Is folic acid good for everyone?1,2
A David Smith, Young-In Kim, and Helga Refsum
Fortification of food with folic acid to reduce the number of neural
tube defects was introduced 10 y ago in North America. Many
countries are considering whether to adopt this policy. When forti-
to an increased intake of folic acid for each neural tube defect preg-
nancy that is prevented. Are the benefits to the few outweighed by
possible harm to some of the many exposed? In animals, a folic
acid–rich diet can influence DNA and histone methylation, which
leads to phenotypic changes in subsequent generations. In humans,
increased folic acid intake leads to elevated blood concentrations of
naturally occurring folates and of unmetabolized folic acid. High
killer cell cytotoxicity, and high folate status may reduce the re-
psoriasis, and cancer. In the elderly, a combination of high folate
levels and low vitamin B-12 status may be associated with an in-
creased risk of cognitive impairment and anemia and, in pregnant
women, with an increased risk of insulin resistance and obesity in
their children. Folate has a dual effect on cancer, protecting against
cancer initiation but facilitating progression and growth of preneo-
plastic cells and subclinical cancers, which are common in the pop-
ulation. Thus, a high folic acid intake may be harmful for some
people. Nations considering fortification should be cautious and
stimulate further research to identify the effects, good and bad,
caused by a high intake of folic acid from fortified food or dietary
supplements. Only then can authorities develop the right strategies
for the population as a whole.
Am J Clin Nutr 2008;87:517–33.
supplements, cancer, antifolates, cognition, epigenetics, public
Folate, folic acid, vitamin B-12, fortification,
In its recent report, “Folate and Disease Prevention,” the UK
mended (1) that mandatory fortification of flour with folic acid
should be introduced, with certain conditions, in the United
defects (NTDs). The report of the SACN provides an expert
assessment of available evidence, starting from the premise that
folic acid can play a role in disease prevention. We do not chal-
a different question: Is the benefit to the relatively few mothers
and children sufficient justification for exposing the entire pop-
by the SACN that ?77–162 NTD pregnancies would be pre-
of 300 ?g folic acid/100 g flour. Thus, between 370 000 and
780 000 people in the United Kingdom will be exposed to extra
folic acid for each infant saved. Can we be sure that, out of
three-quarters of a million people, less than one person will not
suffer serious harm, that ?100 people will not suffer intermedi-
ate adverse effects, and that ?1000 people will not suffer mild
It is now almost 10 y since mandatory folic acid fortification
was introduced in the United States and Canada. Is there any
evidence since then that an increased intake of folic acid might
have caused harm in some people? The purpose of this article is
selected observational studies that are consistent with possible
harm; it is not meant to be a systematic review of the evidence,
but rather to highlight issues for further discussion. We wish to
stimulate an open debate on these issues, with a view to better
informing policy makers in countries that are considering forti-
fication. We will argue that what is urgently needed now is
The plan of the article is as follows: after a brief review of the
biochemistry of folates and a comparison of folic acid with nat-
ural folates, we will look at the evidence that elevated concen-
trations of folates might cause harm in relation to anemia, cog-
nition, the balance between folate and vitamin B-12, natural
review the likely dual role of folate in cancer, followed by a
discussion of the interaction between folates and antifolate
and of histones plays a key role, will be surveyed, because we
a brief discussion of what blood concentrations of folate might
Department of Physiology, Anatomy & Genetics, University of Oxford,
Oxford, United Kingdom (ADS and HR); the Departments of Medicine and
Nutritional Sciences, University of Toronto, Division of Gastroenterology,
St Michael’s Hospital, Toronto, Canada (Y-IK); and the Institute of Basic
Medical Sciences, Department of Nutrition, University of Oslo, Oslo, Nor-
2Reprints not available. Address correspondence to AD Smith, Depart-
Oxford, OX1 3PT, United Kingdom. E-mail: firstname.lastname@example.org.
Received April 3, 2007.
Accepted for publication September 21, 2007.
Am J Clin Nutr 2008;87:517–33. Printed in USA. © 2008 American Society for Nutrition
by guest on March 29, 2014
BIOCHEMICAL ROLES OF FOLATES
Folates are cofactors and cosubstrates for biological methyl-
ation and nucleic acid synthesis and also function as regulatory
enzymatic reactions, not being tightly bound to the apoenzyme,
and carry one-carbon residues. In effect, folates are cosubstrates
for the reactions they are involved in. The intracellular concen-
trations of the different folates are in general much lower than
their Michaelis constant values for the enzymes, and so the rate
of cellular folate concentrations (4). Measurements of the
concentration of plasma total homocysteine, which reflects the
intracellular concentration of homocysteine, can be used as a
surrogate marker of the possible range. Homocysteine can
be converted to methionine by methionine synthase, and
donates the methyl group. The concentration of plasma total
?2 nmol/L to ?15 nmol/L (5, 6). Thus, over at least this range,
methylation potential of tissues of the body. There is no conve-
that this can also vary over a similar range of plasma folate
concentrations (4). For example, the misincorporation of uracil
into DNA, because of the inadequate biosynthesis of thymidine,
a folate-requiring step, is inversely related to the blood concen-
tration of folate (7).
ecules that exert allosteric effects on several enzymes in the
reductase (MTHFR), glycine-N-methyltransferase, and serine
is less well understood, but it will be influenced not only by the
affinity of the enzymes for folate but also by the steady state
concentration of folate in the cell (11).
many regulatory processes (9, 10), plasma concentrations of
folates clearly influence the cellular concentrations of folates.
Because many enzymes using folates have Michaelis constant
cell that use folates.
Folates enter mammalian cells as monoglutamates, but are
rapidly modified by the addition of 4–8 glutamate residues to
form long side chains. Polyglutamation greatly increases the
affinity of folates as both substrates of their own enzyme and
inhibitors of other enzymes in the folate pathway (8, 13). Poly-
glutamation also constitutes a mechanism to trap folates within
poorly accepted by the membrane carriers responsible for efflux
across the cell membrane (9). The consequences of very high
intracellular concentrations of folates are not known, but it may
be significant that many folate-requiring enzymes are inhibited
est increases in cellular concentrations of folates will activate
several folate-dependent enzymes, whereas large increases in
COMPARISON OF FOLIC ACID WITH NATURAL
and is called folic acid (pteroylmonoglutamate); it is different
diet because it is in the oxidized state and contains only one
conjugated glutamate residue (14). The folates that are used as
coenzymes and regulatory molecules in the body are all in the
reduced form (tetrahydrofolates; THF) and are mainly polyglu-
Folic acid has a substantially higher bioavailability than do
natural folates, being rapidly absorbed across the intestine (15).
