Is Natural Folate as Effective as Synthetic Folic Acid in Increasing Serum and Red Blood Cell Folate Concentrations During Pregnancy? A Proof-of-concept Pilot Study
Phase: NA
Conditions: Pregnancy
Sponsor: University of British Columbia

RESEARCH PROTOCOL 
 
 
Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
24/03/2023v11 
 
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Background 
Folate metabolism. Folate is a B-vitamin that plays a critical role in cell growth and division, 
erythropoiesis, and DNA synthesis, repair, and methylation.1 Folate also supports increasing blood 
volume in pregnancy and prevention of anemia (alongside adequate iron and vitamin B12).2,3 Folate 
from natural sources in food is in the reduced form and enters circulation directly as 5-
methyltetrahydrofolate (5-MTHF) (Figure 1 in the Appendix). Folic acid is the synthetic form of 
folate that is used in supplements and food fortification (e.g. fortified flour) due to its high stability and 
bioavailability. Folic acid is different from naturally occurring folate, as it is fully oxidized, has a 
different biochemical structure, and is metabolized differently.2 Folic acid must first be reduced to 
dihydrofolate (DHF) by an enzyme called dihydrofolate reductase (DHFR). It is then reduced further in 
a series of steps to 5-MTHF, before entering circulation.3  
Folate in early pregnancy is critical for preventing neural tube defects (NTDs). Neural tube 
closure is an early developmental event that fuses the cranium and spinal cord.4 Full closure occurs 
between 21 to 28 days post-conception. Failure of proper neural tube closure can cause death soon after 
birth or lead to serious long-term disability. Folate taken before and during early pregnancy reduces the 
risk of an NTD. In a multi-center RCT, folic acid supplementation reduced NTD recurrence by 72% 
(95% CI: 29 to 92%) in babies whose mother had a prior history of NTD.5 Several randomized trials6 
and a large non-randomized trial in China (n=250,000)7 demonstrated significant protection against 
NTD in women without a prior history of an NTD-affected pregnancy with doses of folic acid as low as 
0.4 mg/d. As a result, the Society of Obstetrics and Gynecologists of Canada recommend that all 
women of childbearing age take between 0.4-4 mg/d folic acid, depending on NTD history and 
calculated risk level, starting 2-3 months before conception.8 
Most babies in BC are exposed to synthetic folic acid supplements in utero. Reports indicate that 
94% of pregnant women in BC took a multivitamin containing folic acid during the first three months 
of pregnancy.9 Of these, 92% reported taking it daily.9 Thus, the majority of babies in BC are likely 
exposed to doses of folic acid of >0.4 mg/d, as most prenatal multivitamin supplements contain higher 
doses8 (e.g. Materna® contains 0.6 mg folic acid10).  
Too much folic acid may be harmful. The capacity of the DHFR enzyme to convert synthetic folic 
acid to DHF is limited11 and the gut cannot metabolize more than ~0.2 mg folic acid at a time.12,13 With 
doses as low as 0.2 mg/d in healthy individuals, unmetabolized folic acid has beed detected in 
circulation (Figure 1 in the Appendix).14 The body’s inability to fully metabolize synthetic folic acid 
is a concern, because high synthetic folic acid intakes have been associated with acceleration of some 
cancers, including mammary tumors in rats,15 colorectal cancer in men and women,16 prostate cancer in 
men,17 and breast cancer in women.18 Additionally, folic acid intake and presence of unmetabolized 
folic acid in plasma has been shown to effect immune function (including reduced natural killer cell 
frequency and cytotoxicity) in healthy adults (men and women) and post menopausal women.19,20 
Further, excess synthetic folic acid may interfere with folate metabolism and DNA methylation.,21 For 
example, high synthetic folic acid intakes (resulting in high levels of DHF) could potentially inhibit the 
formation of 5-MTHF, leading to a decreased synthesis of other key methyl donors, methionine and S-

