The Methylenetetrahydrofolate Reductase 677C→T Polymorphism as a Modulator of a B Vitamin Network with Major Effects on Homocysteine Metabolism
2007; Elsevier BV; Volume: 80; Issue: 5 Linguagem: Inglês
10.1086/513520
ISSN1537-6605
AutoresSteinar Hustad, Øivind Midttun, Jörn Schneede, Dan J. Stein, Tom Grotmol, Per Magne Ueland,
Tópico(s)Metabolism and Genetic Disorders
ResumoFolates are carriers of one-carbon units and are metabolized by 5,10-methylenetetrahydrofolate reductase (MTHFR) and other enzymes that use riboflavin, cobalamin, or vitamin B6 as cofactors. These B vitamins are essential for the remethylation and transsulfuration of homocysteine, which is an important intermediate in one-carbon metabolism. We studied the MTHFR 677C→T polymorphism and B vitamins as modulators of one-carbon metabolism in 10,601 adults from the Norwegian Colorectal Cancer Prevention (NORCCAP) cohort, using plasma total homocysteine (tHcy) as the main outcome measure. Mean concentrations of plasma tHcy were 10.4 μmol/liter, 10.9 μmol/liter, and 13.3 μmol/liter in subjects with the CC (51%), CT (41%), and TT (8%) genotypes, respectively. The MTHFR 677C→T polymorphism, folate, riboflavin, cobalamin, and vitamin B6 were independent predictors of tHcy in multivariate models (P<.001), and genotype effects were strongest when B vitamins were low (P≤.006). Conversely, the MTHFR polymorphism influenced B vitamin effects, which were strongest in the TT group, in which the estimated tHcy difference between subjects with vitamin concentrations in the lowest compared with the highest quartile was 5.4 μmol/liter for folate, 4.1 μmol/liter for riboflavin, 3.2 μmol/liter for cobalamin, and 2.1 μmol/liter for vitamin B6. Furthermore, interactions between B vitamins were observed, and B vitamins were more strongly related to plasma tHcy when concentrations of other B vitamins were low. The study provides comprehensive data on the MTHFR–B vitamin network, which has major effects on the transfer of one-carbon units. Individuals with the TT genotype were particularly sensitive to the status of several B vitamins and might be candidates for personalized nutritional recommendations. Folates are carriers of one-carbon units and are metabolized by 5,10-methylenetetrahydrofolate reductase (MTHFR) and other enzymes that use riboflavin, cobalamin, or vitamin B6 as cofactors. These B vitamins are essential for the remethylation and transsulfuration of homocysteine, which is an important intermediate in one-carbon metabolism. We studied the MTHFR 677C→T polymorphism and B vitamins as modulators of one-carbon metabolism in 10,601 adults from the Norwegian Colorectal Cancer Prevention (NORCCAP) cohort, using plasma total homocysteine (tHcy) as the main outcome measure. Mean concentrations of plasma tHcy were 10.4 μmol/liter, 10.9 μmol/liter, and 13.3 μmol/liter in subjects with the CC (51%), CT (41%), and TT (8%) genotypes, respectively. The MTHFR 677C→T polymorphism, folate, riboflavin, cobalamin, and vitamin B6 were independent predictors of tHcy in multivariate models (P<.001), and genotype effects were strongest when B vitamins were low (P≤.006). Conversely, the MTHFR polymorphism influenced B vitamin effects, which were strongest in the TT group, in which the estimated tHcy difference between subjects with vitamin concentrations in the lowest compared with the highest quartile was 5.4 μmol/liter for folate, 4.1 μmol/liter for riboflavin, 3.2 μmol/liter for cobalamin, and 2.1 μmol/liter for vitamin B6. Furthermore, interactions between B vitamins were observed, and B vitamins were more strongly related to plasma tHcy when concentrations of other B vitamins were low. The study provides