Even in countries without mandatory fortification, some mem-
bers of the population, including infants, have detectable unme-
tabolized folic acid in their blood, probably because of the vol-
untary fortification of foods (16) or intake of supplements
containing folic acid (17). After mandatory fortification in the
women had unmetabolized folic acid in the blood (18). Another
the folates in the blood of persons whose total folates are ?50
nmol/L (19). Both of these studies were conducted in small
groups of people and need to be extended to larger populations.
Theoretically, folic acid could interfere with the metabolism,
cellular transport, and regulatory functions of the natural folates
that occur in the body by competing with the reduced forms for
example, the folate receptor has a higher affinity for folic acid
(DNA and RNA)
(DNA and RNA)
FIGURE 1. The methylation and folate cycles within the cytoplasm
(simplified). The methionine cycle is shown at the top left: its function is to
regenerate methionine from homocysteine (Hcy) so that the methyl group
(?CH3) can be donated, via S-adenosylmethionine (SAM), to the many
acceptors in the cell (DNA, proteins, lipids, and metabolites). The intercon-
nected folate cycles interconvert the different forms of reduced folates, and
thymidine. Enzymes: DHFR, dihydrofolate reductase; MS, methionine syn-
thase; MTHFD, 5,10-methylenetetrahydrofolate dehydrogenase; MTHFR,
5,10-methylenetetrahydrofolate reductase; TS, thymidylate synthase. Inter-
mediates: DHF, dihydrofolate; THF, tetrahydrofolate; 5Me, 5-methyl-; 10f,
10formyl-; SAH, S-adenosylhomocysteine; dUMP, deoxyuridine mono-
phosphate; dTMP, deoxythymidine monophosphate.
SMITH ET AL
by guest on March 29, 2014
might inhibit the transport of methyl-THF into the brain. Trans-
port of unmetabolized folic acid into cells can also occur via the
folate receptor, as well as by several transporters (20), but little
is known about the intracellular effects of folic acid itself. A
recent report has described the down-regulation of folate trans-
porters in the membranes of human intestinal and renal cells
cultured with excess folic acid (21).
to dihydrofolate and then to tetrahydrofolate, probably in the
enzyme, dihydrofolate reductase (DHFR), catalyzes both these
(22–24) and that the activity varies markedly between individu-
als (24). Thus, it is possible that the plasma concentration of
between individuals according to their DHFR activity.
reduce the methionine supply at a critical time and whether the
gene expression of the folate-dependent enzymes or influence
metabolic flux in the pathways involving one-carbon units.
IS THERE EVIDENCE THAT ELEVATED BLOOD
FOLATE CONCENTRATIONS MAY CAUSE HARM?
Fortification will raise the concentration of total folates in the
body, not just unmetabolized folic acid, above that occurring
with normal diets (25, 26). In a significant proportion of the
population, the concentrations are likely to be particularly high
because of dietary habits. These groups include children and the
their diet, and the increasing number who take multivitamin
limit for the intake of folate is not known, but it is usually con-
sidered to be 1 mg/d for adults (27), and there is no consensus
about a safe upper concentration of blood folate. Serum folate
concentrations ?45 nmol/L are often considered supraphysi-
ologic. After folic acid fortification, such supraphysiologic con-
centrations were found in 23% of the US population, including
43% of children aged ?5y and 38% of the elderly (26). There is
little research directed at the question: Do these high concentra-
tions of serum folate have any harmful effects? Some examples
of possible harmful effects are given below. For quantitative
to illustrate some diseases that might be influenced by folate
status in the body (Table 1).
Relation between folate and vitamin B-12: anemia and
The concern most commonly raised (28) is that high folate
concentrations will mask the hematologic signs of overt vitamin
B-12 deficiency and lead to a missed diagnosis and to the sub-
acute degeneration of the spinal cord; but, as discussed in the
trations of folate over a period of time might lead to undesirable
consequences in persons with a low vitamin B-12 status (28).
for methyl groups by the growing cells and further depletion of
nervous system (29).
fortification have been reported from the United States (51).
folate status had a 70% increased risk of cognitive impairment,
had an even higher risk of cognitive impairment (OR: 5.1; 95%
trations (52). These findings from a cross-sectional study are
consistent with earlier reports on subjects with vitamin B-12
deficiency that even low doses of folic acid may aggravate the
impairment increases with rising serum folate concentrations
(55). However, in contrast with “masking” of anemia, the new
findings (52) suggest that the high folate concentrations could
also advance hematologic symptoms. Thus, one has to consider
whether such high folate concentrations could impair normal
folate function, not only in nerve cells but in proliferating cells
One possible mechanism is that high concentrations of folic
acid might act as a folate antagonist after the first step in its
metabolism: conversion to dihydrofolate (Figure 1). Accumula-
tion of this folate derivative in its polyglutamated form inhibits
thymidylate synthase (56) and hence the formation of dTMP
required for DNA synthesis. Dihydrofolate also inhibits the
folate-requiring enzymes of purine synthesis (57). In rats, ad-
ministration of folic acid after partial hepatectomy temporarily
slowed DNA synthesis, a finding that was explained by a delay
in the normal elevation of thymidylate synthase and thymidine
kinase (58). Thus, folic acid may have a dual effect and either
inhibit or facilitate normal DNA synthesis by entering the folate
cycle outside the normal pathways.
fore, high concentrations of folic acid could also inhibit the
synthesis. In those with poor vitamin B-12 status, methionine
synthesis is already compromised, so this mechanism would
by Morris et al (52).
