Effects of the Interaction Between the C677T 5, 10

ORIGINAL COMMUNICATION Effects of the interaction between the C677T 5,10-methylenetetrahydrofolate reductase polymorphism and serum B vitamins on homocysteine levels in pregnant women

KN Kim1, YJ Kim2 and N Chang1*

1Department of Food and Nutritional Sciences, Ewha Womans University, Seoul, Korea; and 2Department of Obstetrics and Gynecology, Medical Research Center, Ewha Womans University, Seoul, Korea

Objective: The purpose of this study was to investigate the effect of the interaction between the C677T mutation in the 5,10- methylenetetrahydrofolate reductase (MTHFR) genotypes and serum levels of B vitamins on serum homocysteine levels in pregnant women. Design: A cross-sectional study. Setting: Ewha Womans University Hospital, Seoul, Korea. Subjects: A total of 177 normal pregnant women, 24.671.1 weeks of gestation, in a 6-month period during 2001–2002. Interventions: Serum vitamin B2, vitamin B6, and homocysteine analyses were conducted using high-performance liquid chromatography methods. Serum folate and vitamin B12 concentrations were determined using a radioimmunoassay kit. MTHFR gene mutation was investigated by the polymerase chain reaction of a genomic DNA fragment. Results: Serum homocysteine was higher in women with the T/T genotype than those with the C/T or C/C genotype of the MTHFR gene (Po0.05). Serum homocysteine was negatively correlated with serum folate in all MTHFR genotypes (Po0.001), and the correlation between the two serum levels was the strongest in the T/T genotype. Serum homocysteine was higher in the subjects with the T/T MTHFR genotype only when the serum folate was below the median level. Explanatory power of B vitamin status as predictors of serum homocysteine levels was more pronounced in the T/T genotypes (68.5%) compared with the C/T (37.9%) or C/C genotypes (20.6%). Conclusions: Serum homocysteine levels in pregnant women varied significantly with MTHFR genotype and the serum B vitamin status. Higher serum folate, vitamin B2, and vitamin B12 concentrations may lessen the MTHFR genotypic effect on serum homocysteine levels. Sponsorship: This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (01-PJ1-PG1-01CH15-0009). European Journal of Clinical Nutrition (2004) 58, 10–16. doi:10.1038/sj.ejcn.1601729

Keywords: homocysteine; vitamin B2; folate; vitamin B12; MTHFR genotype; pregnancy

