Folic acid fortification refers to the addition of folic acid to the food supply — most commonly through the enrichment of wheat, rice, and corn — in an attempt to reduce the potential consequences of folate deficiency. Fortification became mandatory in the United States and Canada in 1998, and today over 80 countries follow similar practices.^[32][33]

The rationale for fortification was to prevent NTDs — a group of potentially fatal birth defects that can occur within the first four weeks of pregnancy, particularly in the context of folate deficiency. To reap the benefits, a woman ideally needs to start supplementing at least 4 weeks before conception, and because many pregnancies are unplanned, fortification ensures that women are universally exposed to folic acid. Observational research suggests that countries that introduced mandatory fortification have seen 30 to 60% reductions in the rates of NTDs.^{[32][34][35][36]} Compared to regions without fortification, rates of NTDs are generally observed to be lower in countries with fortification; however, differences in monitoring strategies between countries make it difficult to draw accurate conclusions.^[34]

Fortification may have had other positive consequences, including reductions in rates of anemia and reduced levels of homocysteine, which may have translated to a small reduction in the rate of strokes.^{[37][36][38][39][40]}

Alternatively, fortification may have led to some unintended negative health consequences. Folic acid fortification has been associated with an increased risk of cognitive impairment in older adults with concurrent low vitamin B12 levels.^[41][42] This could be because folic acid can mask the clinical signs of a vitamin B12 deficiency, meaning the deficiency may go untreated, potentially leading to irreversible cognitive impairment in the long term.^[41]

Additionally, when folic acid fortification was being implemented in North America between 1996 and 1998, a short-term increase in the rate of colorectal cancer was observed (approximately 4 to 6 additional cases per 100,000 people).^[43] While research has generally found folic acid intake to have a neutral or beneficial effect on colorectal cancer risk,^[44][45][46] there is evidence that high intakes of folate might exacerbate preexisting precancerous lesions.^[47][48] However, colorectal cancer rates returned to normal after 1998, which calls into question whether fortification was truly the factor driving the short-term increase.^[49]

Lastly, there is concern that increased exposure to unmetabolized folic acid (UMFA) at a population level may have negative health consequences. UMFA occurs when the amount of folic acid ingested exceeds the body's ability to convert it to a usable form of folate, and UMFA has been found in the serum of nearly all Americans post-fortification.^[50] Currently, there is very little evidence for or against the safety of UMFA and further research is needed.

Folate deficiency can occur for several reasons, including:^{[26][2][94][17]}

• Insufficient dietary intake
• Certain medications (e.g., methotrexate, antiepileptics, oral contraceptives)
• Impaired absorption (e.g., celiac disease, gastric bypass, alcohol use disorder)
• Pregnancy (due to an increased biological demand for folate)
• Genetic polymorphisms (e.g., MTHFR, DHFR)

A folate deficiency can be diagnosed by doing a blood test, as the primary clinical sign is a type of anemia characterized by abnormally large and underdeveloped red blood cells (megaloblastic anemia).^[17] Symptoms of deficiency can include fatigue, poor concentration, increased heart rate (tachycardia), irritability, dizziness, pale skin, and a painful red tongue.^[2]

L-methylfolate, also known as methylfolate or 5-methyltetrahydrofolate (5-MTHF), is the primary active form of folate found in the body, and it directly participates as a coenzyme in metabolic pathways. Since L-methylfolate does not require enzymes like DHFR and MTHFR for activation (unlike folic acid), it has been suggested as an alternative way to supplement folate, particularly in people with genetic mutations that reduce the effectiveness of these enzymes.^[33]

Research suggests that L-methylfolate supplementation can increase plasma and red blood cell folate levels to a similar or greater extent than folic acid when given at equivalent doses and can comparably reduce plasma homocysteine levels.^{[102][103][104]} Additionally, unlike folic acid supplementation, L-methylfolate is both less likely to mask a vitamin B12 deficiency and avoids the unknown potential consequences of unmetabolized folic acid in the body.^[105]

Despite the promising features of L-methylfolate, folic acid has been used in the vast majority of clinical trials, and it hasn’t been established that L-methylfolate provides the same benefits as folic acid — including for the prevention of NTDs. An exception to this is folate supplementation for depression: there is evidence to suggest that L-methylfolate may be more effective than folic acid.^[4][5]

It has been hypothesized that autism spectrum disorder (ASD) may have a connection with folate insufficiency or excess, but the evidence is mixed and largely inconclusive.