A prospective study in the United States, conducted after for-
questionnaires completed by ?2000 persons in Chicago, is as-
extra vitamin B-12 than in those consuming the Recommended
Dietary Allowance (RDA) for vitamin B-12. This is consistent
with the cross-sectional findings of Morris et al (52) that high
folate is a risk in persons with a low vitamin B-12 status. On the
other hand, other prospective studies on folate intake in the
United States (61) and on serum folate concentrations in Italy
(62) in the elderly found that low folate intake or status is a risk
SAFETY OF FOLIC ACID
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also possible that the harmful effect of high folate intake in the
Chicago study is related to a combination of folic acid fortifica-
A recent clinical trial showed that those who took folic acid (0.8
tests compared with those who took placebo (63). It is notable
that subjects were excluded from this trial if they had poor vita-
min B-12 status.
elderly in the cohort studied by Morris et al (52) with a high
all elderly in the United States is affected, then ?1.8 million
elderly might be at increased risk of cognitive impairment and
anemia because of an imbalance between folate and vitamin
B-12. In Canada, the proportion of elderly women that had high
serum folate (?45 nmol/L) and vitamin B-12 insufficiency
United Kingdom after folic acid fortification, then 25 000–
170 000 elderly would have this particular combination, and so
may be potentially at high risk of anemia and cognitive impair-
Maternal vitamin B-12 and folate
In India, most of the population eats a vegetarian diet. Such a
diet lacks not only vitamin B-12 but also often methionine. The
Pune Maternal Nutrition Study from India has shown a possible
adverse effect of high maternal folate status (65). It was found
that the children (aged 6 y) whose mothers, during pregnancy,
Furthermore, children at 6 y of age whose mothers had a com-
bination of high blood folate and low vitamin B-12 concentra-
tions during pregnancy were at greater risk of insulin resistance.
This important, though so far isolated, finding raises the possi-
bility that an imbalance between folate and vitamin B-12 during
an effect on DNA methylation (see section on epigenetics be-
Natural killer cell cytotoxicity
A report by Troen et al (18) studied an index of immune
function, NK cell cytotoxicity, in postmenopausal women in the
United States after folic acid fortification. NK cells are an im-
portant part of the nonspecific immune response and can kill
tumor cells and virally infected cells. In this study, the authors
found an inverse U-shaped relation between total folate intake
and NK cytotoxicity. Women in the bottom tertile of dietary
intake of folates (?233 ?g/d) who took daily supplements con-
whose dietary folate intake was ?233 ?g/d and who took ?400
?g/d in supplements had impaired NK cytotoxicity. Although
there was no relation between total plasma folates and NK cy-
totoxicity, there was a highly significant inverse linear associa-
tion between the amounts of unmetabolized folic acid in plasma
only important sources of folic acid are from fortified foods and
dietary supplements. These findings raise the hypothesis that
Some diseases that may be influenced by dietary folate status1
Incidence or prevalence in UK
(population: 60 million)
Effect of increasing
folate status Nature of evidence
Key recent references
Neural tube defects 800 pregnancies/y (1)2
Protective; may reduce NTDs by
up to 162/y (1)
Randomized trials of folic
acid; population studies
Colorectal polyps22% prevalence in 55–64-y-olds (30),
Dual effects: protective and
Prospective cohorts and
trial of folic acid
Colorectal cancer (31)
(32, 37, 38)
Prospective cohorts and
trial of folic acid
Antifolate drug use
?10 million prescriptions/y for
methotrexate; ?6 million
prescriptions/y for trimethoprim
May reduce efficacy of, or
increase resistance to,
methotrexate and theoretically
all other antifolates
(anticancer, antimalarial, and
Folic acid may modify response
Folic acid may modify response
In vitro studies of cancer;
clinical trials (see
Rheumatoid arthritis0.8% prevalence, 500 000 (41) Randomized trials (42)
Psoriasis 1.5% prevalence, 900 000 (43) Randomized trial(44)
Stroke 97 000/y (45) ProtectiveMeta-analysis of trials;
1Cancer incidence data from Office of National Statistics, 2004 (48). Heart disease was omitted because the evidence is conflicting (49, 50).
2This is the middle of an estimate made by Standing Advisory Committee on Nutrition, allowing for underreporting; the actual number of reported NTD
pregnancies averaged 472/y from 2000 to 2004 (1).
SMITH ET AL
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excess folic acid from supplements or fortification could impair
normal immune function. This hypothesis could, and therefore
should, be tested.
disease (49), the evidence from clinical trials is not consistent.
One trial found that folic acid (together with vitamins B-6 and
B-12) reduced the need for revascularization interventions in
patients who had had balloon angioplasty (66), whereas another
study found that a similar treatment (including 1.2 mg/d folic
restenosis in men, although there was a tendency in the opposite
direction for women, diabetics, and those with hyperhomocys-
teinemia (67). Results from 3 large trials of homocysteine-
ease have now been reported (68–70) with overall negative
results, except for a reduction in stroke (46, 69, 71). In 2 of the
trials, a trend for an increase in cardiac events was found in
patients treated with a combination of folic acid (0.8 mg/d) and
None of these trials has convincingly shown that folic acid and
other B vitamins will be beneficial for heart disease. Two of the
trials raise a note of caution about the use of folic acid (67, 70).
Because the trials to date have been underpowered to show any
effect on cardiac outcomes (72), we will have to wait for a
drawing conclusions about the safety, as well as appropriate
doses, of folic acid in the prevention of cardiovascular disease.
DOES FOLATE PLAY A DUAL ROLE IN CANCER?
Folate is critically required for cell division and growth be-
cause it is a cofactor in the de novo synthesis of purines and
thymidylate and thus in nucleic acid synthesis (Figure 1). It is
also required for DNA repair processes. In cancer cells, where
DNA replication and cell division occur at a rapid rate, removal
of folate or a blockade of its metabolism causes inhibition of
tumor growth. This is the basis of the use of antifolate drugs in
that epidemiologic evidence suggests that raised intakes or con-
centrations of folate protect against the development of several
folate status is associated with DNA strand breaks, impaired DNA
repair, increased mutations, and aberrant DNA methylation. Some
tation (76, 77). However, animal studies on colorectal cancer have
shown that the timing and dose of folate intervention are critical:
1) If folate supplementation is started before the establish-
ment of neoplastic foci, the development and progression
of the tumor is suppressed.
are established, it enhances their growth and progression
cells (Figure 2) (33, 73–75, 81).
