The Effects of Folic Acid and 5- Methyltetrahydrofolate Supplementation on Folate Metabolism and One-Carbon Transfer Reactions in a Mouse Model

Home , Levomefolic acid

THE EFFECTS OF FOLIC ACID AND 5- METHYLTETRAHYDROFOLATE SUPPLEMENTATION ON FOLATE METABOLISM AND ONE-CARBON TRANSFER REACTIONS IN A MOUSE MODEL

David Jugyun Im

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Nutritional Sciences University of Toronto

David Jugyun Im

Master of Science

Department of Nutritional Sciences University of Toronto

2018

Abstract

Due to emerging evidence linking high folic acid (FA) intake to certain adverse health effects, many have speculated that 5-methyltetrahydrofolate (5-MTHF), the natural form of folate, confer safer means of supplementation. As such, we investigated the differential effects of FA and 5-

MTHF supplementation on folate status indices, gene expression of enzymes involved in folate metabolism and one-carbon transfer reactions, and tissue-specific global DNA methylation.

Folate concentrations, gene expression differences, and global DNA methylation was assessed using the microbiological microtitre assay, real time RT-PCR, and in vitro methyl acceptance assay, respectively. 5-MTHF was, at least, as effective as FA in increasing plasma and tissue folate concentrations. Additionally, 5-MTHF had differential effects on tissue-specific gene expression, and increased liver global DNA methylation relative to FA. Our data suggests that 5-

MTHF influences gene expression of enzymes involved in the folate pathway, which may potentially affect downstream biochemical processes such as global DNA methylation.

ii ACKNOWLEDGEMENTS

I would like to thank my supervisor, Dr. Young-In Kim, for not only his endless support and guidance throughout the course of my graduate degree, but for his mentorship outside of the workplace. Although I am greatly appreciative for the incredible opportunities to learn and conduct research in your lab, I am just as appreciative for the life lessons you have passed on to me that has helped me mature as a young man. You have been and will continue to be someone I truly admire and respect.

Thank you to Dr. Debbie O’Connor and Dr. Elena Comelli for your undeserving support throughout my project. Words cannot express how thankful I am for your kindness and empathetic understanding of my difficult situations throughout my project.

A sincere thank you to everyone I worked with at Li Ka Shing. Kyoung-Jin Sohn – all my research is owing to your technical expertise and lending hand in conducting experiments. You continually gave me moral support during the toughest times of this project. Dr. Pamela Plant and Danielle Gifford – your passion and expertise in your respective fields are the reasons I have a thesis to present. You both were absolute blessings to work with.

I would like to also thank everyone I worked with in the Kim lab. Shahnaz Anne Fard – you constantly reached out to help me each and every day. I know I could not have done this project without you there since day 1. I never thought the end would ever come, but it feels as though it may have come too quickly. Heajin Kelly Cheon (don’t hurt me) – my older sister in the lab.

Any time I felt doubt, frustration, confusion, or sadness in the lab, you were my go-to person.

Eszter Pigott – possibly the nicest person I have ever met. Thank you for brightening the room with your warm loving smile. To the summer students – Nicolaas Bloemen, Hannah Tateishi,

iii Kelvin Long – you guys were the absolute best to work with. Enough said. I would also like to give a special ‘thanks’ to Joanna Warzyszynska – the greatest lab role model I have known. You are the epitome of what every graduate student should aspire to be like and I appreciate everything you have taught me during my time as your mentee in this lab.

Last, but most of all, thank you to my family. Dad and mom – the president and first lady of my fan club. I consider myself extremely lucky to have such loving parents that would go to the ends of the earth for me. To my sisters, Michelle and Stephanie, you both are the foundational backbone in my life. I have done nothing to deserve such caring sisters as yourselves. Without your perpetual help, encouragement, and support, I would never have been able to finish my project. To my grandparents, I dedicate my thesis to you. Your warm hearts and high expectations continually push me to become a better person. I would also like to thank my

(pseudo) younger brother, Joshua Ahn, who also provided me with endless encouragement during the most stressful times in my graduate degree. Finally, I have to thank my girlfriend,

Miriam Kim, for your endless support in all aspects of my life throughout the past three years.

Dealing with my ups and downs can’t be easy, but you ensured I kept my head high through it all and I am thankful you did… we made it!

iv Table of Contents I. Table of Contents ...... v II. List of Tables ...... vii III. List of Figures ...... viii IV. List of Abbreviations ...... ix

Chapter 1: Introduction ...... 1 Chapter 2: Literature Review ...... 4 2.1 Folate and Folic Acid ...... 4 2.1.1 Overview ...... 4 2.1.2 Chemical Structure ...... 5 2.1.3 Absorption, metabolism, and biochemical functions ...... 6 2.1.4 Dietary requirements ...... 14 2.1.5 Measures of folate status ...... 15 2.2 Effects of folic acid fortification and supplementation in North America ...... 17 2.2.1 Impact of folic acid fortification and supplementation ...... 17 2.2.2 The effects of folic acid on DHFR and UMFA ...... 18 2.2.3 Effects of FA supplementation on the folate pathway ...... 20 2.3 Folate and folic acid in health and disease ...... 24 2.3.1 Anemia ...... 26 2.3.2 Masking of B12 deficiency ...... 27 2.3.3 Cardiovascular disease ...... 27 2.3.4 Neuropsychiatric and cognitive disorders ...... 28 2.3.5 Carcinogenesis ...... 29 2.3.6 Epigenetic changes ...... 35 2.3.7 Pregnancy ...... 37 2.3.8 Fetal development ...... 40 2.4 5-methyltetrahydrofolate ...... 40 2.4.1 5-MTHF-Ca ...... 41 2.4.2 The case for 5-MTHF supplementation ...... 42 2.4.3 5-MTHF and B12 ...... 43 2.4.4 MTHFR polymorphism ...... 45 2.4.5 Effects of 5-MTHF on folate metabolism ...... 46 2.5 Folic acid vs. 5-MTHF ...... 46 2.6 Summary and research gaps ...... 52 Chapter 3: Rationale, Objectives, Hypothesis, and Significance ...... 54 3.1 Rationale ...... 54 3.2 Objectives ...... 55 3.3 Hypothesis ...... 56 3.4 Expected Outcomes ...... 56 3.5 Significance ...... 56 Chapter 4: Comparative Studies on the Biochemical and Molecular Effects of FA vs 5- MTHF Supplementation in a Mouse Model ...... 58 4.2 Introduction ...... 58

v 4.3 Methods and Materials ...... 60 4.3.1 Animals and Dietary Intervention ...... 60 4.3.2 Experimental Diets ...... 62 4.3.3 Sample collection ...... 67 4.3.4 Determination of plasma, hepatic, and small intestinal folate concentrations ...... 67 4.3.5 Gene expression analysis by quantitative real-time reverse transcriptase PCR ...... 71 4.3.6 Global DNA Methylation Analysis ...... 74 4.3.7 Statistical Analysis ...... 75 4.4 Results ...... 77 4.4.1 Animal health and body weight ...... 77 4.4.2 Plasma, hepatic, and small intestine folate concentrations ...... 78 4.4.3 Gene Expression Analysis ...... 80 4.4.4 Global DNA Methylation ...... 91 4.5 Discussion ...... 93 4.6 Conclusion ...... 106 Chapter 5: General discussion, future directions and conclusion ...... 107 5.1 Summary and general discussion ...... 107 5.2 Future directions ...... 109 5.3 Conclusion ...... 110 References ...... 111

vi List of Tables Table 2.1 Folate transporter, carrier, and receptor localization, optimal pH, and pharmacokinetics ...... 8 Table 2.2 Intracellular enzymes involved in folate retention and export ...... 9 Table 2.3 Enzymes involved in folate metabolism and one-carbon transfer reactions ...... 13 Table 2.4: Recommended Dietary Folate Intake (Daily) (1) ...... 15 Table 2.5 Summary of folate supplementation effects on absorptive mechanisms ...... 22 Table 2.6 Strength of evidence for effects of FA on human health and disease ...... 26 Table 2.7: Summary of clinical trials comparing the effects of FA and 5-MTHF supplementation on biomarkers of folate status...... 48 Table 2.8: Summary of clinical trials comparing the effects of FA and 5-MTHF supplementation on folate status indices in individuals with the MTHFR CàT polymorphism ...... 51 Table 4.1: Abbreviations for dietary groups ...... 61 Table 4.2: Nutrient compositions of experimental L-amino acid defined diets for FA ...... 63 Table 4.3: Nutrient compositions of experimental L-amino acid defined diets for 5-MTHF ...... 64 Table 4.4: Salt mix and vitamin mix compositions of experimental L-amino acid defined diets 66 Table 4.5: qRT-PCR Primer sequences ...... 73 Table 4.6: General summary of liver and small intestine mRNA expression ...... 93

vii List of Figures Figure 2.1: Chemical structure of folic acid (a) and folate (b)...... 6 Figure 2.2: Folate absorption, transport, metabolism, and 1-carbon transfer reaction...... 12 Figure 2.3: Dual modulatory role of folate in colorectal carcinogenesis...... 33 Figure 4.1: Effects of dietary folate supplementation on C57BL/6 body weight...... 78 Figure 4.2: Effects of FA and 5-MTHF supplementation on various measures of folate status. . 79 Figure 4.3: Effects of FA and 5-MTHF supplementation on transporters, carriers, and receptors involved with folate absorption in the liver...... 81 Figure 4.4: Effects of FA and 5-MTHF supplementation on enzymes involved with folate retention and efflux in the liver...... 82 Figure 4.5: Effects of FA and 5-MTHF supplementation on enzymes involved with folate metabolism...... 83 Figure 4.6: Effects of FA and 5-MTHF supplementation on enzymes involved with nucleotide biosynthesis in the liver...... 84 Figure 4.7: Effects of FA and 5-MTHF supplementation on enzymes involved with one-carbon transfer reactions and DNA methylation in the liver...... 85 Figure 4.8: Effects of FA and 5-MTHF supplementation on transporters, carriers, and receptors involved with folate absorption in the small intestine...... 87 Figure 4.9: Effects of FA and 5-MTHF supplementation on enzymes involved with folate retention and efflux in the small intestine...... 88 Figure 4.10: Effects of FA and 5-MTHF supplementation on enzymes involved with folate metabolism...... 89 Figure 4.11: Effects of FA and 5-MTHF supplementation on enzymes involved with nucleotide biosynthesis in the small intestine...... 90 Figure 4.12: Effects of FA and 5-MTHF supplementation on enzymes involved with one-carbon transfer reactions and DNA methylation in the small intestine...... 91 Figure 4.13: Effects of FA and 5-MTHF supplementation on global DNA methylation ...... 92

viii List of Abbreviations

5-MTHF 5-methyltetrahydrofolate AMP adenosine monophosphate ANOVA analysis of variance BDR basal dietary requirement CpG cytosine preceding guanine CRC colorectal cancer CT cycle to threshold DHF dihydrofolate DHFR dihydrofolate reductase DNMT DNA methyltransferase DPM disintegration per minute dTMP deoxythymidine monophosphate or thymidylate dUMP deoxyuridine monophosphate FA folic acid FPGS folylpolyglutamate synthase FR folate receptor GCPII glutamate carboxypeptidase II GGH γ-glutamyl hydrolase GMP guanosine monophosphate MAT methionine S-adenosyltransferase or SAM synthase MS methionine synthase MTHFR methylene tetrahydrofolate reductase NTD neural tube defect PABA para-aminobenozic acid PCFT proton-coupled folate transporter RBC red blood cell RFC Reduced folate carrier SAM S-adenosylmethionine SHMT serine hydroxymethyltransferase SNP single nucleotide polymorphism tHcy total homocysteine THF tetrahydrofolate TS thymidylate synthase UL upper tolerable intake UMFA unmetabolized folic acid

ix Chapter 1: Introduction

Folate, a water-soluble B vitamin (B9), and its synthetic form, folic acid (FA), are critical for one-carbon transfer reactions involved in nucleotide biosynthesis and biological methylation reactions (1). Hence, folate plays an important role in human health and disease (1). Folate deficiency has been linked to a number of adverse health outcomes including megaloblastic anemia, birth defects, cognitive disorders, cardiovascular disease, and development of certain cancers (2). As a corollary to this, FA supplementation has been suggested as an effective nutritional strategy to treat and/or prevent the aforementioned disorders (3).

As a public health initiative in 1998, mandatory FA fortification was implemented in

North America due to overwhelming evidence of its beneficial effects in reducing rates of neural tube defects (NTDs) (3). Additionally, Health Canada and the Public Health Agency of Canada recommend that women of child-bearing age consume a daily multivitamin supplement containing 0.4 mg FA (3). It has been estimated that 30-40% of the North American population take FA-containing supplements for possible but, as yet unproven, health benefits (21, 24).

Owing primarily to prevalent FA supplementation and partly to mandatory FA fortification, blood and intake levels of folate and FA in the North American population have drastically increased (4, 5). Data from the Canadian Health Measures Survey conducted between 2007 and

2009 revealed a virtual non-existence of folate deficiency in Canada, as defined by red blood cell

(RBC) folate concentrations < 305 nmol/L (5). Moreover, it was determined that approximately

40% of the population exhibited RBC folate concentrations above the high cut-off of 1360 nmol/L, defined as the 97th percentile of RBC folate concentrations from the National Health and

Nutrition Examination Survey (NHANES) conducted in the U.S. between 1999-2004 (5).

1 Although the protective effect of FA supplementation on NTDs is well-established (6-8), concerns raised about potential harms associated with excessive intake and blood levels of folate and FA merit exploration. Currently, several lines of evidence have linked high FA intake to certain adverse health outcomes including masking of vitamin B12 deficiency, epigenetic modifications, and the progression of (pre)neoplastic cells (9, 10). Furthermore, excessive FA has been shown to saturate a key metabolic enzyme, dihydrofolate reductase (DHFR), which is responsible for the reduction of FA, resulting in the presence of unmetabolized FA (UMFA) in circulation (11). Some have purported that FA supplementation and chronic exposure to high levels of UMFA may have unanticipated physiological consequences and could possibly act as a driving force behind certain adverse health outcomes (12). However, whether these negative effects are due to high levels of FA or high folate status in general remains unresolved (3). It has been further postulated that supplementation with the predominant circulating form of folate, 5- methyltetrahydrofolate (5-MTHF), confers a safer means of providing supplemental folates (3).

Clinical studies have suggested that 5-MTHF supplementation is, at least, as effective as

FA in increasing blood folate levels and reducing plasma concentrations of homocysteine – an accurate and inverse, albeit non-specific, indicator of folate status (10, 13-18). However, studies comparing the effects of FA and 5-MTHF supplementation on folate metabolism and intracellular one-carbon reactions at the cellular level are lacking, and studies are warranted to elucidate biochemical and functional differences. It should be noted that although the tolerable upper limit (UL) for FA is set at 1 mg/day (3), there is currently no established UL for 5-MTHF as high intake levels of folate from naturally-occurring dietary sources alone is both difficult and improbable (3). As 5-MTHF supplements are now commercially available and speculated to

2 provide a safer means of supplementation over FA, it is critical to investigate its cellular and physiological effects for future clinical use and public health recommendations.

Given these considerations, this thesis aims to compare the effects of FA and 5-MTHF supplementation on intracellular folate metabolism and one-carbon transfer reactions in a mouse model to investigate differential cellular and physiologic effects of each folate vitamer. This study will help elucidate the differential impact of FA and 5-MTHF supplementation at various equimolar doses on cellular metabolism, which may provide a framework for future studies aimed at assessing beneficial or harmful clinical outcomes between the two folate forms.

3 Chapter 2: Literature Review

2.1 Folate and Folic Acid

2.1.1 Overview

Folates are water-soluble B vitamins (vitamin B9) that are essential for human development and health through their role in one-carbon transfer reactions involved in nucleotide biosynthesis and biological methylation reactions (1). Folic acid (FA) refers to the synthetic form of folate, which is used in food fortification and supplements (1). As such, folate is critical for processes such as cell division, DNA integrity and maintenance, and epigenetic modifications that regulate gene expression (1). Folate deficiency in humans has been associated with a number of negative health outcomes and diseases including megaloblastic anemia, hyperhomocysteinemia, neuropsychiatric disorders, cognitive impairment, congenital disorders, adverse pregnancy outcomes, and development of certain cancers (2, 9, 10).

Folate is naturally-occurring in a number of dietary food sources, including green leafy vegetables, fruits, sprouts, certain organ meats, and yeast (2). Additionally, FA is found in labelled enriched fortified white wheat flour and specific grain products as an effort to improve folate status in women of child-bearing age and reduce rates of NTDs (19, 20). Since inception,

FA fortification in tandem with prevalent supplemental use have significantly increased FA intakes and blood levels of folate in the general North American population (4, 5, 21). Although undoubtedly considered a public health success in its preventative role for NTDs (6-8), recent concerns have been raised over the potential adverse effects of excessive FA fortification and supplementation in light of the increasing folate status of in the general North American population (9).

4 2.1.2 Chemical Structure

The term folate is used to collectively describe a variety of folate vitamers with similar chemical structures and nutritional properties (1, 22). The scaffold chemical structure is composed of three moieties: a central para-aminobenzoic acid (PABA) joined to an aromatic pteridine ring through a methylene bridge and to a glutamic acid through a γ-peptide bond

(Figure 2.1) (1). The oxidation state and one-carbon substitution of the pteridine ring and

PABA, respectively, are what distinguish different folate vitamers. Naturally-occurring folates are typically in a reduced state, while FA is fully oxidized and thus, confers higher stability (1).

Physiologically, FA and natural folates are metabolized to 5-MTHF, which makes up 95-98% of circulating folates in the blood (3), and represents the only form capable of crossing the blood brain barrier (23).

While FA is monoglutamylated, naturally-occurring folates frequently contain a polyglutamate chain with a varying number of glutamate residues connected by γ-peptide bonds

(1). Folates can further be methylated by one-carbon units that are linked at the N-5 and N-10 positions of tetrahydrofolate (THF) to form an intermediate 5-MTHF structure (1). The 5-MTHF intermediate can then enter the folate pathway without further reduction or methylation.

Levomefolic acid, also known as L-5-MTHF or 6(S)-L-MTHF, refers to the primary biologically-active diastereoisomer (13) and the predominant form of folate found in circulation

(3). L-5-MTHF has become commercially available as a crystalline form of a calcium salt

(Metafolin®, Merck Eprova AG, Switzerland), which provides improved stability and longer shelf-life of L-5-MTHF (18).

(a)

(b)

Figure 2.1: Chemical structure of folic acid (a) and folate (b). a) Folic acid refers to the synthetic form of folate, and is in its fully oxidized and monoglutamylated state. b) Naturally occurring folates refer food folates, and commonly in the reduced and polyglutamylated state (consisting of up to 9 glutamate residues). Adapted and reprinted from the publisher (John Wiley and Sons Ltd): (24). Copyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

2.1.3 Absorption, metabolism, and biochemical functions

2.1.3.1 Folate Absorption and Transport (Refer to Table 2.1)

Folate is primarily absorbed within the acidic microenvironments at the cell surface of the proximal small intestine, specifically in the duodenum and proximal jejunum (24, 25).

Naturally-occurring folates in food exist primarily in the polyglutamate form, and therefore, must be monoglutamylated prior to intestinal absorption (25). The enzyme glutamate carboxypeptidase II (GCPII), an exopeptidase found anchored to the intestinal apical brush border membrane, hydrolyses the polyglutamate chain of folates, as folates with three or more

6 glutamate residues are incapable of crossing the cellular membrane (24). Following hydrolysis, monoglutamylated folates are then transported intracellularly via proton-coupled folate transporters (PCFT) and reduced folate carriers (RFC), although other transporters, such as folate receptors (FR; where FR-α is the predominant form), can also be expressed in the small intestine

(1, 11).

Although the general function of PCFT, RFC, and FR in transporting folate intracellularly is similar, there are distinct differences between each of these transport proteins

(26). Whereas RFC is ubiquitously-expressed in human tissue, PCFT and FR-α are more specifically expressed. PCFT, in particular, is detected at higher levels in the proximal small intestine, kidney, and liver, as well as the choroid plexus, while FR-α is predominantly expressed in the proximal renal tubules, liver, choroid plexus, uterus, and placenta (26). PCFT exhibits equal affinity for both oxidized and reduced folates (25), and functions optimally at low pH environments (pH 5.5) (25). RFC exhibits high affinity for reduced folates and antifolates and very low affinity for FA. RFC functions optimally at physiological pH (7.4) (25) and has a saturable uptake at low folate concentrations (28). FRs (FRα, FRβ, and FRγ), also commonly known as folate-binding proteins, exhibit high affinity for all forms of folate, but most predominantly for FA (1). Interestingly, however, Fr-α, which is not normally expressed in the intestine, becomes overexpressed in the intestines of folate-deficient mice and may contribute to uptake of folate via endocytic process (9, 25).

7 Table 2.1 Folate transporter, carrier, and receptor localization, optimal pH, and pharmacokinetics Transporter Localization Optimal pH Pharmacokinetics

Reduced folate Virtually expressed pH dependent • High affinity for carrier in all tissues and cell (optimal pH = 7.4) reduced folates (Kt of (RFC) lines; particularly in 2-7 µM) the liver, kidney, and Major route of • Low affinity for FA 57-65 kDa jejunum. delivery of folates to (Ki ~ 100-200 µM) cells within systemic • Solute transport carrier SLC19A1 Epithelia: circulation • Anion exchanger Chromosome gastrointestinal cells, 21q22.3 proximal renal tubule, choroid plexus and retinal pigment epithelium Proton-coupled Small intestine, Low pH optimum • Similar high affinity folate brain, kidney, liver, (optimal pH = 5.5) for reduced folates and transporter retina, spleen, colon Activity increases as FA (PCFT) pH decreases until • Km at apical brush pH 5.5 border membrane of 50-65 kDa jejunum is less than 1 Main mechanism of µM SLC46A1 folate transport • Solute transport carrier Chromosome across the apical • Folate-proton 17q11.2 brush-border symporter membrane of the small intestine Folate receptors Kidney, liver, Functions optimally • High affinity for FA (FRα, FRβ, FRγ) choroid plexus, at neutral to mildly (Kd 1-10 nM) retina, uterus, acidic pH • Various affinities for 28-40 kDa placenta, human (pH 5.6-7.2) different folate forms body fluids • Inefficient transport FR relative to RFC and Chromosome PCFT 11q13.2-q13.5 • GPI anchored protein • Receptor mediated endocytosis • Unidirectional

2.1.3.2 Folate Retention and Export (Refer to Table 2.2)

Once transported into the cell, folates are once again polyglutamylated by the enzyme folylpolyglutamate synthase (FPGS) as a mechanism for cellular retention (1, 10). Additionally, polyglutamylated folates are better substrates for intracellular metabolic enzymes involved in

8 folate metabolism (1, 29); however, 5-MTHF and FA are both poor substrates for FPGS (25, 29).

