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BACKGROUND Investigations of folate-mediated one-carbon metabolism (FOCM) genes and gene-nutrient interactions with respect to colorectal cancer (CRC) risk are limited to candidate polymorphisms and dietary folate. This study comprehensively investigated associations between genetic variants in FOCM and CRC risk and whether the FOCM nutrient status modified these associations. METHODS Two hundred eighty-eight candidate and tagging single-nucleotide polymorphisms (SNPs) in 30 FOCM genes were genotyped for 821 incident CRC case-control matched pairs in the Women's Health Initiative Observational Study cohort. FOCM biomarkers (red blood cell [RBC] folate, plasma folate, pyridoxal-5′-phosphate [PLP], vitamin B12, and homocysteine) and self-reported alcohol consumption were measured at the baseline. Conditional logistic regression was implemented; effect modification was examined on the basis of known enzyme-nutrient relations. RESULTS Statistically significant associations were observed between CRC risk and functionally defined candidate SNPs of methylenetetrahydrofolate dehydrogenase 1 (MTHFD1; K134R), 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR; P450R), and PR domain containing 2 with ZNF domain (PRDM2; S450N) and a literature candidate SNP of thymidylate synthase (TYMS; g.676789A>T; nominal P

Folate-mediated one-carbon metabolism is essential for DNA synthesis, repair, and methylation. Perturbations in one-carbon metabolism have been implicated in increased risk of some cancers and may also affect inflammatory processes. We investigated these interrelated pathways to understand their relation. The objective was to explore associations between inflammation and biomarkers of nutritional status and one-carbon metabolism. In a cross-sectional study in 1976 women selected from the Women's Health Initiative Observational Study, plasma vitamin B-6 [pyridoxal-5'-phosphate (PLP)], plasma vitamin B-12, plasma folate, and RBC folate were measured as nutritional biomarkers; serum C-reactive protein (CRP) and serum amyloid A (SAA) were measured as biomarkers of inflammation; and homocysteine and cysteine were measured as integrated biomarkers of one-carbon metabolism. Student's t, chi-square, and Spearman rank correlations, along with multiple linear regressions, were used to explore relations between biomarkers; additionally, we tested stratification by folic acid fortification period and multivitamin use. With the use of univariate analysis, plasma PLP was the only nutritional biomarker that was modestly significantly correlated with serum CRP and SAA (ρ = -0.22 and -0.12, respectively; P < 0.0001). Homocysteine (μmol/L) showed significant inverse correlations with all nutritional biomarkers (ranging from ρ = -0.30 to ρ = -0.46; all P < 0.0001). With the use of multiple linear regression, plasma PLP, RBC folate, homocysteine, and cysteine were identified as independent predictors of CRP; and PLP, vitamin B-12, RBC folate, and homocysteine were identified as predictors of SAA. When stratified by folic acid fortification period, nutrition-homocysteine correlations were generally weaker in the postfortification period, whereas associations between plasma PLP and serum CRP increased. Biomarkers of inflammation are associated with PLP, RBC folate, and homocysteine in women. The connection between the pathways needs to be further investigated and causality established. The trial is registered at clinicaltrials.gov as NCT00000611.

Background: It has been hypothesized that the response of holo-transcobalamin (holo-TC) to oral vitamin B -12 may be used to assess absorption. To develop a reliable clinical absorption test that uses holo-TC, it is necessary to determine the optimal timeline for vitamin B-12 administration and postdose assessment. Objective: The objective of this study was to assess the magnitude and patterns of change in the postabsorption response of holo-TC to oral vitamin B-12. Design: Adult (18 -49 y) male and female participants (n = 21) with normal vitamin B-12 status were given three 9-μg doses of vitamin B-12 at 6-h intervals beginning early morning (baseline) on day 1. Blood was drawn at 17 timed intervals over the course of 3 d for the analysis of holo-TC and other indicators of vitamin B-12 status. Results: Mean holo-TC increased significantly (P < 0.001) from baseline at 6 h (11%) and 24 h (50%). TC saturation increased significantly (P < 0.001) from baseline at 12.5 h (33%) and 24 h (50%). The mean cobalamin concentration changed significantly (P