RESEARCH PROTOCOL 
 
 
Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
24/03/2023v11 
 
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adenosyl-methionine (SAM).22 Imbalances in folate metabolism (e.g. altered levels of key methyl 
donors) could affect DNA methylation and gene expression.23,24 This is especially concerning in 
pregnancy as the baby is undergoing rapid growth and development.  
There is mounting concern that Canadian babies are exposed to too much folic acid in utero. A 
recent study of 368 Canadian mothers who consumed perinatal folic acid (between ~0.4 to 1 mg/d) 
showed that plasma unmetabolized folic acid was detectable in >90% of maternal and newborn cord 
blood samples.25 Authors concluded that doses of folic acid >0.4 mg/d likely exceeded the physiologic 
capacity to metabolize folic acid and should be reconsidered, especially among those consuming folic 
acid fortified foods.25,26 This raises serious concern regarding the policy of perinatal folic acid 
supplementation in Canada.  
A natural form of folate may be safer than synthetic folic acid. A naturally-occurring reduced form 
of folate, (6S)-5-MTHF (Metafolin®), is commercially available in nutrition supplements including 
prenatal multivitamins.27 This form does not require enzymatic activation as it can directly enter 
circulation. Supplementation with (6S)-5-MTHF has shown to increase blood folate in healthy men and 
women,28-30 and lactating women,31 but has yet to be evaluated in pregnancy. Physiological changes 
during pregnancy, such as hemodilution, altered renal function, or increased inflammation, may affect 
how folic acid is metabolized in pregnancy. This may consequently influence blood folate 
concentrations. It is critical to ensure that natural folate can maintain serum  folate concentrations 
during pregnancy, as it is the serum  that supplies the folate to the growing fetus. (6S)-5-MTHF is also 
thought to be safer than folic acid, as it does not produce unmetabolized folic acid, as do doses of folic 
acid ≥0.2 mg/d. Thus, a proof-of-concept trial is needed to assess the effectiveness and safety of (6S)-
5-MTHF, as compared to folic acid, during pregnancy. Given the lack of evidence on the effect of this 
natural folate during pregnancy, we propose to first conduct a trial during pregnancy after neural tube 
closure (to reduce the risk of harm should it prove less effective) to generate pilot data to inform a 
definitive trial.  
RESEARCH PLAN  
Our overarching goal is to determine if the natural (6S)-5-MTHF is as effective as folic acid in 
increasing serum  and RBC folate concentrations during pregnancy, while resulting in lower plasma 
unmetabolized folic acid levels, as compared to folic acid. We hypothesize that supplementation with 
(6S)-5-MTHF will result in comparable levels of serum  and RBC folate, and lower levels blood 
unmetabolized folic acid, as compared to supplementation with folic acid. A randomized non-
inferiority trial is needed to achieve this overarching goal. In order to inform the design of this 
definitive trial, a pilot study of (6S)-5-MTHF during pregnancy is critical.  
The specific aims of this pilot study are: 
1) To establish the mean ± standard deviation change in serum folate, RBC folate, and 
unmetabolized folic acid in each group following supplementation with (6S)-5-MTHF or folic 
acid  

