Abstract
We previously showed that provision of the folate recommended dietary allowance and either 300, 550, 1100, or 2200 mg/d choline for 12 wk resulted in diminished folate status and a tripling of plasma total homocysteine (tHcy) in men with the methylenetetrahydrofolate reductase (MTHFR) 677TT genotype. However, the substantial variation in tHcy within the 677TT genotype at wk 12 implied that several factors were interacting with this genotype to affect homocysteine. As an extension of this work, the present study sought to identify the main predictors of wk-12 plasma tHcy, alone and together with the MTHFR C677T genotype (29 TT, 31 CC), using linear regression analysis. A basic model explaining 82.5% of the variation (i.e. adjusted R2 = 0.825) was constructed. However, the effects of the variables within this model were dependent upon the MTHFR C677T genotype (P for interaction ≤ 0.021). Within the 677TT genotype, serum folate (P = 0.005) and plasma riboflavin (P = 0.002) were strong negative predictors (inversely related) explaining 12 and 15%, respectively, of the variation in tHcy, whereas choline intake (P = 0.003) and serum creatinine (P < 0.001) were strong positive predictors, explaining 19 and 25% of the variation. None of these variables, except creatinine (P = 0.021), correlated with tHcy within the 677CC genotype. Of the 8 additional polymorphisms tested, none appeared to influence tHcy. However, when creatinine was not in the model, the phosphatidylethanolamine N-methyltransferase 5465G→A variant predicted lower tHcy (P < 0.001); an effect confined to the MTHFR 677TT genotype. Thus, in folate-deplete men, several factors with roles in 1-carbon metabolism interact with the MTHFR C677T genotype to affect plasma tHcy.
Introduction
Methylenetetrahydrofolate reductase (MTHFR)7 is a flavoprotein requiring flavin adeninine dinucleotide, a derivative of riboflavin, as its coenzyme. MTHFR is expressed in all mammalian cells and catalyzes the unidirectional conversion of 5,10-methylene-tetrahydrofolate (THF), a product of cytosolic serine hydroxymethyltransferase, to 5-methylTHF (Fig. 1). In doing so, MTHFR directs 1-carbon units derived from 5,10-methyleneTHF toward cellular methylation reactions at the expense of nucleotide biosynthesis. 5-methylTHF is used to convert homocysteine to methionine in a reaction catalyzed by the vitamin B-12–dependent enzyme, methionine synthase. Alternatively, in liver and kidney, betaine derived from the diet and/or from choline can be used for the remethylation of homocysteine to methionine in a reaction catalyzed by betaine homocysteine methyltransferase (BHMT). Once formed, methionine may be activated to S-adenosylmethionine (AdoMet), a methyl donor for over 50 mammalian methyltransferases (1). Quantitatively, the syntheses of creatine by guanidinoacetate methyltransferase (GAMT), of phosphatatidylcholine by phosphatidylethanolamine N-methyltransferase (PEMT), and of sarcosine by glycine N-methyltransferase (GNMT) represent the most important pathways for transmethylation reactions involving AdoMet (1,2) and are likely to be the greatest producers of S-adenosylhomocysteine and homocysteine (3–5).
FIGURE 1 .
Schematic of 1-carbon metabolism with key enzymes and nutrients denoted with light or dark backgrounds, respectively. B2, riboflavin; B6, vitamin B-6; B12, vitamin B-12; CBS, cystathionine β synthase; DHF, dihydrofolate; DMG, dimethylglycine; GAMT, guanidinoacetate methyltransferase; Gly, glycine; Hcy, homocysteine; Met, methionine; MTHFD1, methylenetetrahydrofolate dehydrogenase; MTRR, methionine synthase reductase; Ser, serine; THF, tetrahydrofolic acid; 5-CH3-THF, 5-methyltetrahydrofolic acid; 5,10-CH2-THF, 5,10-methylenetetrahydrofolic acid; 10-CHO-THF, 10-formyltetrahydrofolic acid.