Whether folate status and the methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism interact to affect methionine-cycle metabolite concentrations is uncertain. We evaluated the effects of dietary folate restriction on relations among folate status indices and plasma concentrations of methionine cycle metabolites in women with the MTHFR 677 C/C and T/T genotypes. Healthy, normohomocysteinemic women (n = 18; 20-30 y old) of adequate B vitamin status, and equally divided according to MTHFR 677C-->T genotype (9 C/C and 9 T/T) were recruited. Folate status indices and methionine cycle metabolites were measured in blood samples collected at baseline and after 7 wk of dietary folate restriction (115 microg dietary folate equivalents/d). Significant negative correlations between plasma total homocysteine concentrations and total or 5-methyl folate concentrations (P = 0.041 and 0.023, respectively) in RBCs were found only in T/T subjects. Formylated folates were detected in RBCs of T/T subjects only, and their abundance was predictive of plasma total homocysteine concentration despite no significant alteration by folate restriction. Plasma concentrations of S-adenosylmethionine and S-adenosylhomocysteine were not significantly affected by dietary folate restriction and the MTHFR 677 T/T genotype. In conclusion, plasma total homocysteine concentrations in subjects with the MTHFR 677 T/T genotype were inversely related to 5-methyl folate concentrations and directly related to formylated folate concentrations in RBCs, even though the latter were not significantly affected by moderate folate restriction.

Background: A common genetic polymorphism [transcobalamin (TC) 776C→G] may affect the function of transcobalamin, the protein required for vitamin B-12 cellular uptake and metabolism. Remethylation of homocysteine is dependent on the production of 5-methyltetrahydrofolate and adequate vitamin B-12 for the methionine synthase reaction. Objectives: The objectives were to assess the influence of the TC 776C→G polymorphism on concentrations of the transcobalamin-vitamin B-12 complex (holo-TC) and to determine the combined effects of the TC 776C→G and methylenetetrahydrofolate reductase (MTHFR) 677C→T polymorphisms and vitamin B-12 status on homocysteine concentrations. Design: Healthy, nonpregnant women (n = 359; aged 20-30 y) were screened to determine plasma vitamin B-12, serum holo-TC, and plasma homocysteine concentrations and TC 776C→G and MTHFR 677C→T genotypes. Results: The serum holo-TC concentration for women with the variant TC 776 GG genotype was significantly different (P = 0.0213) from that for subjects with the CC genotype (74 ± 37 and 87 ± 33 pmol/L, respectively). An inverse relation was observed between plasma homocysteine concentrations and both serum holo-TC (P ≤ 0.0001) and plasma vitamin B-12 (P ≤ 0.0001) concentrations, regardless of genotype. Conclusions: These data suggest that the TC 776C→G polymorphism negatively affects the serum holo-TC concentration and provide additional evidence that vitamin B-12 status modulates the homocysteine concentration in this population.

Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) are important for homocysteine remethylation. This study was designed to determine the influence of genetic variants (MTHFR 677C--T, MTHFR 1298A--C, and MTRR 66A--G), folate, and vitamin B-12 status on plasma homocysteine in women (20-30 y; n = 362). Plasma homocysteine was inversely (P0.0001) associated with serum folate and plasma vitamin B-12 regardless of genotype. Plasma homocysteine was higher (P0.05) for women with the MTHFR 677 TT/1298 AA genotype combination compared with the CC/AA, CC/AC, and CT/AA genotypes. Women with the MTHFR 677 TT/MTRR 66 AG genotype had higher (P0.05) plasma homocysteine than all other genotype combinations except the TT/AA and TT/GG genotypes. There were 5.4-, 4.3-, and 3.8-fold increases (P0.001) in risk for plasma homocysteine in the top 5, 10, and 20%, respectively, of the homocysteine distribution for subjects with the MTHFR 677 TT compared with the CC and CT genotypes. Predicted plasma homocysteine was inversely associated with serum folate (P = 0.003) and plasma vitamin B-12 (P = 0.002), with the degree of correlation dependent on MTHFR 677C--T genotype. These data suggest that coexistence of the MTHFR 677 TT genotype with the MTRR 66A--G polymorphism may exacerbate the effect of the MTHFR variant alone. The potential negative effect of combined polymorphisms of the MTHFR and MTRR genes on plasma homocysteine in at-risk population groups with low folate and/or vitamin B-12 status, such as women of reproductive potential, deserves further investigation.

