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American Journal of Clinical Nutrition, Vol. 83, No. 4, 842-850, April 2006
© 2006 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

[6S]-5-Methyltetrahydrofolate is at least as effective as folic acid in preventing a decline in blood folate concentrations during lactation1,2,3

Lisa A Houghton, Kelly L Sherwood, Robert Pawlosky, Shinya Ito and Deborah L O'Connor

1 From the Department of Nutritional Sciences, University of Toronto, Toronto, Canada (LAH, KLS, and DLO); The Hospital For Sick Children, Toronto, Canada (LAH, KLS, SI, and DLO); and the National Institute on Alcoholism and Alcohol Abuse, NIH, Rockville, MD (RP)

2 Supported by Merck Eprova AG (an affiliate of Merck KGaA, Darmstadt, Germany) and the Natural Sciences & Engineering Research Council of Canada. LAH and KLS were funded by the Ontario Student Opportunity Trust Fund, The Hospital for Sick Children Foundation Scholarship Program.

3 Address reprint requests to DL O'Connor, Department of Nutritional Sciences, University of Toronto, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8. E-mail: deborah_l.o'connor{at}sickkids.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Studies in nonpregnant, nonlactating women suggest that folate supplementation in the form of 5-methyltetrahydrofolate ([6S]-5-methylTHF) is at least as effective as folic acid in increasing blood folate indexes. No data, however, are available on the effect of supplemental [6S]-5-methylTHF on blood folate concentrations during lactation.

Objective: We assessed the relative effectiveness of [6S]-5-methylTHF, a placebo, and folic acid in maintaining blood folate indexes during lactation in a sample of healthy Canadian women consuming folic acid–fortified foods.

Design: This study was designed as a 16-wk, randomized, placebo-controlled intervention. Pregnant women (n = 72) advised to consume a folic acid–containing prenatal supplement (1000 µg/d) during pregnancy were enrolled at 36 wk gestation. After delivery, the women were randomly assigned to receive [6S]-5-methylTHF (416 µg/d, 906 nmol/d) or a placebo or were assigned to a folic acid (400 µg/d, 906 nmol/d) reference group.

Results: At 16 wk of lactation, the mean red blood cell (RBC) folate concentration in women in the [6S]-5-methylTHF group (2178; 95% CI: 1854, 2559 nmol/L) was greater than that in the folic acid (1967; 1628, 2377 nmol/L; P < 0.05) and placebo (1390; 1198, 1613 nmol/L; P < 0.002) groups after adjustment for baseline concentrations (36 wk gestation). The distribution of folate forms in RBCs did not differ significantly between the [6S]-5-methylTHF and placebo groups. However, the folic acid group had greater amounts of 5-formylTHF (P < 0.03).

Conclusion: [6S]-5-MethylTHF appeared to be as effective as, and perhaps more effective than, folic acid in preserving RBC folate concentrations during lactation.

Key Words: Folate • lactation • folate supplements • folic acid • 5-methyltetrahydrofolate


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Less than optimal folate nutrition in humans has been implicated as a risk factor for many negative reproductive outcomes, including neural tube defects (NTDs), anemia during pregnancy, low infant birth weight, and cervical dysplasia (1, 2). Because of strong evidence that folic acid deficiency is associated with NTDs, mandatory folic acid fortification of white flour and enriched cereal grain products was implemented in 1998 in North America. Before folic acid fortification, lactating women in North America generally had folate intakes well below recommended amounts, and blood folate concentrations decreased and homocysteine concentrations increased with breastfeeding (3-8). Women were typically advised to consume a prenatal multivitamin and mineral supplement containing 800–1000 µg folic acid for the duration of pregnancy and to finish their supply of prenatal supplements postnatally. Data from nonpregnant, nonlactating women show a significant increase in red blood cell (RBC) folate concentrations and the virtual elimination of folate deficiency since folic acid fortification began (9, 10).