RESEARCH PROTOCOL 
 
 
Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
24/03/2023v11 
 
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2) To determine participation recruitment and retention rate, the most effective recruitment 
strategies for this population, and adherence to study protocol (to inform a definitive trial) 
3) Exploratory aims: To explore differences in proposed clinical effects associated with folic acid 
supplementatation (immunity, gene methylation) and differences in biomarkers that function 
closely with folate in one carbon metabolism (B-vitamins, choline and its metabolites [betaine, 
dimethylglycine]) and which support overall blood health (ferritin, inflammation). In the 
postpartum phase, we will quantify proportion of total breastmilk folate as folic acid in each 
group, evaluate correlation of maternal postpartum plasma unmetabolized folic acid and 
breastmilk folic acid, and to evaluate RBC folate concentrations following delivery in each 
group. Differences in breastmilk biomarkers associated with folate (choline,32,33 human milk 
oligosaccharides,34 and breastmilk microbiome35) will be explored.    
Study design: A pilot study of supplementation of 0.6 mg/d folic acid (the same dose as in Materna®) 
or an equimolar dose (0.625 mg/d) (6S)-5-MTHF in pregnant women for ~16 weeks of their pregnancy 
(beginning after the neural tube closure). Both groups will also receive a prenatal multivitamin (which 
will not contain any additional folic acid/folate), to ensure nutritional adequacy of other micronutrients 
important during pregnancy (e.g. iron, calcium, etc.). See study supplement nutrition facts outlined in 
Table 1 (Appendix). This will also inform women’s acceptability to consume a multivitamin during 
pregnancy for the future trial. A supplement diary will be provided to participants at the baseline 
meeting and will include a calendar to easily record daily supplement intake, any missed 
supplements/the reason for missing a daily dose, and any perceived side effects. This supplement diary 
and any leftover pills will be given back at the endline meeting in order to assess participant adherence 
to the study protocol. 
Optional continuation of study: After the endline visit, women who are planning to breastfeed will 
have the option to continue supplementing with the study supplements until approximately 1 week 
postpartum, at which time they will provide a small (3 mL) breastmilk sample and venous blood 
sample (12 mL).  
Study visits: are outlined in Table 2 (Appendix). A total of two visits will be required (a total of 
approximately 1.5 hours). Enrolled women will be asked to continue any folic acid or other 
micronutrient supplements, as they were previously taking, until their baseline visit, which will take 
place at 8-21 weeks gestation. At the baseline visit, a blood sample will be collected and women will be 
randomized to a folate group (0.6 mg folic acid or 0.625 mg (6S)-5-MTHF). Following 16 weeks of 
supplementation , an endline blood sample will be collected.  
Optional breastmilk and postpartum blood collection: At the endline visit, women will be given 
more of their assigned folate and prenatal vitamins to continue taking until about 1 week postpartum. 
We will provide women with a collection kit to collect a small breastmilk sample (3mL) at home. This 
sample and any remaining supplements will be picked up. At the time of supplement pick-up (3-4 days 
from breastmilk collection), a certified phlebotomist will take a small (12 mL) venous blood sample. 

RESEARCH PROTOCOL 
 
 
Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
24/03/2023v11 
 
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Population and recruitment: Recruitment will be facilitated by Ms. Cochrane and volunteer research 
assistants, who will call clinics to provide information on the trial, and discuss recruitment strategy 
options, including: 
1) Hanging recruitment poster(s) throughout the clinic (e.g. in waiting rooms, patient rooms, etc)  
2) Having Ms. Cochrane (graduate student/research coordinator) present study information to 
clinic health care professionals (e.g. physicians, nurses, medical office assistants, etc.).  
We will also hang the recruitment poster throughout Vancouver in establishments visited frequently by 
pregnant women (e.g. prenatal classes, prenatal fitness/yoga studios, maternity clothing stores, etc), and 
post it electronically on social media sites viewed by pregnant women in Vancouver (e.g. prenatal 
groups on Facebook). 
Ms. Cochrane will present study details at the Vancouver midwife and obgyn monthly meetings; we 
will provide posters for them to display in their clinics. Online advertising on social media platforms 
will also be used. 
Inclusion criteria: i) pregnant woman (singleton pregnancy); ii) living in greater Vancouver area and 
willing to travel to the University of British Columbia for study visits; iii) <21 weeks gestation at time 
of consent; iv) 19-42 years; v) willing to participate  
Exclusion criteria: i) having a pre-existing medical condition known to impact maternal folate status 
(malabsorptive and inflammatory bowel diseases, active celiac disease, gastric bypass surgery, atrophic 
gastritis, epilepsy, advanced liver disease, kidney dialysis, type 1 or 2 diabetes mellitus, sickle cell 
trait/anemia); ii) lifestyle factors known to impact maternal folate status (current smoking, alcohol 
consumption, recreational drug use); iii) are medium to high risk for development of an NTD 
pregnancy (applies to women or their male partner: personal or family history [parents or siblings] of 
other folate sensitive congenital anomalies, personal NTD history or a previous NTD pregnancy); iv) 
are taking medications known to interfere with B-vitamin metabolism (Chloramphenicol, Methotrexate, 
Metformin, Sulfasalazine, Phenobarbital, Phenytoin, Primidone, Triamterene, Barbiturates); v) pre-
pregnancy body mass index (BMI) ≥30kg/m2; or vi) allergy to any study supplement ingredients   
We require 50 women (25 in each group) to reliably estimate the distributions of serum and RBC 
folate. Thus, to account for drop outs or loss to follow up, we will recruit a total of 60 women (30 in 
each group).  
Study Supplements: Bulk ingredients for the folic acid and prenatal multivitamins have been provided 
by Natural Factors (Coquitlam, Canada). Bulk (6S)-5-MTHF (Metafolin®) has been provided by 
Merck & Cie (Schaffhausen, Switzerland). All bulk ingredients have been compounded into vegetable 
gel-capsules at Natural Factors (Coquitlam, Canada). The prenatal vitamin contains the same 
micronutrient formulation as WN Pharmaceuticals Ltd Prenatal (NPN 80025456), except the folic 
acid has been removed. Health Canada has reviewed and approved the use of the study supplements. 
Randomization and double blinding: The randomization sequence was computer-generated using 
blocks of four which each contain two participants per supplement group. The list was provided to a 

