Intended and Unintended Benefits of Folic Acid Fortification—A Narrative Review

Abstract

Inadequate folate intake during pregnancy is the leading cause of the development of neural tube defects (NTDs) in newborns. For this reason, mandatory fortification of folic acid, a synthetic, easily bioavailable form, in processed cereals and cereal products has been implemented in the US since 1 January 1998 to reduce the risk of NTD in newborn children. This report aimed to review the literature related to the impact of mandated folic acid fortification on the intended and unintended benefits to health. Potential adverse effects were also discussed. We searched Pubmed, Google Scholar, Embase, SCOPUS, and Cochrane databases for reports. About 60 reports published between January 1998 and December 2022 were reviewed, summarized, and served as background for this review. The intended benefit was decreased prevalence of NTDs, while unintended benefits were reduction in anemia, blood serum homocysteine, and the risk of developing cardiovascular diseases. Potential issues with folic acid fortification are the presence of unmetabolized folic acid in circulation, increased risk of cancer, and the masking of vitamin B-12 deficiency. From a health perspective, it is important to monitor the impact of folic acid fortification periodically.

Full text

Citation: Ismail, S.; Eljazzar, S.; Ganji,
V. Intended and Unintended Benefits
of Folic Acid Fortification—A
Narrative Review. Foods 2023,12,
1612. https://doi.org/10.3390/
foods12081612
Academic Editor: Billy Hammond
Received: 22 February 2023
Revised: 6 April 2023
Accepted: 8 April 2023
Published: 11 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
foods
Review
Intended and Unintended Benefits of Folic Acid Fortification—A
Narrative Review
Shrooq Ismail, Sereen Eljazzar and Vijay Ganji *
Human Nutrition Department, College of Health Science, QU Health, Qatar University, Doha P.O. Box 2713, Qatar
*Correspondence: vganji@qu.edu.qa
Abstract:
Inadequate folate intake during pregnancy is the leading cause of the development of
neural tube defects (NTDs) in newborns. For this reason, mandatory fortification of folic acid, a
synthetic, easily bioavailable form, in processed cereals and cereal products has been implemented
in the US since 1 January 1998 to reduce the risk of NTD in newborn children. This report aimed to
review the literature related to the impact of mandated folic acid fortification on the intended and
unintended benefits to health. Potential adverse effects were also discussed. We searched Pubmed,
Google Scholar, Embase, SCOPUS, and Cochrane databases for reports. About 60 reports published
between January 1998 and December 2022 were reviewed, summarized, and served as background
for this review. The intended benefit was decreased prevalence of NTDs, while unintended benefits
were reduction in anemia, blood serum homocysteine, and the risk of developing cardiovascular
diseases. Potential issues with folic acid fortification are the presence of unmetabolized folic acid
in circulation, increased risk of cancer, and the masking of vitamin B-12 deficiency. From a health
perspective, it is important to monitor the impact of folic acid fortification periodically.
Keywords: anemia; folic acid; folic acid fortification; homocysteine; cardiovascular diseases; USA
1. Introduction
Folate (vitamin B9) is a water-soluble vitamin that is found in many foods. Folate can
exist in one of two forms, either the reduced, naturally occurring folate or the oxidized,
synthetic folic acid. Folic acid is used in supplements and to fortify cereals and processed
grain products [
1
]. Dietary sources of folate include green leafy vegetables, legumes, beans,
and organ meats. The bioavailability of folic acid is much higher than natural folate because
it is present in the monoglutamate form, while folate requires the enzyme conjugase for
its digestion [
2
]. Folic acid is also more stable in oxidation and heat compared to natural
folate [
2
]. In the past, efforts were made to use methyl tetrahydrofolate (MTHF) as an
alternative to folic acid as a fortificant because it is a natural and biologically active form
with fewer potential health risks. MTHF utility is limited because it is less stable than
folic acid in foods that undergo thermal processing. However, more research is needed to
determine the efficacy and safety of MTHF as a fortificant [3,4].
Folic acid fortification has been implemented in many countries due to the widespread
dietary inadequacy of this vitamin, especially in women of childbearing age [
5
]. The
main objective of folic acid fortification was to prevent neural tube defects (NTDs) in
newborns [
5
]. Since the implementation of folic acid fortification, it was estimated that 18%
of all potential folic acid-preventable NTDs were prevented worldwide in 2017 and 22%
were prevented worldwide in 2019 [6].
Globally, not every country embraced the mandated folic acid fortification policy.
For example, there are significant differences in folic acid fortification policies between
the US and Europe. In Europe, mandatory fortification of food with folic acid is not
widespread. Some European countries such as the UK, have voluntary folic acid fortification
policies, while others have no fortification policy at all. The debate on whether to introduce
Foods 2023,12, 1612. https://doi.org/10.3390/foods12081612 https://www.mdpi.com/journal/foods

