Q J Med 2002; 95: 733-740
© 2002 Association of Physicians
Low-dose folic acid lowers plasma homocysteine levels in women of child-bearing age
From the 1 Coombe Womens Hospital, Dublin, Ireland, 2 Branches of Epidemiology and Statistics, NICHD/NIH, Bethesda, Maryland, USA, Departments of 3 Clinical Medicine and 4 Biochemistry, Trinity College Dublin, Ireland, 5 Biochemistry Department, Veterinary Sciences Division, Belfast, UK and 6 Health Research Board, Dublin, Ireland
Received 4 April 2002 Accepted for publication 15 July 2002.
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Summary |
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Background: Ongoing clinical trials are investigating whether lowering plasma homocysteine reduces the risk of vascular disease. If so, food fortification with folic acid will be the likely result, and sub-optimal amounts are likely to be preferred, for safety reasons. Dose-finding studies are needed before the outcomes of these trials, to establish the benefits and risks of folic acid consumption over the widest intake range likely to be encountered.
Aim: To find the lowest dose of folic acid that effectively reduces plasma homocysteine in premenopausal women.
Design: Double-blind, randomized placebo-controlled trial.
Methods: Women of child-bearing age (n=95) were randomly allocated to 0, 100, 200, or 400 µg/day of folic acid. Red-cell folate and plasma homocysteine were measured at baseline and after 10 weeks supplementation.
Results: Median red cell folate levels increased significantly in the 200 µg (p=0.0001) and 400 µg (p=0.0001) groups; but not in the placebo (0 µg) (p=0.25) or the 100 µg (p=0.5) groups. Only the 200 µg and the 400 µg groups had significant decreases in plasma homocysteine, (p=0.04 and p=0.0008, respectively). However, when subjects whose initial plasma homocysteine was <8 µmol/l (already optimally low) were removed from the analysis, there were significant plasma homocysteine decreases in all three treatment groups, but not the placebo group.
Discussion: In this sub-population, low doses of folic acid significantly lower plasma homocysteine. This could be achieved safely by fortification.
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Introduction |
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Elevated plasma homocysteine (tHcy) has been associated with chronic disease of the vasculature including peripheral vascular, cerebrovascular and coronary heart disease,1–3 as well as cognitive disease.4 New evidence also suggests an association of elevated plasma homocysteine with conditions affecting the vascular system in pregnancy, such as pre-eclampsia.5 Several intervention studies have confirmed that folic acid will reduce homocysteine.6–9 Large randomized clinical trials are now underway to determine whether folic acid supplements can diminish cardiovascular events by reducing plasma homocysteine. These prevention trials recruit patients with pre-existing cardiovascular disease. The risk of recurrence is high enough to justify the use of pharmacological doses of folic acid; low doses may be inadequate and the requirement for high efficacy over-rides residual safety concerns.
One of these trials has already demonstrated a significant reduction in the rate of re-stenosis after coronary angioplasty.10 If this result is confirmed, public health authorities could be presented with a situation regarding homocysteine lowering analogous to that which occurred 10 years ago in relation to folic acid supplementation for the prevention of neural-tube defects. At that time, women of high risk were recruited into randomized studies to determine the effect of high-dose folic acid supplementation on recurrence of neural-tube defects. The clear demonstration of a protective effect presented a public health dilemma. Dose-finding studies became unethical, and public health policymakers had to rely on historic evidence from small or non-randomized trials to aid them in choosing a safe but effective level of folic acid to recommend in a primary strategy for women capable of becoming pregnant. Similarly, positive outcomes from the current clinical trials would offer substantial potential for reducing the burden of chronic disease in developed countries, and public health policymakers will be placed under considerable pressure to fortify the food supply with folic acid. It is therefore crucial to determine the lowest effective dose of folic acid for homocysteine lowering, in advance of the outcomes of the clinical trials.
To date, only three dose-finding studies have considered the homocysteine-lowering effect of very low doses of folic acid.6,8,9 These recruited either elderly subjects,9 patients with cardiovascular disease8 or were not randomized.6 Two further randomized studies examined the effect of consuming cereal fortified with a low level of folic acid.11,12 It has recently been suggested that the efficacy of folic acid might vary with age, gender, etc. Thus a full picture would only emerge after studies on different sectors of the population. Women of child-bearing age are an important group to consider in developing recommendations, not only because of the effectiveness of folic acid in preventing neural tube defects, (which is not known to be mediated by homocysteine lowering) but also because of the recent reports linking elevated plasma homocysteine with increased rates of pre-eclampsia and other poor pregnancy outcomes.5,13 We have previously demonstrated that low doses of folic acid can significantly increase folate status in such women14 and could therefore conceivably reduce plasma homocysteine levels. We investigated whether doses of folic acid as low as 100 µg can reduce plasma homocysteine in women of childbearing age.
