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Supplementation with [6S]-5-methyltetrahydrofolate or folic acid equally reduces plasma total homocysteine concentrations in healthy women^1,2,3

¹ From the Institute of Nutritional Science, Department of Pathophysiology of Human Nutrition, University of Bonn, Germany (YL, RP-L, and KP), and Merck Eprova AG, Schaffhausen, Switzerland (RM).

² Supported by Merck KGaA (Darmstadt, Germany), which contributed financially, and by Merck Eprova AG (Schaffhausen, Switzerland), which provided Metafolin, the synthetic form of [6S]-5-methyltetrahydrofolate, for the supplements.

³ Address reprint requests to K Pietrzik, Institute of Nutritional Science, Department of Pathophysiology of Human Nutrition, University of Bonn, Endenicher Allee 11-13, D-53115 Bonn, Germany. E-mail: k.pietrzik{at}uni-bonn.de.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: Increased plasma total homocysteine (tHcy) is a risk factor for vascular disease and adverse pregnancy outcomes. Health authorities recommend periconceptional supplementation with 400 µg folic acid to prevent neural tube defects. Several countries have implemented food fortification with folic acid. However, excessive intake of folic acid could mask an undiagnosed vitamin B-12 deficiency. The biologically active [6S]-5-methyltetrahydrofolate ([6S]-5-MTHF) may be an alternative to folic acid because it is unlikely to mask vitamin B-12 deficiency symptoms.

Objective: We compared the tHcy-lowering potential of 2 dosages of [6S]-5-MTHF with that of 400 µg folic acid during 24 wk of supplementation.

Design: In this double-blind, randomized, controlled intervention trial, 144 female participants were supplemented daily with 400 µg folic acid, 416 µg [6S]-5-MTHF, 208 µg [6S]-5-MTHF, or placebo. Concentrations of tHcy and plasma folate were measured at baseline and at 4-wk intervals.

Results: After supplementation, there was a significant interaction between time and treatment with respect to changes in tHcy and plasma folate (both P < 0.001 by two-factor repeated-measures analysis of variance). The decrease in tHcy did not differ significantly between the 3 supplemented groups (P > 0.05; Tukey’s post hoc test). The increase in plasma folate in the group receiving 208 µg [6S]-5-MTHF was significantly lower than that in the groups receiving 400 µg folic acid (P < 0.001) or 416 µg [6S]-5-MTHF (P < 0.05).

Conclusions: [6S]-5-MTHF was shown to be an adequate alternative to folic acid in reducing tHcy concentrations. Supplementation with 416 µg [6S]-5-MTHF was no more effective than that with 208 µg [6S]-5-MTHF.

Key Words: [6S]-5-methyltetrahydrofolate • folic acid • homocysteine • plasma folate • long-term effect • supplementation • healthy women

	INTRODUCTION

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Elevated plasma total homocysteine (tHcy) is a risk factor for vascular disease (1, 2), neurodegenerative disorders (3, 4), and adverse pregnancy outcomes such as preeclampsia, neural tube defects (NTDs), and placental abruption (5-7). High concentrations of tHcy are correlated with low blood folate, and increasing folate intake is a highly effective means of lowering tHcy. To reduce the occurrence of NTDs, public health authorities in several countries recommend that women of childbearing age take 400 µg folic acid before and during the first trimester of pregnancy (8, 9). In view of a high number of unplanned pregnancies, some countries have implemented mandatory food fortification with folic acid (10, 11). Folic acid fortification has been associated with a decline in NTDs in the United States and Canada (12-14). In the United States, it has also been associated with a decrease in tHcy in the population (15), which may have an overall beneficial effect, eg, in terms of a reduction in vascular disease risk.