Foods 2023,12, 1612 2 of 15
mandatory folic acid fortification across Europe is still ongoing. Some concerns are the
long-term safety of folic acid, the potential masking of vitamin B-12 deficiency, and a
possible role in cancer are still being discussed [7,8].
In addition to its intended benefit of preventing NTDs, folic acid fortification brought
some unintended benefits. These were a reduction in anemia (improved hemoglobin con-
centrations), decreased risk of cardiovascular diseases (CVD), and improved cognitive
function [
9
]. A few reviews have been published on the health effects of folic acid supple-
mentation. This is the first comprehensive review of the intended and unintended benefits
of mandated folic acid fortification legislation on public health nutrition. It has been
25 years since the folic acid fortification legislation has been implemented in the US. We
have reviewed the literature and synthesized the evidence on the health effects of mandated
folic acid fortification using the studies reported after 1 Jan 1998. Therefore, the purpose of
this overarching review was to assess the intended and unintended benefits of mandated
folic acid fortification of processed cereals and cereal products in the US.
1.1. Biochemical Role of Folic Acid/Folate
Folic acid acts as a precursor, cofactor, and substrate for various biological processes
such as nucleotide synthesis through one-carbon transfer pathways [
1
]. These pathways uti-
lize nutrients such as glucose, vitamins, and minerals to fuel many metabolic processes [
10
].
These biochemical pathways utilize folic acid and methionine to produce methyl groups
that are essential for processes such as DNA synthesis, antioxidant generation, and epige-
netic regulation [
1
]. The production of S-adenosylmethionine, the universal methyl donor
for these methylation relations, is a crucial part and is what mediates these reactions, which
is a critical step in mediating these processes [
10
]. Figure 1shows a brief overview of the
metabolism of folic acid [11].
1.2. Dietary Reference Intakes (DRI), Adequate Intakes (AI), and Tolerable Upper Intake Levels
(UL) of Folate/Folic Acid
DRI is the average daily level of intake that is needed to meet the nutritional require-
ments of almost all healthy individuals. In the absence of DRI, an AI can be estimated to
ensure nutritional adequacy [
8
]. The US Food and Nutrition Board defines these recom-
mendations in terms of Dietary Folate Equivalents (DFEs) to reflect the fact that folic acid is
more bioavailable than natural food folate (85% bioavailability for the former compared
to 50% for the latter. Therefore, 1
µ
g of DFE can either be 1
µ
g of folate from food, 0.6
µ
g
folic acid from supplements or fortified food, or 0.5
µ
g of folic acid taken on an empty
stomach [
8
]. The DRI for adults aged
≥
19 years old is 400
µ
g of DFE for men and women.
During pregnancy and lactation, the DRI is 600 and 500
µ
g DFE, respectively [
8
]. Folic
acid also has a UL which indicates the maximum daily intake that would be unlikely affect
health. The UL for adult men, women, and pregnant and lactating women over the age of
18 years old is 1000 µg. No UL was given for food folate [8].
1.3. Biomarkers of Folic Acid Status
There have been four biomarkers proposed to estimate folate status. These are plasma
or serum folate, red blood cell (RBC) folate, mean corpuscular volume (MCV) concentra-
tions, and plasma total homocysteine (tHcy) [
9
]. A folate deficiency is defined as a serum
folate concentration of <7 nmol/L or an RBC folate concentration of <312 nmol/L [
8
].
A folic acid deficiency causes megaloblastic anemia or macrocytosis. Many factors may
contribute to the deficiency of folic acid, including states of increased energy requirements
such as pregnancy, lactation, alcoholism, and losses during food preparation, as well as the
use of certain medications [2].
Today’s Westernized diets contribute largely to the unmet folic acid requirements [
12
].
The replacement of complex carbohydrates and vegetables with refined sugars and carbo-
hydrates, as well as the absence of whole foods, are the causes of widespread folic acid
deficiency. Fad diets such as low-carbohydrate or ketogenic diets may also contribute to

Foods 2023,12, 1612 3 of 15
folate deficiency due to the decreased intake of commonly fortified foods, such as cereals
and bread [12].
Foods 2023, 12, x FOR PEER REVIEW 3 of 15
Figure 1. Role of folate in one-carbon transfer metabolism. Folic acid from dietary folate or folic acid
from fortified foods/supplements is reduced to DHF and then to THF by DHFR. SHMT converts
THF to 5,10-methylene THF. 5,10-methylene-THF is reduced by MTHFR to 5-methyl-THF. 5-methyl
THF is catalyzed by MS to generate THF and methionine through the remethylation of Hcy. Cobal-
amin is a cofactor for MS. Methionine is then used to form SAM, which serves as a universal methyl
donor for numerous reactions and produces SAH, which then generates homocysteine. Hcy is then
used either to regenerate methionine, or it is converted to β-cystathionine and then to cysteine in
the transsulfuration pathway. In the regeneration of DHF from 5,10-methylene THF, dUMP is con-
verted to dTMP by TS which can be used for DNA synthesis. Abbreviations: CBS, cystathionine-β
synthase; CSE, Cystahionine-γ lyase; DHF, dihydrofolate; DHFR, dihydrofolate reductase; dTMP,
deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; MS, methionine synthase;
MTHFR, methylenetetrahydrofolate reductase; SAH, S-adenosylhomocysteine; SAM, S-adenosyl-
methionine; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; TS, thymidylate syn-
thase.
1.2. Dietary Reference Intakes (DRI), Adequate Intakes (AI), and Tolerable Upper Intake Levels
(UL) of Folate/Folic Acid
DRI is the average daily level of intake that is needed to meet the nutritional require-
ments of almost all healthy individuals. In the absence of DRI, an AI can be estimated to
ensure nutritional adequacy [8]. The US Food and Nutrition Board defines these recom-
mendations in terms of Dietary Folate Equivalents (DFEs) to reflect the fact that folic acid
is more bioavailable than natural food folate (85% bioavailability for the former compared
to 50% for the latter. Therefore, 1 μg of DFE can either be 1 μg of folate from food, 0.6 μg
folic acid from supplements or fortified food, or 0.5 μg of folic acid taken on an empty
stomach [8]. The DRI for adults aged ≥19 years old is 400 μg of DFE for men and women.
During pregnancy and lactation, the DRI is 600 and 500 μg DFE, respectively [8]. Folic
acid also has a UL which indicates the maximum daily intake that would be unlikely affect
Figure 1.
Role of folate in one-carbon transfer metabolism. Folic acid from dietary folate or folic acid
from fortified foods/supplements is reduced to DHF and then to THF by DHFR. SHMT converts THF
to 5,10-methylene THF. 5,10-methylene-THF is reduced by MTHFR to 5-methyl-THF. 5-methyl THF
is catalyzed by MS to generate THF and methionine through the remethylation of Hcy. Cobalamin is
a cofactor for MS. Methionine is then used to form SAM, which serves as a universal methyl donor
for numerous reactions and produces SAH, which then generates homocysteine. Hcy is then used
either to regenerate methionine, or it is converted to
β
-cystathionine and then to cysteine in the
transsulfuration pathway. In the regeneration of DHF from 5,10-methylene THF, dUMP is converted
to dTMP by TS which can be used for DNA synthesis. Abbreviations: CBS, cystathionine-
β
synthase;
CSE, Cystahionine-
γ
lyase; DHF, dihydrofolate; DHFR, dihydrofolate reductase; dTMP, deoxythymi-
dine monophosphate; dUMP, deoxyuridine monophosphate; MS, methionine synthase; MTHFR,
methylenetetrahydrofolate reductase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine;
SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; TS, thymidylate synthase.
1.4. Folic Acid Fortification
To date, more than half of women of childbearing age fail to meet the folic acid re-
quirement for preventing NTDs [
13
]. Therefore, the US Public Health Service recommends
that women of childbearing age consume 400
µ
g of folic acid daily for NTD prevention,
but no more than 1000
µ
g/day because of the unknown health effects of excessive folic
acid. This can be achieved through diet alone, particularly in countries with mandated