comprehensive data on the MTHFR–B vitamin network, which has major effects on the transfer of one-carbon units. Individuals with the TT genotype were particularly sensitive to the status of several B vitamins and might be candidates for personalized nutritional recommendations. The flavoenzyme 5,10-methylenetetrahydrofolate reductase (MTHFR [MIM 236250]) regulates the flow of folates between the production of nucleotides and the supply of methyl groups for methionine synthesis1Kutzbach C Stokstad EL Mammalian methylenetetrahydrofolate reductase: partial purification, properties, and inhibition by S-adenosylmethionine.Biochim Biophys Acta. 1971; 250: 459-477Crossref PubMed Scopus (264) Google Scholar, 2Selhub J Homocysteine metabolism.Annu Rev Nutr. 1999; 19: 217-246Crossref PubMed Scopus (1039) Google Scholar and has major effects on the distribution of intracellular folates.3Bagley PJ Selhub J A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells.Proc Natl Acad Sci USA. 1998; 95: 13217-13220Crossref PubMed Scopus (330) Google Scholar The MTHFR substrate is 5,10-methylenetetrahydrofolate, which is formed from serine and tetrahydrofolate by vitamin B6–dependent serine hydroxymethyltransferase.2Selhub J Homocysteine metabolism.Annu Rev Nutr. 1999; 19: 217-246Crossref PubMed Scopus (1039) Google Scholar, 4Lucock M Folic acid: nutritional biochemistry, molecular biology, and role in disease processes.Mol Genet Metab. 2000; 71: 121-138Crossref PubMed Scopus (613) Google Scholar The product of the MTHFR reaction is 5-methyltetrahydrofolate, which is the methyl donor in the conversion of homocysteine to methionine catalyzed by cobalamin-dependent methionine synthase.2Selhub J Homocysteine metabolism.Annu Rev Nutr. 1999; 19: 217-246Crossref PubMed Scopus (1039) Google Scholar, 4Lucock M Folic acid: nutritional biochemistry, molecular biology, and role in disease processes.Mol Genet Metab. 2000; 71: 121-138Crossref PubMed Scopus (613) Google Scholar Methionine may be incorporated into proteins or may serve as the precursor of S-adenosylmethionine, a universal methyl group donor, which is converted to homocysteine after demethylation.2Selhub J Homocysteine metabolism.Annu Rev Nutr. 1999; 19: 217-246Crossref PubMed Scopus (1039) Google Scholar, 5Finkelstein JD Regulation of homocysteine metabolism.in: Carmel R Jacobsen DW Homocysteine in health and disease. Cambridge University Press, Cambridge, United Kingdom2001: 92-99Google Scholar Homocysteine is metabolized through two vitamin B–dependent pathways and may be either remethylated and recycled as methionine or removed from the remethylation cycle by undergoing irreversible B6-dependent transsulfuration to form cysteine.5Finkelstein JD Regulation of homocysteine metabolism.in: Carmel R Jacobsen DW Homocysteine in health and disease. Cambridge University Press, Cambridge, United Kingdom2001: 92-99Google Scholar This makes homocysteine a key intermediate in one-carbon metabolism and explains why B vitamins involved in the transfer of one-carbon units are related to plasma concentrations of total homocysteine (tHcy).6Vollset SE Refsum H Ueland PM Population determinants of homocysteine.Am J Clin Nutr. 2001; 73: 499-500PubMed Google Scholar The 677C→T transition in the MTHFR gene (dbSNP accession number rs1801133) results in an Ala222Val substitution in the polypeptide chain,7Frosst P Blom HJ Milos R Goyette P Sheppard CA Matthews RG Boers GHJ den Heijer M Kluijtmans LAJ van den Heuvel LP et al.A candidate genetic risk factor for vascular disease: a common mutation at the methylenetetrahydrofolate reductase.Nat Genet. 