Folic Acid and Colon Cancer: Impact of Wheat Flour Fortification With Folic Acid

C H A P T E R

32 Folic Acid and Colon Cancer: Impact of Wheat Flour Fortification With Folic Acid Sandra Hirsch, Maria Pia de la Maza, Gladys Barrera, Laura Leiva, and Daniel Bunout Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile

O U T L I N E Introduction

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Experimental Studies and FA

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Folate Metabolism and Function

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FA Fortification and CRC

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Folate and Cancer

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Summary Points

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Epidemiological Studies

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References

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Clinical Trials and FA

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Further Reading

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Abbreviations 5-MTHF CRC FA KIR MTR NK NTDs SAM SHMT

5-methyltetrahydrofolate colorectal cancer folic acid killer cell Ig-like inhibitory and activator receptor methionine synthase natural killer neural tube defects SAH S-adenosylhomocysteine S-adenosylmethionine serine hydroxymethyltransferase

INTRODUCTION Colorectal cancer (CRC) is the fourth-most-common cancer in men and the third-most-common cancer in women worldwide. The 2007 World Cancer Research Fund/American Institute for Cancer Research report, called Food, Nutrition, Physical Activity, and the Prevention of Cancer: A Global Perspective, was concluded that there was a limited suggestive association of increased risk for CRC with the intake of foods containing low folate concentrations. Folate is a water-soluble B vitamin that participates in one-carbon metabolism, which has a critical function in methylation reactions and in deoxyribonucleic acid (DNA) synthesis and repair. Thus, it could play an important role in the pathogenesis of several disorders in humans, including anemia; cancer; cardiovascular disease; thromboembolic processes; neural tube defects (NTDs) and other congenital defects; neuropsychiatric disorders; and adverse pregnancy outcomes.

Flour and Breads and their Fortification in Health and Disease Prevention https://doi.org/10.1016/B978-0-12-814639-2.00032-0

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© 2019 Elsevier Inc. All rights reserved.

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The association between dietary folate intake, including natural and synthetic folic acids (FAs), and risk of CRC has been evaluated in numerous epidemiologic studies. In general, the results from these analytic investigations have been mixed. Whereas some studies have reported inverse associations, other studies have observed null and positive associations. Based on the negative association between low folate intake and the highest risk of CRC, the total folate intake associated with colorectal adenoma or cancer risk reduction was estimated to be 600 mg/day.1 Although it is plausible that mandatory FA fortification could be associated with a decline in CRC incidence, a temporal association between FA fortification of enriched cereal grains or wheat flour and an increase in the incidence of CRC in the United States, Canada,2 and Chile has been reported.3 Moreover, in a meta-analysis aimed to determine the effect of FA supplementation on 3-year CRC risk, evaluated with a colonoscopy follow-up, FA supplementation did not have an effect on overall adenoma recurrence. However, colonic surveillance beyond 3 years revealed an increased risk of colorectal adenoma, especially advanced adenomas, among those participants randomized to the FA-supplemented group.4

