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`Effects of folic acid and combinations of folic acid and vitamin B-12
`on plasma homocysteine concentrations in healthy, young women1,2
`
`Anja Brönstrup, Monika Hages, Reinhild Prinz-Langenohl, and Klaus Pietrzik
`
`ABSTRACT
`Background: Elevated plasma homocysteine concentrations are
`considered to be a risk factor for vascular disease and fetal malfor-
`mations such as neural tube defects. Recent studies have shown
`that plasma homocysteine can be lowered by folic acid in amounts
`corresponding to 1–2 times the recommended dietary allowance.
`Preliminary evidence indicates that vitamin B-12 may be beneficial
`when included in supplements or in a food-fortification regimen
`together with folic acid.
`Objective: We aimed to compare the homocysteine-lowering
`potential of a folic acid supplement with that of 2 supplements con-
`taining different doses of vitamin B-12 in addition to folic acid.
`Design: Female volunteers of childbearing age (n = 150)
`received a placebo for 4 wk followed by a 4-wk treatment with
`either 400 ␮g folic acid, 400 ␮g folic acid + 6 µg vitamin B-12,
`or 400 ␮g folic acid + 400 ␮g vitamin B-12.
`Results: Significant reductions (P < 0.001) in plasma homocys-
`teine were observed in all groups receiving vitamin treatment.
`The effect observed with the combination of folic acid + 400 ␮g
`vitamin B-12 (total homocysteine, ⫺18%) was significantly
`larger than that with a supplement containing folic acid alone
`(total homocysteine, ⫺11%) (P < 0.05). Folic acid in combina-
`tion with a low vitamin B-12 dose (6 ␮g) affected homocysteine
`as well (⫺15%).
`Conclusions: These results suggest that the addition of vitamin
`B-12 to folic acid supplements or enriched foods maximizes the
`reduction of homocysteine and may thus increase the benefits of
`the proposed measures in the prevention of vascular disease and
`neural tube defects.
`Am J Clin Nutr 1998;68:1104–10.
`
`KEY WORDS
`Folic acid, vitamin B-12, supplementation,
`homocysteine, neural tube defect, cardiovascular disease,
`women
`
`INTRODUCTION
`Homocysteine is being scrutinized as independent risk factor
`for coronary, cerebral, and peripheral vascular diseases. Most
`case-control studies and several, though not all, prospective stud-
`ies have confirmed such an association over a wide range of
`plasma total homocysteine (tHcy) concentrations (1–4).
`In the absence of vitamin B-6 or vitamin B-12 deficiency or
`genetic defects in non-folate-dependent enzymes, folic acid
`intervention lowers plasma tHcy concentrations. This has been
`
`observed even when presupplementation plasma folate concen-
`trations were well within the range of values currently accepted
`as reflecting adequate status (5, 6). In several studies, daily folic
`acid administration in high (pharmacologic) doses of 0.5 (7) to
`10 mg (8) resulted in significant reductions in plasma tHcy.
`However, for both sexes, additional folic acid intakes of 200–400
`␮g/d, corresponding to 1–2 times the recommended dietary
`allowance of 400 ␮g dietary folate equivalents (9), seem to be
`sufficient to lower plasma tHcy concentrations (5, 6, 10). Indi-
`rect evidence for the protective effect of low plasma tHcy con-
`centrations comes from a recent prospective study linking high
`intakes of folate to a considerably diminished risk for coronary
`artery disease in 80 082 US nurses (11). Besides an involvement
`in the pathogenesis of vascular disease, maternal tHcy concen-
`trations may further play a role in the etiology of fetal malfor-
`mations such as neural tube defects (NTDs) (12–14).
`As of January 1, 1998, the US Food and Drug Administration
`ruled that the fortification of grain and grain products with folic
`acid be mandatory to increase folic acid intakes and contribute to
`the prevention of NTDs (15). However, it has been suggested
`that vitamin B-12 be added to foods as well or that supplements
`be offered containing both folic acid and vitamin B-12 (12, 16,
`17). The rationale for this proposition is that the sole addition of
`folic acid may mask pernicious anemia resulting from vitamin B-
`12 deficiency, which may slowly lead to irreversible nerve dam-
`age. Further support for this proposition is that both folic acid
`and vitamin B-12 are cofactors of methionine synthase,
`the
`enzyme catalyzing the formation of methionine from homocys-
`teine. A defect in this enzyme, also resulting in elevated tHcy
`concentrations, was proposed to be the cause for some (although
`not all) NTDs.