Folic acid fortification is not without risk: an excessive intake of folic acid may mask vitamin B-12 deficiency, potentially delaying its diagnosis (10). Folate and vitamin B-12 deficiency have the same hematologic symptom, megaloblastic anemia, which disappears after supplementation with large amounts of folic acid (10). However, the neurologic disorders associated with vitamin B-12 deficiency may progress unchecked (16, 17). Masking of vitamin B-12 deficiency is unique to folic acid. The predominant naturally occurring form of folate, [6S]-5-methyltetrahydrofolate ([6S]-5-MTHF), is unlikely to mask vitamin B-12 deficiency. The conversion of [6S]-5-MTHF into tetrahydrofolate, which is the precursor of folate forms involved in DNA synthesis, is vitamin B-12 dependent, whereas folic acid can be converted to tetrahydrofolate independent of vitamin B-12. Therefore, folic acid may maintain intracellular DNA synthesis and ameliorate megaloblastic anemia (16, 17). As a public health measure, [6S]-5-MTHF could possibly be used in food fortification or supplements as an alternative to folic acid, provided [6S]-5-MTHF has beneficial effects equal to those of folic acid with respect to reducing vascular disease risk and preventing NTDs.

On the basis of the recommendations for periconceptional supplementation with 400 µg folic acid to prevent NTDs (9), the aim of this double-blind, placebo-controlled trial was to compare the efficacy of a daily intake of 416 µg [6S]-5-MTHF with that of an equimolar amount of 400 µg folic acid in lowering tHcy in a group of healthy female subjects. To test whether lower amounts of [6S]-5-MTHF would be as effective as 400 µg folic acid, we assessed the dose response by adding to the study a group of subjects taking 208 µg [6S]-5-MTHF.

	SUBJECTS AND METHODS

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Subjects
Healthy women aged 18-35 y were recruited in December 2001 through advertisement at the University of Bonn, Germany. Inclusion criteria for participation in the study were normal results on routine laboratory tests and adequate vitamin B-12 status (plasma vitamin B-12 concentration 110 pmol/L; 18). Women were not eligible if they were pregnant, lactating, or planning a pregnancy in the next few months. Regular consumers of folic acid, defined as persons taking > 100 µg folic acid/d in vitamin supplements or fortified food during the last 4 mo, were not included. Further exclusion criteria were medical treatment interfering with folate metabolism (ie, cholestyramine, methotrexate, trimethoprim, sulfasalazin, salicylic acid, cotrimoxazol, antacid, and antiepileptic drugs) and abuse of alcohol or drugs. The study was approved by the Ethics Committee of the Medical Association of Hamburg, Germany, and all participants gave written informed consent.

Design
The study was a 24-wk (January-July 2002), double-blind, randomized, placebo-controlled intervention trial with parallel group design. Because the 677CT polymorphism of the gene encoding for 5,10-methylenetetrahydrofolate reductase (MTHFR) has an effect on homocysteine metabolism with respect to the response to folate intake or folic acid supplementation (19, 20), participants were stratified according to their genotype before randomization to ensure equal distribution of the 3 genotype variants in all groups. Participants were randomly assigned to receive either a placebo, 400 µg folic acid, 416 µg [6S]-5-MTHF, or 208 µg [6S]-5-MTHF. Fasting blood samples were collected by venipuncture at baseline and at 4, 8, 12, 16, 20, and 24 wk of the study. Participants were asked to take one capsule every morning before breakfast, except on the blood-sampling days, when they were asked to take the capsule after venipuncture. Possible side effects were monitored by questioning the subjects at each visit and by reviewing routine laboratory variables. Compliance was assessed by counting the pills remaining at weeks 8, 16, and 24. Dietary intakes were assessed by using 3-d diet records completed at baseline and at weeks 8, 16, and 24 and analyzed by using EBISPRO for WINDOWS (version 4; J Erhardt, University of Hohenheim, Germany).

Supplements
The supplements were manufactured by PCI Services (Schorndorf, Germany) as hard gelatin capsules containing a blend of magnesium stearate and microcrystalline cellulose as a filler (placebo), 400 µg (906 nmol) folic acid (Caesar & Loretz GmbH, Hilden, Germany), or 416 µg (906 nmol) or 208 µg (453 nmol) [6S]-5-MTHF as calcium salt (Metafolin; Merck Eprova AG, Schaffhausen, Switzerland). Subjects and investigators were blinded to treatment. Stability tests of the supplements performed at the beginning, middle, and end of the intervention period showed a recovery of 98% of both folate forms.