DNA methylation is critical for normal genomic structure and function and is dependent on adequate folate status. A polymorphism (677C-->T) in a key folate enzyme, methylenetetrahydrofolate reductase (MTHFR), may impair DNA methylation when folate intake is inadequate and may increase the risk of reproductive abnormalities. The present study was designed to evaluate the effect of the MTHFR 677C-->T polymorphism on changes in global DNA methylation in young women consuming a low folate diet followed by repletion with the current Recommended Dietary Allowance (RDA). Women (age 20-30 years) with the TT (variant; n = 19) or CC (n = 22) genotype for the MTHFR 677C-->T polymorphism participated in a folate depletion-repletion study (7 weeks, 115 microg DFE/day; 7 weeks, 400 microg DFE/day). DNA methylation was measured at baseline, week 7, and week 14 using a [3H]methyl acceptance assay and a novel liquid chromatography tandem mass spectrometry assay of the DNA bases methylcytosine and cytosine. [3H]Methyl group acceptance tended to increase (P = 0.08) during depletion in all subjects, indicative of a decrease in global DNA methylation. During repletion, the raw change and the percent change in the methylcytosine/total cytosine ratio increased (P = 0.03 and P = 0.04, respectively) only in the subjects with the TT genotype. Moderate folate depletion in young women may cause a decrease in overall DNA methylation. The response to folate repletion suggests that following folate depletion women with the MTHFR 677 TT genotype have a greater increase in DNA methylation with folate repletion than women with the CC genotype.

This study was designed to evaluate the effect of the methylenetetrahydrofolate reductase (MTHFR) 677C→T polymorphism on folate and homocysteine response in nonHispanic women consuming a low folate diet followed by a diet providing the Recommended Dietary Allowance (RDA) for folate. Women (aged 20-30 y old) with either the TT (n = 19) or CC (n = 22) MTHFR 677C→T genotype participated in a folate depletion-repletion study (7 wk, 115 μg dietary folate equivalents (DFE)/d; 7 wk, 400 μg DFE/d). Overall serum folate decreased (P < 0.0001) during depletion and increased (P < 0.0001) during repletion with lower (P = 0.03) postdepletion serum folate in women with the TT versus CC genotype. Folate status was low (serum folate < 13.6 nmol/L) in more women with the TT (59%) compared with the CC genotype (15%) postdepletion. Red blood cell folate for all subjects decreased during depletion (P < 0.0001) and repletion (P = 0.02) with lower (P = 0.04) red blood cell folate in women with the TT compared with the CC genotype postrepletion. Homocysteine increased (P < 0.0001) for both genotype groups postdepletion and decreased (P = 0.02) postrepletion for the CC genotype group only. Homocysteine concentrations tended to be higher (P = 0.09) in the TT versus CC genotype group postdepletion and postrepletion. These data suggest that the MTHFR 677C→T polymorphism negatively affects the folate and homocysteine response in women consuming low folate diets followed by repletion with the RDA. These results may be important when evaluating the impact of the MTHFR 677C→T polymorphism in countries in which low folate diets are chronically consumed.

Methylenetetrahydrofolate reductase (MTHFR) polymorphisms may negatively influence one-carbon metabolism and increase health risks in women of reproductive age. The effect of MTHFR single nucleotide polymorphisms at bp 677 and/or 1298 and differences in folate and vitamin B-12 status on plasma homocysteine concentration in women of reproductive age (20-30 y; n = 186) were investigated. From the multivariate regression model, homozygotes (n = 23) for the C677T MTHFR variant had plasma homocysteine concentrations that were higher (P < 0.05) than those observed in the other 5 genotype groups, including those who were heterozygous for both variants (677CT/1298AC; n = 32). Plasma homocysteine was negatively associated with plasma vitamin B-12 concentration (P = 0.015) and serum folate (P = 0.049), with the degree of correlation between plasma vitamin B-12 and homocysteine concentrations dependent on MTHFR genotype. The C677T and A1298C MTHFR polymorphisms were significant predictors (P < 0.05) of plasma homocysteine when regression analysis was used to model plasma homocysteine concentration as a function of genotype, supplement use, serum folate and plasma vitamin B-12 concentration. Plasma homocysteine decreased as vitamin B-12 concentration increased (P = 0.0005) in individuals who were heterozygous for both the C677T and A1298C variants with nonsignificant trends (P = 0.114-0.128) in individuals homozygous for either the C677T or A1298C variants. In contrast, within the group of individuals with the wild-type genotype for both the C677T and A1298C MTHFR variants, homocysteine was not associated with changes in plasma vitamin B-12 concentrations. These data suggest that enhancing vitamin B-12 status may significantly decrease homocysteine in young women with C677T and/or A1298C MTHFR polymorphisms, even when vitamin B-12 concentrations are within the normal range.

