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Food Science and Human Nutrition Department, and * Department of Statistics, University of Florida, Gainesville, FL 32611
2To whom correspondence should be addressed. E-mail: LBBailey{at}mail.IFAS.UFL.EDU.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • vitamin B-12 • methylenetetrahydrofolate reductase • polymorphisms • homocysteine • folate • women
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). A common genetic variant of MTHFR occurs when cytosine is replaced by thymine at bp 677, which yields a replacement of alanine with valine in the enzyme (2
). Individuals homozygous for the C677T MTHFR variant have been reported to have decreased enzyme activity (
40% of control) (2
). A recent study using recombinant human MTHFR showed that the catalytic function and regulation of MTHFR are unaffected in the C677T variant (3
).
Yamada et al. (3
) observed that the C677T variant releases its FAD cofactor three times faster than the wild-type enzyme, and the stability of the MTHFR variant is significantly enhanced by the addition of 5-methyl-THF. This observation is consistent with previous studies with bacterial MTHFR mutant forms that showed that full activity of MTHFR is dependent on a complex folate-FAD-MTHFR interaction, that the C677T variant is prone to FAD loss, and that folate derivatives reduce the rate of FAD loss and help maintain MTHFR activity (4
). The reduced enzyme activity previously reported to be associated with the C677T MTHFR variant may relate to the stability of the FAD complex rather than the catalytic properties of the enzyme (2
,3
). These in vitro data support observations that homozygosity for the C677T MTHFR polymorphism is associated with mild hyperhomocysteinemia in humans with plasma folate concentrations below the median (5
,6
).
In contrast to folate, the effect of suboptimal vitamin B-12 status in conjunction with MTHFR polymorphisms has not been widely studied. Vitamin B-12 is essential for the conversion of homocysteine to methionine by methionine synthase, which transfers the 5-methyl group from 5-methyl-THF to vitamin B-12 and then to homocysteine (1
). D’Angelo et al. (7
) evaluated the influence of both folate and vitamin B-12 status on plasma homocysteine concentration in association with the C677T MTHFR polymorphism. These investigators observed a stronger negative correlation between plasma homocysteine and both plasma vitamin B-12 and serum folate in individuals homozygous for the MTHFR variant (677TT) compared with that of a combined group of individuals with either the heterozygous (677CT) or wild-type (677CC) genotype (7
). Both serum folate and vitamin B-12 were independent predictors of plasma homocysteine, and the interaction of both nutrients with the MTHFR genotype additionally contributed to predicting homocysteine concentrations. The mechanism by which changes in vitamin B-12 status may modulate plasma homocysteine concentration in individuals who are homozygous for the C677T MTHFR polymorphism is unknown.
The A1298C polymorphism of MTHFR yields a substitution of alanine for glutamate (8
,9
). The frequency of the homozygous A1298C (1298CC) MTHFR genotype is comparable to that of the 677TT genotype (10% of individuals) and 15–20% of the population is estimated to be doubly heterozygous (677CT/1298AC) (10
). Weisberg et al. (10
) recently used an in vitro expression system to confirm earlier reports (8
,9
) that both the C677T and A1298C MTHFR polymorphisms yield reduced enzyme activity, with the C677T having a more deleterious effect than the A1298C variant. Plasma homocysteine concentrations have been reported to be significantly higher in individuals heterozygous for both the C677T and the A1298C MTHFR polymorphisms compared with individuals heterozygous for only the C677T variant (8.0 vs. 8.9 µmol/L) (10
). It appears that the A1298C polymorphism alone does not significantly affect plasma homocysteine but may have a modest effect when present with the C677T variant (10
). Recently Yamada et al. (3
) evaluated the biochemical properties of recombinant human MTHFR and found that the 1298CC MTHFR variant had no effect on catalytic function or regulation regardless of whether the mutation occurred alone or in association with the 677TT MTHFR variant. Unlike the C677T MTHFR variant, the A1298C MTHFR enzyme appears thermostable and unaffected by changes in in vitro folate concentration (3
).