Hyperhomocysteinemia is a biomarker of perturbed 1-carbon metabolism and is associated with increased risk for numerous chronic and developmental diseases (6). Suboptimal intakes of the B vitamins folate, vitamin B-12, and riboflavin contribute to hyperhomocysteinemia (7,8) as do genetic deficiencies in 1-carbon metabolizing enzymes (7,9). Of the common genetic variants in such enzymes, the MTHFR 677C→T is the most important determinant of plasma total homocysteine (tHcy), especially when folate status is low (9–11).
Our research group recently conducted a 12-wk controlled feeding study in Mexican-American men with the MTHFR 677CC or 677TT genotype and found that the folate recommended dietary allowance (RDA) was inadequate for men with the 677TT genotype (12). Specifically, at the end of this controlled feeding study, which provided the folate RDA, 34% of men with the MTHFR 677TT genotype had serum folate concentrations in the deficient range (<6.8 nmol/L) and 79% had fasting plasma tHcy concentrations > 14 μmol/L compared with 16 and 7%, respectively, of men with the 677CC genotype (12). In men with the 677TT genotype, plasma tHcy (mean ± SD) tripled from 11.5 ± 7.5 μmol/L at baseline to 30.9 ± 16.7 μmol/L at wk 12, an increase that was not attenuated by increased choline intakes up to 2200 mg/d (12). However, the substantial variation in tHcy within the 677TT genotype ranging from 8 to 63μmol/L implied that several factors were interacting with this genotype to affect homocysteine. Thus, this study sought to identify the main nutritional and genetic determinants of plasma tHcy in Mexican-American men with suboptimal folate status, either alone or in combination with the MTHFR C677T genotype, using linear regression analysis.
Participants and Methods
Participants.
The study participants were healthy Mexican-American men preselected for the MTHFR 677CC or TT genotype and recruited from Pomona, California (and surrounding areas) between June 2005 and September 2006. Additional inclusion study criteria were: age 18–55 y; nonsmoking; no use of medication known to interfere with folate metabolism/measurements; no history of chronic diseases; nonanemic with hemoglobin >120 g/L; normal functioning liver and kidney; and a BMI <38 kg/m2. Seventy-two men fulfilled these criteria and participated in the study. However, only 60 of them completed the study and were included in the analysis (12). Informed consent was obtained from all study participants and the experimental procedures were approved by the Cal Poly Pomona Institutional Review Board for Human Subjects.
Study design and diet.
The original study was a 12-wk controlled feeding study providing the folate RDA and involving randomization of the study participants with the MTHFR 677CC (n = 31) or 677TT (n = 29) genotype to 1 of 4 choline intake levels: 300 (n = 14), 550 (n = 17), 1100 (n = 16), or 2200 mg/d (n = 13). The varying levels of choline intake were achieved using a choline chloride supplement (USP, BCP Ingredients) as previously detailed (12). The diet used during the study followed a 5-d menu, which was repeated for 12 wk (12). The study approximated the folate RDA by providing 438 μg/d dietary folate equivalents, with 319 μg/d derived from food folate, measured microbiologically (13) after trienzyme extraction (14), and 70 μg/d (or 119 dietary folate equivalents per day) from supplemental folic acid consumed with a meal. The diet also provided an average of 300 mg/d choline and 173 mg/d betaine measured by liquid chromatography-MS (LCMS) after phenol chloroform extraction (15,16). Other essential nutrients were supplemented to provide at least 90% of the dietary reference intakes (17–20) if they were not met by the diet; for potassium, the 1989 RDA was used (21). Compliance with the study protocol was demonstrated by serum folate and plasma choline response to the feeding and supplemental protocols, respectively (12,22). As an extension of this work, the present study examined the effects of B vitamin status, choline status, and common genetic variants in proteins with roles in homocysteine metabolism (Table 1) on wk-12 plasma tHcy.
TABLE 1.
