l-5-Methyltetrahydrofolate Supplementation Increases Blood Folate Concentrations to a Greater Extent than Folic Acid Supplementation in Malaysian Women

l-5-Methyltetrahydrofolate Supplementation Increases Blood Folate Concentrations to a Greater... Abstract Background Folic acid fortification of grains is mandated in many countries to prevent neural tube defects. Concerns regarding excessive intakes of folic acid have been raised. A synthetic analog of the circulating form of folate, l-5-methyltetrahydrofolate (l-5-MTHF), may be a potential alternative. Objective The objective of this study was to determine the effects of folic acid or l-5-MTHF supplementation on blood folate concentrations, methyl nutrient metabolites, and DNA methylation in women living in Malaysia, where there is no mandatory fortification policy. Methods In a 12-wk, randomized, placebo-controlled intervention trial, healthy Malaysian women (n = 142, aged 20–45 y) were randomly assigned to receive 1 of the following supplements daily: 1 mg (2.27 μmol) folic acid, 1.13 mg (2.27 μmol) l-5-MTHF, or a placebo. The primary outcomes were plasma and RBC folate and vitamin B-12 concentrations. Secondary outcomes included plasma total homocysteine, total cysteine, methionine, betaine, and choline concentrations and monocyte long interspersed nuclear element-1 (LINE-1) methylation. Results The folic acid and l-5-MTHF groups had higher (P < 0.001) RBC folate (mean ± SD: 1498 ± 580 and 1951 ± 496 nmol/L, respectively) and plasma folate [median (25th, 75th percentiles): 40.1 nmol/L (24.9, 52.7 nmol/L) and 52.0 nmol/L (42.7, 73.1 nmol/L), respectively] concentrations compared with RBC folate (958 ± 345 nmol/L) and plasma folate [12.6 nmol/L (8.80, 17.0 nmol/L)] concentrations in the placebo group at 12 wk. The l-5-MTHF group had higher RBC folate (1951 ± 496 nmol/L; P = 0.003) and plasma folate [52.0 nmol/L (42.7, 73.1 nmol/L); P = 0.023] at 12 wk than did the folic acid group [RBC folate, 1498 ± 580 nmol/L; plasma folate, 40.1 nmol/L (24.9, 52.7 nmol/L)]. The folic acid and l-5-MTHF groups had 17% and 15%, respectively, lower (P < 0.001) plasma total homocysteine concentrations than did the placebo group at 12 wk; there were no differences between the folic acid and l-5-MTHF groups. No differences in plasma vitamin B-12, total cysteine, methionine, betaine, and choline and monocyte LINE-1 methylation were observed. Conclusion These findings suggest differential effects of l-5-MTHF compared with folic acid supplementation on blood folate concentrations but no differences on plasma total homocysteine lowering in Malaysian women. This trial was registered at clinicaltrials.gov as NCT01584050. DNA methylation, folic acid, l-5-methyltetrahydrofolate (l-5-MTHF), folate, supplement, vitamin B-12 Introduction Women of childbearing age are recommended to take a supplement providing 0.4 mg/d folic acid to reduce the risk of a neural tube defect (NTD)-affected pregnancy (1). Ensuring adequate folic acid intakes in women of childbearing age prior to conception is important because the neural tube closes early during pregnancy (day 28 postconception). Because many pregnancies are unplanned, mandatory food fortification with folic acid has been introduced in many countries to reduce the incidence of NTDs (2, 3). The term folate refers to a family of chemically and structurally related compounds that are involved in the transfer of methyl groups in a variety of cellular metabolic reactions, including DNA methylation and purine and pyrimidine synthesis (4). Folic acid, a synthetic oxidized form of folate, is used in food fortification and supplements because it is more stable and more readily absorbed than naturally occurring folates. Dietary folates and folic acid are metabolized to 5-methyltetrahydrofolate (5-MTHF), which serves as a methyl donor for the remethylation of homocysteine to methionine (4). This reaction is catalyzed by methionine synthase (MS) (EC 2.1.1.13) and the cofactor, vitamin B-12. Low vitamin B-12 status, even when folate status is adequate, can trap folate as 5-MTHF, leading to impaired purine and pyrimidine synthesis (5, 6). However, folic acid can be metabolized to tetrahydrofolate (THF) and directly participate in nucleotide synthesis. Consequently, folic acid can mask the hematological (megaloblastic anemia) signs of vitamin B-12 deficiency, delaying the diagnosis of vitamin B-12 deficiency and allowing the neurological complications of vitamin B-12 deficiency to progress unchecked (7, 8). A synthetic reduced form of folate, l-5-methyltetra-hydrofolate (l-5-MTHF), may be an alternative option for use in supplements. In order to participate in purine and pyrimidine synthesis, l-5-MTHF must be converted to THF through vitamin B-12–dependent MS (EC 2.1.1.13). As such, during vitamin B-12 deficiency, 5-MTHF will not be able to participate in nucleotide synthesis and is unlikely to mask the hematological consequences of vitamin B-12 deficiency. Moreover, l-5-MTHF is the pure crystalline synthetic derivative of 5-MTHF, the natural circulating form of folate (9). Previous studies have reported that l-5-MTHF is equally bioavailable to folic acid in fortified breads (10). Taken together, these studies indicate that l-5-MTHF may be a better supplement and fortificant than folic acid. However, because of the reliance of l-5-MTHF on vitamin B-12 for metabolism, in vitamin B-12 deficiency the l-5-MTHF will remain trapped, leading to a functional folate deficiency. We report the results of a 12-wk, randomized, placebo-controlled intervention trial to compare the effects of 1 mg (2.27 μmol) of folic acid supplementation with 1.13 mg (2.27 μmol) l-5-MTHF in women of childbearing age in Malaysia, a population without mandatory folic acid food fortification. The level of 1 mg folic acid and the molar equivalent for l-5-MTHF were chosen because standard prenatal supplements contain 1 mg folic acid (1). The primary outcomes of the study were changes in plasma and RBC folate, and plasma vitamin B-12 concentrations. Secondary outcomes of the study were changes in other methyl nutrient metabolites and long interspersed nuclear element 1 (LINE-1) repeat element methylation. Given the role of folate in generating S-adenosylmethionine for methylation reactions, such as DNA methylation (11), LINE-1 methylation was assessed as a functional indicator of the effect of the supplements on DNA methylation. Methods Participants and study design Participants were recruited in February 2012 from Universiti Putra Malaysia in Seri Kembangan, Selangor, Malaysia, a population that is not exposed to mandatory folic acid fortification. Respondents were invited to participate if they were women aged 20–45 y, were nonusers of supplements containing folic acid or vitamin B-12, and were not pregnant and not planning to become pregnant. Exclusion criteria included pregnant or planning to become pregnant; vitamin supplement user; prior NTD-affected pregnancy; or chronic health condition (e.g., diabetes, inflammatory bowel disease, cancer). The study was approved by the Research Ethics Boards of the Universiti Putra and the University of British Columbia. All women provided informed written consent. The study was registered at clinicaltrials.gov as NCT01584050. A 12-wk, double-blind, randomized, placebo-controlled trial was conducted between March and June 2012. Eligible participants were randomly assigned to receive 1 of the following supplements for 12 wk: 1 mg (2.27 μmol) folic acid, 1.13 mg (2.27 μmol) l-5-MTHF as calcium salt (Metafolin; Eprova, Schaffhausen, Switzerland), or a placebo (cellulose). Randomization was done using computer generated random numbers assigned to the women at enrolment. The supplements were manufactured as hard gelatin capsules containing a blend of magnesium stearate and microcrystalline cellulose as a filler. Supplements were coded so that neither the investigators nor the participants were aware of the contents. Supplements were tested and both forms of folate were found to be completely stable (100% recovery) over the period of the study. Height, weight, BMI, and ethnicity were recorded at baseline. Fasting venous blood samples were collected from participants at baseline and at the end of the 12-wk supplementation period for determination of plasma folate and methyl metabolite concentrations, and RBC folate concentrations. Monocytes were isolated from blood samples for LINE-1 methylation (as outlined later in this section). Biochemical analyses Blood samples were collected into 2 ice-chilled vacutainors, 1 that contained EDTA and 1 