`on plasma homocysteine concentrations in healthy, young women 1 •2
`
`Anja Bronstrup, 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(cid:173)
`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 of2 supplements con(cid:173)
`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 f.Lg folic acid, 400 f.Lg folic acid+ 6 11g vitamin B-12,
`or 400 f.Lg folic acid+ 400 f.Lg vitamin B-12.
`Results: Significant reductions (P < 0.001) in plasma homocys(cid:173)
`teine were observed in all groups receiving vitamin treatment.
`The effect observed with the combination of folic acid+ 400 f.Lg
`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(cid:173)
`tion with a low vitamin B-12 dose (6 f.Lg) 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.
`
`Folic acid, vitamin B-12, supplementation,
`KEY WORDS
`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(cid:173)
`ies have confirmed such an association over a wide range of
`plasma total homocysteine (tHey) 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 tHey concentrations. This has been
`
`observed even when presupplementation plasma folate concen(cid:173)
`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 tHey.
`However, for both sexes, additional folic acid intakes of 200-400
`1-1g/d, corresponding to 1-2 times the recommended dietary
`allowance of 400 fLg dietary folate equivalents (9), seem to be
`sufficient to lower plasma tHey concentrations (5, 6, 1 0). Indi(cid:173)
`rect evidence for the protective effect of low plasma tHey con(cid:173)
`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 tHey concen(cid:173)
`trations may further play a role in the etiology of fetal malfor(cid:173)
`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(cid:173)
`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(cid:173)
`teine. A defect in this enzyme, also resulting in elevated tHey
`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(cid:173)
`mended for women capable of becoming pregnant (9) potenti(cid:173)
`ated the tHey-lowering capacity of this regimen. Two different
`
`1 From the Institute of Nutritional Science, Department of Pathophysiolo(cid:173)
`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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`NEPTUNE GENERICS 1019- 00001
`APOTEX 1019-0001
`
`
`
`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 of2.4 1-1g/d (9) and reflects an amount frequently con(cid:173)
`tained in vitamin supplements for adults (18). The high pharma(cid:173)
`cologic dose of vitamin B-12 takes into account the greatly
`reduced absorption rate of vitamin R-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 tHey reduction.
`
`SUBJECTS AND METHODS
`The study was approved by the Ethical Committee of the Uni(cid:173)
`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 f.Lg folic acid/d; group B, 400 f.Lg folic acid+ 6 f.Lg
`vitamin B-12/d; and group C, 400 f.Lg folic acid+ 400 f.Lg vita(cid:173)
`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(cid:173)
`amin B-12 (cyanocobalamin) from Merck (Darmstadt, Ger(cid:173)
`many). Placebo and vitamin capsules were identical in appear(cid:173)
`ance as the result of a colored gelatin cover so that the
`participants were not aware of the identity of the capsules. Cap(cid:173)
`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(cid:173)
`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 X g and 4 oc for 10 min. Plasma was stored at
`-20 oc until analyzed.