Independent variables examined as predictors of wk-12 plasma tHcy concentrations in Mexican-American men with the MTHFR 677CC or TT genotype12
| Genetic variants | Nutritional factors |
|---|---|
| MTHFR C677T | Serum folate |
| PEMT G-744C | Serum vitamin B-12 |
| PEMT G5465A | Plasma riboflavin |
| MTHFD1 G1958A | Plasma choline |
| cSHMT C1420T | Plasma betaine |
| MTR A2756G | Plasma phosphatidylcholine |
| MTRR A66G | Choline intake |
| BHMT G742A | |
| GNMT C1289T |
Age, serum creatinine, and BMI were also included as examined independent variables.
Abbreviations used: MTHFD1, methylenetetrahydrofolate dehydrogenase; MTRR, methionine synthase reductase.
Sample collection and measurements.
Blood samples were collected from fasted (10-h) participants at baseline and then once per week as previously detailed (12). For the purposes of the present study, all measurements took place during wk 9–12. Plasma homocysteine (wk 12) was measured using a modified HPLC method with fluorometric detection (23). Plasma choline, betaine, and phosphatidylcholine were measured at wk 9 by LCMS (15,24) with modifications based on our equipment (16). Serum cobalamin (wk 12) was measured using Immulite 1000 (Siemens), serum creatinine (wk 12) was measured with an automated analyzer (Roche Hitachi pModular), and plasma riboflavin (wk 12) was measured by LCMS as described by Midttun et al. (25).
Genotyping.
DNA for genotyping was extracted from leukocytes using a commercially available kit (QIAmp DNA Blood Mini kit, Qiagen) followed by amplification of the DNA by PCR. Determination of the C677T MTHFR genotype was made by the method of Frosst et al. (26). Determination of all other genotypes (Table 1) was made after purifying the PCR products (QIAquick PCR Purification kit, Qiagen) and sequencing the double-stranded DNA templates with the Applied Biosystems Automated 3730 DNA analyzer.
Statistical analysis.
We used 2-way ANOVA to assess differences in the measured variables at the end of the study (wk 9–12) between the MTHFR 677CC and TT genotypes and between choline intake groups. The Levene's test was employed for testing the assumption of equal variance among the choline intake groups. Because the assumption of homogeneity was not met, we used a Games-Howell correction for mean separation. Linear regression analysis was used to examine how various independent variables influenced plasma homocysteine at wk 12. In the first regression model, plasma homocysteine was entered as the dependent variable; MTHFR C677T genotype and baseline plasma tHcy were included as covariates. To this basic model, nutritional factors as well as age, BMI, and serum creatinine were added (Table 1). During the construction of the basic model, we investigated 2-way interactions between variables. In the final basic model, only significant terms were preserved. Because all of the independent variables interacted with the MTHFR C677T genotype to affect wk-12 plasma tHcy, the data were presented separately for each MTHFR C677T genotype (i.e. CC or TT). To assess the strength of each independent variable contained in the final model within the MTHFR genotypes, the term of interest was removed and the change in the adjusted R2 was used as a measure of the variability explained by this term. In a 2nd regression model, the predictive ability of the genetic variants of interest (Table 1) on plasma tHcy was investigated using model I as a basis. Due to low frequencies of the methionine synthase (MTR), cytosolic serine hydroxymethyltransferase (cSHMT), and BHMT homozygous variant allele genotypes, only the wild-type and heterozygote genotype were examined. The data were analyzed by SPSS software, version 15, and are presented as means ± SEM unless indicated otherwise.
Results
Sixty men with the MTHFR 677 CC (n = 31) or TT (n = 29) completed this controlled feeding study. The concentrations of the measured independent variables stratified by MTHFR C677T genotype and choline intake at the end of the dietary intervention (wk 12) are shown (Table 2). Compared with men with the MTHFR 677CC genotype, men with the 677TT genotype had higher (P < 0.001) concentrations of plasma tHcy and lower (P < 0.05) concentrations of serum folate and serum creatinine; plasma phosphatidylcholine tended (P = 0.056) to be lower (Table 2). In addition, choline intake modified (P < 0.05) the response of plasma choline and plasma betaine (Table 2). For plasma choline, concentrations were highest in the 2200-mg/d group, intermediate in the 1100- and 550-mg/d groups, and lowest in the 300-mg/d group. A similar pattern was observed for plasma betaine, although the 1100- and 2200-mg/d groups did not differ (P > 0.05). The genotype frequencies for the additional genes analyzed in this study are presented in Table 3.