FOLATE METABOLISM AND FUNCTION Folate is present naturally in foods such as green leafy vegetables, asparagus, broccoli, Brussels sprouts, citrus fruit, legumes, dry cereals, whole grains, yeast, lima beans, and liver and other organ meats. Folates are the major donor and acceptor of one-carbon units in metabolic reactions in the tissues known as one-carbon metabolism.5 These one-carbon units can be at the oxidation level of methanol [5-methyltetrahydrofolate (5-MTHF)], formaldehyde (5,10-methyltetrahydrofolate), and formate (5- or 10-formyltetrahydrofolate or 5,-10-methenyltetrahydrofolate). Basically, all the folate forms in the tissue are polyglutamate, in which the glutamate tail is extended via the g-carboxyl of glutamate. The reaction of folate to polyglutamate forms is required for their biological activity because the polyglutamate forms are more effective substrates for folate-dependent enzymes than are monoglutamates. The synthetic form of folate used in food fortification is FA (pteroylmonoglutamate), which differs from the natural compound because it is fully oxidized and contains only one conjugated glutamate residue (monoglutamyl form), and it is not an active form of the coenzyme. In bread fortification, FA has higher stability and bioavailability (100% with empty stomach and 85% with food) than the natural forms (50%) because it is rapidly absorbed across the intestine. On the other hand, food folate must be cut to the monoglutamyl forms by a brush border glutamyl hydrolase before absorption. The process of absorption occurs in the jejunum by a saturable pH-sensitive transporter that transports oxidized and reduced folates. Most absorbed folates and FA are metabolized to 5-MTHF during their passage across the intestinal mucosa. However, when high amounts of FA are consumed, a percentage of unmetabolized folate appears in the peripheral circulation, as occurs with bread fortification. Folate is excreted by urine; however, the renal excretion capacity can be exceeded and, thus, plasma or serum levels can increase. Tissue folate levels increase less than those of plasma due to the limited capability of tissues to metabolize large doses into the polyglutamate form required for cellular inclusion. Folate monoglutamate in plasma is transported to the tissues via the reduced folate universal carrier that has poor affinity for FA. Another high-affinity folate transporter, known as folate- binding protein, is expressed at a high level in specific tissues such as choroid plexus, kidney proximal tubes, and placenta, as well as in human tumors. Low levels of this receptor are expressed in a variety of other tissues. A third cellular transporter has been identified that transports reduced folate monoglutamates into the mitochondria. When the metabolization of folate is saturated through the methionine synthetase (MTR) reaction in the cell, much of the new folate absorbed is not retained by tissues and appears in the circulation, predominantly as 5-MTHF. The biochemical function of folate is mediating the transfer of one-carbon units involved in nucleotide biosynthesis, the methionine cycle, and biological methylation reactions (Fig. 1). In the methionine cycle, 5-MlTHF transfers one methyl group to homocysteine to synthesize methionine, guaranteeing the provision of S-adenosylmethionine (SAM), the primary methyl group donor for most biological methylation reactions, including that of deoxyribonucleic acid (DNA). The remethylation of homocysteine to methionine is catalyzed by MTR, a vitamin B12 (cobalamin)-dependent enzyme. The reductive methylation of the cobalamin cofactor of MTR to its active state is catalyzed by methionine synthase reductase (MTRR). After donating the methyl group, 5-MTHF is converted to THF and is subsequently transformed to 5,10-methylene THF by serine hydroxymethyltransferase (SHMT). SHMT catalyzes the reversible interconversion of serine and THF to glycine and 5,10-methylene THF and serves as a major entry point for one-carbon units

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FOLATE AND CANCER

Dietary folate Dietary protein

DNA

NADPH + H+ Dihydrofolate (FH2)

dTMP

Methionine ATP

Methrotrexate NADPH + H+

PPI + Pi Thymidylate 5-Fluoracll synthetase

S-Adenosylmethionine

Tetrahydrofolate (FH4)

Acceptor

Serine dUMP

Glycine 5,10-Methylene FH4 MTHFR

Methionine adenosyltransferase

Methyltransferase Methionine Methylated acceptor synthase, S-Adenosylhomocysteine MSR and vitamin B12 Adenosine Homocysteine Serine Cystathionine b-synthase +B6

5-Methyl FH4

Cystathionine Transsulfuration pathway

Cystathionase

Cysteine + NH3 + aKetobutyrate

FIG. 1

Folate and methylation cycle.

into the folate pathway. Here, 5,10-methylene THF is a key substrate in folate metabolism, which can be directed toward nucleotide (thymidylate and purines) biosynthesis or toward methionine regeneration. The cellular concentration of 5,10-methylene THF appears to regulate the flux of this substrate into these various pathways. Several lines of evidence indicate that folate metabolism is regulated by SAM synthesis because the end product and inhibitor of these methylation reactions is S-adenosylhomocysteine (SAH); therefore, the SAM:SAH ratio has been termed the methylation index because SAM synthesis takes a metabolic priority over thymidylate biosynthesis. Probably a limited methyl group availability, caused by either folate or methionine deficiency, shifts the flux of one-carbon units among folate-dependent pathways that preferentially shuttle folate cofactors to the methionine cycle to protect methylation reactions and thereby suppress DNA synthesis. Thus, deficient or excess intake of folate due to supplementation or bread fortification is associated with imbalances in one-carbon metabolism that could facilitate the development of several chronic diseases in animals and humans, such as cancer.

