Clinical and Laboratory Researchdoi: 10.1111/cga.12215

Congenital Anomalies 2017; 57, 157–165

157

INVITED REVIEW ARTICLE

Medical cost savings in Sakado City and worldwide achieved by preventing
disease by folic acid fortification
Yasuo Kagawa1, Mami Hiraoka2, Mitsuyo Kageyama3, Yoshiko Kontai4, Mayumi Yurimoto5, Chiharu Nishijima1, and Kaori Sakamoto1
Department of Medical Chemistry, Kagawa Nutrition University, Sakado City, 2Shukutoku University, College of Nursing and Nutrition, Schoolof Nutrition, Chiba City, 3Yamanashi Gakuin University Faculty of Health and Nutrition, Kofu City, 4University of Niigata Prefecture Faculty of
Human Life Studies Department of Health and Nutrition, Niigata City, and 5Yakult Honsha Co., Ltd., Tokyo, Japan

1

ABSTRACT The introduction of mandatory fortification ofgrains with folate in 1998 in the United States resulted in 767
fewer spina bifida cases annually and a cost saving of $603 million
per year. However, far more significant medical cost savings
result from preventing common diseases, including myocardial
infarction, stroke, dementia and osteoporosis. A costeffectiveness analysis showed a gain of 266 649 quality-adjusted
life-years and $3.6 billion saved annually, mainly due to the
reduction of cardiac infarction. The recommended folate intake
in Japan is 240 μg/day whereas it is 400 μg/day internationally.
Our Sakado Folate Project targeted individuals with genetic
polymorphism of methylenetetrahydrofolate reductase or withhyperhomocysteinemia. Using, for example, folate-fortified rice,
resulted in an increase in serum folate and a decrease in serum
homocysteine in the participants, and reduced medical costs wereachieved by decreasing myocardial infarction, stroke, dementia
and fracture. Due to the small population of Sakado City
(approximately 101 000) and small number of births (693) in
2015, a decrease in spina bifida could not be confirmed but there
was a significant decrease in the number of very low birthweight
infants. The genome notification of subjects was effective inmotivating intake of folate, but the increase in serum folate (from17.4 to 22.5 nmol/L, 129%) was less than that observed following
compulsory folic acid fortification of cereals in the USA (from12.1 to 30.2 nmol/L, 149.6%). Mandatory folic acid fortificationis cheap in decreasing medical costs and is thus recommendedin Japan.
Key Words: folate, fortification, genetic polymorphism, medical cost

INTRODUCTION
The authors reviewed the medical literature reporting key studies on
folic acid fortification, policy recommendations from several foreignorganizations, and our own Sakado Folate Project, and drew
conclusions regarding the decrease in the incidence of disease andthe medical cost savings after the folate intake of the population isincreased. Folate refers to a collection of “folates” that is notchemically well-characterized and includes other members of theCorrespondence: Yasuo Kagawa, Department of Medical Chemistry, Kagawa
Nutrition University, 3-9-21 Chiyoda, Sakado City, Saitama 350-0288, Japan.
Email: kagawa@eiyo.ac.jp
Received December 21, 2016; revised and accepted February 7, 2017.

pteroylglutamate family. These family members are differentiatedby the reduced state of the pteridine ring, a one carbon substitution
at the N5 and/or N10 positions (formyl, methyl, methylene, andmethenyl), and the length of the γ-glutamyls. Polyglutamyl 5methyltetrahydrofolate species are the most abundant naturally
occurring folate in vegetables, and 5-formyltetrahydrofolate species
are the second most abundant (Wang et al. 2010). The bioavailabilityof synthetic folic acid (monoglutamyl pteridine) is approximately
70% better than that of folate (mainly polyglutamyl pteridines) fromdietary sources and thus the term dietary folate equivalent (DFE) is
used to describe the effects of different folate molecular species (Joint
FAO/WHO Expert Consultation on Human Vitamin and Mineral
Requirements 1998). Folate prevents megaloblastic anemia (Chanarin1987) and many other diseases because it is essential to the synthesis,
repair (Lindahl 2013), and epigenetic regulation (Janke et al. 2015) of
DNA. Folate is therefore the link between diet, metabolism, andepigenetics (Janke et al. 2015). Thus, the mechanism by which folate
prevents many diseases, such as neural tube defects (NTD), has beenstudied at the molecular level (Ichi et al. 2012). NTDs are the second
most common group of serious birth defects. Each year, 300 000 to
400 000 infants worldwide are born with NTDs. NTDs result fromfailure of the neural tube to close properly approximately 28 days
post-conception. Two of the most common NTDs are spina bifida
and anencephaly. One of the earliest recommendations on folate
intake was the Recommendations of the Clinical Teratology
Committee of the Canadian College of Medical Geneticists, whichstated that primary prevention of NTDs is preferable to treatment or
to prenatal detection and abortion (Van Allen et al. 1993). The useof biomarkers such as homocysteine to detect folate deficiency has
been described in detail by Bailey et al. (2015). There is now
conclusive evidence that most NTDs can be prevented by theingestion of folic acid near the time of conception (Joint
FAO/WHO Expert Consultation on Human Vitamin and Mineral
Requirements 1998). Thus, mandatory folic acid fortification of cereal
products has been implemented in 82 countries. Mandatory
fortification has led to medical cost savings due to the prevention
not only of NTDs (Grosse et al. 2016), but also of many other diseases
described in the next section (Bentley et al. 2009).

DISEASES PREVENTED BY FOLIC ACIDFORTIFICATION
Megaloblastic anemia (Chanarin 1987) and NTDs (Grosse et al.
2016) are well-known conditions caused by inadequate folate intake,but folate is implicated in many other diseases. For example,© 2017 The Authors. Congenital Anomalies published by John Wiley & Sons Australia, Ltd on behalf of Japanese Teratology Society.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is
properly cited.