Folate is undoubtedly crucial for fetal neurodevelopment during early pregnancy. Folic acid supplementation during pregnancy, often in the form of a multivitamin, has generally been associated with neutral to beneficial effects on ASD risk.^[62][63] However, both high and low levels of maternal serum folate during pregnancy have been associated with an increased risk of ASD.^[64][65]

Lending support to the theory of a connection between folate and ASD, a 2021 meta-analysis of observational studies found that children with ASD were 19 times more likely to have cerebral folate receptor autoantibodies, which could indicate a reduced capacity to transport folate into the brain.^[66] Additionally, autism is associated with an increased likelihood of MTHFR polymorphism, suggesting impaired folate metabolism.^[67]

While further research is needed to understand the relationship between folate and autism, it seems fair to suggest that folic acid supplementation at appropriate doses during pregnancy may have a neutral to beneficial effect on the risk of ASD.

Folic acid may have a role in managing depression for some people when taken along with prescription antidepressants. Still, more research is needed to understand this effect and what form of supplemental folate may be most effective.

Mechanistically, folate is involved in the generation of S-adenosylmethionine (SAMe), a compound that is important for synthesizing neurotransmitters like dopamine, norepinephrine, and serotonin. Additionally, low folate levels may lead to high homocysteine, which has been positively correlated with depression symptom severity.^[30] Although research has generally been mixed regarding whether depression is associated with reduced folate status,^[68][69][30] folate deficiency has been associated with an increased risk of depression and longer, more severe episodes of depression.^[30]

Folic acid taken along with antidepressant medications, specifically selective serotonin reuptake inhibitors (SSRIs) or serotonin and norepinephrine reuptake inhibitors (SNRIs), may reduce depression symptoms and increase rates of remission compared to antidepressants alone.^[70][9][71] However, supplementing with L-methylfolate (an active form of folate that can cross the blood-brain barrier) may be more effective than folic acid.^[4][5]

Importantly, there is insufficient research to suggest that folic acid or L-methylfolate taken alone is effective for depression or to determine whether folic acid or L-methylfolate are effective when taken with other types of antidepressants (e.g., tricyclic antidepressants).

Folic acid supplementation may have minor benefits on blood glucose regulation, but these effects are probably not clinically relevant. Research suggests that folic acid may slightly reduce fasting blood glucose, fasting insulin, and HOMA-IR (a marker of insulin resistance), but not in people with type 2 diabetes. Additionally, folic acid does not appear to affect hemoglobin A1c.^[72][73][74]

Folic acid does seem to have anti-inflammatory effects, likely by reducing levels of homocysteine. Research suggests that folic acid supplementation may reduce C-reactive protein and TNF-alpha in a variety of populations including people with metabolic syndrome and type 2 diabetes.^[75][76][77] However, most studies have been small and the clinical relevance of this effect isn’t clear.^[76]

Although it’s mechanistically plausible that folic acid supplementation could improve cognitive function, in clinical trials it does not seem to have a significant effect.

High levels of homocysteine and low levels of folate have been associated with an increased risk of dementia and Alzheimer’s disease.^[78][79] While this doesn’t prove that high homocysteine and low folate can cause cognitive decline, mechanistically such an effect could make sense.

Folate deficiency can lead to high levels of homocysteine which in turn can result in oxidative stress and stimulation of N-methyl-D-aspartate (NMDA) receptors, both of which could have toxic effects on neurons.^[78] Additionally, folate is involved in the development and repair of nerve cells.^[80] Despite this, clinical trials have generally been anticlimactic, and folic acid supplementation has demonstrated little to no benefit on various measures of cognitive function, including memory, in older adults with or without dementia.^[81][82]

In fact, folic acid fortification in North America has been associated with an increased risk of cognitive impairment in older adults.^[41][42] This may be because folic acid can mask the hematological signs of a vitamin B12 deficiency, potentially leading to an undiagnosed deficiency which may result in irreversible cognitive decline in the long term.^[41]