Foods 2023,12, 1612 4 of 15
folic acid fortification legislation. Supplementation with 400
µ
g of folic acid per day is also
recommended for the prevention of NTDs [13].
The USA was the first nation to implement mandatory folic acid fortification for
the prevention of NTDs, which has significantly reduced their incidence [
14
]. Currently,
more than 80 other countries have adopted the US policy of fortifying cereal grains with
folic acid [
14
]. Fortified products include staple items such as foods like bread, four, rice,
pasta, and breakfast cereals [
15
]. Folic acid fortification programs aimed to decrease NTD
incidence among women of childbearing age; however, because folic acid fortification is not
selective and applies to the entire population, other subgroups such as men, children, and
elderly persons have seen a rise in serum folate concentrations. This increase has resulted
in unintended benefits alongside the intended benefit of decreasing NTDs. Unfortunately,
some potential health issues have been reported regarding excessive folic acid consumption,
but more research is needed to confirm these reports [8].
2. Methods
For this narrative review, we obtained reports by searching electronic databases such
as PubMed, EMBASE, SCOPUS, and Google Scholar. We also conducted an additional
free-hand search to ensure that this review includes relevant papers and to reduce the
possibility of missing any relevant papers. Our inclusion criteria were reports published
between 1998 and 2022. We used main keywords such as ‘folic acid’, ‘folate’, ‘fortification’,
‘neural tube defects’, ‘NTDs’, ‘United States’, and ‘U.S.’ to identify relevant studies. We
included only those reports that were based on the US population. Only those studies that
were reported in English were considered in this review. Two reviewers independently
screened all the articles by publication titles and abstracts and then assessed and evaluated
the full text for inclusion. Any disagreements were resolved through a second review and
discussion among the authors.
3. Results and Discussion
3.1. Intended Benefits of Folic Acid Fortification
NTDs are a form of birth defects that results from the failure to close the neural tube
enclosure in the early gestational period. NTDs can be classified as “open” or “closed”
depending on whether the neural tissue is covered. This defect can cause anencephaly,
spina bifida, as well as some other congenital defects and abnormalities [
16
]. The prevalence
of fatality cases due to spina bifida is
≈
7% compared to other NTDs, i.e., encephalocele
(46%) and anencephaly (100%) [
17
]. However, this difference may be due to a higher
pregnancy termination rate after the prenatal diagnosis of spina bifida [17].
Folic acid status contributes to NTDs through several suggested mechanisms and
hypotheses. Some researchers have suggested that the presence of elevated folate receptor
antibodies would limit folate transport in the early stages of embryo development, leading
to developmental impairment [
18
]. Others have hypothesized that folate has a role in DNA
methylation, which results in the over-expression of some genes involved in autoimmunity
associated with the progression of NTDs [
18
]. Studies have observed a dose-dependent
effect between serum folic acid and the risk of NTD incidence. The lower the serum folic
acid, the higher the risk. This explains the higher beneficial effects in countries with higher
NTD prevalence following folic acid fortification [
18
]. Genetics, race-ethnicity, sex-related
epigenetic changes, maternal age, and socioeconomic status play a role in the development
of NTDs, but folic acid also serves as an independent environmental predictor in the
pathogenesis of these diseases [19].
Since 1965, the relationship between folic acid and NTDs has been well established,
and the combination of supplementation and fortification of folic acid has been successful
in fulfilling their main goal of preventing various NTDs. Due to mandatory food forti-
fication programs, the incidence of NTDs has decreased by up to 22% of cases of folic
acid-preventable NTDs in 2019 alone [20,21].