RESEARCH PROTOCOL 
 
 
Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
24/03/2023v11 
 
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research assistant who packaged the supplements into individual packs identified by participant study 
IDs. A research assistant at Natural Factors will store a document outlining codes assigned to (6S)-5-
MTHF and folic acid. Neither the researchers or the participants will know what form of folate each 
code represents until the analysis phase of the trial. A master spreadsheet listing each participant’s 
study ID and the folate code they have been assigned will be kept, and can be used for unblinding at 
any point if needed. 
Concomitant Medications: Once enrolled, all other medications will be permitted during the 
intervention period, but must be reported to the researchers. The only exception will be folate/folic acid 
containing supplements/natural health products, which will not be permitted throughout the 
intervention period. 
Biochemical outcome measures: A 3-hr fasting venous blood sample (~15 mL) will be collected at 
both baseline and endline visits. The blood analysis will include: 
• Serum and RBC folate – Serum folate (nmol/L) reflects recent status or dietary intake; RBC 
folate (nmol/L) indicates longer term status (e.g. previous 3-4 months). We will assess serum  
and RBC total folate using microbiological assay, as globally recommended.36-38  
• Plasma unmetabolized folic acid -  Unmetabolized folic acid (nmol/L) is not incorporated into 
RBCs; rather, it accumulates and circulates in plasma.23 Thus, it will be measured in plasma, 
along with the other folate forms (THF, 5-Methyl-THF, 5-formyl-THF, and 5,10-methenyl-
THF) using LC-MS.39 
• Complete Blood Count: Analysis will be performed using an automated hematology analyzer 
(Sysmex XNL550, Kobe, Japan). 
• Markers of iron and inflammation: This will include measurement of ferritin (µg/L), soluble 
transferrin receptor (mg/L), body iron stores (mg/kg), retinol binding protein (µmol/L), CRP 
(mg/L), and AGP (g/L) in serum using a sandwich ELISA, and hormones that influence iron 
regulation in pregnancy, including serum hecipdin (ng/mL; measured with an ELISA) and 
serum erythropoietin (mIU/mL; measured with an immunoassay).40 
• Vitamin B12, vitamin B6, vitamin B2, choline, betaine, dimethylglycine – Total vitamin B12 
(pmol/L) will be measured in plasma using an architect immunoanalyser.41,42  Choline (µmol/L), 
betaine (µmol/L) and dimethylglycine (µmol/L) will be measured in plasma using LC-MS. 
Pyridoxal-5’-phosphate (nmol/L), a marker of vitamin B6, and vitamin B2 will be measured in 
plasma using LC-MS. 
• Folate metabolites – S-adenosyl-methionine, S-adenosyl-homocysteine, total homocysteine, 
methionine and cysteine will be measured in plasma using LC-MS43-46 
• Peripheral blood mononuclear layer cells (PBMC) – DNA methylation differences will be 
measured in PBMCs and genomic DNA will be extracted from PBMCs for assessment of 
MTHFR (677 C>T, rs1801133, and 1298 A>C, rs1801131) and DHFR (rs1643649 and 
rs70991108) gene variants using PCR.47-49 Frequency and cytotoxicity of immune cells in 