The C677T MTHFR polymorphism coupled with low folate status is associated with an increased risk for neural tube defects (NTD) (11
). Limited data suggest that the 1298AC MTHFR genotype may also be associated with an increased risk for NTD (8
); however, more definitive evidence is required (12
). The pathogenesis of folate-responsive birth defects is likely to be multifactorial and may involve multiple genetic polymorphisms that could alter normal folate metabolism or transport, especially when coupled with inadequate folate and/or vitamin B-12 status (13
). Both folate and vitamin B-12 play pivotal roles in DNA synthesis and production of methionine from homocysteine. Hypotheses regarding the mechanisms by which impaired folate and vitamin B-12 metabolism may increase the risk of NTD-affected pregnancies include neurotoxicity of homocysteine (14
). Alternatively, deficiencies in methionine synthesis and methylation capacity could affect neural development (15
).
There have been no investigations of the association between folate and vitamin B-12 status on plasma homocysteine in individuals on the basis of their A1298C and C677T MTHFR genotype. Considering the relatively high frequencies of these MTHFR polymorphisms and the possibility of moderately low vitamin B-12 status in the population (16
), it is important to determine the effect of vitamin B-12 status coupled with MTHFR polymorphisms in women of reproductive age. The primary objective of the present investigation was to characterize the effect of point mutations in MTHFR at bp 677 and/or 1298 on plasma homocysteine and the influence of both folate and vitamin B-12 status in women of reproductive age.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Women (20–30 y old; n = 186) were screened for possible inclusion in metabolic studies and met the following inclusion criteria: 1) no history of chronic disease or major surgery; 2) nonsmoker; 3) minimal alcohol consumption; 4) no chronic medication use, including oral contraceptives; and 5) < 120% of ideal body weight. The racial composition of the study population group was primarily Caucasian (92%), with smaller numbers of African American (3%), Asian (4%) or other; 4% of Caucasians reported their ethnicity as Hispanic. Information regarding vitamin supplement use was based on a "yes" or "no" response to the following question: "Do you take a multivitamin or other supplement?" The study was approved by the University of Florida Institutional Review Board and all subjects signed informed consent forms.
Specimen collections and analytical methods.
Fasting blood samples were collected and immediately processed and stored frozen before analysis. Folate concentration of blood specimens was determined using the Lactobacillus casei microbiological assay in a 96-well microplate system, adapted from Tamura (17
) and Horne and Patterson (18
). Plasma vitamin B-12 concentration was determined with a radioligand binding procedure (Bio-Rad Quantaphase II, Hercules, CA). A minor modification of the HPLC method reported by Pfeiffer et al. (19
) was used to determine plasma total homocysteine concentrations.
Determination of C677T and A1298C MTHFR genotypes.
Genomic DNA was extracted from freshly collected or EDTA blood stored at -20°C using a commercially available kit (Bio-Rad AquaPure, Hercules, CA). Genotype analyses for MTHFR C677T and A1298C mutations were performed using modifications of recently described polymerase chain reaction methods by Yi et al. (20
), including the use of the same primers. Briefly, template DNA was simultaneously amplified using improved intron primers for both polymorphic sites and then separately cleaved with HinfI and MboII. After heat inactivation of the enzymes, the restriction digests were pooled and analyzed by gel electrophoresis to identify each possible restriction fragment length polymorphism pattern characteristic of the mutations.
Statistical methods.
The primary regression analysis consisted of evaluating the mean homocysteine concentrations as a function of both the C677T and A1298C genotypes, supplement use, serum folate, RBC folate and vitamin B-12 concentrations. All first-order interactions in the regression model such as genotype-by-genotype interactions were also considered. In addition, mean serum folate and RBC folate and plasma vitamin B-12 concentrations were assessed as a function of the factors listed above (excluding serum folate, RBC folate and vitamin B-12 concentrations as factors). All analyses were carried out using analysis of covariance (ANCOVA) initially with a full model followed by reduction of the models in stepwise fashion. All factors not significant at 
= 0.10 were eliminated from the model. A correlation analysis was carried out examining Pearson correlations between plasma homocysteine, vitamin B-12, serum folate and RBC folate concentrations within genotype classifications.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All subjects were healthy on the basis of responses to an extensive medical history questionnaire and all met the inclusion criteria detailed above. Serum and RBC folate, plasma vitamin B-12 and homocysteine concentrations were within normal limits for all subjects (i.e., > 7 nmol/L, >317 nmol/L, >48 pmol/L and < 14 µmol/L, respectively).
Plasma homocysteine association with serum folate and plasma vitamin B-12.