FOLATE AND CANCER Because folate is essential for methylation processes, and any disruption of these metabolic pathways could induce carcinogenesis, normal folate levels are critical for cancer prevention, as explained next6:

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1. Cellular replication: As a cofactor for the synthesis of purines and thymidylate, folate plays a crucial role in DNA synthesis and replication. Therefore, in cancer cells that have an accelerated DNA replication and cell division rate, folate requirements are high. This effect was best demonstrated in the 1940s, when Farber observed that large doses of FA given to individuals with acute leukemia significantly accelerated the rate of expansion of the leukemic clone.7 Therefore, bread fortification with high concentrations of FA could accelerate preexistent cancer cell replication. On the contrary, the absence of folate or the interruption of folate metabolism produces inhibition of tumor growth due to ineffective DNA synthesis. This is the basis of the antifolate agents (methotrexate and 5-fluorouracil) used for cancer chemotherapy in clinical practice. 2. Global and gene-specific methylation status: A consequence of folate deficiency is an increase in intracellular SAH and DNA hypomethylation, which is associated with genome instability, alterations in the expression of specific genes, induction of cellular differentiation, alterations in chromatin conformation, and cell phenotypic changes that contribute to carcinogenesis. In the case of tumor suppressors, for example, promoter hypermethylation can give support to tumorigenesis. We found that high serum folate levels (>45.3 nmol/L) increase the DNA methylation of the tumor suppressor gene p16 and DNA repair genes MLH1 and MGMT in blood.8 Thus, dietary methyl group supplementation or bread fortification with FA could increase genomic and promoter DNA methylation when the enzymatic system is saturated. 3. Protein methylation: SAM is the methyl donor for the methylation of carboxy, histidine, lysine, and arginine residues in proteins, which have a major effect on protein repair, targeting, signal transduction, and modulation of enzyme activity, RNA metabolism, and transcription regulation. Methylation of lysine and arginine residues in histones participates in the regulation of gene expression and in epigenetic silencing, thus promoting the formation of heterochromatin. Folate concentration can influence demethylation and therefore promote epigenetic mechanisms. 4. Natural killer (NK) activity: NK cells are part of the nonspecific immune response and can kill tumor cells (epithelial tumor cells). These cells express killer cell Ig-like inhibitory and activator receptor (KIR) 3. Experimentally, demethylation of KIR genes by 5-aza-2-deoxycytidine leads to rapid induction of KIR expression, whereas in vitro DNA methylation of the CpG cluster leads to inhibition of KIR promoter activity. Thus, folate levels could regulate NK activity, the first barrier to preventing endothelial cancer cell growth. In a noncontrolled intervention, healthy adults responded to a high-dose FA supplement with changes in cytokine messenger ribonucleic acid (mRNA) expression and reduced number and cytotoxicity of NK cells.9 However, we did not validate that high dose of 5-MTHF or FA influences NK cell function in vitro.10 5. Cell proliferation through activation of Notch1 signaling: In vivo, Notch1 overexpression lead to development and growth of implanted colon cancer (CC) and inhibited spontaneous apoptosis.11 We observed that high concentrations of folate stimulate HT29 cell proliferation in vitro. This higher proliferation was dependent on Notch1 signaling. This effect on proliferation was reversible when we blocked this route with an inhibitor of γ-secretase (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, or DAPT)12 Consequently, low intake of folate equivalent or high intake associated with flour fortification may alter the methylation reactions and lead to carcinogenesis. In particular, colorectal neoplasms, both carcinomas and adenomas, show a decreased global DNA methylation level compared to normal tissue. Conversely, studies have shown methylation of the promoter region of specific tumor suppressor genes in colorectal tumors that are increasingly recognized to play an important role in cancer development through silencing of gene transcription. Evidence suggests that DNA hypomethylation and hypermethylation are independent processes and contribute separately to the process of carcinogenesis.13

