As a public health strategy to help prevent neural tube defect-affected pregnancies, enriched flour and pasta in the United States and Canada are being fortified with folic acid, and women are being advised to take supplementary folic acid around the time of pregnancy to ensure an adequate intake. But in spite of the recently published results of a public health campaign in China, the burden of proof that folic acid alone is responsible for a decreased risk of these birth defects rests on the results of a randomized controlled trial conducted by the Medical Research Council of the United Kingdom. Supporting evidence comes primarily from studies that have shown a protective effect of multivitamins containing folic acid as one of many nutrients. Based on a striking and potentially hazardous type of non-compliance observed in an earlier study in which a participant took multiple vitamin doses simultaneously and a suggestion of similar incidents in the Medical Research Council study, we conclude that a further randomized controlled trial of the protective effect of folic acid alone may be necessary.
From 1Bureau of Reproductive and Child Health,2 Cancer Bureau, Health Canada, Ottawa, Ontario, Canada, and 3Department of Biochemistry, London Health Sciences Centre, London, Ontario, Canada.
Address correspondence to: Linda A. Turner, Health Canada, Bureau of Reproductive and Child Health, HPB Building #7, P.L. 0701D, Tunney’s Pasture, Ottawa, Ontario, Canada K1A 0L2.
Submitted March 8, 2000; final version accepted September 1, 2000.
Editor’s note: See related editorial on page 141 of this issue.
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In the early 1990s, publication of the results of two randomized controlled trials convinced many people, including policy makers, that if women take folic acid, a B vitamin, beginning before conception, they will lower their risk of having a baby with a neural tube defect. By the end of the decade, this strategy to reduce these potentially serious birth defects — that occur in about one out of every 1,000 pregnancies — had been translated into mandatory fortification of food with folic acid in the United States and Canada and public education campaigns around the world urging women to take supplementary folic acid before becoming pregnant.
Recently, the results of a public health campaign in China were published in which women who reported having taken supplements containing only folic acid in the periconceptional period had lower prevalences of neural tube defect-affected pregnancies. 1 Before the publication of these results, the only evidence that taking supplementary folic acid alone had this effect — rather than as one component of a multivitamin or multivitamin/multimineral supplement — was derived from the results of one of the randomized controlled trials, that conducted by the Medical Research Council (MRC) in the United Kingdom, in which women who had a previous pregnancy affected by a neural tube defect were given 4 mg of folic acid daily, a very high level. 2 In the other randomized study, conducted in Hungary, 3 reduction in risk of a first occurrence was observed among women who took a multivitamin/multimineral supplement in the periconceptional period that contained 0.8 mg of folic acid — closer to the level found in multivitamin supplements available over the counter — in addition to several other nutrients. With the exception of the results of two earlier, but small, studies that provided suggestive, but inconclusive, evidence of a protective effect of 4 or 5 mg of folic acid alone, 4,5 supporting evidence has come primarily from nonexperimental 6–10 and non-randomized intervention studies. 11,12 These studies showed that a protective effect was achieved when women took multivitamins in the periconceptional period; these multivitamins contained folic acid at low doses, along with several other nutrients. When considering the type of supplement to recommend to women who have not had a previous pregnancy affected by a neural tube defect, a Health Canada working group recently concluded that it was unclear whether supplementary folic acid alone at doses available over the counter has the same proven protective effect as multivitamin supplements that contain folic acid along with other nutrients. 13
While the recent publication of the results of a large public-health campaign in China adds some support to the theory that lower doses of supplemental folic acid alone (in this case, 0.4 mg) can reduce the prevalence of neural tube defects, the evidence is not as conclusive as it would be if the women had been randomized to take or not to take folic acid around the time of conception. The evidence is also weakened because women were required to purchase the pills. The majority of women whose pregnancies were monitored were farmers or factory workers, with presumably low incomes. Women who could afford to purchase supplements and were willing to do so may have differed systematically with respect to unmeasured risk factors from women who could not afford supplements or from women to whom supplements were not offered. Dietary quality, which was associated with prevalence of neural tube defect-affected pregnancies in previous investigations, 14,15 was not measured. Women who agreed to purchase supplements may have had better diets than women who did not or may have differed on other unmeasured characteristics associated with reduced risk; all risk factors for neural tube defects are not known.