RESEARCH PROTOCOL 
 
 
Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
24/03/2023v11 
 
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PBMCs (lymphocytes [natural killer cells, T-cells, B-cells] and monocytes) will be analyzed 
using antibody staining, flow cytometry, and PCR (RNA sequencing for gene expression of 
cytotoxicity and methylation). Overall gene expression, mRNA and microRNA, differences in 
PBMCs as a result of folate form (folic acid or 5-MTHF) will be evaluated using bulk RNA 
sequencing and mi-RNA-sequencing, respectively.  
Breastmilk collection (optional): Women will be instructed to breastfeed from the right breast at some 
point between 1:00-2:50pm (as mean folate concentration during this time appear representative of 24-
h folate concentrations) on day 5-7 postpartum. The sample should be collected 2-3 hours after the last 
full expression. Participants will initiate let down but putting baby on the left breast, and from the right 
breast, they should aim to get a mix of both foremilk and hindmilk into the sterile collection jar (or with 
a breastpump if they prefer). They can either: 
• Completely express breastmilk from the right breast into the collection jar 
• Express milk for 2-3 minutes at the start of the feed to collect a small amount (3-5 mL) of 
foremilk, and again at the end of the feed to collect the same amount of hindmilk 
Once breastmilk is expressed into the collection jar, women will be instructed to mix the milk and then 
to dispense 1 ml breastmilk (using a sterile falcon pipette – provided at the endline visit) into 3 x 2mL 
amber cryovials. Women will place the cryovials into the freezer. They will be picked up frozen, 
brought to the UBC Karakochuk lab, and then frozen at -80°C. The following will be measured:  
• Total breastmilk folate – folate forms including: UMFA, THF, 5-Methyl-THF, 5-formyl-THF, 
and 5,10-methenyl-THF will be quantified via liquid chromatography-tandem mass 
spectrometry50 
• Folate binding protein – to be determined using a competitive binding radioassay procedure as 
described by Selhub et al51 
• Breastmilk fatty acids & choline forms (free choline, betaine, phosphocholine, 
glycerophosophocholine) will be quantified via liquid chromatography-tandem mass 
spectrometry 
• Breastmilk human milk oligosaccharides and breastmilk microbiome will be quantified via 
HPLC-FL and PCR. 
If women also consent to providing a venous blood sample (12mL), this will be collect at the time of 
breastmilk pick-up. Blood will be brought back to the Karakochuk Food, Nutrition and Health lab, 
processed within 2-hours, and then frozen at -80°C.  The following will be measured: 
• Serum and RBC folate – Serum folate (nmol/L) reflects recent status or dietary intake; RBC 
folate (nmol/L) indicates longer term status (e.g. previous 3-4 months). We will assess serum  
and RBC total folate using microbiological assay, as globally recommended.36-38  
• Plasma unmetabolized folic acid - Unmetabolized folic acid (nmol/L) is not incorporated into 
RBCs; rather, it accumulates and circulates in plasma.23 Thus, it will be measured in plasma 
along with the other folate forms (THF, 5-Methyl-THF, 5-formyl-THF, and 5,10-methenyl-
THF) using LC-MS.39  