`Laboratory investigations
`
`All samples for each participant were analyzed within one run
`to minimize measurement errors. EDTA-treated plasma was ana(cid:173)
`lyzed for tHey 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(cid:173)
`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(cid:173)
`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 tHey, folate, and vitamin B-12 were validated exter(cid:173)
`nally through participation in national and international interlab(cid:173)
`oratory comparisons (for tHey: European External Quality
`Assurance Scheme for homocysteine in serum; for the vitamins:
`ringtest of the Gem1an Society for Clinical Chemistry). Vitamin
`B-6 was measured as pyridoxal-? (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(cid:173)
`rithms of tHey, 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 tHey, folate, RBC
`folate, vitamin B-12, and PLP by means of parametric models
`[paired t test for within-subject comparisons and analysis of vari(cid:173)
`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
`AN OVA model was fitted to the In-transformed ratio including a
`treatment effect. To account for the influence of tHey and vita(cid:173)
`min concentrations before treatment on the change in tHey, these
`parameters were also included in the AN OVA as co variates. Post
`hoc tests used the Scheffe test. The age of the groups was com(cid:173)
`pared by using a Kruskal-Wallis one-way ANOVA because a
`skewed distribution of the data remained after logarithmic trans(cid:173)
`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(cid:173)
`ments containing vitamin B-6, vitamin B-12, or folic acid, was also
`not significantly different among groups. An estimation of compli(cid:173)
`ance with intake of the capsules was possible for 146 of 150 par(cid:173)
`ticipants (97.3%). Good compliance, defined as intake of ~6 cap(cid:173)
`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(cid:173)
`centrations ranging from 3.5 to 14.3 f.Lmol!L; the geometric mean
`value of all groups combined was 7.6 f.Lmol/L. tHey 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(cid:173)
`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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`NEPTUNE GENERICS 1019- 00002
`APOTEX 1019 - 0002
`
`
`
`1106
`
`BRONSTRUP ET AL
`
`TABLE 1
`Demographic characteristics of the study participants1
`
`min supplementation with a decrease in tHey after 4 wk. The
`extent of tHey reduction was more pronounced when only the
`subjects with a tHey concentration> 8 !Lmol/L before treatment
`were considered (group A: -16%; group B: -20%; group
`C: -22%), even though the additional effect of vitamin B-12 on
`tHey reduction was smaller (P = 0.08, ANOVA). Below a tHey
`concentration of 8 !Lmoi/L, the extent of tHey 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(cid:173)
`mediate in group B (- 10% ).
`The change in tHey was also dependent, although to a lesser
`extent, on the plasma folate concentration before vitamin sup(cid:173)
`plementation. The largest reductions in tHey were observed in
`women with the lowest initial plasma folate concentrations (Fig(cid:173)
`ure 2). In every subject with a plasma folate concentration <20
`nmol!L, tHey 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 (tHey reduction in
`group C: -17%, group B: -12%) compared with the administra(cid:173)
`tion of folic acid alone (tHey 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 :0::20
`nmol!L, the change in tHey was slightly more pronounced but
`not significantly different across treatment groups.
`When tHey 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 tHey reduction observed in group B was significantly
`larger than that observed in group A (P < 0.03). Change in tHey
`was not related to RBC folate or plasma vitamin B-12 concen(cid:173)
`trations before vitamin supplementation; inclusion of these vari(cid:173)
`ables as co variates in the AN OVA 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(cid:173)
`gate the relation with plasma folate and tHey as well as the
`response to folic acid supplementation. A strong positive associ-
`
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`Group A
`(n~Sl)
`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
`
`Group
`
`Age (y)
`BMI (kg/m2
`)
`Use ofOC (%)
`Smoking(%)
`Previous B vitamin
`supplementation(%)
`1 x ± SD or proportions. Group A received 400 f.Lg folic acid/d, group B
`received 400 f.Lg folic acid+ 6 f.Lg vitamin B-12/d, and group C received 400
`f.Lg folic acid+ 400 f.Lg vitamin B-12/d. The 3 groups did not differ signifi(cid:173)
`cantly with respect to age (P > 0.05, Kruskal-Wallis ANOVA), BMI (P >
`0.05, AN OVA), or prevalence of use of oral contraceptives (OC), smoking,
`and previous B vitamin supplementation (P > 0.05, chi-square test).
`
`After 4 wk of placebo treatment, the geometric mean tHey
`concentration of all study subjects increased slightly but signifi(cid:173)
`cantly by a mean value of 0.28 !Lmol/L (P < 0.01 ). A subgroup
`analysis showed that this was true for groups A and B, although
`no significant change in the mean tHey concentration occurred in
`group C during placebo treatment (Table 2). The change in tHey
`was not significantly different in subjects reporting regular
`intake ofB vitamins from that in those not regularly taking sup(cid:173)
`plements before the washout phase. Despite these fluctuations,
`tHey concentrations at the start of the vitamin treatment period
`did not differ significantly among groups.
