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International Journal of Cardiology 143 (2010) 16 19

www.elsevier.com/locate/ijcard

The effect of folic acid supplementation on carotid intima-media thickness


in patients with cardiovascular risk: A randomized, placebo-controlled trial
George Ntaios , Christos Savopoulos, Dimitrios Karamitsos, Ippoliti Economou,
Evangelos Destanis, Ioannis Chryssogonidis, Ifigenia Pidonia, Pantelis Zebekakis,
Christos Polatides, Michael Sion, Dimitrios Grekas, Apostolos Hatzitolios
First Propedeutic Department of Internal Medicine, AHEPA Hospital, Aristotle University, Thessaloniki, Greece
Received 6 September 2008; received in revised form 17 December 2008; accepted 10 January 2009
Available online 8 February 2009

Abstract
Introduction: Observational studies have suggested a causal relationship between hyperhomocysteinemia and cardiovascular complications
such as stroke and ischemic heart disease. The Homocysteine Lowering Trialists' Collaboration has shown that daily administration of folic
acid can significantly decrease homocysteine levels up to 25%.
Aim of this study was to investigate the effect of daily supplementation of folic acid (5 mg) on IMT after 18 months of treatment in
patients with at least one cardiovascular risk factor.
Methods: We enrolled 103 patients with at least one cardiovascular risk factor who were randomized to receive either a daily dose of 5 mg
folic acid (group I, n = 53) or placebo (group II, n = 50) for 18 months.
Results: After 18 months of folic acid supplementation, homocysteine levels were significantly reduced in the active treatment group
compared to a non-significant increase in the placebo group. Folic acid levels were markedly increased in the former group and nonsignificantly reduced in the latter. Significant regression of carotid IMT was observed (0.961 0.092 to 0.933 0.077 mm, p b 0.001)
compared to significant IMT progression in the placebo group (0.964 0.099 to 0.984 0.094 mm).
Conclusion: Folic acid supplementation results in significant IMT reduction after 18 months in patients with at least one cardiovascular risk.
2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Atherosclerosis; Folic acid; Homocysteine; Intima-media thickness

1. Introduction
Observational studies have suggested a causal relationship
between hyperhomocysteinemia and cardiovascular complications such as stroke and ischemic heart disease [1]. The
Homocysteine Lowering Trialists' Collaboration has shown
that daily administration of folic acid can significantly
decrease homocysteine levels up to 25% [2]. The effect of B12
supplementation is much weaker resulting in a further
reduction of up to 5%, whereas B6 had no significant
Corresponding author. S. Kiriakidi 1, AHEPA Hospital, Thessaloniki,
54636, Greece. Tel.: +30 6972770288; fax: +30 2310993480.
E-mail address: ntaiosgeorge@yahoo.gr (G. Ntaios).
0167-5273/$ - see front matter 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.ijcard.2009.01.023

influence [2]. Currently, several large, prospective, randomized, placebo-controlled, clinical trials are underway to
investigate the effect of homocysteine-lowering therapy on
cardiovascular risk [3]. The results of the trials that have
already been published are controversial, raising the hypothesis that perhaps, homocysteine is just an epiphenomenon of
atherosclerosis and not a causative risk factor.
Carotid intima-media thickness (IMT) is a reliable marker
of early atherosclerosis and has been associated with
increased incidence of cardiovascular events [4]. Intimamedia thickness has been used extensively as a primary end
point in interventional trials which sought to investigate the
effect of antihypertensives [5] and hypolipidemic [6] drugs
on atherosclerosis.

G. Ntaios et al. / International Journal of Cardiology 143 (2010) 1619

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Fig. 1. Adjustment of the normal distribution (red curve) on the histogram of baseline IMT values. The application of chi-square test (X2) in this case shows that
the probability density of these values can be reliably (p b 0.05) approximated by the normal distribution curve.

The aim of this prospective, randomized, double blind,


placebo-controlled trial was to investigate the effect of daily
supplementation of folic acid (5 mg) on IMT after 18 months
of treatment in patients with at least one cardiovascular risk
factor.

value was calculated from the remaining measurements. All IMT


measurements were assessed blindly by a single experienced
ultrasonographer.

2. Methods

The observed values in all continuous variables were found to be


normally distributed (assessed by Chi-square test) and hence, 2-tail
Student's t-test was applied for the statistical analysis of these data.
In particular, Fig. 1 presents the adjustment of the normal

