点击显示 收起
1 From the Food Science and Human Nutrition Department (KMvC-D, GPAK, KPS, JDV, ERG, DRM, and LBB) and the General Clinical Research Center (DWT), University of Florida, Gainesville, FL
2 Supported by Florida Agricultural Experiment Station, USDA-NRI grant #00-35009102, GCRC RR00082, and approved for publication as Journal Series no. R-10350. 3 Address reprint requests to LB Bailey, Food Science and Human Nutrition Department, University of Florida, PO Box 110370, Gainesville, FL 32611. E-mail: lbbailey{at}mail.ifas.ufl.edu.
ABSTRACT
Background: A common genetic polymorphism [transcobalamin (TC) 776CG] may affect the function of transcobalamin, the protein required for vitamin B-12 cellular uptake and metabolism. Remethylation of homocysteine is dependent on the production of 5-methyltetrahydrofolate and adequate vitamin B-12 for the methionine synthase reaction.
Objectives: The objectives were to assess the influence of the TC 776C G polymorphism on concentrations of the transcobalamin-vitamin B-12 complex (holo-TC) and to determine the combined effects of the TC 776CG and methylenetetrahydrofolate reductase (MTHFR) 677CT polymorphisms and vitamin B-12 status on homocysteine concentrations.
Design: Healthy, nonpregnant women (n = 359; aged 20–30 y) were screened to determine plasma vitamin B-12, serum holo-TC, and plasma homocysteine concentrations and TC 776CG and MTHFR 677CT genotypes.
Results: The serum holo-TC concentration for women with the variant TC 776 GG genotype was significantly different (P = 0.0213) from that for subjects with the CC genotype (74 ± 37 and 87 ± 33 pmol/L, respectively). An inverse relation was observed between plasma homocysteine concentrations and both serum holo-TC (P 0.0001) and plasma vitamin B-12 (P 0.0001) concentrations, regardless of genotype.
Conclusions: These data suggest that the TC 776CG polymorphism negatively affects the serum holo-TC concentration and provide additional evidence that vitamin B-12 status modulates the homocysteine concentration in this population.
Key Words: Holotranscobalamin • transcobalamin • polymorphisms • homocysteine • vitamin B-12
INTRODUCTION
Vitamin B-12 is an essential nutrient that functions as a coenzyme for 2 metabolic processes, the conversion of methylmalonyl-CoA to succinyl-CoA and the remethylation of homocysteine to methionine (1). Vitamin B-12, in the form of methylcobalamin, serves as a coenzyme for methionine synthase, acting as a carrier for the methyl group donated by 5-methyltetrahydrofolate. Vitamin B-12 deficiency can impair this remethylation process and result in an elevated homocysteine concentration, which is linked to several clinical abnormalities, including vascular disease and abnormal pregnancy outcomes (2-7).
In circulation, vitamin B-12 is bound to 2 plasma binding proteins: haptocorrin or transcobalamin (8). Transcobalamin, the transport protein required for cellular uptake of vitamin B-12, constitutes only 20% of bound plasma vitamin B-12. In contrast, haptocorrin binds 80% of plasma vitamin B-12 but does not facilitate cellular uptake (8, 9). Specific membrane receptors recognize the protein portion of the transcobalamin-vitamin B-12 complex (holo-TC), whereas free vitamin B-12 or haptocorrin-bound vitamin B-12 (holo-HC) is not taken up by the cell (9, 10).
Several base pair substitutions may occur in the gene that encodes the transcobalamin protein, and investigators have reported that these may affect protein function (11-13). The most common polymorphism is a cytosine-to-guanine transition at base pair 776 (TC 776CG) that results in the replacement of proline with arginine. Homozygosity for the variant allele (GG) is estimated to exist in 20% of the population, with a prevalence of 30% and 50% for the CC and CG genotypes, respectively (13, 14). Previous studies suggest that the TC 776CG polymorphism may affect transcobalamin binding affinity for vitamin B-12 and the ability to transport vitamin B-12 into tissues (12, 14, 15). Afman et al (12) reported lower holo-TC and holo-TC:total transcobalamin ratios in individuals with either the TC 776 GG or CG genotype than in those with the TC 776 CC genotype. Miller et al (14) observed a reduced mean holo-TC concentration, a lower percentage of total vitamin B-12 bound to transcobalamin, and a higher methylmalonic acid (MMA) concentration in individuals with the TC 776 GG genotype than in those with the TC 776 CC genotype. These results indicate that the TC 776CG polymorphism may alter the cellular availability of vitamin B-12 and exacerbate the effects of low vitamin B-12 status.