`After vitamin treatment for 4 wk, the mean tHey concentra(cid:173)
`tion was reduced significantly in all groups (P < 0.001 ). The
`decrease in tHey varied according to the treatment regimen: the
`most pronounced tHey reduction was observed in group C
`( -18% compared with the tHey concentration at week 4, corre(cid:173)
`sponding to a geometric mean ratio of0.82; Table 2). In group B,
`the tHey concentration at week 8 was 15% lower than that at
`week 4. When folic acid was given alone (group A), the reduc(cid:173)
`tion in the plasma tHey concentration was 11%. The difference
`in tHey-lowering effect between group A and group C was signi(cid:173)
`ficant (P < 0.05, AN OVA with Scheffe post hoc test).
`The individual changes in tHey concentration after 4 wk of
`vitamin treatment are shown in Figure 1. The change in tHey
`was dependent on the tHey concentration before vitamin supple(cid:173)
`mentation (week 4); subjects with a high initial tHey concentra(cid:173)
`tion responded to treatment with larger reductions in tHey than
`those in subjects with initially low tHey concentrations. Above a
`tHey concentration of 8 !Lmol!L, each subject responded to vita-
`
`TABLE2
`Response of plasma total homocysteine (tHey) concentrations to placebo (week 4) and supplementation with folic acid or folic acid plus vitamin B-12
`(week 8)1
`
`Plasma tHey
`Week 8
`Week4
`Week 0 (baseline)
`8.13 ± 2.14 (7.84)3
`7.88 ± 2.22 (7.58)
`7.18 ± 1.62 (6.99) 4
`Group A (n ~51)
`0.89
`8.18 ± 2.41 (7.87)3
`7.52 ± 1.78 (7.31)
`6.81 ± 1.46 (6.65)4
`Group B (n ~ 49)
`0.85
`8.12 ± 1.92 (7.91)
`8.18 ± 1.74 (8.01)
`6.59 ± 1.12 (6.50) 4
`Group C (n ~ 50)
`0.82
`1 x ± SD; geometric mean in parentheses. Group A received 400 f.Lg folic acid/d, group B received 400 f.Lg folic acid + 6 f.Lg vitamin B-12/d, and group C
`received 400 f.Lg folic acid+ 400 f.Lg vitamin B-12/d.
`2 Geometric mean ratio of tHey at week 8 divided by tHey at week 4; values < 1 indicate a decrease in tHey after vitamin treatment.
`3 Geometric mean significantly different from week 0 (baseline), I'< 0.05 (paired ttest).
`4 Geometric mean significantly different from week 4, P < 0.001 (paired t test).
`
`Mean ratio, week 8/week 42
`
`NEPTUNE GENERICS 1019- 00003
`APOTEX 1019-0003
`
`
`
`HOMOCYSTEINE LOWERING BY FOLIC ACID AND VITAMIN B-12
`
`1107
`
`1.2
`
`*
`
`0
`1.0 - - - - - - -··--·- -·-·-- ----
`
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`-,------,-----,------,------,---~
`18
`12
`14
`16
`8
`10
`
`Homocysteine before vitamin treatment (IJmoi!L)
`
`FIGURE 1. Change in the total homocysteine (tHey) concentration in relation to the tHey concentration before vitamin supplementation (week 4).
`The ratio was derived by dividing the concentration of tHey at week 8 by the concentration at week 4. Values < I indicate a decrease in tHey after vit(cid:173)
`amin supplementation. *,supplementation with 400 11g folic acid/d; D supplementation with 400 ~folic acid+ 6 11g vitamin B-12/d; e supplemen(cid:173)
`tation with 400 11g folic acid+ 400 11g 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 tHey con(cid:173)
`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(cid:173)
`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, AN OVA) (Table 3).