*Correspondence: N Chang, Department of Food and Nutritional Introduction Sciences, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, Korea. Elevated total serum homocysteine concentration in preg- E-mail: [email protected] nant women is an important issue as it is known to be related Guarantors: Ki Nam Kim, Namsoo Chang. to many adverse pregnancy outcomes including birth defects Contributors: NSC and YJK contributed to the design of the study, (Mill et al, 1995; Rosenquist et al, 1995), pre-eclampsia (Leeda subject recruiting, and manuscript preparation. KNK carried out the data analysis and the biochemical analyses of serum B vitamins and et al, 1998), placental abruption (Goddijn-Wessel et al, 1996), homocysteine. YJK was responsible for clinical data collection and the spontaneous abortion (Wouters et al, 1993), low birth weight analysis of the MTHFR polymorphism. NSC was the main person (Burke et al, 1992), and other maternal or fetal complications responsible for all stages of the study. All took part in the writing of (Aubard et al, 2000; Scholl & Johnson, 2000). the paper. Received 25 July 2002; revised 9 January 2003; Homocysteine is influenced by the nutritional status of accepted 13 January 2003 B vitamins (vitamin B2, vitamin B6, folate, and vitamin B12), MTHFR polymorphism and serum B vitamins in pregnant women KN Kim et al 11 whose metabolic steps are inter-related with each other vitamin B12 were calculated by CANPro II, a computerized (Perry, 1999; Ueland et al, 2001) and nutritional adequacy of nutrient intake assessment software program developed by these vitamins is essential to maintain the plasma homo- the Korean Nutrition Society (2002). cysteine levels within a normal homeostatic range. The Fasting venous blood was drawn from the antecubital vein. conversion step of tetrahydrofolate (THF) to 5,10-methylene Blood samples for vitamins and homocysteine were centri- THF is coupled with a PLP-dependent reaction and that of fuged for 10 min at 3000 Â g to separate the serum. The 5,10-methylene THF to 5-methyl THF, a FAD-dependent serum was divided into several aliquots, which were kept reaction, and the regeneration of THF from 5-methyl THF is a frozen at À701C until assayed. vitamin B12-dependent reaction. The plasma concentration Serum homocysteine analyses were conducted using a of these vitamins in pregnant women is known to decrease modification of a high-performance liquid chromatography (Bruinse & van den Berg, 1995) and such decreases could (HPLC)-fluorescence detection method by Araki and Sako adversely affect homocysteine metabolism during preg- (1987). The interassay coefficient of variance (CV) for nancy. homocysteine was 4.9%. Serum levels of flavin adenine In addition, genetic polymorphism of the enzyme dinucleotide (FAD), flavin mononucleotide (FMN), and MTHFR, which catalyzes the irreversible conversion of riboflavin were measured by the HPLC-UV detection 5,10-methylene THF to 5-methyl THF in the folate cycle, is method of Botticher and Botticher (1987). The CVs of the known to influence homocysteine metabolism resulting in FAD, FMN, and riboflavin assay were 5.8, 6.9, and 4.1%, hyperhomocysteinemia (Jacques et al, 1996). The homozyg- respectively. Serum pyridoxal-5-phosphate (PLP) was ana- osity for the C677T variant MTHFR allele causes higher lyzed with a reagent kit for HPLC analysis for vitamin B6 fasting plasma homocysteine concentrations and lower (Chromsystems, Mu¨nchen, Germany). The CV of the PLP folate status than heterozygous or wild-type groups assay was 3.3%. Serum vitamin B12 and folate concentrations (Brattstrom et al, 1998). A curve of steeper slope in subjects were determined using a radioimmunoassay kit (Diagnostic with T/T than in those of C/C genotype has been reported Products Corporation, Los Angeles, CA, USA). The CVs for for an inverse relation between plasma homocysteine folate and vitamin B12 analyses were 4.0 and 4.4%, and folate (van der Put et al, 1995; Nelen et al, 1998) or respectively. vitamin B12 (Jacques et al, 1996; Woodside et al, 1998; Moriyama et al, 2002). Several studies have compared the relation between the MTHFR mutation analysis MTHFR polymorphism, plasma homocysteine levels, and the DNA was extracted from the whole blood using an Aquapure B vitamin status in pregnant women with pregnancy Genomic DNA blood kit (Biorad Pacific Ltd., Kowloon, Hong complications (Goddijn-Wessel et al, 1996; Leeda et al, Kong) and stored at À201C for analysis. DNA fragments were 1998). However, few studies have been reported on these amplified from the genomic DNA via the polymerase chain relations and on homocysteine metabolism in normal reaction (PCR). Amplication was carried out in the PCR women with uncomplicated pregnancies. buffer with 0.5 U Taq DNA polymerase (Takara Shuzo Co., The purpose of this paper is to report on our investigation Shiga, Japan). PCR conditions comprised of an initial on the effect of the interaction between the C677T MTHFR denaturation step at 951C for 9 min, followed by 33 cycles polymorphism and serum B vitamins on the homocysteine of 951C for 1 min, 601C for 1 min, and 721C for 1 min, and a concentration in normal pregnant women. final extension step at 721C for 10 min (Frosst et al, 1995). The PCR product was digested by using HinfI (Takara Shuzo Co., Shiga, Japan.) The C to T substitution at nucleotide 677 Method creates an extra HinFI restriction site that cleaves the original Subjects 198-bp PCR fragment into 175- and 23-bp fragments. Size A total of 177 consecutive normal Korean pregnant women fractionation of the PCR products was by electrophoresis on in their 24–28 week of gestation participated in this study. 2.5% agarose-gel containing 0.5 mg/ml ethidium bromide The criteria for normal pregnancy are as follows: no and was visualized using the ultraviolet light. pregnancy complications, no medications, and free of pregravid chronic diseases. The research protocol was approved by the Human Investigation Review Committee Statistical analysis of Ewha Womans University, and informed consent forms Data were presented as mean7s.d. and percentages. All data were obtained from all subjects. of serum measures were log transformed to normalize their distributions and were presented as geometric mean7s.d. After the ANOVA, Duncan’s multiple range test was Data collection and biochemical analyses performed to determine the significance of the differences Dietary habits and food intake data were collected from the in the means of serum vitamins and homocysteine among participants by the 24-h recall method. Intakes of energy and pregnant women with different genotypes of the MTHFR 2 nutrients including vitamin B2, vitamin B6, folate, and gene. A w analysis was used to test the significant differences

