Natural Folate vs. Synthetic Folic Acid in Pregnancy

Study Overview
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Brief Summary
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In this two-arm, double-blind randomized pilot study, the investigators will recruit 60 generally healthy, low-risk pregnant women aged 19-42 years living in Vancouver, Canada. Participants will be randomized to supplement with either 0.6 mg/day folic acid or an equimolar dose (0.625 mg/day) of (6S)-5-methyltetrahydrofolic acid for 16-weeks of their pregnancy. Randomization will occur at 8-21 weeks gestation (after neural tube closure) to reduce the risk of harm should the natural folate prove less effective. All participants will also receive a prenatal multivitamin not containing any form of folate, to ensure adequacy of other nutrients (e.g. iron) required during pregnancy. Three-hour fasting venous blood samples will be collected at baseline and endline to measure serum and red blood cell folate, unmetabolized folic acid and other related biomarkers. Women will be given the option to continue supplementing until 1-week postpartum, and provide a small (3mL) breastmilk sample and blood sample in order to measure differences in folates in breastmilk and postpartum folate. These pilot data will be used to inform a definitive trial regarding the most effective form of folate supplementation for mothers and their babies.

Detailed Description
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A sample size of 50 women (25 in each group) are required to reliably estimate the distributions of serum and red blood cell folate. Thus, to account for drop outs or loss to follow up, a total of 60 women (30 in each group) will be recruited. Aim 1: To establish the mean ± standard deviation change in serum folate, red blood cell folate, and unmetabolized folic acid levels in each group following supplementation with (6S)-5-methyltetrahydrofolic acid or folic acid for 16-weeks of pregnancy. Aim 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). 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, human milk oligosaccharides, and breastmilk microbiome) will be explored. Women may undergo informed consent process anytime <21 weeks gestation. Once participants indicate that they are interested in participating in the trial, the participant will be given a study ID, and a baseline visit will be scheduled. The baseline visit will occur between 8-21 weeks gestation, and will involve discontinuation of current folate/prenatal vitamin supplementation, review and signing the informed consent form (a scanned copy will be shared with the participant), randomization to a folate group, provision of study supplements, completion of a baseline questionnaire, completion of a food frequency questionnaire, measurement of weight and height, and a small blood draw (12ml). Intervention: total time: 16 weeks. Participants will supplement daily with the folate and prenatal vitamin supplements. The research coordinator will call the participants half way through the intervention period to serve as a reminder and answer any questions, which will enhance protocol adherence. The endline visit will occur between 24-37 weeks gestation, and will involve collecting any remaining supplements (for capsule counts), a weight measurement, and a small blood draw (12ml), and completion of a short endline questionnaire. 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/or blood sample.

Official Title
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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

Conditions
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Pregnancy

Intervention / Treatment
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* Dietary Supplement: Folic acid
* Dietary Supplement: (6S)-5-methyltetrahydrofolic acid


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Pregnant woman (singleton pregnancy) Living in the Greater Vancouver area and willing to travel to the University of British Columbia for study visits <21 weeks gestation 19-42 years of age willing to participate Exclusion Criteria: Have a pre-existing medical condition known to impact maternal folate status (malabsorptive of irritable bowel disease, active celiac disease, gastric bypass surgery, atrophic gastritis, epilepsy, advanced liver disease, kidney dialysis, type 1 or 2 diabetes mellitus, sickle cell trait/anemia) Lifestyle factors known to impact maternal folate status (smoking, alcohol overuse, non-prescription drug use/abuse) Are medium to high risk for development of an NTD-affected 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-affected pregnancy) Are taking medications known to interfere with B-vitamin metabolism (Chloramphenicol, Methotrexate, Metformin, Sulfasalazine, Phenobarbital, Phenytoin, Primidone, Triamterene, Barbiturates) pre-pregnancy body mass index ≥30 kg/m2 allergic to any of the supplement ingredients