TABLE 2.
Analyte concentrations at the end of the controlled feeding study (wk 9–12) in Mexican-American men with the MTHFR 677CC or 677TT genotype consuming choline intakes of 300, 550, 1100, or 2200 mg/d1
| Choline Intake, mg/d
|
P-values
|
|||||||
|---|---|---|---|---|---|---|---|---|
| Variable | 300 | 550 | 1100 | 2200 | All | Genotype | Choline | Interaction |
| Plasma tHcy, μmol/L | <0.001 | 0.100 | 0.135 | |||||
| 677CC | 11.4 ± 0.6 | 11.7 ± 0.5 | 11.9 ± 0.9 | 11.1 ± 0.8 | 11.6 ± 0.3 | |||
| 677TT | 25.1 ± 5.4 | 26.7 ± 6.1 | 42.5 ± 4.4 | 27.8 ± 8.0 | 30.9 ± 3.1* | |||
| Total | 18.2 ± 3.2 | 18.8 ± 3.3 | 27.2 ± 4.5 | 18.8 ± 4.3 | ||||
| Serum folate, nmol/L | 0.004 | 0.393 | 0.751 | |||||
| 677CC | 10.5 ± 1.9 | 11.9 ± 1.8 | 10.7 ± 1.5 | 12.3 ± 1.9 | 11.4 ± 0.9 | |||
| 677TT | 8.4 ± 1.2 | 7.1 ± 0.8 | 6.7 ± 0.7 | 10.3 ± 1.7 | 8.0 ± 0.6* | |||
| Total | 9.4 ± 1.1 | 9.6 ± 1.2 | 8.7 ± 0.9 | 11.4 ± 1.3 | ||||
| Plasma riboflavin, nmol/L | 0.384 | 0.668 | 0.816 | |||||
| 677CC | 4.2 ± 1.0 | 5.3 ± 1.4 | 6.5 ± 1.8 | 7.0 ± 2.8 | 5.7 ± 0.9 | |||
| 677TT | 3.4 ± 0.7 | 5.3 ± 1.1 | 5.9 ± 3.3 | 3.5 ± 0 0.8 | 4.7 ± 1.0 | |||
| Total | 3.8 ± 0.6 | 5.3 ± 0.9 | 6.2 ± 1.9 | 5.4 ± 1.6 | ||||
| Serum cobalamin, pmol/L | 0.352 | 0.993 | 0.661 | |||||
| 677CC | 350 ± 44 | 358 ± 38 | 323 ± 34 | 319 ± 41 | 339 ± 19 | |||
| 677TT | 289 ± 31 | 301 ± 41 | 324 ± 35 | 335 ± 30 | 312 ± 17 | |||
| Total | 320 ± 27 | 331 ± 28 | 323 ± 23 | 326 ± 24 | ||||
| Plasma betaine, μmol/L | 0.345 | <0.001 | 0.598 | |||||
| 677CC | 32 ± 4 | 58 ± 5 | 69 ± 9 | 109 ± 15 | 67 ± 6 | |||
| 677TT | 32 ± 2 | 47 ± 3 | 73 ± 9 | 93 ± 15 | 60 ± 6 | |||
| Total | 32 ± 2c | 53 ± 3b | 71 ± 6a,b | 102 ± 11a | ||||
| Plasma choline, μmol/L | 0.211 | < 0.001 | 0.855 | |||||
| 677CC | 5.5 ± 0.5 | 8.4 ± 0.6 | 9.7 ± 0.6 | 12.2 ± 1.0 | 8.9 ± 0.5 | |||
| 677TT | 6.0 ± 0.5 | 8.5 ± 0.9 | 10.5 ± 1.1 | 13.8 ± 1.2 | 9.5 ± 0.7 | |||
| Total | 5.7 ± 0.3c | 8.4 ± 0.5b | 10.1 ± 0.6b | 12.9 ± 0.8a | ||||
| Plasma phosphatidylcholine, μmol/L | 0.056 | 0.881 | 0.265 | |||||
| 677CC | 1671 ± 105 | 1936 ± 62 | 1867 ± 109 | 1926 ± 121 | 1856 ± 50 | |||
| 677TT | 1783 ± 161 | 1672 ± 58 | 1690 ± 106 | 1672 ± 100 | 1704 ± 52 | |||
| Total | 1727 ± 94 | 1812 ± 53 | 1778 ± 77 | 1809 ± 85 | ||||
| Serum creatinine, μmol/L | 0.003 | 0.652 | 0.505 | |||||
| 677CC | 99.8 ± 5.4 | 90.3 ± 2.9 | 91.9 ± 3.7 | 89.6 ± 6.2 | 92.6 ± 2.2 | |||
| 677TT | 82.1 ± 3.7 | 84.0 ± 4.1 | 86.2 ± 4.3 | 81.1 ± 4.8 | 83.5 ± 2.0* | |||
| Total | 91.0 ± 4.0 | 87.3 ± 2.5 | 88.9 ± 2.8 | 85.7 ± 4.0 | ||||