Extension of the concept of a dual role for folate in carci-
nogenesis in humans is subject to all the usual caveats. The
FIGURE 2. Postulated dual modulatory role of folate in carcinogenesis. IEN, intraepithelial neoplasia.
SAFETY OF FOLIC ACID
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epidemiologic evidence of a protective role of folate intake
against the development of cancer (82–85) is largely based on
to multiple potential confounders (86). There have been fewer
studies are also subject to confounding, they are closer to the
disease process than food-frequency questionnaires. Accepting
conditions, we asked the question: Is there any evidence of a
harmful effect of elevated blood concentrations of folate in any
of the human studies?
cancer therapy by Farber followed the observation that admin-
istration of folic acid in the form of pteroyltriglutamate to chil-
dren with acute leukemia led to an acceleration of the disease
process (87). Some modern studies on folate concentrations and
cancer illustrate the complexity of the association. Whereas the
New York Women’s Health Study, carried out before fortifica-
associated with a reduced risk of colorectal cancer (88), a study
of Finnish male smokers found no significant association with
of rectal cancer in those with folate concentrations in the second
to fourth quartiles (89). A striking result has been reported from
a large population-based, nested, case-control study in Sweden:
a significant association was found between plasma folate con-
being protective and higher concentrations being related in a
bell-shaped manner to increased risk (90). The novel finding in
this study was that low folate status may inhibit colorectal car-
Some other recent studies also report associations between
elevated folate status and cancer. Another Swedish study found
an increased risk of prostate cancer in men older than 59 y who
women show a 19% higher risk of breast cancer in women who
reported taking supplementary folic acid (?400 ?g/d) and a 32%
intake (?660 ?g/d), particularly from folic acid–containing sup-
plements, than did other cohorts (?300–350 ?g/d), in whom a
protective association with folate intake was found (36, 75, 91). In
the Nurses’ Health Study, there was a trend for an association be-
tween ovarian cancer and increased intake of total folate (but not
dietary folate) in 80254 women followed-up for up to 22 y: the
in the bottom quintile was 1.21 (95% CI: 0.94, 1.63; P for trend
other nutrients (92).
More definitive evidence of beneficial or harmful effects of
term randomized trials, but most of these have not yet been
completed. Smaller trials for shorter periods have produced incon-
clusive results (82). A follow-up of a trial of folic acid (5 mg/d) in
pregnancy, although not originally designed to study cancer,
0.88, 4.72) (93). Two large trials of B vitamins in relation to
cardiovascular disease have also reported outcomes for cancer.
The NORVIT trial originally reported (94) a marginally sig-
P ? 0.08) in those treated with folic acid (0.8 mg) and vitamin
reported to be 1.22 (95% CI: 0.88, 1.70) (70), probably because
the national cancer registry was used for case ascertainment in
the latter report. A similar nonsignificant trend was reported for
(69), in which the RR was 1.36 (95% CI: 0.89, 2.8) in those
treated with a combination of 2.5 mg folic acid, 1 mg vitamin
B-12 and 50 mg vitamin B-6 for 5 y. It will be important to
monitor the future incidence of cancer in these trial subjects.
The results from the first randomized trial of folic acid for the
prevention of colorectal cancer in genetically predisposed pa-
tients (32) showed that treatment with folic acid (1 mg/d) for up
to 6 y did not prevent the recurrence of colorectal adenomas. On
risk of advanced lesions with a high malignant potential (RR:
explanation for this result is that folic acid might have promoted
the progression of already existing, undiagnosed preneoplastic
lesions (eg, aberrant crypt foci or microscopic adenomas) or
adenomas missed on initial colonoscopy in these patients at a
high risk of developing colorectal cancer. Another unexpected
than colorectal cancer was significantly increased in the folic
acid–supplemented group (P ? 0.02); this was largely due to an
excess of prostate cancer (P ? 0.01) (32). The mean age of the
study participants was 57 y (64% were men); therefore, it is
sor lesions in the prostate, which progressed more rapidly with
folic acid supplementation. Thus, overall, the randomized clin-
and preneoplastic lesions.
What is the effect of the dramatically increased folate status
tation on cancer incidence in the United States and Canada? To
address this important public health concern, Mason et al (34)
examined a temporal trend of colorectal cancer incidence in the
United States and Canada after fortification using 2 data sets
from these countries: the Surveillance, Epidemiology and End
Result registry and Canadian Cancer Statistics, respectively.
Their analysis demonstrates that, concurrent with folic acid for-
tification, the United States and Canada experienced abrupt re-
the 2 countries had enjoyed in the preceding decades. Absolute
rates of colorectal cancer began to increase in 1996 (United
States) and in 1998 (Canada) and reached a maximum in 1998
(United States) and in 2000 (Canada), and rates have continued
100 000individuals,ie,some15 000extracasesperyear.These
tion may have been wholly or partly responsible for the observed
rate of colorectal cancer screening endoscopic procedures do not
seem to account for this increase in colorectal cancer incidence.
SMITH ET AL
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However, because there was no control group and because it was
impossible to completely control for all potential confounders in-
between folic acid fortification and increased rates of colorectal
cancer in North America in the late 1990s.
for the apparent contradictions are also possible. For example, it
is biologically plausible that any effect of folate on carcinogen-
the patterns of these risk factors will differ between individuals.
Observational studies have identified many factors, apart from
morphisms in genes coding for enzymes related to one-carbon
metabolism (98–100). The genotype can change a protective
effect of folate into a harmful effect. For example, Ulrich et al
a reduced risk of colorectal adenomas in those homozygous for
thymidylate synthase. In contrast, the same folate intake was
associated with an increased risk in those homozygous for the
risk factors comes from a Norwegian study of the occurrence of
colorectum (102). In the entire cohort, the risk of adenomas was
inversely related to the red blood cell folate concentration, with
an OR of 3.05 (95% CI: 1.34, 6.96) for the bottom tertile versus
the top tertile, consistent with a protective effect of folate status
on colorectal cancer. Overall, there was no association between
the MTHFR 677C3T genotype and adenomas, but 2 distinct
high-risk groups were found when the cohort was divided into
those with folate intakes below and above the median and into
smokers and nonsmokers. In smokers with low folate, those
28.1), whereas in smokers with high folate the T allele was no
the CC genotype had a greatly increased risk (OR: 11.85; 95%
CI: 2.86, 49.1). Another study has shown that sex and the pres-
ence of a truncating mutation in the tumor suppressor gene ad-
enomatous polyposis coli (APC) interacts with folate intake in
against colon cancer in men without the mutation (APC?), but
compared with those in the bottom tertile. No such associations
were found for women. These findings are important, first be-
cause they could account for some of the discrepancies between
different studies of folate and colorectal cancer. Second, these
studies show that it is not justified to assume that the finding of
ily applies to all people within that population.