EPIDEMIOLOGICAL STUDIES Before mandatory FA fortification was begun in many countries, the inverse association with folate intake or serum levels with CRC was shown whether folate was assessed in the diet or in blood. >30 retrospective epidemiologic studies have explored the link between dietary folate and total folate intake (dietary and/or total folate intake, including supplemental FA) and the risk of CRC or adenoma. Most of them reported a significant or ambiguous inverse association. Together, these retrospective studies suggest a 140% reduction in the odds ratio of CRC in subjects with the highest folate intake compared with those with the lowest intake, without clinical evidence of folate deficiency. Moreover, the relationship between blood levels of folate and the risk of CRC and adenoma is less well defined than with folate intake.

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CLINICAL TRIALS AND FA

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In 1996, Tseng et al.14 observed in women that only folate was inversely associated with adenoma risk, even after adjusting for other individual micronutrients. In the Nurses’ Health Study (NHS) and the Health Professionals FollowUp Study (HPFS), colorectal adenoma risk was 30%–40% lower in individuals whose median folate intakes were 711 mg/day in women and 847 mg/day in men, compared to the risk associated with the lowest folate intakes (166 mg/day for women and 241 mg/day for men). In the NHS, women whose total folate intake (i.e., via diet plus supplements) was 2400 mg/day had a 31% decrease in risk of CC compared to women in the lowest folate intake group (<200 mg/day). Further, 86% of the high-folate intake (400 mg/day) group consumed daily multivitamins containing FA. In another study, Ma et al.15 found that serum folate levels <6 nmol/l were associated with higher risk of CRC. Similar inverse associations between folate intake or status and colorectal adenoma risk were found in other studies. In a study of the epidemiology of CRC in Asia, Yee et al..16 demonstrated that folate supplementation is not effective in preventing the disease. A meta-analysis published in 2005 of seven cohort and nine case-control studies that examined the association between folate consumption and CRC risk showed that among cohort studies, there was a significantly lower risk (25%) of CRC among those in the highest category of dietary folate intake compared to those in the lowest category, with no evidence of heterogeneity between the study estimates. However, the association between food folate consumption and low CRC [relative risk (RR) for high versus low intake, 0.75; 95% confidence interval (CI), 0.64e0.89] disappears with total folate intake (folate from foods and supplements; RR for high versus low intake, 0.95; 95% CI, 0.81e1.11).17 In a nested case-control study of the Japanese population (the Japan Public Health Center-based prospective study), a rich plasma folate status did not prevent CRC.18 In contrast, another study established a positive association between folate intake and CRC risk. In the Colorectal Cancer Study conducted in Melbourne, Australia, a case-control study designed as a quantitative food survey to identify dietary factors associated with CRC risk in 715 incident cases compared with 727 age/sex frequency-matched, randomly chosen community controls, folate showed an increased risk at the highest level of consumption.19 In a Dutch case-control study comparing cases with at least one histologically confirmed colorectal adenoma (n ¼ 768) and controls with no history of any type of colorectal polyp (n ¼ 709), folate seemed to be a risk factor for colorectal adenomas, especially when vitamin B2 intake was low.20Finally, in a meta-analysis of data from 50,000 individuals, supplementation with FA did not show an increase or decrease in the incidence of site-specific cancer during the first 5 years of treatment.21 Consequently, epidemiological data show that folate intake has a dual effect on CRC risk. Apparently, low and excessive folate intakes as a result of bread fortification or supplementation are associated with high risk of this cancer.