European Journal of Clinical Nutrition MTHFR polymorphism and serum B vitamins in pregnant women KN Kim et al 12 in the prevalence of hyperhomocysteinemia among the such as income and education level of the subjects were not three genotypic groups. A Spearman’s correlation coefficient significantly different among the three MTHFR genotype analysis was used to evaluate the relation between homo- groups (Table 1). cysteine and B vitamins. Analysis of covariance was The mean concentrations of serum FAD, FMN, riboflavin,

performed to compare the slopes of the three regression PLP, folate, and vitamin B12 were not different among the lines between serum homocysteine and folate by MTHFR carriers of an MTHFR mutant allele (T/T or C/T genotype) genotypes. We performed two-way ANOVA to test for compared with those of no mutant allele (C/C genotype) as differences between genotypes and for interactions between shown in Table 2. The mean vitamin intake was 1.370.5 mg, genotype and vitamin levels. Multiple regression analysis 2.471.0 mg, 398.17198.1 mg and 1.872.4 mg, respectively,

was used to examine the associations between serum for vitamin B2, vitamin B6, folate, and vitamin B12. Dietary homocysteine and other variables and identify the best intake of B vitamins assessed by the 24-h recall method was predictor among variables. The differences were considered not different among the three genotype groups (data not significant at the 5% level in this study. The SPSS software shown). Serum homocysteine levels were significantly high- 11.0 was used in the statistical analyses. er in women with the T/T genotype (9.374.6 m mol/l) than in subjects with the C/T (8.372.9 m mol/l) or the C/C genotype (7.472.6 m mol/L, Po0.05). Results As shown in Table 3, more women with the T/T genotype The MTHFR genotype frequencies for C/C, C/T, and T/T were (17.9%) were assessed as hyperhomocysteinemic 59 (33.3%), 87 (49.2%), and 31 (17.5%). Age, gravidity, (Z15 m mol/l) than those with the C/T (4.9%) or the C/C pregravid body mass index, and the socioeconomic status genotype (0%). Figure 1 shows that serum homocysteine was negatively correlated with serum folate in all MTHFR genotypes

Table 1 General characteristics of study subjects Table 3 Prevalence of hyperhomocysteinemia in subjects according to MTHFR genotypes their MTHFR genotype

C/C C/T T/T Total Homocysteine levels, n (%) n=59 n=87 n=31 n=177 o15 mmol/l Z15 mmol/l Significance Age (y) 30.873.2 30.573.7 30.572.9 30.673.4 7 7 7 7 Gravidity 2.2 1.5 2.1 1.3 2.0 0.9 2.1 1.3 C/C type 57 (100.0) 0 ( 0.0) 7 7 7 7 Pregravid BMI 20.8 3.5 20.7 3.3 20.2 2.5 20.7 3.2 C/T type 78 ( 95.1) 4 ( 4.9) w2=11.8 2 (kg/m ) T/T type 23 ( 82.1) 5 (17.9) df=2 Education (y) 14.872.1 15.272.2 15.572.2 15.172.2 7 7 7 7 Income 231.0 76.5 243.7 78.0 250.0 81.6 240.3 77.7 Total 158 ( 94.6) 9 ( 5.4) P=0.003 (10 000won)

Data are expressed as frequency (percentage) of hyperhomocysteinemic 7 Values are mean s.d. BMI-body mass index. Results as determined by Z15 mmol/h at the 95th confidence upper limit by Cikot et al (2001) subjects ANOVA and Duncan’s multiple range test were not significantly different according to MTHFR genotypes. Chi-square test was used to evaluate the among the MTHFR genotypes (Po0.05). distribution.