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Methods |
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Protocol
This was a double-blind randomized controlled trial, approved by the research ethics committee of the Coombe Women's Hospital, Dublin. The study methods have been previously reported in a trial that evaluated the ability of folic acid supplements over a six-month period to raise folate status in order to protect against neural tube defects.14 All female employees in the hospital were invited to be screened using red-cell folate levels (RCF). Of the total 396 female employees, 323 women took part. The follow-up invitation to take part in the intervention study was on the basis of the screening red-cell folate level. Fourteen women whose RCF was below 150 µg/l were judged to be clinically deficient and excluded. We excluded 137 women with RCF >400 µg/l, because an RCF of >400 µg/l is considered to reflect an adequate folate status. Thus, 172 women whose RCF was between 150–400 µg/l were eligible to be randomized between four study groups (Figure 1
). These were a cross-section of all the women working in the hospital, and were representative of the general working population. The average age of participants was 33.8±9.1 years (mean±SD). Written consent was obtained from all participants.
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Assignment
Randomization was achieved using random number assignment according to rank-ordered red-cell folate (RCF) values to ensure a similar distribution of screening values among the four groups. After randomization, a baseline blood sample was obtained from all participants. Since low vitamin B12 status could have affected the ability of folic acid to reduce plasma homocysteine, baseline plasma vitamin B12 and methylmalonic acid levels were measured on study participants. Participants were allocated to receive 0, 100, 200 or 400 µg folic acid tablets, once daily.
Blinding
Colour-coded blue, green, yellow and red tablet dispensers were situated in the hospital cafeteria. Each participant was asked to take one tablet from the assigned dispenser every day. All the tablets appeared identical and were to be taken with food. The participant indicated compliance by initialling a dated, colour-coded sheet. These sheets were changed every three days to prevent multiple recording of compliance. Additional tablets and compliance sheets were provided for non-work days. There was a seven-day placebo run-in period before the trial began, to monitor both compliance with tablet taking and adherence to the correct colour-coded dispensers for their treatment group. Women who did not comply during this run-in period were excluded from the study. Compliance was monitored throughout the study. Good compliance was defined as taking at least five tablets per week. Participants whose compliance was lower than this were contacted and encouraged to comply. Women were withdrawn from the study if they stopped taking tablets, decided to become pregnant, took non-study tablets, increased their dietary folate during the trial period, or took medications that might interfere with folate status. The homocysteine trial duration was 10 weeks, as this gave adequate time for the RCF to rise and the plasma homocysteine to respond to the additional folate. We measured RCF rather than plasma folate, as it is a more stable indicator of tissue folate status.15
Sample collection and analysis
Non-fasting blood samples were collected into EDTA tubes and processed within 2 h of collection. The samples were well mixed and a 1 ml aliquot was taken for packed cell volume (PCV) estimation. For RCF analysis, 0.1 ml of mixed whole blood was added to 0.9 ml of 1% ascorbic acid. This was incubated at room temperature for 40 min then stored at -20 °C until assayed by a microbiological method.16 The blood plasma samples were also stored at -20 °C until plasma homocysteine (tHcy) was measured by an automated fluorescence polarization method.17 The pre and post-trial samples for each participant were assayed in the same batch. Plasma vitamin B12 was measured on the baseline samples by microbiological assay.18 After other analyses were carried out, there was sufficient plasma to analyse baseline MMA levels in 68 subjects. This was carried out using a high-resolution GC MS method.19 All analyses were carried out by operators unaware of the status of the samples.
Data analysis
The conventional ‘intention-to-treat’ analysis of all randomized subjects was not possible, because final blood samples were not available for women who dropped out of the study. Thus, the primary analysis was a modified intention-to-treat analysis, in that it included all women, regardless of compliance, who completed the trial. The secondary analysis excluded all women whose starting tHcy was <8 µmol/l, since several studies have shown that homocysteine levels lower than this are unlikely to respond to additional folic acid.6,7,20,21 Non-parametric methods were used throughout. A Kruskal-Wallis test was used to analyse differences in baseline values both across the four treatment groups and for each treatment group versus placebo. A Jonckheere one-tailed test was then used to analyse dose-related trends in the post treatment change in RCF and tHcy.22 We chose a one-tailed test because we were not testing the hypothesis that folic acid lowers homocysteine, which is already established, but whether very low doses can produce this effect. Following this analysis, a signed rank test was used to evaluate whether differences between individual pre- and post-treatment values were significantly different from zero. Because the response to treatment was not normally distributed, median and inter-quartile ranges (seventy-fifth minus twenty-fifth percentile) are given for the data. The Fisher's Exact test was used for proportions.