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moderate hyperhomocysteinemia induced by low folate status is anindependent risk factor for cardiovascular disease (CVD) (Ishihara
et al. 2008; Bentley et al. 2009), stroke (Yang et al. 2006; Huo et al.
2015; Li et al. 2016b), colon cancer (Bentley et al. 2009), dementia(Jernerén et al. 2015), fracture (Gjesdal et al. 2007), kidney diseases(Massy 2003) and depression (Bottiglieri 2005; Hiraoka et al.
2014). These diseases can be prevented or alleviated by increased
folate intake, as shown by many epidemiological and interventional
studies. The meta-analysis of 30 randomized controlled trials
involving 82 334 participants indicated a 10% lower risk of stroke
and a 4% lower risk of overall CVD with folic acid supplementation(Li et al. 2016b). Folic acid supplementation provided a greater
benefit for preventing CVD in participants with low plasma folate
levels and without preexisting CVD, and resulted in larger decreases
in homocysteine levels (Li et al. 2016b). Folate is required by thebrain for the synthesis of monoamine neurotransmitters, andnumerous observational studies have suggested an association
between folate deficiency and depression (Bottiglieri 2005). Folate
intake below the Recommended Daily Allowance at study baseline
was associated with increased risk of the incidence of Mild
Cognitive Impairment (MCI)/probable dementia (hazard ratio 2.0,
95% CI 1.3 to 2.9) after controlling for multiple confounders(Agnew-Blais et al. 2015). Plasma homocysteine concentration, if
only moderately elevated, is an independent risk factor for CVD
and stroke (Joint FAO/WHO Expert Consultation on Human
Vitamin and Mineral Requirements 1998). Increased risk has been
associated with values higher than 11 mmol/L, which is well within
what is generally considered to be the normal range (5–15 mmol/L)of plasma homocysteine levels (Joint FAO/WHO Expert
Consultation on Human Vitamin and Mineral Requirements
1998). Randomized control tests on the prevention of CVD anddementia with folate often result in negative data. However, these
diseases develop very slowly, and during a test that lasts only a
few years, endothelial cells or neurons are either already damaged,or the disease has progressed insufficiently for necrosis to havedeveloped.

Y. Kagawa et al.
in MTHFR C677T (17.3%) and RFC-1 A80G (18.6%), and thus may
not affect the overall incidence of disease in large populations. In
contrast, the TT genotype of MTHFR C677T showed significantly
lower serum folate and higher serum Hcy than the CC and CT
genotypes (Hiraoka et al. 2004). The TT genotype of the C677T
polymorphism in MTHFR has been associated with CVD (Morita
et al. 1998), dementia (Kageyama et al. 2008), osteoporosis (Gjesdal
et al. 2007), depression (Bottiglieri 2005) and kidney diseases(Sakamoto et al. 2015). In addition, there are many risk SNPs of
CVD, metabolic syndrome, and other chronic diseases (Kagawa
2012; Kagawa et al. 2012) that affect the MTHFR genotypes andmust be considered for disease prevention and the reduction of
medical costs.
Information obtained from the above studies on SNPs indicates that
folate intake sufficient to reduce plasma Hcy to a minimum level ofless than 7.0 μmol/L is required to reduce disease risk (Joint
FAO/WHO Expert Consultation on Human Vitamin and Mineral
Requirements 1998). In Japan, however, a recommendation for
pregnant women to take a folate supplement was made by the
Ministry of Health, Labor and Welfare only in 2000. The possible
benefit of lowering plasma homocysteine through increased folate
intake can be proven only by an intervention trial with folic acidsupplementation in large populations. Using plasma homocysteine
as a biomarker for folate adequacy can only be done on an individual
basis after the possibility of a genetic mutation or an inadequate
supply of vitamin B6 or vitamin B12 has been eliminated (Joint
FAO/WHO Expert Consultation on Human Vitamin and Mineral
Requirements 1998). The risk of hyperhomocysteinemia in subjects
with the TT genotype of MTHFR C677T was successfully correctedby taking 400 μg/day of folate (Hiraoka et al. 2004), which is identicalto the FAO/WHO and US recommended dietary allowance (RDA) of
folate (Joint FAO/WHO Expert Consultation on Human Vitamin andMineral Requirements 1998).

MEDICAL COST SAVINGS RESULTING FROMFOLIC ACID FORTIFICATIONGENETIC POLYMORPHISMS OF FOLATEMETABOLISM
Serum folate and total homocysteine (tHcy) levels are influenced byboth folate intake and genetic polymorphisms. There are several
single nucleotide polymorphisms (SNPs) that affect folate
metabolism, including the 5, 10-methylenetertahydrofolate reductase(MTHFR) gene C677T. The presence of the C677T mutation in the
MTHFR gene (rs1801133) has been regarded as a genetic risk factor
for coronary artery diseases and neural tube defects (Li et al. 2016b).
The frequency of the T allele of MTHFR is 36.9%, 41.7%, 58.6% and8.1%, in Japan, the USA, Mexico and Indonesia, respectively(Sadewa et al. 2002). The authors also tested the following highfrequency SNPs affecting folate and homocysteine metabolism(Hiraoka et al. 2004): MTHFR A1298C, CBS844ins68(cystathionine-β-synthase), MS A2756G (methionine synthase) (Caiet al. 2014), MTRR A66G (methionine synthase reductase) (Zhiet al. 2016), and RFC-1 G80A (reduced folate carrier-1) (Li et al.
2016a). CBS844ins68 is the 8th exon insertion mutation andcomprises 68 base pairs (Cai et al. 2014). However, these SNPs,
except for MTHFR C677T, did not significantly affect serum Hcy,
folate, vitamin B6 and vitamin B12 levels (Hiraoka et al. 2004). The
frequencies of mutant homozygotes were lower in MTHFRA1298C (1.3%), MS A2756G (5.3%) and MTRR A66G (8.5%) than