In order to be effectively polyglutamylated, 5-MTHF must be metabolized to tetrahydrofolate

(THF) by methionine synthase/methyltetrahydrofolate-homocysteine methyltransferase

(MS/MTR), whereas FA must be metabolized to THF before it can be effectively polyglutamylated and retained (29). The enzyme γ-glutamyl hydrolase (GGH) removes terminal glutamate residues from intracellular folates to facilitate efflux from the cell into the circulation

(1, 24). Folate export is mediated by RFC on the basolateral membrane, where it can then be transported to target tissues via FRs, RFC, or PCFT in varying proportions, depending on the tissue type.

Table 2.2 Intracellular enzymes involved in folate retention and export Enzyme Localization Optimal pH Pharmacokinetics

Folylpolyglutamate Intestine, liver, pH 8.2-8.5 • Time and dose- synthase kidney, cancer dependent (FPGS) cells/tissues, bone • Higher extracellular marrow, among folate results in lower 60-70 kDa other tissues intracellular folate accumulation & FOL3 retention Chromosome • Folate retention 9q34.11 • Favours DHF binding and reduced enzymatic activity as glutamate tail increases γ-glutamyl Lysosome protein pH 4.5-6.0 • Low affinity (nM) hydrolase found in liver, • High turnover (GGH) kidney, serum, placenta, colon, 36-120 kDa brain, and testis

GGH Chromosome 8q12.23-113.1

9 2.1.3.3 Folate Metabolism and One-Carbon Transfer Reactions (Refer to Table 2.3)

Upon entering the cell, folates are retained by FPGS and downstream one-carbon transfer reactions can proceed. FA is found in its fully oxidized state and must be reduced in the liver, and to a lesser degree in the small intestine, by the metabolic enzyme dihydrofolate reductase

(DHFR) (24, 31). DHFR reduces FA to dihydrofolate (DHF) and subsequently to THF, which becomes metabolically active in its methylated 5-MTHF form (24, 31). Saturation of the DHFR enzyme can lead to the presence of unmetabolized FA (UMFA), which enters the circulation (11,

24, 32). Intake levels of >200 µg FA have been associated with the appearance of UMFA in circulation in humans (32).

Folate is critical for development as it directly participates in one-carbon transfer reactions involved in biological methylation reactions and de novo nucleotide biosynthesis. The re-methylation of homocysteine to methionine is catalyzed by the enzyme MS, along with the co-factor cobalamin (vitamin B12), and proceeds by transferring a methyl group from 5-MTHF to homocysteine to generate methionine and THF (1). Methionine can then be activated by adenosine triphosphate (ATP) via the enzyme methionine adenosyltransferase (MAT) to produce

S-adenosylmethionine (SAM), the primary biological methyl donor in the body (1). In turn,

SAM plays a key role in the regulation of gene expression as it becomes a methyl donor for cytosine guanine dinucleotide (CpG) DNA methyltransferases (DNMTs) responsible for CpG

DNA methylation (24, 33, 34). DNA methyltransferase 1 (DNMT1) is required for CpG maintenance methylation patterns following DNA replication (33, 34), whereas DNA methyltransferases 3A and 3B (DNMT3A & DNMT3B) mediate de novo methylation of certain

CpG sequences (33, 34).

10 Folate is also involved in nucleotide biosynthesis (1). Serine hydroxymethyl transferase (SHMT) catalyzes the reversible conversion of THF and serine to form 5,10-methyleneTHF and glycine

(1). A methyl group from 5,10-methyleneTHF can then be donated to deoxyuridine monophosphate (dUMP) to yield dTMP (thymidylate; precursor for pyrimidylate biosynthesis) and DHF by the enzyme thymidylate synthase (TYMS) (1). Folate can also participate in the purine synthesis pathway once THF or 5,10-methyleneTHF is formylated (1). Conversely, 5,10- methyleneTHF can be irreversibly converted to 5-MTHF via the enzyme methylenetetrahydrofolate reductase (MTHFR) (1). Then, 5-MTHF can proceed to enter the biological methylation pathway and transfer its methyl group to homocysteine.

Figure 2.2: Folate absorption, transport, metabolism, and 1-carbon transfer reactions. Briefly, polyglutamylated folates are hydrolyzed by glutamate carboxypeptidase II (GCPII). Folate is transported across the intestinal apical cellular membrane by the proton-coupled folate transporter (PCFT), the reduced folate carrier (RFC), and folate receptors (FR). Upon entry, folate is polyglutamylated by folylpolyglutamate synthase (FPGS). However, during folate export, terminal glutamate residues are hydrolyzed by the enzyme γ-glutamyl hydrolase (GGH). Folic acid is reduced to dihydrofolate (DHF) and subsequently to tetrahydrofolate (THF) by dihydrofolate reductase (DHFR). Folates are critical for one-carbon transfer reactions that are involved in biological methylation reactions and nucleotide biosynthesis. The re-methylation of homocysteine (tHcy) is mediated by the enzyme methionine synthase (MS) and requires the transfer of a methyl group from 5-methyltetrahydrofolate (5-MTHF) to generate methionine and THF. Methionine is then activated by ATP to generate S-adenosylmethionine (SAM), the primary methyl donor for most biological methylation reactions, including DNA methylation. DNA methylation is further mediated by DNA methyltransferase 1 (DNMT1), 3a (DNMT3a), and 3b (DNMT3b). During nucleotide biosynthesis, THF and serine is converted to 5,10- methyleneTHF and glycine by serine hydroxymethyl transferase (SHMT) in a reversible reaction. 5,10-methyleneTHF then transfers a methyl group to deoxyuridine monophosphate (dUMP) to generate deoxythymidine monophosphate (dTMP, thymidylate) and DHF. Formylated THF or 5,10-methyleneTHF can also enter the purine synthesis pathway. Concurrently, 5,10-methyleneTHF can be irreversibly converted to 5-MTHF by the enzyme methylenetetrahydrofolate reductase (MTHFR). Adapted and reprinted by permission from the publisher (John Wiley and Sons Ltd): (24). Copyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Table 2.3 Enzymes involved in folate metabolism and one-carbon transfer reactions Enzyme Localization Pharmacokinetics

Dihydrofolate reductase Expressed abundantly in • NADPH dependent enzyme (DHFR) all tissues • Higher activity in rapidly growing cells 25 kDa • FA is a poor substrate • DHFR activity very low in DHFR humans with high Chromosome 5q14 interperson variability

Serine Cytosolic SHMT • Affinity for glycine and 5- hydroxymethyltransferase Predominantly MTHF varies between (SHMT) expressed in the heart, isoforms liver, and ova • SHMT gene expression and 54 kDa activity may be modulated Mitochondrial SHMT by extracellular folate levels SHMT Evenly distributed in all Chromosome 17p11.2 (cSHMT) tissues Chromosome 12q13.2 (mSHMT) Thymidylate synthase Cytoplasm of human • Homodimer (TYMS) cells and tissues • TYMS competitively inhibits mRNA expression in absence of substrate • 5,10-methyleneTHF competes with TS mRNA and upregulates translation Methionine synthase Almost all tissues, • NADPH and cobalamin (MS/MTR) especially in pancreas dependent • Oxidizes Cob(II)alamin to 140kDa Cob(I)alamin

MTR Chromosome 1q42.3-1q44 Methylene tetrahydrofolate All tissues • NADH or NADPH reductase dependent (MTHFR)

74.5 kDa

MTHFR Chromosome 1p35.3 DNA methyl transferase Normal human tissues, • DNA methylation (DNMT1) fetal tissues, and some maintenance

13 tumour tissues. Highest • Favours hemi-methylated 150-190 kDA mRNA expression in cytosines as substrates heart, brain, and tumour • Regulated post- DNMT-1 tissues transcriptionally and by cell Chromosome 19p13.2-3 cycle DNMT 3a and 3b Found in majority of • De novo DNA methylation human cells • DNA methylation DNMT-3a maintenance Chromosome 2p23.3 • Prefers hemi-methylated cytosines to non-methylated DNMT-3b cytosines Chromosome 20q11.2 • May be regulated by de novo DNA methylation

2.1.4 Dietary requirements

A proportion of bacterially synthesized folates are absorbed across the colon (35-38).

Apart from this, mammals are unable to synthesize folates (1, 10). Mammals lack the enzyme required for coupling the pteridine ring to PABA to synthesize folate (10). Naturally-occurring folates are found in a number of food sources, including green leafy vegetables, asparagus, broccoli, sprouts, fruits, legumes, dry cereals, whole grain, yeast, lima beans, liver, and certain organ meats (2). However, the bioavailability of folates from these different foods depend on a few factors. Approximately 50-75% of folate content is lost through processing and storage methods, as naturally-occurring folates are often unstable and susceptible to oxidation in low pH environments (2). The bioavailability of natural endogenous folates is also affected by the fact they are found inside cells and are polyglutamated, where increased glutamate residues are associated with decreased bioavailability (1). Therefore, relative to food folates, FA is fully oxidized, conferring higher stability, and monoglutamylated, conferring higher bioavailability relative to naturally occurring folates.

Folate intake is described using dietary folate equivalents (DFEs) (Table 2.4). One DFE is equal to 1 µg of naturally-occurring folates, 0.6 µg of FA from fortified foods or supplements

14 taken with food, or 0.5 µg of FA consumed on an empty stomach (2). In North America, the recommended daily allowance (RDA) of folate in adults is 400 µg DFEs/day (2). This amount increases to 600 µg DFEs/day during pregnancy and 500 µg DFEs/day during lactation (2). The tolerable upper limit (UL) for FA has been arbitrarily set at 1 mg/day for adults to reduce the risk of masking vitamin B12 deficiency (2). However, there is currently no UL for naturally-occurring folates in North America, as it is unlikely to consume such high levels through diet alone (2).

Table 2.4: Recommended Dietary Folate Intake (Daily) (1) Females Males Birth to 6 Months 65 µg DFE 7-12 Months Old 80 µg DFE 1-3 Years Old 150 µg DFE 4-8 Years Old 200 µg DFE 9-14 Years Old 300 µg DFE

Adults or >14 Years 400 µg DFE

Pregnancy 600 µg DFE Lactation 500 µg DFE N/A

Tolerable Upper Limit (UL) 1000 µg/day (FA only)

2.1.5 Measures of folate status

Folate status is determined by measuring serum or plasma and RBC folate concentrations. Serum/plasma folate concentrations reflect short-term dietary intake and supplemental usage, whereas RBC folate concentrations are more reflective of chronic dietary intake or folate tissue stores (2). Human serum/plasma folate concentrations rapidly rise after folate consumption and peak at approximately 2 hours post-consumption (39) as the half-life of monoglutamylated folates in serum is only a few hours long (2). RBCs accrue folate during bone marrow maturation; thus, RBC folate is more reflective of bone marrow folate storage (2). The

15 120-day turnover rate of RBCs reflects long-term intake of folate and RBC folate are resistant to short-term variations in consumption (2). Therefore, RBC folate concentrations are used as the primary diagnosis of clinical folate deficiency, and RBC folate concentrations <340 nmol/L are indicative of folate deficiency, according to standards set out by the World Health Organization

(40). Likewise, serum folate concentrations <10 nmol/L are suggestive of folate deficiency if repeatedly observed over a one-month period (40).

Plasma homocysteine concentrations also represent an additional accurate, inverse, albeit non-specific, functional biomarker of folate status (2). Plasma homocysteine concentrations increase with folate deficiency as limited 5-MTHF levels reduce the ability to transfer available methyl groups from one-carbon donors for methionine production (2). As this transfer is catalyzed by a B12-dependent enzyme, inadequate vitamin B12 levels, other one-carbon nutrients

(e.g., choline, betaine, vitamin B6) and additional factors such as renal dysfunction and aging (1), can also contribute to increased levels of homocysteine (1, 2). Plasma homocysteine concentrations >16 nmol/L are generally considered high and may be indicative of folate deficiency, although cut-off levels of 14 and 12 nmol/L have also been used in studies (2).

Although the high cut-offs for RBC and serum/plasma folate concentrations are not yet well-defined, RBC folate concentration of 1360 nmol/L [97th percentile of RBC folate concentrations in the National Health and Nutrition Examination Survey (NHANES) conducted in the U.S. from 1999 to 2004] and serum folate concentration of 45 nmol/L [arbitrarily chosen based on upper end of assay calibration curve for serum folate quantification in NHANES 1991-

1994 (phase II) and 1999 to 2000], respectively, which have generally been used in scientific literature.

16 2.2 Effects of folic acid fortification and supplementation in North America

2.2.1 Impact of folic acid fortification and supplementation

Due to the evidence suggesting the NTD preventative effect of periconceptional FA supplementation, mandatory FA fortification of all white wheat flour and rice, pasta, cornmeal, and other grain products labelled enriched was implemented in Canada (6, 19, 20). Currently, regulations on fortification of grain products exist in more than 50 countries, worldwide (6).

However, mandatory FA fortification has not been implemented in a number of western

European countries (6), although voluntary fortification efforts may still take place (41).

In 1998, Canada introduced mandatory FA fortification of white wheat flour and cornmeal products at a level of 150 ug/100 g, and of enriched pasta at a level of 200 ug/100 g

(19, 20). Similarly, regulations in the U.S. mandate fortification of white wheat flour, cornmeal products, and enriched pasta at a level of 140 ug/100 g (20). In addition to consuming FA- fortified foods, 30-40% of the North American population consume FA-containing supplements

(21), where standard multivitamins contain 400 µg of FA (45). Supplemental consumption is typically associated with the female gender, older age, higher socioeconomic status, and a healthier lifestyle (22). Concurrently, multivitamin supplements are habitually consumed among cancer patients and survivors, with the highest prevalence found in female breast cancer patients

(46). Supplemental usage within this population is typically associated with the female gender and a higher level of education (46). Health Canada recommends for all women of child-bearing age to consume daily multivitamin supplements containing 400 µg of FA in addition to folate- rich foods (47); however, previous prenatal supplements in Canada contained approximately 1 mg of FA (48). Therefore, attributing to increased multivitamin supplement consumption, intakes and blood levels of folate and FA have significantly increased in the post-fortification era in

17 North America (4).

Studies using the 1988-2010 NHANES data reported that mean serum and RBC folate concentrations have increased 2.5- and 1.5-fold, respectively, since mandatory fortification was implemented in the U.S. (4). In addition, folate deficiency, measured by serum folate concentrations <10 nmol/L and RBC folate concentrations <340 nmol/L, both dropped to ≤ 1% from 24% and 3.5%, respectively, in the post-fortification era in the U.S. (4). Data from the

Canadian Health Measures Survey (CHMS) from 2007 to 2009 revealed a virtual non-existence of folate deficiency (defined by the Institute of Medicine in 1998 as RBC folate concentrations <

305 nmol/L) in the Canadian population, and >40% of the Canadian population had RBC folate concentrations above the high cut-off of 1360 nmol/L (defined as the 97th percentile of RBC folate concentrations from the 1999 to 2004 NHANES data) (5).

2.2.2 The effects of folic acid on DHFR and UMFA

The liver is the primary site for FA biotransformation, where FA is reduced to DHF and subsequently to THF by the enzyme DHFR. A study conducted by Bailey et al. determined this process to be extremely inefficient in human relative to rats (11). Human DHFR was also found to have approximately a 5-fold variation in activity between samples (11). The activity of human liver DHFR was observed to be 56 times lower than that of average activity of rat DHFR with

FA as the substrate (11). Additionally, although the affinity of DHFR for DHF is higher than FA, high concentrations of FA could competitively inhibit the conversion of DHF to THF, thereby creating an intracellular deficiency for metabolically active folates (52).

The saturation of DHFR can lead to the presence of UMFA within the circulation, indicated by the increased detectability of UMFA post-fortification, particularly among

18 supplement-users (49). Detection of circulating UMFA also appeared to have increased in the

North American population since FA fortification. Specifically, the prevalence of detectable

UMFA increased from 55.0% pre-fortification to 74.7% post-fortification in supplement nonusers, and 72.5% to 80.7% in supplemental users (49) in the Framingham Offspring Cohort.

According to data from the NHANES from 2001 to 2002, detectable UMFA in the U.S. population aged ≥ 60 years was 38% (50). A study on post-menopausal women aged 50-75 and in good health in the greater Seattle area, revealed an approximate 78% prevalence in detectable

UMFA post-fortification (53). However, it should be noted that detectable UMFA is not only limited to countries with mandatory FA fortification. In a study conducted on a cohort in Ireland, where only voluntary FA fortification is present, detectable UMFA was found in the umbilical cord blood of 17 out of 20 newborn babies, and 18 out of 20 mothers, none of whom were consuming FA supplements at the time of this study (41). The profound effect of FA supplementation and fortification on detectable UMFA has been a cause of concern as the pharmacodynamics properties of UMFA are not known (12). Theoretically, UMFA could interfere with metabolism, cellular transport, and regulatory functions of natural folates by competing with the reduced forms for binding with key enzymes and carrier proteins (9).

Increased blood concentrations of UMFA have also been observed to reduce natural killer cell cytotoxicity in both postmenopausal women (53) and in mice (54).

FA has also been suggested to be a significant inhibitor of the MTHFR enzyme (9, 57,

65). In a mouse model, FA consumption was associated with reduced MTHFR protein expression and reduced activity levels (65). Consistent with this finding, an in vitro model using

MTHFR purified from pig liver found that DHF competitively inhibited MTHFR activity with

5,10-methylenetetrahydrofolate as the substrate (57). Therefore, FA may inhibit 5-MTHF

19 formation, which can lead to reduced generation of methionine (9, 57, 65). In this regard, FA or

UMFA can disrupt key metabolic pathways and lead to an intracellular deficiency in metabolically active folates, which can have profound biochemical effects, such as aberrant

DNA methylation or inhibited nucleotide biosynthesis. Based on these findings, many have speculated that 5-MTHF supplementation would be safer compared to FA, as 5-MTHF is unlikely to produce UMFA in circulation, and bypasses the need for DHFR and MTHFR metabolism.

2.2.3 Effects of FA supplementation on folate metabolism

There have been few studies that have examined the effects of FA supplementation on folate absorption, metabolism, and one-carbon transfer reactions.

2.2.3.1 Folic Acid and 5-MTHF supplementation on absorption and transport (Refer to Table

2.5)

There are few studies that have elucidated the effects of FA supplementation on folate absorption in vivo. In a study using laying hens, FA supplementation (through 10 mg and 100 mg

FA/kg diets) for 28 days was associated with significantly reduced folate uptake in the duodenum, although folate uptake was unaffected in the jejunum (58). However, in both these segments, Pcft and Rfc mRNA expression levels were not affected, suggesting a posttranscriptional or translational regulatory mechanism (58). In another study using laying hens, Jing et al. examined the comparative supplemental effects of FA and 5-MTHF on absorptive mechanisms in the intestine (56, 59). This will be further discussed below.

In a study using Wistar rats, a 20 mg FA/kg supplemented diet for 10 days decreased intestinal folate uptake, which was associated with down-regulated protein expression of the Pcft

20 and Rfc (28). However, when exposed to chronic FA supplementation at the same dose for 60 days, no significant differences were observed in intestinal folate uptake, which can be attributed to the non-significant change in Pcft and Rfc protein expression levels (28). Notably, in both acute and chronic FA supplementations, mRNA expression levels of Pcft and Rfc did not differ

(28). Therefore, in agreement with the previous study (58), it is speculated that post- transcriptional or translational adaptations regulated the observed differences in intestinal folate uptake (28).

In an in vitro model using human Caco-2 colon and HK-2 renal epithelial cells, maintenance in a long-term FA oversupplemented condition (100 µmol FA/L growth medium) was observed to decrease folate uptake relative to maintenance in a FA sufficient condition (0.25

µmol FA/L growth medium), which was associated with a significant decrease in mRNA level, protein level, and promoter activity of the RFC (60). Additionally, decreased folate uptake was also associated with decreased mRNA levels of the PCFT and FR (60). Contrary to the aforementioned studies (28, 58), folate uptake was also suggested to be mediated by a transcriptional mechanism, at least for the RFC system.

Folate deficiency in a mouse model was associated with increased gene expression of the

Pcft in the small intestine, a likely compensatory mechanism to increase folate absorption (61).

Generally, it was found that FA supplementation tended to reduce FA uptake as well as decrease mRNA expression of the Pcft, Rfc, and Fr, whereas folate deficiency resulted in increased FA uptake and mRNA expression of the transporter, carrier, and receptor genes (61).

