodilation) in prepu-
bertal children with elevated homocysteine (Hcy) levels.
Methods: In a cross-sectional study, 35 children with Hcy lev-
els ≥ 7.3 μmol/l were matched by gender and age with 29
children with Hcy levels ≤ 4.1 μmol/l. Our collected data in-
cluded weight, height, waist circumference, systemic arterial
pressure, lipid profile, fasting glycaemia and oxidative stress
markers. A Doppler ultrasound was performed to measure
the carotid intima media thickness and the endothelium-de-
pendent vasodilation of the brachial artery. Results: There
were no statistically significant differences between the
Received: July 15, 2012
Accepted after revision: February 28, 2013
Published online: July 20, 2013
Roseli Oselka Saccardo Sarni
Rua René Zamlutti, No. 94 Apt. 52
Vila Mariana
São Paulo, SP 04116-260 (Brazil)
E-Mail rssarni
@ uol.com.br
© 2013 S. Karger AG, Basel
0250–6807/13/0632–0025$38.00/0
www.karger.com/anm
Downloaded by:
201.69.104.142 - 7/22/2013 12:54:28 PM
SuanodeSouza etal.
Ann Nutr Metab 2013;63:25–31
DOI: 10.1159/000350314
26
sociation include oxidative stress, disruption to the me-
tabolism of nitric oxide, vascular remodelling, the toxic
effect of Hcy on the endothelium and the alteration of
protein structure due to hypomethylation and N-homo-
cysteinylation
[2] .
While normal values of plasma Hcy in adults range
from 5 to 15 µmol/l, the range of normal values for chil-
dren is currently unconfirmed
[3] . Some studies have re-
ported an association between higher Hcy concentrations
in children and adolescents and risk factors for CVD,
such as obesity
[4] and insulin resistance [5] .
A r t e r i o s c l e r o t i c d i s e a s e b e g i n s i n c h i l d h o o d a n d t h e n
progresses through adolescence into adulthood. Impor-
tant advancements in laboratory methods and diagnostic
imaging have enabled the early identification of such vas-
cular disorders in youth
[6] . T h e m e t h o d s m o s t f r e q u e n t l y
employed for the early detection of arteriosclerotic disease
include the measurement of several important blood mark-
ers, including high-sensitivity C-reactive protein (hs-
CRP), intercellular adhesion molecule-1 (ICAM-1), vascu-
lar cell adhesion molecule-1 (VCAM-1), asymmetric di-
methylarginine (ADMA) and oxidative stress markers (e.g.
malondialdehyde and glutathione). Arterial Doppler ultra-
sound can also be used to assess arteriosclerotic disease
[6] .
The aim of this study was to assess the presence of risk
factors for CVDs [alterations in the lipid profile, fasting
glycaemia, high arterial pressure values, oxidative stress,
increased intima media thickness (IMT) and impaired
endothelium-dependent vasodilation (EDV)] in prepu-
bertal children with elevated Hcy levels.
Methods
Study Design
This is a cross-sectional and controlled study conducted from
2008 to 2009 (11 months) at a public school in Santo André in São
Paulo, Brazil. The sample included 64 prepubertal children paired
by gender and age (n = 35 with Hcy ≥ 7.3 μmol/l and n = 29 with
Hcy ≤ 4.1 μmol/l, values corresponding to the 90th and 10th per-
centiles of Hcy plasma concentrations in the total school popula-
tion, respectively). The steps for including subjects are described
in figure 1 .
Children whose parents did not agree to participate were ex-
cluded from the study, as well as those children who exhibited sec-
ondary sexual characteristics (pubertal development) or malnutri-
tion and/or stunted growth. Children with a reported history of
chronic disease (endocrine, kidney, heart or liver disorders, im-
munodeficiency or rheumatologic diseases) were also excluded, as
were those who exhibited infectious or inflammatory conditions
at the time of the study. We also did not include children who were
using drugs or vitamin supplements that could interfere with the
metabolism of Hcy.