Observational studies have found that men with erectile dysfunction (ED) tend to have lower levels of plasma folate compared to men without ED, and folate level seems to negatively correlate with the severity of ED.^[83][84] While research is still preliminary, folic acid supplementation may improve symptoms of ED for some men, including when used in addition to the ED medication tadalafil.^[84]

As it relates to male infertility, limited research suggests that folic acid supplementation may increase sperm motility in infertile men, but does not seem to affect sperm concentration or morphology. It’s currently unknown if this benefit leads to increased rates of pregnancy.^[85] Interestingly, genetic polymorphisms in methionine synthase (MTR) — an enzyme involved in folate metabolism — have been strongly associated with male infertility, most notably in Asian populations.^[8] It’s been suggested that folic acid may improve male fertility through antioxidant mechanisms or by promoting DNA methylation during sperm production.^[85]

Ensuring a mother has adequate amounts of folate during pregnancy is clearly beneficial for fetal development, although there may be adverse effects if taken beyond recommended dosages. Additionally, folate exposure during pregnancy may impact the infants' epigenome (gene expression), potentially influencing health in the long term.^[86]

In the 1990s several large randomized controlled trials (RCTs) conducted in various countries found that folic acid supplementation was effective at lowering the risk of fetal NTDs when taken before conception.^[87][88][89] In response, folic acid supplementation is now routinely recommended to women who are pregnant or trying to conceive. Because of the clear benefits of folic acid supplementation, it’s no longer ethical to perform RCTs where one group of pregnant women is given folic acid and the other is not. As a result, much of the research we have today is from decades-old RCTs or observational studies, providing an overall weaker evidence base.

Limited data from RCTs suggests that folic acid supplementation likely reduces the rate of pregnancy termination due to NTDs and may increase birth weight, although research is mixed on the latter.^[90][91][12] Unfortunately, folic acid has not been found to protect against other types of congenital disorders, such as cleft lip, cleft palate, or congenital heart defects.^[12].

Observational research suggests that folic acid supplementation during pregnancy may be associated with a reduced risk of certain types of childhood cancers,^[92][93] and a reduced risk of autism and attention deficit hyperactivity disorder (ADHD) traits.^[25]

Folic acid at a sufficient dose should still increase folate levels regardless of a methylenetetrahydrofolate reductase (MTHFR) polymorphism, although the response may be slightly less than in someone without an MTHFR polymorphism.^[98]

MTHFR is the key rate-limiting enzyme required for the conversion of folic acid into L-methylfolate, the main active form of folate in the body. MTHFR is prone to genetic mutations called single-nucleotide polymorphisms (SNPs) which can reduce the functioning of the enzyme, leading to impaired production of L-methylfolate and lower overall folate levels.^[29][99] Accordingly, people with MTHFR polymorphisms have an increased need for folate.

Approximately 25% of the global population has an MTHFR polymorphism, and these polymorphisms are associated with an increased risk of various medical conditions including heart and blood vessel disease, infertility, certain types of cancer, and NTDs.^[100][98] However, there’s evidence that when folate levels are adequate, disease risk may be the same as for those without MTHFR mutations.^[101] When comparing how people with and without MTHFR polymorphisms respond to folic acid supplementation, people with polymorphisms do see a slightly lower increase in plasma folate levels. Still, folate increases to an adequate level regardless.^[98]

Supplementing with L-methylfolate bypasses MTHFR and may be more efficient at raising folate levels in people with MTHFR polymorphisms, but this supplemental form of folate is much less researched than folic acid.

There is convincing evidence that folate could play a modulatory role in cancer development, which may be positive or negative depending on the context. While adequate folate levels may be protective, too little may encourage cancer development, and too much may exacerbate preexisting cancer. The effect of supplemental folic acid on cancer risk has generally been found to be neutral, although research results have been mixed.

