Excess Folic Acid and Vitamin B12 Deficiency: Clinical Implications?

Abstract

Background:

In the 1940s to 1950s, high-dose folic acid supplements (>5 mg/d) were used clinically to reverse the megaloblastic anemia of vitamin B12 deficiency caused by pernicious anemia. However, this treatment strategy masked the underlying B12 deficiency and possibly exacerbated its neuropathological progression. The issue of masking and exacerbating B12 deficiency has recently been rekindled with the institution of folic acid fortification and the wide-spread use of folic acid supplements.

Objectives:

The objectives of this review are to describe clinical and epidemiological evidence that excess folic acid exacerbates B12 deficiency, to summarize a hypothesis to explain this phenomenon, and to provide guidance for clinicians.

Results:

Cognitive function test scores are lower and blood homocysteine and methylmalonic acid concentrations are higher in people with low B12 and elevated folate than in those with low B12 and nonelevated folate. High-dose folic acid supplementation in patients with pernicious anemia or epilepsy cause significant reductions in serum B12. It is hypothesized that high-dose folic acid supplements cause depletion of serum holotranscobalamin and thus exacerbate B12 deficiency.

Conclusion:

The evidence for excess folic acid exacerbating B12 deficiency is primarily correlative or from uncontrolled clinical observations, and the hypothesis to explain the phenomenon has not yet been tested. Nonetheless, the evidence is sufficiently compelling to warrant increased vigilance for identifying B12 deficiency in at risk individuals, including older adults and others with low B12 intake or conditions that are associated with B12 malabsorption, who also ingest excessive folic acid or are prescribed folic acid in high doses.

Plain language title

Excess Folic Acid and Vitamin B12 Deficiency: Clinical Implications?

Plain language summary

It has been known for many decades that high doses of the B vitamin supplement, folic acid, can alleviate the anemia of vitamin B12 deficiency, at least temporarily. However, by alleviating the anemia, such folic acid supplements were said to “mask” the underlying vitamin B12 deficiency, thus allowing neurological damage to continue or possibly be exacerbated. Consequently, treating vitamin B12 deficiency with high dose folic acid was discontinued in the 1970s. The issue of whether folic acid supplements can exacerbate vitamin B12 deficiency reemerged in the 1990s with folic acid fortification of cereals and grains in the United States and Canada (and now in over 80 countries around the world) to prevent spina bifida and other birth defects. This narrative review summarizes the results of studies that have assessed the relationships between folic acid and folate and vitamin B12 status in patients and in populations. A recent hypothesis on how folic acid might exacerbate vitamin B12 deficiency is summarized, and recommendations to clinicians are made for increased vigilance in assessing vitamin B12 status in certain groups at risk of vitamin B12 deficiency, including older adults, people with gastrointestinal issues and other factors that cause vitamin B12 malabsorption, people with unexplained neurological problems, and people who follow vegan or vegetarian diets which are naturally low in vitamin B12.

Introduction

In 1997, Charles Brantigan, a physician at Presbyterian/St. Luke’s Medical Center in Denver, Colorado, wrote a letter to the editor of the journal, JAMA, recounting his wife’s experience with the so-called “masking” of vitamin B12 (B12) deficiency by folic acid supplementation. ¹ Brantigan stated that:

Those recommending folate supplementation generally allege that B12 deficiency is easily recognized by most physicians, even when it does not cause anemia and, therefore, the downside of folate supplementation is small…My wife, who had taken folate supplements, developed quite advanced neurologic findings from vitamin B12 deficiency because of the absence of anemia as an alerting symptom. Her experiences with competent physicians at a major medical center convinced me that vitamin B12 deficiency is not a benign condition, and it is not diagnosed easily by general physicians.