The Institutional Research Ethical Committees of the ABC
Medical School and Federal University of São Paulo approved the
study, and written consent for each subject was obtained from the
parents.
Clinical Evaluation, Arterial Blood Pressure and Pubertal
Stage
In stage 3 of the study ( fig.1 ), the parents completed a stan-
dardised questionnaire in which they reported general informa-
tion (personal, familial, gestational, history of past or present dis-
ease and any use of medication or vitamin supplements). A pae-
diatrician performed a complete physical examination, including
pubertal stage evaluations
[7] and a single measurement of arte-
rial blood pressure
[8] .
Nutritional Status and Waist Circumference
Weight and height were measured to classify each child’s nu-
tritional status using the World Health Organization (WHO)
Software (WHO Anthro, version 3.2.2, January 2011), which uses
the reference values suggested by the WHO in 2007
[9] . We con-
sidered a child with a body mass index (BMI) z-score > +1 to be
overweight/obese. Each child’s waist circumference (WC) was
measured and classified into percentiles according to gender and
age
[10] , and values above the 90th percentile were considered
high.
Laboratory Measurements
Blood was collected from the children (15 ml of blood) in the
morning after a 12-hour fast. The samples were immediately re-
frigerated at 4
° C and transported in an appropriate container, pro-
tected from light. Serum and plasma were then separated by cen-
trifugation (10 min at 4,000 r.p.m.) and analysed for the following:
• Hcy and cysteine (Cys): analysed according to the method sug-
gested by Pfeiffer et al.
[11] [high-performance liquid chroma-
tography (HPLC), fluorometric detection];
• Vitamins: vitamin B12 (competitive solid-phase immunoassay,
Immulite, Siemens
), folate (competitive liquid-phase immu-
noassay, Immulite, Siemens) and vitamin B6 (HPLC coupled
to ultraviolet detection)
[12] ;
• Lipid profile: total cholesterol, high-density lipoprotein choles-
terol (HDL-c), low-density lipoprotein cholesterol (LDL-c)
and triglycerides (colorimetric method, Express Plus Bayer
and kit Biotécnica
); for the lipid profile we used the cut-off
points recommended by the American Academy of Pediatrics,
2008
[13] ;
• Glycaemia: colorimetric method (Express Plus Bayer and
kitBiotécnica) was used, and the cut-off suggested by the
American Diabetes Association for fasting glucose was adopted
(adequate <100 mg/dl)
[14] , and
• Oxidative stress: the markers tested were thiobarbituric acid re-
active substances (TBARS) (spectrophotometry)
[15] , lipid
peroxides (ferrous oxidation-xylenol orange; FOX) (spectro-
photometry)
[16] , and the reduced (GSH) and oxidised (GSSG)
glutathione ratio, GSH/GSSG (HPLC coupled to electrochem-
ical detection)
[17] .
Doppler Ultrasonography
A vascular surgeon blinded to the groups performed a single
Doppler Ultrasonography in the morning between 10:
00 and
11:
00a.m. at a controlled and recorded room temperature (22–
Downloaded by:
201.69.104.142 - 7/22/2013 12:54:28 PM
Homocysteine and Cardiovascular Risk in
Prepubertal Children
Ann Nutr Metab 2013;63:25–31
DOI: 10.1159/000350314
27
24 ° C). A General Electric model Logiq 400 device, with a multi-
frequency transducer of 8.2–11 MHz, was used in B mode. Mea-
surement of IMT of the left and right common carotid arteries was
performed in the far wall, approximately 2 cm proximal to the
bifurcation
[18] . EDV was measured in the brachial artery at rest
and for 30, 60, and 90 s
[19] .
Statistical Analysis
The IBM SPSS statistical package for Windows version 19.0
(IBM Corporation
) was used to analyse the data. The normality
of continuous variables was assessed using the Shapiro-Wilk test.
The data on parametric variables are expressed as the mean and
standard deviation, and the nonparametric variables are expressed
using the median and range.