`The present study aimed to determine whether the addition of
`vitamin B-12 to a folic acid supplementation regimen recom-
`mended for women capable of becoming pregnant (9) potenti-
`ated the tHcy-lowering capacity of this regimen. Two different
`
`1 From the Institute of Nutritional Science, Department of Pathophysiolo-
`gy of Human Nutrition, University of Bonn, Germany.
`2 Address reprint requests to K Pietrzik, Institute of Nutritional Science,
`Department of Pathophysiology of Human Nutrition, University of Bonn,
`Endenicher Allee 11-13,
`FRG-53115 Bonn, Germany. E-mail:
`k.pietrzik@uni-bonn.de.
`Received March 2, 1998.
`Accepted for publication June 10, 1998.
`
`1104
`
`Am J Clin Nutr 1998;68:1104–10. Printed in USA. © 1998 American Society for Clinical Nutrition
`
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`HOMOCYSTEINE LOWERING BY FOLIC ACID AND VITAMIN B-12
`
`1105
`
`doses of vitamin B-12 were chosen to explore whether there was
`a dose-response relation with increasing vitamin B-12 dose. The
`low dose was approximately double the recommended dietary
`allowance of 2.4 ␮g/d (9) and reflects an amount frequently con-
`tained in vitamin supplements for adults (18). The high pharma-
`cologic dose of vitamin B-12 takes into account the greatly
`reduced absorption rate of vitamin B-12 from high doses after
`saturation of the active absorption pathway (19). Women of
`childbearing age were chosen as the target population group
`because they may especially benefit from tHcy reduction.
`
`SUBJECTS AND METHODS
`The study was approved by the Ethical Committee of the Uni-
`versity Hospital of Bonn. After a washout phase of 4 wk, 156
`female participants aged 20–34 y received a placebo daily during
`the first 4 wk of the study. For the next 4 wk, the volunteers were
`randomly assigned to one of the following treatment groups:
`group A, 400 ␮g folic acid/d; group B, 400 ␮g folic acid + 6 ␮g
`vitamin B-12/d; and group C, 400 ␮g folic acid + 400 ␮g vita-
`min B-12/d. The vitamin capsules were specially prepared by
`Allpack (Schorndorf, Germany) with synthetic folic acid
`(pteroylmonoglutamic acid) from Takeda (Osaka, Japan) and vit-
`amin B-12 (cyanocobalamin) from Merck (Darmstadt, Ger-
`many). Placebo and vitamin capsules were identical in appear-
`ance as the result of a colored gelatin cover so that the
`participants were not aware of the identity of the capsules. Cap-
`sules were provided in excess and participants were asked to
`return the remaining capsules after each 4-wk period to enable
`pill counting as a measure of compliance.
`At the beginning of the washout phase, all subjects were
`instructed to continue their usual dietary habits for the duration
`of the study but to refrain from intake of other vitamin supple-
`ments or foods enriched with vitamins. Six participants opted to
`withdraw during the study; the 150 participants completing the
`study were included in the statistical analysis.
`After subjects had fasted overnight, blood was drawn from
`each participant at the start of the study (week 0), after the
`placebo period (week 4), and after the treatment period (week 8).
`Blood was immediately cooled on ice and centrifuged within 15
`min at 2000 ⫻ g and 4 ⬚C for 10 min. Plasma was stored at
`⫺20 °C until analyzed.