This letter was a first-person account of a phenomenon that had been well-established since the 1950s ^2,3 that folic acid supplements could correct the megaloblastic anemia of B12 deficiency, thereby masking or obscuring the deficiency. If physicians only look for megaloblastic anemia, they can miss the diagnosis and thus allow neurological damage to progress unabated. Moreover, some clinical evidence suggests that folic acid not only masks B12 deficiency, but also exacerbates or accelerates its neuropathological progression. ⁴

Notably, the year of Brantigan’s letter, 1997, was during the transition period in the United States when folic acid fortification of cereal and grain products was mandated by the US Food and Drug Administration (begun in 1996 and completed by January 1, 1998) to reduce the incidence of neural tube defect (NTD)-affected pregnancies. ⁵ While very effective in its primary purpose of preventing NTDs, there is concern that exposure of the general population to excess folic acid through fortification and supplements may put individuals at increased risk of neurological damage due to undiagnosed B12 deficiency. In the United States, it has been estimated that 6% of adults below the age of 60 years and 20% of adults above the age of 60 years experience B12 deficiency (defined as serum B12 <148 pmol/L). ⁶ These percentages represent millions of people put potentially at risk due to highly prevalent use of vitamin supplements, and to lesser degrees fortification of ready-to-eat cereals and the federally mandated fortification of cereal grains, the latter of which was instituted to prevent what amounts to a few thousand NTD-affected pregnancies per year. With folic acid fortification now instituted in more than 80 countries, ⁵ and low B12 status highly prevalent in all age groups around the world, ⁷ the question of if and how excess folic acid exacerbates B12 deficiency is of potentially major importance to public health not just in the United States but globally as well. It also raises the ethical issue of how to balance the certainty of benefit to a few (i.e., preventing NTDs) with the unproven possibility of harm to many (i.e., masking or exacerbation of B12 deficiency). Of note, Brantigan concluded his letter by saying, “…with patients who have folate deficiency, perhaps it would be best to treat those who have the problem rather than give the entire U.S. population folate supplementation.” ¹

Herein we summarize some of the historical evidence concerning the putative effect of excess folic acid on promoting B12 depletion, as well as our recently proposed mechanistic hypothesis on how this might occur. ⁸ It is important to note, however, that the vast majority of the evidence concerning the excess folic acid/B12 deficiency question is correlative in nature or from clinical observations that were not from controlled clinical trials. Moreover, the proposed mechanistic hypothesis remains to be tested. Accordingly, we conclude this narrative with a list of suggestions for clinicians for what to do in the face of arguably compelling circumstantial evidence, but absence of proof.

Treatment of Pernicious Anemia With High-Dose Folic Acid

Deficiencies of both folate and B12 can cause megaloblastic anemia due to disrupted DNA synthesis in blood cell precursors within the bone marrow. The consequent lack of cellular proliferation of these precursors occurs while cytoplasmic maturation continues relatively unabated leading to enlarged but reduced overall numbers of circulating red blood cells, as well as hypersegmented polymorphonuclear leukocytes and pancytopenia. ^7,9 In folate deficiency, the impairment of DNA synthesis is directly the result of the lack of 2 forms of folate, 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate, which are substrates for the synthesis of thymidylate and purines, respectively. In B12 deficiency, the activity of the B12-dependent enzyme methionine synthase, which catalyzes the conversion of homocysteine (HCY) to methionine and which requires 5-methyltetrahydrofolate as a substrate, is inhibited. Consequently, folate is trapped as 5-methyltetrahydrofolate (the so-called “methylfolate trap”) ¹⁰ and made unavailable for thymidylate and purine synthesis. Thus, a functional folate deficiency is caused by B12 deficiency, which causes megaloblastic anemia.

Pernicious anemia (PA) is an autoimmune disorder in which there is destruction of the parietal cells of the stomach that produce intrinsic factor, a binding protein that is required for the physiological absorption of B12 in the small intestine. ^7,9 This leads to severe malabsorption of B12 and characteristic clinical manifestations of deficiency, including megaloblastic anemia and neurological degeneration in the central and peripheral nervous systems. Treatment of PA typically requires regular, life-long intramuscular injections of B12 that allow the vitamin to be taken up into the blood for delivery to all tissues while bypassing the need for intestinal absorption.