`
`Vitamin B-12
`
`At week 0, the geometric mean plasma vitamin B-12 concen(cid:173)
`tration for all groups combined was 268 pmol/L (Table 3 ). Five
`
`1.2
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`·-,--------------,------,------,------,------,-----~
`10
`20
`50
`60
`70
`80
`30
`40
`
`Plasma folate before vitamin treatment (nmoi/L)
`
`~
`~ 1.1
`Q)
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`1.0
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`u
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`~ 0.6
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`0
`~ 0.5
`a::
`
`FIGURE 2. Change in the total homocysteine (tHey) concentration in relation to the plasma folate concentration before vitamin supplementation
`(week 4). The ratio was derived by dividing the concentration of tHey at week 8 by the concentration at week 4. Values < 1 indicate a decrease in tHey
`after vitamin supplementation. *,supplementation with 400 flg folic acid/d; D supplementation with 400 flg folic acid I 6 flg vitamin B-12/d; e sup(cid:173)
`plementation with 400 11g folic acid + 400 11g vitamin B-12/d.
`
`NEPTUNE GENERICS 1019- 00004
`APOTEX 1019-0004
`
`
`
`1108
`
`BRONSTRUP ET AL
`
`TABLE3
`Response of plasma vitamin indexes to placebo (week 4) and supplementation with folic acid or folic acid plus vitamin B-12 (week 8/
`
`Week 0 (baseline)
`
`Week4
`
`Week 8
`
`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)
`
`27.6 ± 10.0 (25.9)
`
`30.2 ± 10.7 (28.3)
`847 ± 381 (782)
`276 ± 110 (253)'
`
`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)
`
`28.8 ± 8.6 (27.4)
`836 ± 412 (779)
`265 ± 109 (239)
`
`Total group (n ~ 150)
`Folate (nmol/L)
`RBC folate (nmol/L)
`Vitamin B-12 (pmol/L)
`PLP (nmoi!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)
`Folate (nmol/L)
`RBC folate (nmol/L)
`279 ± 125 (250)
`Vitamin B-12 (pmol/L)
`47.0 ± 20.4 ( 43.4)
`PLP (nmoi!L)
`1x ± SD; geometric mean in parentheses. Group A received 400 f1g folic acid/d, group B received 400 f1g folic acid+ 6 f1g vitamin B-12/d, and group C
`received 400 f1g folic acid+ 400 f1g vitamin B-12/d. RBC, red blood cell; PLP, pyridoxal-F.
`2 Geometric mean significantly different from week 4, I' < 0.001 (paired t test).
`3 Geometric mean significantly different from week 0 (baseline), P < 0.01 (paired ttest).
`4 No statistical test performed because of the difference in vitamin B-12 treatment among the 3 groups.
`
`45.6 ± 14.3 (43.5i
`1485 ± 615 (1359)2
`345 ± 144 (317)4
`
`46.6 ± 17.4 ( 43.8)2
`1438 ± 643 (1296i
`259 ± 104 (240)
`
`46.1 ± 13.4 (44.1)2
`1551 ± 593 (1443i
`371 ± 125 (353i
`
`44.0 ± 11.4 ( 42.5)2
`1468 ± 616 (1344)2
`407 ± 158 (377)2
`
`participants had plasma concentrations indicative of a subopti(cid:173)
`mal vitamin B-12 status (< 111 pmol!L, or< 150 pg/mL) (25).
`Their corresponding plasma tHey concentrations ranged from
`7.3 to 12.0 f.Lmol/L.
