Nutrient–Gene Interactions
Methylenetetrahydrofolate Reductase 677C3 T Variant Modulates Folate
Status Response to Controlled Folate Intakes in Young Women1,2
Cheryl L. Guinotte, Michael G. Burns, Juan A. Axume,* Hiroko Hata,† Tania F. Urrutia,*
Aaron Alamilla,* Dale McCabe,* Anny Singgih,* Edward A. Cogger† and Marie A. Caudill3
Human Nutrition and Food Science Department, *Biological Sciences Department and †Animal and
Veterinary Science Department, Cal Poly Pomona University, Pomona, CA 91768
KEY WORDS:
●
folate
●
homocysteine
●
human
●
MTHFR genotype
Methylenetetrahydrofolate reductase (MTHFR)4 is an important regulatory enzyme in folate-dependent one-carbon
metabolism (Fig. 1). MTHFR catalyzes the irreversible reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the major form of folate in plasma and the predominant methyl donor in homocysteine remethylation to
methionine. After activation of methionine to S-adenosylmethionine (SAM), the folate-derived methyl group may be used
to methylate numerous compounds including DNA. Alternatively, the substrate of MTHFR, 5,10-methylenetetrahydrofolate, may serve as a one-carbon donor in the synthesis of
purines and the pyrimidine, thymidine.
The commitment of one-carbon units to methylneogenesis
●
RDA
or nucleotide synthesis is controlled by SAM through its effect
on MTHFR activity (1–3). Under conditions of low SAM,
MTHFR is stimulated to direct the pool of one-carbon units
toward methylneogenesis (1– 4). In contrast, elevations in
SAM inhibit MTHFR activity, thus increasing the availability
of one-carbon units for nucleotide synthesis (3). Dietary deficiencies may also affect MTHFR activity directly (i.e., riboflavin) (5,6) or indirectly (i.e., folate, vitamins B-12 and B-6,
choline, methionine) by altering the cellular concentrations of
methionine-cycle intermediates (1,7).
MTHFR activity is also modulated by common singlenucleotide polymorphisms in the MTHFR gene (8 –12). The
best-characterized MTHFR genetic polymorphism is the cytosine to thymidine (C3 T) transition at nucleotide 677, resulting in a codon that encodes valine rather than alanine (8).
Individuals possessing one or two T alleles have 30 and 65%,
respectively, lower MTHFR activities in vitro than that of the
677 CC genotype (8). The MTHFR 677 TT genotype is
associated with reduced folate status (13–15), DNA hypomethylation (16,17) and plasma homocysteine elevations (8),
particularly in those with low folate status (15,18,19), and may
modulate risk for cardiovascular disease (20 –22), cancers (23)
and developmental anomalies (24 –27).
The MTHFR 677 TT genotype was recognized as a genetic
factor that may alter folate requirements during derivation of
1
Presented at Experimental Biology, April 2002, New Orleans, LA [Caudill,
M. A., Guinotte, C., Axume, J., Hata, H., Urrutia, T., Burns, M., Alamilla, A.,
McCabe, D. & Cogger, E.A. (2002) Effect of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism on folate status when folate intake is controlled. FASEB J. 16: A568.6 (abs.)].
2
Supported by National Institutes of Health grant S06GM53933 and funds
from the California Agricultural Research Initiative.
3
To whom correspondence should be addressed.
E-mail: [email protected].
4
Abbreviations used: C, cytosine; DFE, dietary folate equivalents; DRI, dietary reference intakes; MTHFR, methylenetetrahydrofolate reductase; SAM, Sadenosylmethionine; tHcy, total homocysteine; T, thymidine; RDA, Recommended Dietary Allowance.
0022-3166/03 $3.00 © 2003 American Society for Nutritional Sciences.
Manuscript received 12 November 2002. Initial review completed 17 December 2002. Revision accepted 5 February 2003.
1272
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ABSTRACT A common genetic variant in the methylenetetrahydrofolate reductase (MTHFR) gene involving a
cytosine to thymidine (C3 T) transition at nucleotide 677 is associated with reduced enzyme activity, altered folate
status and potentially higher folate requirements. The objectives of this study were to investigate the effect of the
MTHFR 677 T allele on folate status variables in Mexican women (n ⫽ 43; 18 – 45 y) and to assess the adequacy
of the 1998 folate U.S. Recommended Dietary Allowance (RDA), 400 ␮g/d as dietary folate equivalents (DFE).
Subjects (14 CC, 12 CT, 17 TT genotypes) consumed a low folate diet (135 ␮g/d DFE) for 7 wk followed by repletion
with 400 ␮g/d DFE (7 CC, 6 CT, 9 TT) or 800 ␮g/d DFE (7 CC, 6 CT, 8 TT) for 7 wk. Throughout repletion with 400
␮g/d DFE, the TT genotype had lower (P ⱕ 0.05) serum folate and higher (P ⱕ 0.05) plasma total homocysteine
(tHcy) concentrations than the CC genotype. CT heterozygotes did not differ (P ⬎ 0.05) in their response relative
to the CC genotype. Throughout repletion with 800 ␮g/d DFE, the CT genotype had lower (P ⱕ 0.05) serum folate
concentrations and excreted less (P ⱕ 0.05) urinary folate than the CC genotype. However, there were no
differences (P ⬎ 0.05) in the measured variables between the TT and CC genotypes. Repletion with 400 ␮g/d DFE
led to normal blood folate and desirable plasma tHcy concentrations, regardless of MTHFR C677T genotype.