While it is now recognized that treatment of PA requires B12 replacement therapy, it was observed in the 1940s that positive hematological responses (reversal of megaloblastic anemia) in PA patients could be achieved through high-dose oral supplementation with folic acid (20-50 mg/d). ¹¹ This effect is explained metabolically: folic acid is taken up by cells, converted to dihydrofolate and then tetrahydrofolate by the enzyme, dihydrofolate reductase. The tetrahydrofolate is further converted to 5,10-methylenetetrahydrofolate by serine hydroxymethyltransferase. The 5,10-methylenetetrahydrofolate is then available for thymidylate synthesis or conversion to 10-formyltetrahydrofolate for purine synthesis, thus bypassing the methylfolate trap and inducing DNA synthesis and the reversal of megaloblastic anemia. Thus, for a time in the late 1940s and early 1950s, high-dose folic acid supplementation was used to treat PA patients.

It was soon recognized, though, that PA patients treated with high-dose folic acid were still severely deficient in B12 and thus remained at risk for neurological damage. In 1951, Conley and Krevans ¹² reported neurological manifestations in new PA patients linked to high oral doses of folic acid (20-28 mg/d). In the following years, additional cases were reported of apparent exacerbation of PA-related neurological symptoms after prolonged folic acid supplementation. ³ The practice of treating PA with high-dose folic acid then went out of favor and was discontinued in the early 1970s. Whether and how folic acid exacerbated neurological symptoms in PA was left unresolved.

Folic Acid Fortification and the Rekindling of the High Folate/Low B12 Issue

With the institution of folic acid fortification in the United States and Canada in the mid- to late-1990s, the issue of whether exposure to excess folic acid could have unintended negative consequences was rekindled. Fortification has dramatically reduced the prevalence of folate deficiency, ⁵ but at the same time significantly increased the proportion of individuals exceeding the upper tolerable intake level for folic acid (>1 mg/d), particularly in those also consuming folic acid supplements and fortified ready-to-eat cereals. ¹³ Notably, since the initiation of fortification, evidence for an interaction between high folate and low B12 has accumulated coming largely from cross-sectional epidemiological data.

In 2007, Morris et al ¹⁴ analyzed data from over 1300 older adults (∼70 years of age) who participated in the 1999 to 2002 edition of the National Health and Nutrition Examination Survey (NHANES). Not unexpectedly, participants with evidence of low B12 status (either serum B12 <148 pmol/L or plasma methylmalonic acid [MMA] >210 nmol/L) had elevated odds ratios for having anemia and cognitive impairment. Moreover, the magnitudes of the odds ratios for both anemia and cognitive impairment were greater for those participants with low B12 status and elevated serum folate (≥59 nmol/L) compared with those with low B12 status and nonelevated serum folate. Similar observations were made in 2 additional cohorts who were exposed to folic acid fortification, one in Australia ¹⁵ and the other in Chile, ¹⁶ specifically that there was an interaction between the combination of high folate and low B12 status on Mini-Mental State Examination scores, an indicator of global cognitive function. Similar interactions between high folate and low B12 status on other cognitive function tests were subsequently found in an analysis of data from the 2011 to 2014 edition of NHANES. ¹⁷ These findings are contrasted with one study in the United Kingdom, where no high folate/low B12 interaction on cognitive function scores was observed. ¹⁸ The reason for this discrepancy is unclear, but could be due to the cutoff value for elevated serum folate used in the United Kingdom study being lower (≥30 nmol/L) than in the other studies cited above.