Although concerns about suboptimal folate status during reproduction are well justified, overzealous folic acid supplementation carries risks of its own (11). The main concern about high folic acid intakes has been that they could delay the diagnosis of vitamin B-12 deficiency by correcting the characteristic hematologic signs of this deficiency (1). Undetected vitamin B-12 deficiency could result in the onset and progression of neurologic damage, which can be irreversible (12). Others have noted that synthetic folic acid is directly transported into blood and speculate that although the life-long exposure of adult and fetal cells to these synthetic forms have not been investigated, health risks may exist (13). We speculate that the presence of unmetabolized folic acid may impair folate secretion into milk. The binding affinity of unmetabolized folic acid to the mammary epithelial folate receptor is 10-fold that of the natural circulating form of the vitamer, 5-methyltetrahydrofolate (14). For folate to be transferred into the cytoplasm of milk, we believe that it first must disassociate from the membrane folate receptor (15). This process is likely to be more efficient with 5-methyltetrahydrofolate as the substrate than with unmetabolized folic acid.

Recently, a stable supplemental form of [6S]-5-methyltetrahydrofolate ([6S]-5-methylTHF) as calcium salt (Metafolin; Merck Eprova AG, Schaffhausen, Switzerland) has become available. Studies of nonpregnant, nonlactating adults suggest that it is at least as effective as folic acid in increasing blood folate indexes and lowering plasma homocysteine (16-18). Use of supplemental [6S]-5-methylTHF may have some advantages over folic acid. Specifically, [6S]-5-methylTHF is unlikely to mask vitamin B-12 deficiency, it will not produce unmetabolized folic acid in the circulation, and it may be a more efficient supplemental source of the vitamer during lactation. The purpose, then, of the present study was to compare the relative effectiveness of [6S]-5-methylTHF, a placebo, and folic acid in maintaining folate status during lactation. Our secondary objective was to evaluate the folate status of a sample of healthy Canadian women consuming folic acid–fortified foods.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Participants were healthy pregnant women identified by word-of-mouth and through the Motherisk Program at The Hospital For Sick Children, Toronto, Canada. Motherisk is a counseling program that provides information to women on the safety or risk to a developing fetus and newborn of maternal exposure to drugs, chemicals, and disease. Women at <36 wk gestation were eligible for the study if they did not smoke, were between the ages of 16 and 40 y, and intended to exclusively breastfeed for >4 mo postpartum. In addition, all participants agreed not to plan a pregnancy and take appropriate precautions to prevent a pregnancy during the course of the study. Women who were unlikely to be available for study visits, did not complete food records at 36 wk of pregnancy (baseline), presented with a medical condition, or consumed a drug known to interfere with folate metabolism (eg, antibiotics) were ineligible. The study protocol was approved by the Human Ethics Committee at The Hospital for Sick Children, Toronto, and the participants gave written informed consent.

Study design
A 16-wk, double-blinded, randomized, placebo-controlled trial was conducted between April 2002 and December 2003. All visits were conducted in the Clinical Investigation Unit at The Hospital for Sick Children or in the participant's home. At 36 wk gestation (baseline ± 1 wk), venous blood samples were collected for the measurement of RBC folate, plasma folate, and plasma homocysteine (tHcy) concentrations; to perform a complete blood count; and to determine MTHFR 677C->T genotype. A self-administered questionnaire was used to collect demographic data on each participant's age, reproductive history, education, income, and prenatal supplement consumption. The subjects were asked to complete a 3-d weighed food record. Within 1 wk after the birth of their infants, the participants were asked to discontinue the use of their prenatal supplements and were randomly assigned to 1 of 2 treatment groups: placebo or [6S]-5-methylTHF (416 µg/d, 906 nmol/d). As planned a priori, a reference group of lactating women, who were enrolled according to the same inclusion and exclusion criteria as described above and followed all aspects of the study protocol, were provided with a commercial folic acid supplement containing an equimolar dose of folate (400 µg/d, 906 nmol/d) (Jamieson Laboratories, Montreal, Canada). The folate content of supplements was verified analytically and both forms of folate were found to be stable over the period of the study. In addition, all subjects received a daily multivitamin and mineral supplement, which contained 1 mg vitamin B-6, 3 µg vitamin B-12, and 4 mg ferrous fumurate (Exact; Pharmetics, Quebec, Canada). This multivitamin and mineral supplement contained no folate, and the participants agreed not to consume any other folate-containing vitamin or mineral supplement during the course of the study.