`
`Laboratory investigations
`All samples for each participant were analyzed within one run
`to minimize measurement errors. EDTA-treated plasma was ana-
`lyzed for tHcy by HPLC with fluorescence detection according
`to the method described by Araki and Sako (20) and Vester and
`Rasmussen (21) with minor modifications. The CVs for this
`assay were as follows: within-assay variation < 5.6% and
`between-assay variation < 5.7%. Folate and vitamin B-12 were
`measured in heparin-treated plasma with commercially available
`chemiluminescence kits (Chiron Diagnostics, Fernwald, Ger-
`many; within-assay CV < 4.6% and < 6.5%, respectively, and
`between-assay CV < 13.0% and < 9.1%, respectively). For the
`measurement of red blood cell (RBC) folate, the same chemilu-
`minescence kit as for plasma folate was used (within-assay CV
`< 10.7%, between-assay CV < 13.1%). Hemolysis for this assay
`was achieved by incubating whole blood with 0.2% ascorbic acid
`at room temperature for 90 min before freezing the mixture
`according to the directions of the manufacturer of the kit. The
`
`assays for tHcy, folate, and vitamin B-12 were validated exter-
`nally through participation in national and international interlab-
`oratory comparisons (for tHcy: European External Quality
`Assurance Scheme for homocysteine in serum; for the vitamins:
`ringtest of the German Society for Clinical Chemistry). Vitamin
`B-6 was measured as pyridoxal-P (PLP) by HPLC (within-assay
`CV < 2.3%, between-assay CV < 5.9%) (22). All samples were
`analyzed in duplicate.
`
`Statistical analysis
`Because of positively skewed distributions, the natural loga-
`rithms of tHcy, folate, RBC folate, vitamin B-12, and PLP were
`used in all analyses as continuous variables. Therefore, besides
`presentation of arithmetic means, geometric means for these
`variables are given. The treatment groups were compared with
`respect to body mass index (BMI), plasma tHcy, folate, RBC
`folate, vitamin B-12, and PLP by means of parametric models
`[paired t test for within-subject comparisons and analysis of vari-
`ance (ANOVA) for between-subject comparisons]. The primary
`analysis variable was the change in a plasma index after 4 wk of
`vitamin treatment. This change was expressed as the ratio of the
`concentration at week 8 to that at week 4, and a one-way
`ANOVA model was fitted to the ln-transformed ratio including a
`treatment effect. To account for the influence of tHcy and vita-
`min concentrations before treatment on the change in tHcy, these
`parameters were also included in the ANOVA as covariates. Post
`hoc tests used the Scheffe test. The age of the groups was com-
`pared by using a Kruskal-Wallis one-way ANOVA because a
`skewed distribution of the data remained after logarithmic trans-
`formation. Differences in proportions between the groups were
`tested by using a paired chi-square test. Correlation analysis
`used logarithmically transformed variables for calculation of
`Pearson’s correlation coefficients. Differences were considered
`significant at P < 0.05; all P values are two-tailed. Data analyses
`were performed with the statistical program SPSS (version 6.1.3;
`SPSS Inc, Chicago).
`
`RESULTS
`The demographic characteristics of the study participants are
`summarized in Table 1. No significant differences were observed
`among groups with respect to age, BMI, use of oral contraceptives,
`or prevalence of smoking. Previous use of B vitamin supplements
`before the washout phase, which included regular intake of supple-
`ments containing vitamin B-6, vitamin B-12, or folic acid, was also
`not significantly different among groups. An estimation of compli-
`ance with intake of the capsules was possible for 146 of 150 par-
`ticipants (97.3%). Good compliance, defined as intake of ‡ 6 cap-
`sules/wk, was noted for all groups, ranging from 98.0% in group A
`to 100% in groups B and C.
`
`Total homocysteine
`At week 0 participants were normohomocysteinemic, with con-
`centrations ranging from 3.5 to 14.3 ␮mol/L; the geometric mean
`value of all groups combined was 7.6 ␮mol/L. tHcy concentrations
`in plasma correlated inversely with concentrations of folate and
`vitamin B-12 in plasma (r = ⫺0.2828, P < 0.001, and r = ⫺0.3774,
`P < 0.001, respectively), but not with vitamin B-6 (PLP)
`(r = ⫺0.0771, P = 0.3). The association with RBC folate (com-
`puted from measurements at week 4) was weaker (r = ⫺0.1752,
`P = 0.03) than that observed with plasma folate at week 0.