21 Table 2.5 Summary of folate supplementation effects on absorptive mechanisms Author Model Treatment Results

Ashokkumar Caco-2 5 generations: - Oversupplementation decreased et al. HK-2 cells 1) RFC, PCFT, and FR mRNA 2007 (60) Oversupplementation expression 100 µmol FA/L - Oversupplementation decreased RFC 2) Sufficient protein expression conditions Overall: Chronic oversupplementation 0.25 µmol FA/L of FA leads to downregulation of 9 µmol FA/L folate uptake, associated with decreased mRNA expression of RFC, PCFT, and RFC. Regulation appears to be mediated by transcriptional mechanisms Jing et al. Shaver 21 days: - FA supplementation trended towards 2009 (56) white laying 1) Control diet lower Rfc mRNA expression relative hens Basal diet (BD) – no to control in jejunum, and no supplemental folate difference in duodenum, caecum 2) FA group - 5-MTHF supplementation BD + 10.00 mg/kg FA significantly lowered Rfc mRNA 3) 5-MTHF group expression vs. control in jejunum, but BD + 11.30 mg/kg 5- no difference in duodenum, caecum MTHF - Rfc mRNA expression similarly decreased with FA and 5-MTHF supplementation Overall: FA or 5-MTHF supplementation lowers Rfc expression in the jejunum Jing et al. Shaver 21 days: - FA supplementation had no effect on 2010 (59) white laying 1) Control diet mRNA expression of absorptive hens Basal diet (BD) – no proteins in duodenum, jejunum, or supplemental folate caecum 2) FA group - 5-MTHF supplementation BD + 10.00 mg/kg FA significantly lowered PCFT mRNA 3) 5-MTHF group expression vs. control in jejunum, but BD + 11.30 mg/kg 5- no difference in duodenum, caecum MTHF - No difference between effects of FA and 5-MTHF supplementation on Rfc expression in jejunum Overall: 5-MTHF supplementation may downregulate Pcft mRNA expression in jejunum and also reduce folate intake Dev et al. Albino Acute: 10 days - Acute oversupplementation 2011 (28) Wistar rats Chronic: 60 days decreased protein expression of PCFT

22 1) Control diet and RFC 2 mg FA/kg diet - Acute oversupplementation did not 2) FA group influence mRNA levels 20 mg FA/kg diet Overall: Acute, but not chronic, folate oversupplementation led to downregulation in intestinal folate uptake at acidic pH optima associated with decreased protein expression Tactacan Shaver 28 days: - Plasma folate concentrations et al. white laying 1) Control diet increased & plasma tHcy 2012 (58) hens BD – no supplemental concentrations decreased in FA folate supplemented hens 2) BD + 10 mg/kg FA - Mucosal to serosal uptake of FA was 2) BD + 100 mg/kg decreased in duodenum, but not the FA jejunum of supplemented hens - No difference in mRNA expression of Pcft and Rfc genes between all groups Overall: Increased dietary levels of FA supplementation resulted in decreased transport of FA in duodenum, but not jejunum of laying hens

2.2.3.2 Folic acid supplementation and folate deficiency on metabolism and one-carbon transfer reactions

In addition to the regulation of absorption, previous studies have suggested that FA supplementation can modulate metabolic enzymes as well. In an in vivo study using sows and gilts, FA and glycine supplementation significantly increased mRNA expression levels of Shmt in both endometrial and embryonic tissues, although it was suggested that FA alone is likely to be mainly responsible for the purported effect (62). Another in vivo study reported that when chickens fed folate-deficient diets were injected with FA, Shmt activity increased relative to those not injected with FA (63).

With regard to MTHFR, FA supplementation was observed to modulate MTHFR activity and protein expression levels. In a study using a BALB/c Mthfr+/+ and a Mthfr+/+ mouse model

23 and an in vitro model, it was determined that FA supplementation (20 mg FA/kg diet) reduced

MTHFR protein expression and activity levels in the liver, respectively (65). It should be noted that these results were found in both heterozygous Mthfr male mice and in wild-type mice (65).

In the same study, FA supplementation was associated with decreased Ms expression in the liver in both heterozygous Mthfr male mice and in wild-type mice, suggesting a shift toward folate- independent homocysteine remethylation due to reduced 5-MTHF substrate (65).

Hayashi et al. investigated the effects of folate deficiency on steady-state levels of genes regulating intracellular one-carbon metabolism using human HCT116 and Caco-2 colon adenocarcinoma cell lines (66). In the HCT116 cell line, folate depletion was associated with induced adaptive regulations favouring increased folate uptake and intracellular retention, while appearing to preferentially shuttle the folate pool to the methionine cycle (66). In the Caco-2 cell line, the effects of folate depletion were not as apparent (66); however, the cellular adaptations to folate depletion appeared to also favour folate retention, while shuttling the available folate pool to the nucleotide biosynthesis at the expense of the methionine cycle (66). Notably, in both cell lines, folate depletion was associated with upregulated maintenance DNA methylation and downregulated de novo DNA methylation (66).

2.3 Folate and folic acid in health and disease

Folate plays an important role in human health and disease as it is involved in nucleotide biosynthesis and biological methylation reactions (1, 2, 24). Its deficiency has been linked to many adverse health outcomes including NTDs, megaloblastic anemia, cognitive impairments, and cancer development (24). Folate deficiency can occur due to insufficient dietary intake, impaired folate absorption and metabolism, and/or increased demand and utilization (2, 24).

24 Folate absorption can also be reduced due to certain gastrointestinal disorders, such as celiac disease and Crohn’s disease, where the absorptive lining of the small intestine is diseased, chronic exposure to high amounts of alcohol, certain medical conditions that increase rates of cell turnover, pregnancy, tobacco smoking, or use of drugs, such as antifolates used to treat inflammatory conditions and cancer (2, 10, 24).

FA supplementation has been shown to be an effective strategy in the prevention and treatment of a few of these adverse outcomes, such as megaloblastic anemia and NTD prevention. In particular, a 15-50% decrease in NTD incidence has been observed in the North

American population following FA fortification (6-8), but the effects of FA supplementation on other diseases have yet to be clearly elucidated. FA supplementation has long been regarded as safe and purely beneficial (8); however, a recently emerging body of evidence has suggested that

FA supplementation may be associated with adverse health outcomes, including progression of

(pre)neoplastic lesions, accelerated cognitive impairment, reduced natural killer cell cytotoxicity, resistance to antifolates drugs, and aberrant DNA methylation (24).

Although the role of FA supplementation in health and disease has not been unequivocally established, the U.S. National Toxicology Program and the Office of Dietary

Supplements of the National Institutes of Health concluded no further beneficial effects of supplemental folate beyond adequate folate status on reducing cancer. Certain effects of FA supplementation on health and disease have been well elucidated, while others require stronger evidence (see Table 2.6).

25 Table 2.6 Strength of evidence for effects of FA on human health and disease

Effects

Evidence Beneficial Null Adverse

Convincing • Macrocytic anemia • Masking vitamin B12 • Neural tube defects deficiency

Probable • Prevention of new cancer • Coronary heart • Promotion of • Stroke disease (pre)neoplastic lesions • Cognitive function • Other congenital • Epigenetic ∆ • Congenital heart defects defects • ↓ NK cell activity • Epigenetic ∆ Possible/ • Pregnancy outcomes • Resistance to antifolates Insufficient • Neuropsychiatric disorders • Accelerated cognitive dysfunction with low vitamin B12 status • ↑ risk of obesity, insulin resistance in offspring

2.3.1 Anemia

It is well established that folate deficiency can cause megaloblastic anemia (24).

Megaloblastic anemia is defined by morphological changes in bone marrow and peripheral blood cells. Initially, megaloblasts, or enlarged nucleated erythrocyte precursor cells with noncondensed chromatin, develop as a result of defective DNA synthesis due to folate deficiency, which interferes with appropriate cell proliferation of bone marrow cells involved in erythropoiesis (2, 24). Following the morphological changes in the bone marrow is the appearance of macrocytes and macroovalocytes, as well as hypersegmented neutrophils in the peripheral blood (2, 24). Over time, erythrocyte count, haematocrits, and haemoglobin concentrations decrease, resulting in anemia (2, 24). Symptoms of megaloblastic anemia include weakness, fatigue, irritability, difficulty concentrating, and palpitations (2). FA supplementation has been shown to be an effective treatment for folate-deficiency-associated megaloblastic anemia (2, 24).

26 2.3.2 Masking of B12 deficiency

The tolerable UL for FA of 1 mg/day was set in response to a concern with respect to the potential for high FA intakes of masking hematological symptoms and signs of vitamin B12 deficiency (2). Vitamin B12 deficiency is more commonly observed among elderly individuals in

North America due to reduced absorption and extraction from dietary sources owing partially to gastric atrophy from aging (52). Vitamin B12 deficiency can result in pernicious anemia, cognitive and neurological complications, and certain gastrointestinal symptoms (2). Although high levels of FA intake can correct for vitamin B12 deficiency-associated anemia, presumably through replenished levels of folate for nucleotide biosynthesis, it does not correct for the adverse neurological effects associated with vitamin B12 deficiency (9, 24, 54). The regeneration of SAM is a vitamin B12 dependent process and therefore cannot occur during vitamin B12 deficiency, while nucleotide biosynthesis is not vitamin B12 dependent and can proceed during vitamin B12 deficiency with adequate folate supply (24). Masking vitamin B12 deficiency- associated anemia allows vitamin B12 deficiency-induced neurological deficits to progress, resulting in irreversible subacute combined degeneration (9, 24).

2.3.3 Cardiovascular disease

Many epidemiological studies have shown that elevated levels of homocysteine are associated with cardiovascular diseases, such as coronary heart disease, stroke, peripheral vascular disease, and thrombosis (67, 70, 72). The Homocysteine Studies Collaboration, a meta- analysis which examined data from 30 prospective and retrospective studies, determined that a

25% reduction in homocysteine concentration was associated with an 11% decreased risk of coronary heart disease and a 19% decreased risk of stroke (24, 73). FA supplementation was

27 shown to be an effective treatment in reducing homocysteine concentrations, as folate is directly involved in the remethylation of homocysteine to methionine (73, 74). Hence, FA supplementation has been purported to have a protective effect on cardiovascular disease.

However, a recent meta-analysis examining 8 large randomized double-blind clinical trials of FA supplementation with or without B vitamins involving subjects at higher risk of cardiovascular disease found no significant effect in its association with any adverse vascular outcomes, including major vascular events, major coronary events, or stroke over a median follow-up period of 5 years (72). Findings from these studied predisposed participants suggest a probable null effect of reducing coronary heart disease incidence as a secondary prevention with

FA supplementation (72).

2.3.4 Neuropsychiatric and cognitive disorders

Folate deficiency is often associated with neuropsychiatric disorders, including depression, cognitive impairment, and Alzheimer’s disease or dementia in elderly individuals

(24, 75). Folate deficiency has also been linked to a reduced response to antidepressant treatment in individuals suffering from major depression (75). Symptoms are exacerbated if left untreated, but remain responsive to improvements in folate status (24, 75). Indeed, these adverse effects are also observed in individuals with inborn errors of folate transport and metabolism (24, 75).

It has been suggested that the observed association between folate deficiency and neurological disorders involves the reduced availability of SAM required for methylation in the brain (24, 75). Inadequate SAM levels may contribute to neurological disorders through several mechanisms including epigenetic modifications, which may contribute to demyelination in the nervous system, reduced levels of SAM to monoamine pathways, which is associated with

28 decreased turnover of serotonin, dopamine, and noradrenaline (76), and vascular or neurotoxic mechanisms mediated by hyperhomocysteinemia (75). SAM is a universal methyl donor, available as a prescription drug or nutritional supplement, and shows promise as a treatment option for major depression disorder and Alzheimer’s Disease (77).

Folate supplementation, regardless of folate type (FA or 5-MTHF), has been reported to improve mood and morbidity (78-81). Monotherapy of 5-MTHF has shown promise as evidenced by its antidepressant effect in the absence of folate deficiency (79). However, this effect was observed in patients whose RBC folate concentrations increased with treatment without folate deficiency (79, 82). A proposed mechanism for this phenomenon is analogous to laughing gas, or nitrous oxide, and its effect on euphoria (75). Nitrous oxide inactivates MS

(MTR) in the brain and liver (83), inhibiting SAM and consequently, production of monoamine neurotransmitters, specifically, serotonin, epinephrine, and dopamine (75, 83). Another speculated mechanism is that increased levels of folate increases the levels of SAM, which allows for the methylation of myelin basic protein and membrane phospholipids to maintain myelination in the nervous system (85). Accordingly, certain neuropsychiatric disorders are purported to be attributed to a reduction in availability of SAM required for methylation in the brain (24, 75, 79).

2.3.5 Carcinogenesis

The role of folate supplementation in cancer prevention and progression remains inconclusive and results have shown inconsistent findings. Epidemiological studies have suggested an inverse relationship between folate status, either folate intake or blood levels of folate, and the risk of several human malignancies including cancer of the colorectum,

29 oropharynx, esophagus, stomach, pancreas, lungs, breast, cervix, uterus, and ovary, as well as neuroblastoma and leukemia (10, 45). Corollary to this, FA supplementation has been suggested to reduce the risk of these cancers (10). However, the notion that folate deficiency may increase the risk of cancer appears to be counterintuitive to the role of antifolate in cancer treatment. The conceptual basis for antifolate chemotherapy involves folate deficiency, depleting intracellular folate as a mechanism of disrupting one-carbon transfer reactions and in turn, decreasing the provision of substrates for nucleotide biosynthesis, which prevents cell replication and cancer progression (10, 45). The precise nature and magnitude of the inverse relationship between folate status and cancer risk have not been clearly established for all cancer types (10, 45). The role of folate in carcinogenesis has been best studied in colorectal cancer (CRC) (10, 45), where case- control and prospective studies suggest a 20-40% increased risk of CRC or its precursor, adenoma, in individuals with the lowest folate intake/blood levels relative to those with the highest folate status (86-88). In addition, epidemiologic evidence suggests that nutrient-gene interactions, where one-carbon nutrients and genetic variants of enzymes involved in the folate metabolic pathway, may modulate cancer risk (10). For example, the MTHFR C677T polymorphism appears to decrease risk of certain cancers, but increase the risk of others in a site- specific manner depending on cancer type and folate status (10, 89).

Animal studies have also provided support for the purported inverse association between folate status and cancer risk. However, animal studies have also demonstrated a dual modulatory role of folate in cancer development and progression depending on the dose and stage of cell transformation at the time of folate intervention (Figure 2.3) (10). In animal models of CRC, folate deficiency enhanced, whereas modest levels of FA supplementation suppressed the development of CRC if intervention was initiated prior to the establishment of neoplastic foci in

30 the intestine (90, 91, 92). If folate intervention was initiated after the establishment of neoplastic foci, however, folate status had an opposite effect on development and progression of CRC (90,

91, 92); folate deficiency induced a regression or inhibited the progression, whereas folic acid supplementation promoted the progression of already existing (pre)neoplastic lesions (10, 93).

Interestingly, in a normal colorectum, supraphysiologic supplemental doses (20 x BDR) promoted the development of cancer (10).

The potential mechanisms behind the dual modulatory effects of folate status and cancer development and progression relate to folate metabolism and one-carbon transfer reactions involved in DNA synthesis and DNA methylation (10). In normal tissues, folate deficiency can lead to neoplastic transformation in part due to DNA strand breaks, chromosomal and genomic instability, uracil misincorporation, increased mutations, and defective DNA repair resulting from the lack of available nucleotide substrates (10). FA supplementation may prevent or correct these anomalies in normal tissue. In (pre)neoplastic cells, however, folate deficiency inhibits proliferation and growth of transformed cells by limiting the availability of nucleotide substrates

(10). In contrast, FA supplementation may promote (pre)neoplastic cell proliferation and growth by providing nucleotide substrates (10).

In addition to the mechanisms relating to nucleotide biosynthesis, aberrant DNA methylation (global DNA hypomethylation and hypermethylation of promoter regions of tumour suppressor and cancer-related genes) resulting from folate deficiency and FA supplementation may contribute to carcinogenesis (10). As previously described, folate is integrally related to the generation of SAM, the universal donor for most biological methylation reactions including

DNA methylation (1). Global hypomethylation is associated with chromosomal instability and increased mutations, whereas promoter CpG island hypermethylation is associated with gene

31 silencing, resulting in a loss of function (10). Promoter CpG islands of over 60% of tumour suppressor and mismatch repair genes have been observed to be methylated in cancer (110).

In normal cells, CpG sites in the gene body are generally methylated, whereas CpG islands found proximal to a gene promoter are unmethylated (10). Contrarily, in transformed/cancer cells, CpG sites in the gene body harbor widespread loss of methylation, and gain methylation in CpG islands found on gene promoter regions, including many tumor suppressor genes (10). Folate deficiency has been proposed to promote cancer development in the colorectum by inducing genomic DNA hypomethylation, potentially causing chromosomal instability, while folate supplementation has been suggested to protect against such events (10).

However, the effect of folate on DNA methylation has not been clearly elucidated and studies have indicated inconsistent results.

Clinical studies have reported inconsistent results concerning the effect of FA supplementation on cancer risk (27). Randomized clinical trials have reported conflicting results regarding the effect of FA supplementation on occurrence or recurrence of adenoma or CRC

(10). In the United Kingdom Colorectal Adenoma Prevention Trial, a randomized, double-blind trial using patients younger than 75 years, Logan et al. observed a reduced recurrence for colorectal adenoma associated with 300 mg/d aspirin supplementation, although no effect was observed with 0.5 mg/d FA supplementation (95). In a 24-month double-blind, controlled trial,

Paspatis and Gregorios observed a non-significant lowered risk of colorectal adenoma recurrence with 1 mg/d FA supplementation (97). In a 3-6.5 year, randomized, double-blind trial using participants from the Health Professionals Follow-Up Study and the Nurses’ Health Study, Wu et al. observed no significant effect of FA supplementation (1 mg/d) on colorectal adenoma recurrence, although participants with low baseline plasma folate concentrations receiving FA

32 supplementation experienced a significant decrease in adenoma recurrence (97). Jaszewski et al. reported a significant 64% lowered risk of recurrent adenomas associated with a 3-year FA supplementation of 5 mg/day (98).

Figure 2.3: Dual modulatory role of folate in colorectal carcinogenesis. Animal studies have suggested a dual modulatory role of folate on cancer development and progression contingent on dose and timing of folate intervention. In normal tissues, folate deficiency increases cancer risk, whereas FA supplementation decreases risk of neoplastic transformation. In the presence of preneoplastic lesions, folate deficiency inhibits, whereas FA supplementation promotes the progression of cancer. Possible mechanisms for these effects are attributed to nucleotide biosynthesis, and aberrant DNA methylation patterns. Adapted and reprinted by permission from the publisher (John Wiley and Sons Ltd): (24). Copyright © 2001 John Wiley & Sons, Ltd. All rights reserved.

The Aspirin/Folate Polyp Prevention Study (AFPPS), a randomized, placebo-controlled trial of aspirin and FA supplementation on recurrence of colorectal adenomas in patients aged 21 to 80 years with previously removed adenomas, reported that FA supplementation at 1 mg/day for up to 6 years does not reduce colorectal adenoma risk (99). Using the same cohort from the

AFPPS, Figueiredo et al. reported an increased risk of prostate cancer associated with 1 mg/d FA

33 supplementation (100). Interestingly, baseline dietary folate intake and plasma folate in nonmultivitamin users were inversely associated with the risk of prostate cancer (100). Results from this study supported the observations made in previous animal studies regarding the potential tumour promoting effect of FA supplementation. It is possible that FA supplementation promoted the progression of pre-existing undiagnosed precursor of colorectal and prostate cancers in these highly predisposed participants (93).

Two ecological studies that examined the temporal post-fortification trend of colorectal cancer incidence in the U.S., Canada, and Chile reported increased colorectal cancer rates in these countries post-fortification, suggesting the potential significant role of FA fortification and increased folate status behind this trend (101, 102). In contrast, two large population-based studies using cohorts from the NIH-AARP Diet and Health Study and the Cancer Prevention

Study II Nutrition Cohort, have reported decreasing trends of colorectal cancer incidence post- fortification in the U.S. (103, 104).

Meta-analyses examining the effects of FA supplementation on overall cancer risk or

CRC risk in various cohorts have reported conflicting results. Two meta-analyses of clinical trials examining the effects of FA supplementation alone or in conjunction with other B vitamins on cardiovascular disease outcomes as the primary endpoint reported a null effect on cancer incidence (72), or an increased risk of cancer incidence and mortality (107). More recently, three other meta-analyses of clinical trials examining the effects of FA supplementation on recurrence of adenoma reported null associations (105, 106) or an increased risk (108). The role of FA supplementation on cancer development and progression remains highly controversial and inconsistent. More studies examining the effect of fortification and supplementation on cancer development and progression are warranted to clarify this issue.

34 2.3.6 Epigenetic changes

Epigenetics can be defined as heritable changes in gene expression that are not attributed to any alterations in the DNA sequence (24, 34). Some epigenetic mechanisms include DNA methylation, chemical modifications of histones, and RNA interference (24, 34). Epigenetic changes are potentially reversible and are susceptible to environmental influences like nutritional and pharmacological exposures (24, 34). Such exposures are of particular importance in the in utero environment as maternal exposure, such as nutrition, can potentially alter the intrauterine one-carbon precursor environment, which can affect one-carbon metabolism in the developing offspring (24, 34). During the embryonic stage, parental DNA methylation patterns are erased and a new pattern of DNA methylation is established and re-programmed after fertilization (34)

DNA methylation of cytosines within cytosine-guanine (CpG) sequences is an important mechanism of transcriptional regulation (24), where up to 80% of all CpG sites in human are normally methylated (109). In the mammalian genome, DNA methylation occurs on the cytosine residue that occurs 5’ to a guanosine residue in CpG sites (9). CpG islands refer to CpG dense areas that are clustered within small stretches of DNA (9). These CpG islands are found proximal to the promoter regions of about half of the genes within the genome, and are predominantly found in the unmethylated state (9), thereby allowing for transcription to occur. Upon methylation of CpG islands, stable and heritable transcriptional silencing occurs, which is mediated by the transcriptional repressor, methyl-CpG binding protein-2 (MeCP2) (34, 111).

MeCP2 binding causes the recruitment of corepressors, such as histone deacetylases and histone methyltransferases (111). Histone deacetylation suppresses transcription by allowing tighter nucleosomal packaging, thus rendering an inactive chromatin conformation (34). DNA methylation primarily occurs in the bulk of the genome where CpG density is low, including

35 exons, noncoding regions, and repeat DNA sites, which allows for the appropriate organization of chromatin (24, 110). DNA methylation is a dynamic process mediated by CpG DNA methytransferases 1, 3a, and 3b using SAM as the methyl donor (34). DNA methylation plays an important role in human health and disease as methylation of the promoter CpG islands is associated with gene silencing (34), whereas CpG DNA methylation in the gene body is associated with genomic stability (24). Indeed, aberrant patterns or dysregulation of DNA methylation are associated to the development of several human diseases, including cancer (9,

103).