Folic acid supplementation of the expectant mother’s diet should
result in fewer NTDs among infants and ancillary savings in medical
costs (Van Allen et al. 1993). A minimum dosage of folic acid of
0.8 mg/day and a maximum dosage of 5.0 mg/day was recommended(Van Allen et al. 1993), along with a well-balanced, nutritious diet for
all women who are at increased risk of having offspring with NTDs
and who are planning a pregnancy. The results from economic
evaluations demonstrate that folic acid fortification of food andpreconception folic acid consumption are cost-effective ways toreduce the incidence and prevalence of NTDs (Yi et al. 2011). The
annual direct medical cost per patient was estimated to be €42 943($51 574 in 2003) for NTD and between €11 728 ($11 061 in
1993) to €54 270 ($65 177 in 2003) for spina bifida in the USA (Yiet al. 2011). The prevention of CVD and the medical cost savings
expected from folic acid supplementation was also estimated(Hornberger 1998). Bayesian statistical and decision-analytic
techniques were used to estimate the cost–benefit and sample sizeof a placebo-controlled trial of folate targeted to US citizens aged
35 to 84 years with elevated serum homocysteine levels. The mainend point is event-free survival (i.e., survival without new ischemic
heart disease or stroke) at 5 years. Because the screening and othercosts associated with this study were small compared with theconsequences of stroke, ischemic heart disease, or death, the increasein 5-year event-free survival with folate that should compel the use of© 2017 The Authors. Congenital Anomalies published by John Wiley & Sons Australia, Ltd on behalf of Japanese Teratology Society.

Cost savings by folic acid fortification
folate is just 1.1% which was explained in detail (Hornberger 1998).
The event-free survival rate for the placebo and folate groups wereassumed to lie, with 95% probability, between 83.7% and 87.7%,
and 82.9% and 90.7%, respectively. Such a trial would provide an
expected societal cost–benefit savings exceeding $11 billion within
15 years (Hornberger 1998).
In 1998, compulsory folic acid fortification was started in the US
and Canada, and the FDA mandated that all enriched cereal grain
products be fortified with folic acid at a level of 140 μg/100 g of cereal
product (Ganji and Kafai 2006). The exact human test using 13C- and2
H double labeled folate fortified cereals established this fortificationlevel (Pfeiffer et al. 1997). This fortification mandate is estimated tohave reduced the annual number of US live-birth spina bifida casesby 767 (Grosse et al. 2016). The present value of mean direct lifetime
cost per infant with spina bifida is estimated to be $791 900,
excluding caregiving costs. Folic acid fortification is estimated toreduce the present value of total direct costs for each year’s birthcohort by $603 million above the cost of fortification (Grosse et al.
2016). These estimates of cost savings are larger than thosepreviously reported, even using conservative assumptions.
Following the implementation of mandated folic acid fortification,a decline in stroke mortality was observed in the US between 1990
and 1997, and this decline accelerated in 1998 to 2002 in nearly all
population strata, with an overall change from 0.3% (95%CI,
0.7 to 0.08) to 2.9 (95%CI, 3.5 to 2.3) per year (P = 0.0005)(Yang et al. 2006). The fall in stroke mortality in Canada averaged
1.0% (95%CI, 1.4 to 0.6) per year from 1990 to 1997 andaccelerated to 5.4% (95%CI, 6.0 to 4.7) per year in 1998 to2002 (P ≤ 0.0001). In contrast, the decline in stroke mortality in
England and Wales did not change significantly between 1990 and2002 (Yang et al. 2006). After the implementation of compulsory
folic acid fortification, trends in serum folate, red blood cell (RBC)
folate, and circulating total homocysteine concentrations in the
United States were analyzed in 17 144 participants in the National
Health and Nutrition Examination Surveys (Ganji and Kafai 2006).
Overall, the geometric mean serum folate concentrations (nmol/L)
were 12.1 ± 0.3, 30.2 ± 0.7, and 27.8 ± 0.5 in 1988–1994 (before
fortification), 1999–2000 (after fortification), and 2001–2002 (after
fortification), respectively. This means that the serum folate levels
were 149.6% and 129.8% higher in the survey participants in 1999–
2000 and 2001–2002, respectively, compared to in 1988–1994(P < 0.0001). The RBC folate concentrations (nmol/L) were
391 ± 5.4, 618 ± 11.7, and 611 ± 9.3 in 1988–1994, 1999–2000,
and 2001–2002, respectively. Age-, sex-, and race-ethnicity-adjusted
tHcy declined from 9.5 μmol/L in 1988–1994 to 7.6 μmol /L in
1999–2000 and to 7.9 μmol/L in 2001–2002.
The greatest benefits from fortification were predicted in
myocardial infarction prevention, with 88 172 cases averted per year
in steady state condition after 5 years’ folic acid fortification for a
fortification level of 700 μg/100 g enriched grain (Bentley et al.
2009). This level of fortification, discussed by Dary (2009), was also
projected to avert 38 805 cases of colon cancer and 1423 cases of
NTD. Compared with no fortification, all post-fortification strategies
provided Quality Adjusted Life Year (QALY) gains and cost savings
for all subgroups, with predicted population benefits of 266 649
QALY gained and $3.6 billion saved in the long term by using afortification level of 700 μg/100 g enriched grain.

OUTLINE OF THE SAKADO FOLATE PROJECTThe Japanese RDA is insufficient for persons with polymorphism ingenes involved in folate metabolism and in aged persons with159