`
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`1106
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`BRÖNSTRUP ET AL
`
`TABLE 1
`Demographic characteristics of the study participants1
`
`Group
`
`Group A
`(n = 51)
`24.9 – 3.5
`20.9 – 1.8
`58.8
`9.8
`25.5
`
`Group B
`(n = 49)
`24.0 – 1.9
`21.4 – 2.2
`55.1
`14.3
`22.4
`
`Group C
`(n = 50)
`23.5 – 2.1
`21.3 – 2.5
`54.0
`22.0
`18.0
`
`Age (y)
`BMI (kg/m2)
`Use of OC (%)
`Smoking (%)
`Previous B vitamin
`supplementation (%)
`1 x– – SD or proportions. Group A received 400 ␮g folic acid/d, group B
`received 400 ␮g folic acid + 6 ␮g vitamin B-12/d, and group C received 400
`␮g folic acid + 400 ␮g vitamin B-12/d. The 3 groups did not differ signifi-
`cantly with respect to age (P > 0.05, Kruskal-Wallis ANOVA), BMI (P >
`0.05, ANOVA), or prevalence of use of oral contraceptives (OC), smoking,
`and previous B vitamin supplementation (P > 0.05, chi-square test).
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`min supplementation with a decrease in tHcy after 4 wk. The
`extent of tHcy reduction was more pronounced when only the
`subjects with a tHcy concentration > 8 ␮mol/L before treatment
`were considered (group A:⫺16%; group B:⫺20%; group
`C:⫺22%), even though the additional effect of vitamin B-12 on
`tHcy reduction was smaller (P = 0.08, ANOVA). Below a tHcy
`concentration of 8 ␮mol/L, the extent of tHcy reduction in group
`C (⫺14%) was significantly larger than in group A (⫺5%) (P <
`0.05, ANOVA with Scheffe post hoc test), whereas it was inter-
`mediate in group B (⫺10%).
`The change in tHcy was also dependent, although to a lesser
`extent, on the plasma folate concentration before vitamin sup-
`plementation. The largest reductions in tHcy were observed in
`women with the lowest initial plasma folate concentrations (Fig-
`ure 2). In every subject with a plasma folate concentration < 20
`nmol/L, tHcy concentrations decreased after vitamin treatment,
`whereas this was not always the case for women with higher
`plasma folate concentrations. However, when plasma folate at
`the onset of vitamin treatment was > 20 nmol/L, subjects seemed
`to benefit from the addition of vitamin B-12 (tHcy reduction in
`group C:⫺17%, group B:⫺12%) compared with the administra-
`tion of folic acid alone (tHcy reduction in group A:⫺10%) (A
`compared with C: P < 0.05, ANOVA with Scheffe post hoc test).
`In contrast, for women with a plasma folate concentration £ 20
`nmol/L, the change in tHcy was slightly more pronounced but
`not significantly different across treatment groups.
`When tHcy and plasma folate concentrations before vitamin
`treatment (week 4) were included in the ANOVA as covariates,
`in addition to the significant differences between groups C and
`A, the tHcy reduction observed in group B was significantly
`larger than that observed in group A (P < 0.03). Change in tHcy
`was not related to RBC folate or plasma vitamin B-12 concen-
`trations before vitamin supplementation; inclusion of these vari-
`ables as covariates in the ANOVA did not improve the model.
`
`Folate
`At week 0, participants had plasma folate concentrations in
`the normal range (all but one > 9.9 nmol/L; all > 6.8 nmol/L) (23,
`24). As expected, during the placebo phase, no changes in
`plasma folate concentrations were observed (Table 3), indicating
`also that any effects due to vitamin supplementation before the
`study began could be neglected.
`Because of the laborious procedure for measurement of RBC
`folate, this index was measured only at weeks 4 and 8 to investi-
`gate the relation with plasma folate and tHcy as well as the
`response to folic acid supplementation. A strong positive associ-
`
`After 4 wk of placebo treatment, the geometric mean tHcy
`concentration of all study subjects increased slightly but signifi-
`cantly by a mean value of 0.28 ␮mol/L (P < 0.01). A subgroup
`analysis showed that this was true for groups A and B, although
`no significant change in the mean tHcy concentration occurred in
`group C during placebo treatment (Table 2). The change in tHcy
`was not significantly different in subjects reporting regular
`intake of B vitamins from that in those not regularly taking sup-
`plements before the washout phase. Despite these fluctuations,
`tHcy concentrations at the start of the vitamin treatment period
`did not differ significantly among groups.