1995; 10: 111-113Crossref PubMed Scopus (4935) Google Scholar which is associated with a thermolabile8Kang SS Zhou J Wong PW Kowalisyn J Strokosch G Intermediate homocysteinemia: a thermolabile variant of methylenetetrahydrofolate reductase.Am J Hum Genet. 1988; 43: 414-421PubMed Google Scholar enzyme. MTHFR in lymphocytes from subjects with the TT genotype has ∼30% of the catalytic activity of the wild type, whereas the CT genotype has 65% catalytic activity.7Frosst P Blom HJ Milos R Goyette P Sheppard CA Matthews RG Boers GHJ den Heijer M Kluijtmans LAJ van den Heuvel LP et al.A candidate genetic risk factor for vascular disease: a common mutation at the methylenetetrahydrofolate reductase.Nat Genet. 1995; 10: 111-113Crossref PubMed Scopus (4935) Google Scholar Lower catalytic activity is associated with a redistribution of one-carbon substituted folates away from 5-methyltetrahydrofolate toward more oxidized forms, which may be used for DNA synthesis and repair.3Bagley PJ Selhub J A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells.Proc Natl Acad Sci USA. 1998; 95: 13217-13220Crossref PubMed Scopus (330) Google Scholar, 4Lucock M Folic acid: nutritional biochemistry, molecular biology, and role in disease processes.Mol Genet Metab. 2000; 71: 121-138Crossref PubMed Scopus (613) Google Scholar The MTHFR polymorphism is associated with clinical endpoints; an increased risk of cardiovascular disease9Kluijtmans LA van den Heuvel LP Boers GH Frosst P Stevens EM van Oost BA den Heijer M Trijbels FJ Rozen R Blom HJ Molecular genetic analysis in mild hyperhomocysteinemia: a common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease.Am J Hum Genet. 1996; 58: 35-41PubMed Google Scholar, 10Klerk M Verhoef P Clarke R Blom HJ Kok FJ Schouten EG MTHFR 677C→T polymorphism and risk of coronary heart disease—a meta-analysis.JAMA. 2002; 288: 2023-2031Crossref PubMed Scopus (799) Google Scholar and neural tube defects (MIM 601634)11van der Put NM Steegers-Theunissen RP Frosst P Trijbels FJ Eskes TK van den Heuvel LP Mariman EC den Heyer M Rozen R Blom HJ Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida.Lancet. 1995; 346: 1070-1071Abstract Full Text PDF PubMed Scopus (765) Google Scholar, 12Shields DC Kirke PN Mills JL Ramsbottom D Molloy AM Burke H Weir DG Scott JM Whitehead AS The "thermolabile" variant of methylenetetrahydrofolate reductase and neural tube defects: an evaluation of genetic risk and the relative importance of the genotypes of the embryo and the mother.Am J Hum Genet. 1999; 64: 1045-1055Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar and a lower risk of colorectal cancer (MIM 114500)13Ma J Stampfer MJ Giovannucci E Artigas C Hunter DJ Fuchs C Willett WC Selhub J Hennekens CH Rozen R Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer.Cancer Res. 1997; 57: 1098-1102PubMed Google Scholar, 14Ulvik A Vollset SE Hansen S Gislefoss R Jellum E Ueland PM Colorectal cancer and the methylenetetrahydrofolate reductase 677C→T and methionine synthase 2756A→G polymorphisms: a study of 2,168 case-control pairs from the JANUS cohort.Cancer Epidemiol Biomarkers Prev. 2004; 13: 2175-2180PubMed Google Scholar, 15Kono S Chen K Genetic polymorphisms of methylenetetrahydrofolate reductase and colorectal cancer and adenoma.Cancer Sci. 2005; 96: 535-542Crossref PubMed Scopus (92) Google Scholar are found in subjects with the variant compared with the wild-type enzyme. The MTHFR 677C→T polymorphism is the single most important genetic determinant of plasma tHcy.16Rozen R Polymorphisms of folate and cobalamin metabolism.in: Carmel R Jacobsen DW Homocysteine in health and disease. Cambridge University Press, Cambridge, United Kingdom2001: 259-269Google Scholar, 17Bathum L, Pedersen I, Christiansen L, Konieczna A, Sørensen