Introduction

Elevated total serum homocysteine concentration in pregnant women is an important issue as it is known to be related to many adverse pregnancy outcomes including birth defects (Mill et al, 1995; Rosenquist et al, 1995), pre-eclampsia (Leeda et al, 1998), placental abruption (Goddijn-Wessel et al, 1996), spontaneous abortion (Wouters et al, 1993), low birth weight (Burke et al, 1992), and other maternal or fetal complications (Aubard et al, 2000; Scholl & Johnson, 2000).

Homocysteine is influenced by the nutritional status of B vitamins (vitamin B₂, vitamin B_6, folate, and vitamin B₁₂), whose metabolic steps are inter-related with each other (Perry, 1999; Ueland et al, 2001) and nutritional adequacy of these vitamins is essential to maintain the plasma homocysteine levels within a normal homeostatic range. The conversion step of tetrahydrofolate (THF) to 5,10-methylene THF is coupled with a PLP-dependent reaction and that of 5,10-methylene THF to 5-methyl THF, a FAD-dependent reaction, and the regeneration of THF from 5-methyl THF is a vitamin B₁₂-dependent reaction. The plasma concentration of these vitamins in pregnant women is known to decrease (Bruinse & van den Berg, 1995) and such decreases could adversely affect homocysteine metabolism during pregnancy.

In addition, genetic polymorphism of the enzyme MTHFR, which catalyzes the irreversible conversion of 5,10-methylene THF to 5-methyl THF in the folate cycle, is known to influence homocysteine metabolism resulting in hyperhomocysteinemia (Jacques et al, 1996). The homozygosity for the C677T variant MTHFR allele causes higher fasting plasma homocysteine concentrations and lower folate status than heterozygous or wild-type groups (Brattstrom et al, 1998). A curve of steeper slope in subjects with T/T than in those of C/C genotype has been reported for an inverse relation between plasma homocysteine and folate (van der Put et al, 1995; Nelen et al, 1998) or vitamin B₁₂ (Jacques et al, 1996; Woodside et al, 1998; Moriyama et al, 2002).

Several studies have compared the relation between the MTHFR polymorphism, plasma homocysteine levels, and the B vitamin status in pregnant women with pregnancy complications (Goddijn-Wessel et al, 1996; Leeda et al, 1998). However, few studies have been reported on these relations and on homocysteine metabolism in normal women with uncomplicated pregnancies.

The purpose of this paper is to report on our investigation on the effect of the interaction between the C677T MTHFR polymorphism and serum B vitamins on the homocysteine concentration in normal pregnant women.

Method

Subjects

A total of 177 consecutive normal Korean pregnant women in their 24–28 week of gestation participated in this study. The criteria for normal pregnancy are as follows: no pregnancy complications, no medications, and free of pregravid chronic diseases. The research protocol was approved by the Human Investigation Review Committee of Ewha Womans University, and informed consent forms were obtained from all subjects.

Data collection and biochemical analyses

Dietary habits and food intake data were collected from the participants by the 24-h recall method. Intakes of energy and nutrients including vitamin B₂, vitamin B₆, folate, and vitamin B₁₂ were calculated by CANPro II, a computerized nutrient intake assessment software program developed by the Korean Nutrition Society (2002).

Fasting venous blood was drawn from the antecubital vein. Blood samples for vitamins and homocysteine were centrifuged for 10 min at 3000 g to separate the serum. The serum was divided into several aliquots, which were kept frozen at -70°C until assayed.

Serum homocysteine analyses were conducted using a modification of a high-performance liquid chromatography (HPLC)-fluorescence detection method by Araki and Sako (1987). The interassay coefficient of variance (CV) for homocysteine was 4.9%. Serum levels of flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and riboflavin were measured by the HPLC-UV detection method of Botticher and Botticher (1987). The CVs of the FAD, FMN, and riboflavin assay were 5.8, 6.9, and 4.1%, respectively. Serum pyridoxal-5-phosphate (PLP) was analyzed with a reagent kit for HPLC analysis for vitamin B₆ (Chromsystems, München, Germany). The CV of the PLP assay was 3.3%. Serum vitamin B₁₂ and folate concentrations were determined using a radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, CA, USA). The CVs for folate and vitamin B₁₂ analyses were 4.0 and 4.4%, respectively.

