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Nutrient-Gene Interactions

Combined Marginal Folate and Riboflavin Status Affect Homocysteine Methylation in Cultured Immortalized Lymphocytes from Persons Homozygous for the MTHFR C677T Mutation¹

Lori Lathrop Stern, Barry Shane^*, Pamela J. Bagley, Marie Nadeau, Vivian Shih and Jacob Selhub²

Vitamin Metabolism and Aging Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111; ^* Department of Nutritional Sciences, University of California, Berkeley, CA 94720; and Amino Acid Disorder Laboratory at Massachusetts General Hospital, Charlestown, MA 02129

²To whom correspondence should be addressed. E-mail: jacob.selhub{at}tufts.edu.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor for the synthesis of methionine from homocysteine. A common C677T mutation in the MTHFR gene renders the enzyme 50% less active than the wild-type enzyme as shown in in vitro studies using cell extracts. We developed an immortalized cell culture model to determine whether the lower in vitro activity imparted by the homozygous (T/T) genotype is demonstrated in situ when exposed to adequate and marginal physiologic concentrations of folate and riboflavin. T/T MTHFR activity was compared with that of C/C genotype cell extracts by an in vitro assay and in intact cells by measuring the distribution of folate forms, the accumulation of homocysteine in the medium and the synthesis of methionine from formate and homocysteine. Under adequate nutrient conditions, the in vitro activity of the T/T MTHFR enzyme was approximately half that of the C/C genotype. Similarly, the proportion of 5-methyltetrahydrofolate in cells with the T/T genotype was approximately half that of the cells with wild-type MTHFR. In contrast, homocysteine accumulation in the culture medium was low and not different between genotypes, nor was there a difference in methionine synthetic capacity. Significant differences were observed between genotypes only when the supply of both folate and riboflavin was limited in the medium, which resulted in increased homocysteine accumulation and decreased methionine production in the T/T genotype. These data are consistent with the current understanding of the molecular interaction of the MTHFR mutant with folate substrates and the FAD prosthetic group.

KEY WORDS: • folate • methylenetetrahydrofolate reductase • homocysteine • immortalized cells

Methylenetetrahydrofolate reductase (MTHFR)² (5-methyltetrahydrofolate: acceptor-oxidoreductase, EC 1.7.99.5) is a flavoprotein that catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate [5-CH₃-H₄ folic acid (tetrahydrofolic acid)]. This reaction is physiologically irreversible, committing the one-carbon unit of 5-CH₃-H₄ folic acid to the remethylation of homocysteine for methionine synthesis.

Kang et al. (1) first identified a common variant of the MTHFR enzyme that was characterized by an 50% reduction in in vitro enzyme activity and a greater sensitivity to heat inactivation compared with the normal enzyme. When lymphocyte or fibroblast extracts containing this variant enzyme were preincubated at 46°C for 5 min, the residual enzyme activity was considerably lower than the normal enzyme; thus, this variant was named "Thermolabile MTHFR." These properties were later found to be attributable to a cytosine to thymidine transition at nucleotide 677 (C677T), resulting in an alanine to valine substitution in the protein (2). This mutation has been shown to be common in many populations, with a homozygous (T/T) prevalence of 10% in the North American Caucasian population (2,3).

Of interest is that the T/T genotype is associated with mild elevations of plasma homocysteine compared with the wild-type (C/C) and heterozygous genotypes, indicating that the activity of the thermolabile MTHFR is also impaired in vivo (4). Jacques et al. (5) showed, however, that a significant elevation of plasma homocysteine in T/T genotype individuals occurs only under the condition of low folate status. When folate status was adequate, homocysteine concentrations were low irrespective of MTHFR genotype. This observation, which has since been shown by others (6,7), suggests that the in vivo activity of the T/T MTHFR is protected from inactivation when folate status is adequate despite its in vitro phenotype of a lower enzyme activity.

This interaction between the MTHFR genotype and folate status was further substantiated in two other studies. Lathrop Stern et al. (8), and most recently Friso et al. (9), found that genomic DNA methylation was substantially lower with the T/T MTHFR genotype only in the presence of low folate status. As folate status improved, genomic DNA methylation was higher and not different from that seen in persons with the wild-type (C/C) genotype. Another study found that riboflavin status was also associated with elevated plasma homocysteine levels only in persons who had both the T/T genotype and low folate status. With higher folate status, homocysteine levels were low irrespective of the MTHFR genotype and riboflavin status (10).