decreased folate bioavailability. The average folate intake in 2014
was 284 μg/day, which is higher than the Japanese RDA of
240 μg/day for adults (Ministry of Health, Labor and Welfare2014). However, the folate intake of women aged 20–39 years was
only 243–253 μg/day in 2014, which is much less than the RDA
of 480 μg/day for pregnant women (Ministry of Health, Labor andWelfare 2014). There are three reports on the folate intake ofJapanese pregnant women: 289 ± 150 μg/day (Mito et al. 2007),
336 ± 161 μg/day (Nakano and Ishii 2004) and 283 μg/day (Kondo
et al. 2013, standard deviation not clear, the value of 2011). These
values are much less than the RDA of 480 μg/day. The folate intake
of 50% of the poor pregnant women was less than 260 μg/day (OndaR, reported in the Japanese Society of Nutrition and Dietetics, 2015).
The folate intake higher than these levels among Japanese wasquite effective in preventing cardiovascular diseases wasestablished by Japan Collaborative Cohort Study on a total of
23 119 men and 35 611 women, age 40 to 79 years (Cui et al.
2010). Increased dietary folate intake from < 272 μg/day
to > 536 μg/day resulted in 51% reduction of mortality for men
from heart failure (P < 0.01) and that from ischemic heart diseases(down to 72%, P < 0.05), and for women ischemic heart diseases(down to 56% P < 0.01). These inverse associations did notchange materially after adjustment for cardiovascular risk factors,so, folate intake of 243–253 μg/day by general Japanese is notenough to prevent cardiovascular diseases and thus more folate
intake will proportionally reduce the medical costs.
Moreover, the bioavailability of folate is low in persons withgenetic polymorphism (Hiraoka et al. 2004). About 15% of Japanesewith the TT genotype of MTHFR C677T require 400 μg/day of folate
to increase the serum folate level to that of CC and CT genotypes.
Folate deficiency among persons with dementia was confirmed bymeta-analysis of 31 studies (Lopes da Silva et al. 2014) and ofJapanese dementia studies (Kageyama et al. 2008). The risk of brain
infarction is 3.4-fold higher in persons with the TT type comparedto persons with the CC type (Morita et al. 1998). The randomized
controlled double blind test was performed to confirm the exact folate
requirement of Japanese (Hiraoka et al. 2014).
To prevent the above-described diseases caused by folate
deficiency, Sakado City in Japan made an agreement with Kagawa
Nutrition University called the “Sakado Folate Project” in 2006.
The Project offered participants a lecture, genotyping, blood analysis,
nutrition surveys, and guidance according to the collected data
(Fig. 1). The lecture was similar to that given in the Biomarkers of
Nutrition for Development (BOND) project and was designed toprovide evidence-based advice to anyone with an interest in the roleof folate, vitamin B12, and other micronutrients in health (Bailey
et al. 2015). We provided people with an overview of available
biomarkers (serum folate and homocysteine concentrations) and theinterpretation of these biomarkers across a range of clinical andpopulation-based uses. We also explained the genetic polymorphisms
involved in folate deficiency, and obtained written informed consent
from participants in accordance with the instructions of the
Declaration of Helsinki. The study procedures were approved by
the Kagawa Nutrition University, Human Subjects and Genome
Ethics Committee. There were 1101 participants (male: n = 345,
female: n = 756) aged 61 ± 10 years. All participants completed a
dietary intake analysis by means of a validated, brief selfadministered diet history questionnaire (DHQ-L) (Sasaki 2004)
containing questions about the consumption frequency of 56 foods
and beverages commonly consumed by the Japanese population.
Blood chemistry analysis included serum folate and tHcy
concentration. The serum was isolated and stored at 80°C until
analysis. Serum folate and vitamin B12 concentrations were measured
at an external laboratory (SRL, Inc., Tokyo, Japan) using an Access 2© 2017 The Authors. Congenital Anomalies published by John Wiley & Sons Australia, Ltd on behalf of Japanese Teratology Society.

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Y. Kagawa et al.

Fig. 1 Steps comprising the Sakado Folate Project.

chemiluminescence enzyme immunoassay (Beckman Coulter, Inc.,
Carlsbad, CA). Serum tHcy concentration was determined by enzyme
assay using an Alfressa Auto Hcy kit (Alfressa Pharma, Inc., Osaka,
Japan) (Hiraoka et al. 2014). In order to genotype a large number ofblood samples rapidly and at low cost, we developed an automatedgenotyping machine using a bead array in capillary tubes (Kagawa
et al. 2010) and used this approach for MTHFR and other risk SNPs
(Kagawa et al., 2012).
One month after obtaining the blood samples, the genotype wasannounced by the attending medical doctor to subjects who had
agreed to know their genotypes, and nutritional and exercise guidance
was provided by a registered dietitian. To supply enough folate easily,
we developed “folate fortified rice” containing 26.7 mg folate,
187 mg thiamin, 66.7 mg vitamin B6 and 320 μg vitamin B12 per
100 g rice in collaboration with House-wellness Foods Company.
We also developed a folate fortified bread called Sakado Folate Bread(folic acid 340 ± 21 μg/100 g (215 ± 14.7 μg/slice/64.0 g bread) andsucceeded in improving folate status and depressive states (Hiraoka
et al. 2014). Commercially available Sakado Folate Bread contains
160 μg folic acid/slice.
Serum folate concentrations were significantly increased andserum tHcy concentrations decreased at 4 months after the onset of
this intervention by the Project (Fig. 2). The horizontal lines in panel
A (serum folate), B (serum homocysteine), C (dietary folate intake)and D (dietary green-yellow vegetables) indicate the standard values(7 ng/mL), (6 μg/mL), (300 μg/day) and (200 μg/day), respectively.
In individuals with the TT genotype, these changes were particularlymarked and intake of ‘green vegetables’, which are rich in folate, wasincreased in the TT genotype participants. After the instructional
Project, 86% of the subjects consumed folic acid fortified food, of
which 42% consumed folate-fortified rice every day.
The most important aspects of the Project were the health educationof about 101 000 citizens due to the efforts of volunteers and wider
consumption of folate-fortified food, especially folate-fortified rice.
The above-mentioned BOND project (Bailey et al. 2015) was
designed to easily explain the role of folate and vitamin B12 to thegeneral public by volunteer nutritionists. In Sakado Folate Project,
we also obtained help of 125 nutrition advisors (nutrition mates)
who both visited people’s neighborhoods and worked in numerous

facilities called “Terakoya”. BOND project was similar in theirguidance principle, but the implementation of Sakado Folate Projectusing nutritional advisors, “Terakoya” and other measures were quite
different from those of BOND project. According to the official report
prepared by Sakado City on the nutritional behavior of its citizens,after the start of this Project 80% of the population knew about theimportance of folic acid, 90% tried to consume more vegetables,and 73% wanted to obtain health advice. The population of Sakado
City was only about 101 000 in 2015 and there were 693 births in
2015; consequently, a decrease in neural tube defects could not be
confirmed. However, the birth rate of very low birth weight infants(body weight < 1500 g) decreased from 7.8 (1998–2002) to 6.1(2006–2010) per thousand births after the Project. The total fertility
rate in the first period was 1.13 and second period was 1.20 (Ministryof Internal Affairs. Statics Bureau of Japanese Government, 1990a,b).
There are five reasons for the overall success of the Sakado Folate
Project.
1. Effective organization of volunteers recommended by thegovernment to promote a healthy diet.
2. The invention of a rapid and inexpensive genetic polymorphism
analyzer.
3. The involvement of a well-trained registered dietician in the
nutritional survey who was available throughout the project togive advice.
4. The cooperation of the local government and the mayor of Sakado
City.
5. The development of two folate-fortified foods: Sakado Folate Riceand Sakado Folate Bread.

