COMPARATIVE CARCINOGENIC EFFECTS OF FOLIC ACID VS. 5-
METHYLTETRAHYDROFOLATE SUPPLEMENTATION ON COLON CANCER
PROGRESSION IN A RODENT MODEL
by
Baljit Kaur
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Department of Nutritional Sciences
University of Toronto
© Copyright Baljit Kaur 2021
ii
COMPARATIVE CARCINOGENIC EFFECTS OF FOLIC ACID VS. 5-
METHYLTETRAHYDROFOLATE SUPPLEMENTATION ON COLON CANCER
PROGRESSION IN A RODENT MODEL
Baljit Kaur
Master of Science
Department of Nutritional Sciences
University of Toronto
2021
Abstract
A growing body of evidence has linked high folic acid (FA) intake to adverse health outcomes,
including tumour promotion. 5-methyltetrahydrofolate (5MTHF) has been proposed to be a safer
alternative form of folate supplementation. However, its effects on tumor promotion are
unknown. We investigated the comparative effects of FA versus 5MTHF supplementation on the
progression of aberrant crypt foci (ACF; earliest precursor of colon cancer). Equimolar doses of
FA and MTHF (control=1mg/kg diet; supplemental=10mg/kg diet) were provided to rats injected
a colorectal carcinogen, azoxymethane, following ACF formation. At necropsy, colorectal
lesions and tumour parameters were assessed histologically and compared. 5MTHF resulted in
higher plasma folate concentration compared to FA (p<0.05). Tumor incidence (adenoma, p=0.5;
adenocarcinoma, p=0.60) did not differ between folate forms. However, 5MTHF resulted in a
greater increase in tumor burden (sum of tumor diameters) compared to FA (p<0.05). Our
findings suggest 5MTHF may have a higher tumor promoting effect than FA.
iii
Acknowledgements
I’d like to start off by thanking my supervisor, Dr. Young-In Kim for providing me with
the opportunity to pursue a Masters. Thank you for your endless support and guidance. I am
especially appreciative of your interest towards my education and career and am thankful for all
of the help and insight you have provided.
Thank you to Dr. Debbie O’Connor and Dr. Elena Comelli for your support throughout
my project. I am beyond appreciative of your insight and advice towards my research but also
your compassion and understanding throughout my project.
A sincere thank you to Kyoung-Jin Sohn for your direction throughout my project. I am
grateful for your lab expertise, and support. Thank you for being so willing to lend a helping
hand, even if it meant coming to the lab on weekends to help me conduct my experiments.
To the entire vivarium and histology staff at Li Ka Shing – I am truly grateful for your
insight and knowledge. Thank you for your endless support throughout my project.
To Dr. Medline, thank you for your enthusiasm and insight towards this project and for
taking the time to thoroughly assist in the histological analysis of my many many slides.
To everyone that I’ve met and worked with in the Kim Lab, thank you. To the summer
students and volunteers, Carina Chan, David Park and Samantha Yim – you guys were
absolutely amazing to work with! I could not have completed this project without you guys.
Last but not least, my family and friends – I know the rat stories weren’t your favourite
but thank you for listening. To my cousins, Arshi, Neetu and Kena – thank you for the rides
home, the 2am trips downtown, and most importantly the coffee runs. To my friends Amrita and
Gurmeet – thank you for your endless encouragement especially during the most stressful of
times. I couldn’t have done it without you guys.
iv
These past few years have gone by so quickly, and I am so grateful to have worked with, learned
from and experienced this journey with each and every one of you.
v
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
Chapter 2: Literature Review
2.1 Colon Cancer………………………………………………………………………………..5
2.1.1 An Overview of Colorectal Cancer ………………………………………………..5
2.1.2 Colon Cancer Risk Factors ………………………………………………………..7
2.1.3 Colon Cancer Carcinogenesis …………………………………………………….8
2.1.4 Molecular Genetics of Colorectal Cancer …………………………………………9
2.2 Folate Overview …………………………………………………………………………11
2.2.1 Chemistry of Folate ……………………………………………………………12
2.2.2 Folate Absorption and Metabolism ……………………………………………...15
2.2.3 Folate Metabolism, nucleotide synthesis and the methionine cycle …………….18
2.2.3.1 Thymidylate and purine synthesis ……………………………………19
2.2.3.2 SAM Regeneration, Methionine Cycle and DNA Methylation ………20
2.2.4 Biomarkers of Folate Status ……………………………………………………..21
2.2.5 Folate Intake ……………………………………………………………………..22
2.2.5.1 Folic Acid Fortification ………………………………………………25
2.2.5.2 Folic Acid Supplementation ………………………………………….26
2.2.6 Folate and Health ………………………………………………………………..26
2.2.6.1 Low Folate Status …………………………………………………….29
2.2.6.2 High Folate Status ……………………………………………………30
Folate and Masking of B12 Deficiency ………………………30
Folate and Unmetabolized Folic Acid ………………………..31
Folate and Cancer …………………………………………….32
2.3 Folate and Colon Cancer ………………………………………………………………….36
2.3.1 Evidence from Epidemiological Studies ………………………………………...36
2.3.2 Evidence from Intervention Studies ……………………………………………..42
2.3.3 Evidence from Animal Studies ………………………………………………….47
2.3.4 Summary of Folate and Colon Cancer Risk ……………………………………..49
2.4 5-methyltetrahydrofolate ………………………………………………………………….51
2.4.1 5-methyl-Ca+ ……………………………………………………………………51
2.4.2 Arguments for 5MTHF supplementation ………………………………………..52
2.4.3 5MTHF and 5MTHFR SNP ……………………………………………………..52
2.4.4 5MTHF and B12 ………………………………………………………………...53
vi
2.5 Folic acid vs. 5MTHF ……………………………………………………………………..54
Chapter 3: Rationale, Objectives, Hypothesis and Significance
3.1 Rationale ………………………………………………………………………………….55
3.2 Study Objective …………………………………………………………………………...56
3.3 Study Hypothesis …………………………………………………………………………56
3.4 Significance ……………………………………………………………………………….56
Chapter 4: The Effects of Folic Acid and 5MTHF Supplementation on the Progression
of Colon ACF to Adenomas and Adenocarcinomas in the AOM Rat Mode
4.1 Introduction ……………………………………………………………………………….58
4.2 Materials and Methods ……………………………………………………………………60
4.2.1 The AOM Rodent Model ………………………………………………………..60
4.2.2 Study Design …………………………………………………………………….61
4.2.3 AOM Administration ……………………………………………………………61
4.2.4 Experimental Diets ………………………………………………………………62
4.2.5 Sample Collection ……………………………………………………………….63
4.2.6 Folate Concentrations ……………………………………………………………64
4.2.7 Determination of Aberrant Crypt Foci …………………………………………..66
4.2.8 Histology ………………………………………………………………………...67
4.2.9 Statistics …………………………………………………………………………67
4.3 Results …………………………………………………………………………………….68
4.3.1 Animal Health and Body Weight ………………………………………………..68
4.3.2 Plasma Folate Concentrations …………………………………………………...69
4.3.3 Aberrant Crypt Foci ……………………………………………………………..70
4.3.4 Tumor Incidence and Multiplicity ………………………………………………71
4.3.5 Size of Tumors …………………………………………………………………..74
4.4 Discussion …………………………………………………………………………………76
4.5 Conclusion ………………………………………………………………………………...85
Chapter 5: Summary
5.1 Summary ………………………………………………………………………………….86
5.2 Future Directions ………………………………………………………………………….88
References ………………………………………………………………………………………89
Appendices ……………………………………………………………………………………107
A Nutrient composition of experimental L-amino acid defined diets for FA …….107
B Nutrient composition of experimental L-amino acid defined diets for 5MTHF
…………………………………………………………… ……………………..108
C Salt mix and vitamin mix compositions of experimental L-amino acid defined diet
…………………………………………………………………………………..109
vii
LIST OF TABLES
Table 2.1: Dietary and Lifestyle factors associated with decreased or increased CRC risk ……..7
Table 2.2: Characteristic of genes often associated with CRC …………………………….……10
Table 2.3 Localization and Affinity of main folate carrier, transporter and receptor ……………16
Table 2.4: Recommended daily allowance for folate …………………………………………...23
Table 2.5: Expression of FRα in normal and malignant human tissues …………….…………..35
Table 2.6: Summary of case-control studies of folate and colon cancer across populations ……37
Table 2.7: Summary of case-control studies of folate and adenoma risk ……………………….39
Table 2.8: Summary of cohort studies of folate and colon cancer risk …………………………40
Table 2.9: Summary of RCT of FA supplementation and biomarkers of CRC risk …………….43
Table 2.10: Summary of RCT of FA supplementation and colorectal adenoma recurrence ……44
Table 2.11: Summary of RCT of FA supplementation and cancer incidence as secondary
endpoint …………………………………………………………………………………………45
Table 2.12: Summary of meta-analyses consisting of both epidemiological and intervention-
based studies. ……………………………………………………………………………………46
Table 2.13. Summary of animal studies of FA supplementation and adenoma or colorectal cancer
incidence ………………………………………………………………………………………...50
Table 4.1: Effect of FA and 5MTHF supplementation on the development of colorectal adenoma
and adenocarcinomas. …………………………………………………………………………...71
Table 4.2: Summary of Results ………………………………………………………………….75
viii
LIST OF FIGURES
Figure 2.1: Chemical structure of folic acid and 5MTHF ……………………………………….14
Figure 2.2: Summary of folate absorption and metabolism …..…………………………………17
Figure 2.3: Biochemical functions of folate. …………………………………………………….18
Figure 2.4: Cumulative percentile distribution of red blood folate concentrations by age group
among female participants in the Canadian Health Measures Survey …………………………..24
Figure 2.5: Mean percentiles of dietary and total FA intake in the United States. ………………27
Figure 2.6: The dual modulatory role of folate …………………………………………………..48
Figure 4.1: Experimental Study Design …………………………………………………………64
Figure 4.2: Effect of dietary folate supplementation on body weight …………………………...68
Figure 4.3: Effects of FA and 5MTHF supplementation on plasma folate concentrations. ……..69
Figure 4.4: Effect of FA and 5MTHF supplementation on total number of ACF. ……………...70
Figure 4.5: Effect of FA and 5MTHF supplementation on incidence of colonic neoplasms …..72
Figure 4.6: Distribution of signet ring cell carcinoma among the four dietary groups with respect
to total number of adenocarcinomas. ……………………………………………………………72
Figure 4.7: Effect of FA and 5MTHF supplementation on the mean number of adenomas …….74
Figure 4.8: The Effect of FA and 5MTHF supplementation on the sum of tumor diameter in all
animals. ………………………………………………………………………………………….74
Figure 4.9: The Effect of FA and 5MTHF supplementation on the sum of tumor diameter in
tumor bearing animals. …………………………………………………………………………..75
ix
LIST OF ABBREVIATIONS
5-MTHF 5-methyltetrahydrofolate
ACF Aberrant Crypt Foci
APC Adenomatous Polyposis Coli
ANO VA analysis of variance
BDR Basal dietary requirement
CpG Cytosine preceding guanine
CRC Colorectal cancer
DFE Daily Folate Equivalent
DHF Dihydrofolate
DHFR Dihydrofolate reductase
DNMT DNA methyltransferase
dTMP Ddeoxythymidine monophosphate or thymidylate
dUMP Deoxyuridine monophosphate
FA Folic acid
FAP Familial Adenomatous Polyposis
FR Folate receptor
GCPII Glutamate carboxypeptidase II
GGH γ-glutamyl hydrolase
GMP Guanosine monophosphate
Hcyst Homocysteine
MMR Mismatch Repair
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
THF Tetrahydrofolate
TS Thymidylate synthase
UL Upper tolerable intake
UMFA Unmetabolized folic acid
1
CHAPTER 1: INTRODUCTION
Folate is a water-soluble B vitamin (B9) that is found naturally in green leafy vegetables,
citrus fruits, legumes and organ meats (1). Folates are essential for human health and
development through their role in one-carbon transfer reactions involved in nucleotide
biosynthesis and biological methylation reactions (1, 4). As a result, folates play a critical role in
processes such as DNA replication, integrity and repair as well as DNA methylation (1, 4).
Folate deficiency in humans has been associated with a number of adverse health outcomes
including megaloblastic anemia, cognitive impairments, adverse pregnancy outcomes including
neural tube defects (NTD) and development of certain cancers (2, 9, 10). As such,
supplementation with folic acid (FA), the synthetic form of folate, has been suggested as an
effective way to prevent and treat the aforementioned disorders (3). One example of health
benefits of FA supplementation is the protective effect of periconceptional FA supplementation
on NTD risk (59) as well as the drastic decrease of NTDs in Canada following the mandated
public health initiative of FA fortification in 1998 (3).
FA fortification was intended to provide an additional 100-200 μg/day, but population
data suggest otherwise. Data from the Canadian Health Measures Survey conducted between
2007 and 2009 revealed folate deficiency in Canada was virtually non-existent and that
approximately 40% of the population exhibited red blood cell (RBC) folate concentrations well
above the high cut-off of 1360 nmol/L (97th percentile of RBC folate concentrations) (5). While
fortified foods have contributed to this overall increase in folate status to some degree, a number
2
of studies have shown that widespread supplement use is the most significant predictor of high
folate status post-fortification (5, 12).
It has been estimated that about 30-40% of the general North American population take
FA supplements containing a minimum of 400 μg and up to 1 mg FA, which is the tolerable
upper limit (UL) set by the Institute of Medicine (8, 21-25). Additionally, about 64-81% of
cancer patients and survivors take a dietary supplement in the range of 1-5 mg FA, well above
the tolerable UL (9). Although the protective effects of FA supplementation on NTDs are well
established (6-8), a growing body of evidence suggests that FA, particularly at high doses may
have adverse outcomes on human health, such as masking B12 deficiency, presence of
unmetabolized FA (UMFA) in circulation as well as promotion of existing pre-cancerous lesions
and cancers (13).
Perhaps the most studied relationship between folate and cancer risk is colorectal cancer
(CRC). CRC studies provide the most compelling epidemiological, intervention and animal
evidence supporting the role of folate in carcinogenesis. Animal studies in particular, have
demonstrated that folate plays a dual modulatory role, dependent on dose and stage of cell
transformation at the time of folate intervention (13). In normal cells, folate deficiency
predisposes cells to neoplastic transformation, whereas modest levels of FA supplementation
may prevent neoplastic transformation (13, 15). Once preneoplastic lesions are established, folate
deficiency suppresses, whereas FA supplementation promotes, progression of those lesions (13,
3
16). Whether the observed tumor promoting effect is specific to FA or high folate status in
general remains unresolved (3).