`After 4 wk of placebo treatment, the geometric mean vitamin
`B-12 concentration of the whole group was slightly but signifi(cid:173)
`cantly lower than at week 0 (P < 0.01 ). This was mainly attrib(cid:173)
`utable to changes in group A (P < 0.01). The observed change
`did not correlate with the change in tHey 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, AN OVA 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(cid:173)
`nation of these vitamins had a more pronounced tHey-lowering
`
`effect than supplementation with folic acid alone. Of the young
`women participating in this study, the vast majority had an ade(cid:173)
`quate status of the vitamins involved in homocysteine metabo(cid:173)
`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 tHey
`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 f.Lg folic acid/d) to lower tHey concentrations
`in middle-aged, healthy men. Because in their study the supple(cid:173)
`mentation regimen was increased over the course of the study in
`6-wk intervals, no information on the tHey-lowering effect attrib(cid:173)
`utable solely to supplementation with 400 f.Lg folic acid can be
`obtained. However, as in our study, the tHey-reducing effect was
`clearly dependent on the tHey concentration at baseline. Before
`supplementation, men in the 2 lowest tertiles together had a mean
`plasma tHey concentration of 8.09 f.Lmol!L, which is almost iden(cid:173)
`tical to that observed in group A in our study. After folic acid
`administration, the extent of tHey reduction in group A was com(cid:173)
`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 1-1g/d results on average in a
`reduction in tHey concentration of 4 f.Lmol!L, or 12%. However,
`this rather high effect of folic acid supplementation may be
`restricted to subjects with moderate or intermediate hyperhomo(cid:173)
`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 1-1g/d is
`required for a mean reduction of the plasma tHey concentration
`of 11%. Even though we are still awaiting results from interven-
`
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`NEPTUNE GENERICS 1019- 00005
`APOTEX 1019-0005
`
`
`
`HOMOCYSTEINE LOWERING BY FOLIC ACID AND VITAMIN B-12
`
`1109
`
`tion studies of the protective effect of vitamin supplements in
`lowering plasma tHey 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 f.Lg/d), the relative
`risk for coronary artery disease, controlling for other cardiovas(cid:173)
`cular risk factors, was 0.69 (95% CT: 0.55, O.R7) compared with
`women in the lowest quintile (median intake: 158 f.Lg/d) (11).
`Calculations showed that with each 200-f.Lg 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 f.Lg/d, the
`former recommended dietary allowance for women (29).
`In this study, vitamin B-12 supplementation increased the
`tHey-lowering potential of folic acid; this was especially obvious
`when vitamin B-12 was given in pharmacologic amounts ( 400
`f.Lg). In subgroup analyses, the extent of the tHey reduction was
`significantly higher with the addition of increasing doses of vit(cid:173)
`amin B-12 in women with a plasma folate concentration >20
`nmol/L. Because folate and vitamin B-12 have a synergistic
`function as co factors 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 f.Lg vitamin B-12/d as well.
`Thus, on average, for this group of women, the low dose of vit(cid:173)
`amin B-12 was quite bioavailable. The increase in plasma vita(cid:173)
`min B-12 concentration was even more distinct after supplemen(cid:173)
`tation with the high (pharmacologic) dose of 400 f.Lg vitamin
`B-12. For this dose, the extent of the plasma vitamin B-12
`increase observed was comparable with what Ubbink et a! (28)
`found after supplementing young, healthy men with the same
`dose for 6 wk. However, it seems evident that this intake of vit(cid:173)
`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 f.Lg folic acid or to enriched
`foods maximizes the reduction of tHey through the synergistic
`potential of both vitamins. This was most evident with the dose
`of 400 f.Lg vitamin B-12/d together with folic acid. Others have
`suggested that, in addition to an effect on tHey, combined sup(cid:173)
`plementation with 400 f.Lg folic acid+ 400 f.Lg 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 f.Lg 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 ~g 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(cid:173)
`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 ofNTDs, results
`in significant reductions in the tHey concentration of normoho(cid:173)
`mocysteinemic women without vitamin deficiency. Above a
`
`plasma tHey concentration as low as 8 f.Lmol!L, each subject
`responded to vitamin supplementation with a reduction in tHey.
`Beyond the presumed prevention of nerve damage in persons
`with p