Collectively, these data demonstrate that the MTHFR C3 T variant modulates folate status response to controlled
folate intakes and support the adequacy of the 1998 folate U.S. RDA for all three MTHFR C677T genotypes.
J. Nutr. 133: 1272–1280, 2003.
MTHFR C3 T VARIANT AND FOLATE STATUS
1273
⫽ 43), the feeding phase was conducted in cohorts over 18-mo. To
reduce systematic errors (i.e., selection bias, selection/maturation
interaction and experimental mortality), all three MTHFR C677T
genotypes were enrolled in each cohort.
Breakfast and dinner were consumed daily in the Human Nutrition’s metabolic kitchen at Cal Poly Pomona University. Subjects
were allowed to consume lunch and snacks as well as 14 meals
(breakfast and/or dinner) of their choosing off-site. Weight was
monitored weekly and any deviation of ⫾5% from baseline was
addressed by modifying energy intake with folate-free items such as
sodas, gelatin, whipped topping and margarine. The principal investigator and/or trained graduate students had daily contact with the
subjects to help ensure compliance.
Diet and supplements
the 1998 folate U.S. Recommended Dietary Allowance
(RDA); however, data were insufficient to make recommendations (28). In the U.S. population, the frequency of the 677
TT genotype is ⬃10% overall and as high as ⬃20% in Mexican Americans (25,29). Hence, the folate requirement of a
large minority population may not be met with the current
folate U.S. RDA, 400 ␮g/d as dietary folate equivalents (DFE).
The purpose of this study was to investigate the effect of the T
allele on folate status response in Mexican American women
of childbearing age, under conditions of controlled folate
intake. A second objective was to assess the adequacy of the
1998 U.S. RDA, 400 ␮g/d DFE, and twice that level, 800 ␮g/d
DFE, to achieve normal folate nutriture in all three MTHFR
C677T genotypes.
SUBJECTS AND METHODS
Subjects
Women of self-reported Mexican descent, defined as having two
Mexican parents, were selected for participation in this study and
were recruited from staff and students at Cal Poly Pomona University
and the surrounding community. At the initial screening, potential
subjects completed a health history questionnaire and gave a fasting
blood sample for MTHFR genotype determination. For those with the
appropriate MTHFR genotype, another fasting blood sample was
obtained for clinical chemistry evaluation. Additional inclusion criteria were nonsmoker, nonanemic, nonsupplement users, no chronic
drug use, no alcohol consumption, no antifolate medication use (i.e.,
methotrexate, carbarnazepine, phenytoin, azaribine, isoniazid, nitric
oxide, colestipol), no history of chronic disease such as diabetes or
cardiovascular disease, nonpregnant and not planning a pregnancy,
nonlactating and a normal blood chemistry profile. The screening and
experimental procedures were reviewed and approved by the Institutional Review Board of Cal Poly Pomona University, and informed
consent was obtained from each participant.
A low folate 5-d rotation menu (Table 2), consisting of nonfolic
acid–fortified foods, was designed. The folate content (mean ⫾ SD) of
the diet, as determined by trienzyme methodology on three separate
occasions, was 135 ⫾ 15 ␮g/d. Unfortified flour (Kansas State University, Manhattan, KS) was used to prepare folic acid–fortified foods,
normally obtained commercially, including biscuits, waffles, chocolate chip and oatmeal cookies, pizza dough, chocolate cupcakes and
blueberry muffins. Chicken, ground beef, carrots and green beans
were boiled three times for 10 min each boil to reduce the amount of
folate in these foods, as previously described (30). Menu items were
weighed to the nearest 0.5 g to ensure that all subjects received the
same amount of food folate.
Energy and all other nutrients in the diet, except for folate, were
analyzed by use of the ESHA Food Processor Nutrient Data Base
(version 7.81; ESHA Research, Salem, OR). The diet provided a
mean energy intake of 8828 kJ/d with ⬃60, 10 and 30% of the energy
from carbohydrate, protein and fat, respectively. Subjects were given
supplements to provide the 1998 dietary reference intakes (DRI) for
the B vitamins (except folate) and choline and the 1989 RDA for all
other essential nutrients not met by the diet. The supplements
included a multimineral given every day (LifeTime, Nutritional Specialties, Anaheim, CA); a multivitamin (Trader Darwin’s Stress
Vitamin; Trader Joe’s, South Pasadena, CA), cut into thirds and
given every 4th d; vitamin K (KAL, Nutraceutical Corp., Park City,
UT), given every other day; choline (TwinLab, Twin Laboratories,
Ronkonkoma, NY), given every other day; and iron (TwinLab, Twin
Laboratories) given as needed (based on weekly hematocrit measures). The diet and supplements provided ⬃85% of the 1989 RDA
for calcium and 90 to 120% of the 1989 RDA or 1998 DRI for all
other essential vitamins (except folate) and minerals. The watersoluble vitamins lost during boiling of food items as well as the
fortificants normally in enriched flour were adjusted for in the calculations.