Overall, the evidence reviewed above provides cause for
concern that increasing folate levels in an entire population
may, in some people, increase the risk of cancer. More re-
that interact with folate in the prevention and promotion of
WILL INCREASING THE CONCENTRATION OF
BLOOD FOLATES MODIFY THE EFFECTS OF
Drugs designed to interfere with enzymes in the metabolic
malaria, psoriasis, and ectopic pregnancy.
From basic principles of pharmacology, it is clear that folates
should antagonize the effects of most antifolates (39), and, in-
deed, a folate derivative (folinic acid) is often given as an anti-
dote. However, rather little research has been directed at the
ify the efficacy of antifolate drugs. In a review about antifolates
and she expressed concern that folate may interfere with the
effectiveness of the antifolate treatment and possibly support
particularly because antifolates are widely used: for example,
there are ?10 million prescriptions for methotrexate alone each
tant in the treatment of childhood leukemias, of which there are
nearly 500 new cases each year in the United Kingdom.
There are indications from noncancer chemotherapy that a
patient’s folate status may influence the response to methotrex-
ate. Methotrexate is the most widely used disease-modifying
drug for the treatment of rheumatoid arthritis, which affects
?500 000 people in the United Kingdom (41). A post hoc anal-
ysis of 2 randomized trials found that patients who were taking
1–2 mg folic acid/d had a poorer clinical response to methotrex-
ate (42), and it has been reported that patients with higher con-
methotrexate (106). Supplementation with 5 mg folic acid/d re-
duced the effectiveness of methotrexate in the treatment of pso-
riasis in a randomized trial (44). Finally, a study of 50 women
with ectopic pregnancies treated with methotrexate found that
those with serum folate concentrations ?20.7 ng/mL had a
higher failure rate and needed more methotrexate (107). Al-
though studies with lower doses of folic acid are needed to see
whether the concentrations attained after fortification might an-
tagonize the effects of methotrexate, it is noteworthy that in 2 of
the reports, the poorer efficacy was related to higher blood con-
centrations of folates.
To date, there are few, if any, studies on how folate status
affects the incidence of conditions such as psoriasis and rheu-
matoid arthritis, including the seriousness or frequency of an
to folate concentrations. Such studies should be performed and
could provide critical data about whether folic acid fortification
is safe to use in sections of the population that are likely to use
Many antimalarial drugs are antifolates, and there is evidence
of the reduced efficacy of these drugs after high-dose (2.5–5
mg/d) folic acid treatment (108). This is one of the possible
population at high risk of malaria (eg, Tanzania), where malaria
was treated with antifolates, was associated with an increased
risk of severe illness and death, whereas no such increased risk
was found in children in Nepal, where there is little malaria and
the children were not treated with antifolates (109). It is not
SAFETY OF FOLIC ACID
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known whether lower doses of folic acid have any effect on the
risk of getting malaria and, if already infected, influences the
seriousness or frequency of an attack. We therefore suggest that
folic acid fortification is introduced.
An important problem with the use of antifolates in chemo-
therapy is the development of drug resistance (110). Resistance
can occur at many stages, from drug transport to increased ex-
pression of target enzymes or by metabolism of the drug. In-
creased folic acid levels could theoretically facilitate drug resis-
the cellular folate concentration is a determining factor in the
sensitivity of cells to antifolates (40). Folic acid, being in the
oxidized state, has to be reduced by DHFR before it can act as a
cofactor (Figure 1). Indeed, many antifolate drugs are inhibitors
of DHFR; therefore, folic acid will compete with them for the
drug resistance. Another mechanism of resistance to antifolates
is mediated by proteins of the multidrug resistance family; these
tions of folates and thus facilitate transport of antifolates out of
the cell (111). This finding led Hooijberg et al (112) to state the
following: “The existence of multi-drug resistance transporters
implies that folate supplementation is a double-edged sword,
of their hypothesis is shown in Figure 3.
In relation to proposals to fortify food with folic acid in the
United Kingdom, we can carry out a “thought experiment,” as
follows. The prevalence of psoriasis is 1.5% of the population
(41). Thus, ?1.4 million people in the United Kingdom have
these conditions. A rate of only 1 in 100 of those with psoriasis
or rheumatoid arthritis adversely affected by increased folate
simply, is it acceptable that for every NTD prevented, ?200
people might suffer from an increased severity of their psoriasis
or rheumatoid arthritis?
We conclude that there is some evidence that folates might
research is needed to see whether the expected blood concentra-
tions of folate after fortification have any influence on the re-
tion of whether increasing folate levels will influence the
incidence or natural history of the many diseases that are sensi-
tive to antifolates, ie, could persons with conditions such as
rheumatoid arthritis or psoriasis become dependent on using a
also raise the possibility that a low folate status may provide
natural resistance to malaria and that fortification, therefore,
could increase the risk of infection and the resistance to com-
monly used antimalarial drugs.
DOES FOLIC ACID HAVE ANY GENETIC OR
one-carbon units for purine and thymidine synthesis, and ade-
quate folate status is essential for nucleic acid synthesis and cell
division. Low folate status in humans is associated with the
an increase in DNA strand breaks (7). Another key role of folate
is in the provision of methyl groups for the conversion of homo-
cysteine to methionine, which is incorporated into proteins and
also has other important functions related to methylation reac-
requires N-formylmethionyl-tRNA, and folate is necessary for
the synthesis of the methionyl and formyl residues. Methionine
can be converted to the methyl-group donor S-adenosyl-
methionine (SAM), a molecule with many functions (113–115),
including methylation of cytosine residues in DNA (116) and of
are involved in regulating gene expression (119).