Foods 2023,12, 1612 5 of 15
The cost-effectiveness of folic acid fortification has been studied in several projects.
One study conducted in the US investigated the cost-effectiveness of folic acid fortification
and showed that folic acid fortification decreased the present value of total direct costs
for each year of birth by $603 million more than the cost of fortification. Additionally,
the number of infants born with spina bifida was reduced by 767 each year [
21
]. The
first-year survival rate among 2841 infants with spina bifida and 638 with encephalocele,
who were born between 1995 and 2001, was also studied [
22
]. The survival rate for infants
with spina bifida during the mandatory fortification period was 92.1% (adjusted HR: 0.68;
95%CI: 0.5, 0.91), an improvement compared to the 90.3% survival rate before fortification.
However, no significant improvement was observed in encephalocele for the first-year
survival rate. This evidence shows that folic acid fortification plays a significant role not
only in reducing the prevalence of NTDs but also improving the severity of spina bifida
and first-year survival rate [22].
Post-fortification, it has been shown that the highest reduction in NTDs was observed
among the Hispanic population, who initially had a higher prevalence of NTDs compared to
the African American population within the US [
23
]. Another study showed that although
the rate of spina bifida in the Hispanic population declined with time, it remained constantly
higher when compared to white or black populations [
24
]. This has been explained in part
by the higher mutation rate of the C677T methylenetetrahydrofolate reductase (MTHFR)
homozygous genotype [24].
Other possible reasons for the reduction in NTD cases include medical and surgical
interventions, which have improved with advancements in intensive care support and the
use of antibiotics to treat central nervous system infections, leading to better treatment of
NTDs [
23
]. Additionally, there have been improvements in associated comorbidities, such
as low birth weight and prematurity, which have been identified as strong factors for infant
deaths [
23
]. Improvements in surgical interventions for spina bifida and other related
defects, such as hydrocephalus and cardiac defects, may also contribute to the reduction in
NTD-related death rates, which are unrelated to folic acid fortification [18].
Numerous studies have evaluated optimal serum folate concentrations to reduce the
risk of NTDs. The World Health Organization recommends maintaining a serum folate
concentration of at least 906 nmol/L to decrease this risk [
25
]. Folic acid supplements are
usually recommended in pre-pregnancy due to the greater amounts of folic acid needed for
the rapid rate of cellular and tissue growth during early pregnancy [
25
]. Table 1presents
studies with the intended and unintended benefits of folic acid.
Folate metabolism may differ depending on pregnancy status. One study compared
two diets consisting of two levels of folic acid (450
µ
g from food vs. 850
µ
g from folic
acid fortified foods) given to pregnant and non-pregnant women. This study showed that
pregnant women compared to non-pregnant women are more efficient at conserving folate,
especially when given at a lower dose [
25
]. Although urinary excretion is increased at both
doses, this suggests that both lower and higher levels may maintain the necessary folic acid
concentrations [25].

Foods 2023,12, 1612 6 of 15
Table 1. Summary of studies that assessed health benefits of folic acid fortification 1.
Reference
(Author/s and Year) Study Design Intervention/Data Collection Outcome Measurements Findings Conclusions
Intended benefits of folic acid fortification
Bol et al., 2006 [22] Retrospective cohort Between 1995 to 2001 Spina bifida and encephalocele
↑Dietary Folic acid, ↑First-year
survival rate; no difference
in encephalocele
In addition to preventing the
occurrence of NTDs, folic acid may
have a role in a role in reducing the
severity of NTDs
Ho et al., 2021 [23] Systematic review Evidence searched from 1 January
1990 to 31 August 2020
Spina bifida and infant
mortality rate
↓spina bifida and associated infant
and neonatal mortality rates
Significant declines in spina bifida
associated infant/neonatal mortality
and case fatality. Likely due to folic
acid fortification
Unintended benefits of folic acid fortification
Ganji et al., 2006 [26] Retrospective cohort From 1988 to 2002 pre- and
post-fortification
Serum folic acid, RBC, RBC
folate, and tHcy concertation ↑Dietary folic acid, ↓tHcy Folic acid plays a role in the
reduction in tHcy.
Holmes et al., 2011 [27] Meta-analysis Mean follow-duration of 4.7 y
Risk of stroke; effect modification
by population
↑OR stroke was higher in Asia
than in America, Australia, and
New Zealand
Stroke risk in Asia was higher in
comparison to areas with folate
fortification such as America,
Australia, and New Zealand.
Yang et al., 2006 [28] Cohort study From 1990–2002 Stroke mortality ↓Stroke mortality with
folate fortification
Stroke mortality decreased after
mandatory folic acid fortification.
Morris et al., 2007 [29] Cohort study From 1999–2002; folic acid
fortification in elderly >60 y of age Cognitive impairment
↑High serum folate; ↓B12
deficiency; ↑anemia and cognition;
Normal B12 ↑serum folate,
↓cognitive impairment
Folic acid has two-sided effects on
cognitive health depending on the
serum B12 serum concentration.
Biemi et al., 2021 [30] Retrospective, observational
2004–2010 pre- and
post-fortification in children
5–14 y of age
Hemoglobin, hematocrit, RBC,
MCV, and anemia
No difference in mean hemoglobin;
↑MCV concentrations
post-fortification
No change in anemia in
post-fortification; however, MCV
significantly increased suggesting an
increase in B12 deficiency.
Ganji et al., 2009 [31] Cohort study 1988–2004, pre and post-folic
acid fortification Anemia and macrocytosis ↑Dietary folic acid; ↓anemia
in women
Improvement in hemoglobin and
decreased prevalence of anemia after
folic acid fortification.
Carrasco Quintero et al.,
2013 [32]Randomized control trial In 2010, maize flour fortified with
folic acid Hemoglobin in women ↑Hemoglobin
Fortified flour is a good option for
regionalized women in rural areas
who are underweight,
undernourished, and have anemia.
Li et al., 2018 [33] Cross-sectional study 2011–2012, serum folic acid
Serum folate and insulin
resistance (fasting plasma
glucose, OGTT, serum insulin,
and HOMA-IR)
↑Serum folate; ↓HOMA-IR
Serum folate was inversely associated
with insulin resistance
1
Abbreviations: B12, vitamin B-12; HOMA-IR, homeostatic-model assessment for insulin resistance; MCV, mean corpuscular volume; NTD, neural tube defect; OGTT, oral glucose
tolerance test; OR, odds ratio; RBC, red blood cells; RCT, randomized controlled trials; tHcy, total homocysteine.