CLINICAL TRIALS AND FA A few placebo-controlled studies have demonstrated an increase in cancer outcomes with folate supplementation. The Aspirin/Folate Polyp Prevention Trial found that aspirin, but not FA, reduced recurrence of colorectal adenomas. The trial had a factorial design, with three aspirin (placebo and 81 and 325 mg/day) and two FA (placebo and 1 mg/ day) groups being studied for 6 years. There were 884 subjects who had colonoscopic evaluation for adenomas during the trial. Among individuals who received aspirin plus FA, adenoma recurrence increased significantly, in contrast to those who received aspirin alone, which had a chemoprotective effect on colorectal adenoma. Moreover, a significant excess of prostate cancers was observed in the folate group.22 Other double-blind, randomized trials among participants of two large prospective cohorts, the HPFS and the NHS, evaluated the effect of FA supplementation on recurrent colorectal adenoma. Participants were randomly assigned to receive FA (1 mg/day; n ¼ 338) or a placebo (n ¼ 334) for 3 to 6 years. Among subjects with plasma folate levels at baseline below 17 nmol/l, FA supplementation was associated with a significant decrease in adenoma recurrence (RR, 0.61; 95% CI, 0.42–0.90; P ¼ 0.01), whereas for subjects with high folate concentrations at baseline (>17 nmol/ l) supplemental FA had no significant effect.23 Two randomized, double-blind, placebo-controlled clinical trials (the Norwegian Vitamin Trial and the Western Norway B Vitamin Intervention Trial) were performed between 1998 and 2005. A total of 6837 patients with ischemic heart disease were treated with oral B vitamins (FA, 0.8 mg/day) plus vitamin B12 (0.4 mg/day) and vitamin B6 (40 mg/day) (n ¼ 1708), FA (0.8 mg/day) plus vitamin B12 (0.4 mg/day) (n ¼ 1703), vitamin B6 alone (40 mg/ day) (n ¼ 1705), or placebo (n ¼ 1721). Treatment lasted from 1998 to 2005, and subjects were followed until December 31, 2007. Serum folate concentration increased sixfold, and cancer incidence and cancer mortality were greater in the group that received FA and vitamin B12.24

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EXPERIMENTAL STUDIES AND FA In animal models, there is evidence that supplementation with FA has a promoting effect on carcinogenesis, and that folate deficiency reduces the development of CRC and ileal polyps. In neoplastic cell cultures, interruption of folate metabolism generates an inhibition of tumoral cell replication as a result of ineffective DNA synthesis.25 In old mice, the increase in genomic and p16 promoter DNA methylation, a phenomenon observed in the earliest stages of carcinogenesis, was directly related to dietary folate. Age- related enhancement of p16 expression occurred in folate-repleted (P ¼ 0.001) and folate-supplemented groups (P ¼ 0.041), but not in the folate-depleted groups. This may indicate that adequacy of dietary folate is important to maintain sufficient expression of p16 in the aged colon.26 In summary, the lines of evidence indicate that intracellular folate depletion suppresses the progression of existing neoplasms. In neoplastic cells, in which DNA replication and cell division occur at an accelerated rate, folate depletion or interruption of folate metabolism causes ineffective DNA synthesis, resulting in inhibition of tumor growth.

FA FORTIFICATION AND CRC Many countries have implemented mandatory or voluntary FA fortification of flour and uncooked cereal grain products to reduce the risk of NTDs. The United States and Canada have mandated fortification since 1998. Chile fortifies wheat flour since 2000, and the rest of the American countries (Latin America) started later. As a consequence, the incidence of births complicated by NTDs has declined 20%–50% in the United States, Canada, Chile, and Costa Rica.27 In the United States and Canada, population-based studies showed an approximately twofold increase in plasma folate levels in the adult population. Simultaneously, an increase in CRC rates occurred probably as a result of FA fortification in North America in 1996 and 2000, based on population-based observations from two representative data sets from the United States and Canada (the U.S. Surveillance Research Program and Canadian Cancer Statistics 2006, respectively). Apparently, this change in CRC rate is not a consequence of changes in the rate of colorectal endoscopic procedures, as the authors discussed. These observations alone do not prove causality, but they are consistent with the known effects of folate on existing neoplasms, as shown in both preclinical and clinical studies.2 In Chile, the fortification program showed a 40% reduction in the rate of NTD in 1 year. In women of reproductive age, serum folate levels increased from 9.7 to 37 nmol/L.28 In elderly people, who consumed an average of 220 g of bread/day, equivalent to 185 g of flour and fortified by 410 mg FA, serum folate levels increased from 16.2 to 32 nmol/ L, and homocysteine levels decreased and masked vitamin B12 deficiency. Moreover, serum folate levels increased to >40 nmol/L in 37% of these individuals.29 As in the United States and Canada,2 a temporal association between folate fortification and an increase in CRC hospital discharge was observed. The rate/ratio between the period before and after the fortification for CC in the group aged 45–64 years was 2.6 (99% CI, 2.93–2.58) and for the group aged 65–79 years, it was 2.9 (99% CI, 3.25–2.86), as shown in Figs. 2 and 3.3 FIG. 2 Rate/ratio of hospital discharge because of CC in adults aged 45–64 years, before and after the start of the mandatory program of fortifying flour with 220 mg of synthetic FA/100 g of wheat flour. Rate/ratios are expressed as the rate for each year/the rate for 1992.