We used a χ
2 test to compare categorical variables, Student’s t
test for parametric variables, and a Mann-Whitney test for non-
parametric variables. Analysis of variance (ANOVA) with the
Tukey post-hoc test was used to compare EDV at the 30-, 60-, and
90-second time points within groups. The significance level for the
tests was established at 5%.
Results
Among children with Hcy ≥ 7.3 μmol/l, 20/35 (57.1%)
were female, the mean age was 8.6 ± 1.4 years, and the
household per capita income was 165.00 ± 107.00 USD.
These data did not differ significantly from those of the
group with Hcy levels ≤ 4.1 μmol/l ( table1 ).
PROO
PROO
0LVVHGVFKRROLQDVVHVVPHQWZHHN
,QIRUPHGFRQVHQWZDVQRWVLJQHG
&DUHWDNHUGLGQRWFRQVHQW
0LVVHGVFKRRORQEORRGFROOHFWLRQGD\
/RVVRIVDPSOH
3XEHUWDOGHYHORSPHQW
&DUHWDNHUGLGQRWFRQVHQW
0LVVHGVDPSOHFROOHFWLRQ
/RVVRIVDPSOH
3XEHUWDOGHYHORSPHQW
&DUHWDNHUGLGQRWFRQVHQW
0LVVHGVDPSOHFROOHFWLRQ
/RVVRIVDPSOH
Fig. 1. Description of the study case series.
Downloaded by:
201.69.104.142 - 7/22/2013 12:54:28 PM
SuanodeSouza etal.
Ann Nutr Metab 2013;63:25–31
DOI: 10.1159/000350314
28
The mean Hcy concentration for the group with Hcy
levels ≥ 7.3 μmol/l was twice the mean observed for the
group with Hcy levels ≤ 4.1 μmol/l (8.4 ± 1.0 vs. 3.8 ± 1.3
μmol/l; p < 0.001; table1 ).
Overweight/obese participants were more frequently
found in the group with Hcy ≥ 7.3 μmol/l when com-
pared to the group with Hcy ≤ 4.1 μmol/l [13/35 (37.1%)
vs. 4/29 (13.8%); p = 0.035; fig.2 ]. Only the triglycerides
plasma concentration was higher in the overweight/
obese children when compared to children with a nor-
mal BMI [10/17 (58.8%) vs. 9/47 (19.1%); p = 0.004]. All
children with a WC above the 90th percentile were
found in the group with Hcy ≥ 7.3 μmol/l [9/35 (25.7%)].
The average concentrations in the overweight/obese and
normal BMI groups were 7.3 ± 2.4 versus 5.8 ± 2.6 μmol/l
(p = 0.048) and 427.8 ± 77.7 versus 366.9 ± 72.1 μmol/l
(p < 0.001) for Hcy and Cys, respectively (data not
shown).
In the group with Hcy levels ≥ 7.3 μmol/l, the levels of
vitamin B12 were lower (597.7 ± 302.4 vs. 822.4 ± 332.5
pg/dl; p = 0.007) and the Cys levels were higher (417.8 ±
70.2 vs. 342.2 ± 66.8 μmol/l; p < 0.001) compared to the
group with Hcy levels ≤ 4.1 μmol/l ( table1 ).
Despite the higher prevalence of overweight/obese
children and large WCs in the group with Hcy levels ≥ 7.3
μmol/l compared to the group with Hcy levels ≤ 4.1
μmol/l, there was no difference in the frequency of altera-
tions in LDL-c [4/35 (11.4%) vs. 2/29 (6.9%); p = 0.681],
HDL-c [3/35 (8.6%) vs. 5/29 (17.2%); p = 0.451], triglyc-
erides [12/35 (34.2%) vs. 7/29 (24.0%); p = 0.422], glycae-
mia [8/35 (22.8%) vs. 4/29 (13.7%); p = 0.522] or mean
values of systolic and diastolic arterial pressure (data not
shown).