Values are mean ± SEM, n = 6–9. Means in a row with superscripts without a common letter differ, P < 0.05. *Different from corresponding MTHFR 677CC genotype, P < 0.05 (2-way ANOVA).
TABLE 3.
Frequencies of the tested genotypes in all study participants and within the MTHFR 677CC or 677TT genotype12
| MTHFR C677T
|
|||
|---|---|---|---|
| Genotype | Total | 677CC | 677TT |
| PEMT G-744C | |||
| 744GG | 11 | 7 | 4 |
| 744GC | 23 | 8 | 15 |
| 744CC | 26 | 16 | 10 |
| PEMT G5465A | |||
| 5465GG | 16 | 11 | 5 |
| 5465GA | 23 | 9 | 14 |
| 5465AA | 21 | 11 | 10 |
| MTHFD1 G1985A | |||
| 1958GG | 13 | 7 | 6 |
| 1958GA | 29 | 16 | 13 |
| 1958AA | 18 | 8 | 10 |
| cSHMT C1420T | |||
| 1420CC | 37 | 24 | 13 |
| 1420CT | 21 | 7 | 14 |
| 1420TT | 2 | 0 | 2 |
| MTR A2756G | |||
| 2756AA | 42 | 21 | 21 |
| 2756AG | 18 | 10 | 8 |
| 2756AG | 0 | 0 | 0 |
| MTRR A66G | |||
| 66AA | 30 | 15 | 15 |
| 66AG | 23 | 13 | 10 |
| 66GG | 7 | 3 | 4 |
| GNMT C1289T | |||
| 1289CC | 22 | 10 | 12 |
| 1289CT | 31 | 18 | 13 |
| 1289TT | 7 | 3 | 4 |
| BHMT G742A | |||
| 742GG | 29 | 14 | 15 |
| 742GA | 27 | 15 | 12 |
| 742AA | 4 | 2 | 2 |
For the cSHMT, MTR, and BHMT genotypes, only the wild-type and heterozygotes were examined in the model due to the low frequencies of the homozygous variants.
Abbreviations used: MTHFD1, methylenetetrahydrofolate dehydrogenase; MTRR, methionine synthase reductase.
The independent variables that emerged in model I as the predictors of plasma tHcy included serum folate, plasma riboflavin, plasma betaine, serum creatinine, and choline intake (Table 4). Based on the adjusted R2, these variables explained 82.5% of the variation in homocysteine. However, the effect of the variables tested in model I was dependent upon the MTHFR C677T genotype (P for interaction ≤ 0.021). Specifically, all variables were significant predictors of plasma tHcy within the MTHFR 677TT genotype and together explained 71% of the variation within this genotype, as indicated by a separate linear regression for this genotype. The variability explained by each independent variable as determined by the change in the adjusted R2 value after the singular exclusion of each factor was 6.3% for plasma betaine, 11.7% for serum folate, 14.7% for plasma riboflavin, 19.3% for choline intake, and 25.0% for serum creatinine. Serum folate, plasma riboflavin, and plasma betaine were inversely related to plasma tHcy within the MTHFR 677TT genotype (Fig. 2), whereas serum creatinine (Fig. 2) and choline intake (Fig. 3) were positively associated. In contrast, within the MTHFR 677CC genotype, only serum creatinine was a significant predictor explaining 12.0% of the variation.