5-methyletetrahydrofolate (5MTHF), the predominant circulating form of folate, has been
proposed to be a safer means of providing supplemental folates (3). Clinical studies have
suggested that 5MTHF supplementation is, at least, as effective as FA in increasing blood folate
levels and reducing plasma concentrations of homocysteine, an inverse indicator of folate status
(3, 10, 13-18). A few studies have compared the effects of FA and 5MTHF supplementation on
folate metabolism and intracellular one carbon reactions (128, 129). One study in particular,
conducted on colon cancer cell lines, found greater cellular proliferation and higher intracellular
folate concentrations with 5MTHF supplementation compared to FA (129). It should also be
noted that there is currently no established UL for 5MTHF as high intakes of naturally occurring
folates were generally thought to be improbable and supplemental MTHF was unavailable due to
its unstable chemical structure. However, now that 5MTHF is commercially available in a more
stable form and speculated to provide a safer means of supplementation over FA, it is important
to define safe parameters of use.
Currently, no studies have been conducted to evaluate the comparative effects of FA and
5MTHF supplementation on colon cancer progression. Although animal studies have
demonstrated the effects of FA supplementation on the promotion of pre-existing lesions (2, 3,
58, 59, 105), the effects of 5MTHF are unknown. This is especially problematic considering the
dramatic increase in population folate status, particularly among colon cancer patients and
4
survivors (13). Additionally, about 35-50% of the population above the age of 50 have adenomas
and many more have aberrant crypt foci (ACF; earliest colon cancer precursor), many of which
are asymptomatic (61, 130). Given the dual modulatory role of FA supplementation, this poses a
risk. 5MTHF has been proposed to be a safer alternative supplemental form of folate, however
this is largely without proof and may not be true. This study aims to determine and compare the
effects of 5MTHF versus FA supplementation on the progression of colon cancer. A rodent
model using a chemical carcinogen, AOM, will be used to establish colon cancer precursors,
following which, supplemental diets will be administered, and colorectal tumours and their
parameters will be examined and compared. This study will help to elucidate the potential
differential effects of FA and 5MTHF at various equimolar doses and may provide a framework
for future studies aimed at assessing safe parameters of use.
5
CHAPTER 2: LITERATURE REVIEW
2.1 COLORECTAL CANCER
2.1.1 AN OVERVIEW OF COLORECTAL CANCER
Colorectal cancer (CRC) is the second most commonly diagnosed cancer in Canada
(131). It is the second leading cause of death from cancer in men and third leading cause of death
from cancer in women in Canada (131). According to the Canadian Cancer Society, in 2017
alone, there was an estimated 26, 800 new cases and 8, 400 deaths from colon cancer (1). It is
estimated that 1 in 13 Canadian men and 1 in 16 Canadian women will develop CRC during
his/her lifetime (131).
Since 1985, age-standardized incidence and mortality rates have steadily declined (131)
However, due to the growth and aging of the Canadian population, the number of new cases
continues to increase (130). Mortality rates, however, have steadily declined in both males and
females, likely due to a combination of improved prevention, early detection and treatment of
CRC (131).
The etiology of CRC involves a complex interaction between genetics and environmental
factors and can be classified as either familial or sporadic. Heritable CRC include familial
adenomatous polyposis, hereditary nonpolyposis CRC, Peutz-Jegher syndrome, MYH-associated
polyposis, and juvenile polyposis syndrome (24). Individuals with a family history of these
syndromes are at an increased risk of inheriting germline mutations in genes associated with
mismatch repair (MMR), APC (adenomatous polyposis coli), MYH and overall DNA
6
maintenance and integrity (38). Approximately 25% of CRC patients have a family history of
CRC, however the remaining 75% of CRC cases are said to be sporadic. These are more
spontaneous in nature and are thought to arise from an interaction between genetics, age and
environmental factors.
7
2.1.2 COLON CANCER RISK FACTORS
Non-modifiable risk factors involve genetic predisposition (CRC syndromes and family
history of CRC or other cancers), age, previous history of adenomas or CRC, and chronic
inflammatory bowel disease (21-23). Dietary factors are also thought to play a significant role in
the causation of CRC (13). Other life-style factors, such as physical activity and smoking have
also been noted to impact risk of developing CRC (23, 101).
Table 2.1: Dietary and Lifestyle factors associated with decreased or increased CRC risk
(132, 133)
Evidence Decrease Risk Increase Risk
Convincing Physical activity, dietary fiber Red meat, processed meat,
alcohol (men), body fatness,
abdominal fatness, adult
attained height
Probable Garlic, milk and calcium Alcohol (women)
Limited Non-starchy vegetables, fruit,
vitamin D, folate, selenium,
fish
Iron, cheese, animal fats,
sugars
No Conclusion Cereals, poultry, shellfish and other seafood, total fat, fatty
acid composition, caffeine, vitamin A, retinol, vitamin C,
vitamin E, meal frequency
8
2.1.3 COLON CANCER CARCINOGENESIS
CRC pathogenesis is characterized as a multi-step process. It starts with increased
proliferation and/or decreased apoptosis of epithelial cells, followed by adenoma formation and
dysplasia, invasion and finally metastasis (102).
Most human adenocarcinomas evolve from ACF (aberrant crypt foci), which are the
earliest known precursors of colon cancer (100). These were first observed in rodents as lesions
consisting of large, thick crypts (34). Similar ACF have been reported in human colonic mucosa
(36-38). Being able to quantify these ACF in animals provides useful information into
mechanisms associated with disease progression.
ACF have been classified as either dysplastic or nondysplastic (103, 104). Dysplastic
ACF are equivalent to microadenomas and are estimated to account for about 5% of all ACF
(104). Nondysplastic ACF have the histological appearance of crypt serration in the epithelium
folds which is a recognizable morphological alteration arising as a consequence of inhibition of
apoptosis. However, both dysplastic and nondysplastic ACF confer a high risk of developing
colon cancer with increases in size and crypt multiplicity.
9
2.1.4. MOLECULAR GENETICS OF COLORECTAL CANCER
The transformation of normal human colon epithelial cells to advanced cancer involves a
multitude of molecular genetic changes. In general, patients with familial adenomatous polyposis
inherit APC mutations and develop numerous dysplastic ACF, some of which progress as they
acquire further mutations. Tumors from patients with hereditary nonpolyposis CRC go through a
similar series of mutations. MMR deficiency accelerates the process of tumor progression and
causes microsatellite instability and chromosomal instability. Progression of sporadic CRC is the
result of the accumulation of accidental and sequential genetic events. Genes commonly mutated
in human cancer belong to one of three different classes: oncogenes, tumor suppressor genes and
mismatch repair genes. Table 2.2 outlines some of the characteristics of genes associated with
CRC.
Microsatellite instability and chromosomal instability are the two main types of genomic
instability (68). Chromosomal instability involves the loss or gain of chromosomes which leads
to abnormal DNA content (aneuploidy). This is characteristic of familial adenomatous polyposis
associated cancers due to germline mutations of the APC gene as well as about 80% of sporadic
CRC. The microsatellite instability pathway involves the loss of function of genes that repair
base pair mismatches occurring during the normal process of DNA replication. Hereditary
nonpolyposis CRC develops as a result of germline mutations of the MMR genes that promote
replication errors throughout the genome.
10
Table 2.2: Characteristics of genes associated with different stages of CRC
Gene Function Stage of CRC
Kirsten ras oncogene (K-ras) Encodes protein bound to a
guanine-triphosphate that
plays a role in the
transduction of extracellular
growth signals (116)
Early stage of CRC (117)
APC tumor suppressor gene Encodes APC protein,
regulates cell signal and
growth (118)
Early stage of CRC (119)
P53 tumor suppressor gene Temporarily arrests
progression of G1 cell cycle
to allow for DNA repair prior
to the initiation of DNA
synthesis
Benign progression and
malignant progression (119)
Mismatch repair (MMR) Corrects errors of DNA
replication (120, 121)
Polyps begin to form with
MMR gene mutations
11
2.2 FOLATE OVERVIEW
Folate, a water-soluble B vitamin, refers to a general class of compounds sharing similar
chemical structures and nutritional properties. These compounds are essential for human
development and health through their role in one-carbon transfer reactions involved in nucleotide
biosynthesis and biological methylation reactions (1, 4). Folate is critical for processes such as
cell division, DNA integrity and maintenance, and epigenetic modifications that regulate gene
expression (1, 4). Folate deficiency in humans has been associated with a number of negative
health outcomes and diseases including megaloblastic anemia, neuropsychiatric disorders,
cognitive impairment, congenital disorders, adverse pregnancy outcomes, and development of
certain cancers (2, 9, 10, 60).
Folic acid (FA), the synthetic form of folate is found most commonly in supplements and
in fortified foods. Naturally occurring folates, such as 5-methytetrahydrofolate (5MTHF) are
found naturally in green leafy vegetables, citrus fruits, liver and other organ meats (3).
12
2.2.1 CHEMISTRY OF FOLATE
Folate, a water-soluble B vitamin, refers to a general class of compounds sharing similar
chemical structures and nutritional properties. These compounds are composed of a pteridine
ring, para-aminobenzoic acid (PABA) and varying number of glutamate residues (63) (Figure
2.1). Despite being able to synthesize all of the individual components of folate, mammals lack
the enzyme required to couple the pteridine ring to PABA and are thus are unable to synthesize
folate de novo (1). Mammals must obtain folate from the diet, in food or supplemental form
(1,2).
Most naturally occurring folates have reduced pteridine rings and are therefore
susceptible to oxidative chemical rearrangements which consequently results in a loss of activity
(64). FA (the synthetic form of folate), on the other hand, has a fully oxidized pteridine ring and
one glutamate residue, thus increasing its bioavailability and stability in light and food
processing (65). Due to its low cost and high stability, FA is used most commonly in food
fortification and in supplements (66).
In order for FA to be incorporated in the folate pathway (Figure 2.3) and utilized for
cellular processes, it must first be reduced to dihydrofolate (DHF), then tetrahydrofolate (THF)
and then is methylated to produce 5MTHF (63, 66). 5MTHF, which is a naturally occurring form
of folate, can enter the folate pathway freely without being further reduced (63). FA and
naturally occurring folates (not including 5MTHF) are metabolized to 5MTHF by the enterocytes
and to a larger degree, the liver. The 5MTHF form of folate makes up 98% of the circulating
13
folate in the blood and is also the only form of folate (3) able to cross the blood brain barrier
(16).
5MTHF is also known as L-5-MTHF, (6S)-5-MTHF, and levomefolate. It has recently
become commercially available in supplemental form as a 5MTHF Ca+ salt (Metfolin®, Merk
Eprova AG, Switzerland). A distinction between the two 5MTHF enantiomers exists: L-5MTHF
is the active form of folate whereas D-5MTHF is the inactive form. Previously, both
diasterioisomers of 5MTHF were supplied due to instability. However, the Ca+ salt allows for an
increased stability and shelf life in the active form (17). It is also important to note that the
supplemental form of 5MTHF is manufactured to be monoglutamted (17) and as such is referred
to as synthetic-5MTHF by the FDA.
14
Figure 2.1: Chemical structure of folic acid and 5MTHF. Folic acid is portrayed at the top
with oxidized pteridine rings. The bottom portrays the structure of reduced folates and positions
of one carbon substitutions. In nature, 5MTHF is polyglutamylated but synthetic 5MTHF-Ca
(used commercially in supplements) is monoglutamated. (63)
15
2.2.2 FOLATE ABSORPTION AND METABOLISM
Folates are absorbed in the small intestine, specifically the acidic cell surface of the
duodenum and jejunum (70). Before folates enter the enterocyte, they are hydrolyzed into their
monoglutamated forms by glutamate carboxypeptidase II (GCPII). These monoglutamylated
folate forms are transported into the cell via folate carriers, transporters, and receptors: reduced
folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptors (FR). The
expression of these transport systems varies with tissue, and each transport system has varying
affinities for varying folate derivatives and optimally function at varying pH levels (71).
Once in the enterocyte, the monoglutamylated forms of folate take on one of two paths.
They are either polyglutamylated by FPGS (folylpolyglutamate synthase) which traps the folates
inside the cell, or they enter the portal hepatic circulation for distribution. Polyglutamylated
folates are better retained in cells and are better substrates for intracellular folate-dependent
enzymes, compared to monoglutamated forms (72). 5MTHF and FA are poor substrates for
FPGS (72). 5MTHF and FA must be converted to THF in order to be polyglutamylated (72).
Intracellularly, folates are converted to 5MTHF, which is the primary circulating form of folate.
Metabolism of folates to 5MTHF occurs as folates pass through the enterocytes. However,
reduction and conversion to 5MTHF can also occur in the liver (73). In order to leave the cell,
polyglutamylated folates are hydrolysed by gamma-glutamyl hydrolase (GGH) to the
monoglutamylated form (74). Folate export is mediated by RFC on the basolateral membrane
16
(66). The below table outlines the affinities of FA and reduced folate (5MTHF) to folate carrier,
transporter and receptor.
Table 2.3 Localization and Affinity of main folate carrier, transporter and receptors (62,
63, 66, 70)
Localization Affinity
Reduced folate carrier (RFC) All tissues and cell lines;
specifically, in the liver,
kidney and jejunum
High affinity for reduced
folates
Low affinity for FA
Proton coupled folate
transporter
(PCFT)
Small intestine, kidney,
colon, liver, brain
High affinity for both reduced
folates and FA
Folate receptors
(FRα, FRβ, FRγ)
Uterus, kidney, liver, placenta
choroid plexus
FRα: High affinity for all
forms of folate
FRβ: High affinity for
reduced folate
17
Figure 2.2: Summary of folate absorption and metabolism. Folate hydrolysis occurs via
GCPII (glutamate carboxypeptidase II) activity. Folate uptake occurs via FR (folate receptor),
PCFT (proton coupled folate transporter) and RFC (reduced folate carrier) at the cell membrane.
Folate retention is mediated by FPGS (folypolyglutamyl synthase) while hydrolysis and efflux
are mediated by GGH (y-glutamyl hydrolase). Each green triangle represents a glutamate
residue, which is linked via a peptide bond to form a polyglutamated folate. Adapted from (137)
In addition to the aforementioned carriers, receptors and transporters, the small intestine
also expresses OATP (organic, anion-transporting polypeptide), BCRP (breast cancer resistance
protein) and MRP (multidrug-resistance-associated protein) (141). The OATP2B1 mediates
some folic acid and 5MTHF transport, however antifolates seem to be a better substrate, which
make this family of transporters a topic of increasing interest in the context of drug interaction
and drug delivery (141). The BCRP and MRP2 transporter are both ATP binding transporters
that have the capacity to oppose the inward flux of folates mediated by PCFT (142). These
transporters pump folate from the enterocytes back into the intestinal lumen (142). However, the
extent to which these transporters influence net folate absorption remains unclear (142).
18
2.2.3 FOLATE METABOLISM, NUCLEOTIDE SYNTHESIS AND THE METHIONINE CYCLE
Folates are involved in one-carbon transfer reactions, which are important for cell growth
and division. This process mediates de novo synthesis of thymidylate and purine nucleotides
which are important substrates for DNA methylation and other biological methylation reactions.
Figure 2.3 outlines the biochemical functions of folate.