Background: Folate-mediated one-carbon metabolism is essential for DNA synthesis, repair, and methylation. Perturbations in associated biomarkers have been implicated in increased risk of colorectal cancer. Inflammation, especially related to inflammatory bowel disease, is also implicated as a risk factor for colorectal cancer, and one-carbon nutrients, particularly vitamins B6 and B12, may directly affect inflammatory processes. A concurrent investigation of these two interrelated pathways could yield new information regarding relationships between inflammatory processes and one-carbon metabolism. Objective: To investigate correlations between nutritional, inflammatory, and one-carbon metabolism biomarkers. Methods: A cross-sectional study design was used to explore correlations between biomarkers of nutrition, inflammation and one-carbon metabolism in 1,976 women selected from the 93,676 participants in the Women's Health Initiative Observational Study. Nutritional biomarkers included vitamin B6 (pyridoxal-5'-phosphate [PLP]), vitamin B12, plasma folate, and red blood cell (RBC) folate. C-reactive protein (CRP) and serum amyloid A (SAA) were used as biomarkers of inflammation. Homocysteine and cysteine levels were measured as integrated biomarkers of one-carbon metabolism. All biomarkers were measured using established methods from samples obtained at baseline. SAS 9.2 was used to perform Student's t, Chi-squared, and Spearman rank correlation tests, along with multivariate linear regressions adjusted for age and body mass index (BMI), to explore the relationships between the biomarkers, with statistical significance at p Results: Plasma PLP was the only nutritional one-carbon metabolism biomarker to show significant inverse correlations with both CRP and SAA (r=-0.22, -0.12 respectively; p Conclusions: Biomarkers of inflammation are associated with concentrations of PLP, cysteine and homocysteine. While these associations between the biomarkers of inflammation, diet, and one-carbon metabolism are promising, determining causality is an important next step. Citation Format: Michael J. Paskow, Clare Abbenhardt, Joshua W. Miller, Xiaoling Song, Elissa C. Brown, Ting-Yuan David Cheng, Mark H. Wener, Yingye Zheng, Adetunji T. Toriola, Marian L. Neuhouser, Shirley A. A. Beresford, Karen Makar, Lynn B. Bailey, David R. Maneval, Ralph Green, JoAnn Manson, Linda Van Horn, Cornelia Ulrich. Relationships between biomarkers of diet, one-carbon metabolism, and inflammation within the Women's Health Initiative observational study. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 34.