Table 2 Serum B vitamins and homocysteine concentrations of women according to their MTHFR genotype

MTHFR genotypes

C/C C/T T/T Total n=59 n=87 n=31 n=177

FAD (nmol/l) 10.173.0 10.872.6 11.372.4 10.672.7 FMN (nmol/l) 3.771.8 3.971.7 4.271.2 3.971.6 Riboflavin (nmol/l) 1.972.0 2.177.4 1.871.4 1.975.4 PLP (nmol/l) 19.7713.0 21.4734.4 23.7738.9 21.2729.9 Folate (nmol/l) 18.1711.8 17.1710.4 15.2712.2 17.1711.3 Vitamin B12 (pmol/l) 317.97163.8 278.37164.4 308.97158.2 296.27162.6 Homocysteine (mmol/l) 7.072.6a 7.872.9a,c 8.374.6b 7.673.2

Values are genometric mean7s.d. a,b,c Different superscript letter indicates the comparison with significant differences (Po0.05). The differences between the MTHFR genotypes as determined by ANOVA and Duncan’s multiple range test were not significant (Po0.05).

European Journal of Clinical Nutrition MTHFR polymorphism and serum B vitamins in pregnant women KN Kim et al 13 different from that of the C/C or C/T genotype (T/T: Y ¼À0.464X þ 13.60, C/C: Y ¼À0.21X þ 9.37, C/T ¼À0.21X þ 10.08, Po0.05) Table 4 presents descriptive statistics for serum homocysteine concentrations and the main effects of the MTHFR genotype, serum B vitamin levels, and interaction effects of the MTHFR genotype and serum B vitamin levels. These analyses revealed significant effects of the MTHFR genotype, serum folate levels, and the MTHFR genotype by serum folate level interactions for serum homocysteine concentrations. Serum homocysteine concentrations were significantly higher in the subjects with the T/T type of the MTHFR genes only when the serum folate levels were below the median level. Within the same MTHFR genotype, homocysteine levels were significantly higher among the subjects whose folate levels were below the median com- Figure 1 Correlation between serum folate and homocysteine in pared to those with vitamin levels above the median in all pregnant women according to their MTHFR genotype. Serum three MTHFR genotypes. The MTHFR genotype by serum homocysteine was negatively correlated with serum folate in all FAD vitamin level interactions were also observed. When MTHFR genotypes. The correlation between two serum levels was serum FAD levels were below the median level, homocys- stronger in the T/T type (r ¼À0.624, Po0.001) than in the C/C or C/T genotype (C/C: r ¼À0.359, C/T: r ¼À0.361). The slope of the teine levels in the subjects with the T/T or C/T type were regression line for the TT genotype was significantly different from significantly higher than that of the C/C type. For vitamin that of the C/C or C/T genotype (T/T: Y ¼À0.464X þ 13.60, C/C: B12, higher homocysteine levels in subjects with low vitamin Y ¼À0.21X þ 9.37, C/T ¼À0.21X þ 10.08, Po0.05). status were shown only in the C/C genotype group. We analyzed the relation of serum homocysteine to serum B vitamins concentrations for each MTHFR genotype. As (Po0.001), and the correlation between the two serum levels listed Table 5, serum homocysteine levels were significantly was stronger in the T/T type (r ¼À0.624) than in the C/T or related to serum folate concentrations in all MTHFR C/C genotype (C/T: r ¼À0.361, C/C: r ¼À0.359). The slope of genotypes. Explanatory power of B vitamin status as the regression line for the T/T genotype was significantly predictors of serum homocysteine levels was more pro-

Table 4 Comparison of serum homocysteine levels according to serum B vitamins levels by the MTHFR genotype

MTHFR genotypes Significant factorsa

C/C C/T T/T Genotype Serum level Genotype serum level

FAD (nmol/l) NS NS * omedian (n=69) 6.172.5a 8.173.2b 8.975.5b Zmedian (n=69) 7.772.5 7.572.3 7.173.7 FMN (nmol/l) NS NS NS omedian (n=77) 6.572.4 8.073.2 8.474.6 Zmedian (n=71) 7.472.9 7.672.5 7.274.4 Riboflavin (nmol/l) NS NS NS omedian (n=75) 6.672.6 7.972.9 8.374.8 Zmedian (n=79) 7.472.7 7.772.7 7.875.0 PLP(nmol/l) NS NS NS omedian (n=83) 6.872.5 7.773.1 9.575.2 Zmedian (n=87) 6.972.9 8.072.8 7.074.0 Folate(nmol/l) **** omedian (n=82) 7.872.9a# 8.672.5a# 11.174.5b## Zmedian (n=95) 6.271.9 7.372.5 6.472.9 Vitamin B12(pmol/l) NS * NS omedian (n=72) 8.172.6# 8.273.1 8.974.5 Zmedian (n=105) 6.272.5 7.371.9 7.574.1