At 4 and 16 wk postpartum (±1 wk), blood samples were collected and the mothers were asked to complete 3-d weighed food records. RBC folate, plasma folate, and plasma tHcy concentrations were measured at each time point, and the distribution of the major forms of folate in the RBCs at 36 wk gestation (baseline) and 16 wk postpartum were determined in a subset of 20 participants. Only subjects who consumed 1 mg folic acid/d during pregnancy and were strictly compliant with the study procedures were considered for inclusion in the subsample. Subjects were excluded for noncompliance if they did not have a complete set of blood and breast-milk samples or if they did not return all unused study capsules and, hence, compliance with taking supplements could not be assessed. In total 7, 8, and 6 subjects in the 5-methylTHF, placebo, and folic acid groups, respectively, were excluded. Of the remaining women, 2–3 subjects per genotype (CC, CT, and TT) per treatment were selected for the folate form subanalysis.

Biochemical assessment
Blood sampling and folate analysis
Blood samples were collected in EDTA-treated tubes, transported on ice, and processed within 2 h of collection. A complete blood count was performed on whole blood by using an electronic hematology analyzer (HmX; Beckman Coulter, Miami, FL). For MTHFR 677C->T genotyping, DNA was extracted from whole blood and a polymerase chain reaction and electrophoresis were performed according to the method of Frosst et al (19). For the measurement of total blood folate, aliquots (100 µL) of whole blood were diluted 10-fold with ascorbic acid and doubly distilled water (1%, wt:vol) and incubated at 37°C for 30 min to allow for the deconjugation of folate by plasma pteroylpoly-{gamma}-glutamate hydrolase (EC 3.4.19.9). RBCs and plasma were then separated by centrifugation (1500 x g for 15 min at 4°C) from the remaining blood. For folate form analysis, aliquots of RBCs were diluted 20-fold with ascorbic acid and doubly distilled water (0.5% wt:vol) and incubated at 37°C for 30 min. Plasma was portioned for future folate analyses, and sodium ascorbate (1%, wt:vol) was added to samples to prevent the oxidation of folate. All samples were frozen immediately after processing and was stored at –80°C.

Plasma and whole-blood folate concentrations were measured by microbiologic assay as described by Molloy and Scott (20) by using the test organism Lactobacillus rhamnosus (ATCC 7649; American Type Tissue Culture Collection, Manassas, VA). The accuracy and reproducibility of these assays were assessed by using a whole-blood control standard with a certified value (29.5 nmol/L) (Whole Blood 95/528; National Institute of Biological Standards and Control, Hertfordshire, United Kingdom). Analysis in our laboratory yielded a folate content of 32 ± 4.3 nmol/L with an interassay CV of 13.2%. RBC folate was calculated from whole-blood folate by subtracting PF and correcting for hematocrit. Plasma tHcy was measured by HPLC with electrochemical detection according to the method of Cole et al (21) (interassay CV: 1.6%).

Determination of folate forms
Distribution of major RBC folate forms at 36 wk gestation and 16 wk postpartum in our study were determined by using a modification of a previously described stable-isotope dilution liquid chromatography–mass spectrometry method with electrospray ionization (ESI) (22). Procedures were adapted for analysis of whole-blood lysates and included the determination of tetrahydrofolate (THF) and 5-formylTHF as well as 5-methylTHF and folic acid. Folate standards were obtained from Sigma (St Louis, MO). 13C5-glutamyl-5-methylTHF, 13C5-glutamyl-folic acid, 13C5-glutamyl-THF, and 13C5-glutamyl-5-formylTHF were obtained from Merck Eprova AG (Schaffhausen, Switzerland). The 13C atoms occupied the 5 carbons of the glutamic acid portion of each molecule. Standard stock solutions were prepared according to a previously described procedure (22) and stored at –60°C. Typically, 0.5 mL whole-blood lysate was used for analysis, and the samples were spiked with the labeled internal standards (0.8–1.0 ng/mL) and allowed to equilibrate (30 min at 4°C). Equilibrated lysates were loaded onto a 100-mg BOND-ELUT (Varian Inc, Palo Alto, CA) phenyl solid phase extraction column that had previously been washed with methanol (1 mL) and 0.03 mmol phosphate buffer/L (1 mL). The column was then washed with 0.03 mmol phosphate buffer/L (1 mL) and 0.1% formic acid (1 mL) to remove traces of salt. The analytes were eluted with 250 µL acetonitrile:water:methanol (26:60:14) + 0.1% formic acid, and 40 µL of the extract was injected onto a 150 x 4.26 mm 5µ C18 LUNA column (Phenomenex, Torrance, CA) and samples were analyzed on an Agilent-1100 LC ion-trap mass spectrometer (Agilent Technologies) by ESI in the positive ion mode. The column was maintained at 25°C with a gradient of solvent A (0.1% formic acid in water) and solvent B (acetonitrile:water:methanol, 26:60:14, + 0.1% formic acid). The following gradient was applied: isocratic, 0.175 mL/min, 30% solvent B, 0–9 min; linear, 0.3 mL/min, 100% solvent B, 9–14 min; isocratic, 0.3 mL/min, 100% solvent B, 14–25 min; linear, 0.175 mL/min, 30% B, 25–30 min; isocratic, 0.175 mL/min, 30% B, 30–45 min.