To date the activity of the thermolabile MTHFR variant in whole cells and tissues is not well understood. The majority of studies that describe the activity of the T/T genotype enzyme were performed using cell extracts or are epidemiologic in nature. Therefore, to compare the activity of the human T/T variant with the wild-type enzyme in intact cells, we developed a cell culture model from peripheral lymphocytes of persons with the T/T and wild-type MTHFR genotypes. We used this model in the present study to investigate whether the lower in vitro activity imparted by the T/T genotype is also demonstrated in situ under conditions of either adequate or marginal folate and riboflavin.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Subjects.

The study protocol was approved by the Tufts-New England Medical Center Human Institutional Review Board. We recruited healthy adults according to MTHFR genotype. Three persons homozygous for the C677T mutation (T/T genotype) and 3 persons with wild-type (C/C) MTHFR were selected.

Materials.

Epstein-Barr virus and Cyclosporin A were kindly provided by Dr. Vivian Shih at Massachusetts General Hospital, Boston, MA. Custom minimal essential medium (MEM) devoid of folate and riboflavin was obtained from JRH Biosciences, Denver, PA. 6S-5-Formyltetrahydrofolate was obtained from Shircks Laboratories, Jona, Switzerland. 6R-[¹⁴C]-5-Methyltetrahydrofolate and [¹⁴C]-formic acid, were purchased from Amersham Life Science, Arlington Heights, IL. Dialyzed horse serum was from BioWhittaker, Walkersville MD, and all other cell culture products were obtained from either GibcoBRL (Grand Island, NY), Sigma Chemical (St. Louis, MO) or BioRad (Hercules, CA).

MTHFR genotype.

MTHFR genotype was determined by PCR-restriction fragment length polymorphism analysis based on the method described by Frosst et al. (2). Briefly, a 198-bp fragment was amplified by PCR using the primers 5'-TGAAGGAGAA GGTGTCTGCGGGA-3' and 5'-AGGACGGTGC GGTGAGAGTG-3'. The presence of the mutant T allele creates an HinfI restriction site resulting in two fragments (175 and 23 bp), the larger of which can be visualized under UV light after gel electrophoresis and ethidium bromide staining.

Immortalization of lymphocytes.

Immortalized lymphoblastoid cell lines were developed from peripheral lymphocytes using the method of Anderson and Gusella (11). Peripheral venous blood (16 mL) was drawn into tubes containing acid citrate dextrose and kept at room temperature for 24 h. Mononuclear cells were then isolated using Lymphoprep tubes (GibcoBRL). The isolated cells were resuspended in Iscove’s modified Dulbecco’s medium (10 mL) supplemented with 100 mL/L horse serum and divided among three 25-cm² flasks. To immortalize the cells, a solution containing Epstein-Barr virus and Cyclosporin A was added to the cell suspension and allowed to incubate at 37°C in atmospheric air supplemented with 5% carbon dioxide. This process specifically promoted the outgrowth of immortalized B cells.

Experimental culture conditions.

Cell cultures were maintained in custom-made MEM under the following four treatment conditions: folate and riboflavin-sufficient, reduced folate, reduced riboflavin and combined reduced folate and riboflavin. To represent adequate and low physiologic plasma levels of folate and riboflavin derivatives, the following were used: sufficient medium conditions were supplemented with 25 nmol/L 6S-5-HCO-H₄ folic acid and 70 nmol/L riboflavin. Under reduced vitamin conditions, the media were supplemented with 2 nmol/L 6S-5-HCO-H₄ folic acid and 5 nmol/L riboflavin. 5-HCO-H₄ folic acid was used as the folate source because of its comparability to 5-CH₃-H₄ folic acid, the predominant circulating form of folate, in terms of its availability to the cell via the reduced folate carrier. 5-HCO-H₄ folic acid is more stable than 5-CH₃-H₄ folic acid. We also used dialyzed horse serum (50 mL/L) containing 0.59 nmol/L folate and 45 nmol/L riboflavin to minimize the contribution of vitamins from this source. Viability of the cells was determined by the Trypan blue test. All cultures used for assay were, at a minimum, 75% viable. Cell counting was performed using a Model Z Coulter Counter (Coulter, Miami, FL).