that did not. Tubes were immediately placed on ice following blood collection. Within 30 min, plasma was separated by centrifugation at 2000 × g for 10 min at 4°C, divided into aliquots, and stored at –80°C until further analyses. Plasma folate and whole-blood folate concentrations were quantified as previously described by O'Broin and Kelleher (12) with chloramphenicol-resistant Lactobacillus casei as the test microorganism and folic acid as the stock standard prepared in 0.5% ascorbic acid. RBC folate was calculated from whole-blood folate by subtracting plasma folate and correcting for hematocrit. The interassay CV was 8.7% based on repeated measures of a pooled control sample. Plasma total homocysteine, total cysteine, betaine, choline, and methionine concentrations were quantified by HPLC (13, 14). Plasma vitamin B-12 concentrations were quantified using a commercial microparticle enzyme immunoassay with an AxSym Analyzer (Abbott Laboratories). Monocyte DNA methylation analyses Monocytes were isolated from whole blood using the EasySep Human Monocyte Isolation Kit (STEMCELL Technologies, Vancouver, Canada). DNA was extracted from monocytes using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The 677C→T variant (rs1801133) in the gene encoding methylenetetrahydrofolate reductase (MTHFR) was genotyped using TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA). Bisulfite pyrosequencing was used to quantify LINE-1 repeat element methylation using published primers and methods (15). Genomic DNA was bisulfite-treated using the EZ DNA Methylation Gold Kit (Zymo Research, Irvine, CA). LINE-1 regions were amplified in bisulfite-converted DNA using HotStart Taq DNA Polymerase (Qiagen, Hilden, Germany) followed by bisulfite pyrosequencing. The percentage of methylation of each CpG site was quantified using the PyroMark CpG Software (Qiagen). Results are presented as the mean methylation for all CpG sites analyzed. Statistical analyses Normally distributed variables are presented as means ± SDs. Variables were log (natural)-transformed if they were not normally distributed and are presented as the median (25th and 75th percentiles). The effect of supplementation on continuous variables was determined by unadjusted general linear models in normally distributed data and in log (natural)-transformed data. Post hoc analyses were used to determine differences among the supplement groups. The Kruskal-Wallis H nonparametric test was used to determine the effect of supplementation on continuous variables that were not normally distributed after log (natural) transformations. Results were considered significant at P < 0.05. Analyses were conducted using SPSS software version 22 (IBM, Armonk, NY). Results The participant flow and follow-up of the intervention trial are shown in Figure 1. Of the 142 women that met inclusion criteria and were recruited and randomized for the trial, 108 women attended the baseline visit. Of those, 75 women completed the trial: n = 26 in the placebo group, n = 29 in the folic acid group, and n = 20 in the l-5-MTHF group. There were 33 women lost to follow-up: n = 12 in the placebo group, n = 11 in the folic acid group, and n = 10 in the l-5-MTHF group. There were no differences in the baseline characteristics between the women who completed the trial and the women who were lost to follow-up. FIGURE 1 View largeDownload slide Participant flow and follow-up of supplementation with either placebo, folic acid, or l-5-MTHF in women of childbearing age in Seri Kembangan, Selangor, Malaysia. l-5-MTHF, l-5-methyltetrahydrofolate. FIGURE 1 View largeDownload slide Participant flow and follow-up of supplementation with either placebo, folic acid, or l-5-MTHF in women of childbearing age in Seri Kembangan, Selangor, Malaysia. l-5-MTHF, l-5-methyltetrahydrofolate. The baseline characteristics of the study population are shown in Table 1. The age of participants was 22.0 ± 0.2 y. There were no differences in age, ethnicity, BMI, MTHFR C677T genotype frequencies, or blood folate status among the supplement groups at baseline. In addition, there were no differences among the supplement groups in plasma methyl metabolite concentrations (betaine, choline, total cysteine, total homocysteine, methionine) and monocyte LINE-1 methylation at baseline. TABLE 1 Characteristics of the Malaysian women at baseline1 Intervention group Characteristic Placebo (n = 26) Folic acid (n = 29) l-5-MTHF (n = 20) P value Age, y 21.9 (20.8, 22.9) 21.3 (20.0, 22.5) 22.7 (20.3, 24.2) 0.146 BMI, kg/m2 19.6 (18.7, 25.7) 21.3 (20.4, 24.9) 21.3 (19.3, 26.5) 0.725  OWO, n (%) 6 (23.1) 7 (24.1) 7 (35.0) MTHFR C677T genotype  CC, n (%) 19 (73.1) 24 (82.8) 15 (75.0)  CT, n (%) 7 (26.9) 5 (17.2) 4 (20.0)  TT, n (%) 0 (0.0) 0 (0.0) 1 (5.0) RBC folate, nmol/L 752 ± 167 739 ± 160 700 ± 163 0.551 Plasma folate, nmol/L 13.3 (9.8, 17.0) 11.2 (8.4, 19.6) 12.1 (9.2, 14.7) 0.757 Plasma vitamin B-12, pmol/L 389 (327, 531) 456 (334, 523) 371 (271, 472) 0.468 Plasma betaine, μmol/L 42.9 ± 7.1 40.0 ± 7.8 40.2 ± 7.8 0.329 Plasma choline, μmol/L 8.9 ± 1.5 8.1 ± 1.7 8.7 ± 1.4 0.162 Plasma total cysteine, μmol/L 249 ± 26.2 252 ± 30.1 253 ± 28.2 0.879 Plasma total homocysteine, μmol/L 8.4 ± 2.1 8.7 ± 1.6 9.4 ± 1.9 0.156 Plasma methionine, μmol/L 26.3 ± 3.1 25.6 ± 5.1 26.0 ± 3.5 0.849 LINE-1 methylation, % 79.7 (76.2, 81.9) 79.8 (76.1, 81.0) 76.7 (74.8, 80.7) 0.761 Intervention group Characteristic Placebo (n = 26) Folic acid (n = 29) l-5-MTHF (n = 20) P value Age, y 21.9 (20.8, 22.9) 21.3 (20.0, 22.5) 22.7 (20.3, 24.2) 0.146 BMI, kg/m2 19.6 (18.7, 25.7) 21.3 (20.4, 24.9) 21.3 (19.3, 26.5) 0.725  OWO, n (%) 6 (23.1) 7 (24.1) 7 (35.0) MTHFR C677T genotype  CC, n (%) 19 (73.1) 24 (82.8) 15 (75.0)  CT, n (%) 7 (26.9) 5 (17.2) 4 (20.0)  TT, n (%) 0 (0.0) 0 (0.0) 1 (5.0) RBC folate, nmol/L 752 ± 167 739 ± 160 700 ± 163 0.551 Plasma folate, nmol/L 13.3 (9.8, 17.0) 11.2 (8.4, 19.6) 12.1 (9.2, 14.7) 0.757 Plasma vitamin B-12, pmol/L 389 (327, 531) 456 (334, 523) 371 (271, 472) 0.468 Plasma betaine, μmol/L 42.9 ± 7.1 40.0 ± 7.8 40.2 ± 7.8 0.329 Plasma choline, μmol/L 8.9 ± 1.5 8.1 ± 1.7 8.7 ± 1.4 0.162 Plasma total cysteine, μmol/L 249 ± 26.2 252 ± 30.1 253 ± 28.2 0.879 Plasma total homocysteine, μmol/L 8.4 ± 2.1 8.7 ± 1.6 9.4 ± 1.9 0.156 Plasma methionine, μmol/L 26.3 ± 3.1 25.6 ± 5.1 26.0 ± 3.5 0.849 LINE-1 methylation, % 79.7 (76.2, 81.9) 79.8 (76.1, 81.0) 76.7 (74.8, 80.7) 0.761 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. For plasma vitamin B-12, betaine, choline, total cysteine, total homocysteine, and methionine: placebo, n = 24; folic acid, n = 28; l-5-MTHF, n = 19. For LINE-1 methylation: placebo, n = 24; folic acid, n = 27; l-5-MTHF, n = 19. LINE-1, long interspersed nuclear element-1; l-5-MTHF, l-5-methyltetrahydrofolate; MTHFR, methylenetetrahydrofolate reductase; OWO, overweight (BMI 25.0–29.9) or obese (BMI ≥30.0). View Large TABLE 1 Characteristics of the Malaysian women at baseline1 Intervention group Characteristic Placebo (n = 26) Folic acid (n = 29) l-5-MTHF (n = 20) P value Age, y 21.9 (20.8, 22.9) 21.3 (20.0, 22.5) 22.7 (20.3, 24.2) 0.146 BMI, kg/m2 19.6 (18.7, 25.7) 21.3 (20.4, 24.9) 21.3 (19.3, 26.5) 0.725  OWO, n (%) 6 (23.1) 7 (24.1) 7 (35.0) MTHFR C677T genotype  CC, n (%) 19 (73.1) 24 (82.8) 15 (75.0)  CT, n (%) 7 (26.9) 5 (17.2) 4 (20.0)  TT, n (%) 0 (0.0) 0 (0.0) 1 (5.0) RBC folate, nmol/L 752 ± 167 739 ± 160 700 ± 163 0.551 Plasma folate, nmol/L 13.3 (9.8, 17.0) 11.2 (8.4, 19.6) 12.1 (9.2, 14.7) 0.757 Plasma vitamin B-12, pmol/L 389 (327, 531) 456 (334, 523) 371 (271, 472) 0.468 Plasma betaine, μmol/L 42.9 ± 7.1 40.0 ± 7.8 40.2 ± 7.8 0.329 Plasma choline, μmol/L 8.9 ± 1.5 8.1 ± 1.7 8.7 ± 1.4 0.162 Plasma total cysteine, μmol/L 249 ± 26.2 252 ± 30.1 253 ± 28.2 0.879 Plasma total homocysteine, μmol/L 8.4 ± 2.1 8.7 ± 1.6 9.4 ± 1.9 0.156 Plasma methionine, μmol/L 26.3 ± 3.1 25.6 ± 5.1 26.0 ± 3.5 0.849 LINE-1 methylation, % 79.7 (76.2, 81.9) 79.8 (76.1, 81.0) 76.7 (74.8, 80.7) 0.761 Intervention group Characteristic Placebo (n = 26) Folic acid (n = 29) l-5-MTHF (n = 20) P value Age, y 21.9 (20.8, 22.9) 21.3 (20.0, 22.5) 22.7 (20.3, 24.2) 0.146 BMI, kg/m2 19.6 (18.7, 25.7) 21.3 (20.4, 24.9) 21.3 (19.3, 26.5) 0.725  OWO, n (%) 6 (23.1) 7 (24.1) 7 (35.0) MTHFR C677T genotype  CC, n (%) 19 (73.1) 24 (82.8) 15 (75.0)  CT, n (%) 7 (26.9) 5 (17.2) 4 (20.0)  TT, n (%) 0 (0.0) 0 (0.0) 1 (5.0) RBC folate, nmol/L 752 ± 167 739 ± 160 700 ± 163 0.551 Plasma folate, nmol/L 13.3 (9.8, 17.0) 11.2 (8.4, 19.6) 12.1 (9.2, 14.7) 0.757 Plasma vitamin B-12, pmol/L 389 (327, 531) 456 (334, 523) 371 (271, 472) 0.468 Plasma betaine, μmol/L 