MEDICAL COST SAVINGS RESULTING FROMTHE SAKADO FOLATE PROJECT
We verified the effect of personalized nutrition intervention on thefolate status of participants; this, together with genotype notification,
was effective in motivating individuals to change their dietary habits
and improve their folate intake (Fig. 3). Thus, the subjects with the© 2017 The Authors. Congenital Anomalies published by John Wiley & Sons Australia, Ltd on behalf of Japanese Teratology Society.

Cost savings by folic acid fortification

161

Fig. 2 Effects of the Sakado Folate Project on serum folate and homocysteine, and the intake of dietary folate and green-yellow vegetables, on individuals carrying
three genotypes of MTHFR. (A) Serum folate concentration (ng/mL, y-axis); the horizontal line indicates the standard (7 ng/mL). (B) Serum homocysteine(μmol/mL, y-axis), and the horizontal line indicates the standard (6 μg/mL). (C) Intake of dietary folate (μg/day, y-axis), and the horizontal line indicates thestandard (300 μg/day). (D) Intake of dietary green-yellow vegetables (μg/day, y-axis), and the horizontal line indicates the standard (200 μg/day).

most risky TT genotype showed the highest increase in serum folate
and the largest decrease in serum homocysteine (Figs 2, 3).
Both the prevalence of disease and the medical costs of subjects
participating in the Project, and of the general Sakado population,
were reduced. A nutrition survey conducted by the Saitama Prefecture
Department of Health in 2011 revealed that the prevalence (% of adult
population) of obesity, hypertension, type 2 diabetes anddyslipidemia diagnosed by physicians was significantly lower thanthat in the males of three other cities (Fig. 4). The data on Figure 4
are based on adult population in 2011. The male population showedsignificantly lower prevalence of these diseases than the other threecities (P = 0.04), but female population did not reach significant level.
The average ages of males and females in Sakado city in 2011 were42.4 and 44.4, respectively, while those of three cities (Yoshikawa,

Fig. 3 Effects of genome notification on changes inserum folate and serum homocysteine inindividuals carrying three different genotypesof MTHFR (CC, CT and TT). Bars represent
the means ± SD.

© 2017 The Authors. Congenital Anomalies published by John Wiley & Sons Australia, Ltd on behalf of Japanese Teratology Society.

162

Fig. 4 Prevalence of obesity, hypertension, diabetes and dyslipidemia among
the population in Sakado City and three other cities in Saitama
Prefecture (Saitama Prefecture Local Government 2011). The ordinate
indicates prevalence in percentage. The prevalence in Sakado City
was significantly fewer than that in other three cities in men (P = 0.04).

Asaka and Okegawa cities) on average were younger; 41.4 and 43.1
(Ministry of Internal Affairs. Statics Bureau of Japanese Government,
1990a,b).
This was reflected in the relative decrease in medical costs in
Sakado City immediately after the start of the Project in 2006 (Fig. 5).
The relative decrease of medical costs from 2003–2005 to 2006–2008
in Sakado City plotted in Figure 5 is statistically significant(P = 0.0459, using Wilcoxon rank sum test). The actual medical costsof National Health Insurance per person was ¥303 900 in 2006–2007
compared with those of ¥290 399 in 2001–2005 in Sakado City. The
increment ¥13 501 was the smallest among all cities in Saitama
Prefecture. The abscissa in Figure 5 is the year and the ordinate
indicates the medical costs for each of the four cities being compared
relative to the average total medical costs in Japan (1.00). A few years
may be necessary for this Project to expect a reduction in thesignificant amount of the medical expenditure. However, in the large
population of Sakado city, similar to United States as soon as the

Y. Kagawa et al.
serum folate elevation, rapid decrease in stroke took place within ayear (Yang et al. 2006), because the increase of serum folate anddecrease of serum homocysteine takes place only within a few weeks(Hiraoka et al. 2004, 2014). Moreover, rapid preventive effects offolate on cardiac infarction was reported in detail (Moens et al.
2008). In addition, the subjects of the Project in 2006 and 2007 weremainly local health and nutrition leaders to teach citizens rapidly.
Since there were no rapid demographic change in Sakado city
(Ministry of Internal Affairs. Statics Bureau of Japanese Government,
1990a,b), the rapid reduction in the medical expense is attributed to
the Project. We should show the values to the year 2014 or 2015 inFigure 5, however, unfortunately, there was a large reform of the
medical insurance system during 2008, which is Comprehensive
Reform of Social Security: Advanced Elderly Medical Service
System (Medical care for the elderly over 75 years old) (Ministry of
Health, Labor and Welfare 1990), so we have to discontinue the graphat 2008. Although the total medical cost were changed, in 2014, thatof Sakado city (average age: male 42.8, female 44.7) was 95.7% ofthat of all cities in Saitama (average age: male 42.3, female 44.3),
and 92.5% of that of neighboring Kawagoe city (average age: male
42.6, female 44.7).
The average medical costs saved per person per month (during
May and October before the project from 2003 to 2005 minus thoseafter the project from 2006 to 2008) after the Project for cardiac
infarct, cerebrovascular disease, dementia and osteoporosis were¥364–297 = ¥67, ¥420-¥409 = ¥11, ¥85-¥47 = ¥38 and ¥230¥222 = ¥8, respectively. These data were based on the automaticcomputer output of Japanese Health Care Insurance System of
Sakado city, and the medical costs were analyzed by experts ofmedical epidemiology. However, the results for average citizens
shown in Figures 4 and 5 do not represent the direct effects of folate
intake because we did not measure the serum folate levels of all
citizens.
The relationship between the costs and the changes in serum folate
level was used to estimate the medical cost savings (Bentley et al.
2009). The proportional association between the changes inserum/plasma folate and the intake of folate in the diet based on
information from several studies reporting these two parameters were
calculated using a linear equation that describes their association(Quinlivan and Gregory 2007): change in serum folate(mg/L) = [intake of folic acid as DFE (μg/day) × 0.0145] + 0.132