Foods 2023,12, 1612 7 of 15
3.2. Unintended Benefits of Folic Acid Fortification
3.2.1. Reduction in tHcy
Worldwide, CVDs are among the most prominent global public health challenges
of the 21st century [
34
]. Many studies have established that homocysteine (Hcy) is con-
sidered an independent risk factor for CVD. It has been reported that more than 60%
of cases with CVD were also simultaneously diagnosed with hyperhomocysteinemia, al-
though further research is needed regarding its management and treatment [
34
]. Hcy is
a non-essential, sulfur-containing amino acid that is produced by methionine through a
demethylation reaction [
34
]. In the human body, 50% of the Hcy is remethylated to form
methionine. This reaction requires several enzymes as well as three vitamins that serve as
coenzymes, including folic acid, vitamin B-12, and vitamin B-6 [
34
]. In the conversion of
Hcy into methionine, folate in the form of MTHF provides Hcy with a methyl group in the
remethylation catalyzed in the presence of vitamin B-12 by methionine synthase [35].
Elevations in plasma tHcy are multifactorial and may be affected by factors such as
genetics, enzyme dysfunction, and vitamin deficiencies [
35
,
36
]. A deficiency in pyridoxine
and cyanocobalamin coenzymes can lead to an increase in total plasma tHcy concentra-
tions [
37
]. Genetic defects in enzymes required in the remethylation pathway that may
lead to hyperhomocysteinemia include MTHFR, methionine synthase, and the first step
of the transsulfuration pathway responsible for cystathionine
β
-synthase. Furthermore,
lifestyle factors such as smoking, coffee, and alcohol consumption, and some medical
conditions such as diabetes and renal impairment, may contribute to elevated plasma tHcy
concentrations [37].
Circulating tHcy concentrations can be assessed by measuring serum MTHFR concen-
trations [
34
]. Elevation of circulating tHcy concentrations may cause organ dysfunction,
leading to an increase in the risk of venous thromboembolism, arterial thrombosis, and
early development of CVDs [
36
]. Some studies showed that high concentrations of tHcy
in the blood, along with a folate deficiency, are significantly associated with the risk of
myocardial infarction [
38
]. Other studies have linked plasma tHcy and the risk of fractures
due to osteoporosis in the elderly [38].
One unintended benefit of increasing folic acid in diets through folic acid fortification
has been a reduction in circulating tHcy concentrations. The association between tHcy and
folic acid fortification was previously studied in 1999 in a cohort study [
36
]. The authors
determined the impact of folic acid fortification on serum folate and tHcy concentrations.
They compared individuals exposed to folic acid fortification (between the years 1997–1998)
to those who were not (between the years 1995–1996) [
36
]. They found that those exposed
to folic acid fortification had a significant increase in mean folic acid concentrations, as well
as a reduction in tHcy compared to baseline concentrations, while the control group had
no significant reductions in tHcy [36].
A retrospective study using National Health and Nutrition Examination Surveys
(NHANES) 1988–2002 reported on the temporal association of folic acid fortification and
serum folic acid, RBC folate, and tHcy concentrations [
26
]. This study showed a reduction
in serum folate with a mean concentration of 149.6% in 1999–2000 and 129.8% in 2001–
2002, both significantly higher than in 1988–1994 even after adjusting for age, sex, and
race/ethnicity. Hcy was also reduced from 9.5
µ
mol/L in 1988–1994 to 7.6
µ
mol/L in
1999–2000 and to 7.9
µ
mol/L in 2001–2002 after adjusting for various confounders. The
reduction in serum folate in 2001–2002 compared to 1999–2000 was justified predominantly
in people with high folate intake levels, rather than in those with lower intake levels [
26
].
This shows that folic acid fortification contributed to significant improvements in folate
status. However, over time, serum folate concentrations slightly declined in 2001–2002 due
to lower folic acid intake [26].
One meta-analysis published in 2014 observed the difference in tHcy reduction among
groups with fortification, partial fortification, and without fortification [
39
]. This study
found that the tHcy reductions were 27%, 18.4%, and 21.3% in the subgroups without
folate fortification, with folate fortification, and with partial folate fortification, respectively.