There was a statistically significant variation of EDV
between times 30–60 s and 30–90 s in both groups. The
peak variation of EDV occurred at 60 s (Hcy levels ≥ 7.3
μmol/l: –5.2%, 95% CI 2.9–7.4; p < 0.001, and Hcy levels
≤ 4.1 μmol/l: –5.2%, 95% CI 0.8–9.8; p = 0.024; data not
shown).
*
Table 1. General characteristics, nutritional status, vitamins, homocysteine and cysteine concentrations in chil-
dren with higher and lower Hcy levels
Variable Hcy ≥7.3 μmol/l
(n = 35)
Hcy ≤4.1 μmol/l
(n = 29)
p value
General characteristics
Gender (male:female), n 15:20 14:15 <0.428a
Age, years 8.6±1.4 8.5±1.4 <0.882b
Nutritional status
WC, cm 60.0 (50.0; 79.0)d56.0 (49.0; 71.0)d<0.001c
BMI, z-score 0.9±1.2 0.1±0.9 <0.001b
Vitamins, Hcy and Cys
Vitamin B12, pg/ml 597.7±302.4 822.4±332.5 <0.007b
Folate, ng/ml 14.2±5.15 16.2±5.2 <0.131b
Vitamin B6, nmol/l 30.9±5.4 29.7±5.4 <0.394b
Hcy, μmol/l 8.4±1.0 3.8±1.3 <0.001b
Cys, μmol/l 417.8±70.2 342.2±66.8 <0.001b
Values are means ± SD unless otherwise indicated. aLevel of significance by χ2 test. bLevel of significance by
Student’s t test. cLevel of significance by Mann-Whitney test. dMedian (minimum; maximum).
Fig. 2. Frequency of overweight/obesity in the groups. Level of
significance of χ
2 test.
Downloaded by:
201.69.104.142 - 7/22/2013 12:54:28 PM
Homocysteine and Cardiovascular Risk in
Prepubertal Children
Ann Nutr Metab 2013;63:25–31
DOI: 10.1159/000350314
29
Analyses adjusted for nutritional status did not show
statistically significant differences between the group
with Hcy ≥ 7.3 μmol/l and the group with Hcy ≤ 4.1 μmol/l
regarding the arterial blood pressure (systolic and dia-
stolic), lipid profile (HDL-c, LDL-c and triglycerides),
glycaemia and oxidative stress markers (FOX, TBARS
and the GSH/GSSG ratio) ( table2 ).
Neither the left and right carotid IMT nor the EDV at
30, 60 or 90 s exhibited statistically significant differences
between the groups after adjusting for nutritional status
( table2 ).
Discussion
The present study of prepubertal children found no
difference in the classical risk factors for CVD (dyslipi-
daemia, systemic blood pressure and alterations in gly-
caemia), oxidative stress markers or endothelial altera-
tions when comparing children with an Hcy level ≥ 7.3
μmol/l to those with an Hcy level ≤ 4.1 μmol/l. Only over-
weight/obese children were more frequently identified in
the group with higher Hcy levels.
Studies similar to ours have already reported the rela-
tionship of obesity to higher concentrations of Hcy in
children and adolescents
[4, 5, 20] . Hcy promotes athero-
thrombogenesis via a variety of mechanisms
[21] and
could increase the risk for future development of CVDs
in association with obesity.