2.1. Study population


We enrolled one hundred and three patients who were followed
up in our Internal Medicine Department between October 2005 and
February 2008. Inclusion criteria were the presence of at least one
cardiovascular risk factor, such as diabetes mellitus, arterial
hypertension, coronary artery disease, dyslipidaemia, smoking
and previous ischemic stroke. Exclusion criteria were the use of
drugs which interfere with homocysteine levels (such as cyclosporine, methotrexate, fibrates and antiepileptics), impaired renal
function (glomerular filtration rate b 60 ml/min), renal transplantation, malignancy, pregnancy, vitamin supplementation and prior
carotid endarterectomy. The patients were randomized to receive
either a daily dose of 5 mg folic acid (group I, n = 53) or placebo
(group II, n = 50) for 18 months. The local ethics committee
approved the study protocol and informed consent was obtained
from all patients.
2.2. Carotid ultrasound
The measurement of IMT was performed by B-mode ultrasound
with a Siemens Sonoline Elegra system with a 7.5 MHz transducer.
For each patient, the mean IMT value was calculated from 6
measurements in each carotid artery in the longitudinal plane. These
measurements involved both the near and far carotid wall at the
carotid bifurcation, at the common carotid artery (1 cm proximal to
the bifurcation) and at the internal carotid artery (1 cm distal to the
bifurcation). The measurement of IMT at sites where carotid plaque
was present, was not taken into account and in these cases, the mean

2.3. Statistical analysis

Table 1
Clinical characteristics and biochemical parameters of the patients on
randomization.
Group I (n = 53) Group II (n = 50) p
Female gender (n %)
Age (years SD)
Body mass index (kg/m2 SD)
Coronary artery disease (n %)
Prior stroke (n %)
Arterial hypertension (n %)
Diabetes mellitus (n %)
Smoking (n %)
Statins
Antiplatelets
Beta blockers
Calcium channel blockers
ACE inhibitors/ARB
Homocysteine (mol/l SD)
Folic acid (nmol/l SD)
B12 (pmol/l SD)
Triglycerides (mg/dl SD)
Total cholesterol (mg/dl SD)
LDL- cholesterol (mg/dl SD)
HDL-cholesterol (mg/dl SD)
Creatinine (mg/dl SD)

30 (56.6%)
73.2 4.6
26.26 3.42
17 (32%)
39 (73.6%)
36 (67.9%)
19 (35.8%)
20 (37.7%)
34 (64.1%)
48 (90.5%)
31 (58.5%)
17 (32.1%)
31 (58.5%)
13.9 4.9
19.9 8.7
321 319
160.2 91.7
202.6 39.6
179.2 47.7
51.4 14.9
0.94 0.26

27 (54%)
73.9 4.3
27.57 3.03
16 (32%)
33 (66%)
33 (66%)
19 (38%)
20 (40%)
36 (72%)
42 (84%)
34 (68%)
12 (24%)
24 (48%)
14.1 4.9
18.2 6.1
352 329
172.4 57.1
213.1 46.8
194.1 47.9
49.8 9.2
0.95 0.28

0.94a
0.99b
0.37 b
0.99 a
0.53 a
0.99 a
0.98 a
0.97 a
0.51 a
0.62 a
0.47 a
0.56 a
0.45 a
0.84 b
0.26 b
0.63 b
0.43 b
0.20 b
0.12 b
0.52 b
0.85 b

SD: Standard Deviation, ACE: Angiotensin Converting Enzyme, ARB:


Angiotensin-II Receptor Blockers, aChi-square test, bStudent's t-test.

18

G. Ntaios et al. / International Journal of Cardiology 143 (2010) 1619

distribution curve on the histogram of baseline IMT values; the


application of chi-square test in this case shows that the probability
density of these values can be reliably (p b 0.05) approximated by
the normal distribution curve. Chi square test was applied for
categorical variables. All values were reported as mean standard
deviation. The level of significance was set at 95% (p b 0.05). The
statistical analysis of data was performed with SPSS 14.0 and
Microsoft Excel.
3. Results
The baseline clinical characteristics and biochemical parameters
of the patients are presented in Table 1. There were no statistically
significant differences between the two groups. After 18 months of
folic acid supplementation, homocysteine levels were significantly
reduced in the active treatment group compared to a non-significant
increase in the placebo group (Table 2). Folic acid levels were
markedly increased in the former group and non-significantly
reduced in the latter. Significant regression of carotid IMT was
observed at the end of the study (0.961 0.092 to 0.933 0.077 mm,
p b 0.001) compared to significant IMT progression in the placebo
group (0.964 0.099 to 0.984 0.094 mm) (Table 2).

4. Discussion
Our study confirmed the beneficial effect of folic acid
supplementation on homocysteine levels and demonstrated a
significant regression of IMT after 18 months of treatment in
patients with at least one cardiovascular risk factor. We chose
to supplement patients only with folic acid (and not with B12
and B6 as well) because it has been shown that the main
reduction of homocysteine is due to folic acid, whereas B12
provides only little further decrease [2]. The limitations in
our study were the modest sample size and the medium
duration of follow-up.
The impact of B-vitamins on IMT has been previously
investigated in certain patient groups with conflicting,
Table 2
IMT, folic acid and homocysteine plasma concentrations on randomization
and after treatment.
Group I (n = 53)

Group II (n = 50)

Folic acid
Baseline (nmol/l SD)
Follow up (nmol/l SD)
p (baseline vs. follow up)

19.9 8.7
44.6 1.6
s

18.2 6.1
17.1 5.4
ns

ns
s

Homocysteine
Baseline (mol/l SD)
Follow up (mol/l SD)
p (baseline vs. follow up)