Mass spectrometry conditions
The source was set to 300°C, ionization voltage was set to 3.5 Kv, and nitrogen pressure was set to 15 psi. All tuning parameters were optimized under analytic flow conditions with a focus on the protonated cation at a mass-to-charge ratio (m/z) of 460. The scanning range was from 450 to 480 m/z for the 12C and 13C5 5-methylTHF cations at m/z 460 and 465; 12C and 13C5 THF cations at m/z 446 and 451, respectively; 12C and 13C5 5-formylTHF cations at m/z 474 and 479, respectively; and 12C and 13C5 folic acid cations at m/z 442 and 447, respectively. All analytes were eluted within 30 min. Analytes were quantified by comparing the ratio of the ion current abundances of the internal standards to the analytes and by calculating their concentrations from known concentrations of internal standard that had been added to the sample. A previous determination of method precision was shown to be 10.8%, 11.2%, 15.4%, and 17.8% for THF, 5-methylTHF, 5-formylTHF, and folic acid, respectively.

Dietary folate assessment
Dietary intakes of energy, folate, and vitamin B-12 were determined via 3-d weighed food records completed by participants over 2 nonconsecutive weekdays and 1 weekend day at 36 wk gestation (baseline) and 4 and 16 wk postpartum. The participants were provided with electronic digital scales accurate to 1 g (CS2000; Ohaus Corporation, Pine Brook, NJ) and received oral and written instruction by 1 of 2 registered dietitians on how to weigh and record all food and beverages consumed. Food intake data were then analyzed by using an electronic version of the Canadian Nutrient File (2001b), which is based on the US Department of Agriculture Nutrient Database for Standard Reference and reflects current mandated levels of folic acid fortification in Canada (23, 24). Folate values in the Canadian Nutrient File were not determined after a trienzyme extraction procedure. No adjustment was made to the database to reflect possible overages in folate fortification levels. Folate intake was expressed as the average of 3 d of recorded intake in micrograms of dietary folate equivalents (DFE) as defined by the Institute of Medicine (1).

Statistical analyses
A sample size of 21 per group was estimated for the detection of a 1-SD difference in mean RBC folate between treatment groups ({delta} = 209 nmol/L; SD = 225 nmol/L) with 80% power and {alpha} = 0.0167. We previously reported a one SD difference in RBC folate concentrations at 4 mo postpartum among lactating adolescent mothers consuming 400 µg folic acid/d compared with a placebo (7). Twenty-six subjects were recruited per group in anticipation of a 20% attrition rate. This was an intent-to-treat study, and no subjects were withdrawn by investigators as a result of noncompliance.