MTHFR mutation analysis

DNA was extracted from the whole blood using an Aquapure Genomic DNA blood kit (Biorad Pacific Ltd., Kowloon, Hong Kong) and stored at -20°C for analysis. DNA fragments were amplified from the genomic DNA via the polymerase chain reaction (PCR). Amplication was carried out in the PCR buffer with 0.5 U Taq DNA polymerase (Takara Shuzo Co., Shiga, Japan). PCR conditions comprised of an initial denaturation step at 95°C for 9 min, followed by 33 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min, and a final extension step at 72°C 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 creates an extra HinFI restriction site that cleaves the original 198-bp PCR fragment into 175- and 23-bp fragments. Size fractionation of the PCR products was by electrophoresis on 2.5% agarose-gel containing 0.5 mg/ml ethidium bromide and was visualized using the ultraviolet light.

Statistical analysis

Data were presented as means.d. and percentages. All data of serum measures were log transformed to normalize their distributions and were presented as geometric means.d. After the ANOVA, Duncan's multiple range test was performed to determine the significance of the differences in the means of serum vitamins and homocysteine among pregnant women with different genotypes of the MTHFR gene. A ² analysis was used to test the significant differences in the prevalence of hyperhomocysteinemia among the three genotypic groups. A Spearman's correlation coefficient analysis was used to evaluate the relation between homocysteine and B vitamins. Analysis of covariance was performed to compare the slopes of the three regression lines between serum homocysteine and folate by MTHFR genotypes. We performed two-way ANOVA to test for differences between genotypes and for interactions between genotype and vitamin levels. Multiple regression analysis was used to examine the associations between serum homocysteine and other variables and identify the best predictor among variables. The differences were considered significant at the 5% level in this study. The SPSS software 11.0 was used in the statistical analyses.

Results

The MTHFR genotype frequencies for C/C, C/T, and T/T were 59 (33.3%), 87 (49.2%), and 31 (17.5%). Age, gravidity, pregravid body mass index, and the socioeconomic status such as income and education level of the subjects were not significantly different among the three MTHFR genotype groups (Table 1).

Table 1 - General characteristics of study subjects.

Full table

The mean concentrations of serum FAD, FMN, riboflavin, PLP, folate, and vitamin B₁₂ were not different among the carriers of an MTHFR mutant allele (T/T or C/T genotype) compared with those of no mutant allele (C/C genotype) as shown in Table 2. The mean vitamin intake was 1.30.5 mg, 2.41.0 mg, 398.1198.1 g and 1.82.4 g, respectively, for vitamin B₂, vitamin B₆, folate, and vitamin B₁₂. Dietary intake of B vitamins assessed by the 24-h recall method was not different among the three genotype groups (data not shown). Serum homocysteine levels were significantly higher in women with the T/T genotype (9.34.6  mol/l) than in subjects with the C/T (8.32.9  mol/l) or the C/C genotype (7.42.6  mol/L, P<0.05).

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

Full table

As shown in Table 3, more women with the T/T genotype (17.9%) were assessed as hyperhomocysteinemic (15  mol/l) than those with the C/T (4.9%) or the C/C genotype (0%).

Table 3 - Prevalence of hyperhomocysteinemia in subjects according to their MTHFR genotype.

Full table

Figure 1 shows that serum homocysteine was negatively correlated with serum folate in all MTHFR genotypes (P<0.001), and the correlation between the two serum levels was stronger in the T/T type (r=-0.624) than in the C/T or C/C genotype (C/T: r=-0.361, C/C: r=-0.359). The slope of the regression line for the T/T genotype was significantly different from that of the C/C or C/T genotype (T/T: Y=-0.464X+13.60, C/C: Y=-0.21X+9.37, C/T=-0.21X+ 10.08, P<0.05)

Figure 1.

