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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 non-

significantly reduced in the latter. Significant regression of carotid IMT was observed (0.961 0.092 to 0.933 0.077 mm, pb0.001)compared to significant IMT progression in the placebo group (0.9640.099 to 0.9840.094 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 compli-cations 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 effectof B12supplementation is much weaker resulting in a further

reduction of up to 5%, whereas B6 had no significant

influence [2]. Currently, several large, prospective, rando-

mized, placebo-controlled, clinical trials are underway to

investigate the effect of homocysteine-lowering therapy on

cardiovascular risk [3]. The results of the trials that havealready been published are controversial, raising the hypoth-

esis 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]. Intima-

media 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.

International Journal of Cardiology 143 (2010) 1619

www.elsevier.com/locate/ijcard

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

mailto:ntaiosgeorge@yahoo.grhttp://dx.doi.org/10.1016/j.ijcard.2009.01.023http://dx.doi.org/10.1016/j.ijcard.2009.01.023mailto:ntaiosgeorge@yahoo.gr

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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.

2. Methods

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 cyclos-

porine, methotrexate, fibrates and antiepileptics), impaired renal

function (glomerular filtration rateb60 ml/min), renal transplanta-

tion, 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 obtainedfrom 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

value was calculated from the remaining measurements. All IMT

measurements were assessed blindly by a single experienced

ultrasonographer.

2.3. Statistical analysis

The observed values in all continuous variables were found to be

normally distributed (assessed by Chi-square test) and hence, 2-tail

Student'st-test was applied for the statistical analysis of these data.

In particular, Fig. 1 presents the adjustment of the normal

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 thatthe probability density of these values can be reliably (pb0.05) approximated by the normal distribution curve.

Table 1

Clinical characteristics and biochemical parameters of the patients on

randomization.

Group I (n =53) Group II (n =50) p

Female gender (n %) 30 (56.6%) 27 (54%) 0.94a

Age (years SD) 73.2 4.6 73.9 4.3 0.99b

Body mass index (kg/m2 SD) 26.26 3.42 27.57 3.03 0.37 b

Coronary artery disease (n %) 17 (32%) 16 (32%) 0.99 a

Prior stroke (n%) 39 (73.6%) 33 (66%) 0.53 a

Arterial hypertension (n %) 36 (67.9%) 33 (66%) 0.99 a

Diabetes mellitus (n %) 19 (35.8%) 19 (38%) 0.98 a

Smoking (n %) 20 (37.7%) 20 (40%) 0.97 a

Statins 34 (64.1%) 36 (72%) 0.51a

Antiplatelets 48 (90.5%) 42 (84%) 0.62 a

Beta blockers 31 (58.5%) 34 (68%) 0.47 a

Calcium channel blockers 17 (32. 1%) 12 (24%) 0.56 a

ACE inhibitors/ARB 31 (58.5%) 24 (48%) 0.45 a

Homocysteine (mol/l SD) 13.9 4.9 14.1 4.9 0.84 b

Folic acid (nmol/l SD) 19.9 8.7 18.2 6.1 0.26 b

B12 (pmol/l SD) 321 319 352 329 0.63 b

Triglycerides (mg/dlSD) 160.291.7 172.457.1 0.43 b

Total cholesterol (mg/dlSD) 202.639.6 213.146.8 0.20b

LDL- cholesterol (mg/dlSD) 179.247.7 194.147.9 0.12b

HDL-cholesterol (mg/dlSD) 51.414.9 49.89.2 0.52 b

Creatinine (mg/dl SD) 0.94 0.26 0.95 0.28 0.85 b

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

Angiotensin-II Receptor Blockers, aChi-square test, bStudent'st-test.

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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 (pb0.05) approximated by

the normal distribution curve. Chi square test was applied for

categorical variables. All values were reported as meanstandard

deviation. The level of significance was set at 95% (pb0.05). The

statistical analysis of data was performed with SPSS 14.0 andMicrosoft Excel.

3. Results

The baseline clinical characteristics and biochemical parameters

of the patients are presented inTable 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 0.9330.077 mm,

pb0.001) compared to significant IMT progression in the placebogroup (0.9640.099 to 0.9840.094 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 B12and B6 as well) because it has been shown that the main

reduction of homocysteine is due to folic acid, whereas B12provides only little further decrease[2]. The limitations inour 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,

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 (IMT1 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 patientsafter 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 sig-

nificant 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 similarmanner, 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 treatedwith drugs that have been shown to decrease IMT, such as

statins, angiotensin-converting enzyme inhibitors, angioten-

sin-II receptor blockers and calcium-channels antagonists

[12].

Along with the aforementioned studies, our results add

further controversy on the impact of folic acidsupplementation

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. Arecent 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

sinceat least1990 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

Table 2

IMT, folic acid and homocysteine plasma concentrations on randomization

and after treatment.

Group I (n =53) Group II (n =50) p

Folic acid

Baseline (nmol/l SD) 19.9 8.7 18.2 6.1 ns

Follow up (nmol/l SD) 44.6 1.6 17.1 5.4 sp (baseline vs. follow up) s ns

Homocysteine

Baseline (mol/l SD) 13.9 4.9 14.1 4.9 ns

Follow up (mol/l SD) 11.2 3.6 14.5 4.9 s

p (baseline vs. follow up) s ns

Mean IMT

Baseline (mm SD) 0.961 0.092 0.964 0.099 ns

Follow up (mm SD) 0.933 0.077 0.984 0.094 s

p (baseline vs. follow up) s s

Overall difference (mm SEM) -0.0280.004 0.01980.0024 s

SD: standard deviation, SEM: Standard Error of the Mean, s: p b0.001, ns:

pN0.05.

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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 cardio-vascular complications after B-vitamins supplementation

[18,19]. However, these trials recruited mainly normoho-

mocysteinemic patients and it cannot be excluded that a

beneficial effect might have been observed if the studies

included solely hyperhomocysteinemic patients. The Homo-

cysteine 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 clinicalend 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 ofthe 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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