Folic acid supplements were prepared from commercially available
folic acid (Sigma Chemical, St. Louis, MO) and were consumed at
TABLE 1
Study design
MTHFR2
C677T
Genotype
Depletion
(wk 0–7)
135 ␮g/d
DFE
Repletion1 (wk 8–14)
400 ␮g/d
DFE
800 ␮g/d
DFE
n
Experimental design
This was a 14-wk metabolic feeding study consisting of a 7-wk
folate depletion phase followed by a 7-wk folate repletion phase with
400 or 800 ␮g/d DFE (Table 1). Throughout the 14-wk study,
subjects consumed a low folate diet (135 ␮g/d DFE). At wk 8, subjects
were randomized to consume total folate of 400 or 800 ␮g/d DFE,
consisting of 135 ␮g/d food folate and 156 and 391 ␮g/d supplemental
folic acid, respectively. Because of the large number of subjects (n
CC
CT
TT
14
12
17
7
6
9
7
6
8
1 Subjects were randomized to 400 or 800 ␮g/d dietary folate
equivalents (DFE) derived from 135 ␮g natural food folate plus 156 or
391 ␮g synthetic folic acid.
2 MTHFR, methylenetetrahydrofolate reductase.
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FIGURE 1 The regulatory role of methylenetetrahydrofolate reductase (MTHFR) in folate-dependent one-carbon metabolism. CBS, cystathionine ␤ synthase; DHF, dihydrofolate; DMG, dimethylglycine; DNA,
deoxyribonucleic acid; dTMP, deoxythymidine monophosphate;
dUMP, deoxyuridyne monophosphate; MS, methionine synthase; RNA,
ribonucleic acid; THF, tetrahydrofolate: X, methyl group acceptor.
GUINOTTE ET AL.
1274
TABLE 2
Five-d– cycle menus consumed by all subjects throughout 14-wk study1– 4
D1
Breakfast
Waffle
Blueberries and whipped topping
syrup, margarine
Cranapple juice
Dinner
Tuna casserole
Green beans and carrots
Cranapple juice
Snacks
Popcorn
D3
D4
D5
Granola cereal
Raisins
Mocha mix
Applesauce
Crangrape juice
Waffle
Syrup, margarine
Chunky spiced applesauce
Cranapple juice
Granola cereal
Raisins
Mocha mix
Applesauce
Crangrape juice
2 blueberry muffins
Margarine
Fruit cocktail
Crangrape juice
Ham
Cheddar cheese
Biscuit
Fruit cocktail, canned
Oatmeal raisin cookie
Soda
Tuna
Whole wheat pita
Fruit cocktail, canned
Chocolate chip cookie
Soda
Turkey
Swiss cheese
Biscuit
Fruit cocktail, canned
Oatmeal raisin cookie
Soda
Chicken salad
American cheese
Whole wheat pita
Peaches, canned
Chocolate chip cookie
Soda
Chicken enchiladas
Green beans, canned
Orange sherbet
Cranapple juice
Pizza w/ham
Green beans, canned
Peach crisp
Cranapple juice
Spaghetti
Green beans and
carrots, canned
Chocolate cupcake
Cranapple juice
Chicken breast
Barbeque sauce
Biscuit
Garlic potatoes
Green beans and
carrots, canned
Cranapple juice
Apple and cinnamon
rice cakes
Popcorn
Apple and cinnamon
rice cakes
Popcorn
1 Menus analyzed by trienzyme folate extraction method provided a mean of 135 ␮g/d dietary folate equivalents (DFE).
2 Menu items prepared with unfortified flour were waffles, blueberry muffins, cookies, chocolate cupcakes, pizza dough and biscuits. Unfortified
flour also used in sauces for tuna casserole and chicken enchiladas and for the peach crisp topping.
3 Chicken, hamburger, green beans and carrots were thrice boiled for 10 min each boil to reduce folate content.
4 Foods allowed in unlimited amounts included soda, jello and whipped topping; also two cups of coffee or tea were allowed per day.
the morning and evening meals throughout repletion, under the
supervision of the investigators. To make the folic acid solution for
repletion with 400 and 800 ␮g/d DFE, weighed folic acid was dissolved in a small amount of 0.1 mol/L NaOH and brought to volume
with 0.1 mol/L phosphate buffered saline (pH 7.0). The folic acid
concentration was determined spectrophotometrically at 282 nm,
with a molar absorptivity coefficient at pH 7.0 of 27,600 L/(mol 䡠 cm)
(31). The folic acid stock solution was prepared once during the
study, dispensed into 200-mL containers, wrapped in aluminum foil
and stored at ⫺80°C. Appropriate volumes of the folic acid solution
were dispensed into 50-mL conical tubes to which ⬃40 mL apple
juice was added. The tubes were then wrapped in aluminum foil and
frozen at ⫺20°C. The folic acid supplements for subject consumption
were prepared every 3 mo from the folic acid stock solution. Spectrophotometric analysis demonstrated that losses of folic acid during
storage at ⫺80°C were negligible.
Needham Heights, MA). Plasma and leukocytes were collected from
EDTA blood that was immediately placed on ice and centrifuged
within 1 h of the blood draw at 1800 ⫻ g for 15 min at 4°C. Plasma
was dispensed into 1.5-mL microcentrifuge tubes and stored at
⫺80°C for plasma tHcy analyses. For genotyping, the buffy layer
representing peripheral leukocytes (⬃500 ␮L) was removed, dispensed into 1.5-mL microcentrifuge tubes containing 50 ␮L dimethyl
sulfoxide (DMSO; Sigma Chemical), mixed by inversion and frozen
at ⫺80°C.