Fig. 5 Trend in relative medical costs in Sakado City
compared with four cities in Saitama
Prefecture. The abscissa shows the year andthe ordinate indicates the medical costs
incurred by each city relative to the average
total medical costs in Japan (=1.00) taken fromthe home page of the Ministry of Health, Laborand Welfare (http://www.mhlw.go.jp/topics/
bukyoku/hoken/iryomap/).
The
relativedecrease of medical costs from 2003–2005 to
2006–2008 in Sakado City plotted in Figure 5
is statistically significant (P = 0.0459, using
Wilcoxon rank sum test). Black thick line:
Sakado City, black broken line: average for
Saitama Prefecture. Black dotted line: city A,
gray line: city B, gray broken line: city C, andgray dotted line: city D.

© 2017 The Authors. Congenital Anomalies published by John Wiley & Sons Australia, Ltd on behalf of Japanese Teratology Society.

Cost savings by folic acid fortification

163

Table 1 Estimated medical cost savings resulting from folate intake by the residents of Sakado City

Genotypes
CC (n = 357)
CT (n = 553)
TT (n = 191)
Total (n = 1101)

Serum folate (ng/mL)
Changes
2.11
1.46
5.34
2.24

Changes in synthetic
folate intake (μg/d)

Medical costs saved
per year/person

Total medical costs
saved per year

80.39
54.00
211.47
85.58

¥1644
¥1392
¥2238
¥1669

¥586 900¥769 766¥427 377¥1 784 042(Dary 2009). The formula considers the equivalence of folic acid interms of DFE based on the approximately 70% higher bioavailabilityof monoglutamyl pteridine over dietary sources of folate. Table 1
summarizes the changes in serum folate of three genotypes of
MTHFR before and after the Project, with the intake of folate being
expressed in terms of synthetic folate (monoglutamyl pteridine).
The serum folate concentration in the 1101 subjects increased from
7.68 ng/mL to 9.92 ng/mL, a change of 2.24 ng/mL. However, thesubjects notified of carrying the TT genotype (n = 191) showed serum
folate concertation increased 178% from 6.87 (17.4 nmol/L) to 12.22(22.5 nmol/L). nmol/L). The calculated medical costs saved perperson was ¥1,669 for a total of ¥1,784,042 per year (Sakado City
Local Government 2015) according to the relationship between
folate intake and medical cost reported (Bentley et al. 2009), if
the relationship proposed by Bentley is applicable to the peopleof Sakado.
However, these estimated medical cost savings were limited tocoronary infarction, colon cancer and spina bifida (Bentley 2009),
and therefore we measured the medical cost savings of the subjects
participating in the Project (Fig. 6). To do this, we compared theaverage medical costs of Sakado citizens (expected costs: ¥10 276
to ¥12 196; average: ¥11 076 per person per month) with those ofparticipants in the Project. The average medical costs of theparticipants fluctuated greatly, between ¥4518 to ¥13 793, (average:¥7958 per person per month), due to the small number of participants,
and this caused large skewness of the frequency distribution of
personal medical cost, because some rare medical treatment costs

extremely high and affects the average value. Thus, the median (¥0
to ¥4830, average: ¥2960, per person per month) clearly shows
medical cost savings of ¥8116 per person per month (a cost savingsof 26.7% compared to the control citizens) attributed to the Sakado
Folate Project (Fig. 6). According to the medical cost calculation
system there are always some delay in the exact sum up. So the curveis an expected value considering the recent medical costs, not theexact medical cost.
Finally, mandatory folic acid fortification (Bentley et al. 2009) is
obviously more cost-effective than the Project, because in addition
to the cost of folic acid fortification, the Project incurred other costs
per participant, such as personnel expenses for education on nutrition(¥1500 per person per group lecture), nutritional surveys (forexample, the FFQ type cost ¥1000 per person), genotyping (about¥500 per person), and general biochemical analyses (¥110 = 11analyte per person). Moreover, following supplementation, serum
folate levels increased from 12.1 to 30.2 nmol/L (an increase of
149.6%) in the USA (Ganji and Kafai 2006), but only from 17.4 to
22.5 nmol/L (an increase of 129% averaged between 2006 and2012) in Sakado. Country-specific differences in the medical carecosts for treating patients with each disease, as well as personnel
expenses, fluctuations in the exchange rate between the American
dollar and the Japanese yen, and the numerous assumptions required,together made it difficult to calculate the exact cost benefits of the
Project compared with that of mandatory folic acid fortification.

CONCLUSION

Fig. 6 Expected medical costs and actual medical costs of subjects
participating in the Sakado Folate Project. The abscissa shows theyear and the ordinate indicates the medical costs per person permonth in yen. Black line: expected costs of citizens in Sakado City,
Gray line: average of actual costs of the subjects participating in
the Project, Light gray line: median of the actual costs of the subjects
participating in the Project.

In summary, folic acid fortification was effective in preventing not
only NTDs but also other diseases, including CVD, dementia,
kidney diseases and osteoporosis, irrespective of genetic
polymorphisms such as the MTHFR TT genotype. The medical
costs saved were dependent on the disease involved, the projects
implemented in each country, and the economic evaluation
methods used. The genotyping was effective in high risk approachwith increased motivation (Figs 2, 3). Genome notification played
an important role in the Sakado Folate Project and was effective inmotivating folate intake, especially in participants with the TT
genotype, yet the increase in serum folate (129%) was less thanthat observed following compulsory folic acid fortification in the
USA (149.6%). Evaluation showed that compulsory folic acidfortification is more cost-effective than the genotype-based
educational approach used in the Sakado Folate Project. In
conclusion, compulsory folic acid fortification is the best method
to prevent disease and reduce medical costs.