Figure 1
is a three-dimensional surface graph illustrating the interaction between serum folate and plasma vitamin B-12 on plasma homocysteine, independent of genotype and supplement status. From the surface plot, a subject with low serum folate and plasma vitamin B-12 concentrations would be predicted to have a highly elevated plasma homocysteine concentration relative to all other serum folate and plasma vitamin B-12 combinations. The fold in the plot indicates that there may be a nonlinear relationship between plasma vitamin B-12 and homocysteine concentrations.
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Approximately one half (49%) of the subjects reported supplement use before blood screening, and the proportion of subjects who reported taking supplements was not different among genotype combinations (P > 0.05). No information was obtained regarding the duration, frequency of use or dose of nutrients in the supplements. Significantly higher (P < 0.05) serum folate, RBC folate and plasma vitamin B-12 concentrations were detected in individuals who reported supplement use (n = 92) than in those who did not (n = 93) independent of genotype (Table 1)
.
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The distribution of the 6 genotype combinations found in our population group is shown in Table 2
. Approximately 12% of our subjects were homozygous for either the C677T (677TT) or the A1298C (1298CC) MTHFR polymorphism, whereas 17% were doubly heterozygous (677CT/1298AC). No significant differences (P > 0.05) were detected among all 6 genotype combinations for serum folate, RBC folate or plasma vitamin B-12 concentrations (Table 3)
. The plasma homocysteine concentration was higher (P < 0.05) for individuals who were homozygous for the C677T polymorphism (677TT) relative to the other 5 genotype combinations (Table 3)
.
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Table 4
contains the estimated regression coefficients and their corresponding standard errors and P-values obtained from the model by regressing plasma homocysteine concentration on plasma vitamin B-12, serum folate, vitamin B-12 by C677T genotype interaction and vitamin B-12 by A1298C interaction. The levels of the categorical genotype variable were coded as indicator variables with yes = 1 and no = 0. The homozygous genotype level was arbitrarily set as the reference group. The fitted lines from this model are illustrated in Figures 2
and 3
. Significant differences were detected in predicted mean plasma homocysteine as a function of the C677T (P = 0.017) and A1298C (P = 0.024) polymorphisms. Plasma homocysteine concentration was negatively associated with vitamin B-12 (P = 0.015) and serum folate (P = 0.049) concentrations, with the degree of correlation between plasma vitamin B-12 and homocysteine concentrations dependent on genotype (Figs. 2
and 3)
. The predicted change in plasma homocysteine concentration in relation to serum folate and plasma vitamin B-12 concentrations for the C677T and the A1298C genotypes in general decreased in association with increased serum folate and vitamin B-12 concentrations as illustrated in Figures 2
and 3
, respectively. All genotypes were associated with a clear inverse response between serum folate and plasma homocysteine, but subjects homozygous (TT) for the C677T MTHFR variant exhibited the highest predicted homocysteine across serum folate concentrations (Fig. 2)
.
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The association between plasma vitamin B-12 concentration and predicted plasma homocysteine concentration was significant (P = 0.0005) in the subjects that were doubly heterozygous (677CT/1298AC) for the MTHFR polymorphisms (Fig. 3)
. Similar but nonsignificant trends were observed in the homozygous/wild (677TT/1298AA; P = 0.128) and wild/homozygous (677CC/1298CC; P = 0.114) groups (Fig. 3)
. In contrast, within the wild/wild (677CC/1298AA) group, plasma homocysteine concentration was not predicted to change significantly throughout the range of vitamin B-12 concentrations (Fig. 3)
. Compared with the wild/wild (677CC/1298AA) MTHFR genotype group, the predicted homocysteine concentration was somewhat higher in the remaining 5 genotype combinations when the plasma vitamin B-12 concentration was below the median (345 pmol/L). At the higher plasma vitamin B-12 concentrations, the predicted plasma homocysteine concentrations were lowest for individuals who were heterozygous for both polymorphisms and those with the wild/homozygous (677CC/1298CC) variants compared with all other genotypes (Fig. 3)
. Throughout the range of vitamin B-12 concentrations, predicted homocysteine was higher in individuals with the homozygous/wild (677TT/1298AA) genotype relative to the other groups with the exception of the wild/wild (677CC/1298AA) genotype group.
Correlation between plasma homocysteine, serum folate and vitamin B-12 concentrations.