RESEARCH PROTOCOL 
 
 
Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
24/03/2023v11 
 
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• Vitamin B12, vitamin B6, vitamin B2, choline, betaine, dimethylglycine – Total vitamin B12 
(pmol/L) will be measured in plasma using an architect immunoanalyser.41,42  Choline (µmol/L), 
betaine (µmol/L) and dimethylglycine (µmol/L) will be measured in plasma using LC-MS. 
Pyridoxal-5’-phosphate (nmol/L), a marker of vitamin B6, and vitamin B2 will be measured in 
plasma using LC-MS. 
• Peripheral blood mononuclear layer cells (PBMC) – DNA methylation differences will be 
measured in PBMCs and genomic DNA will be extracted from PBMCs for assessment of 
MTHFR (677 C>T, rs1801133, and 1298 A>C, rs1801131) and DHFR (rs1643649 and 
rs70991108) gene variants using PCR.47-49 Frequency and cytotoxicity of immune cells in 
PBMCs (lymphocytes [natural killer cells, T-cells, B-cells] and monocytes) will be analyzed 
using antibody staining, flow cytometry, and PCR (RNA sequencing for gene expression of 
cytotoxicity and methylation). Overall gene expression, mRNA and microRNA, differences in 
PBMCs as a result of folate form (folic acid or 5-MTHF) will be evaluated using bulk RNA 
sequencing and mi-RNA-sequencing, respectively. 
Dietary assessment: Total folic acid and folate intakes will be assessed using a validated food 
frequency questionnaire: Block Folic Acid/Dietary Folate Equivalents Screener (from 
NutritionQuest).52,53 We will calculate total dietary folate equivalents (DFEs) and adjust final outcomes 
for any differences between groups. 
Anthropometrics: Pre-pregnancy BMI will be calculated using self-reported pre-pregnancy weight 
and height. Participants will be weighed at baseline and endline visits for calculation of gestational 
weight gain throughout the intervention period, and total weight gain throughout pregnancy using the 
reported pre-pregnancy weight. 
Other data to be collected:  
Via a structured questionnaire: At basline we will collect: age, country of birth, ethnicity, education, 
occupation, household income/number of people supported by income, medical and medication history, 
reported pre-pregnancy weight and height, past pregnancies, smoking status, alcohol consumption, diet 
history, dietary supplement use. Participants will also be asked how they hear about the study, to 
inform the most effective recruitment strategies. At endline we will ask for general feedback regarding 
the trial process and confirm any changes in medical conditions or medication use since the baseline 
visit.   
Timeline: The research will be conducted over a period of approximately 2.5 years, with recruitment 
beginning as soon as ethics approval is complete and an intervention period of 16-weeks (or until 1-
week postpartum if breastmilk sample collected).  
Data analysis: Descriptive statistics will be used for participant baseline data. Continuous variables 
will be reported with a mean ± standard deviation (or median and interquartile range if not normally 
distributed) and categorical variables as absolute numbers per group and percentage. Capsule counts 
will be used to calculate participant adherence and the supplement diary will used to provide 
descriptive insight into adherence barriers. Data will be assessed for normality and transformations or 

RESEARCH PROTOCOL 
 
 
Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
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use of equivalent non-parametric tests will be considered if data do not follow a normal distribution. 
Statistical analyses will be performed using Stata (Stata Corp, Texas, USA). All analyses will initially 
be completed on an intention-to-treat basis according to initial group allocation at baseline and without 
any imputation for missing data. Secondary per-protocol analyses will be conducted including those 
who fully completed the study and adhered to the study protocol. Multiple imputation will be used to 
correct for missing data as appropriate in sensitivity analyses.  
Primary analyses: We will calculate the mean ± standard deviation for primary outcomes (serum 
folate, RBC folate and plasma UMFA) in each group at baseline and endline, as well as the mean ± 
standard deviation of the within-woman change in these outcomes in each group. These estimates will 
be used to inform the sample size calculation of the definitive trial.  
Secondary analyses: Exploratory analyses of primary outcomes and exploratory biomarkers will be 
conducted using a multiple linear regression model to investigate if the change from baseline to endline 
differed according to treatment status, adjusting for baseline values and explanatory variables (DFEs, 
exploratory biomarkers, weeks gestation at supplement initiation). The proportion of women with 
UMFA ≥0.2 nmol/L (considered a detectable concentration) at baseline and endline in each group will 
also be calculated. To inform our secondary objective, overall study participation rate will be estimated 
by dividing the total number of women enrolled by the total number of women who were interested in 
participating. Weekly recruitment rate will be estimated by dividing the total number of women 
enrolled by the total number of weeks it took to recruit them. Participant retention rate will be 
estimated by dividing the number of women who complete the full trial by the total number of women 
enrolled. For each recruitment strategy (including posters, social media, word of mouth), total reach 
(social media only), inquires, enrollments and cost will be evaluated. The odds of enrollment after 
inquiring from each strategy will be evaluated with an odds ratio. As available, reasons for not 
enrolling will be recorded.  
Postpartum analyses: We will calculate the mean ± standard deviation for proportion of folic acid in 
breastmilk (dividing concentration of breastmilk folic acid by sum of all breastmilk folate forms) and 
for maternal postpartum RBC folate. Linear regression will be used to estimate the association between 
breastmilk folic acid concentration and maternal postpartum plasma UMFA. Postpartum outcomes will 
be further analyzed using multiple linear regression, adjusting for endline values and explanatory 
variables (DFEs, total weeks supplementing, adherence to breastmilk collection protocol). 
Data privacy and storage: A consent statement that fully clarifies the study’s purpose, risks and 
procedures will be provided and explained to participants and will highlight the participants’ right to 
withdraw at any point from the study. All participants will be given a unique study ID, which will be 
used on all study documents and data collection forms. All electronic documents will be encrypted and 
stored on a secure server at the BC Children’s Hospital Research Institute, and all paper documents will 
be stored in a locked filing cabinet in Dr. Karakochuk’s office at the University of British Columbia. 
Only the principal investigators (Karakochuk and Hutcheon) and the research coordinator (graduate 
student, Kelsey Cochrane) will have access to the data. Blood and breastmilk (if applicable) samples 
will be kept in a secure, locked freezer at the University of British Columiba, and stored for 
approximately 5 years after collection.  