Homocysteine remethylation could be impaired by reduced production of 5-methyltetrahydrofolate due to allelic variants of the methylenetetrahydrofolate reductase (MTHFR) 677CT polymorphism or by limited availability of vitamin B-12 caused by decreased cellular uptake or inadequate dietary intake (14, 16-18). The objectives of the present study were to assess the influence of the TC 776CG polymorphism on the serum holo-TC concentration and to determine the combined effects of both the TC 776CG and MTHFR 677CT homozygous variants (GG and TT, respectively) and vitamin B-12 status on plasma homocysteine concentrations in young women.
SUBJECTS AND METHODS
Subjects and protocol
Subjects were healthy, nonpregnant women (n = 359, aged 20–30 y) screened for possible participation in a controlled metabolic study previously reported by our research group (19). For some indexes, a sample was not available for all subjects; thus, the total subject number for some analyses is <359 and is reported accordingly. Subjects were interviewed before the laboratory screening to determine their eligibility on the basis of the following exclusion criteria: chronic use of tobacco or alcohol products, use of any prescription medications, history of chronic disease or major surgery, and consumption of vegetarian diets. The ethnic distribution of the subjects was 2% Asian, 9% African American, 81% white, 4% Hispanic, and 4% other. The University of Florida Institutional Review Board approved the study, and written informed consent was obtained from all subjects.
Specimen collection and analytic methods
Blood samples were drawn in EDTA-coated tubes and serum separator clot activator tubes. EDTA-coated tubes were kept on ice, and serum tubes were kept at room temperature until processing. EDTA-coated tubes were centrifuged at 2000 x g at 4 °C for 30 min to obtain buffy coat for genotype determinations and plasma for vitamin B-12 and homocysteine analyses. Serum tubes were centrifuged at 650 x g at room temperature for 15 min to obtain serum for holo-TC measurement. Samples were stored at –30 °C until analyzed. Genomic DNA was extracted from the buffy coat layer with the use of a commercial kit (Aquapure; Bio-Rad laboratories, Hercules, CA).
Serum holo-TC was measured by radioimmunoassay (holoTC RIA reagent kit; Axis Sheild, Ulvenveien, Oslo, Norway) based on the method of Ulleland et al (20). Briefly, serum samples were diluted with buffer, and magnetic microspheres coated with monoclonal antibodies to transcobalamin were added. After incubation, the holo-TC bound to microspheres was magnetically separated and washed with buffer. The holo-TC concentration was determined by radioimmunoassay standardized with recombinant human holo-TC, with immobilized intrinsic factor as a binder and 57Co-labeled vitamin B-12 as a tracer. Plasma vitamin B-12 was measured by radioimmunoassay with a commercially available kit (Quantaphase II; Bio-Rad). Plasma homocysteine concentrations were measured by using a modification of the HPLC method reported by Pfeiffer et al (21).
MTHFR 677CT genotype was determined by polymerase chain reaction followed by restriction enzyme analysis with Hinf1 as previously reported (19, 22, 23). Genotypes for the TC 776CG polymorphism were determined by using dynamic allele-specific hybridization methods (24). Briefly, genomic DNA polymorphic sequences were amplified by polymerase chain reaction with one biotinylated primer. The product was bound to streptavidin-coated plates, and the unbound strand was removed with alkali. A 3-carboxy-X-rhodamine (ROX)-labeled probe specific for the G allele was hybridized to the bound DNA at low temperature, thus forming a probe-target duplex. The temperature was increased while both denaturation temperature and diminution of ROX fluorescence via resonant energy transfer from the added double-strand-specific intercalating dye SYBR green (Molecular Probes, Eugene, OR) were recorded. For analysis, the negative derivatives of the melting curves were plotted to show a lower temperature peak for a homozygous allelic mismatch, a higher temperature peak for a homozygous match, or both peaks for a heterozygous sample. Genotype determinations were confirmed for 16 randomly selected samples by using a probe for the alternate C allele. Primers and probes (Table 1) were designed for the TC 776CG polymorphism (National Center for Biotechnology Information Single Nucleotide Polymorphism cluster ID rs1801198), and analyses were performed with dFOLD and DYNASCORE v. 0.65 software, respectively (DynaMetrix Limited, Stockholm, Sweden; Internet: http://www.dynametrix-ltd.com).