TABLE 4.
Model I with the major determinants of plasma tHcy at the end of the controlled feeding study (wk 9–12) in Mexican-American men with the MTHFR 677CC or 677TT genotype1
| MTHFR 677CC Genotype
|
MTHFR 677TT Genotype
|
Interaction with MTHFR
|
|||||
|---|---|---|---|---|---|---|---|
| Variable | β | SEM | P | β | SEM | P | P |
| Intercept | 6.9 | 2.58 | 0.014 | 12.2 | 16.6 | 0.472 | |
| Serum folate | −0.079 | 0.147 | 0.598 | −4.542 | 1.44 | 0.005 | <0.001 |
| Plasma riboflavin | 0.001 | 0.067 | 0.990 | −1.24 | 0.356 | 0.002 | 0.001 |
| Plasma betaine | −0.016 | 0.014 | 0.268 | −0.218 | 0.091 | 0.026 | 0.021 |
| Serum creatinine | 6.29 | 2.52 | 0.021 | 66.4 | 14.8 | 0.009 | <0.001 |
| Choline intake, mg/d | 0.542 | 0.003 | 0.001 | ||||
| 300 | −1.77 | 1.47 | 0.243 | −20.8 | 7.4 | 0.011 | 0.019 |
| 550 | −0.337 | 1.08 | 0.758 | −17.4 | 6.7 | 0.017 | 0.010 |
| 1100 | −0.636 | 1.04 | 0.547 | 2.25 | 5.8 | 0.700 | 0.618 |
| 2200 | 0 | 0 | |||||
Based on the adjusted R2, model 1 explained 82.5% of the variation in homocysteine overall, 0% of the variation in homocysteine in the MTHFR 677CC genotype, and 71% of the variation in homocysteine in the MTHFR 677TT genotype. The main effect of the MTHFR C677T genotype in the presence of the interaction was not significant in the overall model (P = 0.735).
FIGURE 2 .
Partial residual plots for plasma tHcy compared with plasma riboflavin (A), serum folate (B), plasma betaine (C), and serum creatinine (D) in Mexican-American men with the MTHFR 677CC (n = 31) or TT (n = 29) genotype. Measurements were taken at the end of a controlled feeding study (wk 9–12) providing the folate RDA and 300, 550, 1100, or 2200 mg/d choline. These plots were controlled for the main effects of model I, including serum folate, plasma riboflavin, plasma betaine, serum creatinine, and choline intake.
FIGURE 3 .
Estimated marginal means of plasma tHcy (wk 12) grouped by choline intake (n = 13–17/intake group) in Mexican-American men with the MTHFR 677CC (n = 31) or TT (n = 29) genotype. Data were analyzed using linear regression and are presented as estimated marginal means (controlled for serum folate, plasma riboflavin, plasma betaine, and choline intake) ± SEM. Within a genotype, labeled means without a common letter differ, P < 0.05.
None of the genetic variants assessed in this study were significant predictors within the context of model I. However, the PEMT G5465A genotype interacted with the MTHFR C677T genotype to affect plasma tHcy in model II, which did not include serum creatinine (Fig. 4). The PEMT G5465A genotype explained 12.0% of the variability in plasma tHcy within the MTHFR 677TT.
FIGURE 4 .
Estimated marginal means of plasma tHcy (wk 12) stratified by the PEMT G5465A genotype (n = 16 GG, 23 GA, 21 AA) in Mexican-American men with the MTHFR 677CC (n = 31) or TT (n = 29) genotype. Data were analyzed using linear regression and are presented as estimated marginal means (controlled for serum folate, plasma riboflavin, plasma betaine, and choline intake) ± SEM. Within a genotype, labeled means without a common letter differ, P < 0.05.