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Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
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Risk assessment: Folic acid is provided in the dose currently recommended in Canada (0.4-1 mg/d); 
thus, is considered low risk.8 Natural (6S)-5-MTHF is recognized as safe and is included in Canadian 
prenatal vitamins with no established tolerable upper limit (no risk of harm is currently known). 
Randomization will occur between 8-21 weeks gestation (after neural tube closure). Participants will be 
notified of any clinically deficient nutrition biomarkers in their results. 
Methods for reporting adverse and serious adverse events: An adverse event (AE) is defined as any 
untoward occurrence in a participant administered an investigational product. The AE may be: i) a new 
illness, ii) worsening of a concomitant illness, iii) an effect of the study protocol, or iv) a combination 
of two or more of these factors. The use of the term ‘AE’ does not imply a relationship with the study 
product or with the clinical study. Adverse events fall into the categories of ‘non-serious’ and ‘serious’. 
A serious adverse event (SAE) is one that: i) results in death, ii) is life threatening, iii) requires subject 
hospitalization or prolongation of existing hospitalization, or iv) results in persistent or significant 
disability or incapacity. Any other important medical event that may not result in death, be life 
threatening, or require hospitalization, may be considered a SAE when, based upon appropriate medical 
judgment, the event may require medical intervention to prevent one of the outcomes listed above. All 
other AEs are considered non-serious.  
The period of observation for collection of AE/SAEs will begin upon enrolment of participants and will 
continue throughout the duration of the study. Participants will be instructed to report any suspected 
AE/SAEs to the research coordinator immediately. All AE/SAEs will be assessed by the qualified 
investigator for recommendations on next steps. Participants may be withdrawn and instructed to 
discontinue the study supplements immediately if deemed appropriate by the qualified investigator.  
Documentation of an AE/SAEs requires that a separate report be completed by the principal 
investigator in each case. All AEs occurring during the study will be reported and recorded, regardless 
of seriousness or relationship to the study product. The following information will be documented in 
each case: i) participant study ID, ii) description of the event, iii) intensity/severity, iv) seriousness, and 
v) relationship to the study product. If a participant had to discontinue supplements due to an AE/SAE, 
the supplements will be collected immediately by the researcher and re-tested for quality control. If 
there is a safety issue identified with the supplements, all participants will be contacted and instructed 
to immediately discontinue supplement intake.  
All AE/SAEs will be reported to the ethics committee and to Health Canada. Researchers will continue 
to follow participants who are withdrawn from the trial due to an AE/SAE until their symptoms have 
resolved or returned to baseline.  
Withdrawal criteria: Criteria for participant withdrawal includes: miscarriage, use of folate/folic acid 
containing supplements, and following an adverse/serious adverse event if the event in has been 
deemed due to the supplements/trial intervention (as assessed by the qualified investigator). 
Participants may also self-withdraw at any time, may be lost to follow-up, or will be withdrawn if 
recommended by their physician. If participants choose to enter the study and then are withdrawn at a 
later time, all information about them collected up to that point, including data obtained from blood 