Nutrient concentrations.

Intracellular and medium folate concentrations were determined by microbial assay using Lactobacillus casei (12). Intracellular and medium riboflavin concentrations were measured by HPLC with fluorescence detection following precipitation of proteins with trichloroacetic acid (13). The data are the sum of riboflavin, FMN and FAD.

MTHFR specific activity.

MTHFR activity was measured in cell extracts using the method of Kang et al. (1) with modifications. Approximately 8 x 10⁶ cells were rinsed twice with 3 mL cold PBS and suspended in a final volume of 0.5 mL PBS. Cell extracts were prepared by sonicating the cell suspensions three times for 10 s each followed by centrifugation at 10,000 x g for 15 min. Protein in the extract was measured using BioRad dye reagent. Cell extract containing 50 µg protein was then incubated at 37°C for 1 h in a mixture containing 90 µL of 1 mol/L potassium phosphate buffer, pH 6.3; 27 µL of 1 mmol/L FAD; 8 µL of 100 mmol/L EDTA; 10 µL of 10 mmol/L menadione sulfate; 106 µL of water; 5 µL of [¹⁴C]-5-CH₃-H₄ folic acid (925 MBq/L); 4 µL of 2 mmol/L 5-CH₃-H₄ folic acid; and PBS added to a final volume of 250 µL. To stop the reaction, 300 µL of a dimedone solution (6 g/L dimedone, 1 mol/L sodium acetate, pH 4.5, 100 mL/L ethanol) was added to the incubation mixture and heated at 100°C for 5 min. In this process, the dimedone forms an adduct with the radiolabeled formaldehyde, which was extracted in a toluene-based scintifluor (5 mL) and counted. MTHFR activity is expressed as nmol formaldehyde formed/(mg protein · h).

Medium homocysteine.

Medium homocysteine concentrations were determined by HPLC with fluorescence detection (14). Cultured lymphoblasts remain suspended in medium and the volume of medium added for optimal growth should remain approximately proportional to the number of cells; therefore, the volume of medium in each flask will differ. A portion of suspended cells was counted, then centrifuged at 210 x g for 10 min. Subsequently, a 100-µL aliquot of medium was analyzed for homocysteine concentration and expressed as µmol/(L of medium · 10⁶ cells).

Proportions of folate forms.

Folate 1-carbon distributions were measured by the method of Bagley and Selhub (15). Briefly, folates were heat extracted from 10 to 20 x 10⁶ cells in a high pH (9.2) buffer. These extracts were applied onto a 2-column chromatography system for folate binding protein affinity purification, separation by HPLC, and analysis by an ESA 4-channel coulometric electrochemical detector and software for Windows (ESA, Chelmsford, MA). Folate standards were used for the identification and quantification of specific folate forms.

Methionine synthesis.

Methionine synthesis was measured in whole cells using a method derived from Ellegaard et al. (16) and Baum et al. (17). In this assay, cells are incubated with radiolabeled formic acid and an excess of homocysteine to force the synthesis of methionine. Therefore, the capacity of MTHFR to synthesize 5-CH₃-H₄ folic acid for the remethylation of homocysteine to form methionine is assessed. Cell suspensions containing 5 x 10⁶ cells in 0.5 mL PBS were incubated in duplicate at 37°C for 3 h in the following mixture: 2 mL Krebs-Ringer buffer, 10 g/L gelatin and a final concentration of 40 mmol/L DL-homocysteine and 7.4 µmol/L [¹⁴C]-formic acid [37kBq (1 µCi)/incubation]. The reaction was stopped by the addition of 0.25 mL 33% perchloric acid and incubation on ice for 1 h. The acid precipitate was pelleted by centrifugation at 850 x g for 20 min. The supernatant fraction was loaded onto a Dowex, H⁺ form, column (1-mL bed volume). The column was rinsed twice with 1 mL of water to rid the column of unreacted [¹⁴C]-formate. The radioactive methionine was then eluted from the column with 4 mL of ammonium hydroxide (1.5 mol/L). Radioactivity was counted in the eluant and methionine synthesis expressed as Bq/10⁶ cells.