Folate is directly involved in the generation of SAM and as such, folate status has been shown to modulate DNA methylation, particularly in two important life stages: in utero/early postnatal life and aging (34). However, the effect of folate deficiency and FA supplementation on global or gene-specific DNA methylation has not been consistently demonstrated in in vitro models, animal models, or human studies (34). Collectively, these studies suggest that the effects of folate status on DNA methylation are cell, tissue, organ, site, and gene-specific and depend on the stage of cell transformation as well as the timing, degree, and duration of folate exposure (8,

34, 112).

As mentioned earlier, the in utero environment is very susceptible to nutritional exposures as epigenetic programming takes place during this stage of life with the establishment of a new DNA methylation pattern in the fetus (34). As proof-of-concept, a study conducted using yellow agouti mice demonstrated that maternal supplements containing FA and other B vitamins can increase CpG methylation in the promoter region of the agouti gene (120) in offspring, which in turn affected colour and obese phenotypes rendering them obese and yellow.

Another study examining the effects of methyl donor supplementation on Axin(Fu)+ offspring

36 determined that increased methyl donor supplementation increased DNA methylation and in turn, reduced tail kinking (121). A preliminary prospective study in the United Kingdom found an inverse correlation between cord plasma homocysteine concentrations and global DNA methylation in cord lymphocytes in the offspring (119). It should be noted that the significant associations between maternal FA use and cord blood folate and genomic methylation were not reported.

These in utero mice studies suggests that excess maternal folate intake and/or FA supplementation above adequate folate intake may modulate DNA methylation, which can confer adverse developmental and health outcomes in the offspring.

2.3.7 Pregnancy

The requirement for folate increases during pregnancy as uterine, placental, and foetal growth result in a greater dependency on one-carbon transfer reactions involved in nucleotide biosynthesis for DNA synthesis and cell replication (2). Compared to maternal blood folate concentrations, higher cord blood folate concentrations are observed as FRs are greatly expressed in the placenta (122). Therefore, maternal folate is transported to the fetus against a concentration gradient (122). In developing countries, an estimated 35-75% of pregnant women develop anemia (23). Although iron deficiency is the most common cause of anemia during pregnancy, inadequate folate intake during pregnancy may result in maternal folate deficiency, which may manifest as megaloblastic anemia (122). As such, FA supplementation is recommended not only during the periconceptional period, but throughout pregnancy as well

(122).

37 Folate deficiency during pregnancy has also been associated with various adverse complications, including NTDs, placental abruption, preeclampsia, spontaneous abortion, and stillbirth, although findings have been inconsistent (122). Low birth weight arguably constitutes as the most important pregnancy outcome as it is strongly related to high mortality and morbidity

(140). However, the relationship between folate status and fetal growth is inconclusive.

Generally, with regards to birth weight, it is suggested that adequate folate status promotes optimal fetal growth (122, 134). In a study examining a large Norwegian cohort, fetal growth restriction, as defined by birth weights of <10th percentile, was significantly greater in women who were in the higher quartiles of total homocysteine levels than those in the lower quartiles

(137), although this relationship was not confirmed in other studies (138, 139). Moreover, among twelve studies evaluating the effects of prenatal FA supplementation on offspring birth weight

(124-135), 7 studies observed increased birth weights in response to FA supplementation (125,

127, 128, 130, 131, 132-134), and no effect was found in the remaining 5 studies. This consistency is probably attributed to adequate maternal folate status during early pregnancy in some studies and inconsistent timing of FA intervention in these studies (122). Preterm delivery

(delivery before 37 weeks of gestation) is also a leading cause of perinatal morbidity and mortality (122). The effect of maternal folate and homocysteine status on preterm delivery has not equivocal and inconsistent (122).

A few studies have, albeit equivocally, linked FA supplementation during early pregnancy to increased rates of other pregnancy complications, such as twinning and miscarriages (141, 145). Multiple births have also been associated with an increased risk of certain pregnancy complications, as well as infant morbidity and mortality (141). Maternal FA supplementation has also been observed to increase risk of childhood asthma (142, 143),

38 allergies (144), and autism (146) in a limited number of studies. The Pune Maternal Nutrition

Study from India examined the effects of high maternal folate status on adverse health outcomes

(147). It was found that maternal blood folate concentrations during pregnancy was directly associated with total fat mass in children (aged 6), where higher folate concentrations resulted in greater fat mass in children (147). Additionally, it was found that high maternal blood folate concentrations in conjunction with low vitamin B12 concentrations increased the risk of insulin resistance in children (aged 6) (147). It has been suggested that imbalances between maternal folate and B12 concentrations could influence imprinting in the embryo, perhaps through DNA methylation, predisposing the offspring to metabolic syndrome and insulin resistance (147).

Future studies are warranted to elucidate this intriguing interaction between maternal folate and vitamin B12 status in relation to offspring’s health risk including insulin resistance, metabolic syndrome and obesity.

In rodent studies, the effects of maternal FA supplementation included an increased risk of mammary tumors in female offspring (148), and decreased colorectal cancer risk in male pups

(181), an increased glucose concentration regardless of folate level during weaning (149), a reduced femoral area and strength and lower body weight after weaning in female offspring

(150), and an increased insulin resistance in male offspring (151). A study by Cho et al. (152) illustrated a complex role of maternal folate intake and status on offspring health outcomes. The study determined that male pups weaned from dams fed with 10-fold FA diets during pregnancy responded differently depending on their post-weaning diet. Male pups who were fed the recommended vitamin diet showed an increased obesogenic behaviour and physiological response (151).

39 2.3.8 Fetal development

It has been unequivocally established that optimal periconceptional folate status plays a vital role for a proper closure of the neural tube (24), although the underlying mechanisms remain elusive (156). Spina bifida and anencephaly are two common types of NTDs and are most commonly seen in association with suboptimal folate status (157). The first association between folate status and NTDs was made by the Leeds Pregnancy Nutrition (30). Subsequent to this observation, a large body of epidemiologic studies have demonstrated the inverse association between maternal folate status and the risk of NTDs (24). Two large randomised double-blinded clinical trials demonstrated the significant protective effect of periconceptional FA supplementation on NTD risk (123). Although the association between periconceptinal FA supplementation and other congenital defects has not clearly been established, a few studies have shown the protective effect of periconceptional FA supplementation on certain congenital defects such as congenital heart defects, limb defects, and urinary tract anomalies (24, 123).

2.4 5-methyltetrahydrofolate

FA has long been believed to be strictly beneficial as a supplement and fortificant. It has been preferred over naturally-occurring folates due to its greater stability, higher bioavailability, and lower cost. Concerns regarding the safety of FA have recently been raised due to the drastically increased post-fortification intake and blood levels and the potential association with certain adverse health outcomes. The reduced and predominant circulating form of folate, 5-

MTHF, has been commercially available as mixed diastereoisomers (L and D forms). The L form, or L-5-methyltetrahydrofolate (L-MTHF), also known as 6(S)-5-MTHF, refers to the active form (14) while D-5-MTHF is stored in tissues but is not metabolized by the body (158).

40 Therefore, racemic mixtures of these diastereoisomers possess only half the biological activity relative to FA (158). The L-isomer can partake in one-carbon transfer reactions, whereas the D- isomer cannot (158).

2.4.1 5-MTHF-Ca

Both FA and 5-MTHF are commercially available over-the-counter in doses of 200 µg,

400 µg, 800 µg, or 1000 µg. 5-MTHF is commercially available as a crystalline calcium salt, called Metafolin®. Metafolin®, patented and produced by Merck Inc., is a diastereoisomericalls- pure source of L-MTHF containing no more than 1% D-methyltetrahydrofolate (14). Another 5-

MTHF supplement includes Quatrefolic®, a glucosamine salt patented and produced by Gnosis, which has been demonstrated to have enhanced oral bioavailability in comparison to FA and

Metafolin®. Deplin®, patented and produced by Pamlab, Inc., is a prescription product that contains L-MTHF as its active ingredient (159). As a medical food, Deplin® is designated as a

U.S. Food and Drug Administration (FDA) category intended for the clinical management of depression (159). Deplin® is prescribed at two doses of 7.5 mg or 15 mg tablets, to folate deficient subjects with major depressive disorder (159). Pamlab, Inc. also produces other prescription medical products, Neévo® or NeévoDHA®, for pregnant women who are at high risk of developing pregnancy complications, including anemia and pre-eclampsia (160). Other medical foods that are available on the market with L-MTHF as the active ingredient include

Metanx®, Cerefolin®, or CerefolinNAC® among others (Pamlab, Inc.).

Although Metafolin® is advertised as a natural folate, it is actually derived from synthetic FA. The L-5-MTHF is generated through reduction and condensation reactions and finally crystallized as a calcium salt (L-5-MTHF-Ca) (161). Crystalline L-5-MTHF-Ca is stable

41 on storage for approximately 48 months at 40ºC when processed into vitamin and mineral tablets

(161). When Metafolin® is microencapsulated with ascorbate as an antioxidant, it can be used as a food fortificant (14, 161). As Metafolin® is derived from FA, it is found in its monoglutamylated state and therefore, demonstrates similar bioavailability (161, 162).

A panel of scientific experts in the U.S.A. has determined that L-5-MTHF-Ca is generally regarded as safe (GRAS) for its intended use, and may be lawfully used as a dietary ingredient in the U.S. (161). The European Food Safety Authority has also adopted this favourable opinion for

L-5-MTHF-Ca as a source of folate in foods and supplements (161, 164). Due to the lack of comparative data on the influence of FA versus L-5-MTHF on the progression of neuropathy caused by B12 deficiency, the EFSA has considered it prudent to apply the 1 mg/d UL established for FA on L-5-MTHF-Ca when consumed (161, 164).

2.4.2 The case for 5-MTHF supplementation

It has been purported that 5-MTHF is as, if not more effective than, FA in improving folate status and to confer safer means of providing supplemental levels of folate in comparison to FA. As the naturally-occurring and predominant circulating form of folate found in the blood

(112), 5-MTHF is in a completely reduced and methylated state, rendering it bioactive and ready to participate in intracellular one-carbon transfer reactions. In contrast, FA is oxidized and must first be reduced to THF and methylated through a series of reactions before entering one-carbon transfer reactions (9). In blood, 5-MTHF is also unlikely to be associated with the appearance of

UMFA, while FA intake levels as low as 200 µg have been shown to be associated with the appearance of UMFA in circulation (9). In addition, 5-MTHF is unlikely to mask vitamin B12 deficiency as it must participate in the B12-dependent remethylation of homocysteine to

42 methionine before it can enter the nucleotide biosynthesis pathway – the proposed mechanism by which FA can correct for vitamin B12 deficiency-associated anemia (9). Finally, 5-MTHF bypasses the need for metabolic enzymes such as DHFR and MTHFR, which are key enzymes for FA metabolism. As DHFR activity in humans is considerably lower, with higher person-to- person variability relative to rodents (65), and the MTHFR polymorphism, associated with lower activity, is highly prevalent in the North American population, 5-MTHF is potentially a more efficient and effective supplemental form than FA that would allow for continual SAM regeneration.

2.4.3 5-MTHF and B12

The associations between folate and vitamin B12 have been previously discussed in the folate one-carbon pathway in Section 2.1.3.3. Although several clinical studies have compared the effects of 5-MTHF versus FA on folate and homocysteine concentrations (13-18), there is not yet convincing evidence that 5-MTHF is a more advantageous supplemental form over FA.

However, one of the speculated benefits that could arise from 5-MTHF supplementation is during vitamin B12 deficiency. In these instances, a ‘methyl trap’ may occur (16), whereby 5-

MTHF accumulates due to a cease in the pathway, specifically at the B12-dependent, one-carbon transfer reaction of homocysteine to methionine (16). Therefore, during vitamin B12 deficiency,

5-MTHF may accumulate without utilization, thereby rendering it unable to correct for the associated anemic symptoms as 5-MTHF is incapable of entering the nucleotide biosynthesis pathway (1). Contrastingly, high FA intake may correct vitamin B12 deficiency-related anemia, presumably through replenishment of folates for nucleotide biosynthesis, and allow progression of adverse neurological outcomes, such as spinal cord damage (24).

43 Although the presence of neurological complications among patients in the absence of anemia raised concerns that FA may have an adverse effect on vitamin B12 deficiency, a population-based study found no evidence to support such concerns (166). Comparing the

NHANES prefortification data between 1991 and 1994 and postfortification data between 2001 and 2006, Qi et al. observed an unchanged prevalence of serum vitamin B12 deficiency without anemia or macrocytosis in older U.S. adults >50 y (166). Although the prevalence of serum vitamin B12 deficiency without anemia or macrocytosis is low (3.9% and 4.1%, respectively post-fortification) (166), it can be speculated that 5-MTHF supplementation may lower this prevalence even more.

In another study involving participants in the 1999-2002 U.S. NHANES, Morris et al. examined the relation between serum folate and vitamin B12 status and the association to anemia, macrocytosis, and cognitive impairment (67). Low vitamin B12 status, defined as concentrations

<148 pmol/L, in combination with high serum folate (>59 nmol/L) was associated with anemia and cognitive impairment, although when vitamin B12 status was normal, high serum folate was associated with a protective effect against cognitive impairment (67).

Several other studies have also established an inverse relationship between circulating folate concentrations and cognitive impairment and dementia, even after correcting for vitamin

B12 status (165, 167). These results suggest the need for not only normal vitamin B12 status, but also normal folate status in protecting against certain neurological complications.

Given these aforementioned studies, it is possible that 5-MTHF supplementation will not address the issue of masking vitamin B12 deficiency and that more emphasis should be placed on

B12 status in the North American population. However, as a benefit, supplementing 5-MTHF

44 would allow for more accurate diagnosis of B12 deficiency as it is unlikely to correct anemic symptoms.

2.4.4 MTHFR polymorphism

MTHFR polymorphisms are most frequently found in the form of MTHFR C677T and

MTHFR A1298C. The MTHFR C677T is a common mutation with an allele frequency of approximately 35% in the general North American population (168-169). This polymorphism is associated with reduced enzyme activity and increased thermolability (170). However, the prevalence of MTHFR C677T varies among racial and ethnic groups (89). To be specific, studies have found that Caucasian and Asian populations typically showed rates of approximately 12-

15% for homozygous individuals (TT), and up to 50% for heterozygous (CT) individuals (171,

172). In contrast, African-Americans exhibited very low incidence of the TT genotype (89).

A second common polymorphism of the MTHFR gene is the MTHFR A1298C mutation

(89). Similar to the C677T mutation, the A1298C polymorphism results in decreased MTHFR activity, but conversely, does not result in a thermolabile protein (173, 174). Allele frequencies of up to 33% have been reported with this polymorphism (173, 174). In particular, the prevalence of the homozygous CC mutation in North American subjects, including mainly Caucasians, was

7-12% (175). Preliminary evidence has suggested that under conditions of extreme folate depletion, MTHFR A1298C may become clinically relevant (89), although its effects on plasma homocysteine and folate level are inconsistent (89).

Such mutations in the MTHFR gene have been associated with low folate status and hyperhomocysteinemia (89). As such, MTHFR mutations have been connected, albeit inconsistently, to disorders related to folate deficiency and hyperhomocysteinemia, including

45 NTDs, cardiovascular disease, pregnancy outcomes, cancers, cognitive impairment, and psychiatric disorders, particularly in patients with low folate status and vitamin B12 concentrations (89).

2.4.5 Effects of 5-MTHF on folate metabolism

5-MTHF is the active and predominant circulating form of folate in the blood and in tissue stores (59). To date, there have been two studies conducted by Jing et al. that analyzed the effects of FA and 5-MTHF on intracellular mechanisms in in vivo models. Jing et al. compared

FA and 5-MTHF supplementation on Pcft and Rfc mRNA expressions in Shaver white laying hens (56, 59) (refer to Table 2.5). In the study, jejunal Pcft and Rfc mRNA expression, but not duodenal or cecal, was downregulated in response to 5-MTHF supplementation relative to the basal diet. By contrast, FA supplementation had no effect compared with the basal diet (56, 59).

In addition, Jing et al. observed no significant differences between equimolar doses of FA and 5-

MTHF on the mRNA expression of Pcft or Rfc in the intestine (56, 59). Thus, it appears that based on existing studies, intracellular mechanisms may be differentially regulated in response to

5-MTHF supplementation and FA.

2.5 Folic acid vs. 5-MTHF

There are a number of clinical studies that have compared the effects of FA supplementation with 5-MTHF. The majority of these studies focused on biomarkers of folate status and tHcy status. Two studies conducted by Venn et al. examined the effects of 100 µg/d

FA and 113 µg/d 5-MTHF supplementation on plasma and RBC folate concentrations in women of childbearing age and in healthy volunteers (13, 14) (Table 2.7). In these studies, the molar

46 equivalent dose of 5-MTHF supplementation provided for 24 weeks was at least as effective as

100 µg FA in increasing plasma folate concentrations and reducing plasma tHcy concentrations.

These results are consistent with other studies that have compared FA and 5-MTHF supplementation in cohorts of healthy women, healthy male volunteers, healthy pregnant women, and healthy men and women (15-18) (Table 2.7).

Another clinical study conducted by Houghton et al. specifically examined the effects of

5-MTHF versus FA supplementation on folate status in lactating women (17). In this study, pregnant women advised to consume FA-containing prenatal supplement (1000 µg/d) during pregnancy were enrolled at 36-week gestation (17). Upon delivery, women were randomized to receive 400 µg of FA, molar equivalent dose of 5-MTHF, or placebo for 16 weeks (17). At 16 weeks of lactation, RBC folate concentrations in women taking 5-MTHF were higher than those in women taking FA (17). It should also be noted that women in the FA group, relative to the 5-

MTHF group, had higher concentrations of 5-formylTHF, which can directly be used for nucleotide biosynthesis (17). In a 24-week, double-blind, placebo-controlled trial, Lamers et al. also compared the effects of 400 µg/d FA, 416 µg/d 5-MTHF, and 208 µg/d 5-MTHF supplementation on tHcy and plasma folate in healthy women aged 18-35 years (15). After 24 weeks of supplementation, equimolar doses of FA and 5-MTHF supplementation similarly lowered tHcy and raised plasma folate concentrations relative to the placebo group (15).

In a double-blind, crossover study, Pentieva et al. reported no significant differences in short-term bioavailabilities between the FA and 5-MTHF (16). Green et al. compared the effects of bread fortified with either 400 µg FA or microencapsulated equimolar L-MTHF (18).

Although both forms resulted in increased plasma and RBC folate concentrations relative to the

47 placebo, there were no significant differences in the magnitude of increase between the two folate vitamers (18) (Refer to Table 2.7).

Table 2.7: Summary of clinical trials comparing the effects of FA and 5-MTHF supplementation on biomarkers of folate status. Author Study Population Treatment Results

Venn et al. Women of 24-week treatment - Plasma folate and 2002 (13) childbearing age (18- FA groups increased 49 years) Placebo: gelatin for both 5-MTHF and n=104 capsules FA

FA group Placebo + 100 µg (227nmol)

5-MTHF group Placebo + 113 µg (227 nmol) Venn et al. Healthy men and 24-week treatment - 5-MTHF and FA 2003 (14) women (> 18 years) groups both had n=155 Placebo: gelatin significantly lower capsules plasma tHCy - 5-MTHF was more FA group effective than was FA Placebo + 100 µg in lowering tHcy (227nmol) Overall: Low-dose 5-MTHF group 5-MTHF is at least as Placebo + 113 µg effective as is FA in (227 nmol) reducing tHcy concentrations in healthy persons

Lamers et al. Female participants 24-week treatment - No significant 2004 (15) n=114 decrease between the Placebo: gelatin 3 supplemented capsules groups with a decrease in tHcy FA group - Increase in plasma Placebo + 400 µg folate in the group receiving 208 µg 5- 5-MTHF group MTHF was Placebo + 416 µg significantly lower

48 Placebo + 208 µg than 400 µg or 416 µg 5-MTHF groups

Pentieva et al. Males (18-45 years) 3 days of varying - Maximum plasma 2004 (16) n=21 treatment with 1- folate response week interval in (Rmax) did not differ between between FA and 5- MTHF treatments 1) Placebo: capsule Overall: Short-term bioavailabilities of 5- 2) FA treatment MTHF and FA are 500 µg equivalent

3) 5-MTHF treatment 500 µg De Meer et al. Young adults (18-30 5-week treatment - Absorption and 2005 (176) years) and middle initial elimination aged adults (≥ 50) FA group higher for FA n = 24 400 µg supplementation relative to 5-MTHF 5-MTHF group in young adults 454 µg - Lower FA absorption in middle- aged adults relative to young adults

Houghton et al. Healthy pregnant 16-week treatment - RBC folate 2006 (17) women < 36-week concentrations higher gestation Placebo: in 5-MTHF group multivitamin and relative to FA, 1 week after birth, mineral supplement + adjusted to baseline participants 0 µg folate - No difference in discontinue prenatal folate forms between supplements FA group 5-MTHF and placebo 400 µg/d - FA group had 16-week lactating (906 nmol/d) greater amounts of 5- women formylTHF n=72 5-MTHF group 416 µg/d (906 nmol/d) Green et al. Healthy men and 16-week treatment - RBC folate 2013 (18) women (18-45 years (consuming 1 wheat concentration was old) roll per day) higher in 5-MTHF n=45 and FA groups vs. Placebo: wheat rolls placebo No added folate - Plasma folate

49 concentration was FA group higher in 5-MTHF 400 µg/day and FA groups vs. placebo 5-MTHF group - No significant 452 µg/day differences in blood folate concentrations between 5-MTHF and FA group Overall: 5-MTHF and FA similarly increased blood folate concentrations

In another study conducted by de Meer et al., folate absorption and elimination were determined in young and middle-aged healthy individuals supplemented with FA or 5-MTHF for

5 weeks (176). Young adults showed increased folate turnover, or higher absorption and elimination, after FA supplementation compared with 5-MTHF supplementation (176).