RESEARCH PROTOCOL 
 
 
Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
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samples, will be retained for analysis in order to protect the integrity of the research, which may benefit 
future research participants and patients. However, no further information will be collected.  
Compensation for study participation: To compensate participants for their time (approx. 1.5 hours), 
we will provide a detailed summary of their folate analysis and dietary intakes report, prenatal vitamins 
throughout the intervention period, and a $25 gift voucher presented at their baseline and endline visit 
(for a total of $50). Travel and parking costs associated with study visits will be covered (up to 
$20/visit).  
Women who continue the study postpartum will receive an additional $35 (for a total of $85; $75 if 
only breastmilk is collected with no blood draw), supplements to take until ~1 week postpartum, and a 
report of their breastmilk folate results (and lactating blood folate results if providing a blood sample). 
Significance: This trial will provide the proof-of-principle that supplementation with (6S)-5-MTHF 
can effectively increase blood folate concentrations during pregnancy while resulting in lower levels of 
unmetabolized folic acid, as compared to synthetic folic acid. These pilot data are critical for the design 
of an adequately powered definitive trial of (6S)-5-MTHF supplementation, as an alternative to 
synthetic folic acid, during pregnancy. Ultimately, the definitive trial will inform the safest and most 
effective form of folate supplementation for Canadian women and their babies. 
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concentrations during pregnancy? A proof-of-concept pilot study 
 
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concentrations during pregnancy? A proof-of-concept pilot study 
 
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53. 
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Appendix  
 
 
 
 
 
 
 
 
 
 

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Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
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Table 1: Nutrition Facts for Study Supplements 
Folate (participants will be randomized to either): 
Dose: 1 capsule per day  
Group 1: Folic acid 
0.6 mg 
Group 2: Natural folate 
0.625 mg 
*Non-medicinal ingredients: microcrystalline cellulose, magnesium stearate, purified water, hydroxypropyl methylcellulose, 
titanium dioxide  
Prenatal Multivitamin Supplement  
(to be provided to all participants); 
Dose: 2 capsules per day  
Per capsule (full dose) 
Beta-Carotene 
750 mcg (1500 mcg) 
Vitamin A 
            150mcg RAE (300mcg RAE) 
Vitamin E 
6.75 mg (13.5 mg) 
Vitamin D3 
200 IU (400 IU) 
Vitamin C 
42.5 mg (85 mg) 
Niacin 
9 mg (18mg) 
Pantothenic Acid 
3 mg (6mg) 
Vitamin B6 
0.95 mg (1.9 mg) 
Vitamin B1 
0.7 mg (1.4 mg) 
Vitamin B2 
0.7 mg (1.4 mg) 
Biotin 
15 mcg (30 mcg) 
Vitamin B12 
1.3 mcg (2.6 mcg) 
Calcium 
125 mg (250 mg) 
Magnesium 
25 mg (50 mg) 
Iron 
13.5 mg (27 mg) 
Zinc 
3.75 mg (7.5 mg) 
Manganese 
1 mg (2 mg) 
Copper 
0.5 mg (1 mg) 
Iodine 
110 mcg (220 mcg) 
Molybdenum 
25 mcg (50 mcg) 
Chromium 
15 mcg (30 mcg) 
Selenium 
15 mcg (30 mcg) 
*Note the prenatal multivitamin is exactly the same as Prenatal (NPN 80025456), licence holder WN Pharmaceuticals 
Ltd, except that it does not contain any folate. 
**Non-medicinal ingredients: Microcrystalline cellulose, magnesium stearate, stearic acid, silicon dioxide, purified water, 
hydroxypropyl methylcellulose, titanium dioxide 

RESEARCH PROTOCOL 
 
 
Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
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Table 2: Study Visits and Activities 
 
Gestational age 
Study visits (V) 
Study activities 
<21 weeks gestation 
Recruitment: 
In-person, e-mail 
or phone  
▪ Confirmation of eligibility and enrolment 
▪ Informed consent 
8-21 weeks gestation  Visit 1: Baseline 
Time:  
About 60 mins 
▪ Baseline blood collection and questionnaire 
▪ Food frequency questionnaire 
▪ Randomization to folic acid or natural folate 
groups 
▪ Distribution of supplements 
▪ Weight/height 
 
 
▪ Intervention is ongoing (16 weeks total) 
▪ Self-reported adherence diary 
24-37 weeks 
gestation 
V2: Endline 
Time:  
About 30 mins 
▪ Endline blood collection 
▪ Weight 
▪ Capsule count 
▪ Endline Questionnaire 
<1week postpartum 
Optional visit  
▪ Breastmilk collection 
▪ Blood collection 
 
 
 
 
 
 
 
 
 
 
 
 
 

RESEARCH PROTOCOL 
 
 
Is natural folate as effective as synthetic folic acid in increasing serum  and red blood cell folate 
concentrations during pregnancy? A proof-of-concept pilot study 
 
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