`After vitamin treatment for 4 wk, the mean tHcy concentra-
`tion was reduced significantly in all groups (P < 0.001). The
`decrease in tHcy varied according to the treatment regimen: the
`most pronounced tHcy reduction was observed in group C
`(⫺18% compared with the tHcy concentration at week 4, corre-
`sponding to a geometric mean ratio of 0.82; Table 2). In group B,
`the tHcy concentration at week 8 was 15% lower than that at
`week 4. When folic acid was given alone (group A), the reduc-
`tion in the plasma tHcy concentration was 11%. The difference
`in tHcy-lowering effect between group A and group C was signi-
`ficant (P < 0.05, ANOVA with Scheffe post hoc test).
`The individual changes in tHcy concentration after 4 wk of
`vitamin treatment are shown in Figure 1. The change in tHcy
`was dependent on the tHcy concentration before vitamin supple-
`mentation (week 4); subjects with a high initial tHcy concentra-
`tion responded to treatment with larger reductions in tHcy than
`those in subjects with initially low tHcy concentrations. Above a
`tHcy concentration of 8 ␮mol/L, each subject responded to vita-
`
`TABLE 2
`Response of plasma total homocysteine (tHcy) concentrations to placebo (week 4) and supplementation with folic acid or folic acid plus vitamin B-12
`(week 8)1
`
`Plasma tHcy
`Week 8
`Week 4
`Week 0 (baseline)
`7.18 – 1.62 (6.99)4
`8.13 – 2.14 (7.84)3
`7.88 – 2.22 (7.58)
`0.89
`Group A (n = 51)
`6.81 – 1.46 (6.65)4
`8.18 – 2.41 (7.87)3
`7.52 – 1.78 (7.31)
`0.85
`Group B (n = 49)
`6.59 – 1.12 (6.50)4
`8.12 – 1.92 (7.91)
`8.18 – 1.74 (8.01)
`0.82
`Group C (n = 50)
`1 x– – SD; geometric mean in parentheses. Group A received 400 ␮g folic acid/d, group B received 400 ␮g folic acid + 6 ␮g vitamin B-12/d, and group C
`received 400 ␮g folic acid + 400 ␮g vitamin B-12/d.
`2 Geometric mean ratio of tHcy at week 8 divided by tHcy at week 4; values < 1 indicate a decrease in tHcy after vitamin treatment.
`3 Geometric mean significantly different from week 0 (baseline), P < 0.05 (paired t test).
`4 Geometric mean significantly different from week 4, P < 0.001 (paired t test).
`
`Mean ratio, week 8/week 42
`
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`HOMOCYSTEINE LOWERING BY FOLIC ACID AND VITAMIN B-12
`
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`FIGURE 1. Change in the total homocysteine (tHcy) concentration in relation to the tHcy concentration before vitamin supplementation (week 4).
`The ratio was derived by dividing the concentration of tHcy at week 8 by the concentration at week 4. Values < 1 indicate a decrease in tHcy after vit-
`amin supplementation. *, supplementation with 400 µg folic acid/d; ⵧ supplementation with 400 µg folic acid + 6 µg vitamin B-12/d; 䊉 supplemen-
`tation with 400 µg folic acid + 400 µg vitamin B-12/d.
`
`ation was observed between plasma folate and RBC folate at
`week 4 (r = 0.5436, P < 0.001). Even though 3 of 150 subjects
`had low RBC folate concentrations at this time point (< 317
`nmol/L, or 140 ng/mL), their folate status seemed to be normal
`as indicated by their corresponding plasma folate and tHcy con-
`centrations. Mean plasma folate and RBC folate concentrations
`at the start of the vitamin treatment did not differ among the 3
`groups (P > 0.05, ANOVA). After 4 wk of vitamin supplementa-
`tion, all treatment groups showed significant increases in mean
`
`plasma and RBC folate concentrations (P < 0.001 for both). The
`extent of the mean increase varied between 52% and 55%
`(plasma folate) and 69% and 78% (RBC folate) when compared
`with the values at week 4, and were not significantly different
`between the groups (P > 0.05, ANOVA) (Table 3).