TIA, Kyvik KO. Genetic and environmental influences on plasma homocysteine: results from a Danish twin study. Clin Chem (in press)Google Scholar Much of the interest in this polymorphism stems from its association with moderate hyperhomocysteinemia (MIM 603174), which is a risk factor for occlusive arterial disease,18Nygård O Nordrehaug JE Refsum H Ueland PM Farstad M Vollset SE Plasma homocysteine levels and mortality in patients with coronary artery disease.N Engl J Med. 1997; 337: 230-236Crossref PubMed Scopus (1603) Google Scholar cognitive decline,19Duthie SJ Whalley LJ Collins AR Leaper S Berger K Deary IJ Homocysteine, B vitamin status, and cognitive function in the elderly.Am J Clin Nutr. 2002; 75: 908-913PubMed Google Scholar and osteoporosis.20McLean RR Jacques PF Selhub J Tucker KL Samelson EJ Broe KE Hannan MT Cupples LA Kiel DP Homocysteine as a predictive factor for hip fracture in older persons.N Engl J Med. 2004; 350: 2042-2049Crossref PubMed Scopus (482) Google Scholar, 21Herrmann M Widmann T Colaianni G Colucci S Zallone A Herrmann W Increased osteoclast activity in the presence of increased homocysteine concentrations.Clin Chem. 2005; 51: 2348-2353Crossref PubMed Scopus (99) Google Scholar It is still not clear whether these conditions are caused by homocysteine toxicity22Hofmann MA Lalla E Lu Y Gleason MR Wolf BM Tanji N Ferran Jr, LJ Kohl B Rao V Kisiel W et al.Hyperhomocysteinemia enhances vascular inflammation and accelerates atherosclerosis in a murine model.J Clin Invest. 2001; 107: 675-683Crossref PubMed Scopus (445) Google Scholar or if an elevated concentration of plasma tHcy is mainly an epiphenomenon.23Wald DS Law M Morris JK Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis.BMJ. 2002; 325: 1202-1208Crossref PubMed Scopus (1626) Google Scholar, 24Splaver A Lamas GA Hennekens CH Homocysteine and cardiovascular disease: biological mechanisms, observational epidemiology, and the need for randomized trials.Amer Heart J. 2004; 148: 34-40Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 25Bønaa KH Njølstad I Ueland PM Schirmer H Tverdal A Steigen T Wang H Nordrehaug JE Arnesen E Rasmussen K Homocysteine lowering and cardiovascular events after acute myocardial infarction.N Engl J Med. 2006; 354: 1578-1588Crossref PubMed Scopus (1208) Google Scholar Most published work on B vitamins and homocysteine has focused on folate and cobalamin in smaller studies, which do not allow a comprehensive investigation of the various components of the metabolic network related to one-carbon metabolism. The present study included 10,601 middle-aged or elderly men and women from a population-based cohort, and our aim was to assess the MTHFR polymorphism and B vitamins as modulators of one-carbon metabolism, with use of plasma tHcy as the main outcome measure. The Norwegian Colorectal Cancer Prevention (NORCCAP) cohort was established to study the utility of sigmoidoscopy and occult blood testing as screening modalities for early detection of colorectal cancer in men and women aged 50–64 years.26Bretthauer M Thiis-Evensen E Huppertz-Hauss G Gisselsson L Grotmol T Skovlund E Hoff G NORCCAP (Norwegian Colorectal Cancer Prevention): a randomised trial to assess the safety and efficacy of carbon dioxide versus air insufflation in colonoscopy.Gut. 2002; 50: 604-607Crossref PubMed Scopus (171) Google Scholar Study subjects were randomly selected from the population registries in the city of Oslo and the county of Telemark and were included from 1999 to 2001. The study was approved by the Regional Ethics Committee and the Data Inspectorate. The procedures followed were in accordance with the Helsinki Declaration, and written informed consent