Figure 2.3: Biochemical functions of folate. Within the cell, folate exists as one of three active
metabolites (red): tetrahydrofolate (THF), 5,10-methyleneTHF and 5-methylTHF, and have a
role in nucleotide biosynthesis and biological methylation reactions. Dihydrofolate reductase
(DHFR) and serine hydroxymethyltransferase (SHMT) are involved in the maintenance of the
intracellular folate pool. Thymidylate synthase (TS) is involved in nucleotide biosynthesis.
Methytetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are involved in the
methionine cycle. DNA is methylated by DNA methyltransferases (DNMTs) and unmethylated
by DNA demethylase. DHF=dihydrofolate, dUMP=deoxyuridine-5-monophosphate,
dTMP=deoxythymidine-5 monophosphate, DMG=dimethylglycine, SAM=S-
adenosylmethionine, SAH=S adenosylhomocysteine. Adapted from (137).
19
2.2.3.1 THYMIDYLATE AND PURINE SYNTHESIS
DHF (which is produced via the conversion of dUMP to dTMP) is reduced to regenerate
THF (refer to figure 2.3) by DHFR (62). THF can either re-enter the nucleotide biosynthesis
pathway or be used to regenerate 5MTHF via conversion to 5,10-methyleneTHF by serine
hydroxymethyltransferase (SHMT), which is then further reduced by 5,10-
methylenetetrahydrofolate reductase (MTHFR) to yield 5MTHF (66, 62).
5MTHF donates a carbon to the biological methylation pathway (described in section
2.2.3.2.). This yields THF, which plays an important role in the building blocks of DNA and
RNA via its role as a substrate for pyrimidylate and purine synthesis. THF and serine are
catalyzed via SHMT, in a reversible conversion, to form 5,10-methyleneTHF and glycine (66, 8).
5,10-methyleneTHF transfers a methyl group to deoxyuridine monophosphate (dUMP) yielding
dTMP (a precursor of pyrimidylate biosynthesis) and DHF by thymidylate synthase (TS) (75).
The products from the previous cycle, THF and 5,10-methyleneTHF also contribute to purine
synthesis once they are formylated. THF and serine form 5, 10-methyleneTHF and glycine by
SHMT. 5, 10-methyleneTHF can also be converted to 5,10-methyenylTHF and then to 10-
formylTHF by MTHFD1 or MTHFD2 (methylenetetrahydrofolate dehydrogenase) (75). In
purine synthesis, the carboxyl groups of two 10-formylTHFs are required to yield IMP (inosine
monophosphate), a precursor to the purines AMP (adenosine monophosphate) and GMP
(guanosine monophosphate) (75).
20
2.2.3.2 SAM REGERNATION, METHIONINE CYCLE AND DNA METHYLATION
5MTHF is the only folate form able to participate in the regeneration of S-
adenosylmethionine (SAM). SAM is a universal methyl donor for biological methylation
reactions including DNA methylation (37, 72). 5MTHF donates a methyl group for the
transmethylation reaction governing the conversion of homocysteine to methionine. This
reaction is catalyzed by methionine synthase (MS), a vitamin B12-dependent enzyme (8, 63, 72).
Methionine is then converted to SAM by methionine S-adenosyltransferase (MAT) or SAM
synthase (73, 76, 30). DNA methylation is mediated by DNA methyltransferases (DNMT1,
DNMT3a, and DNMT3b) (71). DNMT1 is believed to maintain DNA methylation patterns
following DNA replication (76,29) and DNMT 3a and 3b are capable of de novo methylation
(77). Methyl binding protein 2 (MBD2) can act as both a DNA methylation-dependent repressor
and activator of genes silenced by methylation (78). De novo de-methylation is mediated by ten
eleven translocation (TET) 1 protein which oxidizes methylated cytosines to 5-
hydroxymethylcytosine, the first step in DNA de-methylation (81, 94, 95).
21
2.2.4 BIOMARKERS OF FOLATE STATUS
Serum and RBC folate concentrations, in addition to homocysteine concentrations can be
used to determine folate status (68). Serum folate concentrations are reflective of short-term
dietary intake whereas RBC folate concentrations reflect long-term status. RBC folate
concentrations are considered more reflective of tissue folate stores (1). RBC folate is directly
related to bone marrow folate stores at the time of erythropoiesis and the 120-day turnover rate
of RBC makes this measure resistant to short-term folate variation (1, 4). This measure is also
used to diagnose clinical folate deficiency. According to WHO standards, RBC folate
concentrations of <340 nM or serum folate concentrations of <10 nM observed repeatedly over a
1-month period are indicative of folate deficiency (69). There are currently no established high
cut-offs for RBC and serum folate concentrations. The 97th percentile of RBC folate
concentrations (1360 nM) in the 1994-2004 National Health and Nutrition Examination Survey
(NHANES) has been used as an arbitrary cut off by Colapinto et al. (4, 5).
Homocysteine is a nonspecific inverse functional indicator of folate status and at
concentrations above 16 μM can indicate folate deficiency (1, 3-5). Folate, in the form of
5MTHF, is required to remethylate homocysteine to methionine. During folate deficiency, there
is reduced conversion of homocysteine to methionine, thereby increasing homocysteine
concentrations (1, 4, 5). Elevated homocysteine concentrations can also indicate inadequate
vitamin B12 and vitamin B6 status (1, 4). Renal dysfunction and aging can also raise
homocysteine concentrations (1, 4, 5).
22
2.2.5 FOLATE INTAKE
As mentioned earlier, mammals lack the enzyme required to couple the pteridine ring to
PABA and are thus unable to synthesize folate de novo. For this reason, mammals must obtain
folate from the diet, in food or supplemental form (1, 2, 4). Table 2.3 outlines the recommended
dietary allowance (RDA) as outlined by the Institute of Medicine (4, 5). DFE is a measure for
folate intake which takes into consideration the nearly 50% lower bioavailability of food folate
compared to folic acid (4). One DFE is equal to 1μg of naturally occurring dietary folate, 0.6 μg
of FA when taken with food, or 0.5 μg of FA on an empty stomach (1, 4, 5). There is currently
no tolerable upper limit for folate intake, including 5MTHF and its supplements, however the
Institute of Medicine suggests no more than 1 mg/day FA from fortified foods and supplements.
This guideline is deemed acceptable to prevent masking vitamin B12 deficiency (4, 5). Whether
or not this guideline holds true for 5MTHF supplemental use has not yet been established.
Currently, there is no published guideline for effective conversion factors governing 5MTHF
supplements to their expressed μg DFE equivalents. However, FDA suggests 5MTHF
supplements (both branded and generic calcium salts) to use the same conversion factor as that
used for folic acid (1 μg 5MTHF = 1.7 μg DFE) (142).
More recent recommendations suggest that the dietary reference intakes for
micronutrients be reconsidered to better explain risk factors for chronic disease (137, 143). This
refers to the chronic disease risk reduction index (CDRR) which takes into account the strength
of evidence for both a causal and dose dependent relationship of a micronutrient and chronic
23
disease outcome (137). Currently, there is no CDRR for folate/folic acid (137, 143). However, if
the risk of chronic disease is found to be causally related to intake, the DRI should be updated
and the CDRR should be established.
Table 2.4: Recommended daily allowance for folate.
Population Children Adults
Demographic 1-3 4-8 9-13 14-18 18+ Pregnant Lactating
RDA (μg DFE/day) 150 200 300 400 400 600 500
Note: values are defined as DFE (daily folate requirements), which accounts for the
variability in bioavailability and stability of naturally occurring folate. RDA refers to the
recommended daily allowance, which is the average daily intake sufficient to meet the nutritional
requirements of nearly all healthy individuals in a group.
Dietary intakes and blood folate measurements in the US and Canada have increased
dramatically over the past two decades owing mostly to the widespread use of FA supplements,
but also to some degree, fortification. The Canadian Health Measures Survey (CHMS), reported
that folate deficiency (red blood cell folate concentration < 305 nmol/L) is present in less than
1% of the population, and 40% have high folate concentrations (red blood cell folate
concentration > 1360 nmol/L) (5) (Figure 2.4).
24
Figure 2.4: Cumulative percentile distribution of red blood folate concentrations by age
group among female participants in the Canadian Health Measures Survey. Folate
concentrations for deficiency (305 nmol/L) and high folate concentrations (1360 nmol/L) are
indicated by vertical lines (5). Copied under license from the Canadian Medical Association and
Access Copyright.
25
2.2.5.1 FOLIC ACID FORTIFICATION
FA fortification was first mandated in 1998, after an overwhelming body of evidence
from several epidemiological and intervention studies showing the protective effects of FA
supplementation against neural tube defects (NTDs) (2, 60). The Medical Research Council
Vitamin Study; a multi-centered, double blinded, randomized control trial with pregnant women
considered high-risk of having NTDs, showed a near 72% protective effect with a daily
intervention of 4mg FA (6). As a result, the Canadian and US government-initiated fortification
of all white flours, cornmeal and pasta. In Canada, 150 ug FA per 100g product is added, and is
intended to provide an additional 100-200 ug FA daily. The effectiveness of this can be seen in
the improvements of folate status as well as the significant reductions in the incidences and
prevalence of NTDs in the US and Canada (2, 7-9, 60, 98).
26
2.2.5.2 FOLIC ACID SUPPLEMENTATION
Among the general population, the use of a daily folate acid containing multivitamin or
folate alone has become a habitual practice. According to data from the NHANES and the
Canadian Community Health Survey, up to 50% of adults regularly use a dietary supplement
(10). Data from the United States further state that 70% of the population over 70 years of age
also take at least one dietary supplement per day (11). A number of studies show that individuals
amongst the highest percentile of total folate and FA intake have greater contributions from
dietary supplements (12) (Figure 2.5). In fact, 40% over the age of 60 years have detectable
levels of unmetabolized FA (UMFA) which persist after fasting (12). Studies have also shown
that about 10% of FA supplement users in the general population have daily intake of folate
exceeding the Dietary Reference Intakes upper limit of 1 mg/day (3). A number of studies also
show that supplement use is the most significant predictor of folate status, not diet (5, 12).
27
Figure 2.5: Mean percentiles of dietary and total FA intake in the United States. Dietary FA
is from fortified foods, and total FA includes fortified foods and dietary supplements. As total
FA intake increases, the proportion contributed to by supplement use increases almost
exponentially (12).
The use of FA supplements is widespread among cancer patients and survivors. A
systematic review (13) summarized 32 studies between 1999-2006 and found that regular
supplement use ranged from 64 to 81% among cancer survivors, which is a higher prevalence
than the general population of 50% (14, 15). Furthermore, over 50% of CRC patients report
taking a daily supplement during the course of their treatment (16). Supplemental levels of folic
acid in the range of 1-5 mg/day are routinely provided to certain subgroups of patients who are
taking antifolate based medications (e.g. methotrexate) to prevent adverse effects relating to
folate depletion (3).
28
2.2.6 FOLATE AND HEALTH
Mandated FA fortification and prevalent supplemental use have increased the intake and
blood levels of folate in North America including those of cancer survivors (3). Although FA
fortification has achieved its primary objective, that is decreasing the rate of NTDs, and FA
supplementation may provide several health benefits, an emerging body of evidence suggests
that high folate status, primarily from high FA intake, may be associated with adverse health
outcomes (8, 72).
A large number of epidemiological studies report an association between low folate status
and increased risk of megaloblastic anemia, neural tube defects and other adverse birth outcomes
(3). However, there is a growing body of evidence suggesting that FA supplementation at high
doses has adverse outcomes, including masking of vitamin B12 deficiency as well as the
potential negative effects associated with detectable levels of unmetabolized folic acid (UMFA)
(1, 17, 138). Furthermore, its effects on cancer, and whether or not supplementation is cancer
preventative or promoting, remains a topic of increasing interest.
29
2.2.6.1 LOW FOLATE STATUS
Folate deficiency can occur as a result of any of the following: insufficient dietary intake,
impaired folate absorption and metabolism, and/or increased demand/utilization (1, 15). Well-
known contributors to folate deficiency include: lack of folate in diet, gastrointestinal disorders
such as celiac disease which can impair folate absorption, chronic alcoholism, conditions such as
inflammatory bowel, Crohn’s disease or pregnancy which increase the rates of cell turnover thus
increasing folate requirements, and drugs such as antifolates used in chemotherapy as well as
certain anti-epileptic and anti-inflammatory medications that can interfere with folate
absorption/metabolism (1, 8, 15, 78, 97). Folate is essential for nucleotide biosynthesis and
biological methylation reactions. Folate deficiency has been associated with many diseases such
as megaloblastic anemia, cardiovascular disease, cognitive impairments, neural tube defects
(NTDs), and cancer (66).
30
2.2.6.2 HIGH FOLATE STATUS
FA supplementation has been used to prevent and treat some of the above-mentioned
conditions associated with folate deficiency. FA supplementation is highly effective and
successful in treating megaloblastic anemia (72, 79) and especially preventing NTDs (80-82). FA
supplementation has been generally regarded as safe and has long been presumed to be purely
beneficial (83). However, supplementation, particularly at high doses has been linked to adverse
health outcomes, including the masking B12 deficiencies leading to an accelerated risk of
neurocognitive impairments, accumulation of unmetabolized folic acid (UMFA), decreased
natural killer cell cytotoxicity and cancer promotion (7, 66, 84, 99).
Folate and Masking of B12 Deficiency
Vitamin B12 is an essential cofactor, involved in the conversion of 5MTHF and
homocysteine to THF and methionine (Figure 2.3). When FA levels are high, potential B12
deficiencies can be masked. Although B12 deficiency is uniform among the Canadian population
(41), the Institute of Medicine recommends that adults over the age of 50 years achieve their B12
intake through supplements or fortified foods (1, 4). Due to this recommendation, the elderly are
more likely to be exposed to higher FA intakes from supplements and from their diets, which are
generally high in grain products (5, 66). This poses a risk, as it can lead to a delayed diagnosis
and impaired cognitive function (136). A prospective study by Morris et al showed that rates of
cognitive decline were highest among those taking additional 400 ug FA supplement (135).
31
Proposed mechanisms suggest that an abundance of FA allows for continuous production
of nucleotides (via the 5,10 methylene THF to purine and pyrimidine synthesis pathway) but
impairs DNA methylation pathway since SAM regeneration is dependent on B12, and thus
cannot occur during B12 deficiency (1, 4) (Figure 2.3). 5MTHF has been proposed to be a more
advantageous supplement, however many have also proposed that supplementing with 5MTHF
would result in a methyl folate trap during B12 deficiency (4), resulting in an accumulation of
unusable 5MTHF (Figure 2.3).