However, the middle-aged adults in the same study showed no changes in absorption and initial folic acid elimination upon supplementation with either 5-MTHF or FA supplements (176). FA absorption was found to be 20% lower in middle-aged adults than young adults (176) (Refer to

Table 2.7).

With respect to subjects with the MTHFR C677T polymorphism, a few studies have been conducted to examine the effects of FA and 5-MTHF supplementation on folate biomarkers and tHcy (177, 178) (Refer to Table 2.8). Houghton et al. determined that the polymorphism did not affect blood folate indexes (17). Unlike previous studies, this study did not find that 5-MTHF or

FA affected plasma tHcy concentrations over time, however, baseline tHcy levels were already very low in both groups (177, 178). In addition, Litynski et al. (179) and Fohr et al. (158) compared the effect of low doses of 5-MTHF and FA supplementation on plasma tHcy concentrations in healthy subjects with or without the MTHFR C677T polymorphism. In both

50 studies, 5-MTHF showed comparable efficacy in reducing tHcy during chronic supplementation as FA, regardless of the MTHFR C677T polymorphism (158, 179). Fohr et al. further demonstrated that women who were homozygous for the MTHFR C677T mutation benefited the most from supplementation (158).

Table 2.8: Summary of clinical trials comparing the effects of FA and 5-MTHF supplementation on folate status indices in individuals with the MTHFR CàT polymorphism Author Study Treatment Results population Fohr et al. Non-pregnant 8-week treatment - FA supplementation significantly 2002 (158) healthy women decreased tHcy concentrations in all (19-39 years Placebo group genotype groups n=160) - 5-MTHF supplementation FA group significantly deceased tHcy Wild-type, 400 µg concentrations in heterozygous group homozygous, or - largest nonsignificant reduction heterozygous for 5-MTHF group occurred in women with the TT MTHFR Cà T 480 µg (racemic genotype after week 4 of 5-MTHF mixture) supplementation

Overall: 5-MTHF supplementation is at least as effective as FA in reducing tHcy concentrations in women with homozygous MTHFR C677T polymorphism Litynski et al. Males (n=32) and 7-week treatment - 5-MTHF and FA supplementation 2002 (179) females (n=8) decreased homocysteine levels in (19-69 years) Placebo: none wild-type subjects and in homozygous subjects Wild-type or FA group - 6 months after stopping treatment, homozygous, for 400 µg homocysteine levels remained lower MTHFR C à T than pretreatment levels, only in 5-MTHF group homozygous subjects treated with 5- 400 µg (racemic MTHF mixture) Overall: 5-MTHF supplementation showed comparable efficacy in reducing homocysteine as FA. 5- MTHF showed prolonged effects of lowering total homocysteine levels after 6 months of ceasing treatment unlike FA.

51 Prinz- Healthy females Single oral dose of - Maximum concentration and plasma Langenohl et with TT genotype FA (400 µg) and folate concentration significantly al. 2009 (163) (n = 16) or CC [6S]-5-MTHF higher for 5-MTHF compared to FA genotype (n = 8) (416 µg) in in both genotypes randomized - UMFA in plasma occurs regularly Wild-type or crossover design following FA supplementation but homozygous, for rarely in 5-MTHF MTHFR C à T Overall: 5-MTHF increases plasma folate more effectively than FA irrespective 677CàT mutation of the MTHFR

2.6 Summary and research gaps

Folate is an important B vitamin that is critical for human health, as it participates in one- carbon transfer reactions important for nucleotide biosynthesis and biological methylation reactions. Due to mandatory fortification and prevalent supplemental use, the intakes of folate and FA have significantly increased, resulting in dramatically increased blood levels of folate and appearance of UMFA in circulation in North America. An emerging body of evidence has suggested that high folate status, primarily from FA supplementation, may be associated with certain adverse health effects, including masking B12 deficiency, reducing natural killer cell activities, aberrant epigenetic changes, and cancer progression. In this regard, 5-MTHF, which is the predominant circulating and bioactive form of folate in the body, has been purported to confer a safer means of providing supplemental levels of folate over FA. However, there have been no studies that have examined the comparative effects of each folate vitamer on human health outcomes. Although clinical studies have shown comparable effects of FA and equimolar

5-MTHF on increasing folate status and lowering tHcy levels, there is a conspicuous lack of

52 studies that have compared the influence of these two folate vitamers on intracellular mechanisms involved in folate metabolism and one-carbon transfer reactions as well. The present study seeks to examine the mechanisms underlying 5-MTHF and FA and the manners in which they differ so as to gain insight and determine whether 5-MTHF may be used in the future as a safer and more effective source of folate, rather than the now prevalent FA.

53 Chapter 3: Rationale, Objectives, Hypothesis, and Significance

3.1 Rationale

FA fortification and supplementation have significantly increased intakes and blood levels of folate and FA in North America. Although the health benefits of FA supplementation on the prevention of NTDs and megaloblastic anemia are well established, an emerging body of evidence suggests that high folate status, primarily from high FA supplementation, may be associated with certain adverse health outcomes. Therefore, many have speculated that 5-MTHF would confer safer means of providing supplemental levels of folate over FA. As the predominant circulating and bioactive form of folate, 5-MTHF is unlikely to mask vitamin B12 deficiency and present UMFA in the circulation, whereas FA supplementation has been linked to these outcomes.

The UL for FA was arbitrarily set by the Institute of Medicine at 1 mg/day to prevent the masking of vitamin B12 deficiency. It is extremely difficult to achieve high folate intake from natural food sources alone; hence, no UL has been established for natural folates. Recently, 5-

MTHF supplement has been available on the market as a calcium salt (L-5-MTHF-Ca) at similar doses to FA. The European Food Safety Authority (EFSA) has evaluated calcium L-MTHF as an alternative for FA, and has determined that L-5-MTHF-Ca is generally recognized as safe

(GRAF) (161). Nevertheless, the EFSA recommended that the safety and efficacy of L-5-MTHF-

Ca be more clearly elucidated in future studies.

In order to establish 5-MTHF as a safer alternative form of providing supplemental folate its effects on intracellular folate metabolism and one-carbon transfer reactions involved in nucleotide biosynthesis and biological methylation reactions need to be compared with those of

54 FA supplementation. In this regard, it is unknown whether the purported adverse health outcomes associated with FA supplementation are specific to high FA intake or are related to high folate status in general. If the latter is the case, then 5-MTHF may not be any safer than FA.

Furthermore, because 5-MTHF can enter the methionine cycle more readily than does FA, 5-

MTHF, may have more profound effects on biological methylation reactions than does FA.

Clinical studies that have compared the two folate vitamers to date have only examined their effects on serum/plasma and RBC folate and plasma Hcy concentrations. Very few studies have investigated the effects of equimolar doses of FA and 5-MTHF supplementation on biochemical outcomes or human health outcomes. Therefore, studies are warranted to determine potential differential effects of FA vs. 5-MTHF on nucleotide biosynthesis and biological methylation reactions, which may result in profound consequences.

3.2 Objectives

The overarching objective of this study is to compare the effects of FA and 5-MTHF supplementation on the intracellular folate metabolic pathway and one-carbon transfer reactions in an in vivo model.

Specific aims of this study are:

1) To compare the effects of FA and 5-MTHF supplementation on plasma and tissue folate concentrations;

2) To compare the effects of FA and 5-MTHF supplementation on gene expression of folate transporters, carriers, receptors, and enzymes involved in the folate metabolic pathway and one- carbon transfer reactions; and

3) To compare the effects of FA and 5-MTHF supplementation on global DNA methylation.

55 3.3 Hypothesis

FA and 5-MTHF supplementation will elicit differential effects on folate metabolism and one- carbon transfer reactions.

3.4 Expected Outcomes

1) 5-MTHF supplementation will increase circulating and tissue folate concentrations to a greater extent than FA supplementation;

2) 5-MTHF supplementation will downregulate processes involved in folate absorption and intracellular uptake and retention while favouring processes involved in folate export relative to

FA supplementation;

3) FA supplementation will favour processes involved in nucleotide biosynthesis; and

4) 5-MTHF supplementation will favour processes involved in DNA methylation and will result in higher DNA methylation than FA supplementation.

3.5 Significance

Clinical studies have demonstrated that 5-MTHF supplementation is at least as effective in improving folate status as FA supplementation. Additionally, 5-MTHF may possess biochemical advantages over FA with a lower risk of adverse health effects. Therefore, 5-MTHF has been purported to confer safer means of providing supplemental levels of folate. Recently, 5-

MTHF has been commercially available as a calcium salt and has been promoted as a safer and better alternative to FA for mostly unproven health benefits. To date, however, no studies have comprehensively compared the biochemical and molecular effects between 5-MTHF and FA supplementation on intracellular folate metabolism and one-carbon transfer reactions. This is an

56 important public health issue as a safe and effective means of providing supplemental levels of folate is needed to prevent certain adverse health outcomes in those at risk (e.g., NTDs). Data generated from this study will provide a framework for future clinical and intervention studies that investigate the efficacy and safety of 5-MTHF supplementation on human health.

Specifically, findings from this study may have diverse and widespread implications in the treatments for diseases, such as coronary artery disease (180) and hyperhomocysteinemia (29).

57 Chapter 4: Comparative Studies on the Biochemical and Molecular Effects of FA vs 5-MTHF Supplementation in a Mouse Model

4.2 Introduction

Folate and its synthetic form, folic acid (FA), are critical to human health as they mediate one-carbon transfer reactions involved in nucleotide biosynthesis and biological methylation reactions (1). Intakes and blood levels of folate and FA have significantly increased in the North

American population due primarily to prevalent supplemental use of FA and partly to mandatory

FA fortification (4, 5). Although the beneficial effects of FA supplementation on NTD prevention are well established, an emerging body of evidence has suggested that high FA intake may increase the risk of several adverse health outcomes (6), including cancer progression, masking of vitamin B12 deficiency, decreased NK cytotoxicity, and epigenetic modifications

(24). In addition, FA intake in excess of 200 µg has been shown to saturate the enzyme dihydrofolate reductase (DHFR), wherein unmetabolized FA (UMFA) enters into the circulation

(11). UMFA has been purported to be the main culprit for certain negative health outcomes that high FA intake has been associated with. UMFA may either potentially disrupt or cause shifts in folate metabolic pathways by competing with appropriate substrates for folate-related enzymes, or alter expression of genes involved in folate absorption, metabolism, and one-carbon transfer reactions (1, 60).

As high FA intakes and blood levels have been associated with certain adverse health outcomes, many have advocated for the use of 5-MTHF in supplements. 5-MTHF refers to the predominant circulating and bioactive form of folate in the body. In contrast to FA, 5-MTHF is in its fully reduced and is methylated (1), thereby bypassing FA biotransformation and

58 metabolism. Thus, studies have shown that 5-MTHF intake may exhibit a number of advantages over that of supplemental FA (65, 188). First, the intake of 5-MTHF may be correlated with a decreased interaction with drugs that inhibit DHFR when compared to folic acid, making it a safer alternative (188). Second, the use of 5-MTHF has the suggested potential to prevent masking of haematological symptoms associated with vitamin B12 deficiency (188). Finally, direct supplementation with 5-MTHF reduces the adverse potential of unconverted folic acid remaining in peripheral circulation (65).

At present, studies that have examined the comparative effects of FA and 5-MTHF supplementation on intracellular folate metabolism and one-carbon transfer reactions are lacking.

Jing et al. found that 5-MTHF and FA supplementation was associated with decreased expression of Pcft and Rfc in the jejunum of laying hens to a similar extent (56, 59). Clinical studies that compared the two folate vitamers have demonstrated that 5-MTHF supplementation is at least as effective as FA in increasing folate concentrations and lowering Hcy concentrations

(13-18). Previous animal studies have also observed that FA supplementation and high folate status could potentially modulate mRNA and protein expression levels of enzymes and proteins involved in the folate metabolic pathway (65, 66). However, no studies to date have comprehensively examined potential differential effects of 5-MTHF and FA supplementation on gene expression of enzymes and proteins involved in folate absorption, intracellular uptake, retention and export and one carbon transfer reactions involved in nucleotide biosynthesis, methionine cycle, and biological methylations reactions.

The purported beneficial effects and safety of 5-MTHF over FA supplementation have not been unequivocally demonstrated. Nevertheless, 5-MTHF has been suggested as a safer and perhaps more effective form of providing supplemental levels of folate compared with FA.

59 Optimal folate status via supplementation is an important issue during pregnancy and lactation as well as in the prevention and treatment of certain medical conditions. As such, determining the optimal form of folate supplementation is an important public health and nutrition issue in addition to the dose and timing of supplementation. In addition to studies comparing clinical outcomes of 5-MTHF vs. FA supplementation, it is imperative to elucidate biochemical and molecular differences that 5-MTHF and FA supplementation may have on intracellular folate metabolism and one-carbon transfer reactions before any public health recommendation can be made. Given these considerations, the aim of this study was to compare the potential effects of

FA and 5-MTHF supplementation on downstream folic metabolic pathways in order to provide better insight into the pharmacological and clinical implications of folate supplementation.

4.3 Methods and Materials

4.3.1 Animals and Dietary Intervention

This study was carried out in strict accordance with the Regulation of the Animals for

Research Act in Ontario and the Guidelines of the Canadian Council on Animal Care. The study protocol was approved by the Animal Care Committee of St. Michael’s Hospital (protocol #621,

Toronto, ON). Post-weaning (3-week old) male C57BL/6 mice (n = 66) were purchased from

Charles River Laboratories (St. Constant, QC). C57BL/6 mice are an inbred strain that are widely used as models for human disease. Due to the consistency of their genetic background, significant effects can be attributed to dietary intervention.

Sample size was calculated using a significance level of 0.05 and a power of 80%.

Considering this study is the first study to compare the effects FA and 5-MTHF supplementation in a mouse model, effect size and variance was not available. Therefore, we resorted to the effect

60 sizes proposed by Cohen who suggested a ‘medium’ effect size was one which was equal to 0.5 x standard deviation. An 80% probability of obtaining a significant effect for folate, given that the difference between FA and 5-MTHF is large, we required a sample size of 54 (n = 9/group).

Upon arrival, mice were caged in groups of 3 or 4 and placed on amino-acid defined diets

(Dyets, Bethlehem, PA) containing 2 mg FA/kg diet (the control diet containing the basal dietary requirement [BDR] of folate for rodents) for one week after which mice cages were randomized to receive the same amino-acid defined diets containing 2 (control), 10 (5X BDR), or 20 (10X

BDR) mg FA/kg diet or their corresponding molar equivalent doses of 5-MTHF for 10 weeks.

RBC turnover in rodents is approximately 48-50 days and therefore, 10 weeks can be considered chronic supplementation. Water and diets were provided ad libitum. Diets were replaced every other day to ensure palatability and minimal oxidation of the 5-MTHF in the diet. Animal health was monitored daily and body weights were recorded weekly.

For convenience purposes, dietary groups will be referred to as set abbreviated names

(Refer to Table 4.1). 2 mg FA/kg diet refers to the control diet (Control); 10 mg FA/kg diet refers to the moderate supplemented FA diet (MFA); and 20 mg FA/kg diet refers to the high supplemented FA diet (HFA); 2 mg 5-MTHF/kg diet refers to the control 5-MTHF diet (C5-

MTHF); 10 mg 5-MTHF/kg diet refers to the moderate supplemented 5-MTHF diet (M5-

MTHF); and 20 mg 5-MTHF/kg diet refers to the high supplemented 5-MTHF diet (H5-MTHF).

Table 4.1: Abbreviations for dietary groups Diet Abbreviation

2 mg FA/kg diet Control 10 mg FA/kg diet MFA 20 mg FA/kg diet HFA 2 mg 5-MTHF/kg diet C5-MTHF 10 mg 5-MTHF/kg diet M5-MTHF 20 mg 5-MTHF/kg diet H5-MTHF

4.3.2 Experimental Diets

The aforementioned diets containing varying amounts of FA (Dyets, Bethlehem, PA) constitute a well-established method of providing supplemental levels of folate in a predictable manner in rodents. These levels of folate in diets have been extensively used in previous FA intervention studies within rodent models (91, 93, 112, 113, 181, 183). The detailed composition of the diet is shown in Table 4.2, Table 4.3, and Table 4.4. These diets were consistent in their components and only differed in their supplemented doses of FA and 5-MTHF. The control diet, which provides 2 mg FA/kg diet, is generally accepted as the BDR for rodents (182, 183). This level of FA relative to its caloric content closely approximates the recommended dietary allowance (RDA) of 0.4 mg dietary folate equivalent (DFE) per day in humans consuming an average of 2000 kcal per day (182). The 10 mg FA/kg diet represents FA supplementation at 5X

BDR. Folate intake levels at 5X the RDA (2.0 mg/day) can be commonly found in individuals, particularly in women, in the North American population taking daily multivitamin supplements and consuming high levels of fortified food sources. The 20 mg FA/kg diet represents FA supplementation at 10X BDR. Folate intake levels at 10X RDA (4.0 mg/day) are not commonly observed in the general North American population except in certain medical conditions in which high supplemental levels of FA are given to reduce or prevent the adverse effects of antifolates and in a subgroup of women at reproductive age at high risk of developing NTDs.

Because of inherent differences in folate metabolism between humans and rodents, the selected supplemental levels of FA may not accurately reflect the corresponding levels in humans (11).

FA biotransformation is significantly more efficient in rodents than in humans due to comparatively high DHFR activity (11). Consequently, a higher dose of FA is likely required to

62 elicit similar physiological effects of FA in rodents relative to humans. Therefore, the selected supplemental levels of FA in mice in the present study likely achieved much lower plasma concentrations of UMFA than would have been achieved by the equivalent supplemental levels of FA in humans.

Equimolar dose of 5-MTHF (0.416 g, 2.082 g, and 4.16 g of 5-MTHF Ca+ salt

[Metafolin®, Merck Eprova MW 497.5 g/mol]) for each level of FA was supplemented per kg of diet to generate 5-MTHF diets.

Table 4.2: Nutrient compositions of experimental L-amino acid defined diets for FA

2 mg FA/kg 10 mg FA/kg 20 mg FA/kg Nutrient (g/kg of diet) Cat # 517774 Cat # 517911 Cat # 517801 (control) (5X BDR) (10X BDR)

L-Alanine 3.5 3.5 3.5 L-Arginine free base 11.2 11.2 11.2 L-Asparagine.H20 6 6 6 L-Aspartic Acid 3.5 3.5 3.5 L-Cystine 3.5 3.5 3.5 L-Glutamic Acid 35.0 35.0 35.0 Glycine 23.3 23.3 23.3 L-Histidine free base 3.3 3.3 3.3 L-Isoleucine 8.2 8.2 8.2 L-Leucine 11.1 11.1 11.1 L-Lysine HCl 14.4 14.4 14.4 L-Methionine 8.2 8.2 8.2 L-Phenylalanine 11.6 11.6 11.6 L-Proline 3.5 3.5 3.5 L-Serine 3.5 3.5 3.5 L-Threonine 8.2 8.2 8.2 L-Tryptophan 1.74 1.74 1.74 L-Tyrosine 3.5 3.5 3.5 L-Valine 8.2 8.2 8.2 Total L-amino acid 171.44 171.44 171.44 Dextrin 407 407 407 Sucrose 194.6 193 191

63 Cellulose 50 50 50 Corn Oil (w/0.015% BHT) 100 100 100 Salt Mix #210006 57.96 57.96 57.96 Vitamin Mix #317756 (no Folate) 10 10 10 Choline Chloride 2 2 2 Sodium Bicarbonate 6.6 6.6 6.6 Folic Acid/ sucrose premix 5mg/g 0.4 2 4 Total 1000.000 1000.000 1000.000

Table 4.3: Nutrient compositions of experimental L-amino acid defined diets for 5-MTHF 2 mg 10 mg 20 mg 5-MTHF/kg 5-MTHF/kg 5-MTHF/kg Nutrient (g/kg of diet) Cat # 517774 Cat # 517911 Cat # 517801 (control) (5X BDR) (10X BDR) L-Alanine 3.5 3.5 3.5 L-Arginine free base 11.2 11.2 11.2 L-Asparagine.H20 6 6 6 L-Aspartic Acid 3.5 3.5 3.5 L-Cystine 3.5 3.5 3.5 L-Glutamic Acid 35.0 35.0 35.0 Glycine 23.3 23.3 23.3 L-Histidine free base 3.3 3.3 3.3 L-Isoleucine 8.2 8.2 8.2 L-Leucine 11.1 11.1 11.1 L-Lysine HCl 14.4 14.4 14.4 L-Methionine 8.2 8.2 8.2 L-Phenylalanine 11.6 11.6 11.6 L-Proline 3.5 3.5 3.5 L-Serine 3.5 3.5 3.5 L-Threonine 8.2 8.2 8.2 L-Tryptophan 1.74 1.74 1.74 L-Tyrosine 3.5 3.5 3.5 L-Valine 8.2 8.2 8.2 Total L-amino acid 171.44 171.44 171.44 Dextrin 407 407 407 Sucrose 194.584 192.918 190.84 Cellulose 50 50 50 Corn Oil (w/0.015% BHT) 100 100 100 Salt Mix #210006 57.96 57.96 57.96 Vitamin Mix #317756 (no Folate) 10 10 10

64 Choline Chloride 2 2 2 Sodium Bicarbonate 6.6 6.6 6.6 5-MTHF in sucrose 5mg/g 0.416 2.082 4.16 Total 1000.000 1000.000 1000.000

65 Table 4.4: Salt mix and vitamin mix compositions of experimental L-amino acid defined diets Salt Mix #210006 Ingredients (g/kg of diet) Calcium carbonate 14.60000 Calcium phosphate, dibasic 0.17000 Sodium chloride 12.37000 Potassium phosphate, dibasic 17.16000 Magnesium sulfate, anhydrous 2.45000 Magnesium sulfate, monohydrate 0.18000 Ferric citrate 0.62000 Zinc carbonate 0.05400 Cupric carbonate 0.05400 Potassium iodide 0.00058 Sodium selenite 0.00058 Chromium potassium sulfate 0.01900 Sodium fluoride 0.00230 Molybdic acid, ammonium salt 0.00120 Sucrose 10.27534 Vitamin Mix #317756 Ingredients (g/kg of diet) Thiamin HCl 0.006 Riboflavin 0.006 Pyridoxine HCl 0.007 Nicotinic acid 0.030 Calcium pantothenate 0.016 Cyanocobalamin 0.00005 Vitamin A palmitate (500 000 IU/g) 0.008 Vitamin D3 (400 000 IU/g) 0.0025 Vitamin E acetate (500 IU/g) 0.100 Menadioine sodium bisulfate 0.00080 Biotin 0.00002 Sucrose 9.82363

66 4.3.3 Sample collection

Mice were sacrificed after 10 weeks of dietary intervention by 5% isoflurane inhalation followed by cardiac puncture and cervical dislocation. Mice were fasted for 1 hour prior to sacrifice by removing dietary pellets from cages in order to minimize variation in plasma folate concentrations. Blood was collected by cardiac puncture and stored in EDTA-treated vacutainer tubes (BD Biosciences). Tubes were then placed in a cup on ice while being protected from light.