SAS for WINDOWS (version 9.1; SAS Institute Inc, Cary, NC) was used for statistical analysis, and the results were considered significant at P < 0.05. All data were checked for normal distribution (PROC UNIVARIATE procedure). Skewed data were transformed for statistical analyses and then back transformed to geometric means with 95% CIs for presentation in tables. Baseline characteristics and compliance between treatment groups were compared by using a one-way analysis of variance for continuous variables and chi-square or Fisher's exact test for categorical variables (eg, MTHFR 677C->T genotype). Group differences in blood biochemical indexes at 4 and 16 wk postpartum were analyzed by using repeated-measures analysis of covariance with the respective baseline analyte concentration (36 wk gestation) as the covariate. Group ([6S]-5-methylTHF, folic acid, or placebo) and time (4 or 16 wk postpartum) were treated as main effects, with a group-by-time interaction. Tukey's comparison procedure was used for pairwise comparisons if a statistically significant group-by-time interaction was found. Examination of the relation between group and plasma folate included dietary folate intake (expressed as DFE) as a covariate. No other possible confounding variables, such as parity and MTHFR genotype, were prognostic of blood folate or tHcy concentrations and, therefore, were not included in the final statistical models.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Subject characteristics
Of the 72 women (20–38 y) who were recruited for the study, 69 were assigned to a treatment group within 1 wk after the birth of their infant. Sixty-four women completed the trial (n = 21 in the [6S]-5-methylTHF group, n = 22 in the placebo group, and n = 21 in the folic acid group) (Figure 1Go). Three of the recruited participants were not assigned to a treatment group because they were unable to initiate breastfeeding. Five subjects withdrew (n = 1 in the [6S]-5-methylTHF group, n = 1 in the placebo group, and n = 3 in the folic acid group) during the course of the study because they discontinued breastfeeding or found the study too difficult because of the extra demands of a sick infant. Sixty-seven of 69 randomly assigned participants (97%) reported that they consumed a folic acid supplement ({approx}1000 µg) daily during pregnancy. No significant differences in age, gravidity, parity, income, education, or prenatal folic acid consumption existed between the 3 groups (Table 1Go). Study subjects were well educated [63 of 69 randomly assigned participants (93%) had completed a community college or university degree], and 52 of 69 participants (76%) were predominantly from households with an average income >$75000/y. The mean family income of Canadians in 2001 was $66836 (25). Age, gravidity, parity, income, and education of participants within the subgroup (n = 20) used to investigate the distribution of individual RBC folate forms were not significantly different from those of the women in the larger overall study (data not shown).


Figure 1
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  		FIGURE 1. Outline of subject recruitment, study visit schedule, and sample collection. [6S]-5-methylTHF, synthetic [6S]-5-methyltetrahydrofolate.

 

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  		TABLE 1 Select subject characteristics at 36 wk gestation (baseline)1

 
Average dietary folate intakes before randomization and during the intervention did not differ significantly between treatment groups. At baseline (36 wk gestation), the mean (±SD) dietary folate intake of the study population was 547 ± 133 µg DFE/d. During lactation, dietary folate intakes were 490 ± 128 and 465 ± 152 µg DFE/d at 4 and 16 wk postpartum, respectively. The Recommended Dietary Allowance for pregnancy and lactation is 600 and 500 µg DFE/d, respectively (1). Blood folate but not tHcy concentrations differed between treatments at baseline (36 wk gestation). On average, women consumed 85 ± 14% (x± SD) of the prescribed study capsules during lactation, and no significant differences existed between treatment groups. Both the study and placebo capsules were well tolerated, and no treatment-related adverse side effects were observed.

Biochemical indexes
RBC folate concentrations decreased in all groups postpartum, despite dietary intakes exceeding an average of 450 µg/d (Table 2Go). Although there were no significant differences in mean RBC folate concentrations between treatment groups at 4 wk postpartum, the mean RBC folate concentration among women randomly assigned to the [6S]-5-methylTHF group was significantly greater than that of women in either the folic acid or placebo group at 16 wk postpartum. In contrast, the mean plasma folate concentration of women in either the [6S]-5-methylTHF or folic acid group was significantly greater than that in the placebo group at both 4 and 16 wk postpartum. Plasma tHcy concentrations remained unchanged throughout the intervention and did not differ significantly between treatment groups. The mean plasma tHcy concentrations of the study population at 16 wk postpartum was 8.4 µmol/L (95% CI: 8.2, 8.9 µmol/L). Most of the subjects had values <10.4 µmol/L (56 of 64) and <13 µmol/L (63 of 64) at 16 wk postpartum—2 commonly used cutoffs used to define elevated tHcy concentrations (26, 27). The distribution of subjects with tHcy concentrations >10.4 µmol/L, by treatment group, were as follows: n = 2 in the [6S]-5-methylTHF group, n = 3 in the folic acid group, and n = 3 in the placebo group.