`
`Vitamin B-12
`At week 0, the geometric mean plasma vitamin B-12 concen-
`tration for all groups combined was 268 pmol/L (Table 3). Five
`
`FIGURE 2. Change in the total homocysteine (tHcy) concentration in relation to the plasma folate concentration before vitamin supplementation
`(week 4). The ratio was derived by dividing the concentration of tHcy at week 8 by the concentration at week 4. Values < 1 indicate a decrease in tHcy
`after vitamin supplementation. *, supplementation with 400 µg folic acid/d; ⵧ supplementation with 400 µg folic acid + 6 µg vitamin B-12/d; 䊉 sup-
`plementation with 400 µg folic acid + 400 µg vitamin B-12/d.
`
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`1108
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`TABLE 3
`Response of plasma vitamin indexes to placebo (week 4) and supplementation with folic acid or folic acid plus vitamin B-12 (week 8)1
`
`Week 0 (baseline)
`
`Week 4
`
`Week 8
`
`Total group (n = 150)
`Folate (nmol/L)
`RBC folate (nmol/L)
`Vitamin B-12 (pmol/L)
`PLP (nmol/L)
`Group A (n = 51)
`Folate (nmol/L)
`RBC folate (nmol/L)
`Vitamin B-12 (pmol/L)
`PLP (nmol/L)
`Group B (n = 49)
`Folate (nmol/L)
`RBC folate (nmol/L)
`Vitamin B-12 (pmol/L)
`PLP (nmol/L)
`Group C (n = 50)
`44.0 – 11.4 (42.5)2
`28.8 – 8.6 (27.4)
`27.6 – 10.0 (25.9)
`Folate (nmol/L)
`1468 – 616 (1344)2
`836 – 412 (779)
`—
`RBC folate (nmol/L)
`407 – 158 (377)2
`265 – 109 (239)
`279 – 125 (250)
`Vitamin B-12 (pmol/L)
`47.0 – 20.4 (43.4)
`—
`—
`PLP (nmol/L)
`1 x– – SD; geometric mean in parentheses. Group A received 400 ␮g folic acid/d, group B received 400 ␮g folic acid + 6 ␮g vitamin B-12/d, and group C
`received 400 ␮g folic acid + 400 ␮g vitamin B-12/d. RBC, red blood cell; PLP, pyridoxal-P.
`2 Geometric mean significantly different from week 4, P < 0.001 (paired t test).
`3 Geometric mean significantly different from week 0 (baseline), P < 0.01 (paired t test).
`4 No statistical test performed because of the difference in vitamin B-12 treatment among the 3 groups.
`
`45.6 – 14.3 (43.5)2
`1485 – 615 (1359)2
`345 – 144 (317)4
`—
`
`46.6 – 17.4 (43.8)2
`1438 – 643 (1296)2
`259 – 104 (240)
`—
`
`46.1 – 13.4 (44.1)2
`1551 – 593 (1443)2
`371 – 125 (353)2
`—
`
`29.8 – 11.4 (27.6)
`—
`292 – 119 (268)
`51.5 – 25.0 (46.2)
`
`30.1 – 10.4 (28.2)
`—
`268 – 100 (251)
`53.9 – 27.9 (47.8)
`
`31.7 – 13.4 (28.9)
`—
`329 – 123 (307)
`53.5 – 26.0 (47.7)
`
`30.2 – 10.7 (28.3)
`847 – 381 (782)
`276 – 110 (253)3
`—
`
`30.5 – 10.2 (28.8)
`810 – 295 (759)
`251 – 102 (233)3
`—
`
`31.3 – 12.9 (28.8)
`896 – 428 (812)
`313 – 111 (292)
`—
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`participants had plasma concentrations indicative of a subopti-
`mal vitamin B-12 status (< 111 pmol/L, or < 150 pg/mL) (25).