13.9 4.9
11.2 3.6
s

14.1 4.9
14.5 4.9
ns

ns
s

Mean IMT
Baseline (mm SD)
Follow up (mm SD)
p (baseline vs. follow up)
Overall difference (mm SEM)

0.961 0.092
0.933 0.077
s
-0.028 0.004

0.964 0.099
0.984 0.094
s
0.0198 0.0024

ns
s
s

SD: standard deviation, SEM: Standard Error of the Mean, s: p b 0.001, ns:
p N 0.05.

however, results. Till et al. showed that supplementation


with B-vitamins (folic acid, B12 and B6) resulted in significant
reduction of IMT (1.50 0.44 to 1.42 0.48 mm) in patients at
risk for cerebral ischemia (IMT 1 mm) after 1 year of
treatment, whereas IMT increased in placebo-controlled
patients (1.47 0.57 to 1.54 0.71 mm) [7]. Similar results
were reported by Marcucci et al. in renal transplant patients
after 6 months of B-vitamin supplementation [8]. However,
both studies were modest in sample size (50 and 56 patients
respectively) and had a short follow-up (12 and 6 months
respectively). Recently, Vianna et al. demonstrated a significant IMT reduction in hyperhomocysteinemic, end-stage
renal disease patients after 2 years of intermittent (10 mg, three
times a week) folic acid supplementation [9].
However, the ASFAST trial failed to confirm the results
reported by Vianna et al, since they found no significant
change of IMT in 315 patients with chronic renal failure
compared to controls, after supplementation with high-dose
folic acid for a median duration of 3.6 years [10]. In a similar
manner, Fernandez-Miranda et al. reported that there was no
significant change of IMT in patients with coronary artery
disease treated with folic acid (5 mg) for 3 years compared to
controls [11]. It is interesting to notice that actually, there
was a reduction in IMT (0.71 0.23 to 0.69 0.20 mm) in the
active treatment group, which was not statistically significant
[11]; however, it would not be irrational to consider it as
clinically significant, since the expected change (due to
aging) of IMT over this 3-years' follow-up would be
progression [12]. Moreover, this IMT reduction in the active
treatment group would be possibly statistically significant if
the control group did not exhibit a similar IMT reduction
which was attributed to the fact that patients were also treated
with drugs that have been shown to decrease IMT, such as
statins, angiotensin-converting enzyme inhibitors, angiotensin-II receptor blockers and calcium-channels antagonists
[12].
Along with the aforementioned studies, our results add
further controversy on the impact of folic acid supplementation
on IMT. Obviously, the large, randomized studies of the
clinical effects of B-vitamin supplementation will provide a
definite answer whether hyperhomocysteinemia is indeed a
causative cardiovascular risk factor or just an epiphenomenon
in the atherosclerotic process [3]. However, the results of the
trials that already have been completed are also conflicting. A
recent meta-analysis by Wang et al. on eight randomized trials
of folic acid with stroke as a primary end point, showed a
significant reduction in the risk of stroke by 18% [13].
Similarly, the HOPE-2 reported that fewer patients assigned to
B-vitamins suffered a stroke compared to controls, although
the study yielded negative results regarding all other major
cardiovascular events [14]. Furthermore, a population-based
cohort study by Yang et al. demonstrated that the slowly
declining trend in stroke mortality rates which was observed
since at least 1990 in the United States and Canada, accelerated
significantly after 1998, when folic acid fortification of cereals
became mandatory [15]. On the contrary, there was no

G. Ntaios et al. / International Journal of Cardiology 143 (2010) 1619

improvement in the decline of stroke mortality in England and


Wales, where folic acid fortification is not mandatory [15].
Hence, it is plausible that the dietary supplementation of folic
acid via fortification could offer a long-term cardiovascular
protection [16,17].
On the other hand, the recently published WAFACS and
WENBIT trials reported no significant reduction in cardiovascular complications after B-vitamins supplementation
[18,19]. However, these trials recruited mainly normohomocysteinemic patients and it cannot be excluded that a
beneficial effect might have been observed if the studies
included solely hyperhomocysteinemic patients. The Homocysteine Lowering Trialists' Collaboration concluded that
the folic acid-mediated homocysteine reduction is greater at
higher pretreatment homocysteine concentrations [2].
Hence, if these trials recruited only hyperhomocysteinemic
patients, it would not be unreasonable to expect greater
homocysteine reduction and thence, greater and possibly
significant reduction in primary and/or secondary clinical
end points [20].
Summarizing, our study reported a significant IMT
reduction after 18 months of folic acid supplementation in
patients with at least one cardiovascular risk. Further studies
with larger sample size and longer follow up are necessary to
provide definite answers about the effect of folic acid on
IMT.
Acknowledgements
We would like to express our gratitude to Dr. J.F.
Moschonas for his valuable assistance in the preparation of
the manuscript.
The authors of this manuscript have certified that they
comply with the Principles of Ethical Publishing in the
International Journal of Cardiology [21].
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