The Medical Research Council Study
For the United States Food and Drug Administration, the burden of proof of the effectiveness of folic acid alone in the prevention of neural tube defects has rested most heavily on the results of the MRC study. 16 Closer inspection of the results of the MRC study in conjunction with a review of earlier studies, however, revealed the possibility that the treatment groups in the MRC study may have been contaminated by an unusual but striking type of non-compliance. In the MRC trial, unlike the Hungarian trial in which missed capsules were merely counted, compliance with treatment regimens was measured by blood tests every 3 months following enrollment, which was before conception in most cases. Testing continued through the 12th week of pregnancy. In this multi-center, multi-national study, women were randomized to one of four treatment arms: 4 mg of folic acid alone (a level 10 times higher than the recommended daily intake and the amount being recommended through public education programs), multivitamins containing folic acid (at the same high dose), multivitamins not containing folic acid, and a “control” arm in which capsules contained iron as ferrous sulphate and calcium as di-calcium phosphate. There was no pure placebo group. Among women randomized to receive capsules containing folic acid alone or folic acid in addition to other nutrients, the authors reported that the 90th centiles of serum folate levels in blood drawn at the visit immediately preceding conception were close to 200 ng/ml. These values are exceptionally high — even for values at the 90th centile — and could indicate that some women took several previously missed capsules at the same time — to “catch up” — just before their blood test. We observed that values as high as 200 ng/ml are similar to the serum folate level of a woman participating in one of the earlier inconclusive studies who later confessed to having taken a large number of previously missed capsules (each containing 5 mg of folic acid) just before her blood test. 4 These authors reported that this woman’s serum folate level of 212 ng/ml was more than 10 standard deviations above the mean. (Her pregnancy ended in the spontaneous abortion of an anencephalic fetus).
Further evidence that serum folate levels as high as 200 ng/ml are likely the result of ingesting doses of folic acid well in excess of a single dose of 4 mg is shown in the results of several studies in which volunteers took doses of folic acid of between 2 and 15 mg per day. After 3 weeks of daily supplementation with 2 mg of folic acid, the serum folate level of non-pregnant female volunteers reached a mean level of 18.5 ng/ml (range 17.3 ng/ml to 20.26 ng/ml). 17 The mean level of 18.5 ng/ml is slightly less than half of the median level of 44 ng/ml reported among the participants in the MRC study who received supplements of 4 mg folic acid. 2 The initial mean serum folate level of participants in this study was 5.18 ng/ml, comparable with the median level of 5.0 ng/ml reported for participants in the MRC study who did not receive supplements containing folic acid, 2 evidence that the blood assay techniques in these two studies gave comparable results. In an earlier study a mean serum folate level of 51.0 ng/ml was observed among volunteers after 1 month of daily supplementation with 15 mg of folic acid per day, rising from an initial mean level of 6.8 ng/ml. 18 A mean serum folate level of 120 ng/ml — rising from an initial level of 4.49 ng/ml — was observed in another study in which investigators gave volunteers a daily dose of 15 mg of folic acid. The subjects in this study, however, had been instructed to take two doses simultaneously if an earlier dose had been missed. 19
Adverse Effect of Vitamin A?
If we consider that the high serum folate levels observed among women in the folic acid arms of the MRC trial indicate that some MRC participants attempted to “catch up” by taking missed capsules before their blood tests, we must also consider that a similar proportion of women in the other treatment arms did likewise. Women taking multiple doses simultaneously would have been exposed to very high levels of the nutrients contained in whatever capsules they had been randomized to take; and, among those who conceived close in time to the blood test, the embryo would also have been exposed to potentially harmful effects of some nutrients. Capsules given to women randomized to the two multivitamin groups contained vitamin A. If women in these treatment arms took multiple doses simultaneously just before the blood test, the possibility exists that embryos already conceived or conceived shortly afterward would have been exposed to high levels of vitamin A. High doses of vitamin A taken by women in early pregnancy have been found in previous investigations to be associated with an increased risk of several types of congenital anomalies. 20–22 Although the human teratogenic potential of high doses of vitamin A remains unclear with respect to the dose and timing of exposure associated with specific malformations, an array of congenital anomalies including spina bifida have been reported in several species of laboratory animals when exposed to high single doses of vitamin A during early gestation, with different anomalies associated with maternal exposure at different stages of gestation. 23,24
Although the prevalence of neural tube defect-affected pregnancies among women who received multivitamins in addition to folic acid (1.4%) was lower than that among women who received multivitamins without folic acid (2.6%), with a prevalence ratio of 0.51 (95% CI = 0.16 to 1.68), suggestive of a protective effect of folic acid alone, we cannot rule out chance. We could also expect, on the other hand, a lower prevalence of neural tube defect among the group of women who received multivitamins in addition to folic acid compared with the prevalence among women who received folic acid alone, because previous research has suggested that other components of multivitamins may also have a protective effect, 7 or may act together to produce one. 25 The prevalence of neural tube defects among offspring of women who received multivitamins in addition to folic acid (1.4%) was greater than that among offspring of women who received folic acid alone(0.7%), with a prevalence ratio of 0.49 (95% CI = 0.09 to 2.68); that is, there was a greater prevalence among women who received a combination of vitamins in addition to folic acid, contrary to expectations, but again, we cannot rule out chance. Moreover, when we compare prevalences of the total numbers of anomalies among offspring of women in these three treatment groups (excluding single gene defects), the findings are similar in the multivitamin groups with and without folic acid — at 4.8% and 5.3% — and these prevalences are again somewhat higher than the 3.0% observed for the folic acid alone treatment group. While this result also could have occurred by chance, it is also compatible with the possibility that an increased prevalence of neural tube defects and other anomalies among pregnancies of women in the multivitamin groups occurred because some women in these groups were exposed to high doses of vitamin A.