In the mammalian genome, methylation only occurs on cyto-
sine residues that occur 5' to a guanosine residue in a CpG dinu-
cleotide. CpG dinucleotides are enriched in “CpG islands,”
which are found proximal to the promoter regions of about half
the genes in the genome, but these are predominantly unmeth-
are normally heavily methylated. Methylation of the promoter-
related CpG islands can suppress gene expression by causing
chromatin condensation, whereas methylation of isolated CpG
dinucleotides in coding regions can lead to mutations because
methylcytosine residues are prone to hydrolytic deamination.
DNA methylation is important as an epigenetic determinant of
gene expression, in the maintenance of DNA integrity, in chro-
matin organization and in the development of mutations (120).
Errors in normal epigenetic processes have been called epimu-
tations, defined as epigenetic silencing of a gene that is not
normally silenced or epigenetic activation of a gene that is nor-
mally silent (121). It is now believed that epimutations might
the aberrant methylation of DNA and histones (122–125).
FIGURE 3. Therapeutic window for folate supplementation. With in-
creasing concentration of folates administered during chemotherapy, the
efficacy of single drugs or drug combinations may be improved; simulta-
neously, drug toxicity decreases. Overdose of folates, however, can induce
multiple drug resistance, which decreases drug efficacy. Reprinted with
permission from Hooijberg et al (112).
SMITH ET AL
by guest on March 29, 2014
methylation (126), but sometimes it leads to hypermethylation
substrate inhibition of the enzymes that make use of them, and
modeling studies suggest that high folate levels could, under
certain circumstances, have the same functional effect as low
folate status (4). Other modeling studies imply that DNA meth-
ylation may be relatively protected from changes in folate status
(11). More experimental evidence is needed on this point.
and humans is likely to be tissue-, site-, and gene-specific (77,
128). Changing the folate status in humans has been shown to
yet established whether alterations in DNA methylation after
changes in folate status are harmful in humans, for example, by
regulating the expression of oncogenes or tumor-suppressor
genes (77, 128–130). Often, both CpG island hypermethylation
and genome-wide hypomethylation are found in the same tumor
cells (128, 131). Hypermethylation of CpG islands in promoter
(epimutation), is as common in cancer as are mutations in these
genes (120). In human colonic mucosa cells, methylation of a
CpG island in the putative tumor suppression gene ESR1, which
This is consistent with a role of hypermethylation in aberrant
gene silencing, which could initiate neoplasia when both alleles
are silenced (132). Notably, in the mouse, CpG island DNA
methylation of ESR1in colonic cells can be increased in an age-
related and dose-dependent manner by increasing levels of
dietary folic acid (132). It is, of course, too simplistic to
assume that elevated folate status will inevitably lead to hy-
permethylation of tumor-suppressor gene promoters because
the control of methylation is complex, involving several dif-
ferent DNA-methyltransferases, histones, and other regula-
tory proteins (133–135). The key question is as follows: Does
elevated folate status increase the probability of such hyper-
An epigenetic effect of maternal diets rich in folic acid has
been elegantly shown in the agouti mouse (136, 137), which
displays marked phenotypic variation due to variable cytosine
the promoter is fully active, ectopic agouti expression occurs in
all tissues and results in mice with a yellow coat that have a
tendency for obesity, cancer, diabetes, and a short life. Feeding
acid (and also extra vitamin B-12) results in progeny that are
darkly mottled (Figure 4), leaner, and healthier and that have a
normal life span. These changes are paralleled by increasing
methylation of the promoter: methylated CpG sites occurred in
CpG sites were found in 80% of the cells in the mice with the
darkest coat color, who were born to dams fed the high-folate
tation and the degree of methylation and between methylation
and coat color (136, 137). Thus, merely supplementing a moth-
er’s already nutritionally adequate diet with extra folic acid,
vitamin B-12, choline, and betaine can permanently affect the
offspring’s DNA methylation at an epigenetically susceptible
locus and has a consequential impact on the phenotype.
A similar effect of a high-methylation maternal diet has been
shown on the expression of the AxinFused epiallele in mice:
suppression of the kinked tail was paralleled by increased CpG
methylation in the promoter (138). It was recently shown that
these diet-induced epigenetic changes can be transmitted to fu-
ture generations (139, 140), which led the authors to speculate
that “in light of the roughly 20-year generation time of humans,
our results suggest that current dietary habits may have an influ-
ence on grandchildren who will be born decades from now,
independent of the diets that their parents consume” (139).
Mice carrying teratogenic mutant genes can be protected by
supplying the mother with extra folic acid, even when their diet
is folate-replete (141). There is a long history of animal studies
showing that maternal nutrition can influence the physical and
cognitive health of the progeny (142, 143), but relatively few
studies have specifically looked at folic acid status. A widely
studied model is dietary restriction of protein in the pregnant rat
(144), whose offspring, for example, have a higher blood pres-
sure than do offspring of mothers fed a normal diet. Folic acid
supplementation of the low-protein diet of pregnant rats pre-
vented the elevated blood pressure in the offspring and also
prevented some of the adverse vascular changes (145, 146).
However, some undesirable consequences of folic acid supple-
mentation to pregnant rats have been found in their offspring:
when additional folic acid was fed to mothers on a normal-
protein diet, the blood pressure of the offspring was higher than
(145). When folic acid was fed to rats on a low-protein diet, the
brain concentrations of docosahexaenoic acid than offspring of
mothers on a control diet (147, 148). Persistent changes in the
phenotype of the offspring imply changes in gene expression
decreases in the degree of CpG methylation in the promoters of
Yellow (agouti) Mottled Heavily mottled
methylation of cytosine residues in the transposon promoter that regulates
diet, whereas the mottled mice received supplements containing extra folic
acid, vitamin B-12, choline, and betaine. The increase in methylation at the
agouti promoter locus was graded in the mice from left to right. Reprinted
with permission from Waterland and Jirtle (137).
SAFETY OF FOLIC ACID
by guest on March 29, 2014
genes for peroxisome proliferators–activated receptor ? and for
the glucocorticoid receptor occurred in the livers of offspring of
mothers who had been on a low-protein diet during pregnancy
found (150). The changes in DNA methylation were accompa-
nied by increases in the expression of both genes, as assessed by
mRNA concentrations. The addition of folic acid to the low-
protein diet prevented both the decreased CpG methylation and
the increased gene expression.