Data are expressed as geometric mean7s.d. Statistical significance of serum B vitamin levels and the MTHFR genotypes was analysed by two-way ANOVA. *,**Significant effect of genotype, serum B vitamin level, or their interaction, Po0.05 and Po0.01, respectively NS, not significant. a,b Different superscript letters indicate the comparison with significant differences (Po 0.05). #,## Significant difference according to serum B vitamin levels within the same MTHFR genotypes Po0.05 and Po 0.01, respectively.

European Journal of Clinical Nutrition MTHFR polymorphism and serum B vitamins in pregnant women KN Kim et al 14 Table 5 Coefficients from multiple regression analysis between serum homocysteine and B vitamins concentrations in subjects according to their MTHFR genotype

Independent variables MTHFR genotypes

C/C C/T T/T

b s.e.(b) b s.e.(b) b s.e.(b)

FAD 0.293 0.239 À0.055 0.197 À0.338 0.720 FMN À0.105 0.212 À0.137 0.132 0.358 0.659 Riboflavin À0.048 0.087 0.059 0.046 À0.104 0.176 PLP 0.050 0.107 0.102 0.052 0.204 0.169 Folate À0.260 0.124* À0.294 0.068*** À0.708 0.198** Vitamin B12 0.041 0.135 0.010 0.097 0.317 0.253

b, multiple correlation coefficient. R2 of model, the percentage of variation in serum homocysteine explained by the biochemical indexes serum FAD. FMN, 7 7 riboflavin, PLP, folate and vitamin B12 were 20.6 0.2%, 37.9 0.1% and 68.5+0.1% in the C/C, C/T and T/T genotypic women respectively. *Po0.05, **Po0.01, ***Po0.001.

nounced in the T/T genotypes (68.5%, P ¼ 0.289) compared protected this enzyme against flavin loss and inactivation as with the C/T (37.9%, P ¼ 0.002) or C/C genotypes (20.6%, suggested by Guenther et al (1999).

P ¼ 0.083). In addition to folate, high normal status of vitamin B2 expressed by high serum levels was shown to compensate for the hyperhomocysteinemia associated with homozygosity

for the C677T mutation. Vitamin B2, in the form of FAD, is a Discussion cofactor for MTHFR. An earlier animal study showed a

In our study, serum homocysteine concentrations were significant association between vitamin B2 and MTHFR that found significantly higher in pregnant women with the the enzyme activity and the relative amounts of 5-methyl T/T genotype, as compared to those in the C/T or C/C THF were reduced in the liver of riboflavin-deficient rats genotype, confirming the results of other investigators (Bates & Fuller, 1986). Hustad et al (2000), McNulty et al (Jacques et al, 1996; Brattstrom et al, 1998; Gudnason et al, (2002), and Jacques et al (2002) demonstrated that plasma

1998). Serum homocysteine was negatively correlated with vitamin B2 was an independent determinant of plasma serum folate in our study subjects of all the three MTHFR homocysteine, and this inverse dose–response relation was genotypes, and the correlation between the two serum levels essentially confined to subjects with the C/T or T/T genotype was strongest as had been previously reported by Ueland et al of the MTHFR C677T polymorphism where the requirement (2001) and Guttormsen et al (1996), who had observed a of FAD for maximal catalytic activity might be increased steeper negative slope in plots of serum folate vs serum (Hustad et al, 2000; Ueland et al, 2001). Several investigators

homocysteine concentration in T/T individuals compared to have suggested that vitamin B6 deficiency may lead to those in the C/T or C/C subjects. hyperhomocysteinemia, which is an established risk factor The results of our study suggest that pregnant women with for adverse pregnancy outcome (van den Berg et al, 1994; a combination of decreased serum folate concentrations and Leeda et al, 1998). However, the present study failed to show

the T/T genotype of the MTHFR gene could be at an the effects of serum vitamin B6, PLP on serum homocysteine. increased risk of developing hyperhomocysteinemia, con- Our results showed that although serum vitamin B12 firming the previous findings by Powers et al (1999). In our concentrations were similar among all three genotype study, pregnant women who were homozygotes for the groups, significantly higher serum homocysteine levels were