Statistical analysis.

The Student’s t test for independent samples was used for all mean comparisons. Comparisons were made between genotypes for each medium condition. Within genotype, comparisons were also made for each medium condition compared with its control, i.e., adequate nutrient status. These means were obtained from a minimum of three measurements for each cell line. Values for the methionine synthesis measurement were log-transformed to achieve homogeneity of variance. Differences were considered significant at P < 0.05. All analyses were performed with Systat 7.0.1 for Windows (Evanston, IL).

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Measurements of the intracellular folate and riboflavin coenzyme concentrations in lymphoblasts demonstrated that folate concentrations were markedly reduced when cells were grown in low folate medium, although none of the treatments resulted in a marked reduction of intracellular riboflavin (Table 1). In addition, there were no differences between the two cell types with respect to intracellular vitamin concentrations when grown under any of the medium conditions.

View larger version (36K): [in this window] [in a new window]   FIGURE 1 Methylenetetrahydrofolate reductase (MTHFR) activity in cell extracts (A) and medium homocysteine accumulation (B) from immortalized C/C and T/T MTHFR genotype lymphoblasts grown in control medium, adequate in folate and riboflavin, or in folate-reduced (FR), riboflavin-reduced (RR) and combination folate- and riboflavin-reduced medium (FRR). The bars represent means ± SD (n = 3). Means for each medium condition with different letters differ, P < 0.05. * Different from the respective control, P < 0.05.

Typical chromatograms of the distribution of the cellular folate form from cells of both genotypes grown in nutrient-adequate medium show that the majority of the cellular folates were reduced polyglutamyl derivatives with and without one-carbon substitutions (Fig. 2). When the cell types were compared, peaks representing 5-CH₃-H₄ folic acid polyglutamates were clearly lower, and those representing formylated tetrahydrofolates were higher in cells with the T/T genotype compared with those in wild-type cells. The relative proportion of 5-CH₃-H₄ folic acid polyglutamates in the T/T genotype cells (22.2 ± 1.5%) was approximately half that of the C/C genotype cells (44.3 ± 8.1%) (P < 0.05), whereas the total concentration of intracellular folates did not differ between genotypes (Table 1). There was no difference between genotypes in the relative proportion of H₄ folic acid or in the average polyglutamate chain length (data not shown). For those grown in folate-deficient medium, the chromatographic peaks were too low for accurate detection and interpretation.

View larger version (17K): [in this window] [in a new window]   FIGURE 2 Chromatographic separation of folates in immortalized human lymphocytes by HPLC with electrochemical detection from lymphoblasts with the wild-type methylenetetrahydrofolate reductase (MTHFR) genotype (A) and lymphoblasts homozygous for the C677T mutation in the MTHFR gene (T/T genotype) (B). Each chromatogram represents the response of channels 1–4, which are set at 0, 300, 500 and 600 mV, respectively. Folate derivatives in the various peaks are denoted as: T, H4 folic acid; M, 5-CH3-H4 folic acid; and F, 10-HCO-H4 folic acid. The numbers following the letters denote the number of glutamate residues.

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The basis of this interaction appears to be related to the strength of the association between the mutant enzyme and its prosthetic group, FAD. Guenther et al. (18) recently studied a variant form of the Escherichia coli enzyme expressing a similar modification to the human mutant C677T MTHFR enzyme. The reduced activity of the modified E. coli enzyme is attributable to diminished FAD binding, which affects the equilibrium between the more stable tetramer and the less stable dimeric form of the protein. Folate derivatives protect the wild-type and mutant E. coli enzymes against flavin loss.

The human enzyme is a dimer rather than a tetramer, and contains a domain that is lacking in the E. coli protein. This domain allosterically binds the inhibitor S-adenosylmethionine (SAM). Nevertheless, a recent study of the human enzyme variant expressed in baculovirus showed characteristics similar to the E. coli enzyme whereby the mutation weakened the association of the enzyme with FAD (19). Once FAD is disassociated from the enzyme, there is a rapid conversion of the dimer to monomers, which is associated with the genotype-related loss of activity. Both methyltetrahydrofolate and SAM protect the complex against FAD dissociation.