Discussion
This study sought to examine the influence of nutritional and genetic factors on plasma tHcy concentrations in Mexican-American men with the MTHFR 677CC or TT genotype at the end of a 12-wk controlled feeding study that resulted in diminished folate status, particularly in the 677TT genotype (12). Under such conditions, the results of the present study demonstrate that Mexican-American men with the MTHFR 677TT genotype are particularly sensitive to the availability of nutrients with roles in 1-carbon metabolism. In this regard, serum folate, plasma riboflavin, and plasma betaine were inversely associated with plasma tHcy in men with the 677TT genotype (Fig. 2), whereas choline intake was positively associated (Fig. 3); no associations with plasma tHcy were observed with these nutrients in men with the 677CC genotype.
The predictive ability as measured with the R2 value among the nutrients with inverse associations with plasma tHcy in the MTHFR 677TT genotype was especially strong for riboflavin and folate. Specifically, the singular exclusion of riboflavin, folate, or betaine from model I decreased the predictive value by 15, 12, and 6 percentage points, respectively. The relatively strong influences of riboflavin and folate on plasma tHcy concentrations within the MTHFR 677TT genotype are in agreement with findings from a large epidemiology study showing that B vitamin status, particularly folate and riboflavin, strongly modified the effect of MTHFR 677TT genotype on plasma tHcy (27).
The positive relationship between choline intake and plasma tHcy concentrations within the MTHFR 677TT genotype was unexpected and was not transparent from our previous work published in this group of men, most likely because of differences in the statistical approach and stage of folate depletion (12). Under the conditions of the present study, which utilized plasma tHcy concentrations at the end of the feeding study and included covariates, consumption of either 1100 or 2200 mg/d by men with the MTHFR 677TT genotype resulted in fasting plasma tHcy concentrations that were approximately twice the concentration observed in the 300- or 550-mg/d groups (Fig. 3). Further, the singular exclusion of choline intake from model I decreased the predictive value by 19.3%, showing that choline intake was a stronger determinant of plasma tHcy, albeit in a different direction, than riboflavin, folate, or betaine. Interestingly, like the other nutrients tested in this model, choline intake was not a predictor of plasma tHcy among men with the MTHFR 677CC genotype.
The mechanism by which choline intake interacts with the MTHFR C677T genotype to increase fasting plasma tHcy concentrations in this study is unknown. However, data generated from a tracer study showed that women with the MTHFR 677TT genotype produced more homocysteine via transmethylation than those with the 677CC genotype at the end of a 7-wk period of folate restriction (28). As suggested by these authors (28), the increased synthesis of homocysteine may be explained by increased GNMT activity, an enzyme subject to reciprocal regulation by AdoMet (positive cooperativity) and 5-CH3THF (allosteric inhibition) (29). In the present study, folate status was particularly low in men with the MTHFR 677TT genotype, a finding consistent with GNMT release from inhibition by 5-methylTHF and enhanced production of homocysteine as shown in rats (30). In turn, increased choline intake may have aggravated this metabolic scenario by driving the remethylation of homocysteine through the BHMT pathway at the expense of its permanent removal via the transsulfuration pathway.
The complexity of the relationship between choline intake and plasma homocysteine is further exemplified with our previous finding that increased choline intakes (1100 and 2200 mg/d) attenuated the rise in plasma homocysteine following a methionine load in this group of men (relative to the 300- and 550-mg/d intake levels) (22). A major metabolic difference between fasting and postmethionine load homocysteine metabolism is the activity of the transsulfuration pathway. By increasing AdoMet, the methionine load stimulates cystathionine β synthase, the regulatory enzyme of the transsulfuration pathway (31). The beneficial effect of increased choline intake under these circumstances may simply reflect greater disposal of homocysteine when 2 pathways are working to remove excess homocysteine (i.e. homocysteine remethylation through BHMT + transsulfuration to cysteine) rather than one (i.e. transsulfuration).