Elevated Hcy levels are associated with dietary factors,
such as lower intake, as well as with concentrations of fo-
late and vitamin B12
[21] , and are responsive to treatment
with these vitamins
[22] . We did not find a deficiency of
vitamins involved in Hcy metabolism in either group ex-
cept for vitamin B12 levels, which were lower in the group
with elevated levels of Hcy. A possible explanation for the
Table 2. Analyses adjusted for nutritional status comparisons between arterial blood pressure, lipid profile, gly-
caemia, oxidative stress markers, IMT, and endothelial reactivity in children with higher and lower Hcy levels
Variable Hcy ≥7.3 μmol/l Hcy ≤4.1 μmol/l p value
Arterial blood pressure
Systolic arterial pressure, mm Hg 98.0±10.5 96.6±10.8 0.703a
Diastolic arterial pressure, mm Hg 63.7±12.0 64.8±15.0 0.825a
Lipid profile and glycaemia
HDL-c, mg/dl 47.5±7.8 50.2±11.1 0.323a
LDL-c, mg/dl 97.9±20.1 102.8±20.5 0.409a
Triglycerides, mg/dl 86.7±36.6 79.3±34.4 0.468a
Glycaemia, mg/dl 93.8±7.3 89.4±8.3 0.057a
Oxidative stress
FOX, μmol/l 50.4±13.7 53.3±15.9 0.516a
TBARS, nmol/ml 2.8±1.9 3.6±2.3 0.193a
GSH/GSSG ratio 57.4±30.2 47.3±25.6 0.222a
IMT assessment
Right carotid diameter, cm 0.68±0.6 0.65±0.3 0.109a
Left carotid diameter, cm 0.68±0.6 0.67±0.3 0.450a
Right IMT, mm 0.43 (0.4; 0.5)c0.40 (0.4; 0.5)c0.073b
Left IMT, mm 0.43 (0.4; 0.5)c0.43 (0.4; 0.6)c0.824b
EDV
Basal diameter brachial artery, cm 0.28±0.02 0.28±0.04 0.779a
Dilation 30 s, % 4.4±5.8 4.4±5.3 0.993a
Dilation 60 s, % 10.9±6.6 8.5±7.0 0.385a
Dilation 90 s, % 9.90±5.4 6.6±5.0 0.084a
Values are means ± SD unless otherwise indicated. aLevel of significance by Student’s t test. bLevel of sig-
nificance by Mann-Whitney test. cMedian (minimum; maximum).
Downloaded by:
201.69.104.142 - 7/22/2013 12:54:28 PM
SuanodeSouza etal.
Ann Nutr Metab 2013;63:25–31
DOI: 10.1159/000350314
30
appropriate folate levels is the mandatory fortification of
corn and wheat flours with folic acid in Brazil since 2004
[23] .
Despite the higher frequency of overweight/obese
children observed in the group with Hcy ≥ 7.3 μmol/l, an
association with other morbidities, such as increased ar-
terial blood pressure, dyslipidaemia and alterations of
glycaemia, was not observed, differently to the findings
of other studies
[4, 5, 20] . The following factors may be
considered to explain our results: the young age of the
investigated children, the low frequency of obesity com-
pared to other studies, the low concentrations of Hcy
probably due to mandatory fortification of flours and
the possible participation of other factors, such as Cys
[24] .
Cys, an amino acid involved in Hcy metabolism, has
recently been implicated as a possible factor related to the
negative outcomes associated with elevated Hcy levels. A
recent study
[25] performed on adults suggests that Cys
concentrations are related to BMI, especially when con-
sidering fat mass, insulin resistance, and the type of diet
consumed
[25] .
We found higher Cys levels in the group with Hcy ≥ 7.3
μmol/l (417.8 vs. 342.2 μmol/l; p < 0.001). These values
were even higher than those reported in studies per-
formed on adults (range 273–296 μmol/l)
[25] . The high-
er Cys concentrations observed might be due to the high
protein consumption by the Brazilian population, includ-
ing adolescents, as disclosed by the Pesquisa de Orçamen-
tos Familiares 2008/2009
[26] . No population-based
study has yet been performed assessing high Cys levels in
healthy children and adolescents, which limits the com-
parison of our results.
Endothelial dysfunction is the earliest detectable man-
ifestation of atherosclerosis, and several studies have de-
scribed greater impairment in obese children and adoles-
cents compared to their normal-weight peers
[27] . Peak
EDV occurs approximately 45–60 s after stimulation;
however, alterations in the arterial diameter can be ob-
served up to 10 min after stimulation
[27] . In the present
study, EDV exhibited a vasodilation peak at 60 s after
stimulation in both groups.