View this table:
TABLE 1. Primer and probe sequences for the transcobalamin 776CG polymorphism1
Normal values for indexes of vitamin B-12 status assessment
Serum holo-TC concentrations <35 pmol/L were considered low (25). Plasma vitamin B-12 concentrations between 148 and 221 pmol/L were considered marginally deficient (low-normal), and values <148 pmol/L were considered deficient (26).
Statistical methods
Initial descriptive statistics for serum holo-TC, plasma vitamin B-12, and plasma homocysteine concentrations were calculated and expressed as means and SDs unless otherwise noted. Logarithmic transformation was applied to correct for skewed distribution of homocysteine data. Homocysteine data are reported as antilogs. Multiple comparisons were made only if an overall F test was significant. Analysis of variance (ANOVA) was used to test for differences in mean serum holo-TC, plasma vitamin B-12, and plasma homocysteine concentrations by genotype (TC 776CG and MTHFR 677CT) and by genotype combinations (all 9 TC 776CG and MTHFR 677CT combinations). To ensure an overall type I error rate of 0.05, Bonferroni correction was applied to all pairwise comparisons when evaluating >2 group means. One-way ANOVA was used to detect differences in mean serum holo-TC, plasma homocysteine, and plasma vitamin B-12 concentrations by supplementation status groups (yes or no). For all comparisons, the alpha level was set a priori to 0.05. All statistics were completed by using SAS 8.00 (SAS Institute Inc, Cary, NC).
RESULTS
About 50% of subjects reported supplement use before the study, and the number of subjects taking vitamins did not differ among genotype groups (P > 0.05). Subjects who were supplement users before the study had significantly (P = 0.0299) higher mean serum holo-TC concentrations and significantly (P = 0.0016) higher mean plasma vitamin B-12 concentrations than did nonusers (76 ± 28 compared with 85 ± 30 pmol/L and 349 ± 106 compared with 303 ± 100 pmol/L, respectively).
Overall serum holo-TC, plasma vitamin B-12, and plasma homocysteine concentrations were 81 ± 38, 328 ± 140, and 6.3 ± 1.3 µmol/L, respectively. A small percentage of subjects had values below normal for either serum holo-TC (<35 pmol/L; 7%) or plasma vitamin B-12 (<148 pmol/L; 2%) (25, 27). One subject whose genotype was TC 776 CG/MTHFR 677 TT had an elevated homocysteine concentration (>14 µmol/L), a low vitamin B-12 concentration (155 pmol/L), and a serum folate concentration that may be considered borderline (19 nmol/L) (28). Subjects with low (<35 pmol/L) serum holo-TC concentrations also had significantly higher mean plasma homocysteine concentrations than did subjects with normal (35 pmol/L) serum holo-TC concentrations (7.2 ± 1.1 compared with 6.3 ± 1.0 µmol/L, respectively; Figure 1). A significant inverse relation was observed between plasma homocysteine concentration and both serum holo-TC and plasma vitamin B-12 concentrations (Figure 2), and a direct relation was found between serum holo-TC and plasma vitamin B-12 concentration (data not shown).
FIGURE 1.. Mean (±SD) plasma homocysteine concentrations in women with serum holotranscobalamin (transcobalamin-vitamin B-12 complex) concentrations <35 pmol/L (n = 25) or 35 pmol/L (n = 319). *Significantly different from women with concentrations 35 pmol/L, P < 0.05.
FIGURE 2.. Relation between plasma homocysteine and serum holotranscobalamin (transcobalamin-vitamin B-12 complex) concentrations (r = –0.24, P 0.0001) and between plasma homocysteine and plasma vitamin B-12 concentrations (r = –0.23, P 0.0001) in all women (n = 344). The vertical lines indicate the cutoffs for normal values for serum holotranscobalamin (35 pmol/L) and plasma vitamin B-12 (148 pmol/L).
As shown in Table 2, genotype frequencies were as expected on the basis of data from previous studies (13, 14, 19, 22, 29). Serum holo-TC, plasma vitamin B-12, and plasma homocysteine concentrations by TC 776CG genotype and by TC 776/MTHFR 677 combined genotypes are presented in Table 3 and Table 4, respectively. Subjects with the TC 776 GG genotype had a significantly lower mean serum holo-TC concentration than did those with the CC genotype (Table 3). No significant differences were detected in plasma vitamin B-12 or plasma homocysteine concentrations among TC 776CG genotype groups. Folate data were reported previously (30).