Foods 2023,12, 1612 8 of 15
During the same period, the reduction in stroke mortality decreased slightly in Canada and
the US after folate fortification [
39
]. The causes and effects between folic acid fortification
and decreased risk of CVD are difficult to demonstrate because most trials had participants
who already had a high risk of CVD and may have already received intensive lipid profile
interventions before folic acid fortification. Thus, the impact of tHcy on lowering the risk
of CVD is difficult to demonstrate in these trials. On the other hand, in populations not
taking lipid-lowering medication with a middle to high risk of cerebrovascular events, the
effect of folate might be more evident [39].
Similar results have been found in another meta-analysis including 24 randomized
control trials (RCT) examining the relationship between folic acid supplementation and
glycemic control [
40
]. The results showed significant reductions in fasting insulin [weighted
mean difference (WMD):
−
1.63 pmol/L; 95%CI:
−
2.53,
−
0.73], fasting blood glucose
(WMD:
−
2.17 mg/dL; 95%CI:
−
3.69,
−
0.65), and HOMA-IR (WMD:
−
0.4; 95%CI:
−
0.7,
−
0.09) following folic acid supplementation. However, the effect on HbA1c was not
significant. This concludes that folic acid supplementation may reduce blood glucose-
related biomarkers [
40
]. However, the reductions were minimal and may limit their
clinical applications for adults with diabetes. Unfortunately, most of the studies have been
conducted in regions without mandatory folic acid fortification and focused on studying
the effect of folic acid supplementation [40] (Table 1).
3.2.2. Impact on Stroke and CVD
Folic acid fortification has been shown to decrease tHcy concentration, and folic acid
supplementation is a debatable option for reducing tHcy concentration. Research has
shown that 0.5–5 mg of folic acid can reduce tHcy by 25%, which can lower the risk of
CVDs. It has been established that lowering tHcy concentrations by 3–4
µ
mol/L reduces the
risk of CVD by 30–40% [
36
,
37
]. One meta-analysis of 12 RCTs showed that participants who
received folic acid supplementation had a significantly decreased risk of stroke compared
to those who did not (RR: 0.85; 95%CI: 0.77, 0.94) [
34
]. However, there were no significant
differences in the other outcomes such as CVD mortality, all-cause mortality, and risk of
coronary heart disease (CHD) [
41
]. Consistent with their findings, another meta-analysis
was carried out on 11 RCTs, including 65,790 individuals with CVD, and found that stroke
incidence in patients with pre-existing CVD was significantly reduced (RR: 0.9; 95%CI: 0.84,
0.97; p= 0.005) [42].
Several studies have investigated the mechanism behind lowering the concentration
of tHcy by giving folic acid in cases of the MTHFR 677C
→
T genotype. One meta-analysis
showed that cases with the polymorphisms in MTHFR 677TT had a 16% (OR: 1.16; 95%CI:
1.05, 1.28) higher risk of chronic heart disease compared with individuals with the CC
genotype [
43
]. However, after dividing the genotype groups into high or low folic acid
status, the genotype effects were noticed to be invalid in the high folic acid status group [
43
].
There was a similar risk for CHD in the MTHFR 677TT genotype adults seen in patients
with the CC genotype (OR: 0.99; 95%CI: 0.77, 1.29). Cases with the MTHFR 677TT genotype
who had low folic acid concentrations were at higher risk for CHD (OR: 1.44; 95%CI: 1.12,
1.83) compared to those with high folic acid status and the CC genotype [
43
]. Although all
the included studies were for folic acid supplementation, the phenomena of impacts of the
higher risk genotype are overcome by adequate serum folic acid concentrations which can
be reached by folic acid fortification [43].
Similar results were reported in a study that was conducted to investigate folate’s
role in the association between MTHFR 677C
→
T and stroke in the genetic analyses [
27
]. A
meta-analysis of 13 randomized trials concluded that the effect of the MTHFR 677C
→
T
variant on tHcy concentration in low folate concentration regions such as Asia (difference
between cases of TT vs. CC genotype: 3.12
µ
mol/L; 95%CI: 2.23, 4.01) was higher in
comparison to areas with folate fortification such as the US (high: 0.13
µ
mol/L; 95%CI:
−
0.85, 1.11) [
27
]. In addition, the odds ratio for stroke was higher in Asia (OR: 1.68; 95%CI:
1.44, 1.97) than in the US, Australia, and New Zealand, (OR: 1.03; 95%CI: 0.84, 1.25) [27].

Foods 2023,12, 1612 9 of 15
A population-based cohort study was conducted to compare the stroke mortality
rates in Canada and the US between 1990 to 2002, during the period of fortification, with
the mortality rates in England and Wales, which did not adopt the folic acid fortification
policy [
28
]. This study found that stroke mortality decreased by an average of
−
1.9%
(95%CI:
−
1.4,
−
0.6) per year from 1990 to 1997 and
−
5.4% (95%CI:
−
6,
−
4.7) per year
from 1998 to 2002 (p
≤
0.0001) [
28
]. However, the effect of folic acid fortification on tHcy
concentration and CVD remains controversial due to several factors such as participants’
health condition, the presence of kidney disease, the dose of folic acid, and interaction with
other vitamins such as vitamin B-12 or vitamin B-6. More studies are needed to clarify
this topic.
3.2.3. Impact on Cognitive Health and Depression
The relationship between folic acid and cognitive performance in adults is still not
fully understood. The major role of folate during fetal development has been studied
and linked to neuronal structure and function, cell polarity and plasticity, and vesicular
transportation [
44
]. A study on folic acid fortification assessed the effect of folic acid
fortification from food alone on the intelligence quotient (IQ) in children aged 6 years old
with mothers diagnosed with epilepsy and on anti-seizure medication (ASM) [
45
]. They
found that folic acid from food alone was not associated with the enhancement of IQ in
children aged 6 years old. In contrast, those children who took folic acid supplements
found a 10.1-point higher increment in IQ levels (95%CI: 5.2, 15; p< 0.001) [
45
]. This shows
that dietary folic acid from fortification, even in a country where food is fortified with folic
acid, is not adequate to improve cognitive outcomes for children of women using ASMs
during pregnancy [45].
Another study conducted among 292 youths aged 8 to 18 years old studied the
associations among fetal folic acid exposure, cortical maturation, and psychiatric risk [
46
].
This study showed exposure-associated cortical thickness increases in bilateral frontal and
temporal regions (9.9% to 11.6%; p< 0.001 to p= 0.03) and the emergence of quadratic
age-associated thinning in temporal and parietal regions (
β
=
−
11.1 to
−
13.9; p= 0.002)
and flatter thinning profiles in frontal, parietal, and temporal areas were associated with
inferior odds of psychosis spectrum symptoms [46].
One cohort study assessed folic acid concentrations and their relationship with cogni-
tive function and dementia in Latinos aged >60 years old who were exposed to folic acid
fortification. After assessing cognitive health with a Modified Mini-Mental State Exami-
nation (3MSE), and a cross-culturally validated neuropsychological test, they found that
the concentration of RBC folate was directly associated with cognitive function scores and
inversely associated with dementia, despite folic acid fortification [
47
]. Additionally, RBC
folate concentration was associated with 3MSE and delayed recall scores after adjusting for
several confounding factors. Furthermore, the relative risk of cognitive impairment and
dementia decreased with increasing RBC folate concentration [
47
]. In contrast, a cohort
study using the NHANES data from 1999–2002 in elderly individuals above 60 years old
found a direct association between high serum folate and both anemia and cognitive im-
pairment in those who had low serum vitamin B-12 concentration during the age of folic
acid fortification. On the other hand, a combination of normal vitamin B-12 status and high
serum folate was found to be protective against cognitive impairment [29].
Similar results have been found in another meta-analysis, which indicated a dose-
response relationship between folic acid and the risk of cognitive impairment in older adults
with vitamin B-12 deficiency. The study suggested a “J shape” relationship between serum
folate concentration and cognitive impairment [
48
]. The suggested mechanism behind this
relationship is that elevated folate might mask vitamin B-12 deficiency. Epidemiological
studies have shown that individuals with low serum vitamin B-12 in combination with high
folate present a higher risk of cognitive impairment compared to those with normal serum
folate, especially in the elderly population due to lower absorption of vitamin B-12 [
29
,
48
].
It has been estimated that folic acid fortification might be associated with an increase in the