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Results |
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Figure 1
summarizes the participant numbers, randomization assignments and exclusions. Reasons for not participating in the trial (n=51) or for exclusion before (n=11) and during the trial (n=15) are documented in Table 1. As reported previously,14 women excluded from the study had lower RCF levels at screening than those who completed the trial (p=0.003) and were less likely to have had a tertiary education. Ninety-five women completed the 10-week trial. There was no difference between groups in screening RCF or education level. Good compliance (defined as taking at least five tablets per week and following the protocol) was achieved in 53% of subjects, divided equally among the four groups. Tables 2a and 2b show the baseline RCF and plasma tHcy concentrations for all women who completed the trial (n=95) and for the sub-group whose initial tHcy was 
8 µmol/l (n=77). There were no differences in baseline values of RCF or tHcy across the groups.
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The primary analysis showed a significant trend of increasing RCF (p<0.0001) with increasing dose of folic acid. A significant trend of decreasing plasma tHcy (p=0.033) was also found in the overall group (Table 3a
). Similar results were obtained when those with plasma tHcy <8 were excluded from the analysis (p<0.0001 and p=0.028, respectively). Pre- and post-treatment tHcy concentrations for all subjects who completed the study are shown in Figure 2.
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Tables 3a and 3b
show the median (IQR) change in RCF and plasma tHcy post treatment in the four groups. Median red-cell folate levels increased significantly in the 200 µg (from 311 to 420 µg/l; p=0.0001) and 400 µg (350 to 484 µg/l; p=0.0001) groups, but not in the placebo (0 µg) (335 to 308 µg/l; p=0.25) or the 100 µg (309 to 358 µg/l; p=0.5) groups. Similarly, only the 200 µg and the 400 µg groups had significant decreases in plasma tHcy, (10.2 to 9.4 µmol/l, p=0.04; and 9.8 to 8.9 µmol/l, p=0.0008, respectively). However, when those whose plasma tHcy was <8 µmol/l were excluded, homocysteine fell significantly in all three treatment groups: 100 µg group, 9.0% reduction, p=0.008; 200 µg group, 9.6% reduction, p=0.005; 400 µg group, 15.3% reduction, p=0.0003.
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The effect of baseline folate was analysed by dividing the total group into those whose baseline RCFs were
250 and >250 µg/l. There was a significantly greater fall in tHcy among those whose baseline RCF was 250 µg/l (-2.77 µmol/l), compared to those whose initial RCF was >250 µg/l (-0.68 µmol/l) (p=0.009). Baseline vitamin B12 and MMA values were all within the normal range, having a mean±SD of 351±107 ng/l (n=93) and 0.19±0.065 µmol/l (n=68) respectively, excluding the possibility that our results might have been influenced by overt or functional vitamin B12 deficiency. In summary, the study data showed a trend of decreasing plasma homocysteine levels with increasing folic acid supplementation. When those whose plasma tHcy was above the optimal range were examined separately, even the lowest dose of folic acid, (100 µg/day) was associated with a reduction in homocysteine.
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Discussion |
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This double-blind, placebo-controlled, randomized trial shows that daily intake of low levels of folic acid can significantly reduce plasma homocysteine in women, especially in subjects with higher baseline levels. Although our data indicate that 200 µg folic acid is effective in reducing plasma homocysteine, even as little as 100 µg/day may have significant long-term effects. To date, no study of this type has been carried out specifically on women, perhaps because pre-menopausal women have lower homocysteine levels than age-matched men, and because the risk of cardiovascular disease is low in this group. Despite this background, there is increasing evidence that elevated plasma homocysteine may be associated with adverse pregnancy outcomes;5,13 thus it is important to know what level of folic acid intake will be effective in this relatively young population.