MTHFR mutation had higher fasting homocysteine levels present in pregnant women whose vitamin B12 levels were only when their serum folate levels were below the median, below the median compared to those with vitamin levels but not when serum folate levels were above the median. above the median only in the C/C genotype. Guttormsen This confirms the study of Brattstrom et al (1998) and Bailey et al (1996) made an observation that all hyperhomocystei- and Gregory III (1999), who reported that the association nemic subjects with the C/C genotype had low plasma

between the MTHFR genotype and homocysteine levels was vitamin B12 concentrations, and that vitamin B12 supple- modified by serum folate status. One explanation for these mentation alone had reduced their plasma homocysteine observations could be, as hypothesized by Jacques et al concentrations. Nakamura et al (2002) explained the reason (1996), that a higher folate status may increase the in vivo for the above observation as follows: subjects with the C/T or stability of the MTHFR enzyme, thus reducing the difference T/T genotype have lower folate concentrations than those in enzyme activity between the T/T and C/T or C/C with the C/C genotype because of a reduced production of 5- genotype. Another explanation for this could be that folate methyl THF, a product of MTHFR. Low 5-methyl THF

European Journal of Clinical Nutrition MTHFR polymorphism and serum B vitamins in pregnant women KN Kim et al 15 concentrations in the cytosol might conceal a deficiency of both types of women who are homozygous and hetero- vitamin B12 in patients with the C/T or T/T. Lucock et al zygous for MTHFR. (2001) suggested that a secondary gene–nutrient interaction In conclusion, we have presented and described the between C677T-MTHFR and vitamin B12 could modulate findings of our work suggesting the strong evidence and folate and homocysteine metabolism in an adverse way effect of the interaction between the C677T MTHFR poly- under critical conditions such as poor nutrient intake or high morphism and serum B vitamins on the homocysteine nutrient demand such as in pregnancy. concentration in normal pregnant women. We have found Our data show that the serum homocysteine concentra- that serum homocysteine concentrations varied significantly tions in pregnant women are determined by an interaction with the MTHFR genotype and serum B vitamin levels. between the MTHFR genotype and serum B vitamin status, Higher folate concentrations might have an attenuating and serum B vitamins (vitamin B2, vitamin B6, folate, and effect on serum homocysteine in mid-pregnancy, and high vitamin B12) predicted 68.5% of serum homocysteine levels serum levels of folate and vitamin B2 can also lessen the in the T/T genotype, 37.9% in the C/T genotype, and 20.6% MTHFR genotypic effect on serum homocysteine. Our study in the C/C genotypes. shows that there exists an interaction between MTHFR Here, we have only looked into the interaction between genotype genetics and vitamin B nutrition in homocysteine vitamin B status and the C677T polymorphism in the metabolism in normal pregnancy. This study supports the MTHFR gene and their effects on serum homocysteine suggestion that the folate and other B vitamin deficiencies levels. Another mutation, named A1298C mutation, are more detrimental in pregnant women with the T/T that commonly occurs as frequently as C677T in the homozygous, and may also be harmful for C/T heterozygous same gene when present alone has been shown not to genotype as well in preventing the potential risks of cause elevations in plasma homocysteine. However, the adverse pregnancy outcomes associated with hyperhomo- combined heterozygosity for both C677T and A1298C cysteinemia. mutations resulted in significant reduction of the MTHFR enzyme activity and plasma homocysteine elevations (van der Put et al, 1998). We do not know at this point about whether or not other recently discovered References mutations such as G1793A mutation in the MTHFR Araki A & Sako Y (1987): Determination of free and total gene (Rady et al, 2002), D919G mutation in methionine homocysteine in human plasma by high performance liquid synthase gene (Chen et al, 2001), A66G mutation in chromatography with fluorescence detection. J. Chromatogr. 422, 43–52. et al methionine synthase reductase gene (Gaughan , 2001), Aubard Y, Darodes N & Cantaloube M (2000): Hyperhomocysteine- G80A mutation in the RFC-1 folate transport protein gene mia and pregnancyFreview of our present understanding and might have played a role in the interaction of vitamin B therapeutic implications. Eur. J. Obstet. 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