The 24-h urine collections were obtained at baseline and weekly
throughout the study in acid-washed 2-L brown plastic bottles containing 5 g of ascorbate. Subjects were advised to keep urine refrigerated at all times to prevent bacterial growth and folate breakdown.
Weekly urine samples from each subject were mixed thoroughly,
dispensed into 250-mL containers and stored at ⫺20°C.
Analytical methods
Sample collection and blood processing
Baseline and weekly fasting (10 h) venous blood samples were
collected in serum separator gel and clot-activator tubes (SST, Vacutainer; Becton Dickinson, Rutherford, NJ) and EDTA tubes (Vacutainer). Serum was collected after centrifugation (650 ⫻ g for 15
min at 21°C), dispensed into 1.5-mL microcentrifuge tubes containing 10 –15 mg of ascorbic acid and stored at ⫺80°C. Whole blood
from the EDTA-containing tube was mixed, dispensed (50 ␮L) into
1.5-mL microcentrifuge tubes and diluted 1:20 with 1 g/L ascorbic
acid solution (950 ␮L). After vortexing, the diluted whole blood was
incubated at room temperature for 30 min and stored at ⫺80°C.
Hematocrits were also measured by use of mixed EDTA whole blood.
The blood was drawn up into two microhematocrit tubes (Fisher
Scientific, Pittsburgh, PA), plugged with clay (Critoseal; Oxford
Labware, St. Louis, MO) and centrifuged. The capillary tubes were
then measured on a microcapillary reader (International Equipment,
Folate content of diet. The folate content of the diet was determined before starting the study and twice during the study. Each meal
including beverage was prepared as for the subject’s consumption,
blenderized with 150 mL of cold 0.1 mol potassium phosphate
buffer/L (pH 6.3) containing 57 mmol ascorbic acid/L to preserve the
folate (32), dispensed into 50-mL conical tubes and stored at ⫺20°C.
Duplicates of the blenderized samples were thawed, homogenized and
subjected to trienzyme treatment (33) and double extraction (34) by
use of the Hepes/Ches buffer (pH 7.85) with ascorbate and 2-mercaptoethanol (35). Total food folate was then measured microbiologically (36).
MTHFR C677T genotype. DNA for genotyping was extracted
from leukocytes by use of a commercially available kit (QIAamp
blood kit; Qiagen, Santa Clarita, CA). Determination of the C677T
MTHFR genotype involved polymerase chain reaction (PCR) and
Hinf1 restriction enzyme digestion, as described by Frosst and associ-
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Lunch
Turkey
Swiss cheese
Whole wheat pita
Fruit cocktail
Chocolate chip cookie
Soda
D2
MTHFR C3 T VARIANT AND FOLATE STATUS
ates (8). In the absence of the MTHFR C677T variant, there was a
single uncut PCR product of 198 base pairs. In the presence of the
MTHFR polymorphism, a Hinf1 restriction site was created, resulting
in digestion of the 198-bp fragment into 175 and 23 bp. The PCR
products were separated by gel electrophoresis on a 1.5% agarose gel
and viewed under UV light.
Plasma tHcy. Total homocysteine concentrations were measured
in duplicate by use of a modified HPLC method with fluorometric
detection (37,38) and had an intra- and interassay CV of 3 and 5%,
respectively.
Blood and urinary folate. Folate concentrations of serum, erythrocytes and urine were determined microbiologically by use of the
microtiter plate adaptation with Lactobacillus casei (36). The intraand interassay CV were both 12%, based on the positive control.
Statistical analysis
RESULTS
Subject characteristics and baseline measures
The final study group was composed of 43 women with the
following MTHFR C677T genotype distribution: 14 wild type
(CC), 12 heterozygous (CT), and 17 homozygous for the T
variant (TT). The mean age of the women was 25 y (range
⫽ 18 – 44 y), whereas the mean body mass index (kg/m2) was
25.2 (range ⫽ 19.5–32). No differences (P ⬎ 0.05) were
detected in age or body weight between women representing
the three MTHFR C677T genotypes at the beginning or end
of study. Body weights were maintained within 5% of baseline
in all but nine subjects (3 CC, 3 CT and 3 TT) who lost ⬃8%
(range: 6.5–10.7%) of baseline weight. A total of 11 women (4
CC, 3 CT and 4 TT) reported using oral contraceptives during
the study period. No differences (one-way ANOVA; P
⬎ 0.05) existed in serum folate, urinary folate or homocysteine
concentrations between the MTHFR genotypes at baseline
(Table 3). Red cell folate concentrations were lower (P
ⱕ 0.05) in the TT genotype than in the CC control, although
CT and CC genotypes did not differ (P ⬎ 0.05) (Table 3).