View this table:
TABLE 2. Percentage of transcobalamin (TC) 776CG and methylenetetrahydrofolate reductase (MTHFR) 677CT genotypes among young women1
View this table:
TABLE 3. Vitamin B-12 status indexes according to transcobalamin (TC) 776CG genotype1
View this table:
TABLE 4. Vitamin B-12 status indexes according to transcobalamin (TC) 776CG and methylenetetrahydrofolate reductase (MTHFR) 677CT genotypes1
DISCUSSION
The primary objectives of the present study were to assess the influence of the TC 776CG polymorphism on holo-TC concentrations and to determine the combined effects of the TC 776CG and MTHFR 677CT polymorphisms and vitamin B-12 status on homocysteine concentrations in young women. Women of reproductive age were of particular interest because of the previously reported association between low vitamin B-12 status and both the TC 776CG and MTHFR 677CT polymorphisms and an increased risk of birth defects (12, 31-33). The observation that small reductions in plasma vitamin B-12 concentrations within the normal range may be associated with an increased risk of birth defects illustrates the importance of assessing vitamin B-12 status in women of reproductive age (12, 31, 34, 35). The metabolic basis of vitamin B-12 deficiency and birth defects is unknown; however, vitamin B-12 is essential for normal one-carbon metabolism, including the synthesis of S-adenosylmethionine, which is required for DNA methylation and normal embryonic development (36, 37). Elevated homocysteine concentrations that may result from a vitamin B-12 deficiency have also been associated with an increased risk of neural tube defects (38, 39).
The assessment of vitamin B-12 status has traditionally been based on either plasma or serum vitamin B-12. The plasma vitamin B-12 concentration widely used to define deficient status is <148 pmol/L (27). However, 5–10% of persons with plasma vitamin B-12 concentrations between 148 and 221 pmol/L have been reported to have clear-cut hematologic abnormalities or neurologic damage due to vitamin B-12 deficiency that responds to supplementation (26). This observation led to the suggestion that plasma vitamin B-12 concentrations between 148 and 221 pmol/L be considered marginally deficient (low-normal) (26) and provided the rationale for our use of this criterion to define normal vitamin B-12 status in the present study.
Plasma homocysteine and serum MMA concentrations are considered functional indicators of vitamin B-12 status (40) and both are inversely related to the plasma vitamin B-12 concentration (1). Methylmalonic acid is a more specific functional indicator of vitamin B-12 status than is plasma homocysteine, because folate deficiency also leads to an increase in the plasma homocysteine concentration (41). Assessment of vitamin B-12 status on the basis of serum holo-TC, a relatively new vitamin B-12 status indicator, was one focus of the present study. Total plasma vitamin B-12 includes both holo-TC and holo-HC; however, only holo-TC can be taken up into the cell, and it therefore represents the biologically active portion (10, 42, 43). The holo-TC concentration was reported to be a more sensitive and earlier marker of changes in vitamin B-12 status than is the total plasma vitamin B-12 concentration (25, 44-48). Combined use of holo-TC and serum MMA may be most useful in determining vitamin B-12 status, particularly in marginally deficient persons. In clinical situations, however, caution must be exercised when interpreting low serum holo-TC or plasma vitamin B-12 in conjunction with normal homocysteine concentrations, as was observed here. On the basis of such criteria, the potential for false-positive determinations of vitamin B-12 deficiency to obscure other plausible bases for clinical abnormalities must be considered, especially when detected in low-risk populations such as the one in the present study.
The findings from the present study support those from previous investigations in which an inverse relation between serum holo-TC and homocysteine concentration was reported (25, 45, 48). Subjects with the TC 776 GG genotype had a significantly lower mean serum holo-TC concentration than did subjects with the TC 776 CC genotype, with no corresponding differences in plasma vitamin B-12 concentration. This suggests that less vitamin B-12 was available for cellular uptake and metabolism. Subjects with low serum holo-TC concentrations, regardless of genotype, had significantly higher mean plasma homocysteine concentrations than did those with normal serum holo-TC (P = 0.0118) concentrations. Because our population consisted of healthy young women, none of whom were vitamin B-12 or folate deficient, it is understandable that none had high homocysteine concentrations. Additionally, a genotype effect may be muted by the subjects' replete nutritional status. Our data do suggest that the TC 776CG polymorphism is associated with lower holo-TC concentrations, and that the holo-TC concentration is inversely related to the homocysteine concentration. In populations in whom vitamin B-12 status is more likely to be compromised, such as vegetarians and vegans (46, 49), there may be a more significant genotype effect. Further investigation of the effect of the TC 776CG polymorphism with a focus on populations with low vitamin B-12 intake is warranted.