`Their corresponding plasma tHcy concentrations ranged from
`7.3 to 12.0 ␮mol/L.
`After 4 wk of placebo treatment, the geometric mean vitamin
`B-12 concentration of the whole group was slightly but signifi-
`cantly lower than at week 0 (P < 0.01). This was mainly attrib-
`utable to changes in group A (P < 0.01). The observed change
`did not correlate with the change in tHcy concentration during
`the placebo period (P = 0.2). At week 4, the plasma vitamin B-
`12 concentration of group A was significantly lower than that of
`group B (P < 0.05, ANOVA with Scheffe post hoc test).
`From week 4 to week 8, no further changes in vitamin B-12
`concentrations occurred in the group receiving folic acid only. In
`group B, the vitamin B-12 concentration increased significantly
`by a mean value of 58 pmol/L (P < 0.001). In group C, the
`plasma vitamin B-12 concentration increased by a mean value of
`142 pmol/L (P < 0.001). This increase was significantly higher
`than that in group B or group A (P < 0.05, ANOVA with Scheffe
`post hoc test).
`
`Vitamin B-6
`Because vitamin B-6 was not a target index in this study,
`plasma PLP concentrations were measured only at the beginning
`of the study. PLP concentrations ranged between 16.4 and 168.9
`nmol/L, with a geometric mean across all groups of 46.2 nmol/L.
`
`DISCUSSION
`The role of folic acid in the prevention of NTDs and vascular
`diseases and the potential additional effect of vitamin B-12 is a
`matter of debate. In this study, we investigated whether a combi-
`nation of these vitamins had a more pronounced tHcy-lowering
`
`effect than supplementation with folic acid alone. Of the young
`women participating in this study, the vast majority had an ade-
`quate status of the vitamins involved in homocysteine metabo-
`lism, according to currently accepted guidelines, and they were
`normohomocysteinemic as defined by Kang et al (26). Despite
`this, folic acid supplementation (alone or in combination with
`vitamin B-12) resulted in significant reductions in plasma tHcy
`concentrations.
`Ward et al (6) administered folic acid in amounts that could be
`reached through optimal food selection or use of fortified foods
`(100, 200, and 400 ␮g folic acid/d) to lower tHcy concentrations
`in middle-aged, healthy men. Because in their study the supple-
`mentation regimen was increased over the course of the study in
`6-wk intervals, no information on the tHcy-lowering effect attrib-
`utable solely to supplementation with 400 ␮g folic acid can be
`obtained. However, as in our study, the tHcy-reducing effect was
`clearly dependent on the tHcy concentration at baseline. Before
`supplementation, men in the 2 lowest tertiles together had a mean
`plasma tHcy concentration of 8.09 ␮mol/L, which is almost iden-
`tical to that observed in group A in our study. After folic acid
`administration, the extent of tHcy reduction in group A was com-
`parable with the reduction observed by Ward et al (6) in the 2
`lowest tertiles over a much longer supplementation period.
`Boushey et al (4) and Tucker et al (27) estimated that an
`increase in folic acid intake of ⬇200 ␮g/d results on average in a
`reduction in tHcy concentration of 4 ␮mol/L, or 12%. However,
`this rather high effect of folic acid supplementation may be
`restricted to subjects with moderate or intermediate hyperhomo-
`cysteinemia, as observed previously (10, 28). Our findings and
`results from others (6) indicate that in normohomocysteinemic
`subjects, twice the amount of additional folic acid, ie, 400 ␮g/d is
`required for a mean reduction of the plasma tHcy concentration
`of 11%. Even though we are still awaiting results from interven-
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`HOMOCYSTEINE LOWERING BY FOLIC ACID AND VITAMIN B-12
`
`1109
`
`tion studies of the protective effect of vitamin supplements in
`lowering plasma tHcy and concurrently risk for vascular disease,
`evidence for this association is increasing. A recent prospective
`study in 80 082 US nurses showed that in women in the highest
`quintile of folate intake (median intake: 696 ␮g/d), the relative
`risk for coronary artery disease, controlling for other cardiovas-
`cular risk factors, was 0.69 (95% CI: 0.55, 0.87) compared with
`women in the lowest quintile (median intake: 158 ␮g/d) (11).