Folate is required for the maintenance, repair, and methylation of DNA, all of which serve preventative roles against cancer. Accordingly, a folate deficiency can lead to increased DNA breaks, mutations, and hypomethylation, which may increase cancer risk.^[51][52] Folate is also required for DNA synthesis and cell division, meaning that it could serve as a growth factor for preexisting cancerous or precancerous cells.^[51] Indeed, many cancer cells have increased expression of the membrane receptors responsible for folate uptake,^[53] and antifolate medications (e.g., methotrexate, pemetrexed), which block folate metabolism, are effective treatments for some cancers.^[54]

The vast majority of epidemiological research has shown that higher intakes of folate are associated with a reduced or unchanged risk of various types of cancer compared to lower intakes.^{[55][56][57][58][59][51][46]} Regarding supplemental folic acid, two meta-analyses of randomized controlled trials (RCTs) concluded that supplementation had no impact on the incidence of cancer in general or specific types of cancer.^[44][60][49] Contrary to this, another meta-analysis reported an increased risk of prostate cancer with supplementation.^[60] In 2023, the European Food Safety Authority (EFSA) expert panel stated that there was insufficient evidence to suggest a causal relationship between folic acid and the risk of prostate cancer or any other type of cancer.^[61]

Concern has been raised about the potential negative effects of unmetabolized folic acid (UMFA), but research on the safety of UMFA is lacking.

After consumption, folic acid must be converted into a usable form by the enzyme dihydrofolate reductase (DHFR). However, DHFR can become easily saturated, leading to a build-up of UMFA. The 2007–2008 National Health and Nutrition Examination Survey (NHANES) found that UMFA was detectable in >95% of Americans who provided serum samples.^[50]. UMFA doesn’t behave like natural folate in the body and is generally thought to have no biological effects. However, exactly how UMFA interacts with the body is poorly understood, and it’s unclear whether there are any health risks with long-term exposure.^[95]

Preliminary research has raised the concern that UMFA might negatively affect immune function and have an inhibitory effect on normal folate metabolism, but further research is needed.^[96][1][97]

B vitamins are common ingredients of multivitamins, of course, but also of energy boosters, such as energy drinks. But while they are best known for their role in energy metabolism,^[106] they may play a role in cancer biology through partaking in one-carbon metabolism^[107] and thus in methylation reactions and DNA synthesis.

This hypothesis was substantiated in 2015 when a paper published by the New England Journal of Medicine caused a stir by reporting that nicotinamide (a form of vitamin B3 also known as niacinamide) could reduce the rate of new non-melanoma skin cancers.^[108]

B vitamins had gained an “anti-cancer” reputation.

Yet it was just one study showing that one form of vitamin B3 could reduce the rate of one type of skin cancer; it didn’t preclude the possibility that some B vitamins could worsen at least some types of cancers.

To look for other possible connections between B vitamin supplementation and cancer, Dr. Theodore Brasky at The Ohio State University, in collaboration with colleagues at the Fred Hutchinson Cancer Research Center and at University of Taipei, performed a large observational study.^[109] Since its publication in the Journal of Clinical Oncology, in 2017, this study has taken the supplement world by storm, for it linked the vitamins B₆ and B₁₂ each with a 30–40% increase in overall risk of lung cancer in men.

Let’s take a closer look at the study.

Study design

To look for possible connections between B vitamin supplementation and lung cancer, the researchers analyzed data from the 77,000 participants in the VITamins And Lifestyle (VITAL) prospective cohort study.^[110] The study itself was designed to look for possible associations between cancer risk and vitamin, mineral, and non-vitamin/non-mineral supplementation.

The researchers chose to focus on the vitamins B₆, B₁₂, and B₉, which play an important role in the one-carbon pathways and thus are most likely to affect carcinogenesis. The study participants, all residents of the State of Washington aged 50–76 at the beginning of the study, were classified into five groups based on their average daily dose of supplemental B vitamins over the previous 10 years. Statistical techniques were then used to adjust for confounding factors such as age, education, body size, and family history of lung cancer.

What were the results?

When the data were stratified by sex, B₆ and B₁₂ as individual supplements were each shown to increase lung cancer risk by 30–40% in men (but not in women).

The greatest risk was found among men with the highest average daily dose of B₆ (>20 mg/day was associated with an 82% greater risk) and B₁₂ (>55 mcg/day was associated with a 98% greater risk) over the ten years preceding the study.