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  		TABLE 2 Red blood cell (RBC) folate, plasma folate, and plasma total homocysteine (tHcy) concentrations in women at baseline (36 wk gestation) and after supplementation with [6S]-5-methyltetrahydrofolate ([6S]-5-methylTHF) (n = 21), folic acid (n = 21), or placebo (n = 22) from 1 to 16 wk postpartum1

 
As was the case among subjects in the overall study, the mean RBC folate concentration (measured by microbiological assay) decreased postpartum in the subset of women (n = 20) identified to look at the distribution of different forms of folate in RBCs (Table 3Go). Likewise, the mean RBC folate concentration of women in [6S]-5-methylTHF group was greater than that of women in the placebo and folic acid groups 16 wk postpartum. We detected THF, 5-methylTHF, 5-formylTHF, and folic acid in the RBCs of women in all 3 treatment groups. Of all the forms detected by LC/ESI-MS, THF (54–64%) and 5-methylTHF (18–33%) made up the largest fraction of RBC folate. 5-FormylTHF was the only form of folate to differ significantly between treatment groups at 16 wk; the folic acid group had a greater concentration than did either the [6S]-5-methylTHF or the placebo group. No effect of genotype was seen on total RBC folate content or on the proportion of individual detectable forms.


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  		TABLE 3 Total red blood cell (RBC) folate concentrations as measured by microbiological assay and individual folate forms (nmol/L) as measured by electrospray ionization mass spectrometry in women at baseline (36 gestation) and after supplementation with [6S]-5-methyltetrahydrofolate ([6S]-5-methyTHF), folic acid, or placebo from 1 to 16 wk postpartum1

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results from the present study showed a precipitous decreases in RBC folate concentrations after parturition in a sample of well-educated lactating Canadian women consuming folic acid fortified foods. In addition to the demands of lactation, this decrease in RBC folate concentrations was, no doubt, due in large part to the discontinuation of prenatal supplements, which contained {approx}1000 µg folic acid. Interestingly, RBC folate concentrations fell less dramatically among women supplemented with 416 µg [6S]-5-methylTHF as compared with women supplemented with an equimolar dose of folic acid. By the 16th wk of lactation, women supplemented with [6S]-5-methylTHF had RBC folate concentrations that were higher than those of women supplemented with folic acid or provided with a placebo. In contrast, plasma folate concentrations did not differ significantly between the [6S]-5-methylTHF–and folic acid–supplemented women at 16 wk postpartum; these concentrations were significantly greater than those of women who consumed the placebo. Consistent with reports from other human studies in which folate intakes were adequate, we found that the MTHFR 677C->T polymorphism did not influence blood folate indexes (28-30).

It is likely that the different conclusions obtained on the basis of plasma folate and RBC folate were due, at least in part, to differences in how quickly these 2 indicators reflect changes in dietary and supplemental folate intakes. Plasma folate concentration reflects recent dietary and supplemental folate intakes. In contrast, the RBC folate concentration is thought to primarily reflect the availability of folate early in erythropoiesis. Given that the life span of a mature RBC is {approx}120 d ({approx}16 wk), it will take at least this length of time before the RBC folate concentration completely reflects a shift in dietary and supplemental folate intakes. The trajectory of the decay in blood folate concentration after the consumption of high folate–containing prenatal supplements (1000 µg/d) in the present study suggests that 15 wk after initiation of a lower folate supplementation regimen (400 µg/d), plasma folate reached a new steady state but RBC folate did not. Furthermore, we cannot rule out the possibility that different conclusions obtained on the basis of plasma folate and RBC folate indexes may reflect differences in the relative affinity of influx and efflux transporters of cells in the erythroid lineage for various forms of folate (1, 31, 32).