TABLE 3
Baseline concentrations of serum folate (SF), red cell folate
(RCF), urinary folate (UF) and plasma total homocysteine
(tHcy) in Mexican American women with differing
MTHFR C677T genotypes1,2
MTHFR C677T genotype
Variable
SF, nmol/L
RCF, nmol/L
UF, nmol/d
tHcy, ␮mol/L
CC
33.9 ⫾ 13.5
1202 ⫾ 188a
46.4 ⫾ 40
5.3 ⫾ 0.9
CT
28.6 ⫾ 10.1
1087 ⫾ 190a,b
29.8 ⫾ 30
5.9 ⫾ 0.9
TT
29.8 ⫾ 9.4
993 ⫾ 258b
70.5 ⫾ 121
5.4 ⫾ 0.6
1 Values are means ⫾ SD, n ⫽ 12–17.
2 Means in a row with superscripts without a common letter differ,
P ⱕ 0.05.
Serum folate
Folate depletion decreased (P ⱕ 0.05) the serum folate
concentration 58%, from 30.8 ⫾ 11.0 to 12.9 ⫾ 4.9 nmol/L.
Subjects possessing the TT genotype had lower (P ⱕ 0.05)
serum folate concentrations throughout depletion compared to
the CC controls, whereas those with the CT genotype were
numerically lower (P ⫽ 0.13; Fig. 2A).
Folate repletion with 400 ␮g/d DFE increased (P ⱕ 0.05)
the serum folate concentration 23%, from 13.9 ⫾ 5.3 to 17.1
⫾ 5.2 nmol/L. However, baseline serum folate concentrations
were not obtained and 32% had serum folate concentrations in
the low normal range, 6.8 –13.6 nmol/L. Throughout repletion
with 400 ␮g/d DFE, subjects possessing the TT genotype had
lower (P ⱕ 0.05) serum folate concentrations than either the
CC or CT genotypes (Fig. 2B). At the end of repletion with
400 ␮g/d DFE, 50, 28.5 and 14% of the TT, CT and CC
genotypes, respectively, had serum folate concentrations in the
low normal range. No subjects had serum folate concentrations ⬍ 6.8 nmol/L.
Folate repletion with 800 ␮g/d DFE increased (P ⱕ 0.05)
the serum folate concentration 223%, from 11.9 ⫾ 4.4 to 38.4
⫾ 9.6 nmol/L, which was greater (P ⱕ 0.05) than baseline
concentrations. A genotype effect was evident during repletion with 800 ␮g/d DFE, with the CT genotype exhibiting
lower (P ⱕ 0.05) serum folate concentrations than the CC
controls (Fig. 2C). The response of the TT genotype was
intermediate.
During the repletion phase of the study, there were no
three-way or genotype/week interactions for serum folate.
However, a significant (P ⱕ 0.01) folate intake/week interaction was detected. In the 400 ␮g/d DFE group, serum folate
increased (P ⱕ 0.01) modestly during the 1st wk of repletion
and remained stable, whereas for the 800 ␮g/d DFE group,
serum folate concentrations doubled during the 1st wk and
continued to increase (P ⱕ 0.05) until wk 11 (Fig. 2B, C);
furthermore, there was a significant (P ⱕ 0.05) genotype/folate
intake interaction. The genotype response pattern for serum
folate to 400 ␮g/d DFE was CC ⬎ CT ⬎ TT compared to CC
⬎ TT ⬎ CT for 800 ␮g/d DFE.
Red cell folate
Folate depletion decreased (P ⱕ 0.05) the red cell folate
concentration 25%, from 1088 ⫾ 231 to 820 ⫾ 163 nmol/L.
Throughout depletion, subjects possessing the TT genotype
had lower (P ⱕ 0.05) red cell folate concentrations than those
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All data summarization and analyses were performed by use of
SPSS10.0 for Windows (SPSS, Chicago, IL). To determine whether
baseline differences existed between the MTHFR C677T genotypes
in the measured variables (serum folate, red cell folate, urinary folate
and plasma tHcy), one-way ANOVA was used. Where a significant
genotype effect was detected by ANOVA, the Tukey HSD test was
used for mean separation.
The relationship between the folate status response variable and
MTHFR C677T genotypes throughout the depletion phase was examined by use of the repeated-measures ANOVA facility of the GLM
procedure with one within-factor (weeks) variable and one betweenfactor (genotype) variable. For the repletion phase of the study, a
repeated-measures ANOVA with one within-factor (weeks) variable
and two between-factor (genotype and folate intake) variables was
used to examine the interaction effects. Because there were no
significant three-way interactions, the data were stratified by folate
intake, 400 and 800 ␮g/d DFE, and the relationships between the
folate status response variables and MTHFR C677T genotypes
throughout the repletion phase were examined by use of a repeatedmeasures ANOVA with one within-factor (weeks) variable and one
between-factor (genotype) variable. Significant genotype effects were
examined by use of the Tukey HSD test and significant week effects
were examined by use of simple and repeated contrasts. Where a
significant Mauchly’s test of sphericity was obtained, the Greenhouse–Geisser epsilon was applied to protect against a type 1 error.
Missing values from the red cell folate data (n ⫽ 4) were calculated
by use of weighted averages on the basis of subject, genotype, folate
intake and week grouping. One subject’s urinary folate data were
excluded from the repletion phase because of incomplete collections.
Significance was set at P ⱕ 0.05. Data are presented as means ⫾ SD
in the text and tables and means ⫾ SEM in the figures.