was obtained from all participants. Whole blood collected into EDTA tubes was immediately put on ice and was centrifuged at 11,000 g for 10 min to obtain plasma. Blood samples collected into serum tubes without additives were allowed to clot at room temperature and were centrifuged within 1 h. Plasma and serum samples were stored at −80°C until analysis. tHcy was analyzed in plasma by gas chromatography–mass spectrometry,27Windelberg A Årseth O Kvalheim G Ueland PM Automated assay for the determination of methylmalonic acid, total homocysteine, and related amino acids in human serum or plasma by means of methylchloroformate derivatization and gas chromatography-mass spectrometry.Clin Chem. 2005; 51: 2103-2109Crossref PubMed Scopus (117) Google Scholar whereas riboflavin, vitamin B6 (pyridoxal-5′-phosphate), and creatinine were analyzed in plasma by liquid chromatography–tandem mass spectrometry.28Midttun Ø Hustad S Solheim E Schneede J Ueland PM Multianalyte quantification of vitamin B6 and B2 species in the nanomolar range in human plasma by liquid chromatography-tandem mass spectrometry.Clin Chem. 2005; 51: 1206-1216Crossref PubMed Scopus (88) Google Scholar Folate29Molloy AM Scott JM Microbiological assay for serum, plasma, and red cell folate using cryopreserved, microtiter plate method.Methods Enzymol. 1997; 281: 43-53Crossref PubMed Scopus (338) Google Scholar and cobalamin30Kelleher BP Broin SD Microbiological assay for vitamin B12 performed in 96-well microtitre plates.J Clin Pathol. 1991; 44: 592-595Crossref PubMed Scopus (213) Google Scholar were determined in serum by microbiological methods. MTHFR 677C→T genotyping was performed by real-time PCR with 5V exonuclease (Taqman) probes.31Ulvik A Ueland PM Single nucleotide polymorphism (SNP) genotyping in unprocessed whole blood and serum by real-time PCR: application to SNPs affecting homocysteine and folate metabolism.Clin Chem. 2001; 47: 2050-2053PubMed Google Scholar Means and medians with percentiles were used for descriptive statistics. Correlation analyses were performed using partial Spearman correlation coefficients adjusted for sex, age, and study center. The χ2 test was used to compare proportions. We used simple linear regression models and models with multiple adjustments to identify predictors of tHcy. Independent variables were represented in the model as indicator variables denoting membership in two or more categories for sex, age, the MTHFR 677C→T polymorphism, folate, riboflavin, cobalamin, vitamin B6, and creatinine. Thus, the regression coefficients estimated the difference in mean tHcy between the chosen reference category and the other categories for each factor. Concentrations of tHcy across categories of each factor were tested for linear trend. We investigated the possible interaction between MTHFR 677C→T genotype and other tHcy predictors, by including product terms between genotype and each predictor in a multiple linear regression model with tHcy as the dependent variable, retaining the main effects of all variables in the model. Furthermore, the data were presented according to MTHFR genotype and B vitamin concentrations, and the regression analyses were performed separately for the CC, CT, and TT genotypes, at concentrations of folate, riboflavin, cobalamin, and vitamin B6 below and above the median. All statistical analyses were performed by SPSS version 11.0. Tests were two-tailed, and P values <.05 were considered statistically significant. The study population (n=10,601, 49% male) was predominantly white (>98%) and had a mean age of 56 years (table 1). MTHFR 677C→T genotype frequencies were 50.3% (CC), 41.3% (CT), and 8.3% (TT) for men and 52.5% (CC), 