42.9 ± 7.1 40.0 ± 7.8 40.2 ± 7.8 0.329 Plasma choline, μmol/L 8.9 ± 1.5 8.1 ± 1.7 8.7 ± 1.4 0.162 Plasma total cysteine, μmol/L 249 ± 26.2 252 ± 30.1 253 ± 28.2 0.879 Plasma total homocysteine, μmol/L 8.4 ± 2.1 8.7 ± 1.6 9.4 ± 1.9 0.156 Plasma methionine, μmol/L 26.3 ± 3.1 25.6 ± 5.1 26.0 ± 3.5 0.849 LINE-1 methylation, % 79.7 (76.2, 81.9) 79.8 (76.1, 81.0) 76.7 (74.8, 80.7) 0.761 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. For plasma vitamin B-12, betaine, choline, total cysteine, total homocysteine, and methionine: placebo, n = 24; folic acid, n = 28; l-5-MTHF, n = 19. For LINE-1 methylation: placebo, n = 24; folic acid, n = 27; l-5-MTHF, n = 19. LINE-1, long interspersed nuclear element-1; l-5-MTHF, l-5-methyltetrahydrofolate; MTHFR, methylenetetrahydrofolate reductase; OWO, overweight (BMI 25.0–29.9) or obese (BMI ≥30.0). View Large The primary outcomes of the intervention trial are shown in Table 2. After 12 wk of supplementation, the folic acid and the l-5-MTHF groups had higher (P < 0.001) RBC folate and plasma folate concentrations than the placebo group. Interestingly, supplementation with l-5-MTHF raised blood folate status in the study population to a greater extent than did folic acid supplementation. The l-5-MTHF supplement group had higher (P = 0.003) RBC folate concentrations and higher (P = 0.023) plasma folate concentrations than the folic acid supplement group. Folic acid and l-5-MTHF supplementation had no effect on plasma vitamin B-12 concentrations. TABLE 2 Blood folate and vitamin B-12 status in Malaysian women after 12 wk of supplementation with placebo, folic acid, or l-5-MTHF1 Characteristic Placebo Folic acid l-5-MTHF RBC folate, nmol/L 958 ± 345a 1498 ± 580b 1951 ± 496c n = 26 n = 29 n = 20 Plasma folate, nmol/L 12.6 (8.80, 17.0)a 40.1 (24.9, 52.7)b 52.0 (42.7, 73.1)c n = 26 n = 29 n = 20 Plasma vitamin B-12, pmol/L 365 (309, 512) 404 (322, 458) 341 (260, 439) n = 23 n = 29 n = 18 Characteristic Placebo Folic acid l-5-MTHF RBC folate, nmol/L 958 ± 345a 1498 ± 580b 1951 ± 496c n = 26 n = 29 n = 20 Plasma folate, nmol/L 12.6 (8.80, 17.0)a 40.1 (24.9, 52.7)b 52.0 (42.7, 73.1)c n = 26 n = 29 n = 20 Plasma vitamin B-12, pmol/L 365 (309, 512) 404 (322, 458) 341 (260, 439) n = 23 n = 29 n = 18 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. Labeled values in a row without a common superscript letter differ, P < 0.001. l-5-MTHF, l-5-methyltetrahydrofolate. View Large TABLE 2 Blood folate and vitamin B-12 status in Malaysian women after 12 wk of supplementation with placebo, folic acid, or l-5-MTHF1 Characteristic Placebo Folic acid l-5-MTHF RBC folate, nmol/L 958 ± 345a 1498 ± 580b 1951 ± 496c n = 26 n = 29 n = 20 Plasma folate, nmol/L 12.6 (8.80, 17.0)a 40.1 (24.9, 52.7)b 52.0 (42.7, 73.1)c n = 26 n = 29 n = 20 Plasma vitamin B-12, pmol/L 365 (309, 512) 404 (322, 458) 341 (260, 439) n = 23 n = 29 n = 18 Characteristic Placebo Folic acid l-5-MTHF RBC folate, nmol/L 958 ± 345a 1498 ± 580b 1951 ± 496c n = 26 n = 29 n = 20 Plasma folate, nmol/L 12.6 (8.80, 17.0)a 40.1 (24.9, 52.7)b 52.0 (42.7, 73.1)c n = 26 n = 29 n = 20 Plasma vitamin B-12, pmol/L 365 (309, 512) 404 (322, 458) 341 (260, 439) n = 23 n = 29 n = 18 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. Labeled values in a row without a common superscript letter differ, P < 0.001. l-5-MTHF, l-5-methyltetrahydrofolate. View Large After 12 wk, folic acid and l-5-MTHF supplementation significantly lowered (P < 0.001) plasma total homocysteine concentrations compared with the placebo group (Table 3). No differences between the folic acid and l-5-MTHF groups were observed for plasma total homocysteine concentrations. Supplementation with folic acid or l-5-MTHF did not alter plasma concentrations of betaine, total cysteine, choline, or methionine (Table 3). Similarly, no differences were observed among the supplement groups in monocyte LINE-1 methylation at 12 wk (Table 3). TABLE 3 Methyl metabolites and monocyte DNA methylation in Malaysian women after 12 wk of supplementation with placebo, folic acid, or l-5-MTHF1 Characteristic Placebo (n = 23) Folic acid (n = 29) l-5-MTHF (n = 18) Plasma betaine, μmol/L 39.4 ± 6.8 42.2 ± 11.4 43.4 ± 7.4 Plasma choline, μmol/L 8.6 (7.7, 9.8) 8.7 (7.8, 10.2) 8.9 (7.7, 11.1) Plasma total cysteine, μmol/L 253 ± 38.2 252 ± 34.0 244 ± 41.2 Plasma total homocysteine, μmol/L 8.8 (7.9, 10.7)a 7.3 (6.5, 9.0)b 7.5 (6.6, 8.9)b Plasma methionine, μmol/L 27.0 ± 4.2 27.0 ± 4.3 26.2 ± 4.4 LINE-1 methylation,2 % 79.4 (74.6, 81.5) 80.7 (76.6, 82.6) 80.3 (76.0, 82.0) Characteristic Placebo (n = 23) Folic acid (n = 29) l-5-MTHF (n = 18) Plasma betaine, μmol/L 39.4 ± 6.8 42.2 ± 11.4 43.4 ± 7.4 Plasma choline, μmol/L 8.6 (7.7, 9.8) 8.7 (7.8, 10.2) 8.9 (7.7, 11.1) Plasma total cysteine, μmol/L 253 ± 38.2 252 ± 34.0 244 ± 41.2 Plasma total homocysteine, μmol/L 8.8 (7.9, 10.7)a 7.3 (6.5, 9.0)b 7.5 (6.6, 8.9)b Plasma methionine, μmol/L 27.0 ± 4.2 27.0 ± 4.3 26.2 ± 4.4 LINE-1 methylation,2 % 79.4 (74.6, 81.5) 80.7 (76.6, 82.6) 80.3 (76.0, 82.0) 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. Labeled values in a row without a common superscript letter differ, P < 0.001. l-5-MTHF, l-5-methyltetrahydrofolate; LINE-1, long interspersed nuclear element-1. 2Data analyzed by Kruskal-Wallis H nonparametric test for independent samples. View Large TABLE 3 Methyl metabolites and monocyte DNA methylation in Malaysian women after 12 wk of supplementation with placebo, folic acid, or l-5-MTHF1 Characteristic Placebo (n = 23) Folic acid (n = 29) l-5-MTHF (n = 18) Plasma betaine, μmol/L 39.4 ± 6.8 42.2 ± 11.4 43.4 ± 7.4 Plasma choline, μmol/L 8.6 (7.7, 9.8) 8.7 (7.8, 10.2) 8.9 (7.7, 11.1) Plasma total cysteine, μmol/L 253 ± 38.2 252 ± 34.0 244 ± 41.2 Plasma total homocysteine, μmol/L 8.8 (7.9, 10.7)a 7.3 (6.5, 9.0)b 7.5 (6.6, 8.9)b Plasma methionine, μmol/L 27.0 ± 4.2 27.0 ± 4.3 26.2 ± 4.4 LINE-1 methylation,2 % 79.4 (74.6, 81.5) 80.7 (76.6, 82.6) 80.3 (76.0, 82.0) Characteristic Placebo (n = 23) Folic acid (n = 29) l-5-MTHF (n = 18) Plasma betaine, μmol/L 39.4 ± 6.8 42.2 ± 11.4 43.4 ± 7.4 Plasma choline, μmol/L 8.6 (7.7, 9.8) 8.7 (7.8, 10.2) 8.9 (7.7, 11.1) Plasma total cysteine, μmol/L 253 ± 38.2 252 ± 34.0 244 ± 41.2 Plasma total homocysteine, μmol/L 8.8 (7.9, 10.7)a 7.3 (6.5, 9.0)b 7.5 (6.6, 8.9)b Plasma methionine, μmol/L 27.0 ± 4.2 27.0 ± 4.3 26.2 ± 4.4 LINE-1 methylation,2 % 79.4 (74.6, 81.5) 80.7 (76.6, 82.6) 80.3 (76.0, 82.0) 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. Labeled values in a row without a common superscript letter differ, P < 0.001. l-5-MTHF, l-5-methyltetrahydrofolate; LINE-1, long interspersed nuclear element-1. 2Data analyzed by Kruskal-Wallis H nonparametric test for independent samples. View Large Discussion The objective of this study was to determine the effects of a 12-wk, randomized, placebo-controlled intervention trial of 1 mg (2.27 μmol) folic acid or 1.13 mg (2.27 μmol) l-5-MTHF supplementation on blood folate and vitamin B-12 concentrations, plasma methyl metabolite concentrations, and monocyte DNA methylation in Malaysian women of childbearing age. Our findings demonstrate that l-5-MTHF may be more effective than folic acid in raising blood folate status in these women. Both folic acid and l-5-MTHF raised RBC and plasma folate concentrations after 12 wk and RBC folate concentrations were above the optimal concentrations (906 nmol/L) for prevention of NTDs (16). However, RBC and plasma folate concentrations were higher in the women supplemented with l-5-MTHF than in the women supplemented with folic acid. Folic acid and l-5-MTHF were both effective at lowering plasma total homocysteine concentrations. Previous studies have demonstrated similar increases in blood folate concentrations following supplementation with folic acid or l-5-MTHF. A comparable randomized, placebo-controlled, double-blind, 24-wk intervention trial by Venn et al. reported that folic acid (0.1 mg/d) or l-5-MTHF (0.113 mg/d) supplementation increased plasma and RBC folate concentrations in healthy women aged 18–49 y; however, these authors found that folic acid and l-5-MTHF were equally effective in raising blood folate status, with no differences between the 2 supplement groups (17, 18). Others have reported a similar finding that bread rolls providing l-5-MTHF (0.452 mg/d) had an equal effect on raising blood folate concentrations as do fortified bread rolls providing folic acid (0.4 mg/d) when fed to healthy young adults for 16 wk (10). In contrast, we report here that 1.13 mg/d l-5-MTHF supplementation increased blood folate status to a greater extent than a 1 mg/d folic acid