Few studies have related Hcy levels to endothelial as-
sessment in children. Zhu et al.
[28] compared obese
children to normal-weight controls and found higher
levels of Hcy (7.8 ± 2.8 vs. 5.5 ± 2.1 μmol/l; p = 0.001),
vitamin B12 and folate in the obese children compared
to the controls. However, an independent association
with greater IMT and impaired EDV was only observed
for BMI.
After adjusting for nutritional status, we did not find
differences in IMT and EDV between groups. This find-
ing is possibly due to a similarity in the frequency of mor-
bidities, oxidative stress and folate deficits in the group
with high Hcy levels compared to the group with low lev-
els.
It has been further postulated that folic acid and other
B vitamins have a direct effect on the endothelium. In
particular, some studies describe a mechanism in which
B vitamins improve the endothelial function by increas-
ing the availability of nitric oxide and tetrahydrobiopter-
in (a cofactor of nitric oxide synthase) and, thereby, their
antioxidant activity
[29] .
Epidemiological studies have reported that elevated
Hcy concentrations are associated with higher risks of
coronary heart disease, stroke and peripheral vascular
disease in adults and have been identified as a potentially
important risk factor for CVDs
[30] . A meta-analysis has
suggested that B vitamin supplementation may have a
significant protective effect on stroke
[31] . However, B
vitamin supplementation in adults is not effective in the
secondary prevention of CVDs
[32, 33] . Taking these re-
sults into account along with our own leads us to specu-
late that Hcy concentrations are part of an inflammatory
profile, which includes increases in the levels of CRP and
fibrinogen
[34] associated with the development of CVDs.
The study has several limitations. First, the small sam-
ple size of the cohort may have underpowered the ability
to detect alterations in some of the variables. In addition,
we did not examine insulin resistance or inflammatory
markers related to obesity.
Overall, the present study, which is the first on this
topic, failed to show an association between high Hcy
concentrations ( ≥ 7.3 μmol/l) in prepubertal children and
the presence of cardiovascular risk factors, oxidative
stress, and alterations in IMT and endothelial function.
Only overweight/obese children demonstrated an asso-
ciation.
Acknowledgements
For the financial support to develop this study, we would like
to thank Núcleo de Estudo, Pesquisa e Assessoria em Saúde
(NEPAS) da Faculdade de Medicina do ABC and Fundo de Am-
paro à Pesquisa do Estado de São Paulo (FAPESP).
Disclosure Statement
The authors report no potential conflict of interest.
Downloaded by:
201.69.104.142 - 7/22/2013 12:54:28 PM
Homocysteine and Cardiovascular Risk in
Prepubertal Children
Ann Nutr Metab 2013;63:25–31
DOI: 10.1159/000350314
31
References
1 Abraham JM, Cho L: The homocysteine hy-
pothesis: still relevant to the prevention and
treatment of cardiovascular disease? Cleve
Clin J Med 2010;
77: 911–918.
2 Perła-Kaján J, Twardowski T, Jakubowski H:
Mechanisms of homocysteine toxicity in hu-
mans. Amino Acids 2007;
32: 561–572.
3 Refsum H, Smith AD, Ueland PM, Nexo E,
Clarke R, McPartlin J, Johnston C, Engbaek F,
Schneede J, McPartlin C, Scott JM: Facts and
recommendations about total homocysteine
determinations: an expert opinion. Clin
Chem 2004;
50: 3–32.
4 Papandreou D, Mavromichalis I, Makedou A,
Rousso I, Arvanitidou M: Total serum homo-
cysteine, folate and vitamin B12 in a Greek
school age population. Clin Nutr 2006;
25:
797–802.
5 Martos R, Valle M, Morales R, Canete R,
Gavilan MI, Sanchez-Margalet V: Hyperho-
mocysteinemia correlates with insulin resis-
tance and low-grade systemic inflammation
in obese prepubertal children. Metabolism
2006;
55: 72–77.