1275
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GUINOTTE ET AL.
interaction. In the 400 ␮g/d DFE group, red cell folate declined
slightly and then remained stable at postdepletion levels. For the
800 ␮g/d DFE group, red cell folate concentrations increased by
wk 10 (P ⱕ 0.05) and remained higher (P ⱕ 0.01) than postdepletion concentrations throughout the study (Fig. 3B, C).
Urinary folate
Folate depletion decreased (P ⱕ 0.05) the urinary folate
excretion 79%, from 51.2 ⫾ 81.4 to 10.9 ⫾ 4.8 nmol/d.
Genotype did not affect urinary folate response (Fig. 4A).
After repletion with 400 ␮g/d DFE, urinary folate excretion
increased, but not significantly, 20%, from 11.3 ⫾ 5.8 to 13.6
⫾ 5.1 nmol/d. Baseline concentrations were not achieved and
no genotype effect was detected (Fig. 4B).
Repletion with 800 ␮g/d DFE increased (P ⱕ 0.05) the
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FIGURE 2 Weekly serum folate concentrations in Mexican American women with differing MTHFR C677T genotypes during folate
depletion (14 CC, 12 CT, 17 TT) (A) and repletion with 400 ␮g/d DFE (7
CC, 6 CT, 9 TT) (B) and 800 ␮g/d DFE (7 CC, 6 CT, 8 TT) (C). *,**Weekly
serum folate concentration for the combined MTHFR genotypes (n
⫽ 43) differs from wk 0 (A) or wk 7 (B, C) at P ⫽ 0.05 or P ⫽ 0.01,
respectively. ⫹,⫹⫹Weekly serum folate concentration for the combined
MTHFR genotypes (n ⫽ 43) differs from the preceding week at P ⫽ 0.05
or P ⫽ 0.01, respectively. Values are means ⫾ SEM. a,bMTHFR genotypes with differing superscript letters differ across all weeks, P ⱕ 0.05.
of CC controls (Fig. 3A). The response of the CT genotype
was intermediate.
After folate repletion with 400 ␮g/d DFE, the red cell folate
concentration, 855 ⫾ 146 nmol/L, did not differ (P ⬎ 0.05)
from postdepletion concentrations, 845 ⫾ 144 nmol/L. All
subjects, however, maintained red cell folate concentrations in
the normal range, above 363 nmol/L. The TT genotype tended
(P ⫽ 0.06) to have lower red cell folate concentrations than
those of CC controls (Fig. 3B).
Folate repletion with 800 ␮g/d DFE increased (P ⱕ 0.05)
the red cell folate concentration 31%, from 793 ⫾ 181 to 1039
⫾ 174 nmol/L. Genotype did not affect red cell folate concentrations (Fig. 3C).
During the repletion phase of the study, there were no threeway, genotype/week or genotype/folate intake interactions for red
cell folate; however, there was a significant folate intake/week
FIGURE 3 Weekly red cell folate concentrations in Mexican American women with differing MTHFR C677T genotypes during folate
depletion (14 CC, 12 CT, 17 TT) (A) and repletion with 400 ␮g/d DFE (7
CC, 6 CT, 9 TT) (B) and 800 ␮g/d DFE (7 CC, 6 CT, 8 TT) (C). *,**Weekly
red cell folate concentration for the combined MTHFR genotypes (n
⫽ 43) differs from wk 0 (A) or wk 7 (B, C) at P ⫽ 0.05 or P ⫽ 0.01,
respectively. ⫹,⫹⫹Weekly red cell folate concentration for the combined
MTHFR genotypes (n ⫽ 43) differs from the preceding week at P ⫽ 0.05
or P ⫽ 0.01, respectively. Values are means ⫾ SEM. a,bMTHFR genotypes with differing superscript letters differ across all weeks, P ⱕ 0.05.
MTHFR C3 T VARIANT AND FOLATE STATUS
1277
DFE for urinary folate was CC ⬎ CT ⬎ TT compared to CC
⬎ TT ⬎ CT for 800 ␮g/d DFE.
Homocysteine
FIGURE 4 Weekly urinary folate excretion in Mexican American
women with differing MTHFR C677T genotypes during folate depletion
(14 CC, 12 CT, 17 TT) (A) and repletion with 400 ␮g/d DFE (7 CC, 6 CT,
9 TT) (B) and 800 ␮g/d DFE (6 CC, 6 CT, 8 TT) (C). *,**Weekly urinary
folate excretion for the combined MTHFR genotypes (n ⫽ 43) differs
from wk 0 (A) or wk 7 (B, C) at P ⫽ 0.05 or P ⫽ 0.01,
respectively.⫹,⫹⫹Weekly urinary folate excretion for the combined
MTHFR genotypes (n ⫽ 43) differs from the preceding week at P ⫽ 0.05
or P ⫽ 0.01, respectively. Values are means ⫾ SEM. a,bMTHFR genotypes with differing superscript letters differ across all weeks, P ⱕ 0.05.
urinary folate excretion 294%, from 10.5 ⫾ 3.7 to 42.9 ⫾ 29.1
nmol/d, which was similar (P ⬎ 0.05) to baseline urinary folate
concentrations. Throughout repletion with 800 ␮g/d DFE, the
CT genotype excreted less (P ⱕ 0.05) urinary folate than the
CC controls (Fig. 4C). The urinary folate excretion of the TT
genotype also tended to be lower (P ⫽ 0.06) than the CC
controls.
During the repletion phase of the study, there were no
three-way or genotype/week interactions for urinary folate.