On the basis of Pearson’s correlation analysis, a significant negative association was found between serum folate and plasma homocysteine in the doubly heterozygous (677CT/1298AC) subjects (r = -0.41, P = 0.02). Vitamin B-12 status was also significantly associated with plasma homocysteine on the basis of Pearson’s correlation analysis, which indicated a strong inverse correlation in subjects heterozygous for both MTHFR polymorphisms (677CT/1298AC) (r = -0.59, P < 0.0004). In those subjects who were homozygous/wild for the C677T polymorphism (677TT/1298AA), an inverse association of borderline significance was identified between plasma vitamin B-12 and plasma homocysteine (r = -0.42, P = 0.06).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) identified a similar inverse relationship between plasma homocysteine and plasma vitamin B-12 concentrations in individuals with C677T variants (677TT or 677CT); however, these investigators did not screen for the A1298C polymorphism. In the present study, the predicted mean homocysteine concentration decreased at varying rates as a function of vitamin B-12 concentration for all genotype combinations, with the exception of the wild/wild genotype. The largest rate of decrease in plasma homocysteine concentration as a function of vitamin B-12 was found in the heterozygous/heterozygous group followed by the wild/homozygous group. These data suggest that enhancing vitamin B-12 status may significantly modulate plasma homocysteine concentrations in individuals doubly heterozygous or homozygous for either the C677T or A1298C MTHFR polymorphism, even when vitamin B-12 concentrations are within the normal range.
For serum folate, regression analysis indicated a genotype-independent response of predicted plasma homocysteine as serum folate concentration increased. Subjects who were homozygous for the C677T mutation had a significantly higher plasma homocysteine relative to the other genotypes, which is in accordance with previous reports (21
). Jacques et al. (5
) reported that the effect of the C677T MTHFR polymorphism on homocysteine is significant only when folate status is inadequate. In the present study, it was surprising to observe a small but significant difference in plasma homocysteine concentration between individuals with the homozygous 677TT genotype relative to the other 5 genotypes because folate status was normal in all subjects.
The rationale in the present study for evaluating the effect of MTHFR genotypes on homocysteine in women of reproductive age was the reported associations between these common genetic polymorphisms and elevations in homocysteine with increased risk for birth defects (13
,11
). However, it is unclear whether small differences in homocysteine concentrations within the normal range, such as those observed in this study, have any relationship to health risk. The potential for inadequate folate status to negatively affect homocysteine in individuals with MTHFR polymorphisms in countries in which folic acid fortification is not mandated is a more valid concern. Several reports, including the data from this study, indicate that folate status has been significantly enhanced in the U.S. population since 1997 when folic acid fortification was initiated (22
–24
). It appears that the chronic long-term effect of folic acid fortification may yield even higher blood folate concentrations than those observed immediately postfortification (23
–25
). Data from the 1999 National Health and Nutrition Examination Survey, based on analysis with a radiobinding assay, indicated that mean serum folate concentrations for all women aged 15–44 y increased from 14.2 nmol/L prefortification to 36.6 nmol/L shortly after the initiation of fortification (25
). Caudill et al. (22
) reported serum folate using a microbiological assay and homocysteine concentrations to be 50 ± 19.9 nmol/L and 5.5 ± 1.7 µmol/L, respectively, in young women who reported no supplement use. In contrast is the fact that serum folate and homocysteine concentrations are significantly different in European countries relative to those currently being reported in the United States. For example, in a French population group, mean serum folate and plasma homocysteine were 15.6 ± 8.6 nmol/L and 9.3 ± 3.3 µmol/L, respectively (26
). In an Italian study, D’Angelo et al. (7
) reported mean serum folate and plasma homocysteine concentrations of women to be 13.1 ± 0.23 nmol/L and 12.3 ± 8.2 µmol/L, respectively. The mean serum folate concentrations recently reported in European countries are similar to those reported in the United States during the prefortification period (14 nmol/L) (25
).