When the data were stratified by smoking status, increased risk was associated with smoking. Smokers who had supplemented with high amounts of B₆ had nearly three times the risk of developing lung cancer, and those having supplemented with high amounts of B₁₂ had over three times the risk. The study found no association between supplementation and increased risk in either former smokers or recent smokers. As for never-smokers, the paper states they “were excluded from the smoking-stratified analysis because of the low number of participants with incident lung cancer in that group.”

The study showed that long-term supplementation with B₆ or B₁₂ increased lung-cancer risk in male current smokers, especially in those supplementing with high dosages of either vitamin.

What is the mechanism?

One-carbon chemical groups lack stability, so they need to be attached to larger molecules in a process called one-carbon metabolism. The vitamins B₆, B₉,^[111] and B₁₂ play an important part in one-carbon metabolism, which in turn plays a crucial part in methylation reactions and nucleotide synthesis.

The nucleus of each of your cells contains your complete DNA. In your DNA is encoded the genetic blueprint for every protein in your body. How then do cells maintain a unique identity? By each reading only certain parts of your DNA, so that only the appropriate genes are turned on at the appropriate time.

For that purpose, sections of your DNA can be “marked” with methyl groups that prevent the expression of nearby genes. This type of epigenetic imprinting is critical to keeping cells normal, healthy, and well behaved. When the process becomes dysfunctional, the wrong genes can be turned on at the wrong times, potentially leading to uncontrolled cell growth — to cancer.

So how would high amounts of B₆ or B₁₂ increase cancer risk? We might find some clues in a recent study on DNA methylation,^[112] which found that two years of supplementation with 400 mcg of B₉ and 500 mcg of B₁₂ changed DNA methylation. Thus, the increase in cancer risk seen in the Brasky study could be caused, in part, by changes in DNA methylation from long-term B vitamin supplementation.

Another curious finding from the Brasky study was that only men saw an increase in cancer risk from B₆ or B₁₂ supplementation. Women did not. We know androgens regulate some of the enzymes that participate in one-carbon metabolism,^[113] which might explain the difference.

Androgens and the vitamins B₆, B₉, and B₁₂ interact to play a role in DNA methylation. Since DNA methylation in part determines which genes are activated (or not) at any given time, this could explain the link between long-term B vitamin supplementation and cancer risk in men.

What does this study mean?

The Brasky study was not designed to show causation, but it did reveal a strong correlation between increased risk of lung cancer and long-term B₆/B₁₂ supplementation, especially in high doses and among smokers. There are several ways B vitamins may interact with cancer metabolism; more research is needed to determine the exact mechanisms at work. In the meantime, we are left with three takeaways:

Smoking, as you know, causes lung cancer. If you smoke, stop. If you are unable to stop, avoid supplementing with B vitamins for an extended period of time, especially if you are male. Long-term B vitamin supplementation seems to increase cancer risk in male smokers, possibly by potentiating carcinogenesis in precancerous cells in response to the carcinogens in cigarette smoke (which would explain why only current smokers, not former or recent smokers, seem affected).

The effect of B vitamins on non-smokers is still uncertain. In this study, sample sizes for never-smokers were too small to evaluate associations accurately.

Although observational studies cannot show causation, the associations between B vitamins and cancer risk found in this study raise an important point, which is that high-dose, long-term consumption of any supplement can potentially interact with your biochemistry in unexpected ways. Exceeding the recommended, tested doses of even the most healthful micronutrients may not be innocuous.

Q&A with Dr. Theodore Brasky, PhD, lead author of the study

When this study was published, its finding that B vitamin supplements increased cancer risk in men generated a lot of press. But isn’t there some nuance to that finding, especially with regard to smoking habits? What ultimate take-home message can be extracted from the data?

The nuance is sort of centered around the general idea that once you start chopping up data, you lose precision. In epidemiology, our best estimates come from data reflecting the largest sample sizes. Our most cited finding was that long-term, high-dose supplementation of vitamin B6 and long-term, high-dose supplementation of vitamin B12 were each associated with about a doubling of lung-cancer risk in men. This is an entirely true representation of our results. However, when we drilled down further — and thus lost some precision — we found that this twofold increase in risk was an average across different groups of men, some with no increase in risk (men who had never smoked or had stopped smoking at the time the study began), and some with a threefold to fourfold increase in risk (men who smoked at the time the study began).