Iron and Calcium
Similarly, women in the control group who took multiple doses would have been exposed to high levels of iron and calcium. Accumulated evidence strongly suggests that supplemental iron can reduce plasma zinc and inhibit absorption of zinc from food, with the degree of impact on zinc status being dose-dependent. 26–29 Although the evidence is not as extensive as in the case of iron supplements, calcium supplements have also been shown to inhibit absorption of zinc in humans. 30 Several types of evidence support the likelihood of an association between maternal zinc status and neural tube defects in humans. For example, investigators have observed strikingly high rates of neural tube defects in populations where zinc deficiency is common, 31 an inverse association has been observed between maternal zinc intake and risk of neural tube defects, 32 and plasma zinc concentrations have been found to be lower among infants with neural tube defects or other congenital anomalies compared with levels among control infants. 33 In laboratory animals, transitory zinc deficiency on specific days during early gestation has been found to be associated with neural tube defects and other anomalies. 34,35
Again, the higher prevalence of neural tube defects and other anomalies among pregnancies of women in the MRC study randomized to receive supplements containing iron and calcium compared with women receiving folic acid alone could have occurred because of a protective effect of folic acid. This observation is also compatible, however, with the possibility that a proportion of women in this group took multiple doses simultaneously, thus exposing themselves to high levels of iron and calcium before their blood tests, and increasing their offspring’s risk relative to that among offspring of women who received folic acid alone.
General Population Comparison
We would be reassured if the observed prevalences of recurrence of neural tube defect-affected pregnancies in the folic acid treatment groups (at 0.7 and 1.4%) were lower than the recurrence rate for the underlying population. Participants in the MRC study, however, were drawn from populations in several countries and regions where risks of recurrence have been observed to vary between zero and approximately 6%. 36 Furthermore, it is possible that women who previously had a pregnancy affected by a neural tube defect who presented for care before conceiving a subsequent pregnancy — as did the majority of participants in the MRC study — may have been at lower risk of a recurrence than women with a previously affected pregnancy in the background populations.
While the above speculations do not prove the ineffectiveness of supplemental folic acid alone in providing protection against neural tube defects, a further randomized controlled trial may be necessary to settle the issue. Given that the randomized trial in Hungary showed a protective effect of multivitamins containing folic acid and the widespread publicity about the protective effect of folic acid alone that has followed publication of the MRC study results, clinicians likely would be reluctant to enroll patients in a placebo-controlled trial. While a trial comparing folic acid alone with a multivitamin that contains folic acid may be acceptable to clinicians, it is unlikely that enough conflict currently exists in the clinical community over which is the preferred “treatment.”37 Were a trial to be undertaken, however, the climate of belief in the benefit of folic acid would likely compel investigators to design the trial to answer the question of whether multivitamins provide an additional benefit to an assumed a priori protective effect of folic acid alone rather than whether folic acid alone provides protection. Although we have little upon which to base an estimate, an additional protective effect of multivitamins assumed to be small would support an argument that an unattainably large study size would be required to answer the question. The pragmatic solution for clinicians who have any doubt about the effectiveness of folic acid alone is to advise women planning a pregnancy to take a multivitamin that contains folic acid.
As a postscript, the example of the woman who confessed to trying to “catch up” with missed doses reminds us that in further trials to determine the efficacy of vitamin supplements in preventing disease, investigators should be mindful to caution participants not to take multiple doses. Also, women in the general population wishing to guard against neural tube defects by taking multivitamin supplements, should be clearly and rigorously cautioned not to take more than one supplement daily—no matter how many days have been missed—to avoid ingesting hazardously high levels of potentially teratogenic nutrients in the vulnerable period of early embryonic development.
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