supplement after 12 wk. The different findings may be related to the dose but also the matrix by which the supplement is provided as the prior studies provided the folate forms in fortified wheat whereas we provided the folate forms as a supplement. Similar findings have been reported by Lamers et al. (19), that a 0.416 mg/d l-5-MTHF supplement raised blood folate concentrations to a greater extent than a 0.4 mg/d folic acid supplement in healthy women aged 19–33 y. These findings suggest that at higher doses, l-5-MTHF may be more effective than folic acid in improving blood folate status in women. The RBC folate concentrations observed in both the folic acid and l-5-MTHF supplement groups after 12 wk were high; both groups had mean values >1390 nmol/L. Studies conducted in older men and women have demonstrated an association between high folate status and worse biochemical indicators of vitamin B-12 deficiency (20–22). As such, we included assessment of plasma vitamin B-12 in the trial and found that neither folic acid nor l-5-MTHF affected plasma vitamin B-12 concentrations in the women. It is also important to note that plasma vitamin B-12 concentrations were adequate (>220 pmol/L) in all treatment groups at baseline. The differential effects we observed between folic acid and l-5-MTHF on blood folate status provide evidence that l-5-MTHF could be considered as an alternative to folic acid for use in supplements. Not only is l-5-MTHF more effective in raising blood folate concentrations after 12 wk than folic acid, but it is also unlikely to mask vitamin B-12 deficiency. This is because l-5-MTHF requires the vitamin B-12–dependent enzyme MS (EC 2.1.1.13) for its metabolism (6). Therefore, when vitamin B-12 is deficient, l-5-MTHF is not converted to THF and cannot ameliorate megaloblastic anemia. Given the reliance of l-5-MTHF on vitamin B-12 for metabolism, in vitamin B-12 deficiency l-5-MTHF will remain trapped, leading to a functional folate deficiency. The l-5-MTHF is not a good substrate for folylpolyglutamate synthase and, as such, l-5-MTHF will not be retained within cells and it is proposed that the functional folate deficiency during the initial stages will not be manifested by reductions in plasma folate concentrations but over time will become apparent (23). In contrast, the functional folate deficiency will be detectable by RBC folate concentrations. In this study, we found no effects of folic acid or l-5-MTHF on plasma concentrations of betaine, cysteine, choline, or methionine. In addition, variants in genes that encode enzymes in the folate cycle are known to influence methyl metabolites like plasma total homocysteine (24). For instance, the 677C→T variant in the MTHFR gene encodes a thermolabile form of the enzyme that has an impaired ability to synthesize 5-MTHF from 5,10-methyleneTHF (25). In our study population, we found that 5 out of 29 women in the folic acid supplement group, and 4 out of 20 women in the l-5-MTHF supplement group were heterozygous (CT genotype) for the 677C→T MTHFR variant. We found only 1 woman in the l-5-MTHF supplement group who had the TT genotype. The Institute of Medicine has set the upper tolerable intake level for folic acid intake at 1 mg/d based on the concerns related to masking of vitamin B-12 deficiency (26). However, there is debate regarding the definition of high folic acid intakes and there are inconsistent data on whether folic acid intakes greater than 1 mg/d are associated with adverse health outcomes (27). For example, a meta-analysis of 13 folic acid intervention trials (range of dose 0.5–5 mg/d for >1 y; 1 study gave 40 mg/d) in subjects with a mean age of 64 y reported no effect of folic acid intervention on cancer incidence (28). In contrast, at higher intakes of folic acid, some appears unmetabolized in circulation and the implications of circulating unmetabolized folic acid on health are not known. Circulating unmetabolized folic acid has been reported in populations with mandatory folic acid fortification (29, 30), in populations without mandatory fortification (31, 32), and in serum and cord blood from pregnant women taking supplements and in breast milk (33, 34). Further, adverse effects of folic acid supplementation (5 mg/d) on immune cell function have been attributed to the presence of unmetabolized serum folic acid (35). As such, more research is needed on the metabolic effects of folic acid intakes at levels greater than 1 mg/d and on the combined metabolic effects of folic acid and vitamin B-12 deficiency. In addition to changes in blood folates status, our findings in the present study indicate a significant reduction in plasma total homocysteine concentrations, equally in both the folic acid and l-5-MTHF groups, compared with the placebo group after 12 wk of supplementation. The effectiveness of l-5-MTHF in lowering plasma total homocysteine concentrations by a similar extent to folic acid indicates another benefit of l-5-MTHF for use in supplements. We observed no effect of folic acid or l-5-MTHF on monocyte LINE-1 methylation. Changes in DNA methylation patterns are implicated in various pathologies, and folate and homocysteine are tightly linked to DNA methylation through the methionine cycle and the generation of the methyl donor, S-adenosylmethionine (AdoMet) (11). Numerous studies have investigated the link between folate status, hyperhomocysteinemia, and DNA methylation. Studies in postmenopausal women fed low-folate diets have reported decreases in circulating folate and increases in total homocysteine concentrations, accompanied by lower leukocyte global DNA methylation (36, 37). Furthermore, increases in global DNA methylation in peripheral mononuclear cells were reported in men with renal disease and hyperhomocysteinemia after treatment with 15 mg/d of MTHF (38). We observed no effect on DNA methylation with either folic acid or l-5-MTHF supplementation despite changes in blood folate status and plasma total homocysteine. In summary, our findings provide evidence that l-5-MTHF could be considered as a potential alternative to folic acid for use in supplements owing to its effectiveness in raising folate status and lowering plasma total homocysteine concentrations. Further studies are warranted to determine the long-term effects of folic acid compared with l-5-MTHF supplement use on blood folate status and DNA methylation patterns. However, it must be noted that only trials using folic acid supplementation have been shown to prevent NTDs (39); no trials have been reported on the effects of l-5-MTHF on preventing NTDs. Acknowledgments The authors’ contributions were as follows—TJG and AMD: conceived and designed the experiments; SPL and GLK: coordinated subject recruitment and data collection; REA, MBG, and SH-L: performed the experiments; REA, AMH, and AMD: analyzed the data; AMH and AMD: wrote the article; and all authors: read and approved the final manuscript. Notes Supported by funding from the Advanced Foods and Materials Network Centre of Excellence of Canada (AMD, DDK, TJG) and the Natural Sciences and Engineering Research Council of Canada (NSERC) (AMD). AMD is supported by an Investigator Grant from BC Children's Hospital Research Institute. AMH was supported by a NSERC Canada Graduate Scholarship Master's Program award. MBG was supported by a University of British Columbia doctoral fellowship. Author disclosures: AMH, REA, SPL, GLK, SH-L, MBG, DDK, TJG, and AMD, no conflicts of interest. Abbreviations used: l-5-MTHF, l-5-methyltetrahydrofolate; LINE-1, long interspersed nuclear element-1; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; NTD, neural tube defect; THF, tetrahydrofolate; 5-MTHF, 5-methyltetrahydrofolate. References 1. Wilson RD , Audibert F , Brock JA , Carroll J , Cartier L , Gagnon A , Johnson JA , Langlois S , Murphy-Kaulbeck L , Okun N et al. 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Folic acid supplementation for the prevention of neural tube defects: an updated evidence report and systematic review for the US Preventive Services Task Force . JAMA 2017 ; 317 ( 2 ): 190 – 203 . Google Scholar CrossRef Search ADS PubMed © 2018 American Society for Nutrition. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Nutrition Oxford University Press

l-5-Methyltetrahydrofolate Supplementation Increases Blood Folate Concentrations to a Greater Extent than Folic Acid Supplementation in Malaysian Women

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Oxford University Press
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© 2018 American Society for Nutrition.