Here the scientist is left with two possibilities. Is the real finding (a) based on the larger sample size with more precise data? — men who use these supplements have twice the risk of lung cancer as do men who don’t use these supplements; or (b) based on the subgroups within men with less precise results? — men who currently smoke and who use these supplements have three to four times the risk of lung cancer as do men who currently smoke and don’t use these supplements. To me, the take-home message is the latter.

Supplementation dose, frequency, and duration are all important from a biological standpoint. How were those factors taken into account in the design of the study questionnaire? What were the pros and cons of the different ways of using those factors (and others) to identify meaningful associations with lung-cancer risk?

Put simply, we had a number of options. We could analyze separately a given supplement’s frequency of use (i.e., days per week), duration of use (i.e., number of years in the past 10 [our questionnaire only asked about the past 10 years of use]), and most common dose used, or we could combine those data.

Analyzing separately any single aspect removes the influence of the other two, which is, in my view, not ideal. Combining the data gives two additional options. We could determine a cumulative dose over the past 10 years or an average daily dose over the past 10 years. We chose the latter because it’s easier to understand and because it allowed us to compare risks with what might be expected for intakes at the level of a multivitamin taken daily for the same amount of time.

However, the disadvantage of this option — which, I contend, remains better than the alternatives — is that the 10-year, daily-dose calculation equates short-term, high-dose intakes with long-term, lower-dose intakes. The highest category of intake for supplemental B₁₂, for instance, was >55 mcg/day. This is >55 mcg taken daily, on average, over 10 years. For some people, it may actually have been about that amount daily for 10 years, but for most it was shorter-term use at higher doses that averaged out to this level.

Therefore, >55 mcg is not meant to be interpreted as the actual dose that might confer risk. Indeed, most B₁₂ supplements are sold at much, much higher doses. A standard pill from a bottle at the grocery store might contain between 500 and 2,000 mcg, with instructions that it should be taken daily. This is why the comparison to what might be consumed from a multivitamin (100% RDA) comes in handy.

Although the question “Does B vitamin supplementation increase cancer risk?” is straightforward, extracting a solid answer from a given study population is another matter. Epidemiologists like yourself are experts at identifying risk associations within large study populations. At the other end of the spectrum, basic scientists like myself tend to use defined experimental models to identify important cellular/molecular controls that drive disease processes. Could you comment on how epidemiological studies and basic science (i.e., bench research) fit in the big picture of biomedical science? Do you feel they complement each other?

A better scientist than I could probably comment on this with real nuance. I can only give my interpretation, which is, sadly, not based off any firsthand experience with bench science. I once pipetted something, but my assay didn’t run properly. C’est la vie. Looking broadly across disciplines, I can say that epidemiologists and “basic” scientists have a complementary relationship born out of necessity.

Epidemiologists cite rodent studies because in these experiments a lot of the variables can be controlled. The animals are very similar genetically, they’re all fed the same diet (unless it’s a nutrition study), handled the same way, etc. Moreover, we can perform some trials in animals that are considered unethical in humans — exposing rodents to tobacco smoke, for instance. We often see the results of these studies as hypothesis generating because, after all, the animal is a model for the human. People do not, in fact, have fur or tails, and we are much more genetically diverse than rodents purpose bred for disease models. In some instances, animal models are better approximations than others. Mice have estrous cycles rather than menstrual cycles, so some similarities for reproductive cancers are muddied by physiology here. Similarly, a mouse’s prostate gland is structured differently from a man’s; again, models. The idea is the same for work involving cells in petri dishes, although the contrast is starker. On the other hand, from what I’m told by my colleagues in these fields, epidemiologic research, which is predominantly done in an observational manner, is seen as hypothesis generating.

That we all work together towards the same goal is what’s important. Although we definitely give each other grief, epidemiologists appreciate basic scientists for their explanation of biologic mechanisms, and (I’m assuming) basic scientists appreciate epidemiologists for their findings in need of biologic explanation.