Additional evidence of an interaction between high folate and low B12 comes from studies focused on biochemical markers of B12 status, specifically HCY and MMA which become elevated in blood in B12 deficiency. In 1999, Jacques et al ¹⁹ reported that geometric mean plasma HCY in the Framingham Offspring study increased in supplement users after the initiation of folic acid fortification (i.e., after exposure to high folic acid from both fortification and supplements). Based on this finding, Malinow et al ²⁰ conducted a 3-week trial of 1 to 2 mg folic acid in over 300 participants; while overall mean serum HCY decreased, a paradoxical increase of HCY was observed in ∼20% of the cohort. Though the influence of B12 status on the effect of folic acid on HCY in these studies was not assessed, findings from patients with PA and epilepsy are consistent with these observations. In one study, HCY and MMA were assessed in 5 PA patients mistakenly treated with folic acid before being switched to B12 supplements; in 3 of the 5 patients, both HCY and MMA increased after folic acid supplementation, and then decreased in all 5 patients after they were switched to B12 supplementation. ²¹ In 2 other studies, PA patients treated with high-dose folic acid for a short period (15 mg/d for 5-8 days) ²² and a long period (5 mg/d for 6 months) ²³ exhibited significant reductions in serum B12 concentrations. Similar decreases in serum B12 were observed in 2 separate studies of epilepsy patients treated with 15 mg/d folic acid for 3 months ²⁴ and up to 2 years. ²⁵ Last, evidence of improved B12 status in a small cohort of B12-deficient older adults after a single intramuscular injection of 10 mg B12 was less robust (based on the combined indicator of B12 status) in those that had serum folate above the median for the group (≥33.9 nmol/L) compared with those below the median. ²⁶

Additional epidemiological evidence is consistent with these findings. Homocysteine and MMA were assessed in an NHANES cohort from before mandatory folic acid fortification in the United States (1991-1994) and another cohort post-fortification (1999-2002). ²⁷ Among the B12 deficient patients in the post-fortification NHANES cohort, both HCY and MMA paradoxically increased with increasing serum folate concentrations. A cross-sectional study of 1535 older adults (age ≥ 60 years) participating in the Sacramento Area Latino Study on Aging (SALSA) also demonstrated a high folate/low B12 interaction. ²⁸ Participants were divided into 4 groups based on plasma concentrations of B12 (< or ≥148 pmol/L) and folate (< or ≥45.3 nmol/L). Plasma HCY and MMA were assessed in each group. Like the NHANES data, individuals with normal B12 and elevated folate status had lower HCY and no difference in MMA concentrations compared to the group with nonelevated folate status. In contrast, HCY and MMA were higher in both B12 deficiency groups but were highest in the high folate/low B12 group.

Taken together, these observations provide compelling evidence for exposure to excess folic acid leading to increased depletion of B12. However, a mechanistic explanation for these findings has been lacking. Recently, we proposed a hypothesis to explain these observations.

Excess Folic Acid and Exacerbation of Vitamin B12 Deficiency—A Hypothesis

Our hypothesis ⁸ is based on a putative effect of folic acid on the active form of B12 in serum, holotranscobalamin (holoTC). Transcobalamin is one of two B12 transport proteins in the blood (the other is haptocorrin) and is responsible for transport of newly absorbed B12 in the small intestine to all the body tissues through receptor-mediated endocytosis via the CD320 receptor. We propose that excess intake of folic acid depletes serum holoTC. Depletion of holoTC by folic acid in individuals with overall low B12 status further reduces the capability to deliver the vitamin to B12-dependent enzymes. This results in a more pronounced state of biochemical deficiency as indicated by accentuated elevation of serum HCY and MMA, and reduced availability of B12 for the nervous system and potential neurological impairment.

Consistent with this hypothesis are data from the aforementioned SALSA study. ²⁸ Like HCY and MMA, holoTC concentrations in the SALSA participants were affected by the high folate/low B12 interaction. Participants with low B12 but elevated folate status had ∼50% lower holoTC concentrations compared to B12 deficient participants who did not have elevated folate. Among the participants who were B12 deficient, elevated folate status was associated with 16% lower total B12 concentrations compared with those with an unelevated folate status. When holoTC was subtracted from the total B12 concentrations, holohaptocorrin (holoHC) was found to be unaffected by folate status.