Previously, our laboratory group extensively studied the association between the MTHFR 677CT polymorphism, plasma homocysteine concentrations, and nutrient status. It is recognized that the MTHFR 677CT polymorphism is associated with altered enzyme conformation and activity that may lead to reduced conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (41, 50). This polymorphism has been associated with increased homocysteine concentrations, most significantly when in conjunction with low folate status (16, 23). Jacques et al (51) observed that individuals with the MTHFR homozygous variant (TT) and plasma folate concentrations < 15.4 nmol/L had a mean fasting homocysteine concentration 24% higher (P 0.05) than that in individuals with the CC genotype, with no significant difference among individuals with a folate concentration 15.4 nmol/L. In the present study, we evaluated the potential association between MTHFR 677CT genotype, homocysteine concentrations, and vitamin B-12 status. Although no significant interaction between vitamin B-12 and MTHFR 677CT genotype was detected, a subset analysis was conducted to compare plasma homocysteine concentrations in subjects with the MTHFR 677 TT genotype stratified by vitamin B-12 status. When vitamin B-12 status was low (plasma vitamin B-12 < 148 pmol/L) in subjects with the MTHFR 677 TT genotype, plasma homocysteine concentrations were significantly higher than those observed in subjects with normal vitamin B-12 status (>148 pmol/L). In this subset analysis, there was no significant difference in folate status between the groups with either low or normal vitamin B-12 status. The small number of subjects in the MTHFR 677 TT subgroup (n = 14) may have resulted in insufficient power to detect an overall interaction between plasma vitamin B-12 and MTHFR 677CT genotype. The potential relation between vitamin B-12 status and plasma homocysteine concentration in individuals with the MTHFR 677 TT genotype, independent of folate status, is of interest and warrants further investigation in a larger population.
Increased homocysteine concentrations, associated with low vitamin B-12 status, have been linked to increased disease risk (3-7, 39, 52). Afman et al (12) measured the plasma concentrations of total vitamin B-12, homocysteine, and the apo- and holo- forms of transcobalamin in neural tube defect case and control mothers. Low vitamin B-12 concentrations were associated with a 3-fold increased risk of having a child with a neural tube defect, whereas low holo-TC was associated with a 5-fold increase in risk (12). The TC 776CG and MTHFR 677CT polymorphisms have also been associated with an increased risk of birth defects (12, 14, 31, 53). Future investigations are needed to determine whether functional vitamin B-12 deficiency will result when the TC 776CG polymorphism is coupled with inadequate dietary vitamin B-12 intake. In addition, the observation that individuals with the MTHFR 677 TT genotype and low vitamin B-12 status had significantly higher homocysteine concentrations warrants further study. These findings and those of previous investigations (12, 14, 31, 53) suggest that the risk of an abnormal pregnancy outcome may be exacerbated when the TC 776CG or MTHFR 677CT polymorphism coexists with low vitamin B-12 status. In the present study, none of the women consumed vegetarian diets, and 50% took multivitamin supplements, thereby reducing their risk of inadequate vitamin B-12 status. Studies designed to investigate the potential effect of these polymorphisms on holo-TC and MMA concentrations in young women with low vitamin B-12 status are needed.
ACKNOWLEDGMENTS
KMvC-D was responsible for the design of the experiment, the transcobalamin analysis, data analysis, and writing of the manuscript. GPAK supervised sample collection, supervised homocysteine and vitamin B-12 analysis, and edited the manuscript. KPS was responsible for sample collection and preparation, homocysteine and vitamin B-12 analysis, and editing of the manuscript. JDV was responsible for homocysteine analysis. ERG was responsible for vitamin B-12 analysis. DRM was the overall manager of laboratory operations and was responsible for sample collection and preparation, genotype determination, and editing of the manuscript. DWT was responsible for data coordination and statistical analysis. LBB was responsible for the design of the experiment, supervision of sample collection and analysis, and preparation and editing of the manuscript. None of the authors had any financial or personal conflicts of interest in any organization supporting this study.
REFERENCES