Of the various studies that examined the efficacy of folic acid in reducing plasma homocysteine, five have monitored the effect of daily doses between 100 µg and 400 µg.8 In a non-randomized intervention study in men, Ward et al.6 demonstrated that daily doses of 100 µg caused a drop in the plasma homocysteine of healthy male subjects over a six-week intervention period, especially in those with higher initial homocysteine, but for an optimal effect, 200 µg per day was required. In randomized trials that used fortified cereal as the vehicle for folic acid delivery, Malinow et al.12 reported no effect of an additional 127 µg of folic acid in cereal on the plasma homocysteine of coronary artery disease patients, while Schorah et al.11 found that 200 µg of additional folic acid in fortified cereal resulted in a significant fall in the plasma homocysteine of healthy volunteers. More recently, Wald et al.8 and Rydlewicz et al.9 demonstrated that at least 400 µg per day was necessary to elicit an optimal reduction of plasma homocysteine in patients with ischemic heart disease8 or in the elderly.9
The recent finding that 1 mg/day folic acid combined with vitamins B6 and B12 protects against restenosis after coronary angioplasty, strongly suggests that the association between high plasma homocysteine and cardiovascular disease is real and causal.10 If the ongoing clinical trials confirm this result, there will be considerable pressure on public health authorities to act on reducing the homocysteine in the general population. Supplementation with pharmacological doses of folic acid may be recommended to reduce the risk of disease progression or recurrence in patients with cardiovascular disease, but such doses would never be acceptable in a primary prevention strategy. The recent experience with folic-acid prevention of neural tube defects would indicate that food fortification is the only realistic solution, but it is unlikely that any sensible fortification program could add sufficient folic acid to provide optimal protection for those whose intake of the folic acid-containing staple food is low, while avoiding consumption of excessive amounts in high-intake individuals. Thus it is essential to know the plasma homocysteine responses of different population groups to the suboptimal levels of folic acid likely to be used in practice. These could be used to determine the likely public health benefit over the various ranges of fortification under consideration. A properly informed decision can then be made, balancing the public health benefit of disease reduction against the possible risks of ingesting large amounts of this non-naturally-occurring pro-vitamin.
Our population consisted of women of reproductive age, and the social mix reflected well those working in Irish society. This group would be a primary target in determining recommendations relating to the prevention of birth defects and the reduction of complications during pregnancy. Our previous study on these women demonstrated that an increased intake of 200 µg per day or even 100 µg per day over time, would improve red-cell folate status to a level that would likely be protective against neural tube defects.14,23 The present study confirms that these amounts would have an equivalent homocysteine-lowering effect. If the observed associations between elevated plasma homocysteine and diseases of the vasculature are proven to be well-founded, then women of child-bearing age would have further reason to maintain a high tissue folate status, in order to reduce the prevalence of conditions affecting the vascular system in pregnancy, such as pre-eclampsia.5
There seems to be a plasma homocysteine level below which additional folic acid will have no effect.6,7,20,21 When our subjects whose baseline plasma homocysteine was already low were removed, those who remained in the 100 µg group showed a significant drop in homocysteine over the study period. Furthermore, our trial and others7 have demonstrated that the fall in homocysteine is greater in those with low folate status. Consequently we believe it is necessary to stratify the effect of additional folic acid in terms of initial homocysteine. The marginal effect seen at the lowest dose level in our study may also reflect the lower overall baseline homocysteine levels of premenopausal women.
In summary, our data show that a dose of folic acid as low as 200 µg per day, and perhaps even 100 µg per day can produce a clinically important reduction in homocysteine over time. Cardiovascular disease is still the principal cause of mortality and morbidity in many countries. Other conditions affecting the vasculature, such as stroke, cognitive disease in the elderly and certain complications of pregnancy add a substantial burden of disease in developed countries. If lowering homocysteine turns out to have a beneficial effect in preventing or retarding cardiovascular disease progress, or other vascular-related illnesses, it is likely that a very small change in plasma homocysteine will have very large public health benefit. These results demonstrate that physiological doses of folic acid can be recommended for primary prevention trials of cardiovascular disease and other conditions postulated to be homocysteine-related.
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Acknowledgments |
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This study was funded by the NICHD, who aided in the interpretation of the data as well as the decision to submit the manuscript for publication. We wish to thank the staff of the Coombe Women's Hospital, Dublin Ireland, for their enthusiastic support for this trial and Clonmel Healthcare Ireland for a gift of the folic acid and placebo tablets. EU Demonstration Project BMH 4983549 and Abbott GmbH, Weisbaden-Delkenheim, Germany, contributed to homocysteine assays and Dr James Troendle kindly provided assistance with statistical programming.
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Notes |
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Address correspondence to Professor J.M. Scott, Department of Biochemistry, Trinity College Dublin, Dublin 2, Ireland. e-mail: jscott{at}tcd.ie
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References |
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