Correlation between serum folate and homocysteine in pregnant women according to their MTHFR genotype. Serum homocysteine was negatively correlated with serum folate in all MTHFR genotypes. The correlation between two serum levels was stronger in the T/T type (r=-0.624, P<0.001) than in the C/C or C/T genotype (C/C: r=-0.359, C/T: r=-0.361). The slope of the regression line for the TT genotype was significantly different from that of the C/C or C/T genotype (T/T: Y=-0.464X+13.60, C/C: Y=-0.21X+9.37, C/T=-0.21X+10.08, P<0.05).

Full figure and legend (27K)

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 compared to those with vitamin levels above the median in all three MTHFR genotypes. The MTHFR genotype by serum FAD vitamin level interactions were also observed. When serum FAD levels were below the median level, homocysteine levels in the subjects with the T/T or C/T type were significantly higher than that of the C/C type. For vitamin B₁₂, higher homocysteine levels in subjects with low vitamin status were shown only in the C/C genotype group.

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

Full table

We analyzed the relation of serum homocysteine to serum B vitamins concentrations for each MTHFR genotype. As listed Table 5, serum homocysteine levels were significantly related to serum folate concentrations in all MTHFR genotypes. Explanatory power of B vitamin status as predictors of serum homocysteine levels was more pronounced in the T/T genotypes (68.5%, P=0.289) compared with the C/T (37.9%, P=0.002) or C/C genotypes (20.6%, P=0.083).

Table 5 - Coefficients from multiple regression analysis between serum homocysteine and B vitamins concentrations in subjects according to their MTHFR genotype.

Full table

Discussion

In our study, serum homocysteine concentrations were found significantly higher in pregnant women with the T/T genotype, as compared to those in the C/T or C/C genotype, confirming the results of other investigators (Jacques et al, 1996; Brattstrom et al, 1998; Gudnason et al, 1998). Serum homocysteine was negatively correlated with serum folate in our study subjects of all the three MTHFR genotypes, and the correlation between the two serum levels was strongest as had been previously reported by Ueland et al (2001) and Guttormsen et al (1996), who had observed a steeper negative slope in plots of serum folate vs serum homocysteine concentration in T/T individuals compared to those in the C/T or C/C subjects.

The results of our study suggest that pregnant women with a combination of decreased serum folate concentrations and the T/T genotype of the MTHFR gene could be at an increased risk of developing hyperhomocysteinemia, confirming the previous findings by Powers et al (1999). In our study, pregnant women who were homozygotes for the MTHFR mutation had higher fasting homocysteine levels only when their serum folate levels were below the median, but not when serum folate levels were above the median. This confirms the study of Brattstrom et al (1998) and Bailey and Gregory III (1999), who reported that the association between the MTHFR genotype and homocysteine levels was modified by serum folate status. One explanation for these observations could be, as hypothesized by Jacques et al (1996), that a higher folate status may increase the in vivo stability of the MTHFR enzyme, thus reducing the difference in enzyme activity between the T/T and C/T or C/C genotype. Another explanation for this could be that folate protected this enzyme against flavin loss and inactivation as suggested by Guenther et al (1999).

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 cofactor for MTHFR. An earlier animal study showed a significant association between vitamin B₂ and MTHFR that the enzyme activity and the relative amounts of 5-methyl THF were reduced in the liver of riboflavin-deficient rats (Bates & Fuller, 1986). Hustad et al (2000), McNulty et al (2002), and Jacques et al (2002) demonstrated that plasma vitamin B₂ was an independent determinant of plasma homocysteine, and this inverse dose–response relation was essentially confined to subjects with the C/T or T/T genotype of the MTHFR C677T polymorphism where the requirement of FAD for maximal catalytic activity might be increased (Hustad et al, 2000; Ueland et al, 2001). Several investigators have suggested that vitamin B₆ deficiency may lead to hyperhomocysteinemia, which is an established risk factor for adverse pregnancy outcome (van den Berg et al, 1994; Leeda et al, 1998). However, the present study failed to show the effects of serum vitamin B₆, PLP on serum homocysteine.