Blood sampling and analysis
Blood samples for measurement of plasma folate, tHcy, and plasma vitamin B-12 concentrations were collected into EDTA-coated tubes (S-Monovette; Sarstedt, Nümbrecht, Germany), immediately put on ice, centrifuged within 15 min (3000 rpm for 10 min at 4 °C), and stored at -20 °C before analysis. Plasma folate and tHcy concentrations were measured by using an immunoassay on the Abbott AxSYM analyzer (Abbott, Wiesbaden, Germany). The intraassay and interassay CVs were 5.9% and 5.6% for plasma folate and 2.3% and 3.5% for tHcy, respectively, according to the results of the analyses of plasma pools and controls provided by the manufacturers. To avoid between-run variation, samples from each participant were measured in one run. Before the determination of plasma folate concentrations, blood samples were manually diluted (1:2) with the use of the AxSYM folate specimen diluent. Plasma vitamin B-12 was measured at baseline by using an immunoassay on the Abbott AxSYM analyzer. Routine laboratory variables including serum creatinine concentrations were measured after venipuncture, at baseline and at week 24, by the central laboratory of the University Hospital, Bonn. Serum creatinine concentrations were measured enzymatically by using the method of Jaffé (Dade Behring, Bad Schwallbach, Germany). Identification of the 677CT MTHFR genotype was conducted by using the polymerase chain reaction according to the method of Frosst et al (21).

Statistical methods
Before further analysis, data were checked for normal distribution with the use of the Shapiro-Wilk test. Because they were skewed positively, data were log transformed to normalize distribution for analyses and back-transformed to geometric means with 95% CI for presentation in tables. One-way analysis of variance (ANOVA) was used to compare the 4 groups with respect to baseline characteristics, dietary intake of folate and methionine, and compliance. Chi-square tests were used to evaluate categoric variables such as smoking and the use of oral contraceptives. For within-group comparison of dietary intake of folate and methionine at baseline and at week 24, Student’s paired t test was used.

For further analyses, the natural logarithms of plasma folate and tHcy were used in all statistical tests as continuous variables. Two-factor repeated-measures ANOVA was applied to examine the interaction between time and treatment and to test for changes within time and treatment. Post hoc analysis was carried out with Tukey’s honestly significant difference test. In case of significant time x treatment interaction, the within-group comparison was carried out by using a Bonferroni corrected P value of 0.008 (0.05/6). Furthermore, multiple regression was used to determine mean percentage changes in plasma folate and tHcy after 24 wk relative to the placebo group after adjustment for baseline values (22). Between-group comparisons were carried out with the use of Tukey’s post hoc test. Results were considered significant at P < 0.05. All analyses were undertaken by using SPSS for WINDOWS software (version 11; SPSS Inc, Chicago).

	RESULTS

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A flow chart of the study is presented in Figure 1. One hundred eighty-one women came to the screening blood test, 37 of whom were excluded because of regular consumption of folic acid (n = 20), plasma vitamin B-12 concentration < 110 pmol/L (n = 9), or personal reasons (n = 8). One hundred forty-four women were included in the study. During the intervention period, one participant withdrew for personal reasons. After the exclusion of one outlier (baseline tHcy: >Azx ± 3 SD) and 7 subjects with missing values (absence on blood-sampling days because of vacation or illness), data from 135 subjects were entered into statistical analyses.

View larger version (23K): [in this window] [in a new window]   FIGURE 1.. Flow chart of the study. *One participant withdrew after 4 wk of intervention. MTHFR, methylenetetrahydrofolate reductase; [6S]-5-MTHF, [6S]-5-methyltetrahydrofolate.