Plasma was collected by immediately centrifuging blood at 2000 x g for 15 minutes at 4°C.

Plasma was then stored at -80°C with 5% ascorbic acid for plasma folate analysis. Livers and proximal small intestines were harvested at necropsy and immediately snap frozen in liquid nitrogen and subsequently stored at -80°C for tissue folate analysis, RNA extractions, and global

DNA methylation analysis.

Liver samples were cut into four equal segments prior to flash freezing in liquid nitrogen.

Small intestine samples were spread across to allow eight equal segments to be cut: the first two segments were deemed to make up the duodenum; the middle three segments made up the jejunum; and the last three segments made up the ileum. The first segments of the small intestines were considered the proximal duodenum and stored for RNA extractions for gene expression analyses. The second segments were considered the distal duodenum and stored for small intestinal folate concentrations. The third segments were considered the proximal jejunum and stored for DNA extraction for global DNA methylation analyses.

4.3.4 Determination of plasma, hepatic, and small intestinal folate concentrations

Plasma, hepatic, and small intestinal folate concentrations were determined by the standard microbiological microtitre plate assay using Lactobacillus rhamnosus (formerly known

67 as Lactobacillus casei) (184). This assay is considered the “gold standard” for determining folate concentrations (184). L. rhamnosus is unable to synthesize folate and is therefore dependent on external sources of folate for growth. Within certain ranges of folate concentrations, bacterial growth is proportional to folate levels found in the medium, which allows for folate concentrations of unknown samples to be determined (184). The microbiological microtitre plate assay measures total folate content in a given sample as L. rhamnosus effectively uses all folate vitamers for growth, although the affinity for folates decreases with elongation of the polyglutamate chain.

Folic acid standard preparation

10 mg of FA was dissolved in 10 mL of double-distilled water and 5 µL of 10M NaOH for a final concentration of 1 mg FA/mL. The solution was then adjusted by titration with HCl to a pH of 7-8 and verified using spectrophotometry (282 nm). The solution was finally diluted to

50 µL/mL using methanol to generate the completed standard and stored at -80°C for use.

Lactobacillus rhamnosus stock preparation

L. rhamnosus ATCC 7649 stock was incubated with Lactobacillus MRS Broth (Difco™,

BD Biosciences) (200 µL in 200 mL) for 18 hours at 37°C. Cells were centrifuged and supernatant was discarded under aseptic conditions. Cell pellets were then resuspended in 180 mL of Lactobacillus MRS Broth and 20 mL of cold, autoclaved 100% glycerol. The solution was thoroughly mixed and aliquots were stored at -80°C for use.

68 Chicken pancreas conjugase preparation

Chicken pancreas acetone (Difco™, BD Biosciences) was first dissolved in 0.1M KPO4 buffer (1.05 g KH2PO4, 0.4 g K2HPO4, 0.1 g C6H7NaO6 and 100 mL double distilled water) and incubated under a blanket of toluene for 6 hours at 37°C. After the toluene was removed, the solution was then centrifuged at 10,000 g for 15 minutes, upon which, the supernatant was collected and tricalcium phosphate (BioRad Gel HTP was rehydrated: 1 part HTP to 6 parts

0.1M KPO4 buffer per 10 g HTP) was added at an equal volume. The solution was thoroughly mixed and centrifuged at 10,000 g for 30 minutes at 4°C and the supernatant was collected. An equal volume of 95% ethanol was added, mixed and stored overnight at -20°C.

The overnight solution was centrifuged at 10,000 g for 30 minutes at room temperature, and supernatant was resuspended in 50 mL of cold 1.0M KPO4 buffer. 10 g of Dowex-1 (BioRad

AGI-X*) was added and the solution was mixed for 1 hour at 4°C. Finally, the solution was filtered at 4°C and aliquots were stored at -80°C for later use.

Tissue folate extraction

Liver and small intestine segments were used for the determination of liver and small intestinal folate concentrations. Liver and small intestine samples were weighed, and 1 or 0.25 mL of extraction buffer (0.1 M C₆H₇NaO₆, 0.1M Bis-Tris, and 5mM β-mercaptoethanol) per 0.1 g of tissue were added to each sample, respectively. Samples were boiled for 15-20 minutes and later cooled on ice. Mixtures were then homogenized and centrifuged at 5000 rpm for 20 minutes. After centrifugation, the supernatant was collected and stored at -80°C. Tissue folate extracts were incubated with chicken pancreas and 0.1M KPO4 buffer at a ratio of 4:1:15 for liver samples, and 4:1:5 for small intestine samples. Samples were thoroughly mixed and

69 incubated at 37°C for 2 hours, and later diluted using 0.1M KPO4 buffer. Aliquots were stored at

-80°C for later use.

Determination of plasma and hepatic folate concentrations

1) L. rhamnosus Inoculation

3 µL of L. rhamnosus stock was first inoculated into 3 mL of Lactobacillus MRS Broth and incubated in a 37°C shaker at 250 rpm for 16-18 hours. After incubation, 500 µL of the inoculum was added to 2.5 mL of Lactobacillus MRS Broth and further incubated in a 37°C shaker at 250 rpm for approximately 5 hours. Bacterial growth for the cultured inoculum was determined via optimal density measurements at 650 nm using a spectrophotometer.

2) Loading Phosphate Buffer and FA Standard

During the aforementioned 5-hour incubation time, 150 µL of freshly-prepared KPO4 buffer was added to each well of a sterile 96-well flat-bottom plate. An eight-point standard curve was then generated by adding 150 µL of previously prepared FA standard (diluted to 2 ng/mL) to each of the starting wells (in duplicate) and further 2-fold serially diluted.

3) Loading Plasma and Tissue Samples

Folate concentrations of each sample were assessed in duplicate by adding 5 µL of prepared tissue folate extracts to designated starting wells and adjusted to 300 µL by adding KPO4 buffer.

Samples were then 2-fold serially diluted across 3 (liver extracts) or 7 (small intestine extracts) additional wells resulting in four or eight measurements, respectively.

4) Preparation of L. rhamnosus Culture

After incubation of the L. rhamnosus culture and further confirmation for optical density, the culture was centrifuged for 5 minutes at 5000 rpm. Cell pellets were resuspended in 3 mL of

70 freshly-prepared folic acid casei media (9.4 g Folic Acid Casei Media [Difco™, BD

Biosciences], 0.05 g C6H7NaO6, and 100 mL ddH2O; boiled and filtered) to remove folate remnants from the Lactobaacillus MRS Broth. The culture was washed another 3 times using freshly-prepared folic acid casei media. Step-wise dilution was done using folic acid casei medium to achieve a final 1000X cumulative dilution factor.

5) Loading L. rhamnosus and Analysis

150 µL of this diluted culture was then added to each well of the 96-well plate. The plate was sealed using a Mylar film and incubated for 16-18 hours at 37°C for bacterial growth.

Optical density was determined using a spectrophotometer at 650 nm wavelength. Folate concentrations from samples were estimated using the standard curve of folate concentrations plotted against densities using SoftMax® Pro V5.4.1 software (Molecular Devices, CA).

4.3.5 Gene expression analysis by quantitative real-time reverse transcriptase PCR

Total RNA was extracted from snap-frozen liver and small intestine samples using the

RNeasy® Microarray Tissue Mini Kit (Qiagen) according to the manufacturer’s instructions.

Briefly, the RNeasy® Microarray Tissue Mini kit protocol initially involves homogenizing tissue samples in a phenol/guanidine-based lysis buffer with subsequent guanidine/phenol/chloroform- based RNA extraction. RNA is then recovered using a spin-column, which uses a RNA-binding specific silica membrane. The RNase-free DNase set (Qiagen) was also used directly on spin- column membranes to digest DNA and prevent DNA contamination within samples. After thorough washing steps, RNA was eluted with RNase-free water.

RNA purity was assessed using spectrophotometric measurements from the NanoDrop

2000 (Thermo Scientific) instrument. Pertinent absorbance values and absorbance ratios

71 (A260/A280 and A260/A230) were specifically used to determine RNA purity. A260/A280 ratios of approximately 2.0 are generally accepted as “pure” RNAs, as values appreciably lower may indicate the presence of proteins, phenols, or other contaminants that absorb near 280 nm.

A260/A230 ratios are a general indicator of nucleic acid purity and are expected to be approximately in the range of 2.0-2.2. Although initially the Nanodrop 2000 (Thermo Scientific) was used to assess RNA purity and concentration, the Agilent RNA 6000 Nano Kit and 2100

Bioanalyzer Instrument are considered the industry standard techniques to assess RNA quality and quantitation and hence, were used to further assess sample RNA Integrity Numbers (RIN) using the 2100 Expert Software (Agilent Technologies) according to the manufacturer’s protocols. Only RNA samples with an RNA integrity number (RIN) of 8 or greater were used for gene expression analyses.

Primers were synthesized by the Integrated DNA Technologies, Inc. (Coralville, Iowa,

USA). Reference sequences for primer were searched for each gene of interest in mice.

Reference sequences were then checked on a GenBank through the National Center for

Biotechnology Information (NCBI). Primers for qPCR were designed by Integrated DNA

Technologies, Inc. using the PrimerQuest tool. A list of primer sequences used are shown in

Table 4.5.

cDNA was prepared using the QuantiTect® Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol. cDNA’s were subsequently diluted to a final concentration of 10 ng/µL for further use. Real-time quantitative PCR was performed in triplicate on MicroAmp Optical 384-well plates (Applied Biosystems) using the ViiA7™ Real-

Time PCR System (Applied Biosystems, Life Technologies) according to the manufacturer’s protocol. PCR reactions were conducted in a 10 µL volume including Fast SYBR® Green PCR

72 Master Mix (Applied Biosystems, Life Technologies), 2 µL cDNA template, and 0.5 µL of optimized 50 µM primers. Cycling conditions during PCR included an initial stage of 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles involving 15 seconds of denaturation at

95°C and 1 minute of primer annealing and fragment elongation at 60°C. All samples were run in triplicates per plate and relative expression of genes of interest was calculated using delta Ct values adjusted to an endogenous control, Rpl13a (liver housekeeping gene) or GUSB (small intestine housekeeping gene), and normalized to an internal calibrator (2 mg FA - control).

Table 4.5: qRT-PCR Primer sequences Primer Sense (Primer 1) Anti-Sense (Primer 2) Rpl13a ATG TCC CCT CTA CCC ACA G TGA ACC CAA TAA AGA CTG TTT GC (HK) Gusb ACC ACA CCC AGC CAA TAA AG AGC AAT GGT ACC GGC AG (HK) Pcft GGT AGA TGG AGT TGA AGA TGC C TTC CTG TCA TTG GTC ACG AC

Rfc CCT GCT TTT CTG CCA CCT AAT CCA TTC TAT GCG TGC CT

Fr-α GGG TGT GCT ACG TGA CCT TT ACG GAA CTG ATC ACG GAC TT

Fpgs TGA AGT TCT GCT GGT CTG C TGC TCT TCA ACT CTA CTG GTG

Ggh CTC CAC TAA CCA GGA CAG AAA G AGA TTC AGG TTA TTC CCG AGT G

Dhfr ATT CCT GCA TGA TCC TTG TCA GAC ATG GTT TGG ATA GTC GGA

Shmt ATT CTG TAG TCA TGG CTT GCT ACT ATG AAC TGG AGT CCC TCA

Mthfr AGA GCT GAG TGA TAA TGA AGT CG CCG AGT TTG CTG ACT ATT TTG AC

Tyms ATC CCA GAT TCT CAC TCC CT TCC TCT GCT CAC AAC CAA AC

Mtr TGC ATC TAG GAC GTG GAT CA GGA AAT GGA GAG GTT GGC TAT

Dnmt1 TTC CGC CTC AAT GAT AGC TC GTG GAG AAA CTG GAA GAG GTA AC

Dnmt3a CTC TTC CAC AGC ATT CAT TAC TG CCC CTA CTA CAT CAG CAA ACG

Dnmt3b TCA GAA GCA GCA GAG TCA TTG CAA ACC CAA CAA GAA GCA ACC

73 Results were analyzed using the cycler threshold (Ct) method and normalized relative to the quantification levels of the housekeeping gene RPL32. Fold change was calculated using the following formula:

-∆∆ C 2 t

∆∆ Ct =∆Ct experimental - ∆Ct control

∆Ct experimental = Ct target - Ct housekeeping

∆Ct control = Ct calibrator (Day 0 50 nM FA) - Ct housekeeping

Fold change was then compared between molar equivalent treatments (FA vs. 5-MTHF) and within folate type (FA or 5-MTHF).

4.3.6 Global DNA Methylation Analysis

Genomic DNA Isolation:

Total genomic DNA was extracted from the proximal jejunum and liver segment using the Qiagen DNeasy Blood and Tissue kit and Qiagen RNase A (Qiagen Inc., Mississauga, ON,

Canada). The final preparation had a ratio of A260:A280 of approximately 1.8, indicative of high quality DNA free of RNA and protein contamination. Concentrations of each DNA sample were then determined through spectrophotometric readings. DNA samples were stored at -20ºC until further analysis.

Global DNA Methylation:

DNA CpG site methylation was determined by in vitro methyl acceptance assay by utilizing [3H-methyl] SAM (New England Nuclear, Boston, MA) as a methyl donor and a prokaryotic CpG DNMT, SssI (New England Biolabs, Beverly, MA), as previously described

74 (185, 186). Extracted DNA was incubated with buffer, SssI, and [3H-methyl] SAM for 1 hour at

30ºC. DNA solutions were then pipetted onto Whatman DAEA 81 filter papers and subsequently dried under lamps. Dried filter papers were washed twice with washing buffer to clean excess reagents and leaving radio-labelled DNA on filter paper.

Upon completion, filter papers were put into 10 mL scintillation cocktail to resuspend

DNA in solution. Radioactivity of solution was measured, which are reported as DPM

(disintegration per minute) per 10 ng of DNA. The manner in which this assay is performed results in an inverse relationship between endogenous methylation status and exogenous [3H- methyl] incorporation. Therefore, higher exogenous radioactive incorporation is characteristic of lower endogenous DNA methylation status. Analyses were performed in duplicate for each sample.

4.3.7 Statistical Analysis

The distribution of each variable was assessed using the D’Agostino and Pearson

Omnibus K2 normality test. The D’Agostino and Pearson Omnibus K2 normality test assesses normality (187).

Statistical significance of the effects of form and dose on growth curves (body weight in g) between dietary groups were assessed using repeated measures Two-Way ANOVA (mixed- model Two-Way ANOVA) followed by Tukey’s multiple comparisons test to detect differences between dietary groups at each week of dietary intervention.

Statistical significance for the effects of form and dose on plasma folate concentration, hepatic folate concentration, small intestinal folate concentration, relative gene expression, and global DNA methylation were assessed using Two-Way ANOVA. As an observational study,

75 parametric tests were used over non-parametric tests to assess gene expression data as observing potential associations were the primary focus of this project. Gene expression data were collected for 6 samples per dietary group for liver, and 8 samples per dietary group for small intestine.

Statistical significance for the effects of each independent variable, form and dose, were determined by analyzing ΔCT levels (CT value of the gene of interest – CT value of housekeeping gene) using Two-Way ANOVA.

To adjust for increased false discovery rates due to multiple statistical analyses, significance levels for folate concentrations, relative gene expression, and global DNA methylation were corrected by the Benjamini-Hochberg procedure. Adjusting this rate helps to control for the likelihood of an outcome occurring by chance, and thus avoids Type I errors. The

Benjamini-Hochberg Procedure is calculated by the formula (i/m)Q, where i represents the rank in p-value, m refers to the total number of tests, and Q represents the false discovery rate, which is considered our alpha value (0.05).

Statistical tests were conducted using GraphPad Prism Version 7 software (La Jolla, CA).

All statistical tests were two-sided and all statistics were considered significant if observed significance level (p-value) were less than the adjusted significance levels calculated by the

Benjamini-Hochberg procedure. Post-hoc analysis for dose was done using Bonferroni’s correction to assess significance between supplemental doses. Results are expressed as mean ±

SD unless stated otherwise.

76 4.4 Results

4.4.1 Animal health and body weight

C57BL/6 animal health was assessed daily, and animals remained in general good health throughout the study. However, it should be noted that a mouse in the M-MTHF group and two mice in the H-MTHF group developed issues with low body weights, potentially attributed to fighting and bullying among caged groups. Body weights and growth curves in this study were slightly higher than the standard growth curves of C57BL/6 mice provided by the mouse vendor

(Charles River Laboratories).

Growth curves were not significantly different between any of the 6 dietary groups throughout the study (p = 0.58, n = 11/dietary group) (Figure 4.1). However, body weights differed significantly (p < 0.05) between certain dietary groups in the second and third week of dietary intervention. Specifically, in the second week of intervention, mice fed the HFA diet had lower body weights than mice fed the M5-MTHF diets (p < 0.05), and in the third week of intervention, mice fed the control and HFA diets had lower body weights than mice fed the M5-

MTHF and H5-MTHF diets (p < 0.05). However, early differences in body weight between the groups were no longer apparent after the third week of dietary intervention. A significant interaction effect of diet*time (p < 0.0001) on body weight was observed, most likely attributing to the pronounced variation in body weight among the dietary groups in earlier weeks of growth during dietary intervention relative to the latter end of treatment.

77 50 2 mg FA 40 10 mg FA 20 mg FA 30 2 mg 5MTHF 20 10 mg 5MTHF

Body Weight (g) 10 20 mg 5MTHF

0 1 2 3 4 5 6 7 8 9 10 Weeks of Dietary Intervention

Figure 4.1: Effects of dietary folate supplementation on C57BL/6 body weight. Growth curves for C57BL/6 mice (n = 11/dietary group). Growth curves were not significantly different between dietary groups (p = 0.5864). Body weights were significantly different (p < 0.05) between certain dietary groups in the second and third week of intervention, as assessed by Tukey’s multiple comparisons test. Early differences in body weights were no longer apparent in after the third week of dietary intervention.

4.4.2 Plasma, hepatic, and small intestine folate concentrations

A two-way ANOVA was used to examine the effects of the dose and form of folate supplementation on plasma, hepatic and small intestinal folate concentrations after 10 weeks of dietary intervention (n = 10-11/group) (Figure 4.2).

There was a significant interaction between the effects of dose and form on plasma folate concentrations (p = 0.0007) (Figure 4.2A). However, although a significant interaction effect was observed, a clear direct association between FA and 5-MTHF dose and plasma folate concentrations was observed. Simple effect analysis showed that within each folate form (FA &

5-MTHF), increasing doses (2 mg/kg, 10 mg/kg, 20 mg/kg) resulted in significantly increased plasma folate concentrations with both FA and 5-MTHF supplementation (p-values < 0.0001).

Simple effect analyses within supplemental doses showed a significantly higher plasma folate concentration associated with H5-MTHF relative to HFA (p < 0.0001), although no differences

78 were observed between control and L5-MTHF (p > 0.9999) or between MFA and M5-MTHF (p

= 0.2098).

There were no significant interactions between the effects of dose and form on hepatic or small intestinal folate concentrations (p = 0.52; p = 0.27, respectively). A clear, direct association between supplemental doses and tissue folate concentrations were observed in the liver and small intestine. Main effect analyses showed a significant effect of dose on tissue folate concentrations, whereby hepatic (p < 0.0001) and small intestinal (p < 0.0001) folate concentrations were reflective of increasing supplemental doses, but did not show a significant effect of folate form on tissue folate concentrations, after adjusting with the Benjamini-Hochberg procedure (p = 0.37, p = 0.05, respectively).

. 100 *** f f

e e 50 d d Concentration (ng/mL) 0 FA 5MTHF B C 20 2000 c c . b . b 15 a 1500 a

10 1000

5 500 Concentration (ng/g tissue) Concentration (ng/g tissue) 0 0 FA 5MTHF FA 5MTHF

Figure 4.2: Effects of FA and 5-MTHF supplementation on various measures of folate status. Increasing supplemental levels of FA and 5-MTHF on [A] plasma (ng folate/mL), [B] hepatic

79 (ng folate/g tissue) and [C] small intestinal (ng folate/g tissue) folate concentrations; n = 10-11/ dietary group. Data are presented as mean +/- SD; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, and blue bars = 20 mg equimolar dose. Significance for the main effects of dose and form were determined using Two-Way ANOVA. Different letters (abc) denote significant main dose effects, and asterisks (*) denote significant main form effects. However, if an interaction effect is significant, different letters (def) denote significant differences between doses within each folate form, and asterisks denote significant differences between folate forms within doses.

4.4.3 Gene Expression Analysis

4.4.3.1 Liver Gene Expression

Comparative gene expression analyses were conducted on transporters, receptors, carriers, and enzymes involved in folate absorption, intracellular uptake and export, intracellular retention and hydrolysis, metabolism, nucleotide biosynthesis and methylation reactions in the liver. A two-way ANOVA was conducted on a sample of 36 mice (n = 6/group) to examine the effects of dose and form on mRNA expression the selected gene in the liver.