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FA FORTIFICATION AND CRC

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FIG. 3 Rate/ratio of hospital discharge because of CC in adults aged 65–79 years, before and after the start of the mandatory program of fortifying flour with 220 mg of synthetic FA/100 g of wheat flour. Rate/ratios are expressed as the rate for each year/the rate for 1992.

One possible explanation for this finding is that this increase is causally related to FA fortification. Other explanations for the increase in the discharge rates for CRC could be an increase in the incidence of risk factors such as obesity, low intake of fiber and calcium, and high intake in fat and red meat. The prevalence of obesity increased from 19.7% in 1997 to 22% in 2003.30 Unfortunately, there are no data on other attributable risks of these factors, but according to Food and Agricultural Organization (FAO) data, the supply of calories and protein has not changed significantly. The FA fortification program has had an effect on homocysteine levels.29 We therefore expected a decline in the rates of cardiovascular disease because hyperhomocysteinemia is considered a cardiovascular risk factor.31, 32 However, the discharge rates for cardiovascular diseases did not change in the two study periods. This is consistent with FA supplementation trials, which have not reported a reduction in the incidence of cardiovascular events.33 There is no cancer registry in Chile. Therefore, the only means of studying the impact of fortification on CRC incidence is to use indirect data. Because a form indicating the diagnosis and other variables must be completed for every discharge from every hospital in Chile, this information, which is complete and reliable, can be used as a proxy for disease incidence. Thus, we used it to study the trends in the incidence of CRC and compare it with that of other diseases as a control. The changes in disease frequency detected using hospital discharge data coincided with mortality trends for breast and gastric cancer and CRC. This gives further support to the validity of hospital discharge data as a proxy for disease incidence. This agreement in the observational studies in three countries after 10 years of the fortification program does not prove causality, but it is consistent with the known effects of folate on existing neoplasms (adenomas), as demonstrated in experimental and clinical studies. Another plausible explanation for the increase in CRC associated with FA fortification is that supraphysiologic fortification of bread or supplementation increases circulating unmetabolized FA, and the real consequence of this is unknown. There is evidence that daily ingestion of 400 mg, or plasma levels >40 nmol/l, produces a sustained appearance of unmetabolized FA in the blood. Troen et al.33 observed that increasing concentrations of plasma FA among postmenopausal women who took FA supplements were inversely associated with decreases in the cytotoxicity of circulating NK cells, which play a role in the destruction of arising clones of endothelial cancer cells. However, we did not demonstrate in vitro that supraphysiological levels of folate were associated with impaired NK cell activity.10 Global methylation status also may be altered with high folate plasma levels. We observed that healthy male subjects in the fortification era, without vitamin supplementation, with plasma folate levels >45 nmol/l had higher SAM and SAH concentrations than those of subjects with normal folate levels.34 In summary, there is evidence that an FA fortification program with 150 or 220 mg of synthetic FA/100 g of wheat flour may be associated with an additional risk of CRC. Thus, it is crucial to evaluate this finding to determine the safe upper limit for folate intake, as well as the safe upper folate concentration and the amount of FA necessary to prevent NTDs and to minimize possible adverse effects.