Folate and Unmetabolized Folic Acid
High doses of FA can also result in the appearance of and accumulating levels of UMFA
in circulation (19, 20, 90). When FA is metabolized, it undergoes a series of reductions, one of
which is governed by the enzyme dihydrofolate reductase (DHFR) (1). This enzyme is
responsible for converting FA to dihydrofolate (DHF) and subsequently to THF, which becomes
the metabolically active 5MTHF form (1, 4-6). The DHFR enzyme, however, has a relatively
low capacity for FA biotransformation (150). This ultimately results in an accumulation of
UMFA in the circulation which does not occur after consumption of naturally occurring food
folates (150, 138). It has been suggested that high levels of UMFA could interfere with the
metabolism, cellular transport and regulatory functions of the natural folates by competing with
the reduced forms for binding with enzymes, carrier proteins, and binding proteins (1, 4). Other
reported adverse effects associated with UMFA include epigenetic instability, decreased natural
32
killer cell cytotoxicity, progression of preneoplastic lesions and disruptions in methylation
patterns (1, 4, 90, 139). Mechanistic explanations are currently lacking, however, UMFA’s have
been suggested to be converted to the reduced forms of folates by peripheral tissues (137). It has
also been proposed that the presence of folate receptor 4 (a subtype of folate receptor) in the
immune systems’ regulatory T cells may be involved in the mechanism governing decreased
natural killer cell cytotoxicity in response to high levels of UMFA (144). However, more studies
are needed to confirm this mechanism of action.
Folate and Cancer
Perhaps the most controversial relationship between folate and health is cancer risk.
Cancers of the lung, prostate, esophagus, stomach, pancreas, breast and colorectum have been
investigated in relation to folate intake (89). In general, epidemiologic studies have suggested
folate deficiency to increase the risk of several of the aforementioned cancers, while FA
supplementation may reduce the risk (86, 3). However, counterintuitive to this idea, is the nature
of antifolate based medications for cancer treatments, which are based on the idea of folate
depletion to disrupt intracellular folate mechanisms resulting in decreased substrates for
nucleotide biosynthesis and ultimately preventing cell replication and cancer proliferation (3).
Additionally, details of this inverse relationship are not yet well established for all cancer types
(3). CRC is without a doubt the most studied cancer and provides the most compelling
epidemiologic, clinical and animal evidence supporting the role of folate in carcinogenesis.
33
There are several proposed mechanisms relating to the role of folate, its role in one-
carbon transfer reactions, DNA synthesis and epigenetic regulations all of which aim to detail the
dual effects of folate on cancer development and progression. The first is folate’s role in DNA
synthesis and repair (8, 40). In normal tissues, folate ensures the fidelity of DNA replication and
stability (8, 72). However, folate deficiency can lead to DNA strand breaks, chromosomal and
genomic instability, uracil misincorporation, increased mutations, and impaired DNA repair (8,
91-93). FA supplementation can prevent some of these defects (8, 91-93). In contrast, in
preneoplastic cells (or transformed cells) where DNA replication and cell division are occurring
at an accelerated rate, folate deficiency can result in ineffective DNA synthesis, resulting in
inhibition of tumor growth and progression (8, 93, 105-107). The most likely mechanism by
which FA supplementation may promote the progression of established preneoplastic lesions is
by providing the nucleotide precursors needed for accelerated cell proliferation and thus cancer
progression (66).
The second mechanism relates to folate’s role in DNA methylation (86). The 5MTHF
form of folate, is involved in the remethylation of homocysteine to methionine, which is a
precursor of SAM, the primary methyl group donor for most biological reactions including DNA
methylation (15). Aberrant DNA methylation due to folate status can contribute to
carcinogenesis through its effects of global DNA hypomethylation, gene-specific
hypermethylation and/or hypermutability (86). Global DNA hypomethylation is associated with
genomic instability (86). DNA hypermethylation at promotor and/or regulatory regions of tumor
34
suppressor and MMR genes can result in gene silencing (86). Hypermutability of methylated
cytosine can also contribute to carcinogenesis (86).
In normal cells, folate deficiency can result in global DNA hypomethylation resulting in
genomic instability, leading to neoplastic transformation (83, 86). In transformed cells, however,
folate deficiency can suppress tumor progression by reversing promoter cytosine-guanine (CpG)
island DNA methylation of tumor suppressors and other cancer related genes leading to the
reactivation of these epigenetically silenced genes (86, 94). In normal cells, FA supplementation
can prevent global DNA hypomethylation, thus reducing the risk of neoplastic transformation by
ensuring genomic stability (86). In transformed cells, however, FA supplementation can promote
tumor progression by inducing de novo methylation of promoter CpG islands of tumor
suppressors and cancer related genes, in turn silencing these genes (86). However, the effects of
folate deficiency and FA supplementation on DNA methylation are highly complex as they are
gene and site-specific and dependent on species, cell type and stage of transformation as well as
timing and duration of folate intervention (83, 86, 63, 94).
The role of folate receptors in cancer development, progression and treatment has
become a topic of increasing interest. Folate receptors are membrane found proteins that have
high levels of affinity for binding and transporting folate into the cell. These receptors have three
isoforms; FRα, FRβ, FRγ, however it is the alpha isoform that has been most widely studied
(122). The FRα has been suggested to have a greater growth advantage to tumors via regulation
of folate uptake or via cell signaling pathways. Increased growth and folate accumulation by
35
cancer cells with elevated FRα suggests that this may be another mechanism by which folate
may promote the progression of neoplastic cells in humans (122, 123). In vitro studies have
suggested that the expression of the FRα gene is regulated by extracellular folate depletion,
increased homocysteine accumulation, and possibly genetic mutations (122, 124). This concept
has led to the development of more targeted therapies involving the FRα. The overexpression of
this isoform in conjunction with its relatively restricted distribution in normal tissues renders it a
viable therapeutic target (125). The below table outlines the expression of FRα in human
malignant tissues.
Table 2.5: Expression of FRα in normal and malignant human tissues (126)
Normal tissues Malignant tissue
(high expression)
Malignant tissue
(low expression)
Uterus, kidney, liver, placenta
choroid plexus
Cervical carcinoma, uterine
carcinoma, metastatic
endometrial carcinoma,
pancreatic carcinoma, renal
cell carcinoma
Lung carcinoma and
adenocarcinoma, breast
carcinoma, colorectal
carcinoma, liver carcinoma,
prostate carcinoma
36
2.3 FOLATE AND COLON CANCER
2.3.1 EVIDENCE FROM EPIDEMIOLOGICAL STUDIES
Although epidemiological evidence has been inconsistent, in general, the association
between folate intake and CRC appear to be an inverse relationship where the greater the intake
the lower the risk (Table 2.5-2.7). Overall, there is a 20-40% decreased risk of CRC and its
precursor, adenoma when those with highest intake of folate are compared to those with the
lowest (21-23, 104, 105, 107). The relationship between blood levels of folate and risk of CRC
and adenoma is less well defined than that between dietary intake and risk of CRC and adenoma
(2, 3, 105, 106).
37
Table 2.6: Summary of case-control studies of folate and colon cancer across populations
Reference Characteristics Cases/Controls Folate source OR, HR (95% CI) Significance
Freudenheim et al
1991
USA
Caucasian
428/428 Dietary folate
<210 vs. >340 ug/d
M: 1.03 (0.56-1.89)
F: 0.69 (0.36, 1.30)
N.S.
N.S.
Benito et al
Majorca, Spain
50-74 yr
286/296 Dietary folate
<141 vs. >222.3 ug/d
0.27 (N.A.)
P<0.01
Ferraroni et al
1994
Italy
20-74 yr
Median age 62 years
828/1189 Dietary folate
<162 vs. >227 ug/d
0.52 (0.40-0.68)
P<0.05
Slattery et al
1997
USA
30-79 yr
men and women
1993/2410 Dietary folate
<120 vs. >210 ug/d
1.20 (0.90-1.60)
N.S.
N.S.
Jiang et al
2004
China 73/343 Dietary folate
<115.64 vs. >172 ug/d
0.91 (0.70-1.19)
N.S.
White et al
1997
USA
30-62 yr
M: 251/233
F: 193/194
Supplemental folate
0 vs. 400 ug/d
M: 0.59 (0.34-1.01)
F: 0.44 (0.24-0.80)
P=0.04
P=0.007
Le Marchand et al
2002
USA
The Multiethnic Cohort Study
45-75 yr
822/2021 Total folate
<297 vs. >2430 ug/d
Dietary folate
<252 vs. >406 ug/d
0.80 (0.58-1.10)
0.90 (0.62-1.30)
N.S.
N.S.
Lightfoot et al
2010
United kingdom
45-80 yr
124/128 Total folate
267 vs. >397 ug/d
1.08 (0.78-1.50)
N.S.
Sharp et al
2008
United kingdom 255/398 Total folate
<263.9 vs. >348.6 ug/d
1.37 (0.80-2.36)
N.S.
Kim et al
2009
Korea
30-79 yr
787/656 Total folate
<209.69 vs. >282.72 ug/d
0.64 (0.49-0.84)
P=0.002
Kato et al
1999
Women’s Health Study
45 yr
Female
105/523 Serum folate
12.23 vs. 31.04 nM/L
Total folate
224 vs. 626 ug/d
0.52 (0.27-0.97)
0.88 (0.46-1.69)
P=0.04
N.S.
Otani et al
2005
Japan
40-69 yr
375/750 Plasma folate
<5.6 vs. >8.6 ng/mL
M: 0.86 (0.45-1.60)
F: 1.00 (0.56-1.90)
N.S.
N.S.
38
Glynn et al
1996
Finland
ATBC Cancer Prevention
Study
50-69 yr
Male Smokers
144/276 Serum folate
2.9 vs. >5.2 ng/mL
0.51 (0.20-1.3)
4.79 (1.36-16.93)
N.S.
P<0.05
Satia-Abouta et al
2003
USA /North Carolina
30-80 yr
Caucasian
African American
C: 337/596
AA: 276/400
Total folate
<196 vs. >741 ug/d
C: 0.8 (0.5-1.2)
AA: 0.9 (0.5-1.6)
N.S.
N.S.
Van Guelpen et al
2006
Northern Sweden Health and
Disease Cohort
226/437 Plasma folate
<5.7 vs. >13 nmol/L
3.87 (1.52-9.87)
P=0.007
Baron et al
USA
Median age 60 yr
260/449 Dietary folate
<214 vs. >388 ug/d
Total folate
<243 vs. >391 ug/d
0.94 (0.53-1.67)
1.11 (0.69-1.78)
N.S.
N.S.
Meyer et al
1993
USA
30-62 yr
M: 238/224
F: 186/190
Total folate
M: <151 vs. >281 ug/d
F: <131 vs. 276 ug/d
1.00 (0.81-1.24)
0.81 (0.66-1.00)
N.S.
P<0.05
Pufulete et al
2005
United kingdom 28/76 Total folate
<260 vs. >348 ug/d
0.09 (0.01-0.57)
P=0.01
Levi et al
2000
Switzerland
27-74 yr
119/491 Total folate
<173 vs. >403 ug/d
1.54 (0.80-3.1)
N.S.
Murtaugh et al
2007
United states
Men and women
751/979 Plasma folate
<441 vs. >742
0.66 (0.47, 0.92)
P<0.05
39
Table 2.7: Summary of case-control studies of folate and adenoma risk
Reference Characteristics Cases/Controls Folate source OR (95% CI) p-trend
Pufulete et al United Kingdom 35/76 Total folate
<282 vs. >359 ug/d
0.98 (0.30-3.22)
N.S.
Han et al USA
Prostate, Lung,
Colorectal, Ovarian
Cancer Screening Trial
Study
55-74 yr
1331/1501 Dietary folate
262 vs. >466 ug/d
1.46 (1.17-1.92)
P<0.001
Giovannucci et al
1998
USA
Nurses’ Health Study
Health Professionals’
Follow-Up Study
M: 331/9159
F: 564/15420
Total folate
M: <241 vs. >847 ug/d
F: <166 vs. >711 ug/d
0.63 (0.41- 0.98)
0.66 (0.46-0.095)
P=0.03
P=0.04
Bird et al
USA
50-75 yr
M: 180/189
F: 152/161
Erythrocyte folate
<165 vs. >315 ng/mL
Plasma folate
3 vs. 16.9 ng/mL
Total folate intake
<242 vs. >576 ug/d
M: 0.47 (0.24-
0.90)
F: 1.26 (0.65-2.43)
M: 0.65 (0.45-
0.95)
F: 0.95 (0.69-1.30)
M: 0.70 (0.36-
1.34)
F: 1.47 (0.73-2.95)
P=0.02
N.S.
P=0.04
N.S.
N.S.
N.S.
40
Boutron-Ruault et
al
1996
France
30-79 yr
1: <10mm adenoma
(n=154)
2: large adeoma (n=208)
3: polyp free (n=426)
subjects recruited after
colonoscopy
Total folate
<218 vs. >402.5 ug/d
1 vs. 3: 0.5 (0.3-
1.0)
2 vs. 1: 0.9 (0.4-
1.9)
2 vs. 3: 0.5 (0.3-
1.0)
P=0.3
N.S.
P=0.04
Benito et al Majorca 101/242 Dietary folate
<146 vs. >227 ug/d
0.27 (N.A.)
P<0.01
Table 2.8: Summary of cohort studies of folate and colon cancer risk
Reference Characteristics # Cases Outcome Folate source RR (95% CI) P-value
Su et al
2000
NHANES I Epidemiologic
Follow-up study
Men
United states
219 CC Dietary folate
103.3 vs. >249 ug/d
M: O.40 (0.18-
0.88)
F: 0.74 (0.36-1.51)
P=0.03
N.S.
Giovannucci
et al
1995
Health Professionals
Follow-up study
51 529 men
40-75 yr
205
6 yr follow
up
Dietary folate
<269 vs. >646 ug/d
0.86 (0.50-1.47)
N.S.
Zhang et al
2005
WHS
39 876 women
>45 yr
220
10.1 yr
follow up
CRC Total folate
<259 vs. 614 ug/d
Dietary folate
<244 vs. 285 ug/d
Dietary folate (excluding
supplement users)
<244 vs. 385 ug/d
1.16 (0.76-1.79)
0.67 (0.43-1.03)
0.46 (0.26-0.81)
N.S.
N.S.
P=0.02
Konings et
al
2002
Netherlands Cohort Study
120 852
55-69 yr
1171
7.3 yr
follow up
CRC Total folate
<168 vs. >266 ug/d
M: 0.73 (0.46-1.17)
F: 0.68 (0.39-1.20)
P=0.03
N.S.
Harnack et
al
2002
Iowa Women’s Health
Study
41 836
598 Total folate
32.1 vs. >2555.2 ug/d
1.12 (0.77-1.63)
N.S.
41
55-69 yr
Flood et al
2002
Breast Cancer Detection
Demonstration
Project Follow-up Cohort
45 264 women
490
8.5 yr
follow up
Total folate
<188 vs. >633 ug/d
Dietary folate
<142 vs. >272 ug/d
1.01 (0.75-1.35)
0.86 (0.65-1.13)
N.S.
N.S.
Larsson et al
2005
Swedish Mammography
Cohort
66 651 women
40-75 yr
805
14.8 yr
follow up
Dietary folate
<150 vs. 212 ug/d
0.80 (0.60-1.06)
N.S.