There are no other published reports on the effect of 5-methylTHF supplementation on blood folate concentrations of lactating women. As summarized by Brouwer et al (33), acute studies that examined the bioefficacy of oxidized and reduced folates with or without various one-carbon units at other stages of the life cycle have produced mixed results. For example, Perry and Chanarin (34) reported a greater increase in serum folate concentrations after ingestion of a single dose of 5-methylTHF than after ingestion of folic acid. In contrast, Brown et al (35) and Tamura and Stokstad (36) found no differences in the bioefficacy of 5-methylTHF and folic acid after ingestion of single doses of these vitamers. Furthermore, Gregory et al (37) reported greater bioefficacy of folic acid than of 5-methylTHF using single-dose oral and intravenous stable-isotope tracers of folate.

More recently, Venn et al (16, 17) examined the efficacy of daily supplementation with [6S]-5-methylTHF (104 µg/d, 227 nmol/d) or an equimolar amount of folic acid in improving blood folate (n = 104) and homocysteine (n = 167) concentrations in nonpregnant, nonlactating women of childbearing age from New Zealand. After 24 wk of supplementation, the authors reported that [6S]-5-methylTHF and folic acid increased plasma and RBC folate concentrations to a similar extent (16). Furthermore, plasma tHcy concentrations decreased by 14.6% and 9.3% among women in the [6S]-5-methylTHF and folic acid groups, respectively (17). The mean reduction in plasma tHcy in the [6S]-5-methylTHF group was greater than that in the folic acid group (P = 0.045).

Unlike Venn et al (17), we found no differences in plasma tHcy values either over time or between treatment groups. This observation likely reflects the different physiologic state of the women in our study, the practice of folic acid fortification in Canada but not in New Zealand, and the fact that most women in our study consumed prenatal supplements containing {approx}1000 µg folic acid/d before study initiation. Before folic acid fortification of the food supply was initiated, Mackey and Picciano (8) reported an increase in tHcy concentration (6.7–7.4 µmol/L) between 3 and 6 mo of lactation in women not consuming supplemental folate postpartum. In contrast, no statistically significant increase in tHcy concentration (7.4–8.5 µmol/L) was observed between lactating women who consumed a 1-mg/d folic acid supplement (8). Our plasma tHcy values reported herein are similar to those of nonpregnant women of reproductive age (n = 204) from the province of Newfoundland after fortification of the Canadian food supply with folic acid (9.2 µmol/L; 95% CI: 8.8, 9.6 µmol/L) (38).

We found that the RBCs of pregnant and lactating women in our study were composed of significant quantities of THF, 5-methylTHF, and 5-formylTHF; {approx}1–2% of RBC folate was composed of unmetabolized folic acid. The presence of THF, 5-methylTHF, and 5-formylTHF in RBCs and whole-blood lysates was previously reported by Fazili and Pfeiffer (39) and Lucock et al (40, 41), although the proportion of THF in the RBCs of women in our study was significantly greater than in these reports. Given the significantly greater exposure of women in our study to folic acid, as evident by RBC folate concentrations that were ≥3 times those reported by Fazili and Pfeiffer (39) and Lucock et al (40, 41), we speculate that the high concentration of THF in RBCs reported in the present study reflect the availability and uptake by cells early in the erythroid lineage of high concentrations of circulating folic acid. Both folate uptake and the enzyme responsible for the reduction of folic acid to dihydrofolate, dihydrofolate reductase, have been identified in cells in the erythroid lineage including mature RBCs, albeit in the latter at much lower concentrations (32, 42, 43). Alternatively, the high concentration of THF could reflect elevated concentrations of high-affinity binding proteins during pregnancy, as was shown to occur during pregnancy (44, 45). These binders have been shown to protect the very labile THF from degradation. THF easily degrades when high-affinity binders are removed from the plasma of pigs by ultrafiltration (46).