The significant inverse relationship between vitamin B-12 status and plasma homocysteine in young women with the C677T and/or A1298C MTHFR polymorphism was unexpected because plasma vitamin B-12 concentrations were well within the "normal range" (>148 pmol/L) (16
). The mechanism by which changes in vitamin B-12 status modulate homocysteine in individuals with MTHFR polymorphisms is unknown. Because vitamin B-12 is not a required coenzyme for the MTHFR reaction, we hypothesize that the MTHFR polymorphisms may coexist with other common polymorphisms that are vitamin B-12 dependent such as the recently identified methionine synthase reductase (MTRR) mutation. The homozygous A66G MTRR variant is estimated to affect 25% of the population and is associated with significant elevations in homocysteine when vitamin B-12 status is less than optimal (15
,27
). Additional screening of genomic DNA samples from subjects in the current study for the coexistence of the vitamin B-12–dependent A66G MTRR variant will clarify this issue. National survey data suggest that impaired vitamin B-12 status is often undiagnosed and may affect a large proportion of the U.S. population (16
). Future investigations appear warranted to investigate the association between vitamin B-12 status and homocysteine response in individuals with common folate and vitaminB-12–related genetic polymorphisms.
The birth defect risk associated with the C677T MTHFR polymorphism is modulated by folate status (28
). Findings from recent investigations suggest that multiple folate and vitamin B-12–responsive genetic polymorphisms may coexist, and the effect of these polymorphisms on birth defect risk may be significantly influenced by changes in folate or vitamin B-12 status. For example, Wilson et al. (15
) reported data that support the hypothesis that the risk for NTD increased significantly when the C677T MTHFR homozygous polymorphism was combined with a second mutation affecting the MTRR enzyme and vitamin B-12 status. Wilson et al. (15
) also found that the vitamin B-12 concentrations were significantly lower (298 pmol/L) in women who had previous NTD-affected pregnancies (case mothers) compared with controls (352 pmol/L). Our research group (29
) also evaluated the vitamin B-12 status in NTD case mothers compared with controls and found that vitamin B-12 concentrations were significantly lower (261 pmol/L vs. 380 pmol/L) in case mothers. These data suggest that even small differences in vitamin B-12 status within the normal range can have a significant effect on NTD risk. The observations by our research group related to the effect of vitamin B-12 status on plasma homocysteine concentrations in young women with the combined MTHFR polymorphisms, coupled with the data from D’Angelo et al. (7
), suggest that the vitamin B-12 requirement may be higher in individuals with certain polymorphisms affecting folate and vitamin B-12–dependent pathways.
In summary, these data suggest that enhancing vitamin B-12 status may significantly influence plasma homocysteine in young women with C677T and/or A1298C MTHFR polymorphisms, even when vitamin B-12 concentrations are within the normal range.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: 5,10-methylene-THF, 5,10-methylenetetrahydrofolate; 5-methyl-THF, 5-methyltetrahydrofolate; MTHFR, methylenetetrahydrofolate reductase; MTRR, methionine synthase reductase; NTD, neural tube defects.
Manuscript received 25 January 2002. Initial review completed 13 February 2002. Revision accepted 8 April 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Bailey, L. B., Moyers, S. & Gregory, J. F., III. (2001) Folate. Bowman, B. A. Russell, R. eds. Present Knowledge in Nutrition 8th ed. 2001 ILSI Press Washington, DC. .
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4. Guenther, B. D., Sheppard, C. A., Tran, P., Rozen, R., Matthews, R. G. & Ludwig, M. L. (1999) The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia. Nat. Struct. Biol. 6:359-365.[Medline]
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12. Scott, J. M. (2001) Genetic diversity and disease: opportunities and challenge. Proc. Natl. Acad. Sci. USA 98:14754-14756.
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15. Wilson, A., Platt, R., Wu, Q., Leclerc, D., Christensen, B., Yang, H., Gravel, R. A. & Rozen, R. (1999) A common variant in methionine synthase reductase combined with low cobalamin (vitamin B-12) increases risk for spina bifida. Mol. Genet. Metab. 67:317-323.[Medline]
16. Institute of Medicine (1998) Folate. Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B-6: Folate, Vitamin B-12, Pantothenic Acid, Biotin, and Choline 1998:196-305 IOM, National Academy Press Washington, DC. .
17. Tamura, T. (1990) Microbiological assay of folate. Picciano, M. F. Stokstad, E. R. Gregory, J. F. eds. Folic Acid Metabolism in Health and Disease 1990:121-137 Wiley-Liss New York. .