Absorption, intracellular folate uptake and export

No interactions were observed between the effects of dose and form on the proton- coupled folate transporter (Pcft) (p = 0.68), reduced folate carrier (Rfc) (p = 0.45), or folate receptor-α (Fr-α) (p = 0.19) mRNA expression (Figure 4.3). Main effect analyses showed no significant effect of dose on Pcft (p = 0.29) and Rfc (p = 0.22) mRNA expression, but did show a significant effect on Fr-α mRNA expression (p = 0.0304) although significance disappeared after adjusting with the Benjamini-Hochberg procedure. Main effect analyses showed no significant effect of form on Pcft (p = 0.86) and Fr-α (p = 0.21) mRNA expression, but had a significant effect on Rfc (p = 0.0066) mRNA expression, where 5-MTHF supplementation was associated with higher mRNA expression.

A. B. Pcft Rfc 1.5 3 *

1.0 2

0.5 1

0.0 0 Relative mRNA Fold Change Relative mRNA Fold Change FA 5MTHF FA 5MTHF

C. 2.5 Fr-α

2.0

1.5

1.0

0.5

0.0 Relative mRNA Fold Change FA 5MTHF

Figure 4.3: Effects of FA and 5-MTHF supplementation on transporters, carriers, and receptors involved with folate absorption in the liver. Graphs represent relative mRNA expression of [A] Pcft, [B] Rfc, and [C] Fr-α; n = 6/dietary group; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, blue bars = 20 mg equimolar dose. Data are shown as relative fold change normalized to housekeeping genes and the control group, but are statistically analyzed using ∆Ct values. Asterisks (*) denote significant main form effects.

Intracellular retention & hydrolysis

No interactions were observed between the effects of dose and form on the folylpolyglutamate synthase (Fpgs) (p = 0.82) and γ-glutamyl hydrolase (Ggh) (p = 0.38) mRNA expression (Figure 4.4). Main effect analyses showed significant effects of dose and form, individually, on Fpgs mRNA expression (p = 0.0034, p = 0.0112, respectively), where 5-MTHF was associated with higher Fpgs mRNA expression, although no significant effect was observed

81 in Ggh mRNA expression (p = 0.41, p = 0.15, respectively). Post-hoc analysis for the effect of dose on Fpgs gene expression showed an inverse association between increasing dose and Fpgs mRNA expression (p < 0.05).

A. B. Fpgs Ggh * 2.5 3 a 2.0 ab b 2 1.5

1.0 1 0.5

0 Relative mRNA Fold Change 0.0 Relative mRNA Fold Change FA 5MTHF FA 5MTHF

Figure 4.4: Effects of FA and 5-MTHF supplementation on enzymes involved with folate retention and efflux in the liver. Graphs represent relative mRNA expression of [A] Fpgs and [B] Ggh; n = 6/dietary group; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, blue bars = 20 mg equimolar dose. Data are shown as relative fold change normalized to housekeeping genes and the control group, but are statistically analyzed using ∆Ct values. Different letters (abc) denote significant main dose effects, and asterisks (*) denote significant main form effects.

Metabolism

There were no significant interactions between the effects of dose and form on the dihydrofolate reductase (Dhfr) (p = 0.25), methylenetetrahydrofolate reductase (Mthfr) (p =

0.92), and serine hydroxymethyltransferase (Shmt) (p = 0.64) mRNA expression after adjusting with the Benjamini-Hochberg procedure (Figure 4.5). Main effect analyses showed that dose did not have a significant effect on Dhfr (p = 0.58) and Shmt (p = 0.097) mRNA expression, but had a significant effect on Mthfr (p = 0.0008) mRNA expression. Post-hoc analysis showed that

Mthfr mRNA expression was inversely related to the dose of folate supplementation (p = 0.0007;

Figure 4.5C). Main effect analyses for form showed no significant effect on Smht (p = 0.20) and

Mthfr (p = 0.37) mRNA expression, but significant effect on Dhfr (p = 0.0106) mRNA

82 expression, where 5-MTHF supplementation was associated with higher Dhfr mRNA expression than FA supplementation (Figure 4.5A).

A. B. Dhfr Shmt 3 * 2.0

2 1.5

1.0 1 0.5

0.0 0 Relative mRNA Fold Change Relative mRNA Fold Change FA 5MTHF FA 5MTHF

C. Mthfr a 2.0 ab b 1.5

1.0

0.5

0.0 Relative mRNA Fold Change FA 5MTHF

Figure 4.5: Effects of FA and 5-MTHF supplementation on enzymes involved with folate metabolism. Graphs represent relative mRNA expression of [A] Dhfr, [B] Shmt, and [C] Mthfr; n = 6/dietary group; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, blue bars = 20 mg equimolar dose. Data are shown as relative fold change normalized to housekeeping genes and the control group, but are statistically analyzed using ∆Ct values. Different letters (abc) denote significant main dose effects, and asterisks (*) denote significant main form effects.

Nucleotide biosynthesis

There was no significant interaction between the effects of dose and form on thymidylate synthase (Tyms) (p = 0.53) mRNA expression (Figure 4.6). Furthermore, there were no significant main effects of dose and form, individually, on Tyms mRNA expression (p = 0.30, p

= 0.68, respectively).

Tyms 2.0

1.5

1.0

0.5

0.0 Relative mRNA Fold Change FA 5MTHF

Figure 4.6: Effects of FA and 5-MTHF supplementation on enzymes involved with nucleotide biosynthesis in the liver. Graphs represent relative mRNA expression of Tyms; n = 6/dietary group; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, blue bars = 20 mg equimolar dose. Data are shown as relative fold change normalized to housekeeping genes and the control group, but are statistically analyzed using ∆Ct values.

Methionine Cycle & DNA methylation

There were no significant interactions between the effects of dose and form on the methionine synthase (Ms) (p = 0.97), DNA methyltransferase 1 (Dnmt1) (p = 0.61), 3A

(Dnmt3a) (p = 0.50), and 3B (Dnmt3b) (p = 0.68) mRNA expression (Figure 4.7). Main effect of dose did not have a significant effect on Ms (p = 0.88), Dnmt1 (p = 0.80), and Dnmt3a (p = 0.93) mRNA expression, but had a significant effect on Dnmt3b mRNA expression (p = 0.0004), where an inverse association was observed (Figure 4.7D). Main effect analysis on form showed no significant effect on Dnmt3a (p = 0.10) and Dnmt3b (p = 0.47), but showed significant effect on Ms (p < 0.0001) and Dnmt1 (p = 0.0006) mRNA expression, where in both cases, 5-MTHF supplementation was associated with higher gene expression relative to FA supplementation

(Figure 4.7A, Figure 4.7B, respectively).

A. B. Ms Dnmt1 * 2.5 * 2.0 2 1.5

1.0 1

0.5

0 Relative mRNA Fold Change 0.0 Relative mRNA Fold Change FA 5MTHF FA 5MTHF C. D. Dnmt3a Dnmt3b 2.5 a 2.0 ab 2 b 1.5

1.0 1

0.5

0.0 Relative mRNA Fold Change 0 Relative mRNA Fold Change FA 5MTHF FA 5MTHF

Figure 4.7: Effects of FA and 5-MTHF supplementation on enzymes involved with one-carbon transfer reactions and DNA methylation in the liver. Graphs represent relative mRNA expression of [A] Ms, [B] Dnmt1, [C] Dnmt3a, and [D] Dnmt3b; n = 6/dietary group; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, blue bars = 20 mg equimolar dose. Data are shown as relative fold change normalized to housekeeping genes and the control group, but are statistically analyzed using ∆Ct values. Asterisks (*) denote significant main form effects.

85 4.4.3.2 Small Intestine Gene Expression

Comparative gene expression analyses were conducted on transporters, receptors, carriers, and enzymes involved in folate absorption, intracellular uptake and export, intracellular retention and hydrolysis, metabolism, nucleotide biosynthesis and methylation reactions in the small intestine. A two-way ANOVA was conducted on a sample of 48 mice (n = 8/group) to examine the effects of dose and form on mRNA expression of the selected genes in the small intestine.

Absorption, intracellular uptake and export

There were no significant interactions between the effects of dose and form on Pcft (p =

0.95), Rfc (p = 0.77), and Fr-α (p = 0.50) mRNA expression (Figure 4.8). Analyses on the main effect of dose showed no significant effect on Pcft (p = 0.12), Rfc (p = 0.24), and Fr-α (p = 0.46) mRNA expression. Analyses on the main effect of form showed no significant effect on Pcft (p =

0.44) and Rfc (p = 0.94) mRNA expression. However, form did have a significant effect on Fr-α

(p < 0.0001) mRNA expression, where 5-MTHF supplementation was associated with lower Fr-

α mRNA expression than FA supplementation (Figure 4.8C).

86 A. B. Pcft Rfc 2.0 1.5

1.5 1.0

1.0

0.5 0.5

0.0 Relative mRNA Fold Change 0.0 Relative mRNA Fold Change FA 5MTHF FA 5MTHF C. Fr-α 2.0 *

1.5

1.0

0.5

0.0 Relative mRNA Fold Change FA 5MTHF

Figure 4.8: Effects of FA and 5-MTHF supplementation on transporters, carriers, and receptors involved with folate absorption in the small intestine. Graphs represent relative mRNA expression of [A] Pcft, [B] Rfc, and [C] Fr-α; n = 8/dietary group; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, blue bars = 20 mg equimolar dose. Data are shown as relative fold change normalized to housekeeping genes and the control group, but are statistically analyzed using ∆Ct values. Asterisks (*) denote significant main form effects.

Intracellular folate retention and hydrolysis

There were no significant interactions between the effects of dose and form on Fpgs (p =

0.27) and Ggh mRNA expression (p = 0.73) (Figure 4.9). Main effect analyses on dose showed no significant effect on Ggh mRNA expression (p = 0.66) or Fpgs mRNA expression (p =

0.0291), after adjusting with the Benjamini-Hochberg procedure. Main effect analysis on form showed no significant effects on Fpgs (p = 0.93) mRNA expression, or Ggh (p = 0.0255) mRNA expression after adjusting with the Benjamini-Hochberg procedure.

87 A. B. Fpgs Ggh 1.5 2.5

2.0

1.0 1.5

1.0 0.5 0.5

0.0 0.0 Relative mRNA Fold Change Relative mRNA Fold Change FA 5MTHF FA 5MTHF

Figure 4.9: Effects of FA and 5-MTHF supplementation on enzymes involved with folate retention and efflux in the small intestine. Graphs represent relative mRNA expression of [A] Fpgs and [B] Ggh; n = 8/dietary group; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, blue bars = 20 mg equimolar dose. Data are shown as relative fold change normalized to housekeeping genes and the control group, but are statistically analyzed using ∆Ct values.

Metabolism

There were no significant interactions between the effects of dose and form on Dhfr (p =

0.40), Shmt (p = 0.55), and Mthfr (p = 0.60) mRNA expression (Figure 4.10). Main effect analyses on dose showed no significant effect on Dhfr (p = 0.84) and Shmt (p = 0.72) mRNA expression, but showed a significant effect on Mthfr (p = 0.0035) mRNA expression. Post-hoc analysis showed that folate supplementation was inversely associated with Mthfr mRNA expression (p = 0.0044; Figure 4.10C). Main effect analyses on form showed no significant effect on Shmt (p = 0.13) and Mthfr (p = 0.68) mRNA expression, but had significant effect on

Dhfr (p = 0.0002) mRNA expression, where 5-MTHF supplementation was associated with significantly lower Dhfr mRNA expression relative to FA supplementation (Figure 4.10A).

88 A. B. Dhfr Shmt 2.0 2.0 *

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0 Relative mRNA Fold Change Relative mRNA Fold Change FA 5MTHF FA 5MTHF C. Mthfr 2.0 a a 1.5 b

1.0

0.5

0.0 Relative mRNA Fold Change FA 5MTHF

Figure 4.10: Effects of FA and 5-MTHF supplementation on enzymes involved with folate metabolism. Graphs represent relative mRNA expression of [A] Dhfr, [B] Shmt, and [C] Mthfr; n = 8/dietary group; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, blue bars = 20 mg equimolar dose. Data are shown as relative fold change normalized to housekeeping genes and the control group, but are statistically analyzed using ∆Ct values. Different letters (abc) denote significant main dose effects, and asterisks (*) denote significant main form effects.

Nucleotide biosynthesis

There was no significant interaction between the effects of dose and form on Tyms gene expression (p = 0.72) (Figure 4.11D). Furthermore, there were no significant main effects of dose (p = 0.61) and form (p = 0.21), individually, on Tyms mRNA expression.

89 2.0 Tyms

1.5

1.0

0.5

0.0 Relative mRNA Fold Change FA 5MTHF

Figure 4.11: Effects of FA and 5-MTHF supplementation on enzymes involved with nucleotide biosynthesis in the small intestine. Graphs represent relative mRNA expression of Tyms; n = 8/dietary group; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, blue bars = 20 mg equimolar dose. Data are shown as relative fold change normalized to housekeeping genes and the control group, but are statistically analyzed using ∆Ct values.

Methionine cycle and DNA methylation

There were no significant interactions between the effects of dose and form on Ms (p =

0.74), Dnmt1 (p = 0.74), Dnmt3a (p = 0.28), and Dnmt3b (p = 0.79) mRNA expression (Figure

4.12). Main effect analyses on dose showed no significant effect on Ms (p = 0.26), Dnmt1 (p =

0.63), Dnmt3a (p = 0.18), and Dnmt3b (p = 0.66) mRNA expression. Main effect analyses on form showed no significant effect on Ms (p = 0.96), Dnmt1 (p = 0.45), and Dnmt3b (p = 0.12) mRNA expression, but showed significant effect on Dnmt3a (p = 0.0075) mRNA expression, where 5-MTHF supplementation was associated with a higher DNmt3a mRNA expression relative to FA supplementation (Figure 4.12C).

90 A. B. Ms Dnmt1 2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0 Relative mRNA Fold Change Relative mRNA Fold Change FA 5MTHF FA 5MTHF C. D.

4 Dnmt3a 2.5 Dnmt3b * 2.0 3 1.5 2 1.0 1 0.5

0 0.0 Relative mRNA Fold Change Relative mRNA Fold Change FA 5MTHF FA 5MTHF

Figure 4.12: Effects of FA and 5-MTHF supplementation on enzymes involved with one-carbon transfer reactions and DNA methylation in the small intestine. Graphs represent relative mRNA expression of [A] Ms, [B] Dnmt1, [C] Dnmt3a, and [D] Dnmt3b; n = 8/dietary group; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, blue bars = 20 mg equimolar dose. Data are shown as relative fold change normalized to housekeeping genes and the control group, but are statistically analyzed using ∆Ct values. Asterisks (*) denote significant main form effects.

4.4.4 Global DNA Methylation

The effects of FA and 5-MTHF supplementation on global DNA methylation were determined in the liver and small intestine (Figure 4.13). A two-way ANOVA was conducted on a sample of 62 mice (n = 7-11/group) in the liver and 64 mice (n = 10-11/group) in the small intestine to examine differences in global DNA methylation determined by the in vitro methyl acceptance assay using [3H-methyl] SAM as a methyl donor and a prokaryotic CpG DNMT,

SssI.

91 There was no significant interaction between the effects of dose and form on global DNA methylation in the liver (p = 0.18). Individually, main effect analysis on dose showed no significant effect on liver global DNA methylation (p = 0.013), after adjusting with the

Benjamini-Hochberg procedure. Main effect analysis on form, however, showed significant effect on liver global DNA methylation (p < 0.0001), where 5-MTHF supplementation was associated with a higher degree of global DNA methylation (or [3H-methyl] incorporation into

DNA) compared with FA supplementation (Figure 4.13A).

There was no significant interaction between the effects of dose and form on global DNA methylation in the small intestine (p = 0.10) (Figure 4.13B). There were also no individual significant main effect of dose or form on small intestinal global DNA methylation (p = 0.30, p =

0.97, respectively).

A B 150000 100000 . * . 80000 100000 60000

40000 50000 20000

0 SI DNA Methylation (DPM) 0 Liver DNA Methylation (DPM) FA 5MTHF FA 5MTHF

Figure 4.13: Effects of FA and 5-MTHF supplementation on global DNA methylation on [A] liver and [B] small intestine DNA methylation were determined by the in vitro methyl acceptance assay using [3H-methyl] SAM as a methyl donor and a prokaryotic CpG DNMT, SssI. The manner in which this assay is performed produces an inverse relationship between the endogenous DNA methylation status and exogenous incorporation [3H-methyl] incorporation.; n = 7-11/ dietary group. Data are presented as mean +/- SD; red bars = 2 mg equimolar dose, green bars = 10 mg equimolar dose, blue bars = 20 mg equimolar dose. Significance for the main effects of dose and form were determined using Two-Way ANOVA. Asterisks (*) denote significant main form effects.

92 Table 4.6: General summary of liver and small intestine mRNA expression. Significant main effects of dose and form are illustrated below. ↓ indicates a significant dose effect, where increasing supplementation incrementally decreased mRNA expression. 5-MTHF or FA indicates significant form effect, where 5-MTHF represents higher mRNA expression relative to FA, and FA represents higher mRNA expression relative to 5-MTHF. Dose Effect Form Effect Liver Small Int. Liver Small Int.

Pcft ------Rfc -- -- 5-MTHF ------FA Fr-α Fpgs ↓ ↓ (NS) 5-MTHF -- Ggh ------Dhfr -- -- 5-MTHF FA Ts ------Shmt ------Mthfr ↓ ↓ -- -- Mtr/Ms -- -- 5-MTHF -- Dnmt1 -- -- 5-MTHF -- Dnmt3a ------5-MTHF Dnmt3b ↓ ------

4.5 Discussion

Due to recent increases in folate status in North America, understanding its effects on the intracellular mechanisms of intrinsic, downstream pathways is necessary to elucidate a safer, more effective means of folate supplementation. Currently, many pre-natal FA supplements have reduced doses from 1 mg to 600 µg due to the growing concerns of FA intake. However, with increasing speculations about the safety concerns of FA, in tandem with the gradual promotion for 5-MTHF, it is important to distinguish the differential effects they have on a genetic and molecular scale, as excessive FA supplementation has been associated with adverse health

93 outcomes. As such, we assessed the effects of FA and 5-MTHF supplementation on folate absorption, intracellular uptake and export, intracellular retention and hydrolysis, metabolism and one-carbon transfer reactions involved in nucleotide biosynthesis and DNA methylation using a 10-week dietary intervention in a C57BL/6 model.

Folate Status Indices

Dietary intervention with amino-acid defined diets containing either 2 (control), 10

(MFA; 5X BDR), or 20 (HFA; 10X BDR) mg FA/kg, or their respective equimolar 5-MTHF doses (C-5-MTHF, M-5-MTHF, H-5-MTHF), were relected plasma and tissue folate concentrations in our mice in a predictable manner. We observed significantly higher plasma folate concentrations with increasing supplemental levels of both FA and 5-MTHF. Additionally,

H-5-MTHF was associated with significantly higher plasma folate concentrations relative to the equimolar FA dose (HFA). Hepatic and small intestinal folate concentrations also increased with increasing supplemental levels of FA and 5MHTF.

Previous studies have established a positive association between supplemental levels of both FA and 5-MTHF and plasma and tissue folate concentrations in both animals and humans

(13-17, 176). However, the novel finding in the present study is that no significant differences in plasma or tissue folate concentrations were observed between FA and 5-MTHF, except that 5-

MTHF resulted in higher plasma folate concentrations than FA at the 20 mg supplemental level.

Clinical studies that have found similar results, where 5-MTHF had a greater effect in increasing plasma folate concentrations, attribute these findings to differences in metabolism of the two folate vitamers (162). 5-MTHF is biologically active and can be stored in the body and can therefore, directly induce change in plasma folate concentrations without any first-pass effect.

94 FA requires metabolism by DHFR, which can become saturated at higher doses leading to lower levels of circulating bioactive folates.

Additionally, in the present study, although non-significant after adjusting with the

Benjamini-Hochberg procedure, higher intestinal Ggh expression was associated with 5-MTHF supplementation relative to FA. In this regard, intracellular intestinal folates would be exported from the cell and enter circulation, hence, increasing plasma folate concentrations.

Our findings observed similar increases in plasma, hepatic and small intestinal folate concentrations with FA and 5-MTHF supplementation. Therefore, our findings suggest that 5-

MTHF supplementation is at least, as effective as FA in increasing plasma and tissue folate concentrations. In agreement with these findings, numerous clinical studies using various cohorts comparing the effects of FA and 5-MTHF supplementation on blood folate status indices have suggested that 5-MTHF supplementation is, at least, as effective as FA supplementation in increasing plasma and RBC folate concentrations, and decrease plasma homocysteine concentrations (13-18).

Folate Absorption and Transport

Based on previous in vivo studies (56, 59), we expected that mRNA expression of transporters, carriers, and receptors involved in folate absorption would be down-regulated in response to 5-MTHF supplementation relative to FA supplementation. Therefore, it was further expected that 5-MTHF supplementation would elicit differential effect on mRNA expressions of genes encoding folate transporters, carriers, and receptors relative to FA supplementation.

Previous studies have indicated inverse relationships between folate supplementation and mRNA expression of the Pcft, Rfc, and Fr-α (28, 56, 58, 59).

95 In the present study, 5-MTHF supplementation was associated with increased Rfc expression in the liver, but a decreased Fr-α expression in the small intestine. Interestingly, Pcft expression in the small intestine and liver was not associated with 5-MTHF or FA supplementation. Previous in vivo studies using the jejunum of laying hens fed on a 5-MTHF supplemented diet showed significant downregulated Pcft and Rfc expression relative to a folate- deficient diet (56, 59), but found no effect of dose or form in the duodenum (56, 59).

In the present study using a mouse model, Rfc mRNA expression in the liver, however, was shown to be greater with 5-MTHF supplementation than with FA supplementation. The increased expression of the Rfc in the liver could be a response to the elevated plasma folate concentrations observed. As the liver represents the primary storage organ for folates, Rfc expression may have been upregulated to facilitate storage in the liver. Additionally, as the RFC has a characteristic high affinity for reduced folates (25), increased levels of 5-MTHF would also increase demand for RFCs to facilitate folate transport into certain tissues such as the liver.