Of all the variables included in model I, serum creatinine was the strongest predictor of plasma homocysteine, explaining 12 and 25% of the variation in plasma tHcy concentrations in the MTHFR 677CC or TT genotype, respectively. Interestingly, compared with men with the MTHFR 677CC genotype, the rise in plasma tHcy with increased serum creatinine was greater in men with the MTHFR 677TT genotype. Because kidney function was normal in all of the study participants, these data suggest that, within the context of this study, men with the MTHFR 677TT genotype were less able to process the extra homocysteine generated by a more active GAMT.
Of the genetic variants tested, none were predictors of plasma tHcy at wk 12 either alone or together with the MTHFR C677T genotype when examined within the context of model I. This lack of association may be due in part to the small number of subjects spread over many genotypes. However, when serum creatinine was not in the model, the PEMT G5465A genotype emerged as a significant predictor of plasma tHcy among men with the MTHFR 677TT genotype, explaining 12% of the variation in this group. Specifically, in men with the MTHFR 677TT genotype, carriers of the PEMT 5465 variant A allele had significantly lower plasma tHcy concentrations than the 5465GG genotype (Fig. 4). As stated earlier, PEMT is a major consumer of AdoMet and producer of S-adenosylhomocysteine and homocysteine (1,3). Thus, the lower plasma tHcy in carriers of the A allele is consistent with in vitro work showing diminished PEMT activity in the variant form of the protein (32). Why an effect of the PEMT G5465A genotype was observed only among men with the MTHFR 677TT genotype is unknown. However, it is possible that the variant form of the PEMT protein was less able to compete for AdoMet, especially in a folate deplete-environment.
It is of interest to note that when baseline plasma tHcy concentrations (wk 0) were used in a similar model, none of the independent variables (measured at baseline) emerged as predictors (P ≤ 0.05) either alone or interacting with the MTHFR C677T genotype (data not shown). Although the effect of choline intake could not be assessed at baseline, analyses conducted with wk 3, 6, and 9 plasma tHcy showed no effects until wk 9 despite marked elevations in plasma concentrations of choline and betaine by wk 3 (22). Taken together, these data strongly suggest that Mexican-American men with the MTHFR 677TT genotype are less able to respond to disruptions in B vitamin homeostasis caused by the dietary and choline treatments administered in this study.
To conclude, the findings of this study demonstrate complex interactions between the nutritional status of these men and the MTHFR C677T genotype. Interestingly, the effects of the nutritional factors investigated in this study were confined to men with the MTHFR 677TT genotype whose folate status was particularly compromised. Against this backdrop of low folate, plasma riboflavin (beneficial) and choline intake (adverse) emerged as strong modifiers of plasma tHcy in men with the 677TT genotype. The unanticipated homocysteine-raising effect of increased choline intake (in the form of supplemental choline chloride) may have arisen from folate-induced metabolic alterations in men with the MTHFR 677TT genotype; however, definitive answers await the results of future investigations. Although the PEMT G5465A genotype is linked to fatty liver disease (32), this study is the first, to our knowledge, to show interactions with the MTHFR C677T genotype and effects on plasma tHcy.
Supported in part by NIH grant number S06GM053933, the California State University Agricultural Research Initiative with master grant funding from the Federal Agricultural Appropriations Bill allocation, grant number 2006-38908-17681, and the USDA/Cooperative State Research, Education, and Extension Service Hatch program.
Author disclosures: M. A. Caudill, N. Dellschaft, C. Solis, S. Hinkis, A. A. Ivanov, S. Nash-Barboza, K. E. Randall, B. Jackson, G. N. Solomita, and F. Vermeylen, no conflicts of interest.
Abbreviations used: AdoMet, adenosylmethionine; BHMT, betaine-homocysteine methyltransferase; cSHMT, cytosolic serine hydroxymethyltransferase; GAMT, guanadinoacetate methyltransferase; GNMT, glycine N-methyltransferase; LCMS, liquid chromatography-MS; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; PEMT, phosphatidylethanolamine N-methyltransferase; RDA, recommended dietary allowance; THF, tetrahydrofolate; tHcy, total homocysteine.
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