Table 1

Table 2

	DISCUSSION

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This study is the first to investigate the long-term effect of different amounts of [6S]-5-MTHF on tHcy in healthy females. Relative to the placebo group, tHcy decreased by 15%, 19%, and 19% in the groups receiving 400 µg folic acid or 416 µg or 208 µg [6S]-5-MTHF, respectively, after 24 wk. The tHcy reduction did not differ significantly between the groups receiving supplementation with 400 µg folic acid and the equimolar amount of [6S]-5-MTHF. As expected in a group of healthy young women, tHcy concentration decreased by 20% (23, 24) after supplementation with 400 µg folic acid. Intervention with 416 µg [6S]-5-MTHF was no more effective than was that with 208 µg [6S]-5-MTHF in lowering the tHcy concentration. Therefore, a 208 µg dose of [6S]-5-MTHF seems sufficient to reduce tHcy concentrations in a group of healthy female subjects.

The effect of [6S]-5-MTHF supplementation on lowering tHcy was not investigated until recently. Venn et al (25) reported the effect of 113 µg [6S]-5-MTHF on tHcy concentrations. Twenty-four weeks of that intervention led to a 15% reduction in tHcy, which was significantly greater than the 9% reduction in the group receiving 100 µg folic acid (25). Throughout the study period of 24 wk, tHcy concentrations decreased continuously in both supplemented groups; it therefore remains unknown whether the maximum reduction in tHcy was achieved by supplementation with 113 µg [6S]-5-MTHF after 24 wk. In the present study, the decrease in tHcy concentrations reached a maximum after 4 wk (P < 0.001), and there were no further reductions until 24 wk, irrespective of the form and dose of folate. Baseline tHcy concentrations in both study populations were similar [8.6 µmol/L (25) and 8.2 µmol/L, respectively], which allowed us to compare the reductions in tHcy concentration resulting from supplementation with 113, 208, and 416 µg [6S]-5-MTHF after 24 wk. These reductions were 15% (25), 19%, and 19%, respectively, which indicated that there was no significant difference in tHcy reduction between the 3 interventions with [6S]-5-MTHF. However, further trials are needed to compare the efficacy of 113, 208, and 416 µg [6S]-5-MTHF in one study population.

Supplementation with folate derivatives improves folate concentration in plasma. In the present study, plasma folate concentrations increased significantly by 151%, 164%, and 101% in the groups receiving 400 µg folic acid or 416 µg or 208 µg [6S]-5-MTHF, respectively, after 24 wk, relative to the control group. Supplementation with 416 µg [6S]-5-MTHF and an equimolar amount of folic acid affected plasma folate equally. This finding is in accordance with the results of Venn et al (26). In the present study, a steady state was defined as no significant increase in plasma folate beyond that reached after 24 wk of intervention. However, that same concentration was reached after only 8 wk of supplementation with 400 µg folic acid. Thus, plasma folate was stabilized at an earlier stage by folic acid than by supplementation with 208 µg and 416 µg [6S]-5-MTHF, with which a steady state in plasma folate concentrations was reached after 12 and 20 wk, respectively. In the study by Venn et al (26), no steady state plateau was reached after supplementation with 113 µg [6S]-5-MTHF and 100 µg folic acid over 24 wk. However, the amount of folate derivatives given may have been too low, or the observation period may have been too short.