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22. Caudill, M. A., Le, T., Moonie, S. A., Esfahani, S. T. & Cogger, E. A. (2001) Folate status in women of childbearing age residing in Southern California after folic acid fortification. J. Am. Coll. Nutr. 20(Suppl.):129-134.
23. Jacques, P. F., Selhub, J., Bostom, A. G., Wilson, P. W. & Rosenberg, I. H. (1999) The effect of folic acid fortification on plasma folate and total homocysteine concentrations. N. Engl. J. Med. 340:1449-1454.
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27. Leclerc, D., Wilson, A., Dumas, R., Gafuik, C., Song, D., Watkins, D., Heng, H.H.Q., Rommens, J. M., Scherer, S. W., Rosenblatt, D. S. & Gravel, R. A. (1998) Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in persons with homocystinuria. Proc. Nat. Acad. Sci. USA 95:3059-3064.
28. Molloy, A. M., Daly, S., Mills, J. L., Kirke, P. N., Whitehead, A. S., Ramsbottom, D., Conley, M. R. & Weir, D. G. (1997) Thermolabile variant of 5,10-methylenetetrahydrofolate reductase associated with low red-cell folates: implications for folate intake recommendations. Lancet 349:1591-1593.[Medline]
29. Boddie, A. M., Dedlow, E. R., Nackashi, J. A., Opalko, F. J., Kauwell, G. P. A., Gregory, J. F., III. & Bailey, L. B. (2000) Folate absorption in women with a history of neural tube defect-affected pregnancy. Am. J. Clin. Nutr. 72:154-158.
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 C. Solis, K. Veenema, A. A. Ivanov, S. Tran, R. Li, W. Wang, D. J. Moriarty, C. V. Maletz, and M. A. Caudill Folate Intake at RDA Levels Is Inadequate for Mexican American Men with the Methylenetetrahydrofolate Reductase 677TT Genotype J. Nutr., January 1, 2008; 138(1): 67 - 72. [Abstract] [Full Text] [PDF]
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B. Beagle, T. L. Yang, J. Hung, E. A. Cogger, D. J. Moriarty, and M. A. Caudill The Glycine N-Methyltransferase (GNMT) 1289 C->T Variant Influences Plasma Total Homocysteine Concentrations in Young Women after Restricting Folate Intake J. Nutr., December 1, 2005; 135(12): 2780 - 2785. [Abstract] [Full Text] [PDF]
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K. M von Castel-Dunwoody, G. P. Kauwell, K. P Shelnutt, J. D Vaughn, E. R Griffin, D. R Maneval, D. W Theriaque, and L. B Bailey Transcobalamin 776C->G polymorphism negatively affects vitamin B-12 metabolism Am. J. Clinical Nutrition, June 1, 2005; 81(6): 1436 - 1441. [Abstract] [Full Text] [PDF]
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J. D. Vaughn, L. B. Bailey, K. P. Shelnutt, K. M. v.-C. Dunwoody, D. R. Maneval, S. R. Davis, E. P. Quinlivan, J. F. Gregory III, D. W. Theriaque, and G. P. A. Kauwell Methionine Synthase Reductase 66A->G Polymorphism Is Associated with Increased Plasma Homocysteine Concentration When Combined with the Homozygous Methylenetetrahydrofolate Reductase 677C->T Variant J. Nutr., November 1, 2004; 134(11): 2985 - 2990. [Abstract] [Full Text] [PDF]
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 J. L. Mills, C. C. Signore, E. P. Quinlivan, J. F. Gregory III, A. E. Czeizel, and N. J. Wald Folic Acid and the Prevention of Neural-Tube Defects N. Engl. J. Med., May 20, 2004; 350(21): 2209 - 2211. [Full Text] [PDF]
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C. L. Guinotte, M. G. Burns, J. A. Axume, H. Hata, T. F. Urrutia, A. Alamilla, D. McCabe, A. Singgih, E. A. Cogger, and M. A. Caudill Methylenetetrahydrofolate Reductase 677C->T Variant Modulates Folate Status Response to Controlled Folate Intakes in Young Women J. Nutr., May 1, 2003; 133(5): 1272 - 1280. [Abstract] [Full Text] [PDF]
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E. P Quinlivan and J. F Gregory III Effect of food fortification on folic acid intake in the United States Am. J. Clinical Nutrition, January 1, 2003; 77(1): 221 - 225. [Abstract] [Full Text] [PDF]
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