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0022-3166
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1541-6100
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10.1093/jn/nxy057
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Abstract

Abstract Background Folic acid fortification of grains is mandated in many countries to prevent neural tube defects. Concerns regarding excessive intakes of folic acid have been raised. A synthetic analog of the circulating form of folate, l-5-methyltetrahydrofolate (l-5-MTHF), may be a potential alternative. Objective The objective of this study was to determine the effects of folic acid or l-5-MTHF supplementation on blood folate concentrations, methyl nutrient metabolites, and DNA methylation in women living in Malaysia, where there is no mandatory fortification policy. Methods In a 12-wk, randomized, placebo-controlled intervention trial, healthy Malaysian women (n = 142, aged 20–45 y) were randomly assigned to receive 1 of the following supplements daily: 1 mg (2.27 μmol) folic acid, 1.13 mg (2.27 μmol) l-5-MTHF, or a placebo. The primary outcomes were plasma and RBC folate and vitamin B-12 concentrations. Secondary outcomes included plasma total homocysteine, total cysteine, methionine, betaine, and choline concentrations and monocyte long interspersed nuclear element-1 (LINE-1) methylation. Results The folic acid and l-5-MTHF groups had higher (P < 0.001) RBC folate (mean ± SD: 1498 ± 580 and 1951 ± 496 nmol/L, respectively) and plasma folate [median (25th, 75th percentiles): 40.1 nmol/L (24.9, 52.7 nmol/L) and 52.0 nmol/L (42.7, 73.1 nmol/L), respectively] concentrations compared with RBC folate (958 ± 345 nmol/L) and plasma folate [12.6 nmol/L (8.80, 17.0 nmol/L)] concentrations in the placebo group at 12 wk. The l-5-MTHF group had higher RBC folate (1951 ± 496 nmol/L; P = 0.003) and plasma folate [52.0 nmol/L (42.7, 73.1 nmol/L); P = 0.023] at 12 wk than did the folic acid group [RBC folate, 1498 ± 580 nmol/L; plasma folate, 40.1 nmol/L (24.9, 52.7 nmol/L)]. The folic acid and l-5-MTHF groups had 17% and 15%, respectively, lower (P < 0.001) plasma total homocysteine concentrations than did the placebo group at 12 wk; there were no differences between the folic acid and l-5-MTHF groups. No differences in plasma vitamin B-12, total cysteine, methionine, betaine, and choline and monocyte LINE-1 methylation were observed. Conclusion These findings suggest differential effects of l-5-MTHF compared with folic acid supplementation on blood folate concentrations but no differences on plasma total homocysteine lowering in Malaysian women. This trial was registered at clinicaltrials.gov as NCT01584050. DNA methylation, folic acid, l-5-methyltetrahydrofolate (l-5-MTHF), folate, supplement, vitamin B-12 Introduction Women of childbearing age are recommended to take a supplement providing 0.4 mg/d folic acid to reduce the risk of a neural tube defect (NTD)-affected pregnancy (1). Ensuring adequate folic acid intakes in women of childbearing age prior to conception is important because the neural tube closes early during pregnancy (day 28 postconception). Because many pregnancies are unplanned, mandatory food fortification with folic acid has been introduced in many countries to reduce the incidence of NTDs (2, 3). The term folate refers to a family of chemically and structurally related compounds that are involved in the transfer of methyl groups in a variety of cellular metabolic reactions, including DNA methylation and purine and pyrimidine synthesis (4). Folic acid, a synthetic oxidized form of folate, is used in food fortification and supplements because it is more stable and more readily absorbed than naturally occurring folates. Dietary folates and folic acid are metabolized to 5-methyltetrahydrofolate (5-MTHF), which serves as a methyl donor for the remethylation of homocysteine to methionine (4). This reaction is catalyzed by methionine synthase (MS) (EC 2.1.1.13) and the cofactor, vitamin B-12. Low vitamin B-12 status, even when folate status is adequate, can trap folate as 5-MTHF, leading to impaired purine and pyrimidine synthesis (5, 6). However, folic acid can be metabolized to tetrahydrofolate (THF) and directly participate in nucleotide synthesis. Consequently, folic acid can mask the hematological (megaloblastic anemia) signs of vitamin B-12 deficiency, delaying the diagnosis of vitamin B-12 deficiency and allowing the neurological complications of vitamin B-12 deficiency to progress unchecked (7, 8). A synthetic reduced form of folate, l-5-methyltetra-hydrofolate (l-5-MTHF), may be an alternative option for use in supplements. In order to participate in purine and pyrimidine synthesis, l-5-MTHF must be converted to THF through vitamin B-12–dependent MS (EC 2.1.1.13). As such, during vitamin B-12 deficiency, 5-MTHF will not be able to participate in nucleotide synthesis and is unlikely to mask the hematological consequences of vitamin B-12 deficiency. Moreover, l-5-MTHF is the pure crystalline synthetic derivative of 5-MTHF, the natural circulating form of folate (9). Previous studies have reported that l-5-MTHF is equally bioavailable to folic acid in fortified breads (10). Taken together, these studies indicate that l-5-MTHF may be a better supplement and fortificant than folic acid. However, because of the reliance of l-5-MTHF on vitamin B-12 for metabolism, in vitamin B-12 deficiency the l-5-MTHF will remain trapped, leading to a functional folate deficiency. We report the results of a 12-wk, randomized, placebo-controlled intervention trial to compare the effects of 1 mg (2.27 μmol) of folic acid supplementation with 1.13 mg (2.27 μmol) l-5-MTHF in women of childbearing age in Malaysia, a population without mandatory folic acid food fortification. The level of 1 mg folic acid and the molar equivalent for l-5-MTHF were chosen because standard prenatal supplements contain 1 mg folic acid (1). The primary outcomes of the study were changes in plasma and RBC folate, and plasma vitamin B-12 concentrations. Secondary outcomes of the study were changes in other methyl nutrient metabolites and long interspersed nuclear element 1 (LINE-1) repeat element methylation. Given the role of folate in generating S-adenosylmethionine for methylation reactions, such as DNA methylation (11), LINE-1 methylation was assessed as a functional indicator of the effect of the supplements on DNA methylation. Methods Participants and study design Participants were recruited in February 2012 from Universiti Putra Malaysia in Seri Kembangan, Selangor, Malaysia, a population that is not exposed to mandatory folic acid fortification. Respondents were invited to participate if they were women aged 20–45 y, were nonusers of supplements containing folic acid or vitamin B-12, and were not pregnant and not planning to become pregnant. Exclusion criteria included pregnant or planning to become pregnant; vitamin supplement user; prior NTD-affected pregnancy; or chronic health condition (e.g., diabetes, inflammatory bowel disease, cancer). The study was approved by the Research Ethics Boards of the Universiti Putra and the University of British Columbia. All women provided informed written consent. The study was registered at clinicaltrials.gov as NCT01584050. A 12-wk, double-blind, randomized, placebo-controlled trial was conducted between March and June 2012. Eligible participants were randomly assigned to receive 1 of the following supplements for 12 wk: 1 mg (2.27 μmol) folic acid, 1.13 mg (2.27 μmol) l-5-MTHF as calcium salt (Metafolin; Eprova, Schaffhausen, Switzerland), or a placebo (cellulose). Randomization was done using computer generated random numbers assigned to the women at enrolment. The supplements were manufactured as hard gelatin capsules containing a blend of magnesium stearate and microcrystalline cellulose as a filler. Supplements were coded so that neither the investigators nor the participants were aware of the contents. Supplements were tested and both forms of folate were found to be completely stable (100% recovery) over the period of the study. Height, weight, BMI, and ethnicity were recorded at baseline. Fasting venous blood samples were collected from participants at baseline