6 Magnussen CG, Thomson R, Juonala M,
Viikari JS, Dwyer T, Raitakari OT, Venn A:
Use of B-mode ultrasound to examine pre-
clinical markers of atherosclerosis: image
quality may bias associations between adipos-
ity and measures of vascular structure and
function. J Ultrasound Med 2011;
30: 363–369.
7 Marshall WA, Tanner JM: Variations in pat-
tern of pubertal changes in girls and boys.
Arch Dis Child 1969;
44: 291–303.
8 National High Blood Pressure Education Pro-
gram Working Group on High Blood Pres-
sure in Children and Adolescents: The fourth
report on the diagnosis, evaluation, and treat-
ment of high blood pressure in children and
adolescents. Pediatrics 2004;
114(2 suppl 4th
report):555–576.
9 de Onis M, Onyango AW, Borghi E, Siyam A,
Nishida C, Siekmann J: Development of a
WHO growth reference for school-aged chil-
dren and adolescents. Bull World Health Or-
gan 2007;
85: 660–667.
10 Freedman DS, Serdula MK, Srinivasan SR,
Berenson GS: Relation of circumferences and
skinfold thicknesses to lipid and insulin con-
centrations in children and adolescents: the
Bogalusa Heart Study. Am J Clin Nutr 1999;
69: 308–317.
11 Pfeiffer CM, Huff DL, Gunter EW: Rapid and
accurate HPLC assay for plasma total homo-
cysteine and cysteine in a clinical laboratory
setting. Clin Chem 1999;
45: 290–292.
12 Sharma SK, Dakshinamurti K: Determination
of vitamin B6 vitamins and pyridoxic acid in
biological samples. J Chromatogr 1992;
578:
45–51.
13 Daniels SR, Greer FR, Committee on Nutri-
tion: Lipid screening and cardiovascular
health in childhood. Pediatrics 2008;
122: 198–
208.
14 American Diabetes Association: Standards of
medical care in diabetes – 2011. Diabetes Care
2011;
34(suppl 1):S11–S61.
15 Ohkawa H, Ohishi N, Yagi K: Assay for lipid
peroxides in animal tissues by thiobarbituric
acid reaction. Anal Biochem 1979;
95: 351–
358.
16 Gay CA, Gebicki JM: Measurement of protein
and lipid hydroperoxides in biological sys-
tems by the ferric-xylenol orange method.
Anal Biochem 2003;
315: 29–35.
17 Rogriguez-Ariza A, Toribio F, López-Barea J:
Rapid determination of glutathione status in
fish liver using high-performance liquid chro-
matography and electrochemical detection. J
Chromatogr B Biomed Appl 1994;
656: 311–
318.
18 Raitakari OT, Juonala M, Kähönen M, Tait-
tonen L, Laitinen T, Mäki-Torkko N, Järvisa-
lo MJ, Uhari M, Jokinen E, Rönnemaa T, Ak-
erblom HK, Viikari JS: Cardiovascular risk
factors in childhood and carotid artery inti-
ma-media thickness in adulthood: the Car-
diovascular Risk in Young Finns Study.
JAMA 2003;
290: 2277–2283.
19 Corretti MC, Anderson TJ, Benjamin EJ, Cel-
ermajer D, Charbonneau F, Creager MA,
Deanfield J, Drexler H, Gerhard-Herman M,
Herrington D, Vallance P, Vita J, Vogel R, In-
ternational Brachial Artery Reactivity Task
Force: Guidelines for the ultrasound assess-
ment of endothelial-dependent flow-mediat-
ed vasodilation of the brachial artery: a report
of the International Brachial Artery Reactivity
Task Force. J Am Coll Cardiol 2002;
39: 257–
265.
20 Gallistl S, Sudi K, Mangge H, Erwa W,
Borkenstein M: Insulin is an independent
correlate of plasma homocysteine levels in
obese children and adolescents. Diabetes Care
2000;
23: 1348–1352.