Our results showed that although serum vitamin B₁₂ concentrations were similar among all three genotype groups, significantly higher serum homocysteine levels were present in pregnant women whose vitamin B₁₂ levels were below the median compared to those with vitamin levels above the median only in the C/C genotype. Guttormsen et al (1996) made an observation that all hyperhomocysteinemic subjects with the C/C genotype had low plasma vitamin B₁₂ concentrations, and that vitamin B₁₂ supplementation alone had reduced their plasma homocysteine concentrations. Nakamura et al (2002) explained the reason for the above observation as follows: subjects with the C/T or T/T genotype have lower folate concentrations than those with the C/C genotype because of a reduced production of 5-methyl THF, a product of MTHFR. Low 5-methyl THF concentrations in the cytosol might conceal a deficiency of vitamin B₁₂ in patients with the C/T or T/T. Lucock et al (2001) suggested that a secondary gene–nutrient interaction between C677T-MTHFR and vitamin B₁₂ could modulate folate and homocysteine metabolism in an adverse way under critical conditions such as poor nutrient intake or high nutrient demand such as in pregnancy.

Our data show that the serum homocysteine concentrations in pregnant women are determined by an interaction between the MTHFR genotype and serum B vitamin status, and serum B vitamins (vitamin B₂, vitamin B₆, folate, and vitamin B₁₂) predicted 68.5% of serum homocysteine levels in the T/T genotype, 37.9% in the C/T genotype, and 20.6% in the C/C genotypes.

Here, we have only looked into the interaction between vitamin B status and the C677T polymorphism in the MTHFR gene and their effects on serum homocysteine levels. Another mutation, named A1298C mutation, that commonly occurs as frequently as C677T in the same gene when present alone has been shown not to cause elevations in plasma homocysteine. However, the combined heterozygosity for both C677T and A1298C 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 mutations such as G1793A mutation in the MTHFR gene (Rady et al, 2002), D919G mutation in methionine synthase gene (Chen et al, 2001), A66G mutation in methionine synthase reductase gene (Gaughan et al, 2001), G80A mutation in the RFC-1 folate transport protein gene might have played a role in the interaction of vitamin B status and homocysteine levels in our study subjects (Chango et al, 2000).

It has been reported that elevated maternal serum homocysteine concentrations may be linked to many adverse pregnancy outcomes including early pregnant loss (Ray & Lastin, 1999), placental infarcts and abruptions (de Falco et al, 2000), and fetal growth restriction (Leeda et al, 1998). Our data suggest that, if a low homocysteine concentration proves critical to preventing pregnancy complications, and if lowering maternal blood homocysteine is the key to the prevention, securing the optimum B vitamin status will become an important issue in pregnant women with the T/T genotype of the MTHFR gene. Maintaining serum vitamin B levels high enough is also critical to pregnant women with the C/T genotype, considering the fact that homocysteine concentration differs in the C/C and C/T groups depending on the level of folate and other B vitamins, although not as greatly as in the case of the T/T. Moreover, the gene frequency of the C/T type is high (49.2%), and although the homocysteine concentration in the C/T type is lower than that in the T/T but still greater than C/C, even a small increase of homocysteine may still lead to pregnancy complications. Ozcan et al (2002) reported that the incidence of poor pregnancy outcomes is similar in both types of women who are homozygous and heterozygous for MTHFR.

In conclusion, we have presented and described the findings of our work suggesting the strong evidence and effect of the interaction between the C677T MTHFR polymorphism and serum B vitamins on the homocysteine concentration in normal pregnant women. We have found that serum homocysteine concentrations varied significantly with the MTHFR genotype and serum B vitamin levels. Higher folate concentrations might have an attenuating effect on serum homocysteine in mid-pregnancy, and high serum levels of folate and vitamin B₂ can also lessen the MTHFR genotypic effect on serum homocysteine. Our study shows that there exists an interaction between MTHFR genotype genetics and vitamin B nutrition in homocysteine metabolism in normal pregnancy. This study supports the suggestion that the folate and other B vitamin deficiencies are more detrimental in pregnant women with the T/T homozygous, and may also be harmful for C/T heterozygous genotype as well in preventing the potential risks of adverse pregnancy outcomes associated with hyperhomocysteinemia.

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