Recent epidemiologic data from our laboratory support this interpretation. Low plasma concentrations of riboflavin were associated with high plasma homocysteine levels only in those homozygous for the C677T mutation that have low plasma folate levels. At higher levels of folate, plasma homocysteine was low irrespective of the riboflavin status and the C677T MTHFR genotype (10).

In the present study, we used immortalized lymphocytes from donors with T/T and C/C MTHFR genotypes to further investigate this interaction between the polymorphic enzyme and folate. Our data show that these lymphocytes maintain the phenotypic expression of the C677T mutation and that the in vitro activity of the enzyme from T/T lymphocyte extracts was considerably lower than that from extracts of the C/C genotype cells. The data on the in situ synthesis of methionine and medium homocysteine accumulation also justified the use of such a model. We found that lowering the folate concentration in the growth medium decreased methionine synthesis in both cell types, and that methionine synthesis differed significantly between the two genotypes when the growth medium contained limited amounts of both folate and riboflavin.

The epidemiologic studies showing that homozygotes for the C677T MTHFR mutation have normal plasma total homocysteine levels when their folate status is adequate can be interpreted in two ways. One paradigm assumes that the activity of the mutant enzyme, under these conditions, is comparable to that of the wild-type enzyme. The other possibility is that the activity of the T/T MTHFR remains lower than that of the C/C MTHFR, yet is sufficient to synthesize 5-CH₃-H₄ folic acid at a rate that meets the cellular demands for homocysteine methylation.

The data presented here are consistent with the second interpretation. We showed that under conditions of adequate folate and riboflavin concentrations in the growth medium, the in situ capacity of immortalized human lymphoblasts with the T/T genotype to synthesize methionine from added formate and excess homocysteine is not different from that of lymphoblasts with the C/C genotype. There also was no difference between genotypes in the quantity of homocysteine released into the growth medium. Nevertheless, the T/T and C/C cells differed with respect to distribution of the intracellular folate form. Cells with the T/T genotypes had approximately half the concentration of 5-CH₃-H₄ folic acid polyglutamates and nearly double the concentration of 5-HCO-H₄ folic acid polyglutamates of the C/C genotype cells. Therefore, these data show that under conditions of adequate folate and riboflavin status, the mutant enzyme is less active than the wild-type enzyme. Yet, despite this lower activity, the mutant enzyme provides sufficient 5-CH₃-H₄ folic acid to meet the demands for methionine synthesis and homocysteine methylation.

Undoubtedly, other tissues may respond differently to these and other nutritional perturbations. Nevertheless, the data presented here are consistent with some of the interpretations of recent epidemiologic data, which suggest that under conditions of adequate folate status, the C677T mutation in the MTHFR gene protects against the development of certain cancers, particularly colorectal cancer (20,21). It has been suggested that this protection is due to the greater availability of nonmethylated folates, which serve as substrates for the synthesis of the purines and pyrimidines that are necessary for DNA synthesis and repair (21).

	ACKNOWLEDGMENTS

 
We thank Robert Russell from Tufts University, Boston, MA for providing technical advice throughout this project and for reviewing the manuscript. Thanks to Antoinette Edmondson from Tufts University for her assistance in the one-carbon distribution measurements. We also thank Roseann Mandell from the Neuroscience Laboratory at Massachusetts General Hospital, Boston, MA for her assistance in immortalizing the cell lines.

	FOOTNOTES

 
¹ Supported in part by National Institutes of Health Training Grant number AG00209–07 (L.L.S.) and the Mary L. Efron Fund for Research (V.S.); this paper is also based upon work supported by the U.S. Department of Agriculture under agreement No. 58–1950-9–001. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.

³ Abbreviations used: C/C, homozygous for the wild-type allele of the methylenetetrahydrofolate reductase gene; C677T, cytosine to thymidine transition at nucleotide 677; 5-CH₃-H₄ folic acid, 5-methyltetrahydrofolic acid; H₄ folic acid, tetrahydrofolic acid; 5-HCO-H₄ folic acid, 5-formyltetrahydrofolic acid; MTHFR, methylenetetrahydrofolate reductase; SAM, S-adenosylmethionine; T/T, homozygous genotype for the C677T transition in the MTHFR gene.

Manuscript received 20 February 2003. Initial review completed 19 March 2003. Revision accepted 24 June 2003.

	LITERATURE CITED

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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