and at the end of the 12-wk supplementation period for determination of plasma folate and methyl metabolite concentrations, and RBC folate concentrations. Monocytes were isolated from blood samples for LINE-1 methylation (as outlined later in this section). Biochemical analyses Blood samples were collected into 2 ice-chilled vacutainors, 1 that contained EDTA and 1 that did not. Tubes were immediately placed on ice following blood collection. Within 30 min, plasma was separated by centrifugation at 2000 × g for 10 min at 4°C, divided into aliquots, and stored at –80°C until further analyses. Plasma folate and whole-blood folate concentrations were quantified as previously described by O'Broin and Kelleher (12) with chloramphenicol-resistant Lactobacillus casei as the test microorganism and folic acid as the stock standard prepared in 0.5% ascorbic acid. RBC folate was calculated from whole-blood folate by subtracting plasma folate and correcting for hematocrit. The interassay CV was 8.7% based on repeated measures of a pooled control sample. Plasma total homocysteine, total cysteine, betaine, choline, and methionine concentrations were quantified by HPLC (13, 14). Plasma vitamin B-12 concentrations were quantified using a commercial microparticle enzyme immunoassay with an AxSym Analyzer (Abbott Laboratories). Monocyte DNA methylation analyses Monocytes were isolated from whole blood using the EasySep Human Monocyte Isolation Kit (STEMCELL Technologies, Vancouver, Canada). DNA was extracted from monocytes using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The 677C→T variant (rs1801133) in the gene encoding methylenetetrahydrofolate reductase (MTHFR) was genotyped using TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA). Bisulfite pyrosequencing was used to quantify LINE-1 repeat element methylation using published primers and methods (15). Genomic DNA was bisulfite-treated using the EZ DNA Methylation Gold Kit (Zymo Research, Irvine, CA). LINE-1 regions were amplified in bisulfite-converted DNA using HotStart Taq DNA Polymerase (Qiagen, Hilden, Germany) followed by bisulfite pyrosequencing. The percentage of methylation of each CpG site was quantified using the PyroMark CpG Software (Qiagen). Results are presented as the mean methylation for all CpG sites analyzed. Statistical analyses Normally distributed variables are presented as means ± SDs. Variables were log (natural)-transformed if they were not normally distributed and are presented as the median (25th and 75th percentiles). The effect of supplementation on continuous variables was determined by unadjusted general linear models in normally distributed data and in log (natural)-transformed data. Post hoc analyses were used to determine differences among the supplement groups. The Kruskal-Wallis H nonparametric test was used to determine the effect of supplementation on continuous variables that were not normally distributed after log (natural) transformations. Results were considered significant at P < 0.05. Analyses were conducted using SPSS software version 22 (IBM, Armonk, NY). Results The participant flow and follow-up of the intervention trial are shown in Figure 1. Of the 142 women that met inclusion criteria and were recruited and randomized for the trial, 108 women attended the baseline visit. Of those, 75 women completed the trial: n = 26 in the placebo group, n = 29 in the folic acid group, and n = 20 in the l-5-MTHF group. There were 33 women lost to follow-up: n = 12 in the placebo group, n = 11 in the folic acid group, and n = 10 in the l-5-MTHF group. There were no differences in the baseline characteristics between the women who completed the trial and the women who were lost to follow-up. FIGURE 1 View largeDownload slide Participant flow and follow-up of supplementation with either placebo, folic acid, or l-5-MTHF in women of childbearing age in Seri Kembangan, Selangor, Malaysia. l-5-MTHF, l-5-methyltetrahydrofolate. FIGURE 1 View largeDownload slide Participant flow and follow-up of supplementation with either placebo, folic acid, or l-5-MTHF in women of childbearing age in Seri Kembangan, Selangor, Malaysia. l-5-MTHF, l-5-methyltetrahydrofolate. The baseline characteristics of the study population are shown in Table 1. The age of participants was 22.0 ± 0.2 y. There were no differences in age, ethnicity, BMI, MTHFR C677T genotype frequencies, or blood folate status among the supplement groups at baseline. In addition, there were no differences among the supplement groups in plasma methyl metabolite concentrations (betaine, choline, total cysteine, total homocysteine, methionine) and monocyte LINE-1 methylation at baseline. TABLE 1 Characteristics of the Malaysian women at baseline1 Intervention group Characteristic Placebo (n = 26) Folic acid (n = 29) l-5-MTHF (n = 20) P value Age, y 21.9 (20.8, 22.9) 21.3 (20.0, 22.5) 22.7 (20.3, 24.2) 0.146 BMI, kg/m2 19.6 (18.7, 25.7) 21.3 (20.4, 24.9) 21.3 (19.3, 26.5) 0.725  OWO, n (%) 6 (23.1) 7 (24.1) 7 (35.0) MTHFR C677T genotype  CC, n (%) 19 (73.1) 24 (82.8) 15 (75.0)  CT, n (%) 7 (26.9) 5 (17.2) 4 (20.0)  TT, n (%) 0 (0.0) 0 (0.0) 1 (5.0) RBC folate, nmol/L 752 ± 167 739 ± 160 700 ± 163 0.551 Plasma folate, nmol/L 13.3 (9.8, 17.0) 11.2 (8.4, 19.6) 12.1 (9.2, 14.7) 0.757 Plasma vitamin B-12, pmol/L 389 (327, 531) 456 (334, 523) 371 (271, 472) 0.468 Plasma betaine, μmol/L 42.9 ± 7.1 40.0 ± 7.8 40.2 ± 7.8 0.329 Plasma choline, μmol/L 8.9 ± 1.5 8.1 ± 1.7 8.7 ± 1.4 0.162 Plasma total cysteine, μmol/L 249 ± 26.2 252 ± 30.1 253 ± 28.2 0.879 Plasma total homocysteine, μmol/L 8.4 ± 2.1 8.7 ± 1.6 9.4 ± 1.9 0.156 Plasma methionine, μmol/L 26.3 ± 3.1 25.6 ± 5.1 26.0 ± 3.5 0.849 LINE-1 methylation, % 79.7 (76.2, 81.9) 79.8 (76.1, 81.0) 76.7 (74.8, 80.7) 0.761 Intervention group Characteristic Placebo (n = 26) Folic acid (n = 29) l-5-MTHF (n = 20) P value Age, y 21.9 (20.8, 22.9) 21.3 (20.0, 22.5) 22.7 (20.3, 24.2) 0.146 BMI, kg/m2 19.6 (18.7, 25.7) 21.3 (20.4, 24.9) 21.3 (19.3, 26.5) 0.725  OWO, n (%) 6 (23.1) 7 (24.1) 7 (35.0) MTHFR C677T genotype  CC, n (%) 19 (73.1) 24 (82.8) 15 (75.0)  CT, n (%) 7 (26.9) 5 (17.2) 4 (20.0)  TT, n (%) 0 (0.0) 0 (0.0) 1 (5.0) RBC folate, nmol/L 752 ± 167 739 ± 160 700 ± 163 0.551 Plasma folate, nmol/L 13.3 (9.8, 17.0) 11.2 (8.4, 19.6) 12.1 (9.2, 14.7) 0.757 Plasma vitamin B-12, pmol/L 389 (327, 531) 456 (334, 523) 371 (271, 472) 0.468 Plasma betaine, μmol/L 42.9 ± 7.1 40.0 ± 7.8 40.2 ± 7.8 0.329 Plasma choline, μmol/L 8.9 ± 1.5 8.1 ± 1.7 8.7 ± 1.4 0.162 Plasma total cysteine, μmol/L 249 ± 26.2 252 ± 30.1 253 ± 28.2 0.879 Plasma total homocysteine, μmol/L 8.4 ± 2.1 8.7 ± 1.6 9.4 ± 1.9 0.156 Plasma methionine, μmol/L 26.3 ± 3.1 25.6 ± 5.1 26.0 ± 3.5 0.849 LINE-1 methylation, % 79.7 (76.2, 81.9) 79.8 (76.1, 81.0) 76.7 (74.8, 80.7) 0.761 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. For plasma vitamin B-12, betaine, choline, total cysteine, total homocysteine, and methionine: placebo, n = 24; folic acid, n = 28; l-5-MTHF, n = 19. For LINE-1 methylation: placebo, n = 24; folic acid, n = 27; l-5-MTHF, n = 19. LINE-1, long interspersed nuclear element-1; l-5-MTHF, l-5-methyltetrahydrofolate; MTHFR, methylenetetrahydrofolate reductase; OWO, overweight (BMI 25.0–29.9) or obese (BMI ≥30.0). View Large TABLE 1 Characteristics of the Malaysian women at baseline1 Intervention group Characteristic Placebo (n = 26) Folic acid (n = 29) l-5-MTHF (n = 20) P value Age, y 21.9 (20.8, 22.9) 21.3 (20.0, 22.5) 22.7 (20.3, 24.2) 0.146 BMI, kg/m2 19.6 (18.7, 25.7) 21.3 (20.4, 24.9) 21.3 (19.3, 26.5) 0.725  OWO, n (%) 6 (23.1) 7 (24.1) 7 (35.0) MTHFR C677T genotype  CC, n (%) 19 (73.1) 24 (82.8) 15 (75.0)  CT, n (%) 7 (26.9) 5 (17.2) 4 (20.0)  TT, n (%) 0 (0.0) 0 (0.0) 1 (5.0) RBC folate, nmol/L 752 ± 167 739 ± 160 700 ± 163 0.551 Plasma folate, nmol/L 13.3 (9.8, 17.0) 11.2 (8.4, 19.6) 12.1 (9.2, 14.7) 0.757 Plasma vitamin B-12, pmol/L 389 (327, 531) 456 (334, 523) 371 (271, 472) 0.468 Plasma betaine, μmol/L 42.9 ± 7.1 40.0 ± 7.8 40.2 ± 7.8 0.329 Plasma choline, μmol/L 8.9 ± 1.5 8.1 ± 1.7 8.7 ± 1.4 0.162 Plasma total cysteine, μmol/L 249 ± 26.2 252 ± 30.1 253 ± 28.2 0.879 Plasma total homocysteine, μmol/L 8.4 ± 2.1 8.7 ± 1.6 9.4 ± 1.9 0.156 Plasma methionine, μmol/L 26.3 ± 3.1 25.6 ± 5.1 26.0 ± 3.5 