Key to this hypothesis are 2 possible mechanisms by which folic acid may cause serum depletion of holoTC. The first is related to redistribution of holoTC to the bone marrow to support the burst of blood cell precursor production (specifically reticulocyte formation) induced by folic acid in B12 deficiency. Support for this comes from a study by Bok et al ²² in which the percent decrease in serum B12 concentrations in PA patients after being treated with high-dose folic acid was directly proportional to the percent increase in reticulocyte formation. It is surmised that this increase in reticulocyte formation includes an increase in CD320 receptors that diverts serum holoTC to the bone marrow to support the large increase in blood cell formation. The second mechanism involves the kidney. Under normal circumstances, holoTC is filtered by the renal glomeruli and then taken up by the kidney via the megalin/cubilin/amnionless receptor complex in the renal proximal tubule cells. ^29,30 In this way, B12 is recycled with minimal losses in the urine. It is hypothesized that excess folic acid interferes with the uptake of B12 in the renal tubules, thus leading to excessive loss of B12 in the urine. Evidence for this hypothesis is limited, but it is known that folate receptors (FR-α and FR-β), which have high affinity for folic acid, colocalize with megalin in the renal proximal tubules and that megalin mediates the uptake of folic acid bound to folate receptors. ^29,30 Excessive folic acid uptake via folate receptor/megalin interaction could inhibit uptake of other megalin ligands, including holoTC.

Clinical Considerations

Importantly, whether either of the proposed mechanisms by which excess folic acid exacerbates B12 deficiency occur remains to be tested. This leads to the clinical question of what to do in the context of arguably compelling circumstantial evidence, but in the absence of proof. Here are suggestions for clinicians:

Never rely solely on the presence of megaloblastic anemia to suspect B12 deficiency. B12 deficiency often presents with a vast array of symptoms—including neurological manifestations—in the absence of anemia.

Never treat megaloblastic anemia due to B12 deficiency with folic acid. Determine the cause of the megaloblastic anemia before deciding a course of treatment.

The circumstantial evidence suggests that the effect of excess folic acid on B12 status is not confined to PA patients erroneously treated with very high doses of folic acid (i.e., >5 mg/day). Other at-risk populations should be screened (and treated as needed) for B12 deficiency, particularly if exposed to folic acid fortification and supplements, including:

Adults >60 years who often have low B12 intake, as well as B12 malabsorption due to atrophic gastritis. Approximately 50% of elder Americans regularly consume vitamin supplements containing folic acid, ³¹ while estimates of the percentage of older adults in the United States with low B12 status range from 10% to 35% depending on age-group and how low B12 status is defined (e.g., different cutoff values for serum B12 and/or MMA). ⁷ Clinicians should therefore confirm with their older patients that the amount of folic acid contained in their supplements does not exceed 1 mg/d; the exception being the patient who has an underlying medical condition dictating a larger dose.

Unexplained neurological symptoms, including peripheral neuropathy, cognitive impairment, and depression.

Patients who have undergone total or partial gastrectomy.

Individuals taking drugs that inhibit B12 absorption, including proton pump inhibitors and H2-receptor antagonists (treatment of acid reflux), and metformin (treatment of diabetes). ³² The prevalence of use of these drugs is very high in the United States: a national survey found that ∼44% of adults age ≥18 years have experienced symptoms of gastroesophageal reflux disease with ∼35% of this group taking acid blockers of some kind to control those symptoms; ³³ and it is estimated that close to 92 million prescriptions for metformin were filled in 2021 alone (increased more than 2-fold since 2004). ³⁴

Individuals who consume vegan or vegetarian diets.

Individuals in whom high-dose folic acid (1-5 mg/d) is often prescribed based on standard practice. These include pregnant women with diabetes or epilepsy, ³⁵ as well as individuals with folate-deficiency anemia or who are taking the drug, methotrexate, for rheumatoid arthritis or cancer treatment. ³⁶

It is noted that these suggestions for clinicians are generally true regardless of whether individuals are exposed to excess folic acid or not. The key take home message is that the circumstantial evidence of an effect of excess folic acid exposure, despite the absence of proof, is sufficient to warrant extra vigilance in people at risk of B12 deficiency in this era of folic acid fortification and supplementation.

Supplemental Material

Supplemental Material, sj-pdf-1-fnb-10.1177_03795721241229503 - Excess Folic Acid and Vitamin B12 Deficiency: Clinical Implications?

Supplemental Material, sj-pdf-1-fnb-10.1177_03795721241229503 for Excess Folic Acid and Vitamin B12 Deficiency: Clinical Implications? by Joshua W. Miller, Andre Smith, Aron M. Troen, Joel B. Mason, Paul F. Jacques and Jacob Selhub in Food and Nutrition Bulletin