However, a significant (P ⱕ 0.01) folate intake/week interaction was detected. In the 400 ␮g/d DFE group, urinary folate
excretion did not change (P ⬎ 0.05) from postdepletion
concentrations throughout the repletion phase. For the 800
␮g/d DFE group, urinary folate excretion increased (P ⱕ 0.05)
by wk 8 and again at wk 9 before stabilizing (Fig. 4B, C).
Moreover, there was a significant (P ⱕ 0.05) genotype/folate
intake interaction. The genotype response pattern to 400 ␮g/d
FIGURE 5 Weekly plasma total homocysteine (tHcy) concentrations in Mexican American women with differing MTHFR C677T genotypes during folate depletion (14 CC, 12 CT, 17 TT) (A) and repletion
with 400 ␮g/d DFE (7 CC, 6 CT, 9 TT) (B) and 800 ␮g/d DFE (7 CC, 6
CT, 8 TT) (C). *,**Weekly plasma tHcy concentration for the combined
MTHFR genotypes (n ⫽ 43) differs from wk 0 (A) or wk 7 (B, C) at P
⫽ 0.05 or P ⫽ 0.01, respectively. ⫹,⫹⫹Weekly urinary folate concentration for the combined MTHFR genotypes (n ⫽ 43) differs from the
preceding week at P ⫽ 0.05 or P ⫽ 0.01, respectively. Values are
means ⫾ SEM. a,bMTHFR genotypes with differing superscript letters
differ across all weeks, P ⱕ 0.05.
Downloaded from jn.nutrition.org at TIMMINS & DISTRICT HOSPITAL on June 13, 2012
Folate depletion increased (P ⱕ 0.05) the plasma tHcy
concentration 31%, from 5.5 ⫾ 0.83 to 7.2 ⫾ 1.2 ␮mol/L.
Throughout depletion, tHcy concentrations between the
MTHFR C667T genotypes did not differ (P ⬎ 0.05) (Fig. 5A).
Repletion with 400 ␮g/d DFE decreased (P ⱕ 0.05) the
tHcy concentration 22%, from 7.1 ⫾ 1.2 to 5.6 ⫾ 0.93
␮mol/L, and all subjects had plasma tHcy concentrations in
the desirable range (i.e., ⬍10 ␮mol/L). Throughout repletion
with 400 ␮g/d DFE, the TT genotype had higher (P ⱕ 0.05)
tHcy concentrations than that of the CC control but the CT
and CC genotypes did not differ (P ⬎ 0.05) (Fig. 5B).
Repletion with 800 ␮g/d DFE decreased (P ⱕ 0.05) the
plasma tHcy concentration 24%, from 7.4 ⫾ 1.2 to 5.5 ⫾ 0.81
GUINOTTE ET AL.
1278
␮mol/L. Throughout repletion with 800 ␮g/d DFE, the
MTHFR C677T genotypes did not differ (P ⬎ 0.05) (Fig. 5C).
During the repletion phase of the study, there were no
three-way or genotype/week interactions for plasma tHcy;
however, there was a significant (P ⱕ 0.05) folate intake/week
interaction. In the 400 ␮g/d DFE group, plasma tHcy decreased (P ⱕ 0.01) initially at wk 9 but increased (P ⱕ 0.01)
to postdepletion concentrations at wk 11 and 13. In the 800
␮g/d DFE group, the initial decline (P ⱕ 0.01) in plasma tHcy
concentrations at wk 8 was maintained throughout the repletion phase (Fig. 5B, C); in addition there was a significant (P
ⱕ 0.05) genotype/folate intake interaction. The genotype
response pattern to 400 ␮g/d DFE for plasma tHcy was CC
⬍ CT ⬍ TT compared to TT ⬍ CC ⬍ CT for 800 ␮g/d DFE.
DISCUSSION
Downloaded from jn.nutrition.org at TIMMINS & DISTRICT HOSPITAL on June 13, 2012
Data derived from observational and intervention studies
suggest that the MTHFR C3 T polymorphism negatively influences folate and homocysteine status (8,13,15,19,39 – 41).
We designed the present study to test the hypothesis that the
MTHFR 677 C3 T variant modulates folate status and to
assess the adequacy of the 1998 folate U.S. RDA. Unique
aspects of the study design included strict control of folate
intake; repletion with the 1998 folate U.S. RDA, 400 ␮g/d
DFE, and a higher level, 800 ␮g/d DFE, after a moderate folate
depletion phase; and inclusion of premenopausal women of
Mexican descent representing all three MTHFR C677T genotypes.
The results of this study provide definitive data that the
MTHFR C3 T polymorphism negatively influences folate and
homocysteine status responses. Throughout folate depletion,
the TT genotype had lower (P ⱕ 0.05) serum folate and red
cell folate concentrations compared to those of the CC control. Throughout repletion with 400 ␮g/d DFE, the TT genotype had lower (P ⱕ 0.05) serum folate concentrations and
higher (P ⱕ 0.05) plasma tHcy concentrations than those of
the CC control. During repletion with 800 ␮g/d DFE, the CT
genotype had lower (P ⱕ 0.05) serum folate concentrations
and excreted less (P ⱕ .05) urinary folate than those of the CC
control.