0.849 LINE-1 methylation, % 79.7 (76.2, 81.9) 79.8 (76.1, 81.0) 76.7 (74.8, 80.7) 0.761 Intervention group Characteristic Placebo (n = 26) Folic acid (n = 29) l-5-MTHF (n = 20) P value Age, y 21.9 (20.8, 22.9) 21.3 (20.0, 22.5) 22.7 (20.3, 24.2) 0.146 BMI, kg/m2 19.6 (18.7, 25.7) 21.3 (20.4, 24.9) 21.3 (19.3, 26.5) 0.725  OWO, n (%) 6 (23.1) 7 (24.1) 7 (35.0) MTHFR C677T genotype  CC, n (%) 19 (73.1) 24 (82.8) 15 (75.0)  CT, n (%) 7 (26.9) 5 (17.2) 4 (20.0)  TT, n (%) 0 (0.0) 0 (0.0) 1 (5.0) RBC folate, nmol/L 752 ± 167 739 ± 160 700 ± 163 0.551 Plasma folate, nmol/L 13.3 (9.8, 17.0) 11.2 (8.4, 19.6) 12.1 (9.2, 14.7) 0.757 Plasma vitamin B-12, pmol/L 389 (327, 531) 456 (334, 523) 371 (271, 472) 0.468 Plasma betaine, μmol/L 42.9 ± 7.1 40.0 ± 7.8 40.2 ± 7.8 0.329 Plasma choline, μmol/L 8.9 ± 1.5 8.1 ± 1.7 8.7 ± 1.4 0.162 Plasma total cysteine, μmol/L 249 ± 26.2 252 ± 30.1 253 ± 28.2 0.879 Plasma total homocysteine, μmol/L 8.4 ± 2.1 8.7 ± 1.6 9.4 ± 1.9 0.156 Plasma methionine, μmol/L 26.3 ± 3.1 25.6 ± 5.1 26.0 ± 3.5 0.849 LINE-1 methylation, % 79.7 (76.2, 81.9) 79.8 (76.1, 81.0) 76.7 (74.8, 80.7) 0.761 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. For plasma vitamin B-12, betaine, choline, total cysteine, total homocysteine, and methionine: placebo, n = 24; folic acid, n = 28; l-5-MTHF, n = 19. For LINE-1 methylation: placebo, n = 24; folic acid, n = 27; l-5-MTHF, n = 19. LINE-1, long interspersed nuclear element-1; l-5-MTHF, l-5-methyltetrahydrofolate; MTHFR, methylenetetrahydrofolate reductase; OWO, overweight (BMI 25.0–29.9) or obese (BMI ≥30.0). View Large The primary outcomes of the intervention trial are shown in Table 2. After 12 wk of supplementation, the folic acid and the l-5-MTHF groups had higher (P < 0.001) RBC folate and plasma folate concentrations than the placebo group. Interestingly, supplementation with l-5-MTHF raised blood folate status in the study population to a greater extent than did folic acid supplementation. The l-5-MTHF supplement group had higher (P = 0.003) RBC folate concentrations and higher (P = 0.023) plasma folate concentrations than the folic acid supplement group. Folic acid and l-5-MTHF supplementation had no effect on plasma vitamin B-12 concentrations. TABLE 2 Blood folate and vitamin B-12 status in Malaysian women after 12 wk of supplementation with placebo, folic acid, or l-5-MTHF1 Characteristic Placebo Folic acid l-5-MTHF RBC folate, nmol/L 958 ± 345a 1498 ± 580b 1951 ± 496c n = 26 n = 29 n = 20 Plasma folate, nmol/L 12.6 (8.80, 17.0)a 40.1 (24.9, 52.7)b 52.0 (42.7, 73.1)c n = 26 n = 29 n = 20 Plasma vitamin B-12, pmol/L 365 (309, 512) 404 (322, 458) 341 (260, 439) n = 23 n = 29 n = 18 Characteristic Placebo Folic acid l-5-MTHF RBC folate, nmol/L 958 ± 345a 1498 ± 580b 1951 ± 496c n = 26 n = 29 n = 20 Plasma folate, nmol/L 12.6 (8.80, 17.0)a 40.1 (24.9, 52.7)b 52.0 (42.7, 73.1)c n = 26 n = 29 n = 20 Plasma vitamin B-12, pmol/L 365 (309, 512) 404 (322, 458) 341 (260, 439) n = 23 n = 29 n = 18 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. Labeled values in a row without a common superscript letter differ, P < 0.001. l-5-MTHF, l-5-methyltetrahydrofolate. View Large TABLE 2 Blood folate and vitamin B-12 status in Malaysian women after 12 wk of supplementation with placebo, folic acid, or l-5-MTHF1 Characteristic Placebo Folic acid l-5-MTHF RBC folate, nmol/L 958 ± 345a 1498 ± 580b 1951 ± 496c n = 26 n = 29 n = 20 Plasma folate, nmol/L 12.6 (8.80, 17.0)a 40.1 (24.9, 52.7)b 52.0 (42.7, 73.1)c n = 26 n = 29 n = 20 Plasma vitamin B-12, pmol/L 365 (309, 512) 404 (322, 458) 341 (260, 439) n = 23 n = 29 n = 18 Characteristic Placebo Folic acid l-5-MTHF RBC folate, nmol/L 958 ± 345a 1498 ± 580b 1951 ± 496c n = 26 n = 29 n = 20 Plasma folate, nmol/L 12.6 (8.80, 17.0)a 40.1 (24.9, 52.7)b 52.0 (42.7, 73.1)c n = 26 n = 29 n = 20 Plasma vitamin B-12, pmol/L 365 (309, 512) 404 (322, 458) 341 (260, 439) n = 23 n = 29 n = 18 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. Labeled values in a row without a common superscript letter differ, P < 0.001. l-5-MTHF, l-5-methyltetrahydrofolate. View Large After 12 wk, folic acid and l-5-MTHF supplementation significantly lowered (P < 0.001) plasma total homocysteine concentrations compared with the placebo group (Table 3). No differences between the folic acid and l-5-MTHF groups were observed for plasma total homocysteine concentrations. Supplementation with folic acid or l-5-MTHF did not alter plasma concentrations of betaine, total cysteine, choline, or methionine (Table 3). Similarly, no differences were observed among the supplement groups in monocyte LINE-1 methylation at 12 wk (Table 3). TABLE 3 Methyl metabolites and monocyte DNA methylation in Malaysian women after 12 wk of supplementation with placebo, folic acid, or l-5-MTHF1 Characteristic Placebo (n = 23) Folic acid (n = 29) l-5-MTHF (n = 18) Plasma betaine, μmol/L 39.4 ± 6.8 42.2 ± 11.4 43.4 ± 7.4 Plasma choline, μmol/L 8.6 (7.7, 9.8) 8.7 (7.8, 10.2) 8.9 (7.7, 11.1) Plasma total cysteine, μmol/L 253 ± 38.2 252 ± 34.0 244 ± 41.2 Plasma total homocysteine, μmol/L 8.8 (7.9, 10.7)a 7.3 (6.5, 9.0)b 7.5 (6.6, 8.9)b Plasma methionine, μmol/L 27.0 ± 4.2 27.0 ± 4.3 26.2 ± 4.4 LINE-1 methylation,2 % 79.4 (74.6, 81.5) 80.7 (76.6, 82.6) 80.3 (76.0, 82.0) Characteristic Placebo (n = 23) Folic acid (n = 29) l-5-MTHF (n = 18) Plasma betaine, μmol/L 39.4 ± 6.8 42.2 ± 11.4 43.4 ± 7.4 Plasma choline, μmol/L 8.6 (7.7, 9.8) 8.7 (7.8, 10.2) 8.9 (7.7, 11.1) Plasma total cysteine, μmol/L 253 ± 38.2 252 ± 34.0 244 ± 41.2 Plasma total homocysteine, μmol/L 8.8 (7.9, 10.7)a 7.3 (6.5, 9.0)b 7.5 (6.6, 8.9)b Plasma methionine, μmol/L 27.0 ± 4.2 27.0 ± 4.3 26.2 ± 4.4 LINE-1 methylation,2 % 79.4 (74.6, 81.5) 80.7 (76.6, 82.6) 80.3 (76.0, 82.0) 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. Labeled values in a row without a common superscript letter differ, P < 0.001. l-5-MTHF, l-5-methyltetrahydrofolate; LINE-1, long interspersed nuclear element-1. 2Data analyzed by Kruskal-Wallis H nonparametric test for independent samples. View Large TABLE 3 Methyl metabolites and monocyte DNA methylation in Malaysian women after 12 wk of supplementation with placebo, folic acid, or l-5-MTHF1 Characteristic Placebo (n = 23) Folic acid (n = 29) l-5-MTHF (n = 18) Plasma betaine, μmol/L 39.4 ± 6.8 42.2 ± 11.4 43.4 ± 7.4 Plasma choline, μmol/L 8.6 (7.7, 9.8) 8.7 (7.8, 10.2) 8.9 (7.7, 11.1) Plasma total cysteine, μmol/L 253 ± 38.2 252 ± 34.0 244 ± 41.2 Plasma total homocysteine, μmol/L 8.8 (7.9, 10.7)a 7.3 (6.5, 9.0)b 7.5 (6.6, 8.9)b Plasma methionine, μmol/L 27.0 ± 4.2 27.0 ± 4.3 26.2 ± 4.4 LINE-1 methylation,2 % 79.4 (74.6, 81.5) 80.7 (76.6, 82.6) 80.3 (76.0, 82.0) Characteristic Placebo (n = 23) Folic acid (n = 29) l-5-MTHF (n = 18) Plasma betaine, μmol/L 39.4 ± 6.8 42.2 ± 11.4 43.4 ± 7.4 Plasma choline, μmol/L 8.6 (7.7, 9.8) 8.7 (7.8, 10.2) 8.9 (7.7, 11.1) Plasma total cysteine, μmol/L 253 ± 38.2 252 ± 34.0 244 ± 41.2 Plasma total homocysteine, μmol/L 8.8 (7.9, 10.7)a 7.3 (6.5, 9.0)b 7.5 (6.6, 8.9)b Plasma methionine, μmol/L 27.0 ± 4.2 27.0 ± 4.3 26.2 ± 4.4 LINE-1 methylation,2 % 79.4 (74.6, 81.5) 80.7 (76.6, 82.6) 80.3 (76.0, 82.0) 1Data are means ± SDs or medians (25th, 75th percentiles). Data were analyzed by general linear models. Labeled values in a row without a common superscript letter differ, P < 0.001. l-5-MTHF, l-5-methyltetrahydrofolate; LINE-1, long interspersed nuclear element-1. 