In contrast, Bagley and Selhub (47) and Davis et al (48) reported that 5-methylTHF polyglutamates are the only folate form found in RBCs from persons with the wild-type MTHFR 677C->T genotype. Formylated THF, in addition to methylated derivatives, are found in the RBCs of persons with the T/T genotype. As described previously (39, 47), the methods by which folates are extracted from RBCs differ between studies and may account for the reported discrepancies between RBC folate forms. Similar to the method used by Fazili and Pfeiffer (39), we prepared RBC lysates at a low pH (4–4.5) to obtain folate monoglutamates by the action of pteroyl-{gamma}-glutamyl hydrolase. Bagley and Selhub (47), however, extract folates from frozen RBCs at a high pH to prevent deconjugation of folate polyglutamates. Fazili and Pfeiffer (39) hypothesize that, perhaps at a more acidic pH, 5,10-methyleneTHF may break down to THF. If this is indeed the case, the proportion of THF in our study could be an overestimate. Using whole-blood samples from rats spiked with either 13C5 5-methylTHF or THF that were processed according to the conditions described herein to prepare RBC lysates, we found no interconversion of the label between the 2 folate forms as analyzed by LC/ESI-MS. We know that other forms of folate, specifically 10-formylTHF, convert rapidly at an acidic pH to 5,10-methenylTHF; hence, these 2 forms are indistinguishable in all the aforementioned studies because an acid mobile phase is used in the chromatography.

The only RBC folate form to differ between the groups was 5-formylTHF; the folic acid group had a higher proportion of formylated folates than did the [6S]-5-methylTHF and placebo groups. This finding was consistent with our understanding of how folic acid can mask vitamin B-12 deficiency. In the event of vitamin B-12 deficiency, methionine synthase activity is impaired, which inhibits the regeneration of THF (Figure 2Go). The latter is required in the synthesis and repair of DNA and hematopoiesis (49). Supplemental folic acid can produce THF by bypassing methionine synthase to resolve hematologic signs of anemia created by impaired DNA synthesis. A higher proportion of formylated folates, as shown in the present study, could favor DNA synthesis and repair because of the dependence of this system on nonmethylated forms of folate.


Figure 2
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  		FIGURE 2. Overview of how high folic acid intakes correct DNA synthesis but not homocysteine remethylation during vitamin B-12 deficiency. DHF, dihydrofolate; THF, tetrahydrofolate; 5-methyTHF, 5-methyltetrahydrofolate; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; 1, methionine synthase; 2, methylenetetrahydrofolate reductase; 3, glycine-N-methyltransferase; B12, vitamin B-12.

 
In summary, because of the discontinuation of prenatal folic acid–containing supplements and the demands of lactation, blood folate concentrations decreased dramatically postpartum in our sample of well-educated breastfeeding women from Southern Ontario who consumed folic acid–fortified foods. Interestingly, [6S]-5-methylTHF appears to be as and perhaps more effective in preserving maternal folate stores during lactation than an equimolar amount of folic acid. Furthermore, the lower concentration of 5-formylTHF in the RBCs of women supplemented with [6S]-5-methylTHF than in those supplemented with folic acid provides direct evidence to support the hypothetical contention that supplemental [6S]-5-methylTHF may be less likely to mask vitamin B-12 deficiency than supplemental folic acid.


   ACKNOWLEDGMENTS
 
We thank the Motherisk program at The Hospital For Sick Children for assisting with recruitment and are grateful to all the mothers who participated. We also thank Anja Martina Bohlmann (Merck KGaA, Darmstadt, Germany) and Rudolf Moser (Merck Eprova AG, Schaffhausen, Switzerland) for ongoing support, for enthusiasm for the project, and for ensuring that both implementation of the study protocol and data collection efforts complied with applicable regulatory requirements. We appreciate the patient and expert advice of Cristina Goia (biostatistician at the Clinical Research Support at The Hospital for Sick Children) and thank Glynnis Dubois for lactation support, Jimao Yang for laboratory assistance, and the research nurses from the Clinical Investigation Unit for blood sample collection.

DLO and LAH designed the study and wrote the manuscript. LAH and KLS recruited the subjects and were responsible for the sample collection, laboratory analysis, and statistical analysis. RP conducted the folate form analysis using LC/ESI-MS and interpreted these data. SI was responsible for the medical support and safety review. KLS, RP, and SI provided editorial assistance. None of the authors had any personal or financial conflicts of interest with the sponsoring institutions.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
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Received for publication October 24, 2005. Accepted for publication December 20, 2005.




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