Roswall et
al
Diet Cancer and Health
Study
Denmark
56, 332
study duration = 10.6 years
men and women
50-64 years
465 CC
cases
283 RC
cases
CC
RC
CC: FA supplement
Per 100 mcg folate
0 vs. >0-83.2 mcg
0 vs. >83.2-142.8 mcg
0 vs. >142.8 mcg
RC: FA supplement
Per 10 mcg folate
0 vs. >0-83.2 mcg
0 vs. >83.2-142.8 mcg
0 vs. >142.8 mcg
1.01 (0.96, 1.06) 0.79 (0.55, 1.13)
0.90 (0.61, 1.30)
0.83 (0.58, 1.20)
0.98 (0.97, 1.06)
0.56 (0.34, 0.91)
0.82 (0.51, 1.33)
0.60 (0.36, 0.99)
Stevens et al
2011
Cancer Prevention Study II
Nutrition Cohort
USA
99, 528
8 year duration
Men and women
50-74 years
1023 CRC
cases
CRC FA supplementation,
fortification during the last
year, and CRC
<101 vs. 101-182 mcg/d
<101 vs. 182-<452 mcg/d
<101 vs. 452-<560 mcg/d
<101 vs. 560 mcg/d
1.02 (0.86, 1.24) 0.95 (0.78, 1.16)
0.96 (0.79, 1.18)
0.84 0.68, 1.03)
42
2.3.2 EVIDENCE FROM INTERVENTION STUDIES
In summary, the majority of small clinical trials examining the effect of FA
supplementation on colon cancer risk as the primary endpoint suggest that FA supplementation
plays a preventative role in developing surrogate end point biomarkers of CRC (Table 2.8 and
2.9). Intervention trials investigating the effects of FA supplementation in the prevention of
cardiovascular disease and/or related events have also determined CRC incidence and/or
adenoma recurrence as a secondary endpoint (Table 2.10). These studies in general do not
support a chemopreventative effect of FA supplementation and instead suggest a possible tumor
promoting effect.
43
Table 2.9: Summary of RCT of FA supplementation and biomarkers of CRC risk
Reference Characteristics FA Dosage/Duration Endpoint Outcome
Cravo et al
1994
CRC or adenoma
N=22
10mg/d
6 months
Rectal mucosa
genomic DNA
methylation
FA increased DNA
methylation
(p<0.002)
Biasco et al
1997
Chronic UC
N=24
15mg/d
3 months
Rectal cell
proliferation
FA reduced (44%
decrease) cell
proliferation in the
upper 40% of
crypts (p<0.01)
Cravo et al
1998
Adenoma
N=20
5mg/d
3 months
Rectal mucosa
genomic DNA
methylation
FA increased (37%
increase) DNA
methylation in
patients with single
adenoma (p=0.05)
Kim et al
2001
Adenoma
N=20
5mg/d
1st: 6 months
2nd: 1 year
Rectal mucosa
genomic DNA
methylation
P53 strand breaks
FA increased
genomic DNA
methylation at 1st
and 2nd follow-up
(P=0.001)
FA decreased
strand breaks at 1st
and 2nd follow up
(P<0.02)
Khosraviani
et al
2002
Adenoma
N=11
2mg/d
3 months
Rectal mucosal cell
proliferation
FA decreased cell
proliferation
(20% decrease,
most pronounced
decrease in upper
1/3 of crypt)
Pufulete et al
2005
Adenoma
N=31
400ug/d
10 weeks
Rectal mucosal
genomic DNA
methylation
FA increased (25%
increase) DNA
methylation
(P=0.09)
Bruce et al
2005
CRC or adenoma
N=98
3mg/d + calcium
carbonate + X-3 fish oil
28 days
Biomarkers of
insulin resistance,
fecal calprotectin,
C-reactive protein
18% decrease in
free fatty acid
(p=0.013)
No effects on other
biomarkers
44
Table 2.10: Summary of RCT of FA supplementation and colorectal adenoma recurrence
Reference Study/Subjects
Previous
Diagnoses
FA
dosage
Duration
Endpoint RR (95% CI) P-value
Paspatis et
al
1994
Adenoma
N=60
1mg/f FA
1st: 1 year
2nd: 2
years
Recurrence Recurrence in Tx vs.
control:
1st: 23% vs. 38%
2nd: 13% vs. 28%
N.S.
N.S.
Cole et al
2007
Aspirin/Folate
Polyp Prevention
Study
21-80 years
Adenoma
N=1021
1mg/d FA
1st: 3 years
2nd: 3-5
years
1˚: recurrence
2˚: advanced
lesions
1st/1˚: RR = 1.04
(0.90-1.20)
1st/2˚: RR = 1.32
(0.90-1.92)
2nd/2˚: RR = 1.13
(0.93-1.37)
2nd/2˚: RR = 1.67
(1.00-2.80)
N.S.
N.S.
N.S.
P=0.05
Jaszewski
et al
Adenoma 5mg/d FA
3 years
Multiplicity OR = 2.77 (0.06-
0.84)
P=0.03
Logan et
al
2008
ukCAP Trial
(United Kingdom
Colorectal
Adenoma
Prevention)
<75 years
Adenoma
0.5 mg/d
FA
3 years
Recurrence RR = 1.07 (0.85-
1.34)
N.S.
45
Table 2.11: Summary of RCT of FA supplementation and cancer incidence as secondary
endpoint
Reference Study/Subjects
Previous
Diagnoses
Intervention
Duration
Endpoint RR or HR
(95% CI)
p-value
Zhu et al Atrophic gastritis 1st year: 20 mg
FA
2nd year: 20 mg
FA
(twice weekly) +
B12
Gastrointestinal
cancer
incidence
OR = 0.12
(0.03-0.51)
P=0.004
Toole et
al
VISP (vitamin,
intervention for
stroke prevention
trial)
Preexisting CVD
20 ug FA + B6 +
B12
2.5 mg FA + B6
+ B12
1.7 years
Cancer
incidence
RR = 0.98
(0.74-1.30)
N.S.
Lonn et al HOPE-2
Preexisting CVD
2.5 mg FA + B6
+ B12
5 years
Cancer
incidence
Cancer
mortality
RR = 1.06
(0.92-1.21)
RR = 0.99
(0.75-1.31)
N.S.
N.S.
Jamison et
al
HOST
Chronic renal
disease
40 mg FA + B6
+ B12
3.2 years
Cancer
incidence
RR = 0.90
(0.65-1.24)
N.S.
Zhang et
al
WAFAC study
≥42 years, women
Preexisting CVD
or ≥3 coronary risk
factors
2.5 mg FA + B6
+ B12
7.3 years
Cancer
incidence
HR = 0.97
(0.79-1.18)
N.S.
Ebbing et
al
NORVIT,
WENBIT
Ischemic heart
disease
0.8 mg FA +
B12 + B6
3.25 years
Cancer
incidence
Cancer
mortality
HR = 1.21
(1.03-1.41)
HR = 1.38
(1.07-1.79)
P=0.02
P=0.01
Armitage
et al
SEARCH
Preexisting CVD
2.0 mg FA +
B12
6.7 years
Cancer
incidence
Cancer
mortality
RR = 1.06
(0.96-1.17)
RR = 1.03
(0.87-1.22)
N.S.
N.S.
46
Table 2.12: Summary of meta-analyses consisting of both epidemiological and intervention-
based studies.
Reference Number/type of
studies included
Outcome Measurement Inverse Positive N.S.
Kim et al
2010
13 cohort studies CRC risk Dietary folate
Total folate X
Kennedy
et al
2011
18 case control
9 cohort studies
CRC risk Dietary folate
Total folate X
Park et al 3 cross sectional
studies
4 case control
studies
4 clinical trial
studies
Adenoma Plasma folate (no
polyp)
Plasma folate
(polyp) X
Qin et al
2013
7 clinical trials Cancer
incidence
Supplement FA X
Figueredi
et al
2009
3 clinical trials Adenoma
recurrence
Plasma folate
<11 nmol/L
>29 nmol/L
X
Wien et al
2012
10 clinical trials Cancer
incidence
Supplement FA
(0.4 to 1.0 mg)
Supplement FA
(>1.0 mg)
X
Vollset et
al
2013
13 clinical trials Cancer
Incidence
Supplement FA
X
Zhou et al 16 clinical trials Cancer
incidence
Supplement FA X
47
2.3.3 EVIDENCE FROM ANIMAL STUDIES
Animal studies conducted in colorectal cancer models have shown that FA supplementation
prevents the development of cancer in normal tissues but promotes the progression of established
neoplastic lesions (2, 3). Table 2.12 summarizes animal studies looking at the effects of FA
supplementation on adenoma or colorectal cancer incidence. Collectively, these studies suggest
that folate possesses dual modulatory effects on cancer development and progression. These
studies also highlight the importance of dose and stage of cell transformation at the time of FA
supplementation (Figure 2.6).
As mentioned, folate deficiency in preneoplastic tissues results in tumor inhibition, which is
also the main rationale behind the use of antifolate medications. Limiting availability of
substrates for DNA synthesis can result in inefficient replication, thus suppressing tumor growth.
On the other hand, in tissues with neoplastic transformation, FA supplementation can promote
tumor growth by providing the substrates necessary for DNA synthesis and replication at the
accelerated rate characteristic of cancer cells. Lindzon et al, showed this promoting effect of FA
supplementation on established colon cancer precursors, using a rodent model. In this model,
aberrant crypt foci (ACF), the earliest precursors of colon cancer, were induced in the animals
using azoxymethane (AOM). Following the induction of ACFs, FA intervention was given, and a
promoting effect was seen, where supplementation promoted transformation to neoplastic cells.
The effect of FA supplementation in normal tissue, prior to preneoplastic transformation has
the opposite effect to that described above. Supplementation in normal epithelia is thought to be
48
cancer preventative due to the sufficient supply of substrates for DNA synthesis, one carbon
metabolism and biological methylation reactions (3, 25). On the other hand, folate deficiency can
result in DNA and chromosome breaks due to uracil misincorporation (3, 26). The limitation of
substrates can also lead to impairments in DNA repair mechanisms, resulting in mutations and
loss of DNA integrity (28).
Figure 2.6: The dual modulatory role of folate deficiency and folate supplementation on
colon cancer progression (3). Copied under licence from the John Wiley and Sons Copyright
Clearance Centre.
49
2.3.4 SUMMARY OF FOLATE AND COLON CANCER RISK
Evidence from animal studies, suggests dual modulatory effect where outcome of folate
supplementation is dependent on dose and time of intervention. Epidemiological and intervention
studies present inconsistent results and suggest that it is unclear whether these adverse effects are
due to high FA or high folate status. Due to the dramatic increase in folate status in the North
American population, in conjunction with the many individuals harbouring asymptomatic
colonic lesions (29), understanding the effects of both low and high dose folate forms is
necessary to interpret safer parameters of use.
50
Table 2.13: Summary of animal studies of FA supplementation and adenoma or colorectal cancer incidence
Reference N; model mg FA/kg diet Duration Endpoint Outcome
Cravo et al
1992
Male Sprague
Dawley rats
DMH injection
0
8
20
weeks
Tumor incidence
- Microadeomas
- Macroadenomas
100% fed 0 mg 29% fed
8mg
86% fed 0 mg
43% fed 8mg
Kim et al
1996
N=40
Sprague Dawley
rats
DMH injections
0
2
8
40
15
weeks
Tumor incidence
- Microscopic (%) - Macroscopic (%)
NS
0mg (70%)
2mg (40%)
8mg (10%)
20mg (50%)
(p<0.03)
Reddy et al
1996
N=72
Fischer-344 rats
AOM injection
Group 1:
control
Group 2: 2000
50
weeks
Tumor incidence
Tumor size
Tumor multiplicity
NA
Group 1 > group 2
Group 1 > group 2
Wargovich et al
1996
N=20
Fischer-344 rats
AOM injections
0
2.5
5
2
weeks
Mean # ACFs Increase vs. control
Le Leu et al
200l
AOM injection
Sprague Dawley
rats
0
8
12
weeks
Tumor incidence
Multiplicity:
Adenomas
Adenocarcinoma
Increase with FA (SI &
colon, and CI alone)
NS increase with FA
Le Leu et al
2000
AOM injection
Sprague Dawley
rats
0
8
12
weeks
ACF Increase with FA
Lindzon et al
2007
N=152
Sprague Dawley
rats
AOM injection
0
2
5
8
34
weeks
Tumor incidence
Size
Multiplicity
ACF
NS
Increase with FA
Increase with FA
Increase with FA
51
2.4 5-METHYLTETRAHYDROFOLATE
FA has been considered more advantageous in food fortification and supplementation
compared to reduced natural folate forms primarily due to its greater stability allowing for a
longer shelf-life. Until recently, 5MTHF, a reduced folate, was available as mixed
diastereoisomers (L and D forms), which allowed for a longer shelf-life but possessed half the
biological activity compared to FA (119). Now, 5MTHF is available commercially as a calcium
salt in the L (active) form (24) and has been shown to be as stable as the diasterioisomers (121).
2.4.1 5-METHYL-CA+
FA and 5MTHF are available commercially, over the counter in doses of 200 μg, 400 μg,
800 μg, and 1000 μg. As a supplement, FA is widely distributed and is easily accessible at drug
stores. 5MTHF is also found commercially, in the form of a crystalline calcium salt
(Metafolin®) and is likely to be found in organic or natural health product stores, or online,
mainly in supplemental form. Metafolin®, patented by Merck Inc., is distributed to supplement
(eg. Genestra Brands, ProThera Inc. Pure encapsulations, and Thorne) and pharmaceutical
companies (eg. PamLab and Bayer Schering Pharma). These supplement brands are available for
purchase in Canada as well as the US and are widely available online.
In the US, 5MTHF-Ca is regarded as GRAS (Generally Recognized as Safe) for its
intended use (67, 124) and can be used as a dietary ingredient (67, 24). The EFSA (European
52
Food Safety Authority) has also supported the use 5MTHF-Ca in foods for specific nutritional
uses with an upper limit of 1 mg/day for adults (125).
2.4.2 ARGUMENTS FOR 5MTHF SUPPLEMENTATION
Albeit little evidence to support, it has been proposed that 5MTHF supplementation may
be beneficial for those with MTHFR polymorphisms or those presenting with B12 deficiency.
However, other arguments for 5MTHF as a better supplemental form of folate to FA are: 1)
5MTHF is immediately available for cellular use (whereas FA must be reduced twice to become
THF) and thus making it more bioavailable (2); 2) supplementation of 5MTHF results in much
less UMFA in the blood; and 3) 5MTHF prevents masking of B12 deficiency (24). Studies have
shown the bioavailability of FA and 5MTHF to be approximately equivalent with comparable
physiological activity (24). Furthermore, long-term supplementation in human studies showed
that 5MTHF and FA similarly decreased homocysteine concentrations and similarly increased
RBC and plasma folate concentrations (74).