The animal studies reviewed here establish the principle that
varying the folate content of the diet of the pregnant mother can
dietary supplementation with folic acid can have similar epige-
netic effects in humans.
Clues might come from studies on imprinting in humans. Im-
printed genes are an example of an epigenetic phenomenon in
which an allele from one parent is preferentially expressed. An-
imal studies have led to the concept that the “marking” of these
genes in the sperm or ova can occur by specific methylation of
certain CpGs (151). These imprints control the epigenetic
changes that ensure monoallelic expression in the developing
been identified (152). Disregulation of imprinted genes can in-
fluence placental and embryonic development and later pheno-
type. Thus, it is important to identify environmental factors that
might influence imprinting. Numerous animal studies have es-
tablished that the preimplantation embryo is sensitive to envi-
ronmental conditions, both in relation to maternal diet and to in
ture media for mouse embryos can alter allelic methylation and
the expression of imprinted genes in the embryo and fetus (154,
155). Manipulation of human embryos in vitro also appears to
induce imprinting changes, as shown by the increased risk of
Beckwith-Wiedemann syndrome in children born after in vitro
fertilization (156). This syndrome is accompanied by epigenetic
alterations in 2 imprinted genes, LIT1 and H19 (157). It is not
known what components of the media might influence imprint-
ing, but, because of its role in DNA methylation, folate is one of
the likely candidates. More research is needed on which factors
used in assisted reproductive technologies can lead to these ef-
in lymphocytes, has been found in ?10% of the normal popula-
tion and was associated with hypomethylation of the differen-
ing for IGF2, H19, and SYBL1 in blood cells has been found in
patients with hyperhomocysteinemia due to renal failure (159).
Hyperhomocysteinemia leads to inhibition of methylation reac-
erful inhibitor of DNA methyltransferases (160). In these pa-
tients the abnormal biallelic expression could be converted to
monoallelic expression by administration of a high dose (15
mg/d) of 5-methyl-THF for 8 wk, which was accompanied by
increased methylation of the promoter regions of these genes.
Whether more usual concentrations of folate can influence epi-
genetic phenomena in humans is not known, but a recent report
from India is suggestive (65). These authors found that the 6-y-
old children of mothers who had high folate status and a low
vitamin B-12 status in pregnancy were more obese and had a
programming” of childhood features might be related to epige-
ilar to the folate-sensitive nutritional programming in animals
discussed above (149).
There is thus strong evidence that DNA methylation in divid-
ing cells and during development of the fetus is a dynamic pro-
cess that can be influenced by the folate or methylation status in
The dogma has been that DNA methylation state in such tissues
is laid down during embryonic development and is stable (161).
However, there is now evidence that this is not always the case.
An early report showed that DNA methylation in anterior pitu-
and lactation (162). The adult brain contains very high concen-
trations of DNA methyltransferase (163), whose expression can
emia can alter the rate of incorporation of methyl groups into rat
brain DNA (165). A diet low in folic acid induced hyperhomo-
cysteinemia in rats and was paradoxically associated with in-
creased CpG methylation of the differentially methylated do-
main of the H19 gene in brain (166). In neither of the 2 latter
studies is it known whether changes in DNA methylation oc-
curred in postmitotic neurons or in glial cells, which can divide.
There is, however, more direct evidence that DNA methylation
can occur in adult neurons. A cell culture study has shown that
depolarization of neurons, which leads to increased synthesis of
in the level of CpG methylation in the regulatory region of the
islands in the promoter region of the gene for reelin from the
onine to rats, and this was accompanied by a decreased expres-
sion of the gene as shown by a fall in the mRNA concentrations
(168). Because reelin is expressed exclusively in GABAergic
interneurons in the frontal cortex, this result is strong evidence
cells. Such a conclusion is supported by the finding that DNA
methyltransferase is expressed in cortical GABAergic neurons
(169) and that inhibition of DNA methyltransferase leads to
decreased methylation of specific CpG islands in the promoter
Striking evidence of the dynamic nature of DNA methylation
in neurons in vivo is provided by a series of studies on the
mechanism by which maternal behavior in the rat can influence
of the offspring (174–176). It was found that a highly specific
5'CpG site in the promoter of the gene for the glucocorticoid
displayed strong licking and grooming behaviors. The demeth-
ylation persisted into adulthood and was associated with in-
adulthood. Remarkably, these biological effects could be re-
SMITH ET AL
by guest on March 29, 2014
site (177, 178). The authors concluded that “DNA methylation
patterns are dynamic and potentially reversible even in adult
the estrogen receptor in the hypothalamus, in which maternal
grooming led to demethylation of the promoter in the pups that
was associated with increased expression of the estrogen recep-
also displayed the same kind of maternal grooming behavior as
their mothers had given them (179). The authors proposed that
“epigenomic changes serve as an intermediate process that im-
prints dynamic environmental experiences, such as variations in
parental care, on the fixed genome resulting in stable alterations
in phenotype” (176).
It is not just maternal diet and behavior that can influence the
offspring: it has been known for some time that the diet of the
well-being (142). It is thus of interest that Waterland et al (180)
have shown that the composition of the postweaning diet in the
rat can influence the degree of methylation of CpG sites in the
paternal allele of the Igf2 gene in the kidney, which leads to loss
of imprinting. These authors speculated that “persistent differ-
ences observed between formula-fed and human milk-fed indi-
viduals (142) are the result of epigenetic alterations induced by
subtle nutritional differences between human milk and infant
Methylation of carboxy-, histidine-, lysine-, and arginine-
residues in proteins, for which SAM is the methyl donor, has
wide-ranging effects on protein repair, protein targeting, signal
transduction, modulation of enzyme activity, RNA metabolism,
and transcription regulation (114, 118). Methylation of lysine
and arginine residues in histones plays multiple and complex
roles in the regulation of gene expression and also in epigenetic
silencing by promoting the formation of heterochromatin (117–
is the social avoidance that follows exposure to chronic defeat
increase in the dimethylation of lysine 27 in histone-3 in the
hippocampus (182). This methylation persisted for ?1 mo,
which led the authors to suggest that chronic stress can mark a
repressive state that cannot easily be reversed and that “hyper-
methylation may represent a stable stress-induced scar in the
effectively irreversible (183), recent work has shown that spe-
cific enzymes exist that can remove the methyl residues (118,
184, 185). The dynamic state of histone methylation raises the
methylation, there do not appear to be any studies that have
addressed this question for histones. However, in theory, folate
status could affect histone methylation by influencing both the
availability of SAM and the level of the product, S-adenosyl-
homocysteine, which shows strong product inhibition of many
methyltransferases (114). Further research is needed to test this
mediated by changes in the methylation status of DNA or his-
cern if applicable to humans because folate concentrations can
potential to cause long-lasting changes in the functioning of
critical organs, such as the brain.