Plasma folate is a strong determinant of tHcy concentration (27). Although supplementation with 208 µg [6S]-5-MTHF resulted in a different plasma folate concentration than did that with 416 µg [6S]-5-MTHF, both decreased tHcy equally. Assuming [6S]-5-MTHF to be the active form of folate in the metabolism (28) and the main form occurring naturally in food (29, 30), the finding of an equal tHcy-lowering potential of 400 µg folic acid and 208 µg [6S]-5-MTHF, despite a lesser increase in plasma folate after supplementation with the latter is comparable to the finding of the study by Ashfield-Watt et al (19). In their investigation, a diet high in natural folate did not increase plasma folate to the same extent as did supplementation with 400 µg folic acid over a period of 4 mo. However, the decrease in tHcy observed in the group consuming the folate-rich diet equalled that in the group receiving folic acid supplements (19). Boushey et al (2) reported that a lower plateau of tHcy is reached when plasma folate concentrations exceed 15 nmol/L. These findings match results of the present study, as plasma folate between baseline and week 4 increased to > 15 nmol/L in all 3 supplemented groups, whereas tHcy concentrations decreased concurrently. However, the study of Venn et al (25) found a reduction in tHcy even though plasma folate concentrations at baseline were already > 15 nmol/L. In the present study, plasma folate concentrations continued increasing in all 3 supplemented groups after 4 wk, and tHcy stabilized at lower concentrations after 4 wk in this supplemented group of healthy women. The effect of plasma folate concentrations on tHcy seems to diminish at plasma folate concentrations that signify a sufficient folate supply.

[6S]-5-MTHF may be an adequate alternative to folic acid because it is unlikely to mask the hematologic symptoms of vitamin B-12 deficiency. In view of the increasing numbers of elderly in the population and the high prevalence of vitamin B-12 deficiency in that group (31), the possible masking of the symptoms by high folic acid intakes is exceptionally relevant. Where mandatory food fortification with folic acid has been implemented, some people reach a daily intake of folic acid > 1 mg (32), which is the tolerable upper intake level above which masking of vitamin B-12 deficiency has been determined to occur (10). [6S]-5-MTHF could be an alternative derivative for supplementation or fortification. In the supplemental form offered as calcium salt in the present study, [6S]-5-MTHF was stable throughout the study period. Good stability of [6S]-5-MTHF in some processed foods (cereals, white bread) was reported by the manufacturer. However, further tests of stability should be performed for different kinds of processed foods (26). The safety of [6S]-5-MTHF was shown in the study by Bostom et al (33) that investigated daily supplementation with 17 mg [6S]-5-MTHF over 12 wk with respect to reducing tHcy in hemodialysis patients. No side effects were reported. With respect to food fortification with [6S]-5-MTHF, intervention trials are needed to evaluate the minimum effective dose of supplemented [6S]-5-MTHF out of food to lower tHcy concentrations.

In conclusion, the present study showed that supplementation with both 208 and 416 µg [6S]-5-MTHF significantly decreased tHcy and significantly increased plasma folate concentrations. [6S]-5-MTHF did not differ in tHcy-lowering potential from an equimolar amount of folic acid. Supplementation with 416 µg [6S]-5-MTHF was no more effective in lowering tHcy concentrations than was that with 208 µg [6S]-5-MTHF. In addition to the observed beneficial effect on tHcy and plasma folate, [6S]-5-MTHF is unlikely to mask vitamin B-12 deficiency as folic acid does. Because this folate form is also stable and is as bioavailable as folic acid, it may be an adequate alternative to folic acid for use in food fortification or supplements. Further studies should be undertaken to investigate the effect of [6S]-5-MTHF in women and men of other age groups and especially in those with elevated tHcy concentrations. Such trials should include dose-response assessments as were used in the present study.

	ACKNOWLEDGMENTS

 
We acknowledge the women who participated in the study. We thank S Braemswig, A Brönstrup, P von Bülow, S Deneke, I Fohr, M Hages, M Schüller, P Pickert, G Puzicha, and O Tobolski for excellent technical assistance and valuable discussions.

YL and KP had the original idea for the study. All authors were responsible for designing and planning the study. YL and KP recruited subjects. YL was responsible for sample collection and laboratory and statistical analyses. All authors contributed to writing the manuscript. RM is the Chief Scientific Officer for Merck Eprova AG, Switzerland, the company that manufactures Metafolin, which it provided for this study. RM contributed to the study by participating in planning the project and writing the manuscript. The stability tests for the supplements were performed by Merck Eprova AG under RM’s supervision. RM was not involved in any decisions about laboratory work and statistical analysis or publication. YL, RP-L, and KP declared no conflict of interest.

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TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

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