2.4.3 5MTHF AND 5MTHFR SNP
Studies have shown polymorphisms of several genes involved in folate metabolism to
greatly affect health (2, 61). The MTHFR C677T polymorphism has been studied extensively.
The frequency of 677TT genotype varies across ethnic groups and geographical locations.
Approximately 10% of the Caucasian and Asian population are homozygous carriers while the
53
Hispanic population has a higher frequency (24). The primary concern for those with MTHFR
C677T polymorphism is the reduced ability to recycle folate within the cell. Studies have shown
the potential for increases in total homocysteine levels, and DNA hypomethylation in those with
the MTHFR C677T polymorphism (40). Many believe that 5MTHF supplementation allows the
folate cycle to bypass the need for MTHFR, allowing for consistent SAM regeneration and DNA
methylation (Figure 2.3). However, the effects of the MTHFR C677T polymorphism on folate
biomarkers have not been consistently observed (125, 127).
2.4.4 5MTHF AND B12
The relationship between B12 and FA has been discussed earlier. In terms of B12 and
5MTHF, there is not yet convincing evidence of and advantage for 5MTHF over FA as a
supplement or fortificant. Many propose that supplementing with 5MTHF will create a ‘methyl
trap’ during B12 deficiency. The ‘methyl trap’ refers to a halt in the folate pathway at MS during
B12 deficiency. Methionine synthase requires B12 to function. Without methionine synthase
activity, 5MTHF will not be able to participate in the methylation of homocysteine to methionine
in the generation of SAM nor will it be able to be involved in nucleotide biosynthesis (Figure
2.3). This could possibly result in megaloblastic anemia, a clinical marker of B12 deficiency.
54
2.5 FOLIC ACID VS. 5MTHF
There are currently no studies that have compared the effects of FA and 5MTHF
supplementation on cancer risk including that of colon cancer. However, human studies
comparing FA and 5MTHF in the general healthy population have shown 5MTHF
supplementation to be as effective as FA supplementation at raising blood concentrations of
folate and lowering homocysteine concentrations (30). There have also been a few animal and in
vivo studies comparing the effects of FA and 5MTHF supplementation on folate absorption (30,
32). Jing et al investigated comparative effects on folate absorption in the jejunum of laying
hens. They found that 5MTHF supplementation significantly lowered RFC and PCFR mRNA
expression compared to control diet. However, this was no different from the results observed
via FA supplementation. Wang et al (33) conducted an in vitro study, comparing the effects of
FA and 5MTHF supplementation on DNA damage and cell death in human lymphocytes.
5MTHF was not more effective than FA in preventing human lymphocyte genomic instability in
vitro (33). Another in vitro study, using human colon cancer cell lines, reported a decreased RFC
and PCFT expression with increasing FA supplementation, but not with 5MTHF (129). 5MTHF
was also shown to have higher global DNA methylation compared to FA in one cell line, while
the opposite was shown in another (130). This study also found faster growth with 5MTHF
supplementation in comparison to equimolar concentrations of FA (130), which suggests that
further research is required in order to determine a safe parameter of use.
55
CHAPTER 3: RATIONALE, OBJECTIVES, HYPOTHESIS AND SIGNIFICANCE
3.1 RATIONALE
Folate status has increased dramatically in the Canadian and US population, mostly from
supplementation but also some from fortification. Many adults in the US and Canada regularly
use dietary supplements for potential health benefits, which are largely unproven. This is
especially problematic for populations at risk for developing colorectal cancer, particularly due
to the growing body of evidence suggesting that folate supplementation can promote progression
of pre-existing lesions (3, 140). Furthermore, it remains unclear whether these adverse effects are
attributable specifically to FA or to high folate status in general. Although 5MTHF is being
promoted as a safer alternative to FA supplementation, convincing evidence supporting this
claim is lacking. Only few studies have compared the effects of 5MTHF supplementation with
FA supplementation on biomarkers of folate status but no studies comparing the effects of
5MTHF versus FA supplementation on health outcomes such as colon cancer risk exit to date.
Given these considerations, this study was undertaken to address these gaps in knowledge, in
particular the potential effects of 5-MTHF supplementation in comparison to FA
supplementation on the progression of colon cancer precursors.
56
3.2 STUDY OBJECTIVE
To compare the effects of FA and 5MTHF supplementation in a rodent model on promotion of
established colon cancer precursors induced by the chemical carcinogen azoxymethane.
3.3 STUDY HYPOTHESIS
5MTHF supplementation will have a greater tumor promoting effect than FA supplementation in
a rodent model of colon cancer in which the precursors of colon cancer was induced by the
chemical carcinogen azoxymethane.
3.4 SIGNIFICANCE
Colon cancer patients and survivors are exposed to high levels of folate, particularly in
the form of FA (13). Furthermore, 35-50% of the population above the age of 50 have adenomas
and many more have ACFs (61, 130). Given the dual modulatory role of folate supplementation,
this high intake of folate poses a risk. 5MTHF has been proposed to be a safer alternative
supplemental form of folate, however the evidence to support this is lacking and this may not be
true given its supplemental effects on cellular proliferations (3). This project will determine
whether supplementation of 5-MTHF, the predominant form of naturally occurring folate,
influences the progression of colon cancer differently to FA. This study will help to elucidate the
effects of 5-MTHF in a colon cancer model to facilitate better understanding of its potential
57
adverse health effects and will also help to provide information for future research studies on
mechanisms associated with carcinogenesis.
58
CHAPTER 4: THE EFFECTS OF FOLIC ACID AND 5MTHF SUPPLEMENTATION
ON THE PROGRESSION OF COLON ACF TO ADENOMAS AND
ADENOCARCINOMAS IN THE AOM RAT MODEL
4.1 INTRODUCTION
Folate plays an important role in human health and disease. Widespread FA supplement use
and FA fortification have increased folate intake (in the form of FA) and blood folate levels in
the North American population. Although there are suggested benefits to taking FA supplements
and FA fortification such as reduced NTD rates, high folate intake, in particular from FA
supplementation have been purportedly associated with adverse health outcomes including a
potential tumor promoting effect. These effects have been well documented in animal studies,
particularly in the context of CRC (2, 9, 10, 142). The major finding from these studies is that
folate possesses a dual modulatory effect on the development of CRC, depending on both the
dose and timing of intervention (3). Folate deficiency has an inhibitory effect whereas folate
supplementation has a promoting effect on the progression of established colorectal neoplasms
(3). In contrast, folate deficiency in normal colorectal mucosa appears to predispose it to
neoplastic transformation, whereas supplementation suppresses transformation (3). In addition to
these tumor promoting effects, high levels of FA have also been shown to mask B12 deficiency
which can progress to irreversible neurological deficits and cognitive impairments as well as
result in the accumulation of UMFA which can compete with the metabolized form of folate (13,
141). For this reason, 5MTHF has been proposed to provide as a safer alternative to FA
supplementation. Some reasons for this conjecture include: 1) the idea that it would benefit
those with 5MTHFR polymorphisms as 5MTHF is immediately available for use by the cells
59
(whereas FA must be reduced), 2) supplementation of 5MTHF will result in less UMFA in the
blood and 3) 5MTHF supplementation will prevent masking B12 deficiency. Although studies
looking at bioavailability of FA and 5MTHF have shown equivalent physiological activity, there
is not yet convincing evidence for 5MTHF as an advantageous supplemental/fortificant over FA.
Additionally, no studies have compared its effects with FA on tumor progression. Thus, the
objective of this study was to elucidate the potential cancer promoting effects of 5MTHF in
comparison to FA on CRC precursors (ACFs) in an animal model to clarify the role of 5MTHF
in CRC. We hypothesized that 5MTHF would have a greater tumor promoting effect compared
to FA in the animal model of CRC.
60
4.2 MATERIALS AND METHODS
4.2.1 THE AOM RODENT MODEL
The use of azoxymethane (AOM) has been demonstrated in a number of studies, as a
potent inducer of carcinomas in the colon. AOM is a methylating agent that results in a loss of
colonic cells via apoptosis followed by an increase in proliferation and mutations of colonic
epithelial cells (133). The resulting epithelial lesions are similar to those found in the human
colorectum (134). This model, like humans, develop tumours that bear K-ras mutations (24, 58).
Although rats do not possess Apc mutations, like human Apc mutated tumours, rat tumours
accumulate beta-catenin in the nucleus (24, 58). This happens as a result of decreased beta-
catenin degradation (Ctnnb1 mutation and GSK3B phosphorylation motif mutation of the beta-
catenin gene) (133, 134). COX-2 and iNOS are also overexpressed in rat tumours, similar to
humans (24, 58, 133, 134).
AOM-induced tumours in rats share many histopathological characteristics with human
tumours (24, 58). The ACF develop 4-6 weeks post injection, followed by adenoma (often
polyps) and carcinoma (microadenocarcinomas at 12-18 weeks, macroadenocarcinomas at 12-18
weeks post injection) (24, 58). This model has been extensively used to study the relationship
between folate and colorectal carcinogenesis and has well documented results showing ACF and
adenoma development (24, 133, 134). The well-established timeline of tumour development also
allows for accurate intervention, to study nutritional factors in colon carcinogenesis.
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4.2.2 STUDY DESIGN
Three-week-old male Sprague-Dawley rats (n=124) were purchased from Charles Rivers
(St. Constant, QC). Rats were housed 2-3 per cage and cages and were changed twice weekly.
Upon arrival, the rats were tagged and placed on the control diet in the form of pellets (1mg/kg
FA diet or 5MTHF molar equivalent) (Dyets, Bethlehem, PA) for 7 weeks (Appendix A, B & C).
At 5 and 6 weeks of age, the rats were given a subcutaneous injection of Azoxymethane (AOM)
(15mg/kg body weight). The animals were randomized at 10 weeks of age to either remain on
their respective controls or receive a 10mg/kg FA diet or a 5MTHF molar equivalent. The rats
were then sacrificed at either 28 or 29 weeks of age.
4.2.3 AOM ADMINISTRATION
Animals were randomized into 5 groups (Monday-Friday) to determine which day of the
week the AOM injection would be administered. The animals received the AOM injection on the
same day at both 5 and 6 weeks. Within the fume hood, 180 mL of double distilled water
(ddH2O) was added to 20 mL of PBS solution and then shaken. From 200 mL, 50 mL was
transferred into a falcon tube to which 70 uL of AOM was added. AOM is light sensitive, thus,
the falcon tube was wrapped in tin foil and then put on ice. Animals were weighed the morning
of the injection and injected with 15 mg of AOM/ kg of their body weight.
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4.2.4 EXPERIMENTAL DIETS
Amino acid-defined diets were used throughout the study (Dyets, Bethlehem, PA). Four
weeks after the last AOM injection the rats were randomized into a total of four experimental
groups (n=30/group). Diets contained 1mg/kg FA or 5MTHF molar equivalent, or 10mg/kg FA
or 5MTHF molar equivalent. Both diet and water were provided ad libitum and diets were stored
at 4°C. Detailed composition of the diet is shown in tables 4.1, 4.2 and 4.3.
The control diet provides 1 mg FA/kg diet. Although 2 mg FA/kg diet is generally
accepted as the BDR for rodents (183, 184) and is the most commonly used control diet in rodent
studies, the 1 mg FA/kg diet provides an alternative dose in the study and has also been observed
to be equally as effective as the 2 mg FA/kg diet in preventing folate deficiency and maintaining
normal metabolic functions. The control level of FA 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 (183). 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 in the North American population taking daily multivitamin supplements
and consuming high levels of fortified food sources. 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). These differences include body
weight, lifespan as well as gene regulation, all of which translate to differences in metabolism
(11). One of the major metabolic differences being the substantially lower DHFR activity in
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humans compared to rodents (11). Equimolar dose of 5-MTHF (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.
Food and water were provided ad libitum and diets were replenished every other day to ensure
palatability. Food intake was not measured as previously conducted studies have shown no
difference in food consumption among animals placed on similar diets containing varying
amounts of folate for similar intervention period (24).
4.2.5 SAMPLE COLLECTION
At the end of dietary intervention period (28/29 weeks of age), rats were sacrificed by
isoflurane overdose followed by T-61 injection. Blood samples were collected from the heart by
cardiac puncture using pre heparinized 16-gauge needles and sterile 1 ml syringe. Blood was
taken off of ice and left at room temperature for 30 minutes. The blood samples were then
centrifuged at 25000x g for 10 minutes at room temperature. Plasma (475µl) was aliquoted into
vacutainers containing 25µl 0.5% ascorbic acid for serum folate assays. The samples were stored
at -80°C. The liver was removed, snap frozen in liquid nitrogen and stored at -80 °C for liver
folate determination. The colon was promptly excised and flushed with PBS solution to eliminate
fecal debris. The entire length of the colon was opened longitudinally. Two centimeters from the
distal end of the colon (rectum) was cut and placed in a cassette with a foam cushion and stored
in 10% buffered formalin for immunohistochemistry. The colon was stored in 10 cm tissue
culture dishes between two pieces of Whatman filter paper and preserved in Bouin’s solution for
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ACF enumeration & tumor parameter analysis. All macroscopic lesions were identified,
measured along the longest diameter, harvested and stored in glass vials containing formalin.
Figure 4.1: Experimental Study Design. Supplemental diets were administered at Week 7 followed by
necropsy at week 24. Body weight measurements were taken weekly.
4.2.6 FOLATE CONCENTRATIONS
Folate concentrations were measured using a standard microbiological Lactobacillus
rhomnosus microtiter plate technique (136). The media becomes proportionally turbid as the
Lactobacillus rhomnosus bacteria grow (136). The bacteria growth rate is proportional to the
amount of folate in the media. Therefore, the turbidity measured by the spectrophotometer
indicates the folate concentration in the samples tested (136).
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FOLIC ACID STANDARD PREPARATION
FA was dissolved in ddH2O with 0.1 M NaOH to a concentration of 10 mg FA/ml. The
concentration was verified using a spectrophotometer at 282 nm. The solution was diluted to 2
ng/ml in sterile filtered 0.1 KPO4 buffer containing sodium ascorbate (0.5%). Standard was
stored in aliquots at -80°C.
LACTOBACILLUS RHOMNOSUS STOCK PREPARATION
Lactobacillus rhomnosus bacterial stock (250µL) was added to 200mL of Lactobacillus MRS
Broth (Difco™, BD Biosciences) and incubated at 35-37°C for 18 hours. Under aseptic
conditions, the tube was centrifuged to sediment the cells and the supernatant was removed. The
pellet was resuspended in 180mL of Lactobacillus MRS Broth and 20mL of 100% autoclaved
cold glycerol was added. The solution was mixed well, aliquoted and stored at -80°C.
DETERMINATION OF FOLATE CONCENTRATION
Lactobacillus rhomnosus stock (3µL) was inoculated in 3 mL o f Lactobacillus MRS
Broth at 37 °C for 16-18 hours. Five hundred µL of the overnight culture was then inoculated in
2.5mL of Lactobacillus MRS Broth for 5 hours (O.D. should be approximately 1.8 at this point).