`Calculations showed that with each 200-␮g increase in folate, the
`relative risk was 0.89 (95% CI: 0.82, 0.96); this held true (relative
`risk = 0.91; 95% CI: 0.84, 0.99) even for intakes > 180 ␮g/d, the
`former recommended dietary allowance for women (29).
`In this study, vitamin B-12 supplementation increased the
`tHcy-lowering potential of folic acid; this was especially obvious
`when vitamin B-12 was given in pharmacologic amounts (400
`␮g). In subgroup analyses, the extent of the tHcy reduction was
`significantly higher with the addition of increasing doses of vit-
`amin B-12 in women with a plasma folate concentration > 20
`nmol/L. Because folate and vitamin B-12 have a synergistic
`function as cofactors of methionine synthase, sufficiency of both
`seems to be important to increase enzyme activity, whereas a
`higher availability of only one cofactor, especially in subjects
`with an already good supply of this cofactor, might lead to only
`a limited increase in enzyme activity.
`After 4 wk of supplementation with vitamin B-12, the mean
`plasma concentration of vitamin B-12 clearly increased. This
`was observed with the dose of 6 ␮g vitamin B-12/d as well.
`Thus, on average, for this group of women, the low dose of vit-
`amin B-12 was quite bioavailable. The increase in plasma vita-
`min B-12 concentration was even more distinct after supplemen-
`tation with the high (pharmacologic) dose of 400 ␮g vitamin
`B-12. For this dose, the extent of the plasma vitamin B-12
`increase observed was comparable with what Ubbink et al (28)
`found after supplementing young, healthy men with the same
`dose for 6 wk. However, it seems evident that this intake of vit-
`amin B-12 can be reached neither by use of commonly available
`multivitamin supplements nor through diet, unless vitamin B-12
`is included in the food-fortification scheme along with folic acid.
`The results of this study suggest that the addition of vitamin
`B-12 to supplements containing 400 ␮g folic acid or to enriched
`foods maximizes the reduction of tHcy through the synergistic
`potential of both vitamins. This was most evident with the dose
`of 400 ␮g vitamin B-12/d together with folic acid. Others have
`suggested that, in addition to an effect on tHcy, combined sup-
`plementation with 400 ␮g folic acid + 400 ␮g vitamin B-12/d
`could counter the higher prevalence of vitamin B-12 deficiency
`in the elderly and the possibility of masked pernicious anemia
`with supplementation of folic acid alone (16, 28, 30). Doses of
`200–400 ␮g vitamin B-12/d are generally regarded as safe and
`effective in case of loss of intrinsic factor (28, 31), but amounts
`as low as 25 µg vitamin B-12/d have been proposed as well (16).
`A combined additional uptake of vitamin B-12 and folic acid
`may also be beneficial for at least a portion of women with an
`NTD child. There is evidence that, at comparable plasma concen-
`trations of vitamin B-12 and folate, some of these women are less
`efficient at metabolizing homocysteine than are control women,
`thus probably pointing to a higher demand for vitamin B-12 (12).
`In summary, the present study showed that additional folic
`acid intake, as recommended for the prevention of NTDs, results
`in significant reductions in the tHcy concentration of normoho-
`mocysteinemic women without vitamin deficiency. Above a
`
`plasma tHcy concentration as low as 8 ␮mol/L, each subject
`responded to vitamin supplementation with a reduction in tHcy.
`Beyond the presumed prevention of nerve damage in persons
`with pernicious anemia, the dose of 400 ␮g vitamin B-12/d
`along with the folic acid supplementation regimen showed the
`largest additional effect in lowering tHcy. With current public
`health measures, however, the additional uptake of both 400 ␮g
`folic acid/d and 400 ␮g vitamin B-12/d may not to be realized.
`Thus, further efforts should aim to optimize public health strate-
`gies to effectively reduce the risk of vascular disease and prevent
`a considerable number of birth defects.
`
`We thank P von Bülow, G Puzicha, M Schüller, and B Thorand for excel-
`lent technical assistance as well as valuable discussions. We are also grateful
`to the women who participated in the study.
`
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