Introduction: There is strong evidence that chronic inflammation plays a role in colorectal carcinogenesis. Despite this, epidemiological studies that have investigated this association using C-reactive protein (CRP) as an inflammatory biomarker have reported conflicting results, especially among women. Limitations of previous studies include the small number of cases and the fact that CRP measurements were performed at only one point in time. Likewise, no previous study has investigated the association of serum amyloid A (SAA), a related marker of inflammation, and colorectal cancer (CRC) risk. We investigated the associations of CRP and SAA with CRC risk, using repeat assessments, in a case-control study nested within the Women's Health Initiative Observational Study cohort (WHI-OS). We also explored the impact of changes in the two biomarkers (from baseline to 3rd year of follow-up) on CRC risk. Methods: The WHI-OS is a prospective cohort study that enrolled 93,676 post-menopausal women between 1993 and 1998 at 40 U.S. clinical institutions. We identified 988 women with CRC and matched 988 cancer-free control women based on age (±3 years), clinical center, race/ethnicity, and time of blood draw (±6 months). CRP and SAA measurements were performed at baseline and during the 3rd year of follow-up. Conditional logistic regression models were used to estimate the multivariate-adjusted odds ratios and 95% confidence intervals (OR, 95% CI) of CRC (adjusted for age, body mass index, post-menopausal hormone use (HRT) and previous history of colonoscopy). Results: Elevated CRP, but not SAA concentrations were positively associated with CRC risk. Women in the highest quintiles of CRP and SAA concentrations had an odds ratios of 1.28 (95% CI 0.94–1.76, p-trend=0.03) and 1.19 (95% CI 0.88–1.62, p-trend=0.17) respectively, compared to women in the lowest quintiles. Women who had high levels of both biomarkers had increased risk of colorectal cancer (OR=1.35, 95% CI 1.06–1.73, p-value=0.02), particularly colon cancer (OR=1.49, 95% CI 1.13–1.97, p-value=0.005) compared to those with low concentrations. Neither CRP nor SAA was positively associated with rectal cancer risk in any of the analyses. There was a non-significant increased risk of CRC (OR 1.31, 95% CI 0.91–1.89, p-trend=0.14) among women who had the largest increase in CRP concentration (from baseline to 3rd year of follow-up), while the risk associated with changes in SAA concentration was close to unity (OR 1.05 95% CI 0.73–1.49, p-trend 0.94). The associations between CRP and CRC risk were not modified by HRT use. Conclusions: Our study supports the hypothesis that inflammation is associated with colorectal carcinogenesis and that CRP and SAA used together may better predict CRC risk than either used alone. Citation Information: Cancer Prev Res 2011;4(10 Suppl):PR-05.

Introduction: The role of one-carbon metabolism related nutrients (folate, riboflavin (B2), B6, and B12) in colorectal carcinogenesis is still not fully understood. Folate and other B vitamins are essential for DNA methylation and repair and higher levels have been associated with a decreased risk for colorectal cancer (CRC) in some studies; however, non-linear relationships have been observed. We investigated the dietary intake of one-carbon nutrients and CRC risk in the WHI Observational Study, a large cohort of postmenopausal women. Methods: Dietary intake of folate and other B vitamins was determined via a Food Frequency Questionnaire and supplement inventory in 88,045 healthy women (aged 50 – 79 years, recruited between 1994 – 1998). Multivariate Cox proportional hazards regression models was used to estimate associations between nutrient intake and CRC risk and to evaluate differences in the associations by tumor site and stage. Hazard ratios (HR) for the 4th vs. 1st quartile were estimated. Results: In age-adjusted analyses significantly reduced risks for CRC were observed for the total (supplemental plus dietary) intake of folate (HR=0.82, 95%CI=0.68-0.97), vitamin B12 (HR=0.82, 0.69-0.99), vitamin B6 (HR=0.77, 0.65-0.92), and riboflavin (HR=0.75, 0.63-0.90). After multivariate adjustment (age, BMI, race, prior colonoscopy, smoking, physical activity, postmenopausal HT use) only the total intake of riboflavin (HR=0.80, 0.66-0.96) remained associated with a reduced risk for CRC. For the intake of vitamin B6 a borderline significance was observed (p=0.06, HR=0.85, 0.70-1.02). A site-specific observation was notable for supplemental and total riboflavin intake with a reduced risk for cancer of the distal colon (HRsupp=0.56, 0.33-0.96; HRtotal=0.69, 0.46-1.04) but not in proximal and rectal cancer. In regards to tumor stage, no significant difference was detected for localized and distant disease; the inverse association between the supplemental and total intake of riboflavin and vitamin B6 was limited to regionally spread disease (HRB2;supp=0.66, 0.45-0.96; HRB2;total=0.73, 0.54-0.99; HRB6;supp=0.69, 0.49-0.98; HRB6;total=0.72, 0.53-0.97). Conclusion: Higher riboflavin and vitamin B6 intake was linked to decreased risk for colorectal cancer in a large cohort of postmenopausal women, with some suggestion for stage- and site-specific associations. Our study provides limited support for an association of other B vitamins with CRC risk. (NIH R01 CA120523, N01WH22110) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1904. doi:10.1158/1538-7445.AM2011-1904