Folic acid media (9.5 g of folic acid media, 0.05 g NaAscorbate, l00mL of ddH20, boiled for 1-2
minutes, cooled and filter sterilized) and potassium phosphate buffer were made fresh each day
prior to starting the assay. Potassium phosphate buffer was added to all the wells of a 96 well
microtiter plate (150 µL per well). In columns 1 and 2 of the plate, 150 µL of folic acid standard
(2µg/mL) was added and then serially diluted along the plate to create a standard curve. Plasma
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samples were thawed and 5 µL was used. Potassium phosphate buffer was added to each well
and the samples were serially diluted three times per plate. All samples were run in duplicates.
The Lactobacillus rhomnosus was diluted lmL in 24mL of folic acid media (l00x dilution) then
375 µL of the diluted inoculum was added to 15 mL of folic acid media and 150µL was used to
inoculate each of the 96 well plate. Each plate was covered with a mylar sealer, mixed and
incubated at 37°C for 16 to 18 hours allowing it to grow. The plate was then read on a
spectrophotometer at 650 nm and using SoftMax software. The standard curve of folate
concentrations plotted against the densities, as generated by the SoftMax software, was used to
estimate the folate concentrations from the samples.
4.2.7 DETERMINATION OF ABERRANT CRYPT FOCI
Colons were promptly removed, cut open along the longitudinal axis and flushed with
phosphate-buffered saline (PBS) to remove fecal debris. The colons were prepared using the
swiss roll technique and further processed using an automatic tissue processor, paraffin
embedded, and cut into sections for hematoxylin and eosin staining using a rotary microtome.
The resulting slides were evaluated for microscopic lesions, conducted by a gastrointestinal
pathologist (Dr. Alan Medline) blinded to the different experimental groups.
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4.2.8 HISTOLOGY
All macroscopic neoplasms were excised at necropsy and stored in 10% formalin. The
samples were then embedded in paraffin, and further processed using an automatic tissue
processor, paraffin embedded, and cut into sections for hematoxylin and eosin staining using a
rotary microtome. The resulting slides were evaluated for microscopic lesions, conducted by a
gastrointestinal pathologist (Dr. Alan Medline) blinded to the different experimental groups.
4.2.9 STATISTICS
Statistical significance of the effects of folate dose and form on body weight, tumor
incidence, tumor multiplicity and tumor parameters were tested using a repeated measured Two-
Way ANOVA followed by a post hoc Tukey’s multiple comparisons test to detect differences
between dietary groups at each week of dietary intervention. Spearman’s rho correlation test was
used to assess correlation between variables to help explain the observed outcome. Two-way
ANOVA was conducted to examine the effects of the dose and form of folate supplementation
on plasma folate concentrations (n=30/dietary group).
Statistical analysis was conducted using SPSS 26.0 and graphs were prepared using SPSS
output. All tests were two-sided and considered significant if the observed significance level (p-
value) was less than 0.05.
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4.3 RESULTS
4.3.1 ANIMAL HEALTH AND BODY WEIGHT
Rats were weighed weekly, on a predetermined day of the week. Growth curves among the four
dietary groups were not significantly different. The mean body weights (in grams) of the rats in each
group did not exhibit any significant differences at any time point throughout the experiment (p=0.076)
(Figure 4.1). At the time of sacrifice, there was no significant difference among the body weights of the
animals on the four diets (p=0.36). The mean weight at the end of the intervention, in each dietary group
was 744.5, 757.2, 766.6 and 772.4 for rats on the 1mg FA, 10 mg FA, 1 mg 5MTHF and 10 mg 5MTHF
per kg diet, respectively.
Figure 4.2: Effect of dietary folate supplementation on body weight (p=0.076, n=30/dietary group).
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4.3.2 PLASMA FOLATE CONCENTRATIONS
There was a significant interaction between the effects of dose and form on plasma folate
concentrations (p<0.05) (Figure 4.2). Within each folate form (FA & 5MTHF), increasing dose
(1mg/kg, 10mg/kg) resulted in significantly increased plasma folate concentrations with both FA
and 5MTHF supplementation (p<0.05). Between equimolar doses, 10 mg 5MTHF showed a
significantly higher plasma folate concentration relative to 10 mg FA (p<0.05), but no
differences were observed between the 1 mg 5MTHF and 1 mg FA. These results are comparable
to previously conducted studies comparing FA and 5MTHF.
Figure 4.3: Effects of FA and 5MTHF supplementation on plasma folate concentrations. Solid
Grey = 1 mg FA, solid black bars = 10 mg FA, hatched grey = 1 mg 5MTHF, hatched black = 10
mg 5MTHF. Significance for the effects of dose and form were determined using two-way
ANOVA. Asterisks (*) denote significant differences between folate forms within doses. Letters
(ab) denote significant differences between folate doses within forms.
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4.3.3 ABERRANT CRYPT FOCI
There was no difference in the total number of ACF developed between equimolar folate
doses. Within each folate form (FA & 5MTHF), increasing dose resulting in significantly greater
number of total ACF (p<0.05, n=30/dietary group) (Figure 4.4).
Figure 4.4: Effect of FA and 5MTHF supplementation on total number of ACF (p=0.023). .
Solid Grey = 1 mg FA, solid black bars = 10 mg FA, hatched grey = 1 mg 5MTHF, hatched
black = 10 mg 5MTHF. Means with different letters differ at p<0.05.
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4.3.4 TUMOR INCIDENCE AND MULTIPLICITY
There was no significant difference in the incidence of adenomas (p=0.57) and
adenocarcinomas (p=0.60) between folate forms (FA & 5MTHF) (Figure 4.5). Furthermore,
there was no significant difference within folate groups in the incidence of adenomas (p=0.68)
and adenocarcinoma (p=0.53) (Figure 4.5). In order to determine tumour incidence, the most
severe histological diagnosis was considered the final diagnosis. For example, if a rat presented
adenoma and adenocarcinoma, the final diagnosis for this rat was adenocarcinoma. However, if a
rat only present adenoma, then the final diagnosis for this rat was adenoma. However, out of the
total 68 adenocarcinomas observed, 8 signet ring cell carcinomas, a more advanced form of
adenocarcinoma, were identified in both the 10mg FA and 10mg 5MTHF dietary group (Figure
4.6). When comparing 10mg FA and 10mg 5MTHF, no significant difference was observed in
the incidence of signet ring cell carcinoma (p=0.074) (Figure 4.6).
Group Normal Adenoma Adenocarcinoma
1 mg FA 8 (26.7%) 7 (23.3%) 15 (50.0%)
1 mg 5MTHF 6 (20.0%) 10 (33.3%) 14 (46.7%)
10 mg FA 5 (16.7%) 6 (20.0%) 19 (63.3%)
10 mg 5MTHF 4 (13.3%) 6 (20.0%) 20 (66.7%)
Total 23 29 68
Table 4.1: The effect of folic acid (mg FA/kg diet) and 5-methyltetrahydrofolate (mg
5MTHF/kg diet) on the development of colorectal neoplasm.
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Figure 4.5: Effect of FA and 5MTHF supplementation on incidence of colonic neoplasms. Solid
bars = FA, hatched bars = 5MTHF. Tumor incidence was not significantly different between
folate forms (adenomas, p=0.57; adenocarcinomas, p=0.60). Tumor incidence was not
significantly different within folate groups (adenomas, p=0.68; adenocarcinoma, p=0.53)
Figure 4.6: Distribution of signet ring cell carcinoma among the four dietary groups with respect
to total number of adenocarcinomas. Solid bars = FA, hatched bars = 5MTHF. Incidence of
signet ring cell carcinoma was not significantly different between folate forms (p=0.074).
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The mean number of adenomas in all rats did not differ significantly among the four
dietary groups (Figure 4.7A). The mean number of adenocarcinomas in all rats did not differ
significantly between the two folate forms; however, a significant effect within each group was
observed where high dose FA and 5MTHF had significantly greater number of adenocarcinomas
compared to their respective low doses, at p<0.05 (Figure 4.7B)
Figure 4.7: Effect of FA and 5MTHF supplementation on the mean number of adenomas (A,
p=0.19) and adenocarcinomas (B). Means with different letters differ at p<0.05. Solid Grey = 1
mg FA, solid black bars = 10 mg FA, hatched grey = 1 mg 5MTHF, hatched black = 10 mg
5MTHF.
When correlation analysis was conducted between plasma folate with number of tumors in
all animals, a statistically significant, positive correlation was observed between plasma folate
and number of adenocarcinomas (r=0.076, p=0.023). However, no relation was observed with
number of adenomas in all animals (r=0.043, p=0.478). The same correlation analysis was then
conducted on tumor-bearing animals only (excluding rats without tumors). The positive
correlation between plasma folate and adenocarcinoma strengthened when only animals bearing
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tumors were considered (r=0.24, p=0.01) and no relation was observed between adenomas in
tumor bearing animals only and plasma folate (r=0.037, p=0.37).
4.3.5 SIZE OF TUMORS
There was a significant interaction between the effects of dose and form on the sum of all
tumor diameter (adenoma + adenocarcnoma). Within each folate form (FA & 5MTHF),
increasing dose (1mg/kg to 10mg/kg) resulted in significantly higher tumor diameter in both FA
and 5MTHF groups. Within folate doses, 10 mg 5MTHF showed a significantly higher sum of
tumor diameter relative to 10 mg FA (p<0.05), however no differences were observed between
the 1 mg 5MTHF and 1 mg FA.
Figure 4.8: The Effect of FA and 5MTHF supplementation on the sum of tumor diameter in all
animals. Means without a common letter differ at p<0.05. Solid Grey = 1 mg FA, solid black
bars = 10 mg FA, hatched grey = 1 mg 5MTHF, hatched black = 10 mg 5MTHF.
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Figure 4.9: The Effect of FA and 5MTHF supplementation on the sum of tumor diameter in
tumor bearing animals. Means without a common letter differ at p<0.05. Solid Grey = 1 mg FA,
solid black bars = 10 mg FA, hatched grey = 1 mg 5MTHF, hatched black = 10 mg 5MTHF.
Table 4.2 outlines and summarizes the findings from this study. The novel findings here are the
significant differences presented between high dose 5MTHF and FA (10mg), where 5MTHF
supplementation resulted in a significantly greater tumour diameter relative to FA.
DOSE FORM
PLASMA FOLATE ↑ 5MTHF > FA
MEAN # ACF ↑ NS
INCIDENCE OF TUMORS Adenoma NS Adenoma NS
Adenocarcinoma NS Adenocarcinoma NS
MULTIPLICITY OF TUMORS Adenoma NS Adenoma NS
Adenocarcinoma ↑ Adenocarcinoma NS
TUMOR DIAMETER IN ALL
ANIMALS ↑ 5MTHF > FA
TUMOR DIAMETER IN TUMOR
BEARING ANIMALS ↑ (FA only) 5MTHF > FA
Table 4.2: Summary of results. “↑” indicates a significant dose effect, where increasing dose
resulted in the corresponding increased outcome. NS indicates no significant effect.
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4.4 DISCUSSION
Due to the dramatic increase in folate status in the North American population, dual role of
folate in colorectal carcinogenesis, and a significant portion of the population harbouring
asymptomatic colonic lesions with neoplastic potential, understanding the effects of different
supplemental forms of folate is necessary to interpret safer parameters of use (61, 130). A
growing body of evidence has suggested adverse health outcomes with high doses of FA,
including tumor promoting effects. 5MTHF, or metafolin®, is now being promoted as a safer
alternative supplemental form of folate compared to FA, however this is largely without proof.
Although studies have compared the effects of 5MTHF and FA metabolically, its effects on
colon cancer progression still remain unknown (128, 129). Given the growing body of evidence
showing tumor promoting effects of FA, particularly in high doses, this poses a risk. As such, we
conducted a comparative study to evaluate the effects of FA and 5MTHF supplementation on
colon cancer progression using an AOM rat model. Dietary interventions with amino-acid
defined diets containing either 1 mg FA or 10 mg FA, or their respective equimolar 5MTHF
concentrations were selected to study these effects. Measures include plasma folate status, ACF
analysis, tumor incidence and multiplicity as well as tumor size.
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Folate Status
We observed significantly higher plasma folate concentrations with increasing
supplemental levels of both FA and 5MTHF. Furthermore, 10 mg 5MTHF was associated with a
significantly higher plasma folate concentration relative to the equimolar FA dose. However,
when 1 mg FA and 1 mg 5MTHF, no differences were seen in plasma folate concentration. The
difference was only noted with the high folate diets (10 mg).
Previously conducted animal and human studies have shown similar results, in that a
positive association between supplemental levels of FA and 5MTHF and plasma folate
concentration has been recognized (30, 109, 113). The novel finding from this study, however, is
that 5MTHF resulted in a higher plasma folate concentration relative to FA at the 10 mg
supplemental level. A study conducted in mice, comparing FA and 5MTHF found similar results
where 5MTHF resulted in a higher plasma folate concentration compared to FA at the 20 mg
supplemental level (128). This study compared the folate forms at the 2mg, 10mg and 20mg
supplemental level (128). This study also found no significant difference between FA and
5MTHF on tissue folate concentrations observed in the liver and small intestine (128). The
differences in these results could be attributed to the difference in animal study model.
Clinical studies observing similar results, where 5MTHF increased plasma folate
concentrations to a greater extent than FA (163), attribute the findings to differences in
metabolism of the two folate forms (163). 5MTHF bypasses many of the metabolic steps which
are otherwise necessary for FA and can thus act directly to alter plasma folate concentrations. In
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particular, the metabolism step governed by the DHFR enzyme, which is easily saturated at
higher doses, can lead to reduced conversion of FA to 5MTHF (115). 5MTHF is a biologically
active form folate and bypasses this metabolic step and can also be stored in the body. For this
reason, it can have direct effects on plasma folate concentrations.
Aberrant Crypt Foci
This study observed an increasing number of ACF with increasing supplemental levels of
both FA and 5MTHF. However, the mean number of ACF did not differ between the FA and
5MTHF groups. Although we did not see 5MTHF to have a greater effect on increasing number
of ACF relative to FA, our results support the idea that 5MTHF is equally as effective as FA in
regard to ACF proliferation. For this reason, it is important to consider the potential
consequences of marketing 5MTHF as a safer alternative to FA.
Tumor Parameter Outcomes
Supplemental folate levels did not affect the incidence of colorectal adenoma or
adenocarcinoma. Additionally, an advanced form of adenocarcinoma known as signet ring cell
carcinoma was observed in only the 10 mg supplemental folate groups. Supplemental folate
levels did not affect the number of colorectal adenomas; however, number of colorectal
adenocarcinomas did increase with increasing level of supplemental folate in both FA and
5MTHF groups. Plasma folate concentrations were also positively correlated with number of
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adenocarcinomas in all animals and this positive association was further strengthened when only
tumor bearing animals were analyzed. Tumor diameter was seen to increase with increasing
supplemental levels of both FA and 5MTHF. The novel finding in this study, however, is that
5MTHF resulted in a significantly higher tumor diameter relative to FA at the 10 mg
supplemental level. When plasma folate concentrations were correlated with sum of tumor
diameter in all animals as well as in tumor bearing animals, a statistically significant positive
association was observed. Although plasma folate concentrations could explain the differential
effects on tumor diameter, it may not be the only explanation, as no differential effects were
observed in other tumor parameter measures between folate forms. However, the positive
correlation between plasma folate concentrations and number of adenocarcinomas within FA and
5MTHF groups suggests that 5MTHF is as effective as FA in promoting adenocarcinoma.