Foods 2023,12, 1612 10 of 15
risk of cognitive impairment in up to 4% of older adults in the US [
29
]. Current regulations
and fortification policies should be addressed to analyze the risk-benefit to weigh public
health risks and benefits.
3.2.4. Depression
About 8% of the US population has depression [
49
]. Folate plays an important role in
the nervous system functioning through one-carbon transfer reactions. Although no study
has directly linked folic acid fortification with decreased depression, a few studies investi-
gated the relationship between folic acid supplementation and depression. Subjects with
depression had low folate status and elevated tHcy leading to a reduced supply of methyl
groups. This further leads to decreased methylation of monoamine neurotransmitters such
as serotonin, dopamine, and nor-epinephrine in the brain, which have been implicated in
mood disorders [
50
]. A study found that folate deficiency was observed in up to a third of
individuals with severe depression. A collective analysis of six RCTs demonstrated that
supplementation of folic acid resulted in a decrease in depression scores, as measured by
the Beck Depression Inventory (WMD: −3.9; 95%CI: −5.3, −2.4). Additionally, it resulted
in a lower depression score on the Hamilton Depression Inventory (WMD:
−
3.5; 95%CI:
−
4.6,
−
2.4); p< 0.001) compared to the control group [
51
]. A meta-analysis consisting of
data from 15,315 participants (1769 subjects with depression and 13,546 control subjects)
revealed a significant association between folate status and depression (pooled adjusted
OR:1.42; 95%CI: 1.1, 1.83) [
52
]. Therefore, it is recommended that individuals with de-
pression or at risk of depression ensure that they are getting adequate amounts of folate
through diet or supplements.
3.2.5. Reduction in Anemia
Anemia is a major public health concern in both developing and developed countries,
caused by several micronutrient deficiencies, including ferrous sulfate, vitamin B-12, and
folate [
53
]. Iron deficiency anemia is the most common form, accounting for over half of all
anemia cases. Viral infections and inflammation are also the causes of anemia [
53
]. The
prevalence of anemia in developing countries is almost 43%, compared to 9% in developed
countries [
53
]. Anemia has many adverse effects on health, including compromising
immunity, reducing cognitive performance, and lowering productivity. Moreover, severe
anemia is a major risk factor for maternal and infant morbidity and mortality [
54
]. Folate
deficiency is typically associated with macrocytic anemia, which can cause megaloblastic
changes in the bone marrow and macrocytosis in red blood cells [54].
In an observational study that compared the prevalence of anemia in children before
and after double fortification of wheat flour with folic acid and iron, no significant differ-
ence was found in mean hemoglobin concentrations between pre-fortification and post-
fortification. However, MCV concentrations increased significantly in the post-fortification
period from the pre-fortification period (from 76.8 fL to 79.1 fL; p= 0.02) [
30
]. Another study
used NHANES data to evaluate the prevalence of anemia and macrocytosis before and
after folic acid fortification (1988–1994 vs. 1999–2004) [
31
]. The study found a significant
increase in hemoglobin from 1988–1994 to 1999–2004 with a rise from 15.1 to 15.4 mg/dL
(p< 0.0001) in men and 13.3 to 13.6 g/dL (p< 0.0001) in women [
31
]. MCV status also
improved during this time frame, with significant increments observed in men (from 90.2 to
90.7 fL; p= 0.0123), in senior men >60 years (from 91.6 to 92.4 fL; p= 0.0105), and in women
(from 90.7 to 91.4 fL; p= 0.0141). The prevalence of anemia was only significant in women,
with a 27.9% reduction (p= 0.0005) in 1999–2004 compared to 1988–1994. Furthermore,
the odds of having anemia in post-fortification compared to pre-fortification were 0.64
in women (95%CI: 0.54, 0.75; p< 0.0001) and 0.79 in men (95%CI: 0.62, 0,99; p< 0.0433).
However, no significant effects were observed on the odds of macrocytosis after folic acid
fortification [31].
In contrast, Hirsch et al. found an increase in MCV in the post-fortification period com-
pared to the pre-fortification period, suggesting an increase in vitamin B-12 deficiency [
55
].