2Data analyzed by Kruskal-Wallis H nonparametric test for independent samples. View Large Discussion The objective of this study was to determine the effects of a 12-wk, randomized, placebo-controlled intervention trial of 1 mg (2.27 μmol) folic acid or 1.13 mg (2.27 μmol) l-5-MTHF supplementation on blood folate and vitamin B-12 concentrations, plasma methyl metabolite concentrations, and monocyte DNA methylation in Malaysian women of childbearing age. Our findings demonstrate that l-5-MTHF may be more effective than folic acid in raising blood folate status in these women. Both folic acid and l-5-MTHF raised RBC and plasma folate concentrations after 12 wk and RBC folate concentrations were above the optimal concentrations (906 nmol/L) for prevention of NTDs (16). However, RBC and plasma folate concentrations were higher in the women supplemented with l-5-MTHF than in the women supplemented with folic acid. Folic acid and l-5-MTHF were both effective at lowering plasma total homocysteine concentrations. Previous studies have demonstrated similar increases in blood folate concentrations following supplementation with folic acid or l-5-MTHF. A comparable randomized, placebo-controlled, double-blind, 24-wk intervention trial by Venn et al. reported that folic acid (0.1 mg/d) or l-5-MTHF (0.113 mg/d) supplementation increased plasma and RBC folate concentrations in healthy women aged 18–49 y; however, these authors found that folic acid and l-5-MTHF were equally effective in raising blood folate status, with no differences between the 2 supplement groups (17, 18). Others have reported a similar finding that bread rolls providing l-5-MTHF (0.452 mg/d) had an equal effect on raising blood folate concentrations as do fortified bread rolls providing folic acid (0.4 mg/d) when fed to healthy young adults for 16 wk (10). In contrast, we report here that 1.13 mg/d l-5-MTHF supplementation increased blood folate status to a greater extent than a 1 mg/d folic acid supplement after 12 wk. The different findings may be related to the dose but also the matrix by which the supplement is provided as the prior studies provided the folate forms in fortified wheat whereas we provided the folate forms as a supplement. Similar findings have been reported by Lamers et al. (19), that a 0.416 mg/d l-5-MTHF supplement raised blood folate concentrations to a greater extent than a 0.4 mg/d folic acid supplement in healthy women aged 19–33 y. These findings suggest that at higher doses, l-5-MTHF may be more effective than folic acid in improving blood folate status in women. The RBC folate concentrations observed in both the folic acid and l-5-MTHF supplement groups after 12 wk were high; both groups had mean values >1390 nmol/L. Studies conducted in older men and women have demonstrated an association between high folate status and worse biochemical indicators of vitamin B-12 deficiency (20–22). As such, we included assessment of plasma vitamin B-12 in the trial and found that neither folic acid nor l-5-MTHF affected plasma vitamin B-12 concentrations in the women. It is also important to note that plasma vitamin B-12 concentrations were adequate (>220 pmol/L) in all treatment groups at baseline. The differential effects we observed between folic acid and l-5-MTHF on blood folate status provide evidence that l-5-MTHF could be considered as an alternative to folic acid for use in supplements. Not only is l-5-MTHF more effective in raising blood folate concentrations after 12 wk than folic acid, but it is also unlikely to mask vitamin B-12 deficiency. This is because l-5-MTHF requires the vitamin B-12–dependent enzyme MS (EC 2.1.1.13) for its metabolism (6). Therefore, when vitamin B-12 is deficient, l-5-MTHF is not converted to THF and cannot ameliorate megaloblastic anemia. Given the reliance of l-5-MTHF on vitamin B-12 for metabolism, in vitamin B-12 deficiency l-5-MTHF will remain trapped, leading to a functional folate deficiency. The l-5-MTHF is not a good substrate for folylpolyglutamate synthase and, as such, l-5-MTHF will not be retained within cells and it is proposed that the functional folate deficiency during the initial stages will not be manifested by reductions in plasma folate concentrations but over time will become apparent (23). In contrast, the functional folate deficiency will be detectable by RBC folate concentrations. In this study, we found no effects of folic acid or l-5-MTHF on plasma concentrations of betaine, cysteine, choline, or methionine. In addition, variants in genes that encode enzymes in the folate cycle are known to influence methyl metabolites like plasma total homocysteine (24). For instance, the 677C→T variant in the MTHFR gene encodes a thermolabile form of the enzyme that has an impaired ability to synthesize 5-MTHF from 5,10-methyleneTHF (25). In our study population, we found that 5 out of 29 women in the folic acid supplement group, and 4 out of 20 women in the l-5-MTHF supplement group were heterozygous (CT genotype) for the 677C→T MTHFR variant. We found only 1 woman in the l-5-MTHF supplement group who had the TT genotype. The Institute of Medicine has set the upper tolerable intake level for folic acid intake at 1 mg/d based on the concerns related to masking of vitamin B-12 deficiency (26). However, there is debate regarding the definition of high folic acid intakes and there are inconsistent data on whether folic acid intakes greater than 1 mg/d are associated with adverse health outcomes (27). For example, a meta-analysis of 13 folic acid intervention trials (range of dose 0.5–5 mg/d for >1 y; 1 study gave 40 mg/d) in subjects with a mean age of 64 y reported no effect of folic acid intervention on cancer incidence (28). In contrast, at higher intakes of folic acid, some appears unmetabolized in circulation and the implications of circulating unmetabolized folic acid on health are not known. Circulating unmetabolized folic acid has been reported in populations with mandatory folic acid fortification (29, 30), in populations without mandatory fortification (31, 32), and in serum and cord blood from pregnant women taking supplements and in breast milk (33, 34). Further, adverse effects of folic acid supplementation (5 mg/d) on immune cell function have been attributed to the presence of unmetabolized serum folic acid (35). As such, more research is needed on the metabolic effects of folic acid intakes at levels greater than 1 mg/d and on the combined metabolic effects of folic acid and vitamin B-12 deficiency. In addition to changes in blood folates status, our findings in the present study indicate a significant reduction in plasma total homocysteine concentrations, equally in both the folic acid and l-5-MTHF groups, compared with the placebo group after 12 wk of supplementation. The effectiveness of l-5-MTHF in lowering plasma total homocysteine concentrations by a similar extent to folic acid indicates another benefit of l-5-MTHF for use in supplements. We observed no effect of folic acid or l-5-MTHF on monocyte LINE-1 methylation. Changes in DNA methylation patterns are implicated in various pathologies, and folate and homocysteine are tightly linked to DNA methylation through the methionine cycle and the generation of the methyl donor, S-adenosylmethionine (AdoMet) (11). Numerous studies have investigated the link between folate status, hyperhomocysteinemia, and DNA methylation. Studies in postmenopausal women fed low-folate diets have reported decreases in circulating folate and increases in total homocysteine concentrations, accompanied by lower leukocyte global DNA methylation (36, 37). Furthermore, increases in global DNA methylation in peripheral mononuclear cells were reported in men with renal disease and hyperhomocysteinemia after treatment with 15 mg/d of MTHF (38). We observed no effect on DNA methylation with either folic acid or l-5-MTHF supplementation despite changes in blood folate status and plasma total homocysteine. In summary, our findings provide evidence that l-5-MTHF could be considered as a potential alternative to folic acid for use in supplements owing to its effectiveness in raising folate status and lowering plasma total homocysteine concentrations. Further studies are warranted to determine the long-term effects of folic acid compared with l-5-MTHF supplement use on blood folate status and DNA methylation patterns. However, it must be noted that only trials using folic acid supplementation have been shown to prevent NTDs (39); no trials have been reported on the effects of l-5-MTHF on preventing NTDs. Acknowledgments The authors’ contributions were as follows—TJG and AMD: conceived and designed the experiments; SPL and GLK: coordinated subject recruitment and data collection; REA, MBG, and SH-L: performed the experiments; REA, AMH, and AMD: analyzed the data; AMH and AMD: wrote the article; and all authors: read and approved the final manuscript. 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Journal of NutritionOxford University Press

Published: Jun 7, 2018

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