39.8% (CT), and 7.7% (TT) for women. The genotype distribution was in Hardy-Weinberg equilibrium for each sex and for the whole study group (P≥.69). Mean concentrations of metabolites and vitamins measured for all genotypes combined were 10.8 μmol/liter for plasma tHcy, 17.1 nmol/liter for serum folate, 18.1 nmol/liter for plasma riboflavin, 331 pmol/liter for serum cobalamin, 62.8 nmol/liter for plasma vitamin B6, and 70 μmol/liter for plasma creatinine (table 1). Plasma tHcy was higher in subjects with the T allele as compared with the wild type. In subjects with plasma tHcy ≥20 μmol/liter (n=209), the prevalence of the TT genotype was 48% (data not shown). Serum folate decreased according to the number of T alleles. Concentrations of riboflavin, cobalamin, and vitamin B6 were independent of genotype (table 1).Table 1Characteristics of the Study PopulationAllMTHFR 677C→T Genotype (Median [1st–99th Percentile])CharacteristicMeanMedian (1st–99th Percentile)CC (n=5,452)CT (n=4,299)TT (n=850)P for trendAge, years5655 (50–64)55 (50–64)55 (50–64)55 (50–64).11Plasma tHcy, μmol/liter10.810.2 (5.8–24.2)9.9 (5.7–19.2)10.4 (5.9–22.8)11.2 (5.7–43.3)<.001Serum folate, nmol/liter17.113.7 (4.7–59.8)14.5 (5.2–60.8)13.4 (4.8–59.9)10.5 (3.5–50.1)<.001Plasma riboflavin, nmol/liter18.110.4 (3.0–135)10.4 (3.0–150)10.4 (2.8–135)10.7 (2.8–119).06Serum cobalamin, pmol/liter331307 (128–732)308 (127–734)308 (130–730)300 (113–766).19Plasma vitamin B6, nmol/liter62.848.0 (12.7–295)47.9 (13.3–280)49.0 (12.6–336)44.4 (11.9–286).34Plasma creatinine, μmol/liter7069 (44–105)69 (44–106)70 (45–104)66 (45–101).01 Open table in a new tab Simple relationships between variables were calculated as nonparametric Spearman correlation coefficients, which were adjusted for sex, age, and study center. Concentrations of several B vitamins were positively related (table 2). The riboflavin-folate and the vitamin B6–folate relationships were modified by MTHFR genotype. Plasma tHcy was inversely related to all B vitamins, and the tHcy–B vitamin relationships were modified by the MTHFR polymorphism and were strongest in subjects with the TT genotype, particularly for folate and riboflavin (table 2). A positive relationship (r=0.21) was observed between tHcy and creatinine.Table 2Spearman Correlation CoefficientsVariable and GenotypetHcyFolateRiboflavinCobalaminFolate:−.44 CC−.38 CT−.45 TT−.58Riboflavin:−.18.26 CC−.15.23 CT−.19.28 TT−.38.35Cobalamin:−.24.16.20 CC−.23.14.19 CT−.25.19.22 TT−.28.16.17Vitamin B6:−.24.39.35.18 CC−.20.35.34.16 CT−.24.41.36.20 TT−.38.47.43.17Note.—Nonparametric Spearman correlation coefficients adjusted for sex, age, and study center are shown for the entire population (n=10,570) and separately for the MTHFR 677CC (n=5,433), 677CT (n=4,282), and 677TT (n=843) genotypes. All correlations were highly significant (P<.001). Open table in a new tab Note.— Nonparametric Spearman correlation coefficients adjusted for sex, age, and study center are shown for the entire population (n=10,570) and separately for the MTHFR 677CC (n=5,433), 677CT (n=4,282), and 677TT (n=843) genotypes. All correlations were highly significant (P<.001). The MTHFR polymorphism, folate, riboflavin, cobalamin, vitamin B6, and creatinine were independently related to plasma tHcy in regression models adjusted for sex, age, and study center (P<.001; data not shown) and in models with multiple adjustments (P<.001) (table 3).Table 3Determinants of Plasma tHcy according to MTHFR 677C→T GenotypeMTHFR GenotypeAllCC (n=5,452)CT (n=4,299)TT (n=850)VariableEstimated tHcy DifferenceaValues are given as means (95% CIs), in μmol/liter.P for trendEstimated tHcy DifferenceaValues are given as means (95% CIs), in