ACKNOWLEDGMENTS
This study was supported by Grants-in-Aid for Scientific Researchfrom the Ministry of Education, Science, and Culture of Japan, JSPS

© 2017 The Authors. Congenital Anomalies published by John Wiley & Sons Australia, Ltd on behalf of Japanese Teratology Society.

Y. Kagawa et al.

164
KAKENHI Grant Number JP16K00865, and grants for Private
Universities No. S0903001.

DISCLOSURES
None.

REFERENCES
Agnew-Blais JC, Wassertheil-Smoller S, Kang JH et al. 2015. Folate, vitamin
B-6, and vitamin B-12 intake and mild cognitive impairment and probabledementia in the Women’s Health Initiative Memory Study. J Acad Nutr Diet
115 (2):231–241.
Bailey LB, Stover PJ, McNulty H et al. 2015. Biomarkers of nutrition fordevelopment-folate review. J Nutr 145 (7):1636S–1680S.
Bentley TG, Weinstein MC, Willett WC, Kuntz KM. 2009. A costeffectiveness analysis of folic acid fortification policy in the Unites States.
Public Health Nutr 12 (4):455–467.
Bottiglieri T. 2005. Homocysteine and folate metabolism in depression. Prog
Neuropsychopharmacol Biol Psychiatry 29:1103–1112.
Cai W, Yin L, Yang F, Zhang L, Cheng J. 2014. Association between Hcylevels and the CBS844ins68 and MTHFR C677T polymorphisms withessential hypertension. Biomed Rep 2 (6):861–868.
Chanarin I. 1987. Megaloblastic anemia, cobalamin, and folate. J Clin Pathol
40 (9):978–984.
Cui R, Iso H, Date C, Kikuchi S, Tamakoshi A, Japan Collaborative Cohort
Study Group. 2010. Dietary folate and vitamin B6 and B12 intake in relation
to mortality from cardiovascular diseases: Japan collaborative cohort study.
Stroke 41 (6):1285–1289.
Dary O. 2009. Nutritional interpretation of folic acid interventions. Nutr Rev
67 (4):235–244.
Ganji V, Kafai R. 2006. Trends in serum folate, RBC folate, and circulating
total homocysteine concentrations in the United States: analysis of data from
National Health and Nutrition Examination Surveys, 1988–1994, 1999–
2000, and 2001–2002. J Nutr 136:153–158.
Gjesdal CG, Vollset SE, Ueland PM, Refsum H, Meyer HE, Tell GS. 2007.
Plasma homocysteine, folate, and vitamin B12 and the risk of hip fracture:
the Hordaland homocysteine study. J Bone Miner Res 22 (5):747–756.
Grosse SD, Berry RJ, Tilford MJ, Kucik JE, Waitzman NJ. 2016.
Retrospective assessment of cost savings from prevention: Folic acidfortification and spina bifida in the U.S. Am J Prev Med 50 (5S1):S74–S80.
Hiraoka M, Fukushima A, Sakuma K et al. 2014. Folic acid supplementation
reduced depression score in young Japanese women with depressionsusceptible genetic polymorphisms. Nutr Health 1 (2):1–10.
Hiraoka M, Kato K, Saito Y, Yasuda K, Kagawa Y. 2004. Gene-nutrient andgene–gene interactions of controlled folate intake by Japanese women.
Biochem Biophys Res Commun 316:1210–1216.
Hornberger J. 1998. A cost–benefit analysis of a cardiovascular disease
prevention trial, using folic acid supplementation as an example. Am J
Public Health 88 (1):61–67.
Huo Y, Li J, Qin X et al. 2015. Efficacy of folic acid therapy in primaryprevention of stroke among adults with hypertension in China: The CSPPT
Randomized Clinical Trial. JAMA 313 (13):1325–1335.
Ichi S, Nakazaki H, Boshnjaku V et al. 2012. Fetal neural tube stem cells from
Pax3 mutant mice proliferate, differentiate, and form synaptic connections
when stimulated with folic acid. Stem Cells Dev 21 (2):321–330.
Ishihara J, Iso H, Inoue M et al. 2008. Intake of Folate, Vitamin B6 and Vitamin
B12 and the Risk of CHD: The Japan Public Health Center-Based
Prospective Study Cohort I. J Am Coll Nutr 27 (1):127–136.
Janke R, Dodson AE, Rine J. 2015. Metabolism and epigenetics. Annu Rev
Cell Dev Biol 31:473–496.
Jernerén F, Elshorbagy AK, Oulhaj A, Smith SM, Refsum H, Smith AD. 2015.
Brain atrophy in cognitively impaired elderly: the importance of long-chain
ω-3 fatty acids and B vitamin status in a randomized controlled trial. Am J
Clin Nutr 102 (1):215–221.
Joint FAO/WHO Expert Consultation on Human Vitamin and Mineral
Requirements. Vitamin and mineral requirements in human nutrition: reportof a joint FAO/WHO expert consultation. Bangkok, Thailand, 21–30
September 1998. pp. 289–302.
Kagawa Y. 2012. From clock genes to telomeres in the regulation of thehealthspan. Nutr Rev 70 (8):459–471.
Kagawa Y, Hiraoka M, Miyashita-Hatano Y et al. 2010. Automated single
nucleotide polymorphism typing using bead array in capillary tube. J Biosci
Bioeng 111:505–508.
Kagawa Y, Yanagisawa Y, Hasegawa K et al. 2012. Single nucleotide
polymorphism of thrifty genes for energy metabolism: evolutionary origins
and prospects for intervention to prevent obesity-related diseases. Biochem
Biophys Res Commun 295:207–222.
Kageyama M, Hiraoka M, Kagawa Y. 2008. Relationship between genetic
polymorphism, serum folate and homocysteine in Alzheimer’s Disease.
Asia Pac J Public Health 20 (Suppl):111–117.
Kondo A, Iwagaki S, Kidaira M et al. 2013. Changes in life styles of pregnant
women and risks for having a pregnancy afflicted with spina bifida. Jpn J
Urol 104:598–604 (in Japanese).
Li X, Hu M, Li W et al. 2016a. The association between reduced folate carrier1 gene 80G/A polymorphism and methotrexate efficacy or methotrexaterelated toxicity in rheumatoid arthritis: A meta-analysis. Int
Immunopharmacol 38:8–15.
Li Y, Huang T, Zheng Y, Muka T, Troup J, Hu FB. 2016b. Folic Acid
Supplementation and the Risk of Cardiovascular Diseases: A MetaAnalysis of Randomized Controlled Trials. J Am Heart Assoc 5 (8) pii:
e003768.
Lindahl T. 2013. My journey to DNA repair. Genomics Proteomics
Bioinformatics 11 (1):2–7.
Lopes da Silva S, Vellas B, Elemans S et al. 2014. Plasma nutrient status ofpatients with Alzheimer’s disease: Systematic review and meta-analysis.
Alzheimers Dement 10 (4):485–502.
Massy ZA. 2003. Potential strategies to normalize the levels of homocysteinein chronic renal failure patients. Kidney Int Suppl 2003 (84):S134–S136.
Ministry of Health, Labor and Welfare. 2014. The National Health andNutrition Survey Japan, 2014 (partly in English). ww.mhlw.go.jp/bunya/
kenkou/eiyou/h24-houkoku.html. Accessed in 2015.
Ministry of Health, Labor and Welfare. 1990–2014. Health and Medical
Services. Health Care Insurance System. http://www.mhlw.go.jp/english/
wp/wp-hw9/dl/02e.pdf. Accessed in 2015.
Ministry of Internal Affairs. Statics Bureau of Japanese Government 1990a–
2015. Average age of all cities and towns in Saitama Prefecture. https://www.pref.saitama.lg.jp/a0206/a009/11-heikin.html. Accessed in 2016.
Ministry of Internal Affairs. Statics Bureau of Japanese Government 1990b2015. Statistics of Child birth and fertility rates in Saitama Prefecture
1990–2014.
https://www.pref.saitama.lg.jp/a0701/hokentoukei/gotoku.
html. Accessed in 2016.
Mito N, Takimoto H, Umegaki K et al. 2007. Folate intakes and folate
biomarker profiles of pregnant Japanese women in the first trimester. Eur
J Clin Nutr 61 (1):83–90.
Moens AL, Champion HC, Claeys MJ et al. 2008. High-dose folic acidpretreatment blunts cardiac dysfunction during ischemia coupled tomaintenance of high-energy phosphates and reduces postreperfusion injury.
Circulation 117 (14):1810–1819.
Morita H, Kurihara H, Tsubaki S et al. 1998. Methylenetetrahydrofolate
reductase gene polymorphism and ischemic stroke in Japanese. Arterioscler
Thromb Vasc Biol 18 (9):1465–1469.
Nakano T, Ishii K. 2004. Dietary Survey of Japanese Women during
Pregnancy and Lactation. Jpn J Nutr Diet 62 (2):103–110 (in Japanese).
Pfeiffer CM, Rogers LM, Bailey LB, Gregory JF 3rd. 1997. Absorption of
folate from fortified cereal-grain products and of supplemental folate
consumed with or without food determined by using a dual-label stableisotope protocol. Am J Clin Nutr 66 (6):1388–1397.
Quinlivan EP, Gregory JF 3rd. 2007. Reassessing folic acid consumption
patterns in the United States (1999-2004): potential effect on neuraltube defects and overexposure to folate. Am J Clin Nutr 86(6):1773–1779.
Sakamoto K, Kanno Y, Hiraoka M, Hayashi M, Kontai Y, Kagawa Y. 2015.
Maintenance of activities of daily living despite risk from genetic
polymorphism in hemodialysis patients under nutritional management
who survived an average of 30 years. J Renal Replacement Therapy 1:6.