Does folate status in the mother influence the child’s
it converts 5,10-methylene-THF, a precursor for nucleic acid
synthesis, into 5-methyl-THF, the substrate that provides the
The common MTHFR (677C3T) polymorphism is associated
show a shift in folate metabolism away from methyl group syn-
flavin adenine dinucleotide (187, 188). In Spain, the prevalence
of the TT genotype has reportedly approximately doubled in the
population since the introduction in 1982 of folic acid supple-
ments for women in early pregnancy (189, 190). The authors
speculated that infants with the T allele normally have a greater
chance of spontaneous abortion (191) because of elevated ho-
mocysteine concentrations in the mother (192) and that folate
supplementation stabilizes the MTHFR enzyme, lowers the ho-
mocysteine concentration, and reduces the risk of abortion, thus
leading to an increased proportion of children born with the T
allele. Although it has been suggested that the result may have
arisen from sampling bias (193), the finding is potentially im-
of the T allele are at increased risk of stroke (194), and it has
been suggested of other diseases, such as depression (195, 196),
(198–200), neural tube defects (201), some but not all cancers
(202, 203), and possibly Down syndrome (204, 205). These
considerations led Lucock and Yates (206) to suggest that folic
acid fortification and supplement use might be “a genetic time
bomb.” The first premise of this dramatic claim, that folic acid
use increases the proportion of children born with the T allele of
MTHFR, is as yet poorly documented and is clearly in urgent
need of further study. Studies of the MTHFR genotype frequen-
in countries planning fortification of food with folic acid.
Thus, saving fetuses that have a genetic constitution that fa-
vors abortion or nonsurvival could lead to children being born
with genotypes that favor increased disease during life. This
important question needs more research, but it is also an ethical
issue for which there is no easy answer.
Folic acid in pregnancy and twins
ceptual use of folic acid supplements increases the likelihood of
twin pregnancies. The consensus appears to be that some of the
earlier reports did not take into account those pregnancies that
followed assisted reproduction methods and that, for normal
pregnancies, there is no increase in twins (207, 208). It is there-
SAFETY OF FOLIC ACID
by guest on March 29, 2014
vitro fertilization has found a positive relation between blood
the United Kingdom at the same level in food as in the United
States, then an additional 600 twin births would occur in preg-
nancies resulting from assisted reproduction methods. Notably,
although twin births often are considered a positive event, twin
pregnancies are associated with an increased risk for both the
mother and the child. Twin pregnancies have a higher risk of
complications, including pregnancy-induced hypertension, ane-
have low birth weight, and to have greater perinatal mortality
special populations such as those undergoing in vitro fertiliza-
tion, may counterbalance the beneficial effect of folic acid for-
tification on NTDs.
WILL BLOOD FOLATE CONCENTRATIONS AFTER
FORTIFICATION BE ABOVE THE THRESHOLD TO
Although a safe upper limit of folic acid intake of 1 mg/d for
adults and 300–800 ?g/d for children, depending on age, has
been proposed by the Institute of Medicine in the United States
(27), there is no consensus about what blood concentrations of
?59 nmol/L might be associated with harm in a subset of the
elderly (52). However, this value was arbitrarily defined and
represents the beginning of the top quintile of folate concentra-
people of different ages and races has shown that the effect of
folic acid fortification on blood folate concentrations in the
United States is quite marked (26). The shift in folate concen-
trations after fortification in 1998 is shown in Figure 5. As can
children and the elderly, who consume large amounts of bread
supplements in the United States is very common; surveys sug-
(17, 18, 52, 60, 81, 211).
folate concentrations ?45.3 nmol/L. Ten percent of these chil-
dren had concentrations ?77.3 nmol/L. We can estimate the
intake of folic acid equivalents needed to achieve these concen-
trations from the formula provided by Quinlivan and Gregory
?780 ?g folic acid/d, ie, double the proposed tolerable upper
limit (300–400 ?g/d) for children of that age. It is striking that
10% are consuming ?1320 ?g folic acid/d, which is well above
blood concentrations were found in children aged 6–11 y; the
third highest concentrations occurred in those aged ?60y, of
these concentrations may cause harm, but it must be of concern
that such high concentrations occur, particularly in children at a
rapid stage of development, when it likely that epigenetic
changes are occurring in many tissues. A recent study in mice
showed that varying the methyl donor status of the postweaning
diet in mice could influence the methylation status and the ex-
pression of imprinted genes (180). Could the same thing happen
in young children?
The question of whether the higher folate concentrations that
occur in significant sections of the population after fortification
can cause harm needs much further research (51), as does the
question of whether the presence of unmetabolized folic acid in
the blood (18, 19) could interfere with folate-dependent metab-
olism. The studies reviewed above show that, in animals, many
crucial biological processes depend on methylation reactions.
Furthermore, dietary intake of methyl donors and/or the folate
status of the diet can influence these processes. In addition, in-
creasing evidence points to the harmful effect of an imbalance
between folate and vitamin B-12 status, something that is likely
to occur in vegetarians, certain ethnic minorities, and in the
elderly with vitamin B-12 malabsorption. Fortification was in-
troduced specifically to prevent NTDs, and we all believe that
improved folate status achieved by increasing folic acid intakes
influences biological processes related to the neural tube. How-
ever, we have to ask the following question: What other biolog-
ical processes are these concentrations of folate capable of in-
fluencing, and are the effects always beneficial or could they
sometimes be harmful?
The authors’ responsibilities were as follows—ADS: helped develop the
final manuscript; HR: helped develop the concept for the article and revised
the manuscript; and Y-IK: helped plan and revise the manuscript. No con-
flicts of interest were reported.
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