Additionally, although tumor incidence holds greater clinical relevance compared to tumor
diameter, exploring the potential mechanistic differences between folate forms to study the
tumor diameter results from our study warrants further research.
The present study is the first to investigate the comparative effects of FA and 5MTHF
supplementation on the progression of established preneoplastic lesions of colon cancer (ACF) to
colorectal adenomas and adenocarcinomas. Previous studies have provided a considerable
amount of data on the tumor promoting effects of FA as well as the dual modulatory effects of
FA (Table 2.12). However, there has been limited research on the effects of 5MTHF on
colorectal carcinogenesis. An in vitro study conducted in colon cancer cell lines found 5MTHF
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supplementation to have significantly greater effects on growth rates compared to FA (129). This
study highlighted the potential negative outcomes of providing equimolar doses of a more
readily available form of folate (129). Based on the aforementioned data, we hypothesized that
5MTHF would have a greater tumor promoting effect relative to FA. Although our findings are
not unequivocally in line with our hypothesis, this study does suggest that both FA and 5MTHF
provided after the establishment of colorectal ACF appear to promote the progression of ACF to
adenocarcinomas. However, 5MTHF was shown to have a greater effect on tumor diameter
relative to FA at the 10 mg supplemental level, which is supportive of our hypothesis.
Some mechanistic explanations can be applied to these results. Firstly, folate
supplementation may promote the progression of established preneoplastic lesions by facilitating
nucleotide precursors to the replicating preneoplastic cells resulting in accelerated proliferation
and growth (27, 28). Thymidylate synthase (TS), is the enzyme responsible for thymidylate
biosynthesis (Figure 2.2). TS uses the substrate 5,10-methyleneTHF and dUMP to generate DHF
and dTMP. A limited number of comparative in vivo studies have been conducted to assess the
effects of FA and 5MTHF on enzymes such as TS, and the findings have shown varying results.
A recent comparative mechanistic study conducted in mice found no difference between FA and
5MTHF supplementation on TS gene expression (128). However, another study conducted in a
regenerating rat liver found FA supplementation to reduce TS gene expression and decrease
DNA synthesis initially but upregulate DNA synthesis later (142). The study also noted that the
mRNA expression did not directly translate to protein expression suggesting post-transcriptional
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and translational modifications to have effects on TS. Although our study did not assess gene or
protein expression, it is clear that future studies are necessary to better understand the effects of
FA and 5MTHF on nucleotide biosynthesis, particularly in the context of colon cancer.
Secondly, folate supplementation may promote the progression of preneoplastic lesions via
its effects on de novo methylation of promoter CpG islands of tumor suppressor and other
critical genes involved in carcinogenesis resulting in possible gene inactivation and then tumor
progression. This hypothesis needs to be tested in future studies. Lastly, folate supplementation
may promote carcinogenesis through hypermutability of methylated cytosines in CpG
dinucleotide sequences (86). The mutations observed in these sites (majority cytosine to
thymine) are governed by the enzyme DNMT (particularly DNMT3A and DNMT3B), which is
an important enzyme for de novo methylation patterns. (34, 35). These mutational hot spots can
lead to inactivation of critical genes resulting in cancer progression. Comparative studies have
suggested 5MTHF supplementation to be associated with greater DNMT3A mRNA expression
relative to FA and have also shown a greater contribution to upregulating genes involved in the
methionine cycle (129). FA supplementation studies using in vitro, animal models and human
models have suggested that the effects of methylation tend to be site and gene specific and
depend on a variety of factors including duration of folate exposure and stage of cell
transformation (127). The mechanistic differences between FA and 5MTHF in the context of
DNA methylation, seem to be the most plausible mechanism to describe the findings from our
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study. Further research assessing DNA methylation in a colon cancer model is necessary to
elucidate and clarify these potential mechanisms of action for cancer progression.
Strengths and Limitations
One of the strengths of this study comes from the amino acid defined diets provided to the
rats in each group. The rats in each group were fed diets that were identical in nutritional content
but only differed in the supplemental amount of FA and 5MTHF. This makes sure that the results
and effects seen in the study are the result of the dietary intervention.
The AOM rat model is a well-established model used to study colon carcinogenesis. This
model has its strengths and limitations. AOM is a methylating agent used to induce colon cancer
via increased apoptosis of colonic cells, followed by an increase in proliferation of mutations in
colonic cells. The use of AOM produces colonic epithelial lesions that are similar to those
observed in humans. The AOM-induced tumors in rats share histopathological characteristics
with human tumors. They develop similarly in terms of timeline (ACF → adenoma/polyp →
carcinomas) and the regional distribution of these tumors is also comparable to that in humans as
the induced tumors are predominantly observed in the distal colon. However, there exist some
genetic differences; the rat model lacks the p53 mutation and unlike Apc mutations noted in
human colonic tumors, rats exhibit an accumulation of β-catenin in the nucleus. Given these
inherent limitations, and differences from human colon cancer, the AOM rat model is still the
best model of CRC due to the similarities in histology and the timeline of cancer progression.
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Although rats and humans share many biological functions, which is another reason that
this model is valuable for colon cancer studies, it is important to note that these findings are not
entirely translatable to the human situation. Major differences between rats and humans include
body weight, lifespan as well as gene regulation, all of which can translate to differences in
metabolism. One of the major metabolic differences between humans and rodents in the context
of this study is the substantially lower DHFR activity in humans compared to rodents. However,
this is the first study of its kind to assess the comparative effects of FA and 5MTHF on colon
cancer progression. Furthermore, our study did not report UMFA concentrations which could
highlight some potential differences between the two folate forms.
Additional limitations, that can be mended for future studies is the use of plasma folate as a
measure to assess folate status, the lack of mechanistic endpoints and the measure of dietary
folate levels in the experimental diets. Plasma concentrations are reflective of short-term dietary
intake and supplement usage (2). Red blood cell folate concentrations should be considered for
future studies as they are more reflective of tissue stores and chronic dietary intake (2). The 120-
day turnover period of red blood cells allow for a measure that is resistant to short term
variations in consumption. The lack of mechanistic endpoints in the study leaves questions
regarding the nature of the differential effects observed. Analyzing gene and protein expression,
DNA methylation and nucleotide biosynthesis could have given insight into the differential
tumor diameters observed between FA and 5MTHF groups. Dietary folate levels in the
experimental diets were not measured throughout the study period. Although similar diets have
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been used in previously conducted studies, it is possible to have slight variation between diet
batches. Future studies should assess folate levels in experimental diets to ensure consistency
between batches.
This study observed the differential effects of FA and 5MTHF on plasma folate
concentrations as well as tumor parameters. The results of this study can be used as a framework
for future studies to elucidate potential biochemical mechanisms and further study the
comparative effects of these two folate vitamers on cancer progression.
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4.5 CONCLUSION
Based on previously conducted comparative studies looking at the effects of FA and
5MTHF on the folate pathway as well as blood folate concentrations, we expected FA and
5MTHF to elicit differential effects on colon cancer progression (128, 129). Our data suggests
that FA and 5MTHF supplementation work similarly in terms of their effects on ACF
proliferation and adenocarcinoma proliferation. The novel finding of this study is their
differential effect on tumor diameter. 5MTHF supplementation resulted in a significantly greater
tumor diameter relative to FA. Therefore, the hypothesis of our study was supported. For this
reason, the findings of this study are in line with previously conducted comparative studies
which show that 5MTHF is indeed more readily available than FA. The findings of this study
also suggest that 5MTHF supplementation may have significant public health implications.
Based on previously conducted studies in conjunction with the findings of this study, it is
possible that the proliferative effects differ in their effects on biological methylation reactions.
For this reason, further studies are necessary to better understand the effects on DNA
methylation and health outcomes, to ultimately conclude on safer parameters of use.
86
CHAPTER 5: SUMMARY
5.1 SUMMARY
FA supplementation has been linked with a number of adverse health outcomes, and a
growing body of evidence supports the idea of FA supplementation having tumor promoting
effects particularly in the context of colon cancer (141). 5MTHF has been marketed as a safer
folate supplemental alternative to FA and has been speculated to have beneficial effects over FA.
This comparative study attempted to elucidate the differential effects of FA and 5MTHF
supplementation on the progression of colon cancer using an AOM rat model. The outcomes of
measure included plasma folate concentration, ACF analysis, tumor incidence and multiplicity as
well as tumor diameter. We hypothesized that 5MTHF would have a greater tumor promoting
effect relative to FA.
Our study supports the findings of previously conducted studies which suggest that
5MTHF is at least as effect as FA in raising plasma folate concentrations. However, at the higher
dose (10mg) 5MTHF increased plasma folate concentrations to a greater degree relative to FA,
which is also in line with previously conducted studies. In terms of ACF enumeration, the
number of ACF were shown to increase with dose of supplemental folate in both groups. Tumor
incidence was not affected by folate form or dose; however, the number of adenocarcinomas was
shown to increase with dose of supplemental folate in both groups. Plasma folate concentrations
were also positively correlated with the number of adenocarcinomas. The novel finding,
concerning the differential effects of FA and 5MTHF supplementation was the differences in
87
tumor diameter. Tumor diameter was seen to increase with increasing level of supplemental
folate in both groups. Plasma folate concentrations were also positively correlated with increases
in tumor diameter. The difference between the two folate forms was seen in the 10mg
supplemental folate groups, where 5MTHF showed a greater increase in tumor diameter
compared to FA. These results provide evidence to support that 5MTHF as a supplement may in
fact not be more beneficial than FA. An important implication of this is the idea that DHFR
activity in rodents is greater than that in humans, and as a results rats metabolize folates more
efficiently than do humans. Taking into consideration the findings of this study, while also
considering the idea that humans are less effective in folate metabolism compared to rodents, it is
possible that the differential effects observed in rodents may be even more adverse in humans.
As such, future studies are warranted to conclude on safe measures for use.
In previous studies, investigators hypothesized that 5MTHF is a more readily available
form of folate. It is possible that due to this, it is also a better substrate for cell proliferation and
growth. Mechanistic studies have eluted to emphasizing the differential effects of 5MTHF on
biological methylation reactions compared to FA. Increases in DNA methylation can lead to
increases in aberrant DNA methylation which can in turn result in the progression of disease (9,
104). However, these studies are quite limited, and findings presented display gene expression
data rather than protein expression data. Despite this limitation, the results of these studies in
conjunction with the results from our study provide framework for further investigations.
88
5.2 FUTURE DIRECTIONS
There are a number of ways to follow up with the findings of our study in terms of cancer
progression, protein and gene expression, and public health translatability. From this study we
were able to confirm that 5MTHF is effective as FA at increasing plasma folate concentrations,
at low doses. However, 5MTHF differentially impacted plasma folate concentrations compared
to FA at the higher supplemental level of 10mg. This study also observed increasing
adenocarcinoma with increasing supplemental dose of both folate forms as well as a significantly
greater tumor diameter shown with 10mg 5MTHF compared to equimolar FA. This is
particularly important as 5MTHF is advertised as being a safer supplemental form of folate
relative to FA. Given the widespread evidence suggesting FA as having cancer promoting
properties, it is important to establish whether or not 5MTHF acts in a similar or differential
manner. A follow up in vivo real time study monitoring the effects of FA and 5MTHF on colon
cancer progression with mechanistic endpoints, involving DNA methylation, nucleotide
biosynthesis would give further insight into the potential differential effects of FA and 5MTHF.
Since it is true that 5MTHF resulted in greater tumor diameter and was at least as effective as FA
in increasing the number of adenocarcinoma when equimolar doses were compared, further
studies are warranted to understand these effects such that safe parameters of use be established.
89
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APPENDICES
Appendix A: Nutrient composition of experimental L-amino acid defined diets for FA
Nutrient (g/kg of diet) 1 mg FA/kg 10 mg FA/kg
L-Alanine 3.5 3.5
L-Arginine free base 11.2 11.2
L-Asparagine.H20 6 6
L-Aspartic Acid 3.5 3.5
L-Cystine 3.5 3.5
L-Glutamic Acid 35.0 35.0
Glycine 23.3 23.3
L-Histidine free base 3.3 3.3
L-Isoleucine 8.2 8.2
L-Leucine 11.1 11.1
L-Lysine HCl 14.1 14.1
L-Methionine 8.2 8.2
L-Phenylalanine 11.6 11.6
L-Proline 3.5 3.5
L-Serine 3.5 3.5
L-Threonine 8.2 8.2
L-Tryptophan 1.74 1.74
L-Tyrosine 3.5 3.5
L-Valine 8.2 8.2
Total L-amino acid 171.44 171.44
Dextrin 407 407
Sucrose 194 193
Cellulose 50 50
Corn Oil (w/0.015% BHT) 100 100
Salt Mix #210006 57.96 57.96
Vitamin Mix #317756 (no Folate) 10 10
Choline Chloride 2 2
Sodium Bicarbonate 6.6 6.6
Folic Acid in sucrose premix 1mg/g 1 10
Total 1000.000 1000.000
108
Appendix B: Nutrient composition of experimental L-amino acid defined diets for 5MTHF
Nutrient (g/kg of diet) 1 mg 5-MTHF/kg 10 mg 5-MTHF/kg
L-Alanine 3.5 3.5
L-Arginine free base 11.2 11.2
L-Asparagine.H20 6 6
L-Aspartic Acid 3.5 3.5
L-Cystine 3.5 3.5
L-Glutamic Acid 35.0 35.0
Glycine 23.3 23.3
L-Histidine free base 3.3 3.3
L-Isoleucine 8.2 8.2
L-Leucine 11.1 11.1
L-Lysine HCl 14.1 14.1
L-Methionine 8.2 8.2
L-Phenylalanine 11.6 11.6
L-Proline 3.5 3.5
L-Serine 3.5 3.5
L-Threonine 8.2 8.2
L-Tryptophan 1.74 1.74
L-Tyrosine 3.5 3.5
L-Valine 8.2 8.2
Total L-amino acid 171.44 171.44
Dextrin 407 407
Sucrose 193.96 184.59
Cellulose 50 50
Corn Oil (w/0.015% BHT) 100 100
Salt Mix #210006 57.96 57.96
Vitamin Mix #317756 (no Folate) 10 10
Choline Chloride 2 2
Sodium Bicarbonate 6.6 6.6
5-MTHF in sucrose 1mg/g 1.04 10.41
Total 1000.000 1000.000
109
Appendix C : 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