Foods 2023,12, 1612 11 of 15
The study suggested that higher doses of folic acid supplements (more than 1000
µ
g) could
mask hematologic symptoms associated with vitamin B-12 deficiency. This could lead to a
missed diagnosis of vitamin B-12 deficiency as irreversible neurological impairment may
occur even in the presence of normal hematology [55].
One RCT found that six months after the fortification of maize flour, women who
consumed the fortified flour had a significant increase in hemoglobin concentrations (mean
13.3 g/dL compared to 13.1 g/dL at baseline) [
32
]. However, a study carried out in China
found no significant effects in the prevalence of anemia among women who consumed folic
acid-fortified wheat flour compared to the control group after 36 months of intervention
(RR: 0.87; 95%CI: 0.68, 1.11) [56].
The effect of folic acid fortification remains controversial due to several reasons,
such as the target population, the amount of folic acid fortification, the cut-off point of
hemoglobin, and whether the difference in hemoglobin is clinically significant. Moreover,
the heterogeneity and residual confounders of studies are limitations. Further research is
needed to explore the effect of folic acid fortification on populations with anemia, especially
macrocytic anemia.
3.2.6. Diabetes
Diabetes can cause some complications related to macro- and micronutrients. The
implication of folic acid in the pathogenesis of type 2 diabetes (T2DM) is associated with
vitamin D deficiency and its consequence in hyperhomocysteinemia [
57
]. A case-control
study found that low intakes of folate and vitamin B-12 in T2DM were associated with
hyperhomocysteinemia and DNA damage, as measured by the presence of micronuclei.
Folic acid supplementation was found to revert the effects of oxidative stress in diabetic
patients [
57
]. Additionally, folic acid supplementation has been shown to reduce glycosy-
lated hemoglobin (HbA1C), fasting blood glucose, insulin resistance, and homocystinuria
in T2DM [58].
One meta-analysis showed that folic acid supplementation was associated with lower
tHcy concentrations, compared to placebo, in patients with T2DM (WMD: −3.52 µmol/L;
95%CI:
−
4.40,
−
2.6; p< 0.001), but not HbA1c (I2 = 43.7%; p= 0.149) [
59
]. Another study
found that high RBC folate was a significant predictor of insulin resistance and other
metabolic complications in obese patients [
33
]. A dose-response meta-analysis of RCTs
showed that folic acid supplementation significantly reduced fasting blood glucose (WMD:
−
2.17 mg/dL; 95%CI:
−
3.69,
−
0.65; p= 0.005) and Homeostatic Model Assessment for In-
sulin Resistance (HOMA-IR) (WMD:
−
0.4; 95%CI:
−
0.70,
−
0.09; p= 0.011). However, these
reductions were considered small, and their clinical implications may not be significant. In
the same analysis, investigators found no effect of folic acid supplementation on HbA1C
concentrations [
40
]. In another meta-analysis of RCTs, Lind et al. [
60
] found that folic acid
supplementation, compared to placebo, reduced fasting insulin and HOMA-IR, but did not
affect the fasting glucose and HbA1c. Based on the evidence, the association between folic
acid supplementation and markers of glucose regulation is somewhat inconsistent. To date,
no studies have been conducted on the effect of folic acid fortification on markers of glucose
regulation. Therefore, further studies are needed to determine whether mandated folic acid
fortification has any favorable impact on glucose regulation in healthy individuals and
those with diabetes.
Emerging evidence suggests that the gut microbiome may play a role in the develop-
ment of T2DM [
61
]. An imbalance in the gut microbiome can lead to inflammation and
metabolic dysfunction, contributing to the development of insulin resistance and subse-
quent development of T2DM [
62
]. Potential mechanisms include alterations in gut perme-
ability, production of microbial metabolites, altered lipid metabolism, and modulation of
host gene expression [
63
]. On the other hand, probiotic bacteria such as Bifidobacterium and
Lactobacillus have been studied as a source of folic acid for the host [
64
]. Vitamins such as
folic acid from the unabsorbed diet can serve as a growth factor for colonic bacteria [
65
,
66
].
It has been suggested that folate may help to promote the growth of beneficial gut bacteria

Foods 2023,12, 1612 12 of 15
while suppressing harmful ones, regulating the host’s immune cell function, and improving
drug efficacy [
66
]. While the relationship between folate fortification, the gut microbiome,
and diabetes is still being explored, some studies have suggested that increasing folate
intake may have a positive impact on glycemic control in individuals with diabetes [
40
].
However, more research is needed to fully understand the mechanisms underlying this
three-way intricate relationship between microbiome, diabetes, and folic acid fortification.
4. Conclusions
The current implementation of mandatory folic acid fortification has achieved its
intended benefit of reducing the incidence of NTDs in newborns. Along with this, there
have been a few unintended benefits that have improved the health status of some popula-
tions, including a reduction in tHcy concentrations, which is considered an independent
risk factor for CVD. Other unintended benefits of folic acid fortification include virtually
eliminating folic acid deficiency from the US, reducing the prevalence of anemia, and
decreasing hemoglobin concentrations. However, the effect of folic acid fortification on
cognitive health, stroke, and diabetes is not very clear. Further research is needed to clarify
these unintended benefits.
While mandated folic acid fortification has been effective in reducing the incidence of
NTDs, there are also some concerns associated with mandatory fortification. Some of the
concerns are as follows:
a.
Increased intake of folic acid: excessive consumption of folic acid can mask the
symptoms of vitamin B-12 deficiency, which can lead to vitamin B-12-induced anemia
and neurological damage due to possible delayed diagnosis.
b.
Increased cancer risk: some studies have suggested that high folic acid intakes may
increase the risk of certain types of cancer. However, the evidence for this is not
very conclusive.
c.
Unmetabolized folic acid (UMFA) concentrations: there are some concerns that
UMFA may have adverse effects on health, although the evidence for this is not very
conclusive yet.
d.
Fortification of unhealthy foods: mandatory folic acid fortification may lead to the
fortification of unhealthy foods or the displacement of other essential nutrients.
e.
Individual choice: some individuals may choose to avoid foods that have been
fortified with folic acid due to personal beliefs or dietary restrictions.
f.
Socioeconomic disparities: Fortification may not be accessible to all segments of the
population leading to socioeconomic disparities in folic acid intakes.
It is important to note that the benefits of folic acid fortification in reducing the
incidence of NTDs outweigh the potential risks associated with its mandatory folic acid
fortification. However, the long-term effects of folic acid fortification need to be evaluated
in non-target populations such as the elderly, children, men, post-menopausal women, and
patients who are on antimetabolites for cancer treatment. Specifically, the elderly tend to
consume more supplements, and children tend to consume more breakfast cereals, which
are known to contain high levels of folic acid. These two populations should be monitored
more closely for any long-term health effects.
Author Contributions:
S.I. and S.E. equally contributed to this review; V.G. contributed to the
conception and design; S.I. and S.E. contributed to the acquisition of literature and analysis of
the data; S.I., S.E., and V.G. contributed to the interpretation of the data; and S.I., S.E., and V.G.
drafted and revised the manuscript. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

Foods 2023,12, 1612 13 of 15
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