Lastly, RFC is present in the basolateral membrane of cells and would be upregulated during 5-

MTHF supplementation to facilitate folate transport out of cells.

We did not examine the effects of FA or 5-MTHF supplementation on Pcft, Rfc, and Fr-α protein expression or activities. Given post-transcriptional, translational and post-translational modifications, the effects of FA or 5-MTHF on protein expression or activities of these folate transporters, carriers, and receptors may not parallel those on mRNA expression. For the aforementioned transporter, carrier, and receptor, previous FA-supplemented in vivo model studies have failed to observed a direct association between mRNA and protein expression. Dev et al. found no effect of acute or chronic FA supplementation on mRNA expression of Pcft and

Rfc, but found a significant down-regulation of both Pcft and Rfc protein expression during acute

96 supplementation (28). Therefore, future in vivo studies are necessary to the investigate the effects of FA and 5-MTHF supplementation protein expression as well as the pharmacokinetic and pharmacodynamic activities of the folate transporter, carrier, and receptor.

Intracellular folate retention and hydrolysis

FPGS is responsible for polyglutamylating folates for retaining intracellular folates and making them better substrates for metabolic enzymes. In the present study, Fpgs mRNA expression was down-regulated in response to both FA and 5-MTHF supplementation in both the liver and small intestine. There is a paucity of in vivo studies that investigated the effects of folate supplementation on Fpgs mRNA expression. However, previous in vitro studies investigating the effects of folate-deficiency on gene expression using cancer cell lines suggested that Fpgs expression is up-regulated in response to folate deprivation to increase folate retention

(66). It can be reasoned that in high extracellular folate environments, Fpgs is downregulated as a homeostatic mechanism for maintaining optimal level and regulating metabolism of intracellular folates. In addition, 5-MTHF supplementation was associated with higher Fpgs expression relative to FA supplementation in the liver. This observation could be attributed to the higher levels of metabolically active monoglutamated folates associated with 5-MTHF supplementation. As the liver represents the primary storage organ for folate, increased FPGS may be necessary to retain and store the high levels of reduced folates. Alternatively, FPGS expression may be differentially regulated in response to FA and 5-MTHF supplementation, although further studies are required to support this conjecture.

GGH is responsible for hydrolyzing polyglutamylated folates for cellular export (1). In our study, the dose and form of folate had no effect on Ggh mRNA expression. However, study

97 conducted by Hayashi et al. using the HCT116 colon adenocarcinoma cell line found a decreased

GGH mRNA expression associated with folate-deficiency (66), suggesting that GGH may be down-regulated in the setting of folate deficiency to decrease folate export out of the cell.

However, whether or not folate supplementation increases Ggh expression as a mechanism to facilitate folate export when exposed to excessive extracellular folate concentrations has not been well established. Although our study found no significant effect of dose or form of folate supplementation on Ggh mRNA expression, post-transcriptional, translational, or post- translational regulations may modulate Ggh. As such, the effect of FA and 5MHTF supplementation on GGH protein expression and activity needs to be investigated in future studies.

Intracellular folate metabolism

DHFR is a metabolic enzyme responsible for reducing FA to DHF, and DHF to THF. In the present study, Dhfr was differentially expressed in response to folate form in a tissue- dependent manner. In the present study, Dhfr mRNA expression in the liver was higher with 5-

MTHF supplementation than with FA supplementation. In contrast, in the small intestine, 5-

MTHF supplementation was associated with a lower Dhfr mRNA expression compared with FA supplementation. The DHFR enzyme has been thoroughly studied by Bailey and Ayling in human and rat liver (11). In human, the reaction with FA, as a substrate of DHFR, is 1300 times slower than with the natural substrate 7,8-DHF (11). In the same study, FA supplementation was associated with competitive inhibition of DHFR’s ability to reduce DHF to THF. Therefore, 5-

MTHF supplementation in the liver may have been associated with a higher Dhfr mRNA expression, as Dhfr activity would be much greater due to the lack of competitive FA inhibition.

98 Additionally, it is unlikely that 5-MTHF supplementation will show presence of FA in the body, but rather high levels of DHF. As a much better substrate for Dhfr, increasing DHF concentrations can potentially increase Dhfr expression in the liver. In the small intestine, FA supplementation was associated with higher Dhfr expression relative to 5-MTHF supplementation. FA is primarily transported to the liver for metabolism, but is also metabolized in the small intestine to a lesser degree. Remaining levels of FA in the small intestine can therefore elicit an increased expression, relative to 5-MTHF, where metabolism is unneeded, to increase bioactive folates in the intestine.

SHMT is a metabolic enzyme responsible for converting THF and serine to 5,10- methyleneTHF, a precursor for thymidylate synthesis, and glycine in a reversible manner. In the present study, Shmt mRNA expression was not affected by dose or form of FA or 5-MTHF supplementation in either tissue. This observation may be in part explained by the fact that

SMHT-mediated folate metabolic pathways are not rate-limiting steps. Previous studies have suggested that the reducing activity of DHFR act as the rate-limiting step in folate metabolism

(189). As such, DHFR may be most sensitive to folate status. DHFR might only reduce the appropriate amount of folates for downstream enzymes to optimally function. In contrast, enzymes such as SHMT, which are involved in intermediate steps in folate metabolism may not be susceptible to folate status, which could explain the lack of effect of dose and form of FA and

5-MTHF supplementation on Shmt mRNA expression observed in the present study. In the in vitro study by Hayashi et al. using HCT116 and Caco-2 human adenocarcinoma cell lines, folate deficiency in HCT116 was associated with a downregulation of SHMT transcript relative to the control, whereas in Caco-2 cells, folate deficiency was associated with an upregulation of SHMT transcript (66).

99 The MTHFR irreversibly converts 5,10-methyleneTHF to 5-MTHF and is a critical enzyme in channelling folate to the biological methylation reaction. Dependent on folate status, it preferentially shuttles folate to either the nucleotide biosynthesis pathway or the biological methylation pathway. Our findings have shown that Mthfr mRNA is significantly downregulated with increased FA and 5-MTHF supplementation in both the liver and small intestine.

In contrast, we did not find any effect of folate form on Mthfr mRNA expression. Previous studies conducted by Christensen et al. using mice, found that Mthfr mRNA expression, protein expression, and activity were all decreased in response to postweaning FA supplementation (65,

191). In addition, another study reported a significant MTHFR inhibition in pig liver with DHF supplementation, although FA, THF, and 5-MTHF were also observed to inhibit MTHFR, albeit to a lesser degree (192). The findings from the present study are consistent with these previous observations. Therefore, decreased Mthfr mRNA expression in response to FA and 5-MTHF supplementation may be a potential homeostatic mechanism to deter folates from entering the biological methylation pathway and preventing aberrant biological methylations. One of the studies conducted by Christensen et al. also determined that FA supplementation increased phosphorylation of the MTHFR protein, thereby decreasing its activity (65). Future studies are warranted to examine the effect of FA and 5-MTHF on MTHFR protein expression and activity as well as their functional ramifications.

Nucleotide Biosynthesis

TYMS is an enzyme responsible for thymidylate biosynthesis using the substrate 5,10- methyleneTHF and dUMP to generate DHF and dTMP. Contrary to our hypothesis, there were no differences between the effect of FA and 5-MTHF supplementation on Tyms gene expression

100 in the liver or small intestine. A previous study that examined the effects of FA supplementation on TYMS, in a model of regenerating rat liver after partial hepatectomy found that FA supplementation reduced Tyms mRNA expression and decreased DNA content in an unpredictable manner, initially delaying DNA synthesis and later upregulating synthesis (190).

However, in the same study, the decrease in Tyms mRNA expression did not translate to decreased Tyms protein expression, but rather increased protein expression. Therefore, TYMS may be more prominently regulated with post-transcriptional or translational modifications, although further studies are necessary to elucidate this.

Biological methylation reactions: DNA Methylation

The MS (also known as MTR in human) is a B-12 dependent enzyme responsible for mediating the methyl transfer from 5-MTHF to homocysteine in generating methionine, the precursor of SAM, the universal methyl group donor for over 100 biological methylation reactions including DNA methylation (34). Although our study did not observe any dose effect on Ms mRNA expression in the liver or small intestine, we did observe a significant form effect on Ms mRNA expression in the liver. 5-MTHF supplementation was associated with higher Ms mRNA expression compared with FA supplementation in the liver but not in the small intestine.

As a substrate for MS, it can be expected that 5-MTHF supplementation would elicit an increased demand for MS, thereby up-regulating Ms in the liver. A previous study has illustrated a down-regulation in Ms mRNA associated with FA supplementation in a mouse model (65). In that study, it was reported that FA supplementation led to decreased expression and activity of the MTHFR protein, which effectively reduced 5-MTHF concentrations compared with the control. Therefore, due to the decreased levels of metabolically active folates for biological

101 methylation reactions (i.e., 5-MTHF), Ms expression would also expectedly decrease in response to the pseudo-folate deficiency, marked by low intracellular concentrations of metabolically active folates. Ironically, this study supported our findings of a direct association between 5-

MTHF supplementation and Ms mRNA expression. In the aforementioned mouse study, FA supplementation was associated with decreased proportional levels of 5-MTHF, which in turn, downregulated Ms expression, protein expression, and protein activity levels (65).

DNMTs are responsible for methylating CpG sites in the genome, which in turn, regulate expression of certain genes. DNMT1 is a methyltransferase that is considered to be the key methyltransferase in mammals. DNMT3A and DNMT3B are the other main methyltransferases that are mainly responsible for de novo CpG methylation. In the liver, Dnmt1 was more highly expressed with 5-MTHF supplementation than with FA supplementation. Additionally, Dnmt3b expression was inversely associated with increased levels of folate supplementation.

Supplementing at the 20 mg level resulted in significantly lower levels of Dnmt3b mRNA expression relative to the 2 mg level. To my knowledge, there are no previous studies that have investigated the effects of folate supplementation on Dnmt1, Dnmt3a, or Dnmt3b mRNA expression in vivo. However, it can be inferred that the observed increase in Dnmt1 and Dnmt3b expression is associated with increased Ms expression. Therefore, supplementation of reduced folates can effectively up-regulate genes of enzymes involved in the biological methylation pathway when compared to FA supplementation. To support the purported effects of folate form, we also investigated the comparative effects of FA and 5-MTHF supplementation on global

DNA methylation. Although no significant effects of dose or form were found in the small intestine, which was expected given our gene expression data, 5-MTHF supplementation was associated with greater global DNA methylation in the liver. Previous studies have also indicated

102 a positive association between FA supplementation and global DNA methylation in the liver

(191). The higher liver global DNA methylation associated with 5-MTHF supplementation relative to FA can be speculated to be attributing to the increased expression of Ms and Dnmt1 in the liver; both genes showed greater expression associated with 5-MTHF relative to FA.

Increasing Dnmt1 expression is likely to increase protein expression, which may potentially increase methylation in the DNA. Global DNA methylation is associated with increased genomic and chromosomal stability, which can potentially reduce cancer risk. Therefore, supplementation with 5-MTHF may decrease cancer risk and progression.

The DNMT1 is a critical enzyme for mammalian development as it is responsible for maintenance, or baseline, methylation patterns in replicating cells (33). The DNMT3A is particularly important for de novo methylation patterns involved in cell differentiation during development prior to birth (33, 34). It is also of critical importance for its involvement in methylation patterns for hematopoiesis. The DNMT3B is also important for de novo methylation patterns, although it has been shown to be associated with maintenance methylation as well (33,

34). It has been shown to be associated with X-chromosomal inactivation, embryonic development, and imprinting (33, 34). The observed increase in gene expression of enzymes involved in the biological methylation cycle is something that should not be misunderstood as positive, but rather used as a foundation for which more studies can help confirm, elucidate, and further build upon. Aberrant DNA methylation can have adverse effects and should be more thoroughly studied before any recommendations are made.

103 Strengths and Limitations

One of the strength to our study comes from our rigorous and highly controlled methodology. Our lab used an inbred C57BL/6 mouse model. The biggest advantage of using an inbred strain is the consistency of the genetic background between individual mice. Therefore, detection of significant differences can be attributed to dietary interventions.

In addition to genetic consistency, our mice were fed diets that were identical in nutritional content and differed only in the amount of FA or 5-MTHF supplemented, further attributing significant effects to dietary treatments. Mice were fed on FA or 5-MTHF supplemented diets for 10 weeks. Considering the RBC turnover in mice is approximately 48-50 days, 10 weeks can be considered a chronic supplementation in our mouse study.

Another strength to our observational study is the vast number of genes that were analyzed. Given the lack of studies that have compared FA and 5-MTHF on folate metabolism and one-carbon transfer reactions, this study provides biochemical evidence for the association between folate supplementation and tissue-specific gene expression of enzymes involved in the folate pathway. In addition to gene expression, our study looked at global DNA methylation and therefore, potential associations can be made between gene expressional changes and global

DNA methylation.

One of the limitations of our observational assessment of FA and 5-MTHF supplemental differences include small sample sizes within groups; in future analyses a larger sample size may be used for clarification of observed effects. In addition, the relevance of any differential effects that folate form or dose has on gene expression should be assessed by examining protein levels and activity. Previous studies have shown a disassociation between gene expression levels and protein expression levels, as well as protein expression levels and activity levels. However, our

104 data provides support for our hypotheses suggesting that FA and 5-MTHF supplementation can similarly increase folate status indices and differentially modulate gene expression of enzymes involved in the folate pathway, which can contribute to the dysregulation of folate dependent processes, such as DNA methylation. Alternatively, modulation of expression may reflect homeostatic mechanisms. Additionally, mice antibodies, required for protein expression, for some enzymes involved in the folate pathway are lacking. Therefore, considering primers are well established in folate metabolism and one-carbon transfer reaction, gene expressional analysis provided more comprehensive pathway associations for this study.

Another limitation in this study is the translatable association between a mouse and human model. Considering that one of the major known difference between them is the substantially lower DHFR activity in humans, we supplemented our mice with up to 20 mg of folate, as an effort to mimic the presence of UMFA in our model. However, given that this was a preliminary observational study, our objective was to first assess differential intracellular effects of FA and 5-MTHF. In conjunction with this limitation is our lack of analyzed and reported

UMFA concentrations, which represents one of the key speculated differences between our two folate vitamers.

Although our study has shown differential effects of FA and 5-MTHF on folate metabolism and one-carbon transfer reactions, its downstream effects on human health must first be assessed before recommendations on safer means of supplementation are provided. However, given that studies assessing folate biochemical processes are lacking, this study can be used as a foundational framework for future studies elucidating the relationship between these findings and health outcomes.

105 4.6 Conclusion

Based on previous studies, it was expected for 5-MTHF and FA to elicit similar effects on folate status indices. However, our data also suggest that 5-MTHF and FA supplementation have differential effects on folate uptake, metabolism, and one-carbon transfer reactions. Of particular interest is their dissimilar effects on the DNA methylation cycle, where 5-MTHF supplementation was associated with increased activity relative to FA. Therefore, the hypothesis of our study that FA and 5-MTHF supplementation would elicit differential effects on folate metabolism and one-carbon transfer reactions is supported. Although this study helps to discern purported beneficial effects of 5-MTHF over FA, these findings should not be interpreted as being advantageous to supplement with 5-MTHF. In reality, these findings only implore more studies to be conducted to determine the biological ramifications on a broader scale as well.

Aberrant DNA methylation has been linked to certain human diseases, including cancer, and therefore, the drastic difference that was observed in liver global DNA methylation should continually be attempted for further elucidation.

Based on the findings of this study, 5-MTHF may have significant public health implications. Apart from the unlikeliness of masking vitamin B12 deficiency, 5-MTHF increases plasma folate concentrations to a greater extent than FA. 5-MTHF also increases global DNA methylation in a tissue-specific manner. As cancer is associated with global DNA hypomethylation and gene-specific CpG island hypermethylation, 5-MTHF may potentially have significant impact in reversing the effects of cancer on DNA methylation, thereby inhibiting progression.

106 Chapter 5: General discussion, future directions and conclusion

5.1 Summary and general discussion

FA has been potentially associated with a number of adverse health effects including masking vitamin B12 deficiency, cancer progression, reduced immune system function, and epigenetic modifications. 5-MTHF refers to the biologically active and reduced form of folate that has been speculated to have beneficial effects over FA. Keeping this in mind, we attempted to conduct a comparative intervention study to determine the differential effects between FA and

5-MTHF supplementation on intracellular mechanisms in a C57BL/6 mouse model. Specifically, the effects of FA and 5-MTHF supplementation on folate status indices, gene expression of folate-associated enzymes, and global DNA methylation were assessed in the liver and small intestine. The dietary FA and 5-MTHF interventions employed in our study similarly modulated folate status in our animals as assessed by plasma folate, hepatic folate, and small intestinal folate concentrations. In conjunction with previous in vivo and clinical studies, our findings suggest that 5-MTHF is, at least, as effective in raising blood and tissue folate concentrations.

Our findings also suggest a differential effect between FA and 5-MTHF supplementation on gene expression of enzymes involved in folate absorption. In the liver, 5-MTHF supplementation was associated with an increased expression of the Rfc gene relative to FA. As

RFC refers to the major folate transporter in mammalian cells and tissues (25), the observed increase in gene expression may translate to a smaller required RDA in 5-MTHF supplements.

RFC has a high affinity for reduced folates, which is beneficial for the transport of folates into numerous tissues. As a calcium salt (Metafolin®), 5-MTHF is also present in a monoglutamated

107 state and hence, bypasses the need for GCPII hydrolysis. Thus, this particular feature allows 5-

MTHF to possess a similar bioavailability to FA.

Regarding metabolic enzymes, 5-MTHF and FA supplementation have been also observed to have differential effects on gene expression. DHFR is of particular importance to FA as it is responsible for FAs complete reduction to THF. However, DHFR has also been suggested to be inhibited by FA as well (11). Human DHFR has been shown to be saturated with intakes of

> 200 µg FA, indicated by the presence of UMFA in the circulation (9). In our study, 5-MTHF supplementation was associated with a greater expression of Dhfr in the liver, but a lower expression in the small intestine relative to FA supplementation. Additionally, increased levels of folate supplementation were linked with decreased expressions of the Mthfr gene. Prevalent

Mthfr polymorphisms are associated with reduced activity of the enzyme leading to decreased levels of metabolically active folates capable of one-carbon transfer reactions (170). 5-MTHF can therefore be beneficial to the aforementioned concerns with DHFR inhibition and Mthfr polymorphisms as this folate vitamer bypasses the need for both enzymes and can directly participate in the one-carbon transfer reaction pathway.

In our study, 5-MTHF was observed to favour biological methylation reactions. In particular, 5-MTHF supplementation was associated with increased Mtr and Dnmt1 gene expression in the liver, which we speculate to have direct contributions to the observed higher global DNA methylation. However, increased global DNA methylation is not necessarily advantageous, as aberrant DNA methylation levels have been associated with the onset and progression of adult-onset diseases (9, 103), but only warrants for more studies to elucidate the biological ramifications on a broader scale.

108 5.2 Future directions

Due to the lack of studies examining the comparative differences between FA and 5-

MTHF supplementation on intracellular mechanisms, more in vivo studies are warranted to confirm our findings. To the best of our knowledge, our study is the first to assess the differential effects of each folate vitamer on folate absorption, metabolism, and one-carbon transfer reactions using an animal model. However, future studies exploring the comparative effects of FA and 5-

MTHF supplementation should include analyzing protein expression and protein activity as well.

Attaining these results would give a thorough understanding of the effects of 5-MTHF supplementation on the functional ramifications of enzymes involved in the folate pathway.

UMFA has also been associated with certain adverse health outcomes, including reduced chemosensitivity in natural killer cells and dysregulation of folate absorption and metabolism.

Considering the lack of reported measures in our study, determining the effects that 5-MTHF supplementation has on UMFA levels in the circulation can also help elucidate some of the adverse effects associated with FA supplementation. The definitive approach to elucidate this is supplementing FA or 5-MTHF in an animal study or clinical trial. However, using a rodent model necessitates over-supplementation to account for the significantly greater DHFR activity relative to humans. Further analyses investigating the effect of UMFA presence on gene expression, protein expression, and protein activity can also provide a foundation to elucidate speculations regarding associated negative health effects.

Lastly, considering the dual modulatory effect that FA has on cancer development and progression, studying the effects that 5-MTHF supplementation has on cancer progression may be interesting. As 5-MTHF must participate in the one-carbon transfer reaction pathway prior to partaking in the nucleotide biosynthesis pathway, its supplemental effects on cancer progression

109 can provide insight on its consumption safety. Specifically, this can be studied by inducing polyp into mice via azoxymethane injection, a potent carcinogen used to induce colon cancer in rodents. Upon cancer development, mice would be randomized to receive a basal diet supplemented with 0 mg folate (folate deficient), 2 mg FA (control), 2 mg 5-MTHF, 10 mg FA, or 10 mg 5-MTHF. After 12 weeks of intervention, tumor size and weight would be measured, and signs of metastasis would be analyzed. Investigating the purported study would help clarify speculations about the supposed benefits of 5-MTHF supplementation over FA.

5.3 Conclusion

1) Our study compared the effects of FA and 5-MTHF supplementation on plasma and tissue folate concentrations. 5-MTHF supplementation was observed to be at least, as effective, as FA supplementation in increasing plasma and tissue folate concentrations.

2) Our study compared the effects of FA and 5-MTHF supplementation on gene expression of folate transporters, carriers, receptors, and enzymes involved in the folate pathway and one- carbon transfer reaction pathways. In the liver, 5-MTHF supplementation was associated with higher folate retention and a preferential shift towards the biological methylation reactions pathway.

3) Our study compared the effects of FA and 5-MTHF supplementation on global DNA methylation in mice liver and small intestine. In agreement with our gene expression findings, 5-

MTHF supplementation was associated with greater genomic DNA methylation in the liver relative to FA supplementation.

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