The lower (P ⱕ 0.05) folate status observed in the TT
genotype relative to the CC genotype was confined to those
consuming 400 ␮g/d DFE or less. On the highest folate intake,
800 ␮g/d DFE, no differences (P ⬎ 0.05) in folate status were
observed between the TT and CC genotypes. These data are
consistent with previous studies that have demonstrated an
interaction between folate status/intake and the MTHFR 677
TT genotype (18,19,39). In the present study, the genotype
response on 400 ␮g/d DFE followed the expected pattern, TT
⬍ CT ⬍ CC for serum and urinary folate and TT ⬎ CT ⬎ CC
for plasma tHcy concentrations. A different (P ⱕ 0.05) pattern emerged, however, between the MTHFR C677T genotypes repleted with 800 ␮g/d DFE, CT ⬍ TT ⬍ CC for serum
and urinary folate and CT ⬎ CC ⬎ TT for plasma tHcy. The
data regarding plasma tHcy, however, must be interpreted with
caution since plasma tHcy concentrations for the TT genotype
were numerically the highest and lowest at the beginning of
the repletion phase with 400 and 800 ␮g/d DFE, respectively.
Possible explanations for the apparent enhanced sensitivity of
the 677 TT genotype to increased folate intake observed in the
present study and others (15,18,39,40,42,43) may include decreased loss of the MTHFR flavin cofactor in the presence of
high folate (44), upregulation of MTHFR and/or upregulation
of hepatic betaine:homocysteine methyltransferase (45).
The relatively low plasma tHcy concentrations in all three
MTHFR C677T variants after folate depletion (7.2 ␮mol/L),
although unexpected, may be attributable to the low initial
tHcy concentrations (5.5 ␮mol/L). Other studies have reported an ⬃2 ␮mol/L increase in plasma tHcy after a low
folate diet (15,46), which is similar to the increase in the
present study. Race or ethnicity may also be a factor because
lower plasma tHcy concentrations have been reported in persons of Mexican descent compared to Caucasians (47). Sufficient dietary intake of the lipotropes, methionine and choline,
as well as the B vitamins that serve as cofactors for homocysteine metabolizing enzymes (i.e., riboflavin, vitamins B-12 and
B-6), also provide a plausible explanation for the lack of
hyperhomocysteinemic individuals after low folate intake.
Choline, for example, is utilized as a methyl donor when folate
intake is low (48).
When establishing the 1998 folate U.S. RDA, 400 ␮g/d
DFE, red cell folate concentration was used as the primary
indicator of folate status, whereas serum folate and plasma
tHcy concentrations served as ancillary markers (28). Red cell
folate concentration reflects tissue stores and is considered a
long-term indicator of folate status (49,50). Serum folate reflects recent dietary folate intake (50); however, under conditions of controlled folate intake, repeated measurements of
serum folate concentrations in the same person over time
reflect changes in folate status (28). Plasma tHcy is a sensitive
functional indicator of folate status and increases under conditions of folate inadequacy (30,32,46). However, plasma tHcy
is a nonspecific marker of folate status, because other vitamin
deficiencies, genetic variations and exogenous factors may
influence it (51,52). In the present study, repletion with 400
␮g/d DFE achieved or maintained serum folate concentrations
⬎ 6.8 nmol/L, red cell folate concentrations ⬎ 317 nmol/L
and plasma tHcy concentrations ⬍ 12 ␮mol/L, concentrations
reported by the IOM 1998 (28) to reflect folate sufficiency.
However, 50% of women possessing the 677 TT genotype and
32% overall had serum folate concentrations indicative of
marginal deficiency (between 6.8 and 14 nmol/L) at the end of
the repletion period with 400 ␮g/d DFE. This may be physiologically unimportant because plasma tHcy concentrations
were low (5.6 ⫾ 0.9 ␮mol/L) after folate repletion with 400
␮g/d DFE and similar (P ⬎ 0.05) to plasma tHcy concentrations in the 800 ␮g/d DFE group, regardless of MTHFR C677T
genotype. Together, these data suggest that the 1998 folate
U.S. RDA is sufficient for all three MTHFR C677T genotypes.
The role of folate in reducing the risk of vascular disease,
cancer and psychiatric mental disorders was also considered
when devising the 1998 folate U.S. RDA. Data, however, were
not sufficient to serve as a basis for setting folate recommendations (28). Of the folate status indicators, the functional
indicator, plasma tHcy, has garnered the most attention and is
considered a risk factor for vascular disease when elevated
(20). In the present study, the 1998 folate U.S. RDA was
effective in preventing even mild elevations in plasma tHcy
concentrations in all subjects, regardless of MTHFR C677T
genotype, and will provide useful information if future folate
U.S. RDAs are based on risk reduction of chronic diseases or
developmental disorders.
To summarize, these data demonstrate that the MTHFR
C3 T variant modulates folate status response to controlled
folate intakes. The lack of relationship between the TT genotype and folate nutriture at the highest intake, 800 ␮g/d DFE,
indicates that folate intake plays a critical role in the ultimate
determination of phenotype. Importantly, the 1998 folate U.S.
RDA, 400 ␮g/d DFE, led to normal blood folate concentrations and desirable plasma tHcy concentrations in women of
Mexican descent, regardless of MTHFR C677T genotype.
MTHFR C3 T VARIANT AND FOLATE STATUS
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