μmol/liter.P for trendEstimated tHcy DifferenceaValues are given as means (95% CIs), in μmol/liter.P for trendEstimated tHcy DifferenceaValues are given as means (95% CIs), in μmol/liter.P for trendInteraction Term (P)bP for the product term between MTHFR genotype and the various tHcy predictors.Sex (vs. female; n=5,386): Male (n=5,213).4 (.2–.5)<.001.3 (.1–.5)<.001.4 (.2–.6)<.0011.0 (−.1–2.0).13.04Age, years (vs. 50–53; n=2,540): 54–55 (n=3,052).5 (.3–.7).5 (.4–.7).4 (.1–.6)1.0 (−.2–2.2) 56–59 (n=2,637).7 (.5–.9)<.001.7 (.5–.9)<.001.7 (.4–1.0)<.001.5 (−.7–1.7).16.34 60–64 (n=2,370)1.2 (1.0–1.3)1.2 (1.0–1.4)1.0 (.8–1.3)1.5 (.2–2.7)Serum folate, nmol/liter (vs. 20.2–151; n=2,647): 13.7–20.2 (n=2,647).8 (.6–1.0).8 (.6–1.0).9 (.6–1.1)1.3 (−.2–2.9) 10.1–13.7 (n=2,646)1.2 (1.0–1.4)<.0011.3 (1.1–1.5)< .0011.3 (1.0–1.6)<.0011.1 (−.4–2.7)<.001<.001 1.5–10.1 (n=2,651)3.0 (2.8–3.2)2.3 (2.1–2.5)3.0 (2.7–3.3)5.4 (4.0–6.8)Plasma riboflavin, nmol/liter (vs. 18.1–596; n=2,644): 10.4–18.1 (n=2,656).1 (−.1–.3).1 (−.1–.3).0 (−.3–.3)−.1 (−1.4–1.1) 6.8–10.4 (n=2,649).2 (.0–.4)<.001.1 (−.1–.3)< .001.2 (−.1–.5)<.0011.0 (−.2–2.3)<.001<.001 .9–6.8 (n=2,652).8 (.6–1.0).4 (.2–.6).7 (.4–.9)4.1 (2.7–5.5)Serum cobalamin, pmol/liter (vs. 380–6,500; n=2,644): 307–380 (n=2,650).3 (.1–.5).2 (.0–.4).5 (.2–.7).6 (−.7–1.8) 245–307 (n=2,646).5 (.3–.7)<.001.4 (.2–.5)<.001.6 (.3–.8)<.0011.4 (.1–2.6)<.001<.001 34–245 (n=2,645)1.5 (1.3–1.6)1.2 (1.0–1.4)1.5 (1.3–1.8)3.2 (2.0–4.5)Plasma vitamin B6, nmol/liter (vs. 73.2–1,093; n=2,651): 48.0–73.1 (n=2,655).1 (−.1–.3).2 (.0–.4).1 (−.2–.4)−.5 (−1.9–.8) 32.6–47.9 (n=2,642).1 (−.1–.3)<.001.2 (.0–.4)<.001.0 (−.3–.3)<.01−.4 (−1.8–1.0).01.006 4.3–32.6 (n=2,653).7 (.5–.9).5 (.3–.8).5 (.2–.8)2.1 (.6–3.7)Serum creatinine, μmol/liter (vs. 30–61; n=2,650): 61–69 (n=2,646).3 (.1–.5).5 (.3–.7).1 (−.1–.4).4 (−.8–1.5) 69–78 (n=2,659).8 (.7–1.0)<.0011.0 (.8–1.2)<.001.5 (.2–.8)<.0011.5 (.2–2.9).003.29 78–571 (n=2,645)1.7 (1.5–1.9)1.8 (1.6–2.0)1.5 (1.2–1.8)2.5 (1.0–4.0)MTHFR 667C→T genotype (vs. CC; n=5,254): CT (n=4,299).4 (.3–.5)<.001 TT (n=850)2.4 (2.2–2.6)Note.—Data were analyzed by multiple regression with tHcy as the dependent variable. All variables in the table and study center were included in the model.a Values are given as means (95% CIs), in μmol/liter.b P for the product term between MTHFR genotype and the various tHcy predictors. Open table in a new tab Note.— Data were analyzed by multiple regression with tHcy as the dependent variable. All variables in the table and study center were included in the model. The estimated difference in mean plasma tHcy between subjects with the TT genotype compared with the CC genotype was 2.4 μmol/liter (table 3). Folate was a strong tHcy predictor, and tHcy was 3.0 μmol/liter higher in subjects in the lowest compared with the highest quartile of folate concentrations (table 3). MTHFR genotype strongly modified folate effects, and, in TT subjects, plasma tHcy was 5.4 μmol/liter higher in the lowest compared with the highest folate quartile. Riboflavin was only a weak tHcy predictor in subjects with the CC and CT genotypes, but, in the TT group, riboflavin was the second strongest tHcy predictor, and the difference between extreme riboflavin quartiles was 4.1 μmol/liter of plasma tHcy (table 3). The cobalamin-tHcy and vitamin B6–tHcy relationships were similarly but less strongly related to genotype. The tHcy difference between extreme vitamin quartiles in subjects with the TT genotype was 3.2 μmol/liter for cobalamin and 2.1 μmol/liter for vitamin B6 (table 3). Sex, but not age or creatinine, interacted with genotype. Predictors of plasma tHcy were studied in subjects with the CC, CT, and TT genotypes at