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SUMMARY POINTS • Folate has a critical function in methylation reactions, as well as in DNA synthesis and repair. • Folate may accelerate tumor cell growth. • In the mandatory era of FA fortification, approximately 40% of the population has supraphysiologic levels of serum or plasma folate. • Low and high folate intake or plasma concentration are related with the risk of CRC. • The upper limit for FA intake, as well as the safe upper folate concentration, must be determined to prevent adverse effects.

References 1. Bailey LB. Folate, methyl-related nutrients, alcohol, and the MTHFR 677C-T polymorphism affect cancer risk: intake recommendations. J Nutr 2003;133(11 Suppl. 1):S3748–53. 2. Mason JB, Dickstein A, Jacques PF, Haggarty P, Selhub J, Dallal G, et al. A temporal association between folic acid fortification and an increase in colorectal cancer rates may be illuminating important biological principles: a hypothesis. Cancer Epidemiol Biomark Prev 2007;16:1325–9. 3. Hirsch S, Sanchez H, Albala C, de la Maza MP, Barrera G, Leiva L, et al. Colon cancer in Chile before and after the start of the flour fortification program with folic acid. Eur J Gastroenterol Hepatol 2009;21:436–9. 4. Fife J, Raniga S, Hider PN, Frizelle FA. Folic acid supplementation and colorectal cancer risk: a meta-analysis. Colorectal Dis 2011;13:132–7. 5. Smith AD, Kim YI, Refsum H. Is folic good for everyone? Am J Clin Nutr 2008;87:517–33. 6. Kim Y. Folate and colorectal cancer: an evidence-based critical review. Mol Nutr Food Res 2007;51:267–92. 7. Farber S. Some observations on the effect of folic acid antagonists on acute leukemia and other forms of incurable cancer. Blood 1949;4:160–7. 8. Sanchez H, Hossain MB, Lera L, Hirsch S, Albala C, Uauy R, Broberg K, Ronco AM. High levels of circulating folate concentrations are associated with DNA methylation of tumor suppressor and repair genes p16, MLH1, and MGMT in elderly Chileans. Clin Epigenetics 2017;9:74–85. 9. Paniz C, Bertinato JF, Lucena MR, De Carli E, Amorim PMDS, Gomes GW, Palchetti CZ, Figueiredo MS, Pfeiffer CM, Fazili Z, Green R, GuerraShinohara EM. A daily dose of 5 mg folic acid for 90 days is associated with increased serum unmetabolized folic acid and reduced natural killer cell cytotoxicity in healthy Brazilian adults. J Nutr 2017;147:1677–85. 10. Hirsch S, Miranda D, Muñoz E, Montoya M, Ronco AM, de la Maza MP, Bunout D. Natural killer cell cytotoxicity is not regulated by folic acid in vitro. Nutrition 2013;29:772–6. 11. Zhang Y, Li B, Ji ZZ, Zheng PS. Notch1 regulates the growth of human colon cancers. Cancer 2010;116:5207–18. 12. Rodriguez JM, Miranda D, Bunout D, Ronco AM, de Ia Maza MP, Hirsch S. Folates induce colorectal carcinoma HT29 cell line proliferation through Notch1 signaling. Nutr Cancer 2015;67(4):706–11. 13. Mathers JC. Folate intake and bowel cancer risk. Genes Nutr 2009;4:173–8. 14. M1 T, Murray SC, Kupper LL, Sandler RS. Micronutrients and the risk of colorectal adenomas. Am J Epidemiol 1996;144(11):1005–14. 15. Ma J, Stampfer MJ, Giovannucci E, et al. Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer. Cancer Res 1997;57:1098–102. 16. Yee YK, Tan VP, Chan P, Hung IF, Pang R, Wong BC. Epidemiology of colorectal cancer in Asia. J Gastroenterol Hepatol 2009;24:1810–6. 17. Sanjoaquin MA, Allen N, Couto E, Roddam AW, Key TJ. Folate intake and colorectal cancer risk: a meta-analytical approach. Int J Cancer 2005;113:825–8. 