21 Tzotzas T, Evangelou P, Kiortsis DN: Obesity,
weight loss and conditional cardiovascular
risk factors. Obes Rev 2011;
12:e282–e289.
22 Toole JF, Malinow MR, Chambless LE, Spen-
ce JD, Pettigrew LC, Howard VJ, Sides EG,
Wang CH, Stampfer M: Lowering homocys-
teine in patients with ischemic stroke to pre-
vent recurrent stroke, myocardial infarction,
and death: the Vitamin Intervention for
Stroke Prevention (VISP) randomized con-
trolled trial. JAMA 2004;
291: 565–575.
23 Scorsatto M, Uehara SK, Luiz RR, de Oliveira
GM, Rosa G: Fortification of flours with folic
acid reduces homocysteine levels in Brazilian
women. Nutr Res 2011;
31: 889–895.
24 Moat SJ, Bonham JR, Cragg RA, Powers HJ:
Elevated plasma homocysteine elicits an in-
crease in antioxidant enzyme activity. Free
Radic Res 2000;
32: 171–179.
25 El-Khairy L, Ueland PM, Nygård O, Refsum
H, Vollset SE: Lifestyle and cardiovascular
disease risk factors as determinants of total
cysteine in plasma: the Hordaland Homocys-
teine Study. Am J Clin Nutr 1999;
70: 1016–
1024.
26 Pesquisa de orçamentos familiares 2008–
2009: análise do consumo alimentar pessoal
no Brasil/IBGE, Coordenação de Trabalho e
Rendimento [Survey of Family Budgets 2008–
2009: analysis of personal dietary consump-
tion in Brazil/IBGE (Brazilian Institute of Ge-
ography and Statistics – Instituto Brasileiro de
Geografia e Estatística), Coordination for
Work and Performance]. Rio de Janeiro,
IBGE, 2011, p 150.
27 Lamotte C, Iliescu C, Libersa C, Gottrand F:
Increased intima-media thickness of the ca-
rotid artery in childhood: a systematic review
of observational studies. Eur J Pediatr 2011;
170: 719–729.
28 Zhu W, Huang X, Li M, Neubauer H: Elevated
plasma homocysteine in obese schoolchildren
with early atherosclerosis. Eur J Pediatr 2006;
165: 326–331.
29 Verhaar MC, Wever RM, Kastelein JJ, van
Loon D, Milstien S, Koomans HA, Rabelink
TJ: Effects of oral folic acid supplementation
on endothelial function in familial hypercho-
lesterolemia. A randomized placebo-con-
trolled trial. Circulation 1999;
100: 335–338.
30 Nygård O, Vollset SE, Refsum H, Brattström
L, Ueland PM: Total homocysteine and car-
diovascular disease. J Intern Med 1999;
246:
425–454.
31 Lee M, Hong KS, Chang SC, Saver JL: Efficacy
of homocysteine-lowering therapy with folic
acid in stroke prevention: a meta-analysis.
Stroke 2010;
41: 1205–1212.
32 Huang T, Chen Y, Yang B, Yang J, Wahlqvist
ML, Li D: Meta-analysis of B vitamin supple-
mentation on plasma homocysteine, cardio-
vascular and all-cause mortality. Clin Nutr
2012;
31: 448–454.
33 Durga J, Bots ML, Schouten EG, Grobbee DE,
Kok FJ, Verhoef P: Effect of 3 years of folic
acid supplementation on the progression of
carotid intima-media thickness and carotid
arterial stiffness in older adults. Am J Clin
Nutr 2011;
93: 941–949.
34 Park CS, Ihm SH, Yoo KD, Kim DB, Lee JM,
Kim HY, Chung WS, Seung KB, Kim JH: Re-
lation between C-reactive protein, homocys-
teine levels, fibrinogen, and lipoprotein levels
and leukocyte and platelet counts, and 10-
year risk for cardiovascular disease among
healthy adults in the USA. Am J Cardiol 2010;
105: 1284–1288.
Downloaded by:
201.69.104.142 - 7/22/2013 12:54:28 PM