© 2017 The Authors. Congenital Anomalies published by John Wiley & Sons Australia, Ltd on behalf of Japanese Teratology Society.

Cost savings by folic acid fortification
Sadewa AH, Sunarti, Sutomo R et al. 2002. The C677T mutation in the
methylenetetrahydrofolate reductase gene among the Indonesian Javanese
population. Kobe J Med Sci 48 (5–6):137–144.
Saitama Prefecture Local Government. 2011. Health and Nutritional Survey
Data 2011. https://www.pref.saitama.lg.jp/a0704/data/documents/505961.
pdf. Accessed in 2012.
Sakado City Local Government. 2015. Report on the Sakado Folate Project to
Saitama Prefecture.
Sasaki S. 2004. Development and evaluation of dietary assessment methods
using biomarkers and diet history questionnaires for individuals. Tokyo,
Japan: Ministry of Health, Labor and Welfare (in Japanese).
Van Allen MI, Fraser FC, Dallaire L et al. 1993. Recommendations on the useof folic acid supplementation to prevent the recurrence of neural tube
defects. Clinical Teratology Committee, Canadian College of Medical
Geneticists. CMAJ 149 (9):1239–1243.

165

Wang C, Riedl KM, Schwartz SJ. 2010. A liquid chromatography-tandem
mass spectrometric method for quantitative determination of native 5methyltetrahydrofolate and its polyglutamyl derivatives in raw
vegetables. J Chromatogr B Analyt Technol Biomed Life Sci 878(29):2949–2958.
Yang Q, Botto LD, Erickson JD et al. 2006. Improvement in stroke mortality in
Canada and the United States, 1990 to 2002. Circulation 113(10):1335–1343.
Yi Y, Lindemann M, Colligs A, Snowball C. 2011. Economic burden of neuraltube defects and impact of prevention with folic acid: a literature review. Eur
J Pediatr 170 (11):1391–1400.
Zhi X, Yang B, Fan S et al. 2016. Gender-specific interactions of MTHFR
C677T and MTRR A66G polymorphisms with overweight/obesity on
serum lipid levels in a Chinese Han population. Lipids Health Dis 15(1):185.

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