Ages Eligible for Study
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Minimum Age: 19 Years
Maximum Age: 42 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Other
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: Folic acid<br>0.6 mg/day | Dietary Supplement: Folic acid<br>* Participants will supplement with 0.6mg/day for 16 weeks.<br>|
| Experimental: (6S)-5-methyltetrahydrofolic acid (Metafolin)<br>0.625 mg/d (an equimolar dose to folic acid) | Dietary Supplement: (6S)-5-methyltetrahydrofolic acid<br>* Participants will supplement with 0.625mg/day for 16 weeks.<br>* Other names: Metafolin;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Concentration of red blood cell folate levels | nmol/L; Reflects longer term status (e.g. previous 3-4 months) | concentrations at both baseline (8-21 weeks gestation), endline (24-37 weeks gestation), and postpartum | 
| Concentration of serum folate levels | nmol/L; Reflects recent status or dietary intake | concentrations at both baseline (8-21 weeks gestation), endline (24-37 weeks gestation), and postpartum | 
| Concentration of unmetabolized folic acid (and other folate forms: THF, 5-Methyl-THF, 5-formyl-THF, and 5,10-methenyl-THF) | nmol/L; unmetabolized folic acid is not incorporated into RBCs, rather it circulates in plasma | concentrations at both baseline (8-21 weeks gestation), endline (24-37 weeks gestation), and postpartum | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Concentration of total vitamin B-12 | pmol/mL; closely involved in folate metabolism and facilitating methionine cycles | concentrations at both baseline (8-21 weeks gestation), endline (24-37 weeks gestation) | 
| Concentration of pyridoxal-5'-phosphate | nmol/L; closely involved in folate metabolism and facilitating methionine cycles | concentrations at both baseline (8-21 weeks gestation), endline (24-37 weeks gestation) | 
| Concentration of vitamin B2 | nmol/L; closely involved in folate metabolism and facilitating methionine cycles | concentrations at both baseline (8-21 weeks gestation), endline (24-37 weeks gestation) | 
| Concentration of betaine | µmol/L; closely involved in facilitating methionine cycles | concentrations at both baseline (8-21 weeks gestation), endline (24-37 weeks gestation) | 
| Concentration of choline | µmol/L; closely involved in facilitating methionine cycles | concentrations at both baseline (8-21 weeks gestation), endline (24-37 weeks gestation) | 
| Concentration of dimethylglycine | µmol/L; closely involved in facilitating methionine cycles | concentrations at both baseline (8-21 weeks gestation), endline (24-37 weeks gestation) | 
| Concentration of S-adenosyl-methionine | µM; Metabolite produced in methionine cycles | concentrations at both baseline (8-21 weeks gestation) and endline (24-37 weeks gestation) | 
| Concentration of S-adenosyl-homocysteine | µM; Metabolite produced in methionine cycles | concentrations at both baseline (8-21 weeks gestation) and endline (24-37 weeks gestation) | 
| Concentration of total homocysteine | µmol/L; Metabolite produced in methionine cycles | concentrations at both baseline (8-21 weeks gestation) and endline (24-37 weeks gestation) | 
| Concentration of methionine | µmol/L; Metabolite produced in methionine cycles | concentrations at both baseline (8-21 weeks gestation) and endline (24-37 weeks gestation) | 
| Concentration of cysteine | µmol/L; Metabolite produced in methionine cycles | concentrations at both baseline (8-21 weeks gestation) and endline (24-37 weeks gestation) | 
| Collection of peripheral blood mononuclear layer cells | Gene variant assessment of MTHFR (677 C>T, rs1801133, and 1298 A>C, rs1801131) and DHFR (rs1643649 and rs70991108) and differences in DNA methylation, and frequency and cytotoxicity of immune cells in PBMCs. | Collection at both baseline (8-21 weeks gestation), endline (24-37 weeks gestation) | 
| Concentration of unmetabolized folic acid in breastmilk (and other folate forms: THF, 5-Methyl-THF, 5-formyl-THF, and 5,10-methenyl-THF) | nmol/L; folic acid that is unmetabolized and enters breastmilk as such | Collection at 1 week postpartum | 
| Folate binding protein in breastmilk | nmol folate binding per liter of milk | Collection at 1 week postpartum | 
| Breastmilk fatty acids & choline forms (free choline, betaine, phosphocholine, glycerophosophocholine) | Quantified via LC-MS/MS | Collection at 1 week postpartum | 
| Breastmilk human milk oligosaccharides and breastmilk microbiome | Quantified via HPLC-FL and PCR | Collection at 1 week postpartum | 
| Complete blood count | Analysis will be performed using an automated hematology analyzer (Sysmex XNL550, Kobe, Japan) | Baseline (8-21 weeks gestation), endline (24-37 weeks gestation), and postpartum | 
| Markers of Iron and Inflammation | This will include measurement of serum 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) | Baseline (8-21 weeks gestation),and endline (24-37 weeks gestation) | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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folate, folic acid, nutrition, pregnancy