18. Otani T, Iwasaki M, Sasazuki S, Inoue M, Tsugane S, Japan Public Health Center-based Prospective Study Group. Plasma folate and risk of colorectal cancer in a nested case control study: the Japan Public Health Center-based prospective study. Cancer Causes Control 2008;19:67–74. 19. Kune G, Watson L. Colorectal cancer protective effects and the dietary micronutrients folate, methionine, vitamins B6, B12, C, E, selenium, and lycopene. Nutr Cancer 2006;56:11–21. 20. van den Donk M1, Buijsse B, van den Berg SW, Ocke MC, Harryvan JL, Nagengast FM, Kok FJ, Kampman E. Dietary intake of folate and riboflavin, MTHFR C677T genotype, and colorectal adenoma risk: a Dutch case-control study. Cancer Epidemiol Biomark Prev 2005;14:1562–6. 21. Vollset SE, Clarke R, Lewington S, Ebbing M, Halsey J, Lonn E, et al. Effects of folic acid supplementation on overall and site-specific cancer incidence during the randomised trials: meta-analyses of data on 50,000 individuals. Lancet 2013;381:1029–36. 22. Cole BF, Baron JA, Sandler RS, et al. Polyp prevention study group. Folic acid for the prevention of colorectal adenomas: a randomized clinical trial. JAMA 2007;297:2351–9. 23. K1 W, Platz EA, Willett WC, Fuchs CS, Selhub J, Rosner BA, Hunter DJ, Giovannucci E. A randomized trial on folic acid supplementation and risk of recurrent colorectal adenoma. Am J Clin Nutr 2009;(6):1623–31. 24. Ebbing M, Bønaa KH, Nygard O, Arnesen E, Ueland PM, Nordrehaug JE, et al. Cancer incidence and mortality after treatment with folic acid and vitamin B12. JAMA 2009;302:2119–26. 25. Bashir O, Fitzgerald AJ, Goodlad RA. Both suboptimal and elevated vitamin intake increase intestinal neoplasia and alter crypt fission in the ApcMin/þ mouse. Carcinogenesis 2004;25:1507–15. 26. Keyes MK, Jang H, Mason JB, Liu Z, Crott JW, Smith DE, et al. Older age and dietary folate are determinants of genomic and p16-specific DNA methylation in mouse colon. J Nutr 2007;137:1713–7. 27. Dary O. Nutritional interpretation of folic acid intervention. Nutr Rev 2009;67:235–44. 28. Hertramf E, Cortes F. Folic acid fortification of wheat flour: Chile. Nutr Rev 2004;62:S44–9. 29. Hirsch S, de la Maza P, Barrera G, Gattas V, Petermann M, Bunout D. The Chilean flour folic acid fortification program reduces serum homocysteine levels and masks vitamin B12 deficiency in elderly people. J Nutr 2002;132:289–91. 30. Vio F, Albala C, Kain J. Nutrition transition in Chile revisited: mid-term evaluation of obesity goals for the period 2000-2010. Public Health Nutr 2008;11:405–12.

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31. Bostom AG, Silbershatz H, Rosenberg IH, et al. Nonfasting plasma total homocysteine levels and all-cause and cardiovascular disease mortality in elderly Framingham men and women. Arch Intern Med 1999;159:1077–80. 32. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 1991;324:1149–55. 33. Troen AM, Mitchell B, Sorensen B, Wener MH, Johnston A, Wood B, et al. Unmetabolized folic acid in plasma is associated with reduced natural killer cell cytotoxicity among postmenopausal women. J Nutr 2006;136:189–94. 34. Jenkins DJA, Spence JD, Giovannucci EL, Kim YI, Josse R, et al. Supplemental vitamins and minerals for CVD prevention and treatment. Am Coll Cardiol 2018;71:2570–84.

Further Reading 35. Hirsch S, Ronco AM, Guerrero-Bosagna C, de la Maza MP, Leiva L, Barrera G, et al. Methylation status in healthy subjects with normal and high serum folate concentration. Nutrition 2008;11(12):1103–9.

4. METABOLIC RESPONSES TO FLOUR AND BREAD FORTIFICATION