folate concentrations below and above the median, with the use of regression models with multiple adjustments (fig. 1). There was an inverse relationship between riboflavin and plasma tHcy, which was modified by MTHFR genotype, both at low and high folate concentrations (fig. 1). The relationship was strongest at low folate levels, however, and, for subjects in the TT group, the estimated tHcy difference between extreme riboflavin quartiles was 5.0 μmol/liter, whereas the corresponding difference was 1.8 μmol/liter at high folate levels. At low folate levels, cobalamin was also strongly and inversely related to tHcy (fig. 1). At high folate concentrations, this relationship was dramatically weakened, particularly in the TT group. Vitamin B6 was related to tHcy both at low and high folate concentrations, but at high folate levels no genotype effect was observed (fig. 1). MTHFR genotype and B vitamin effects were similarly studied at concentrations of plasma riboflavin below and above the median (fig. 2). Folate was robustly related to plasma tHcy, both at low and high riboflavin concentrations, but high riboflavin levels weakened the folate-tHcy relationship in subjects with the TT genotype. Cobalamin was related to plasma tHcy at low and high riboflavin levels, but the relationship was much weaker and was not significantly modified by genotype when riboflavin levels were high (fig. 2). Vitamin B6 was moderately related to plasma tHcy at low and high riboflavin levels, particularly in subjects with the variant enzyme, but the genotype dependency of the vitamin B6–tHcy relationship was weaker when riboflavin levels were high. Concentrations of cobalamin below and above the median had a similar but less pronounced effect on the folate-tHcy, riboflavin-tHcy, and vitamin B6–tHcy relationships in subjects with different MTHFR genotypes (fig. 3). High levels of cobalamin weakened the genotype dependency of the B vitamin–tHcy associations. Vitamin B6 had a similar impact on the effects of other B vitamins and the MTHFR genotype (fig. 4). Folate, riboflavin, and cobalamin were determinants of plasma tHcy both at low and high levels of vitamin B6, but high levels of vitamin B6 attenuated the relationships between the other B vitamins and tHcy, and the cobalamin-tHcy relationship showed no genotype dependency at high vitamin B6 levels.Figure 4B vitamins as determinants of plasma tHcy according to vitamin B6 concentrations and MTHFR 677C→T genotype. The population was stratified according to levels of plasma vitamin B6 (below and above the median) and MTHFR 677C→T genotype. The folate-tHcy, riboflavin-tHcy, and cobalamin-tHcy relationships were then studied in a regression model, which included these vitamins in addition to sex, age, creatinine, and study center. Means with upper limits of 95% CIs and P for trend across quartiles are shown in each panel. Nutrient-gene interaction terms were calculated as the product between MTHFR genotype and the various B vitamins.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We studied the MTHFR 677C→T polymorphism and several B vitamins that are involved in one-carbon metabolism in a large population-based cohort (n=10,601), using plasma tHcy as the main outcome measure. The MTHFR polymorphism, folate, riboflavin, cobalamin, and vitamin B6 were independently related to plasma tHcy. The MTHFR polymorphism had the strongest impact when B vitamin levels were low. Conversely, MTHFR genotype modified B vitamin–tHcy relationships, which were strongest in subjects with the T allele, particularly for folate and riboflavin. Finally, interactions between B vitamins were
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